Hindawi

Evidence-Based Complementary and Alternative Medicine

Volume 2020, Article ID 8104538, 11 pages
https://doi.org/10.1155/2020/8104538

Research Article
Phytochemical Analysis, Cytotoxic, Antioxidant, and Antibacterial
Activities of Lichens

Noura Aoussar,1 Fatima Ezzahra Laasri,2 Mohammed Bourhia,3 Nedeljko Manoljovic,4

Rajaa Ait Mhand,1 Naima Rhallabi,1 Riaz Ullah ,5 Abdelaaty A. Shahat,5,6
Omar M. Noman ,5 Fahd A Nasr ,5 Omer M. Almarfadi,5 Mohammed El Mzibri,2
Perica Vasiljević,7 Laila Benbacer,2 and Fouad Mellouki1
1
RU Microbiology, Hygiene and Bioactive Molecules, LVMQB/EB, University Hassan II of Casablanca,
Faculty of Sciences and Techniques of Mohammedia, Casablanca 20650, Morocco
2
Unit of Biology and Medical Research, National Center for Energy, Nuclear Science and Technology, Rabat, Morocco
3
Laboratory of Chemistry-Biochemistry, Environment, Nutrition, and Health, Faculty of Medicine and Pharmacy,
University of Hassan II, B.P, Casablanca 5696, Morocco
4
Department of Pharmacy, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia
5
Department of Pharmacognosy (MAPPRC), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
6
Chemistry of Medicinal Plants Department, National Research Centre, P. No. 33 El Bohouth St. (former El Tahrirst.),
Giza 12622, Dokki, Egypt
7
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Nis, Nis 18000, Serbia2811

Correspondence should be addressed to Riaz Ullah; [email protected]

Received 18 June 2020; Revised 8 October 2020; Accepted 25 November 2020; Published 3 December 2020

Academic Editor: Shun-Wan Chan

Copyright © 2020 Noura Aoussar et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Lichens present a complex symbiotic relationship between a filamentous fungus, photoautotrophic partner (algae or
cyanobacteria), and bacterial community. The Objective of the Study. This study aimed at investigating the chemical composition
and cytotoxic, antioxidant, and antimicrobial activities of acetone extracts of Moroccan Evernia prunastri (E. prunastri), Ramalina
farinacea (R. farinacea), and Pseudevernia furfuracea (P. furfuracea). Materials and Methods. The phytochemical analysis was
carried out by HPLC-UV. The cytotoxic effect was assessed on human prostate cancer (22RV1), human colon carcinoma (HT-29),
human hepatocellular carcinoma (Hep-G2), and Hamster ovarian cancer (CHO) cells lines by WST1 assay. The antioxidant power
was assessed by DPPH and FRAP assays. The antibacterial effect was obtained using the broth microdilution method. Results. The
findings of phytochemical analysis showed that the lichens studied possess interesting bioactive molecules such as physodalic acid,
evernic acid, and usnic acid, as well as protocetraric acid. According to the American National Cancer Institute guidelines, the
WST-1 test showed that all crude extracts did not show significant cytotoxic effects against all concerous cell lines, and IC50 values
ranged from 42.30 to 140.24 µg/mL. Regarding the antioxidant activity, P. furfuracea extract showed the highest free-radical-
scavenging ability (IC50 � 498.40 µg/mL). The most potent antibacterial extract was recorded for P. furfuracea extract with a
minimum inhibitory concentration (MIC) ranging from 0.039 to 0.31 mg/mL. Conclusion. In this research work, we report that
the studied lichen extracts exhibit an important biological effect, supporting that lichens represent a hopeful source of original
natural products for the research of new bioactive molecules having a pharmaceutical interest.

