Advances in The Characterization of Usnea Barbata

applied
sciences
Article
Advances in the Characterization of Usnea barbata (L.) Weber ex
F.H. Wigg from Călimani Mountains, Romania
Violeta Popovici 1 , Laura Bucur 2, *, Cerasela Elena Gîrd 3, * , Suzana Ioana Calcan 4 , Elena Iulia Cucolea 4 ,
Teodor Costache 4 , Dan Rambu 4 , Mădălina Ungureanu-Iuga 5,6, * , Mircea Oroian 5, *, Silvia Mironeasa 5,† ,
Verginica Schröder 7, * , Emma Adriana Ozon 8,† , Aureliana Caraiane 9,† and Victoria Badea 1,†
1 Department of Microbiology and Immunology, Faculty of Dental Medicine, Ovidius University of Constanta,
7 Ilarie Voronca Street, 900684 Constanta, Romania; violeta.popovici@365.univ-ovidius.ro (V.P.);
victoria.badea@365.univ-ovidius.ro (V.B.)
2 Department of Pharmacognosy, Faculty of Pharmacy, Ovidius University of Constanta, 6 Capitan Al.
Serbanescu Street, 900001 Constanta, Romania
3 Department of Pharmacognosy, Phytochemistry, and Phytotherapy, Faculty of Pharmacy,
Carol Davila University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956 Bucharest, Romania
4 Research Center for Instrumental Analysis SCIENT, 1E Petre Ispirescu Street, 077167 Ilfov, Romania;
suzana.calcan@scient.ro (S.I.C.); iulia.cucolea@scient.ro (E.I.C.); teodor.costache@scient.ro (T.C.);
dan.rambu@scient.ro (D.R.)
5 Faculty of Food Engineering, Stefan cel Mare University of Suceava, 13th University Street,
720229 Suceava, Romania; silviam@fia.usv.ro
6 Integrated Center for Research, Development, and Innovation in Advanced Materials, Nanotechnologies and
Distributed Systems for Fabrication and Control (MANSiD), Stefan cel Mare University of Suceava,
13th University Street, 720229 Suceava, Romania
7 Department of Cellular and Molecular Biology, Faculty of Pharmacy, Ovidius University of Constanta,
6 Capitan Al. Serbanescu Street, 900001 Constanta, Romania
8 Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, Carol Davila University of
Citation: Popovici, V.; Bucur, L.; Gîrd,
C.E.; Calcan, S.I.; Cucolea, E.I.; Medicine and Pharmacy, 6 Traian Vuia Street, 020956 Bucharest, Romania; emma.budura@umfcd.ro
9 Department of Oral Rehabilitation, Faculty of Dental Medicine, Ovidius University of Constanta, 7 Ilarie
Costache, T.; Rambu, D.;
Voronca Street, 900684 Constanta, Romania; aureliana.caraiane@365.univ-ovidius.ro
Ungureanu-Iuga, M.; Oroian, M.;
* Correspondence: laurabucur@univ-ovidius.ro (L.B.); cerasela.gird@umfcd.ro (C.E.G.);
Mironeasa, S.; et al. Advances in the
madalina.iuga@usm.ro (M.U.-I.); m.oroian@fia.usv.ro (M.O.); verginica.schroder@univ-ovidius.ro (V.S.)
Characterization of Usnea barbata (L.) † These authors contributed equally to this work.
Weber ex F.H. Wigg from Călimani
Mountains, Romania. Appl. Sci. 2022,
Abstract: Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) is a medicinal representative of the
12, 4234. https://doi.org/10.3390/
lichens from the Usnea genus (Parmeliaceae, lichenized Ascomycetes), containing bioactive secondary
app12094234
metabolites. The aim of this study is a comparative analysis between two separated parts of the thallus
Academic Editor: Luca Mazzoni layers: medulla–cortex (mcUB) and central cord (ccUB) and the whole dried U. barbata thallus (dUB).
Received: 17 February 2022
These three samples were examined regarding color differences. The U. barbata thallus morphology
Accepted: 19 April 2022 was examined through fluorescent microscopy (FM) and scanning electron microscopy (SEM). The
Published: 22 April 2022 mineral content was measured using inductively coupled plasma mass spectrometry (ICP-MS),
and Fourier transform infrared spectroscopy (FT-IR) preliminarily established the differences in
Publisher’s Note: MDPI stays neutral
the metabolite content. Finally, extracts in different solvents (ethanol and acetone) were obtained
with regard to jurisdictional claims in
from all studied samples, and their total phenolic content (TPC) and free radical scavenging activity
published maps and institutional affil-
iations.
(antiradical activity, AA) were evaluated by spectrophotometry. The ICP-MS results showed that
from 23 elements analyzed, 18 minerals were quantified in mcUB, 13 in dUB, and only 12 in ccUB. The
ccUB fraction recorded the lowest mineral content, color intensity (chroma), luminosity (L*), and TPC
value, followed in increasing order by dUB and mcUB. FT-IR spectra displayed different peaks in ccUB
Copyright: © 2022 by the authors. and dUB samples compared to mcUB. The mcUB fraction also showed the highest TPC, significantly
Licensee MDPI, Basel, Switzerland. correlated with AA. However, dUB had the highest antiradical activity, followed by mcUB and ccUB,
This article is an open access article
with noticeable differences in the acetone extract. The final correlation between all variable data
distributed under the terms and
obtained indicates that 99.31% of the total variance was associated with all minerals, total phenolics,
conditions of the Creative Commons
and color parameters and was also related to the antiradical activity. These obtained results complete
Attribution (CC BY) license (https://
our previous studies on autochthonous U. barbata. Moreover, being a source of bioactive metabolites,
creativecommons.org/licenses/by/
4.0/).
extracting them from the mcUB fraction could increase this process’s yield and selectivity.
Appl. Sci. 2022, 12, 4234. https://doi.org/10.3390/app12094234 https://www.mdpi.com/journal/applsci

Appl. Sci. 2022, 12, 4234 2 of 20
Keywords: Usnea barbata (L.) Weber ex F.H. Wigg; morphology; color; minerals; phenolic secondary
metabolites; free radical scavenging activity
1. Introduction
Lichens (also known as lichenized fungi [1]) are symbiont organisms between fungi
and algae/cyanobacteria [2]. They could be considered a potential source of new drugs [3]
due to their bioactive secondary metabolites with various pharmacological effects [4]. One
of the most significant representatives in the lichens world is the Usnea genus (Parmeliaceae,
lichenized Ascomycetes), a potent phytomedicine with various pharmacological activities [5].
It has more than 600 species, endemic to Asia, Africa, Europe, and America and is used
worldwide for traditional medicines (except in Australia) and food preparation [6]. Us-
nea sp. can be transplanted for air monitoring [7], and, at the same time, their culture
conditions could be optimized, aiming for biomass increasing and antioxidant metabolite
production [8]. Due to their significant diversity, the Usnea lichens are mainly studied in
various Earth regions: Europe [9], India [10], Russia [11], Taiwan [12], Turkey [13], and
New Zeeland [14]. U. barbata is a valuable representative of the Usnea genus, used in
ethnomedicine, homeopathy, and the cosmetic industry [15]. Moreover, Redzic et al. [16]
proved that U. barbata was used as human food (mush and lichen bread) for survival in
four-year-long (1992–1995) war conditions in Bosnia.
All U. barbata benefits are due to its chemical composition, which is strongly dependent
on growth conditions [17]. Bazarnova et al. [18] quantified in U. barbata various primary and
secondary metabolites. The most well-known phenolic secondary metabolites reported are
oligosaccharides, monosaccharides (glucose and galactose), citric, succinic, alpha-linolenic,
and stearic acids. According to Salgado et al. [19], various polyhydroxylated lipids, fatty acids,
and other undetected compounds can be added to the U. barbata phytochemical profile.
The fungal partner (mycobiont) synthesizes these specific compounds mainly due
to symbiosis. As a response to biological and abiotic stress, the secondary metabolites
are extracellular and are found within the lichen thalli as crystals on the fungal hyphae
surface [20]. The primary metabolites are required for lichen growth and nutrition. They
are intracellular in fungal or algal (photobiont) cells [20]. By weight, the mycobiont pre-
dominates in U. barbata, compared to photobionts, assuring the form of the thallus built
due to both symbiotic partners’ activity. U. barbata is a fruticose lichen; its thallus has a
heteromerous structure with three layers: cortex, medulla, and central axis (central cord).
As a cover, U. barbata has a thin cortex (external layer) with a subcortical, low represented
algal zone. These structures are followed by a medulla and a cartilaginous central axis
(central cord) [9]. The upper cortex has a compact structure formed by a dense pack of
fungal hyphae, with a protective role. Algal cells interweaved with fungal hyphae compose
the green algal zone [9]. The medulla is thick and loose, with rare fungal hyphae. Finally,
the central axis (central cord) is formed by longitudinal fungal hyphae that are densely
packed, forming a compact cartilaginous structure [21]. Around this central cord, the
other layers (medulla and cortex) and algal zone have a radial disposition. Both associated
organisms function in the symbiotic process. The algal partner provides carbohydrates
resulting from photosynthesis; they are directly transferred to the lichen-forming fungus in
a specific form, thus increasing the resistance to desiccation [22]. The mycobiont assures the
nutrients and metals required for algal metabolic processes. Moreover, the fungal partner
synthesizes secondary metabolites to protect the photobiont against extreme temperatures,
UV exposure, desiccation, and herbivores [23]. When a high heavy metal accumulation in
the lichen thallus occurs, these lichen metabolites form complexes, increasing lichen metal
tolerance [24]. When heavy metals pass intracellularly, they are expected to influence the
lichen metabolic processes.
Numerous authors analyzed the lichen thallus components in their studies for various
reasons. Hájek et al. [25] showed the influence of low temperature on the Usnea sp. thalli
Appl. Sci. 2022, 12, 4234 3 of 20
structure. Zverina et al. [26] and Carreras et al. [27] described heavy metal accumulation
in Usnea sp. and the lichen thallus destruction by heavy metal stress. Bubach et al. [21]
performed a correlation between the matrix of the biological elements (Na, Mg, K, Ca, Fe)
and geographical parameters in both fractions (cortex–medulla and central axis) of Usnea
sp. affected by a volcanic eruption.
Our study’s novelty consists of separating and analyzing these two different parts
of the U. barbata thallus (medulla–cortex and central cord). We aimed to explore the mor-
phology of U. barbata lichen native to the volcanic Călimani Mountains using fluorescent
microscopy (FM) and electronic scanning microscopy (SEM) images. The obtained mi-
crographs provided the U. barbata integral thallus details compared to ground samples
with different particle sizes. The mineral content, phenolic secondary metabolites, and free
radical scavenging activity evaluation was completed with an overview of the correlation
between the data obtained for all lichen samples. By establishing the fraction with the
highest content of phenolic compounds, our study could increase the extraction yield of
pharmacologically active secondary metabolites.
2. Materials and Methods