1. Introduction lichens to form a third partner [3, 4]. There are about 18,500
species of lichens worldwide that can survive in various
Lichens are naturally arising from an alliance between extreme environmental conditions due to their exceptional
fungus and algae [1, 2]. Moreover, bacteria can also colonize resistance capacity that makes them pioneer species. The
2 Evidence-Based Complementary and Alternative Medicine

intrinsic resistance of lichen is mainly due to the production 2.2. Preparation of Lichen Extracts. Thalli of three lichen
of a wide range of compounds derived typically from sec- species were dried and ground into a fine powder. The
ondary metabolites of fungal components which build up in powder was extracted by maceration (24 h) using acetone at
the cortex or the medullary layer [5, 6]. Approximately 1050 ambient temperature [13]. Extracts of species were filtered
chemical substances are identified in lichens including then concentrated at 40°C under reduced pressure. The
depsides, depsidones, and dibenzofurans [2]. Lichens are extraction yield obtained 4.52%, 1.32%, and 4.32% for
known for their several medicinal virtues, and their me- P. furfuracea, R. farinacea, and E. prunastri extracts, re-
tabolites have been described for their multiple biological spectively. The extracts obtained were kept at −20°C until
properties [5, 7, 8]. further analysis.
Currently, in cancer treatment, several anticancer drugs
have been used with important side effects due to their close
2.3. HPL Analysis. HPLC-UV analysis was performed
therapeutic margin and high toxicity. Moreover, the risks for
according to the method adopted by Huneck and Yoshimura
infection are increased due to the small number of white
[14]. Extracts were solubilized in acetone (500 μL), and the
blood cells (neutropenia) arising from the chemotherapy
analysis was performed using HPLC (Agilent Technologies,
toxic effect on the bone marrow [9]. Due to its immuno-
1200 Series). An injection volume of 10 μL of the extract was
compromised status, antimicrobial therapy is often under-
analyzed using a mobile phase consisting of methanol-wa-
taken in hospitalized cancer patients. However, the
ter-phosphoric acid in the presence of a detector of UV
significant increase in use of antibiotics is associated with the
spectrophotometer (254 nm). Deionized water was purified
appearance of multidrug-resistant pathogens such as
using a purification system (Milli-Q.). HPLC-grade meth-
Staphylococcus aureus. This bacterium is the main noso-
anol was purchased from Merck (Darmstadt, Germany). The
comial pathogen agent worldwide and the most worrisome,
identification of polyphenolic compounds contained in
particularly S. aureus resisting the methicillin (MRSA), as
extracts was carried out by comparing retention times (tR)
well as it is easily capable to develop in biofilms in hospi-
and absorption spectra (200–400 nm) with those of the
talized patients [10].
authentic substances isolated early from other lichen species.
Furthermore, oxidative stress induced by the excessive
Previous studies have shown that the three tested lichens
production of free radicals is associated with different
contain certain phenolic acids (evernic acid, fumarproto-
chronic diseases and also to almost many cancers; namely, in
cetraric acid, atranorin, usnic acid, physodalic acid, chlor-
tumor progression [11], the search for natural antioxidant
oatranorin, and protocetraric acid), and that is why we chose
compounds is of great interest to preserve the physiological
them to be used as reference compounds. The standards used
performances of the body.
in this study were acquired from the following sources:
To overcome these issues, the researchers are ardently
evernic acid and atranorin are isolated from the Evernia
seeking alternative bioactive molecules (antimicrobial, an-
prunastri [15], fumarprotocetraric acid was purified from
tioxidant, and anticancer), with high efficacy and fewer
C. rangiferina and usnic acid from Cladonia foliacea [16],
secondary effects. Lichen secondary metabolites have been
physodalic acid and chloroatranorin from Hypogymnia
documented widely for their effectiveness against different
physodes [17], and protocetraric acid from Toninia candida
tumor cells and also for their bacterial resistance potential.
[18].
As far as we can tell, a few studies have evaluated the an-
ticancer activity of Evernia prunastri, Pseudevernia furfur- 2.4. In Vitro Cytotoxic Activity
acea, and Ramalina farinacea. However, the Moroccan
lichens have not yet been studied in terms of pharmaco- 2.4.1. Cell Lines and Culture. Human prostate cancer
logical effects. (22RV1) cells were kindly provided by Dr. Belharazem,
The present research study aimed to investigate in vitro Institute of Pathology, Medical Faculty of Mannheim
antioxidant potency and antimicrobial, as well as cytotoxic, University, Heidelberg. Human colon carcinoma (HT-29),
effects of organic extracts from E. prunastri, P. furfuracea, human hepatocellular carcinoma (Hep-G2), and hamster
and R. farinacea growing in Moroccan soil. ovarian cancer (CHO) cell lines were kindly given by Dr.
L’Houcine, OUAFIK, APHM, North Hospital, Transfer
Laboratory, Marseille 13015, France. These cell lines were
2. Materials and Methods maintained and cultured as a monolayer in a DMEM me-
dium with the following components: inactivated fetal calf
2.1. Lichen Material. Thallus samples of R. farinacea (L.)
serum with 10%, glutamine with 1%, and antibiotics with
Ach., E. prunastri (L.) Ach., and P. furfuracea (L.) Zopf. were
1%, except for CHO cell lines that were maintained in
collected from Khenifra, Morocco. The collected lichens
McCoy’s 5 A medium. The cells were grown at 37°C in a wet
were identified based on morphological characteristics de-
atmosphere with air (95%) and CO2 (5%).
termined by macroscopic and microscopic studies, as well as
on the basis of colorful reactions by chemical reagents [12].
Voucher specimens of collected species (P. furfuracea # 2501, 2.4.2. Cell Viability Assay. The cytotoxic effect of the acetone
E. prunastri # 2502, and R. farinacea # 2503) have been put at extracts of P. furfuracea, R. farinacea, and E. prunastri
the Herbarium of Moroccan Scientific Institute. against cancer cell lines was estimated using the WST1 test
Evidence-Based Complementary and Alternative Medicine 3

[19]. All cell lines were regularly seeded in 96-well micro- 2.5.4. Determination of Total Flavonoid Content. Total fla-
plates. After cell adhesion (24 h), the five different extract vonoid content (TFC) in extracts was evaluated using
concentrations, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, protocols as previously described [23]. An aliquot of 500
and 12.5 μg/mL, were added in duplicate to the wells and microliters of each lichen extract (1 mg/mL) was added to
reincubated. After incubation at 37°C for 72 h, 100 μL of the 75 µL of sodium nitrite solution (5%) mixed with 150 µL of
medium was replaced with 10 μ L of WST1 and incubated aluminum chloride (10%), after 5 min at ambient temper-
again for further time. Mitomycin was used as a drug ref- ature, 500 µL of NaOH reagent (1 M) was added, and then,
erence, and results were presented as the percentage of cell the absorbance was recorded at 510 nm. TFC was presented
viability, which was determined via the following equation: as catechin equivalent (CE) (µg CE/mg of dry extract).
Asample
Cell viability(%) � 􏼠 􏼡 × 100. (1) 2.6. Antimicrobial Activity
AControl

Asample and AControl with and without extract, respec- 2.6.1. Bacterial Strains. The antibacterial activity of lichen
tively, were read for the assessment of absorbance. The test extracts was assessed against 11 bacterial strains including
was evaluated in duplicate. Gram-positive bacteria: S. aureus (ATCC 25923), five
clinical Methicillin-Resistant S. aureus (MRSA) isolates from
burn wounds of patients at IbnRochd University Hospital of
2.5. Antioxidant Activity Casablanca (Morocco), Listeria innocua (CECT 4030),
B. subtilis (DSM 6633), and Gram-negative bacteria, namely,
2.5.1. FRAP Assay. The ferric-reducing powers of lichen Escherichia coli (ATCC 25922), P. aeruginosa (CECT 118),
extracts were evaluated according to the method described P. mirabilis.
in the early literature [20]. In brief, 1 mL of extract The S. aureus clinical isolates were identified as multi-
(50–1000 µg/mL) was mixed with 2.5 mL of phosphate buffer drug resistant by testing their antibiotic susceptibility
and then added to 2.5 ml of the solution of potassium fer- according to the EUCAST 2016 guidelines [24], as described
ricyanide (1%). Afterward, the mixture was incubated for by Achmit et al. [25].
30 min at 50°C and then centrifuged at 3000 rpm. 2.5 mL of
the supernatants were added to 2.5 mL of distilled water and
mixed with 0.1% FeCl3. Finally, the absorbance of the 2.6.2. Determination of MIC and MBC. The MICs (Mini-
resulting solutions was recorded at 700 nm. In this assay, mum Inhibitory Concentrations) were determined
trolox and ascorbic acid were used as standards. Increasing according to data by Satyajit et al. with some modifications
the absorption of the sample is an indication of increasing [26]. Wells of the plate were filled with both culture medium
reducing power. All experiments were executed in triplicate. and extracts (v/v: 100/100 µL) at concentrations ranging
from 5 to 0.002 mg/mL; to each well, bacterial inoculum at
5 × 106 CFU/mL was added followed by resazurin solution
2.5.2. DPPH Assay. The measurement of the antiradical (0.015%) as a marker of microbial growth. The plates were
effect of extracts from the studied lichen species was carried incubated again for 24 h at 37°C. The lowest effective con-
out by the DPPH test as described by Kosanić et al. [21]. centration was considered as a minimal inhibitory con-
Briefly, 1 mL of extract (50–1000 µg/mL) was mixed with centration (MIC) [27]. Experiments were realized in
2 mL of DPPH aliquot (0.12 mM). The reaction mixture was duplicate.
incubated for 25 min in the dark at ambient temperature. Regarding the MBC (Minimum Bactericidal Concen-
The absorbance of the mixture was recorded at 517 nm. The tration), 10 µL from purple wells of the MICs test were
percentage of inhibition of the DPPH radical was performed subcultured on nutrient agar in Petri plates. MBC was
using the equation given below. considered as the lowest effective concentration with no
Scavenging of DPPH (%) � 100 × [(Absorbance of bacterial growth after reincubation. Moreover, for each
blank − Absorbance of the sample)/Absorbance of blank]. extract, the ratio CMB/MIC was calculated to assess its
IC50 values were obtained from the percentage inhibition antibacterial ability, the extract has a bactericidal effect when
vs. concentration plot, using Regtox software, and expressed CMB/MIC � 1–2 and a bacteriostatic effect when CMB/
in μg/mL. All measurements were conducted in triplicate. CMI � 4–16 [28].

2.7. Statistical Analysis. Data were reported as mean ± (SD).

2.5.3. Determination of Phenols. Total phenolic content One-way ANOVA and post hoc t-tests were used for sta-
(TPC) in extracts was meticulously assessed using the tistical analysis. The correlation coefficient was defined by
Folin–Ciocalteu method [22], with some modifications. the Pearson test using SPSS-22. The differences were ac-
Briefly, 100 µL of extracts (1 mg/mL) was diluted up to cepted as significant at p < 0.05.
4.6 mL and then added to100 µL of the reagent of
Folin–Ciocalteu. Afterward, the mixture was left for 3 min 3. Results
and then added to Na2CO3 (300 µL, 2%). After incubation
for 90 min at 25°C, the absorbance was read at 760 nm. 3.1. HPLC Analysis. The HPLC-UV analysis of extracts of
Results were expressed as µg GAE/mg dry extract. R. farinacea, E. prunastri, and P. furfuracea was used to
4 Evidence-Based Complementary and Alternative Medicine