2.1. Lichen Sample Preparation
U. barbata is native to Calimani Mountains, Romania (900 m altitude, 47◦ 290 N,
25◦ 120 E). The lichen harvesting was performed in March 2020 from a conifer forest belong-
ing to a natural peat bog region [28]. The freshly collected thalli were cleaned and naturally
dried in an airy room sheltered from direct sunlight at 18–25 ◦ C [29].
The dried lichen preservation for an extended period was performed in large paper
bags under similar conditions. At the Ovidius University of Constanta, Department of
Pharmaceutical Botany, Faculty of Pharmacy, U. barbata identification was realized using
standard methods [9]. Samples are preserved at Ovidius University of Constanta, in the
Herbarium of Pharmacognosy Department, Faculty of Pharmacy (Popovici 2/2020, Ph-
UOC) [29]. The dried lichen thalli were ground in a laboratory mill, LM 120 (Perkin Elmer,
Waltham, MA, USA) for 5 min [30]. After grinding, the dried U. barbata lichen was passed
through a sieve (no. 5 [31]) and separated into two fractions. The first fraction of the dried
lichen (dUB)—medulla–cortex (mcUB)—was passed again through the meshes of the same
sieve and separated as a moderately fine (315 µm) powder. The central cord represents the
second fraction (ccUB).
All lichen parts were ground again in the laboratory mill and passed through a sieve
(no. 7), thus obtaining a fine powder with particle size < 180 µm. The samples were kept in
small paper bags until analysis (Supplementary Material, Figure S1).
2.2. Lichen Morphology

2.2.1. Fluorescent Microscopy
Fluorescent microscopy images were obtained using an OPTIKA B-350 microscope
(Ponteranica, BG, Italy) blue filter (λex = 450–490 nm; λem = 515–520 nm) and green filter
(λex = 510–550 nm; λem = 590 nm). The dried U. barbata lichen thalli (Figure 1a) were
washed with deionized water (DIW, Merck Millipore, Burlington, MA, USA). The hand-
cut cross-sections were hydrated with phosphate saline buffer (Thermo Fisher Scientific,
Waltham, MA, USA), pH = 7.4, and stained with 3% acridine orange (Merck Millipore,
Burlington, MA, USA) for 5 min. The samples were rinsed with DIW and placed on
the microscope slides. The FM images were obtained at 100× and 400× magnification
and processed with Optikam Pro 3 Software (OPTIKA S.R.L., Ponteranica, BG, Italy). All
observations were performed in triplicate.
R PEER REVIEW 4 of 20
processed with Optikam Pro 3 Software (OPTIKA S.R.L., Ponteranica, BG, Italy). All
Appl. Sci. 2022, 12, 4234 4 of 20
(a) (b) (c) (d)

Figure 1. U. barbata dried lichen—entire
Figure thallus
1. U. barbata dried (a) and ground—dUB
lichen—entire (b); both separated
thallus (a) and ground—dUB fractions:fractions:
(b); both separated
medulla–cortex—mcUBmedulla–cortex—mcUB
(c) and central cord—ccUB (d). cord—ccUB (d).
(c) and central
2.2.2. Scanning Electron Microscopy

We aimed to recognize the specific U. barbata morphology by examining the ground sam-
We aimed to recognize
ples, dUB,the specific
mcUB, U.with
and ccUB, barbata morphology
particles by examining
of different sizes: approximatelythe
315 ground
µm (Figure 1b–d)
and 180 µm (Figure S1, Supplementary Material) using scanning
samples, dUB, mcUB, and ccUB, with particles of different sizes: approximately 315 μm electron microscopy.
Scanning electron microscopy (SEM) images were obtained using a VEGA II LSH
(Figure 1b–d) and 180 μm (Figure S1, Supplementary Material) using scanning electron
(Tescan, Czech Republic) device. The samples were fixed on double-sided carbon adhesive
microscopy. bands, and the acceleration tension was 30 kV. The SEM images were obtained at differ-
ent magnifications (200×, 300×, 1 k×, 2 k×, 5 k×) and scale bars (200 µm–5 µm). All
Scanning electron microscopy
observations were (SEM)
performedimages were obtained using a VEGA II LSH
in triplicate.
(Tescan, Czech Republic) device. The samples were fixed on double-sided carbon
2.3. Color Evaluation
adhesive bands, and the Theacceleration tension was 30 kV. The SEM images were obtained
color properties of U. barbata dried lichen and both parts (mcUB and ccUB) as a
at different magnifications (200×,
fine powder 300×, size
(particle 1 k×,< 2180
k×,
µm)5 k×)
wereand scale bars
determined (200 μm–5
in triplicate μm).
in the Allsystem
CIELab
by using a Konica Minolta CR-400 (Konica Minolta, Tokyo, Japan) colorimeter. The color
properties in terms of L* (lightness, 0 for absolute black, 100 for absolute white), a* (red-
2.3. Color Evaluation green intensity), and b* (yellow-blue intensity) were recorded in triplicate. The hue angle
(0◦ —red, 90◦ —yellow, 180◦ —green, and 270◦ —blue) and chroma value (0—gray, 100—pure
The color properties
color) of U.calculated
were barbata dried lichen and
using Equations both(2):parts (mcUB and ccUB) as a
(1) and
fine powder (particle size <180 μm) were determined in triplicate ∗ in the CIELab system
b
by using a Konica Minolta CR-400 (Konica Minolta, h abTokyo,
= arctanJapan) colorimeter. The color (1)
a∗
properties in terms of L* (lightness, 0 for absolute black, q
100 for absolute white), a* (red-
green intensity), and b* (yellow-blue intensity) were C ∗ =recorded
( a∗ )2 + in
(b∗triplicate.
)2 The hue angle (2)
(0°—red, 90°—yellow, 180°—green, and 270°—blue) and chroma value (0—gray, 100—
where hab —hue angle, C*—Chroma, L*—lightness, a*—positive values describe red and
pure color) were calculated
negative,using Equations
green nuance, (1) andvalues
b*—positive (2): represent yellow and negative, blue nuance.
𝑏∗
ℎ
2.4. Elemental Analysis = 𝑎𝑟𝑐𝑡𝑎𝑛 (1)
𝑎∗
ICP-MS analyzed twenty-three elements in lichen samples: calcium (Ca), iron (Fe),
(magnesium (Mg), manganese (Mn), zinc (Zn), aluminum (Al), silver (Ag), barium (Ba),
cobalt (Co), chromium𝐶 ∗ =(Cr), copper
𝑎∗ +(Cu), 𝑏 ∗ lithium (Li), nickel (Ni), thallium (Tl), (2)
vanadium
(V), molybdenum (Mo), palladium (Pa), platinum (Pt), antimony (Sb), arsenic (As), lead
where hab—hue angle, C*—Chroma,
(Pb), cadmium (Cd),L*—lightness,
and mercury (Hg). a*—positive
Our previous values
study describe redmethod
described this and [28],
negative, green nuance,
and b*—positive values
detailed data can represent
be found yellow and
in Supplementary negative,
Material, Tablesblue
S1–S4.nuance.
The NexION™ 300S inductively coupled plasma mass spectrometer (PerkinElmer,
2.4. Elemental AnalysisInc., Hopkinton, MA, USA) was the platform for elemental analysis; dried lichen sample
digestion was performed with 65% HNO3 and 30% H2 O2. [28].
ICP-MS analyzed twenty-three elements
The obtained results in ICP-MS
from the lichen analysis
samples:werecalcium
processed(Ca),
with iron (Fe),
Syngistix Software
(magnesium (Mg), manganese
(PerkinElmer, (Mn), zinc (Zn),MA,
Inc, Hopkinton, aluminum (Al),2.3.
USA) Version silver
This(Ag), barium (Ba),
determination was done in
triplicate, and the mineral concentrations were expressed as the
cobalt (Co), chromium (Cr), copper (Cu), lithium (Li), nickel (Ni), thallium (Tl), vanadium mean (n = 3) ± SD [28].
(V), molybdenum (Mo), palladium (Pa), platinum (Pt), antimony (Sb), arsenic (As), lead
(Pb), cadmium (Cd), and mercury (Hg). Our previous study described this method [28],
and detailed data can be found in Supplementary Material, Tables S1–S4.
Appl. Sci. 2022, 12, 4234 5 of 20
2.5. FT-IR Analysis