identify their main phenolic acids by matching their re- P. furfuracea and E.prunastri-R.farinacea (p < 0.05) but not
tention times (tR) and absorbance maxima (nm) UV between R. farinacea and E. prunastri (p > 0.05). TFC of
spectrum with the reference compounds. The chromato- these extracts was calculated from the catechin calibration
grams of eleven standards and extract samples are given in curve (R2 � 0.97). The TFC of tested extracts ranged from
Figures 1 and 2. The structures of the identified molecules 12.23 to 17.63 μg CE/mg of the dry extract with a significant
are shown in Figure 3. The obtained data confirmed that the difference between R. farinacea and P. furfuracea-
main compounds in extracts of P. furfuracea were phys- E. prunastri (p < 0.05), but no significant difference between
odalic acid (PHY), atranorin (ATR), and chloratranorin P. furfuracea and E. prunastri was reported (p > 0.05). The
(CHL). PHY was the most abundant substance. Evernic acid highest total flavonoid content was registered for the extract
(EVE), usnic acid (USN), atranorin (ATR), and chlora- of R. farinacea (Table 2).
tranorin (CHL) were identified, with EVE being the most The ferric reducing power of the studied crude extracts
abundant compound in E. prunastri. Protocetraric acid was reported in a dose-dependent manner. As shown in
(PRO), fumarprotocetraric acid (FUM), EVE, USN, and Figure 6, the highest activity was obtained for R. farinacea
ATR were identified, with PRO being the predominant extract with absorbance increased from 0.01 to 0.22.
phenolic compound in R. farinacea (Figure 2). However, no significant difference between the extract of
R. farinacea and P. furfuracea was observed. This activity
remains lower compared to the positive controls (ascorbic
3.2. Cytotoxic Activity. The cytotoxic effect of R. farinacea, acid and Trolox) (Figure 6).
E. prunastri, and P. furfuracea extracts against different cell The DPPH test of lichen extracts was performed, and the
lines was assessed using the WST1. The results revealed that obtained results are reported in Figure 7. All lichen extracts
the extracts demonstrated a relatively low cytotoxic effect exhibited strong scavenging ability which varied from 6.63%
against all cells in a dose-dependent manner (Figure 4). Loss to 72.12% for concentrations ranged from 50 to 1000 μg/mL,
of cell viability was revealed by the morphological and with a significant correlation with TPC (r � 0.69). Among the
aggregation changes depending on the concentration of tested extracts, P. furfuracea extract showed the best scav-
extracts as shown, for example, by the extracts of enging effect (IC50 � 498.40 µg/mL), which was significantly
R. farinacea, E. prunastri, and P. furfuracea against HT-29 different than R. farinacea and E. prunastri (p < 0.05). The
cell lines (Figure 5). As shown in Figure 5, the number of results also showed that the standards (ascorbic acid and
dead cells positively correlates with the concentration of the Trolox) demonstrated stronger DPPH radical-scavenging
extracts. At high concentrations of the extracts, cells started activity than the tested extracts (Table 3).
to get a more enlarged shape and a formation of blebs in the
cell’s membranes. We also noticed the appearance of apo-
ptotic bodies, large vacuoles in the cell cytoplasm, and 3.4. Antibacterial Activity. The antibacterial effect of
rounded shape of the cells that start to detach from the R. farinacea, E. prunastri, and P. furfuracea extracts was
surface and float in the medium indicating cell death. The evaluated by the microdilution method with resazurin vs.
IC50 values of organic extracts from lichens ranged from eleven bacterial strains including 5 clinical isolates of
42.30 to 140.24 µg/mL (Table 1) with no significant differ- methicillin-resistant S. aureus. The MIC and the MBC of
ence between the sensitivity of cancer cells treated by extracts were determined, and the results are presented in
E. prunastri and by R. farinacea (p > 0.05), and for Table 4. These findings revealed that all extracts exhibited a
P. furfuracea, we found a significant difference between higher antibacterial effect vs. Gram-positive bacteria.
22RV1 cells and the other cell lines (p < 0.05). Furthermore, However, no effect was recorded for Gram-negative bacteria.
a significant difference was observed between P. furfuracea P. furfuracea exhibited an antibacterial effect with MIC
and R. farinacea in the inhibition of all tested cell lines and values of 0.039–0.15 mg/mL and MBC 0.625 mg/mL for all
between P. furfuracea and E. prunastri for HT-29 and 22RV1 strains. The extract from E. prunastri presented a MIC
cells (p < 0.05). Among extracts studied, P. furfuracea extract ranged from 0.039 to 0.15 mg/mL and MBC from 0.625 to
was found to induce the largest effect towards all cancer cell 2.5 mg/mL; also, the extract of R. farinacea possessed MIC in
lines tested, especially against 22RV1 (human prostate the range of 0.078–0.625 mg/mL, while its MBC was at
cancer) cells. As can be seen in Table 1, the cytotoxic effect of 0.625–1.25 mg/mL.
extracts studied was lower compared to that of mitomycin The lower MIC value was demonstrated for P. furfuracea
(positive control). and E. prunastri in the SARM strain N°1 (0.039 mg/mL), and
the higher MBC value was found for E. prunastri in Listeria
innocua (2.5 mg/mL). Overall, the MIC values obtained for
3.3. Antioxidant Activity. The total phenolic contents (TPC) the acetone extract of P. furfuracea were lower than those
of E. prunastri, P. furfuracea, and R. farinacea extracts were obtained with extracts of E.prunastri and R.farinacea.
calculated using the gallic acid curve (R2 � 0.99). As shown in From the obtained ratio, MBC/MCI, it can be noticed
Table 2, the TPC of the three lichen extracts ranged from that the extract from P. furfuracea showed a bactericidal
167.67 to 328.67 μg GAE/mg of dry extract. P. furfuracea effect against Listeria innocua and for R. farinacea against
extract showed the highest TPC (328.67 μg GAE/mg of crude strains of MRSA N°2, 3, 4, and 5. For the rest of the strains, a
extract). We found a significant difference between bacteriostatic effect was recorded.
Evidence-Based Complementary and Alternative Medicine 5