Ground lichen samples (particle size ≤180 µm) were analyzed in triplicate by Fourier-
transform infrared spectroscopy (FT-IR), by attenuated total reflection (ATR), on a Thermo
Scientific Nicolet iS20 (Waltham, MA, USA) spectrometer. The spectra were recorded at
4 cm−1 intervals, in the range of 650 cm−1 to 4000 cm−1 . ATR correction was applied, and
the average spectra were extracted using Omnic software.
2.6. Total Phenolic Content

2.6.1. Dried Lichen Extracts in Ethanol and Acetone
Two series of approximately 1 g dUB, mcUB, and ccUB were refluxed for 1 h with
100 mL solvent (96% ethanol and acetone). The resulting extractive solutions for each lichen
part (dUB, mcUB, and ccUB) were filtered and then made up to 100 mL in a volumetric flask
with each corresponding solvent (Figure S3, Supplementary Material).
2.6.2. Folin–Ciocâlteu Method

According to a previously described method, the total phenolic content was deter-
mined using the Folin–Ciocâlteu reagent (Merck, Darmstadt, Germany) [32]. Pyrogallol
(Merck, Darmstadt, Germany) was used as a standard, and the TPC values were calculated
as µg of pyrogallol equivalents (PyE) per g dried sample. For this analysis, in three vol-
umetric flasks of 25 mL, 2 mL of each ethanol extract (from dUB, mcUB, and ccUB) was
added, and then 1 mL of Folin–Ciocâlteu reagent, 10 mL water, and 12 mL 290 g/L of
Na2 CO3 solution up to the mark. In each volumetric flask, a blue coloration appeared.
After 30 min of reaction in a dark place at room temperature, the absorbance values (each
value being A1 in the calculation formula) were read at 760 nm using a Jasco V630 UV-Vis
Spectrophotometer (JASCO Corporation, Tokyo, Japan) with Spectra Manager™ Software.
A similar determination of phenolic contents was performed for dUB, mcUB, and ccUB
acetone extracts. The total polyphenol content (TPC) determination was done in triplicate,
and the obtained data were expressed as the mean (n = 3) ± SD.
2.7. Free Radical Scavenging Activity

The free radical scavenging activity of the dUB, mcUB, and ccUB ethanol and acetone
extracts was determined on a Jasco V630 UV-Vis Spectrophotometer (JASCO Corporation,
Tokyo, Japan) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging
assay [28]. The DPPH solution was obtained by dissolution of DPPH (Sigma Aldrich, St.
Louis, MO, USA) in methanol to assess an absorbance value of 0.80 ± 0.02; then, 3.90 mL
of DPPH solution with 0.1 mL of each dried lichen extract was vortexed for 30 s. The
reaction time in a dark place at room temperature was 30 min; finally, the absorbances
values at 515 nm were measured. The DPPH solution in methanol with no added dried
lichen extract was used as a standard, while methanol was the blank. Two dilutions in
ethanol and acetone were obtained (1:2 and 1:4) for both lichen extracts, and the DPPH
radical scavenging activity was calculated according to Equation (3).
A control − A sample
Scavenging of DPPH free radicals (%) = 100 × (3)
A control
A control and A sample are the absorbance values at 515 nm for the DPPH and sample
solutions. This determination was performed in triplicate; the obtained data are expressed
as the mean (n = 3) ± SD.
2.8. Statistical Analysis

Mean values of three determinations were compared using analysis of variance
(ANOVA) with the Tukey test and Student’s t-test. The differences were considered signifi-
cant when p < 0.05. Principal component analysis (PCA) was performed using XLSTAT for
Excel software, 2022 version (Addinsoft, New York, NY, USA).
3. Results
3.1. Lichen Samples
Appl. Sci. 2022, 12, 4234
Dried U. barbata lichen (Figure 1a) had a green-gray color; after grinding
6 of 20
for
the ground U. barbata thallus (dUB) obtained is shown in Figure 1b. After passing t
the meshes of sieve no. 5, two fractions were separated: mcUB (Figure 1c) as a m
fine green-gray powder and ccUB (Figure 1d) as filiform, white-yellow fragments
3. Results
3.1. Lichen
5 mm long. Samples
Dried U. barbata lichen (Figure 1a) had a green-gray color; after grinding for 5 min, the
ground U. barbata
3.2. Lichen thallus (dUB) obtained is shown in Figure 1b. After passing through the
Morphology
meshes of sieve no. 5, two fractions were separated: mcUB (Figure 1c) as a moderate fine
The powder
green-gray U. barbata lichen
and ccUB thallus
(Figure morphology,
1d) as filiform, which
white-yellow allows
fragments, aboutthe
5 mmseparation
long.
fractions (mcUB and ccUB), was visualized using two modern microscopy techniqu
3.2. Lichen Morphology
performed fluorescent microscopy (FM) for the entire dried lichen and scanning e
The U. barbata lichen thallus morphology, which allows the separation of both fractions
microscopy (SEM) for ground lichen and fractions with different particle sizes.
(mcUB and ccUB), was visualized using two modern microscopy techniques. We performed
fluorescent microscopy (FM) for the entire dried lichen and scanning electron microscopy
3.2.1.
(SEM) Fluorescence Microscopy
for ground lichen and fractions with different particle sizes.
Fluorescence microscopy is an efficient method for viewing the lichen i
3.2.1. Fluorescence Microscopy
anatomy [33]. The FM images (Figure 2) were obtained using inherent autofluor
Fluorescence microscopy is an efficient method for viewing the lichen internal anatomy [33].
and
The FMacridine-orange-induced
images (Figure 2) were obtainedfluorescence.
using inherentThus, the U. barbata
autofluorescence layers (Figure 2a
and acridine-orange-
their
inducedcellular and subcellular
fluorescence. structures
Thus, the U. barbata (Figure
layers (Figure 2a,b)2c)
andcan
theirbe highlighted.
cellular and subcellular
structures (Figure 2c) can be highlighted.
(a) (b) (c)

Figure
Figure 2. 2. Microstructure
Microstructure of unground
of unground U. thallus—FM
U. barbata lichen barbata lichen
imagesthallus—FM images with d
with different magnifica-
100 × (a) and 400 ×
magnifications: 100× (a) and 400× (b,c).
tions: (b,c).
Figure 2a indicates the three layers of the lichen thallus: cortex, medulla, and central
cord. Figure
Due to 2a
the indicates the three
thick and loose layers
medulla, the of thelayers
three lichen thallus:into
separating cortex, medulla, and
two distinct
cord. Due to the thick
parts—medulla–cortex andand
(mcUB) loose medulla,
central the three
cord (ccUB)—is clear,layers separating
as shown into two
in Figure 2a,b.
parts—medulla–cortex (mcUB) and central cord (ccUB)—is clear, as shown in Figu
With rare fungal hyphae, the loose medulla is the morphological property of U. barbata and
With Usneafungal
severalrare cornuta, U. the
sp. (U. hyphae, fragilescens). Usnea lichens
loose medulla can morphological
is the have a compact medulla (U. of U.
property
longissima, U. rubicunda) or a dense one (U. ceratina, U. florida) [9].
and Theseveral Usnea sp. (U. cornuta, U. fragilescens). Usnea lichens can have a c
algal zone between the cortex and medulla in U. barbata is visible in Figure 2c. It
medulla (U. longissima,
is a subcortical U. rubicunda)
structure formed by algal cellsorintertwined
a dense one with(U. ceratina,
fungal hyphaeU. florida)
(Figure 2c).[9].
The algal zone between the cortex and medulla in U. barbata is visible in Figure
a subcortical structure formed by algal cells intertwined with fungal hyphae (Figure
Scanning electron microscopy (SEM) is an optimal hybrid method for examining lichen
thalli at high resolution [34], showing the micro-morphology of U. barbata thallus layers [35].
The obtained SEM images combined the optical microscopy visualization performance and
facility with high-resolution electron microscopy [36].
Thus, the morphological characteristics were easily identified in all ground U. bar-
bata samples with particle sizes around 315 µm. The SEM micrographs are displayed
in Figures 3 and 4.
medulla–cortex, mcUB (particle size ≤ 315 μm). The SEM micrographs of ccUB (Figure 3a–
d) were obtained at the following magnifications and scale bars: 300× (scale bar = 100 μm),
1 k× (scale bar = 20 μm), 2 k× (scale bar = 20 μm), 5 k× (5 μm). The cartilaginous structure of
the central cord can be observed and, on its surface, free fungal hyphae (Figure 3a,b). This
Appl. Sci. 2022, 12, 4234
particular structure of the central axis and the aspect of free fungal hyphae are detailed in
7 of 20
Figure 3c,d at 2 k× and 5 k× magnification (scale bar = 20 μm and 5 μm).
(a) (b) (c) (d)
(e) (f) (g) (h)

Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 20
Figure 3. (a–d) Microstructure of the central cord (ccUB) with particle size > 315 μm, in SEM images
Figure 3. (a–d) Microstructure of the central cord (ccUB) with particle size > 315 µm, in SEM
with different magnifications: (a) 300× (scale bar = 100 μm), (b) 1 k× (scale bar = 20 μm), (c) 2 k×
images with different magnifications: (a) 300× (scale bar = 100 µm), (b) 1 k× (scale bar = 20 µm),
(scale bar = 20 μm), (d) 5 k× (scale bar = 5 μm); (e–h) SEM micrographs of mcUB (medulla–cortex)
(c)
with × dUB
In
2 kparticle (particle
(scalesize
bar≤=315 size
20 µm), >(d)315
μm: (e) × (scale
5 kμm),
cortex webar
parts can=rare
with identify
5 µm); the
(e–h)
fungal morphology
SEM
hyphae of U.
micrographs
on the surface, mcUB
of barbata thallus:
(medulla–
1 k× (scale bar =
central
cortex) cord
with parts,
particle cortex
size ≤ fragments,
315 µm: and
(e) rare
cortex fungal
parts hyphae
with rare (Figure
fungal 4a–c).
hyphae on
20 μm), (f–h) cortex fragment at different magnifications: (f) 1 k× (scale bar = 20 μm), (g) 2 k× An
the isolated
surface,
(scale
bar k=×20 μm),
fragment
1 of the
(scale central
(h) 5bar = axis
k× (scale barcan
20 be observed
=µm),
5 μm). at different
(f–h) cortex magnifications
fragment at different(Figure 4d–f).
magnifications:
(f) 1 k× (scale bar = 20 µm), (g) 2 k× (scale bar = 20 µm), (h) 5 k× (scale bar = 5 µm).
In mcUB (particle size ≤ 315 μm), cortex fragments with a compact structure and rare
fungal hyphae can be seen (Figure 3e–h). On the surface of fungal hyphae, secondary
metabolite crystals can be observed (Figure 3e). In Usnea lichens, usnic acid is the most
common secondary metabolite in high concentrations; for this reason, usnic acid is
commonly indicated in the corresponding figures [37]. Figure 3f–h displays a cortex
fragment at different magnifications: 1 k× (scale bar = 20 μm), 2 k× (scale bar = 20 μm), 5
k× (scale bar = 5 μm). At high magnifications, the structural differences between the
central axis and cortex (both constituted by fungal hyphae)—which are the basis of their
different properties—can be observed.
(a) (b) (c)
(d) (e) (f)

Figure
Figure 4.
4. SEM
SEM micrographs
micrographs inin dUB
dUB (particle
(particle size
size >> 315
315 μm):
µm): (a–c)
(a–c) cortex
cortex fragments,
fragments, central
central cord,
cord, and
and
fungal
fungal hyphae, at different magnifications: (a) 1 k× (scale bar = 20 µm), (b) 2 k× (scale bar =20
hyphae, at different magnifications: (a) 1 k× (scale bar = 20 μm), (b) 2 k× (scale bar = 20 μm),
µm),
(c) 5 k× (scale bar = 5 μm); (d–f) central cord with fungal hyphae between cortex fragments, at
(c) 5 k× (scale bar = 5 µm); (d–f) central cord with fungal hyphae between cortex fragments,
different magnifications: (d) 200 k× (scale bar = 200 μm), (e) 1 k× (scale bar = 20 μm), (f) 5 k× (scale
at different magnifications: (d) 200 k× (scale bar = 200 µm), (e) 1 k× (scale bar = 20 µm),
bar = 5 μm).
(f) 5 k× (scale bar = 5 µm).
The structures of both layers (central cord and cortex) formed by mycelial hyphae are
essential, supporting dried lichen separation in ccUB and mcUB fractions. Their
longitudinal arrangement and interweaving in the central axis give it a particular strength
and elasticity. This property explains that it separates after a short grinding time and
cannot be finely ground unless the grinding time is long.
Appl. Sci. 2022, 12, 4234 8 of 20
Figure 3 shows SEM images of the central cord, ccUB (particle size > 315 µm), and
medulla–cortex, mcUB (particle size ≤ 315 µm). The SEM micrographs of ccUB (Figure 3a–d)
were obtained at the following magnifications and scale bars: 300× (scale bar = 100 µm),
1 k× (scale bar = 20 µm), 2 k× (scale bar = 20 µm), 5 k× (5 µm). The cartilaginous structure
of the central cord can be observed and, on its surface, free fungal hyphae (Figure 3a,b). This
particular structure of the central axis and the aspect of free fungal hyphae are detailed in
Figure 3c,d at 2 k× and 5 k× magnification (scale bar = 20 µm and 5 µm).
In mcUB (particle size ≤ 315 µm), cortex fragments with a compact structure and
rare fungal hyphae can be seen (Figure 3e–h). On the surface of fungal hyphae, sec-
ondary metabolite crystals can be observed (Figure 3e). In Usnea lichens, usnic acid is the
most common secondary metabolite in high concentrations; for this reason, usnic acid
is commonly indicated in the corresponding figures [37]. Figure 3f–h displays a cortex
fragment at different magnifications: 1 k× (scale bar = 20 µm), 2 k× (scale bar = 20 µm),
5 k× (scale bar = 5 µm). At high magnifications, the structural differences between the
central axis and cortex (both constituted by fungal hyphae)—which are the basis of their
different properties—can be observed.
In dUB (particle size > 315 µm), we can identify the morphology of U. barbata thallus:
central cord parts, cortex fragments, and rare fungal hyphae (Figure 4a–c). An isolated
fragment of the central axis can be observed at different magnifications (Figure 4d–f).
The structures of both layers (central cord and cortex) formed by mycelial hyphae are
essential, supporting dried lichen separation in ccUB and mcUB fractions. Their longitudinal
arrangement and interweaving in the central axis give it a particular strength and elasticity.
This property explains that it separates after a short grinding time and cannot be finely
ground unless the grinding time is long.
The SEM micrographs of all ground lichen samples (ccUB, mcUB, and dUB) as a
fine powder (particle size ≤ 180 µm) are shown in Figure 5. SEM images displayed in
Figure 5 highlighted the microstructure of ccUB (Figure 5a–c) and mcUB (Figure 5d,e)
achieved at different magnifications: 1 k× (scale bar = 20 µm), 2 k× (scale bar = 20 µm), and
5 k× (scale bar = 5 µm). A noticeable difference can be observed between the central cord
fragments (Figure 5a–c) and the cortex ones (Figure 5d–f). Due to the compact cartilaginous
structure of the intertwined longitudinal mycelial hyphae, the fragments of the central
axis appear as pieces of a broken vessel, with a glossy, deep surface, irregular sharpness,
or rounded edges. The structure from Figure 3d can be recognized in Figure 5c. The
cortex fragments are porous, and the compact structure’s rupture during grinding occurs
along with a series of pores on its thickness. The images of dUB powder (Figure 5g–i)
show differentiated fragments belonging to both layers of the lichen thalli (central axis and
cortex) and fungal hyphae.
3.3. Color Evaluation

The data obtained for the color parameters of U. barbata fractions are displayed in
Table 1. Significant differences (p < 0.05) between all samples were observed for all color
parameters considered, supported by the images from Figure 1. The highest luminosity was
observed for ccUB (70.29), while the mcUB fraction presented the lowest value (59.08). The
samples containing the medulla–cortex (dUB and mcUB) exhibited a green nuance indicated
by the negative values of the a* parameter. In contrast, the positive values observed for
ccUB suggested a red nuance. Both fractions and integral lichen showed a yellow nuance,
as the positive values of the b* parameter showed, the nuance being more pronounced in
ccUB (13.92). Compared to the ccUB fraction, the mcUB sample showed a lower hue angle
and chroma value.
Appl. Sci. 2022, 12, 4234 9 of 20
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)