60
500 EVE PHY ATR
400 USN ATR 40

mAU
mAU

300 CHL
200 20 CHL
100
0 0

5 10 15 5 10 15
min min

(a) (b)
PRO
FUM
40
30
mAU

20
10
0
5 10 15
min

(c)

Figure 1: HPLC chromatograms of the standards used at 254 nm. EVE—evernic acid, USN—usnic acid, ATR—atranorin,
CHL—chloratranorin, PHY—physodalic acid, PRO—protocetraric acid, and FUM—fumaprotocetraric acid.

2000
150 PHY EVE
1500
mAU
mAU

100 1000
ATR CHL
50 500
USN ATR CHL
0 0
5 10 15 5 10 15
min min

(a) (b)
PRO
70
60
50
mAU

40
30 EVE
20 FUM
10 USN
0
5 10 15
min

(c)

Figure 2: HPLC chromatograms of extracts of Pseudevernia furfuracea (a), Evernia prunastri (b), and Ramalina farinacea (c) at 254 nm.

4. Discussion In this study, the studied extracts demonstrated a rel-

atively low cytotoxic activity against all cell lines used. This
In the present research work, we examined chemical profiles activity did not differ significantly from one line to another
and the in vitro cytotoxic, antioxidant, and antimicrobial treated by the same extract, which means that these extracts
activities of organic extract from Moroccan lichens, reacted in the same way regardless of the cell used. These
R. farinacea, E. prunastri and P. furfuracea. The studied findings agree with the literature [14, 29, 30]. Among the
extracts affirmed the presence of evernic acid, physodalic extracts, P. furfuracea extract was found to induce the largest
acid, and protocetraric acid as major phenolic compounds. effect against all cancer cell lines tested, especially on human
The biological activities of evernic and protocetraric acids prostate cancer (22RV1) cell lines at exposure time 72 h. This
have been reported in previous studies, where they showed result agrees with those reported in the previous study which
higher activity than the one obtained for our examined li- showed that the extracts of E. prunastri and P. furfuracea
chen extracts including the antioxidant effect by DPPH possess cytotoxic effects against human melanoma (FemX)
assay. Also, they were found to exhibit strong antimicrobial and human colon carcinoma (LS174) with similar IC50
activity against different microorganisms and anticancer values to ours (55.09–120.89 µg/mL). Moreover, the stron-
activity against various cell lines [15, 29] while there are no gest cytotoxic activity was presented by P. furfuracea. It was
available data on the antioxidant, antibacterial, and anti- also determined that these extracts induced cell death caused
cancer activities of physodalic acid [30]. by a strong arrest of the sub-G1 phase in the cell cycle of
6 Evidence-Based Complementary and Alternative Medicine