Figure
Figure 5.
5. SEM
SEMmicrographs
micrographsofofccUB, mcUB,
ccUB, and
mcUB, dUB
and as aasfine
dUB powder
a fine (particle
powder size ≤size
(particle 180≤μm):
180 ccUB
µm):
(a–c); mcUB (d–f); dUB (g–i) with different magnifications: 1 k× and scale bar = 20 μm (a,d,g), 2 k×
ccUB (a–c); mcUB (d–f); dUB (g–i) with different magnifications: 1 k× and scale bar = 20 µm (a,d,g),
and scale bar = 20 μm (b,e,h), and 5 k× and scale bar = 5 μm (c,f,i).
2 k× and scale bar = 20 µm (b,e,h), and 5 k× and scale bar = 5 µm (c,f,i).
3.3. Color
Table Evaluation
1. Color evaluation of U. barbata samples.
The data obtained for the color parameters of U. barbata fractions are displayed in
Property dUB mcUB ccUB
Table 1. Significant differences (p < 0.05) between all samples were observedF-Value for all color
Color Properties
parameters considered, supported by the images from Figure 1. The highest luminosity
was observed
L* (adim.) for ccUB (70.29), b c thea lowest5703.14
63.02 ± 0.11while the mcUB
59.08 fraction
± 0.15 presented
70.29 ± 0.13 value (59.08).
***
b −1.83 ±(dUB c 2.12 ± 0.17 a
The a*
samples −1.29the
(adim.) containing ± 0.02
medulla–cortex 0.03 and mcUB) exhibited 1414.01nuance
a green ***
b* (adim.)
indicated by the negative 0.02 b of the
13.60 ± values 0.04 c
12.52a*±parameter. 13.92 ± 0.05 a the positive
In contrast, 1086.01 values
***
◦)
hab (for 178.52 ± 0.00 c
178.57 ± b 181.42
0.00 fractions 0.01 a
± integral 165,195.71 *** a
observed ccUB suggested a bred nuance. Both and lichen showed
C* (adim.) 13.66 ± 0.02 12.65 ± 0.05 c 14.08 ± 0.04 a 1125.62 ***
yellow nuance, as the positive values of the b* parameter showed, the nuance being more
dUB—dried U. barbata integral lichen, mcUB—medulla–cortex, ccUB—central cord, L*—lightness, a*—positive
pronounced in ccUB (13.92). Compared to the ccUB fraction, the mcUB sample showed a
values describe red and negative, green nuance, b*—positive values represent yellow and negative, blue nuance;
lower
hab —huehue angle
angle, and chroma
C*—Chroma, value.
adim.—adimensional. Mean values followed by different superscript letters are
significantly different, *** significant at p < 0.001.
The green color is due to chlorophyll from the algal layer [22], and the yellow one to
usnic acid, the most well-known phenolic secondary metabolite [38].
Chlorophyll degradation significantly increases in prolonged sunlight and heavy metal
exposure of lichens [39], and their color can be modified. Due to usnic acid’s photoprotective
effect, Usnea species show a moderate level of photoinhibition [40]. Zulaini et al. [41]
Appl. Sci. 2022, 12, 4234 10 of 20
reported that U. diffracta thalli change color chane from greyish-green to brownish-green
through massive heavy metal accumulation due to chlorophyll breakdown.
3.4. Elemental Analysis

The elemental content of dried U. barbata lichen and both fractions is presented in Table 2.
Table 2. Mineral composition of Usnea barbata (L.) Weber ex F.H. Wigg dried lichen (dUB) and both
fractions: medulla–cortex (mcUB) and central cord (ccUB).
Element Content Sample

(µg/g) F-Value
dUB mcUB ccUB
Al 87.879 ± 1.152 b 285.828 ± 2.748 a 21.111 ± 0.608 c 18,444.13 ***
As ND 0.219 ± 0.005 ND -
Ba 3.782 ± 0.052 b 12.113 ± 0.604 a 0.596 ± 0.004 c 866.03 ***
Ca 979.766 ± 12.285 b 1549.600 ± 18.406 a 434.269 ± 22.222 c 2846.40 ***
Cd ND 0.164 ± 0.001 ND -
Co ND 0.225 ± 0.001 ND -
Cr 1.002 ± 0.008 b 2.494 ± 0.010 a 0.864 ± 0.007 c 34,521.18 ***
Cu 1.523 ± 0.013 b 2.246 ± 0.005 a 1.286 ± 0.014 c 5771.15 ***
Fe 52.561 ± 2.582 b 282.468 ± 1.149 a 33.107 ± 0.322 c 21,398.25 ***
Li ND 0.185 ± 0.007 ND -
Mg 172.721 ± 0.647 b 524.239 ± 2.419 a 53.482 ± 0.597 c 81,353.06 ***
Mn 101.425 ± 1.423 b 354.041 ± 1.083 a 29.098 ± 0.347 c 78,943.06 ***
Ni 0.449 ± 0.011 b 1.033 ± 0.005 a 0.355 ± 0.007 c 5658.89 ***
Pb 1.296 ± 0.007 b 2.177 ± 0.014 a 0.584 ± 0.004 c 21,958.36 ***
V 0.241 ± 0.004 b 1.237 ± 0.005 a ND 92,152.36 ***
Zn 20.536 ± 0.125 b 33.223 ± 0.164 a 13.588 ± 0.097 c 17,179.89 ***
Hg 0.671 ± 0.020 b 0.708 ± 0.005 a 0.539 ± 0.017 c 99.48 ***
Sb ND 0.152 ± 0.003 ND -
The analysis was performed in triplicate. The results are shown as the mean ± standard deviation (SD). ND—non-
detected, Al—aluminum, As—arsenic, Ba—barium, Ca—calcium, Cd—cadmium, Co—cobalt, Cr—chromium,
Cu—copper, Fe—iron, Li—lithium, Mg—magnesium, Mn—manganese, Ni—nickel, Pb—lead, V—vanadium,
Zn—zinc, Hg—mercury, Sb—antimony. Mean values followed by different letters are significantly different,
*** significant at p < 0.001.
It can be noted that Ca, Fe, Mg, Mn, and Zn had the highest LOQ values (5 µg/g), followed
by Al (1 µg/g) and the other metals (most numerous), which had LOQ = 0.1 µg/g. Generally,
the LOQ value was directly proportional to their content (Table S5, Supplementary Material).
Comparing both fractions (mcUB and ccUB) with dried lichen (dUB), the data reg-
istered in Table 2 highlight other notable aspects. Thus, mcUB reported the highest
metal contents and ccUB the lowest ones; these differences were significant for macro-
elements (mcUB > dUB > ccUB): Al (285.828 > 87.879 > 21.111 µg/g), Ca (1549.600 > 979.766
> 434.269 µg/g), Fe (282.468 > 52.561 > 33.107 µg/g), Mg (524.239 > 172.721 > 53.482 µg/g),
and Mn (354.041 > 101.425 > 29.098 µg/g).
Generally, trace elements showed a similar distribution in dUB and both fractions,
but the differences were significantly lower: Ba (12.113 > 3.782 > 0.596 µg/g), Cr (2.494
> 1.002 > 0.864 µg/g), Cu (2.246 > 1.523 > 1.286 µg/g), Ni (1.033 > 0.449 > 0.355 µg/g),
Pb (2.177 > 1.296 > 0.584 µg/g), Zn (33.223 > 20.536 > 13.588 µg/g), Hg (0.708 > 0.671 >
0.539 µg/g). Another metal (V) was quantified only in mcUB and dUB (1.237 > 0.241 µg/g),
being undetected in ccUB. Moreover, five trace elements undetected in dUB (As, Cd, Co,
Li, Sb) were quantified in mcUB: As (0.219 µg/g), Cd (0.164 µg/g), Co (0.225 µg/g), Li
(0.185 µg/g), and Sb (0.152 µg/g)
3.5. FT-IR Analysis

The molecular characteristics of U. barbata fractions are presented in Figure 6. Notice-
able differences in absorbance values between fractions were observed for almost all peaks,
undetected in ccUB. Moreover, five trace elements undetected in dUB (As, Cd, Co, Li, Sb)
were quantified in mcUB: As (0.219 μg/g), Cd (0.164 μg/g), Co (0.225 μg/g), Li (0.185 μg/g),
and Sb (0.152 μg/g)
Appl. Sci. 2022, 12, 4234 3.5. FT-IR Analysis 11 of 20

The molecular characteristics of U. barbata fractions are presented in Figure 6.
Noticeable differences in absorbance values between fractions were observed for almost
all peaks,
with with intensities
the highest the highest intensities
obtained obtained
for mcUB, for mcUB,
followed followed
by dUB by Furthermore,
and ccUB. dUB and ccUB.
in
Furthermore, in the regions
− 1 1380–1700 cm –1 and−2310–2380
1 cm–1, different peaks appeared
the regions 1380–1700 cm and 2310–2380 cm , different peaks appeared in ccUB and
in ccUB
dUB and dUB
samples samples
compared compared to mcUB.
to mcUB.
Figure6.6. Overlay
Figure Overlay of
of FT-IR
FT-IR spectra
spectra of
ofU.
U.barbata
barbatadried
driedlichen
lichen(dUB),
(dUB),medulla–cortex
medulla–cortex (mcUB),
(mcUB), and
and
central cord (ccUB).
central cord (ccUB).
3.6. Total
3.6. TotalPhenolic
PhenolicContent
Content
Thevalues
The valuesof
ofthe
thetotal
totalphenolic
phenoliccompounds
compoundsextracted
extractedfrom
fromdUB,
dUB,mcUB,
mcUB,and
andccUB
ccUBin
in
ethanol
ethanoland
andacetone
acetoneare
aredisplayed
displayedin
inTable
Table3.3.
Table 3. Total phenolic content (TPC, expressed as mg PyE/g dried sample) and free radical scav-
enging activity (AA, expressed as % DPPH radical scavenging) of U. barbata dried lichen and both
fractions (mcUB and ccUB) extracted in ethanol and acetone.
Parameter dUB mcUB ccUB F-Value

b,x 25.212 ± 0.084 a,y 18.383 ± 0.004 c,x
TPC ethanol 23.481 ± 0.039 13200.49 ***
TPC acetone 22.675 ± 0.108 b,y 36.243 ± 0.093 a,x 15.170 ± 0.129 c,y 27784.01 ***
t-value 12.16 *** 152.42 *** 43.12 **
16.878 ± 0.204 a,x b,y 11.336 ± 0.174 c,y
AA acetone (% DPPH scavenging) 12.747 ± 0.221 10.96 **
AA ethanol (% DPPH scavenging) 15.985 ± 0.197 a,y 15.735 ± 0.185 a,x 15.080 ± 0.326 b,x 618.35 **
t-value 5.45 ** 17.96 *** 17.55 ***
dUB—dried U. barbata lichen, mcUB—medulla–cortex, ccUB—central cord, TPC—total phenolic content (mg
PyE/g dried sample), mgPyE/g—milligram equivalent pyrogallol per 1 g lichen sample, AA—antioxidant activity.
The mean values followed by superscript letters are significantly different: a–c in the same row for sample
comparison, x–y in the same column for solvent comparison, *** significant at p < 0.001, ** significant at p < 0.01.
Phenolic values of both extracts decreased in the order mcUB, dUB, and ccUB. TPC
values for dUB and ccUB were higher in ethanol extracts (23.481 and 18.383 mg PyE/g)
than in acetone ones (22.675 and 15.170 mg PyE/g). The TPC value in mcUB acetone
extract was 36.243 mg PyE/g, significantly higher than that in ethanol (25.212 mg PyE/g).
These differences are due to the nature and amount of phenolic compounds in each sample
extracted by each solvent. The coloration of the ethanol and acetone extracts from each of
the three lichen samples (Figure S2, Supplementary Material) also supports this observation.
3.7. Free Radical Scavenging Activity

The obtained results are displayed in Table 3. Both extracts’ free radical scavenging
activity decreased in the order dUB, mcUB, and ccUB (Table 3). In ethanol extracts, %
DPPH radical scavenging values registered were not significantly different between dUB
(15.985%) and mcUB (15.735%), while ccUB showed a significantly different value (15.080%).
Appl. Sci. 2022, 12, 4234 12 of 20
Moreover, only mcUB had AA highly correlated with TPC (R2 = 0.976); the others showed a
moderate correlation: R2 = 0.776 (dUB) and R2 = 0.738 (ccUB). Acetone extracts displayed
considerable differences: 16.878% (dUB), 12.747% (mcUB), and 11.336% (ccUB). In this case,
ccUB (R2 = 0.997) showed the highest correlation between antiradical activity and the TPC
value, followed by mcUB (R2 = 0.969) and dUB (R2 = 0.883). Only dUB acetone extract had
AA higher than the ethanol one; mcUB and ccUB acetone extracts had lower antiradical
activity compared to ethanol ones (Table 3). Finally, comparing the AA and TPC for all
lichen samples in each solvent, we can see that only the antiradical activity of ethanol
extracts had a moderate correlation with TPC (R2 ethanol = 0.755).
3.8. Relationships between Variables

The similarities and dissimilarities between the characteristics of U. barbata samples
are displayed in Figure 7.
Figure7.7.The
Figure ThePrincipal
PrincipalComponents
ComponentsAnalysis
Analysis(PCA)
(PCA)plot.
plot.
The
Thetwo
twoprincipal
principalcomponents
componentsexplained
explained99.31%
99.31%ofofthethetotal
totaldata
datavariance,
variance,with
with
85.46% attributed to the first (PC1) and 13.85% to the second (PC2).
85.46% attributed to the first (PC1) and 13.85% to the second (PC2). The PC1 was The PC1 was asso-
ciated with with
associated all minerals, totaltotal
all minerals, phenolics, andand
phenolics, color parameters,
color parameters,while
whilePC2
PC2waswasrelated
related
totoscavenging activity. Luminosity (L*), b* parameter, and chroma (C*)
scavenging activity. Luminosity (L*), b* parameter, and chroma (C*) were negativelywere negatively
correlated(r(r>>−−0.768,
correlated 0.768, pp < 0.05) with
< 0.05) with all
all metals
metals and
and the
the total
totalphenolic
phenoliccontent
content(TPC)
(TPC)ininboth
both
solvents; the b* parameter was negatively correlated (r > 0.706, p < 0.05)
solvents; the b* parameter was negatively correlated (r > 0.706, p < 0.05) only with only withAl,Al,
Ba,
Ba, Ca, Cu, Mg, Mn, Ni, Pb, V, Zn, Hg, Sb, and TPC. TPC values
Ca, Cu, Mg, Mn, Ni, Pb, V, Zn, Hg, Sb, and TPC. TPC values in both solvents werein both solvents were
positively
positivelycorrelated 0.696,pp<<0.05)
correlated(r(r>>0.696, 0.05)with
withallallmineral
mineralcontents.
contents.
An opposition between TPC in ethanol extracts and the a* parameter and between b*
An opposition between TPC in ethanol extracts and the a* parameter and between b*
and C* with some minerals (Cu, Mg, Al) was observed. The scavenging activity in ethanol
and C* with some minerals (Cu, Mg, Al) was observed. The scavenging activity in ethanol
extracts was positively correlated with the TPC content (r > 0.775, p < 0.05).
extracts was positively correlated with the TPC content (r > 0.775, p < 0.05).
4. Discussion
4. Discussion
Using advanced microscopical analysis, the present study provides an accurate mor-
Usingcharacterization
phological advanced microscopical analysis,
of U. barbata, offeringthe present structural
a complete study provides an accurate
investigation. The
morphological characterization of U. barbata, offering a complete structural investigation.
The microstructure of U. barbata integral and fractions displayed in our study agreed with
those reported by Ivanovic et al. [37] and Bubach et al. [21]. The whole lichen consists of
90% mycobiont and only 10% photobiont [24]. Of the separated fractions, one is formed
exclusively of mycelial hyphae (ccUB), and the other (mcUB) has both symbionts. It is
Appl. Sci. 2022, 12, 4234 13 of 20
microstructure of U. barbata integral and fractions displayed in our study agreed with
those reported by Ivanovic et al. [37] and Bubach et al. [21]. The whole lichen consists of
90% mycobiont and only 10% photobiont [24]. Of the separated fractions, one is formed
exclusively of mycelial hyphae (ccUB), and the other (mcUB) has both symbionts. It is
known that, in symbiotic partnerships, only the fungus synthesizes the lichen’s secondary
metabolites. Thus, we wanted to know which lichen fraction had phenolic metabolites and
a mineral content higher than the entire lichen. Being a source of bioactive metabolites,
extracting them from the fraction with the highest content could increase the process yield
and selectivity.
Bubach et al. [21] revealed an entire micrograph of a cross-section of Usnea sp. thallus
obtained by SEM-EDS analysis. These authors separated only two U. barbata thallus
fractions (cortex–medulla and axis), similar to those described in our study (mcUB and
ccUB). The cortex consisted of densely packed thick-walled fungal hyphae in their image,
and the central cord had a compact, cartilaginous structure [21].
Most numerous organic compounds found in lichens are secondary metabolites of
fungal origin [20]. Their amount is usually 0.1–10% in dried lichen and can reach 30% in
some cases. These substances are mostly phenolic compounds, poorly soluble in water, and
can be extracted using organic solvents [42]. They are deposited in the cortex and medulla
as crystals on the hyphae surface. The lichen metabolites are differentially distributed in
the thallus layers, correlated with their bioactivities [20]. Thus, the compounds from the
upper cortex act as a light filter, having a photoprotective effect [40]. The most common
cortical metabolites are usnic acid, atranorin, xanthones, pulvinic acid derivates, and
anthraquinones. With few exceptions (atranorin), they are colored compounds (yellow,
orange), giving the lichen thalli specific coloration [40]. These cortical constituents can be
distinguished from numerous medullary ones [20].
Bioactive metabolites localization in the lichen thallus was explored using various
methods. Some researchers used spot tests (directly applying suitable reagents to the
lichen thallus) or after extraction and analyses of specific lichen parts [43]. Other studies
described classical fluorescent techniques based on the molecule chromophore, where
metabolites are only differentiated by their emission wavelength; then, the metabolite
localization in living plant cells was obtained using multiphoton microscopy [44]. In 2016,
based on the auto-ionisability of all main classes of lichenic bioactive compounds [45],
Le Pogam et al. [46] achieved histolocalisation of the lichen Ophioparma ventosa by laser
desorption ionization coupled to mass spectrometry imaging (LDI-MSI). They applied
different slicing procedures to obtain lichen thalli transverse sections (cryosectioning and
hand-cutting), fixed them on a carbon-conductive adhesive tape, and examined the metabo-
lites’ spatial distribution. MSI displayed the molecular images of usnic acid, thamnolic acid,
divaricatic acid, miriquidic acid, haemoventosin, and all overlaid ions in cryosectioned
pieces of the O. ventosa thallus [46]. Using the same method, Gadea et al. [43] obtained
spatial mapping that revealed phenolic metabolites in the lichen Pseudocyphellaria crocata.
Moreover, for optimal extraction of localized lichen metabolites, different methods of
sample preparation were elaborated. Komaty et al. [47] proposed two types of preparations
for atranorin extraction from Pseudevernia furfuracea. In the first case, the lichen sample
was milled (with a planetary ball mill), resulting in a homogeneous powder. The lichen
thallus was ground in a blender in the second case, obtaining a mixture of medulla pieces
and fine cortex powder. The higher extraction yield with acetone under microwave irradia-
tion was obtained using cortex powder because atranorin is mainly a cortical metabolite.
Ivanovic et al. [37] optimized usnic acid supercritical fluid extraction with CO2 from U.
barbata, using various grinding methods and conditions (temperature, CO2 pressure).
In the case of pharmaceutical applications, either supplements or drugs, the regulatory
agencies approve only one or a maximum of two unpolluted sources for the contained
ingredients. The results are reproducible only for raw materials developed under the same
conditions; therefore, restricting the harvesting area leads to accurate results, essential for
human use formulations. However, only a few data on metal concentrations in U. barbata
Appl. Sci. 2022, 12, 4234 14 of 20
from unpolluted zones were found in previous studies. Culicov et al. [48] investigated
U. barbata from the Mountain Natural Park in Bulgaria, Jayasekera et al. [49] examined U.
barbata from the Sri Lanka rain forest, and Conti et al. [50] analyzed U. barbata from central
and southern Tierra del Fuego, Patagonia, Argentina. Arsenic (As) was detected only in
mcUB (0.219 µg/g), not in the whole dried lichen sample. However, this element was
quantified in lichens belonging to the three mentioned regions: 0.134 µg/g in Sri Lanka,
0.823 µg/g in Tierra del Fuego, and 1.100 µg/g in Bulgaria Natural Park. Cadmium (Cd)
was quantified in increased values in U. barbata from all three zones: 0.096 µg/g in Sri
Lanka, 0.174 µg/g in Tierra del Fuego, and 0.590 µg/g in Bulgaria; in our study, Cd was
detected only in mcUB (0.164 µg/g). The cobalt (Co) content in mcUB was 0.225 µg/g;
however, U. barbata from all other zones displayed different Co contents: 0.522 µg/g in
Tierra del Fuego, 0.260 µg/g in Sri Lanka, and 0.130 µg/g in Bulgarian Mountain Park. We
found the highest lead content in dried U. barbata from Călimani Mountains (1.296 µg/g).
In contrast, the lead amounts in the lichen from other zones were significantly lower (0.426
and 0.221 µg/g Pb in U. barbata from Sri Lanka and Tierra del Fuego). The zinc content in
autochthonous lichen (20.536 µg/g) was lower than 42 µg/g (Tierra del Fuego) and 51 µg
(Bulgaria) and higher than 15 µg/g (Sri Lanka). The U. barbata from Tierra del Fuego had a
0.827 µg/g nickel content, higher than the Călimani Mountains one (0.449 µg/g). Finally,
an amount of 0.056 µg/g Sb was quantified by Conti et al. in U. barbata from Tierra del
Fuego; only in the mcUB was 0.152 µg/g Sb detected.
From their native zones, U. barbata and other lichen species can be transplanted to
various polluted zones for biomonitoring reasons [51]. Conti et al. [52] confirmed the
considerable ability of U. barbata to reflect the levels of environmental elements on a global
scale, signaling, at distant places, volcanic emissions. Previous studies reported that Usnea
sp. was used in this scope, and their action was compared with other lichen species. Thus,
U. hirta transplanted to a city in northern Italy had a similar capacity to accumulate various
metals as H. physodes and P. furfuracea [53]. Meli et al. [54] showed that lichens, obtaining
numerous nutrients from the air, significantly accumulate different air pollutants through
the thallus surface. Lichens take in metals in different ways. The common ones are the
physical passing of metal particles in the medulla intercellular spaces and their binding to
extracellular sites of the mycobiont and photobiont [55]. Hence, the highest mineral content
in the mcUB fraction, followed, in decreasing order, by dUB and ccUB, can be justified.
Accumulation of heavy/trace metals in lichens is essential for their use for nutrition
and/or therapeutical purposes [56]. Moreover, usnic acid, the most bioactive metabolite
from Usnea sp., has high hepatotoxicity [57]; that aspect represents another reason that
further restricts their possible use in these scopes.
Heavy metal accumulation by food intake [58] disturbs various biochemical processes [59]
in the human body. Thereby, permissible limits of heavy metals were established for edible
plants by the Food and Agriculture Organization of the United Nations (FAO) and the World
Health Organization (WHO, 1996) [60,61]. These values are displayed in Table 4.
As a culinary plant for bread and mush ingredients, U. barbata was used in the Balkan
region [64]. In dUB and ccUB, the metal content values (Table 4) show that Cr, Pb, Cu, and
Zn were lower than the allowable limit. Other heavy metals, Hg, Ni, and V, were found in
higher contents compared to permissible ones. The other toxic elements (As, Cd, and Co)
were not detected. On the other hand, U. barbata arsenic contents of 0.823 µg/g in Tierra del
Fuego and 1.100 µg/g in Bulgaria Natural Park were higher than the acceptable limit. The
cadmium and cobalt contents were higher than the permissible limit in U. barbata from all
three zones compared with the Călimani mountains, while zinc and nickel contents were
only measured in the Natural Park of Bulgaria and in Tierra del Fuego. It can be seen that
the heavy metal content in U. barbata from other unpolluted earth zones was significantly
higher than in our one. In mcUB, Cd and Co were higher than the permissible limits. The
same fraction had other heavy metals higher than the allowable limits: Cr, Cu, Ni, Pb, Hg,
and V; only Zn and As contents were lower than the permissible limits (Table 4).
Appl. Sci. 2022, 12, 4234 15 of 20
Table 4. Permissible limits for heavy metals in edible and medicinal plants and their content in U.
barbata whole lichen (dUB) and fractions (mcUB and ccUB).
Legislative WHO/EU WHO Eu.Ph. Ch.Ph. Heavy Metals Content

Authority [60,61] [62] [63] [59] in U. barbata (µg/g)
Permissible Limits (µg/g)
Heavy Metal dUB mcUB ccUB
Edible Plants Medicinal Plants
As 0.5 10 2 ND 0.219 ND
Cd 0.02 0.2 1 0.3 ND 0.164 ND
Cr 1.3 2 ND 0.225 ND
Co 0.01 1.002 2.494 0.864
Cu 10 20 20 1.523 2.246 1.286
Hg 0.03 1 0.1 0.2 0.671 0.708 0.539
Ni 0.1 0.449 1.033 0.355
Pb 2 10 5 1.296 2.177 0.584
V 0.03 0.241 1.237 ND
Zn 50 50 20.536 33.223 13.588
WHO = World and Health Organisation, EU = European Union, Eu.Ph.= European Pharmacopoeia, Ch.Ph. = Chinese
Pharmacopoeia, dUB—dried U. barbata lichen, mcUB—medulla–cortex, ccUB—central cord, ND = not detected.
The approvable limits for heavy metals in medicinal plants are higher compared to
edible ones (Table 4).
Dobrescu et al. [65] reported that U. barbata is commonly used as an antiseptic (in Spain
and USA). According to the same authors, it is a good remedy for various symptoms (insomnia,
whooping cough, bleeding, jaundice, and nausea; in Europe). Our results showed that most
heavy metals in autochthonous U. barbata were present lower amounts than permissible
limits values. However, the Cr content in mcUB exceeded the permissible limit according to
European Pharmacopoeia; the mercury contents in all three lichen fractions were lower than
the WHO’s and the FDA’s acceptable limits [62] and higher than the ones according to the
Chinese Pharmacopoeia [59], and European Pharmacopoeia [63] (Table 4).
The metals can generate complexes with polysaccharides, lichen secondary metabo-
lites, and organic acids [55]. Bačkor and Fahselt (2004) [24] proved that, in C. pleurota, usnic
acid might be associated with Fe, Cu, Ni, and Al. Thus, the significant positive correlation
(p > 0.05) between the total phenolic and mineral contents highlighted in the final PCA plot
could be explained.
As a preview of lichen constituents from all analyzed samples, the FT-IR spectra of
mcUB showed different peaks compared to the ccUB fraction, while dUB presented all
peaks found in both previous fractions. The peaks observed at 3340 cm−1 given by the
O-H and N-H stretching vibration and 2852 and 2920 cm−1 due to the C-H stretching
vibration could depict the presence of phenols, water, carbohydrates, polysaccharides [66],
and lipids [67] in U. barbata fractions. Similar results were reported by Zaini et al. [68] for
usnic acid extracted from Usnea sp. The peak at 3320 cm−1 , along with the ones at 2920
and 2852 and 1700 cm−1 , may be related to the placodiolic and usnic acid characteristics
of U. barbata lichen [69]. The bands observed at 1633, 1700, and 1736 cm−1 attributed
to the N-H and C=O bending vibration could indicate amino acids and esters [67]. The
peaks found at 1700 and 1736 cm−1 can be attributed to the C=O vibrations in lipids, their
presence being suggested by the bands observed at 1323 and 1377 cm−1 given by the CH3
bending [67]. The peaks observed at 1633 and 1700 cm−1 can be assigned to the C=O,
C-N, and/or C-O-NH- vibrations which may be due to proteins [67]. The aromatic and
N-H bending vibrations found at 1511, 1541, 1560, and 1576 cm−1 can depict the presence
of amino acids [67]. The bands at 1511 and 1576 cm−1 were observed only for ccUB and
dUB, indicating the differences in amino acids and proteins compared to the mcUB sample;
this hypothesis is supported by the appearance of the peak at 1440 cm−1 . The peaks in
the regions 1377–1460 cm−1 can also be assigned to the CH3 lipids/proteins and COO- of
amino acids, while the vibrations of N-H and C-N had peaks at 1460–1576 cm−1 [64] and
Appl. Sci. 2022, 12, 4234 16 of 20
reveal the presence of proteins in U. barbata fractions. The bands observed in the region
1323–1440 cm−1 could be attributed to the primary or secondary O–H bending (in-plane)
and phenol or tertiary alcohol (O–H bend) [67]; the peak at 1440 cm−1 was observed only in
ccUB and dUB samples. The secondary aromatic amine and CN stretching vibration could
be responsible for the peaks obtained at 1293 and 1323 cm−1 , while the band observed at
1253 cm−1 , attributed to the C-O stretching vibrations, could be due to acids or esters [67].
The specific bands at 1293 and 1323 cm−1 could be associated with usenamine A, a detected
compound in U. barbata lichen [70]. The peaks obtained at 1042 and 1073 cm−1 can be due
to the C-O stretching vibrations of carbohydrates and glycoproteins.
The most important phenolic secondary metabolites (usnic acid, for Usnea sp.) synthe-
sized by mycobionts are deposited on the hyphae surface in the cortex and medulla [37,71].
Therefore, the highest TPC values in mcUB in both solvents, followed by dUB and ccUB,
could be justified. Usnic acid and other specific secondary metabolites have a higher solu-
bility in acetone than ethanol; this property can explain that mcUB has a higher TPC value
in acetone extract than ethanol extract [32]. The free radical scavenging activity [72] was
evaluated, and, in both extracts, dUB recorded the most significant antiradical potential [73],
followed by mcUB and ccUB. However, due to the various free radical scavenging ability
of phenolic metabolites found in each lichen extract (ethanol and acetone), AA values
considerably differed. Consequently, the correlation between antiradical activity and TPC
recorded significant variations. The highest AA of dUB in both extracts could be induced
by the synergic interaction between the primary metabolites with a non-phenolic structure,
the secondary ones, and the metals found in the whole lichen.
5. Conclusions
Our study could complete the scientific database, which must be constantly updated
with the characteristics of U. barbata separated fractions (medulla–cortex and central cord)
compared to whole lichen. Proving that the medulla–cortex fraction has the highest phenolic
metabolite content, the obtained results could help increase their extraction yield. Further
studies could quantify usnic acid and other bioactive metabolites in each separated lichen
fraction. Moreover, this separation process could be investigated as a potential pre-treatment
method for U. barbata dried lichen thalli, aiming for their extraction process optimization.
Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/app12094234/s1, Figure S1. Ground U. barbata samples, as a
fine powder with particle size < 180 µm (Dub—dried U. barbata lichen, mcUB—medulla–cortex,
ccUB—central cord); Figure S2. The calibration curves of 23 metals analyzed in U. barbata dried
samples using the ICP-MS method; Figure S3. Extracts in ethanol 96% (A) and acetone (B) from
dried U. barbata lichen—dUB (a), medulla–cortex—mcUB (b), and central cord—ccUB (c). Table S1.
Dried U. barbata sample digestion conditions for ICP-MS mineral analysis; Table S2. ICP-MS Standard
solutions; Table S3. Preparation of calibration standard solutions (E1–E5); Table S4. Concentrations of
calibration standard solutions (E1–E5) for different elements; Table S5. Calibration Curve Range, R2 ,
LOD, and LOQ (µg/L and µg/g) for each element.
Author Contributions: Conceptualization, V.P. and M.U.-I.; methodology, L.B., C.E.G., D.R., S.I.C.,
E.I.C., T.C., M.U.-I., M.O. and V.S.; software, V.P., L.B., D.R., S.I.C., E.I.C., T.C., M.U.-I., M.O., S.M.
and V.S.; validation, L.B., D.R., S.I.C., E.I.C., T.C., M.U.-I. and M.O.; formal analysis, V.P. and V.S.
investigation, V.P., E.A.O. and A.C.; resources, V.P., L.B., M.U.-I. and M.O.; data curation, C.E.G.,
S.M., E.A.O. and A.C.; writing—original draft preparation, V.P., D.R., S.I.C. and M.U.-I.; writing—
review and editing, V.P., C.E.G., L.B., M.U.-I., M.O. and S.M.; visualization, L.B., C.E.G., M.O., S.M.,
V.S., E.A.O., A.C. and V.B.; supervision, E.I.C., T.C., L.B., C.E.G., M.O., S.M., V.S., E.A.O., A.C. and
V.B.; project administration, V.B.; funding acquisition, V.P. All authors have read and agreed to the
published version of the manuscript.
Funding: This work is supported by the project ANTREPRENORDOC, in the framework of the
Human Resources Development Operational Programme 2014–2020, financed by the European Social
Fund under the contract number 36355/23.05.2019 HRD OP/380/6/13—SMIS Code: 123847.
Appl. Sci. 2022, 12, 4234 17 of 20
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This study was performed in collaboration with the Research Centre of Instru-
mental Analysis SCIENT, Ilfov, Romania, Faculty of Food Engineering and Integrated Center for
Research, Development, and Innovation in Advanced Materials, Nanotechnologies and Distributed
Systems for Fabrication and Control (MANSiD), Stefan cel Mare University of Suceava, Romania,
and Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania.
Conflicts of Interest: The authors declare no conflict of interest.
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Advances in The Characterization of Usnea Barbata

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Appl. Sci. 2022, 12, 4234. https://doi.org/10.3390/app12094234 https://www.mdpi.com/journal/applsci

2. Materials and Methods

2.2. Lichen Morphology

(a) (b) (c) (d)

2.2.2. Scanning Electron Microscopy

2.5. FT-IR Analysis

2.6. Total Phenolic Content

2.6.2. Folin–Ciocâlteu Method

2.7. Free Radical Scavenging Activity

2.8. Statistical Analysis

(a) (b) (c)

(a) (b) (c) (d)

(e) (f) (g) (h)

(d) (e) (f)

3.3. Color Evaluation

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

3.4. Elemental Analysis

Element Content Sample

3.5. FT-IR Analysis

Appl. Sci. 2022, 12, 4234 3.5. FT-IR Analysis 11 of 20

Parameter dUB mcUB ccUB F-Value

3.7. Free Radical Scavenging Activity

3.8. Relationships between Variables

Legislative WHO/EU WHO Eu.Ph. Ch.Ph. Heavy Metals Content

Supplementary Materials: The following supporting information can be downloaded at https:

Institutional Review Board Statement: Not applicable.

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