COOH
O

OH OH H3C OH O
O O
H3C O
O H3C
OH O O
OH HO

O CH3 O OH
O O
H3C HO COOH
CHO H3C CH3
O CH3

Evernic acid Fumarprotocetraric acid Usnic acid

O
CH3
CH3 O
O
H 3C O CH3 H3C
OH
O O OH
HO
OH O
OH O
HO CH3
O O
O O
CHO COOH OH
COOH HO O
H3C CH3
CHO H3C

Protocetraric acid Physodalic acid Atranorin

CI
CH3 H3C
OH
HO
O
O
CH3
O
O
OH
CH3
Chloroatranorin
Figure 3: The chemical structures of the identified compounds.

LS174 and FemX cells [15]. The literature also pointed out polyphenolic compounds, which is in accordance with other
that either raw lichen extracts or their purified components studies carried out in acetone extract of P. furfuracea and
were effective against different cancer cell lines even at low E. prunastri harvested in Serbia and Turkey showing in the
concentrations [16, 31, 32]. However, according to the same way that P. furfuracea extract had a largest antioxidant
American National Cancer Institute guidelines, the IC50 activity and the highest quantity of phenols than E.prunastri
values of the three lichen extracts found in this study did not extract [15, 35]. Our results indicated higher antioxidant
indicate strong cytotoxic activity IC50 > 30 µg/mL [31], while capacity and phenolic content than those reported by
the strong cytotoxic effect of physodic acid isolated from Kosanić and Bı̀lgı̀n Sokmen in their studies. Moreover, our
P. furfuracea vs. FemX and LS174 cancer cells with IC50 of results showed that R. farinacea extract had the highest ferric
19.52 and 17.89 µg/mL, respectively, was already reported reducing power, but the lowest phenols content, which
[15]. suggests that this activity of this tested extract can be due to
Lichens have been involved in several studies looking for the presence of nonphenolic compounds.
new natural antioxidants and their potential protective ef- Türka The antibacterial activity of R. farinacea,
fects vs. chronic diseases [33, 34]. In the present work, our P. furfuracea, and E. prunastri extracts was evaluated by the
findings showed that the tested extracts had a potent in vitro microdilution method against bacterial strains including
antioxidant effect which correlated to content in total clinical isolates of methicillin-resistant S. aureus. The results
phenols. This result was in accordance with another pub- relieved that all extracts exhibited a potent antibacterial
lished work which showed a positive correlation between the effect vs. Gram-positive bacteria. However, no effect was
phenolic content and the antioxidant activity [14, 15]. observed for Gram-negative bacteria. These results are in
Furthermore, no significant correlation between the flavo- harmony with those carried out by et al. [36] and Tay et al.
noid content in the lichen extract and the antioxidant effect [37] that reported a great activity of P. furfuracea and
was reported therein. This means that lichen components R. farinacea against only Gram-positive bacteria, and they
(depsides, depsidones, and dibenzofurans) are the principal also found that physodic acid and (+)-usnic acid isolated
agents responsible for the antioxidant activities. Among the from these species, respectively, were inactive against Gram-
tested extracts, P. furfuracea extract showed the best anti- negative strains [28, 29]. In recent a study, Gültekin and
oxidant power with the greatest concentration of Özyiğitoğlu showed that acetone extract of P. furfuracea had
Evidence-Based Complementary and Alternative Medicine 7

P.F E.P
120 120

100 100

80 80
Viability %

Viability %
60 60

40 40

20 20

0 0
0 50 100 150 200 0 50 100 150 200
Concentration (μg/mL) Concentration (μg/mL)

CHO HEPG2 CHO HEPG2

HT-29 22RV1 HT-29 22RV1

R.F
120

100

80
Viability %

60

40

20

0
0 50 100 150 200
Concentration (μg/mL)

CHO HEPG2
HT-29 22RV1
Figure 4: : Percentage of cell viability of CHO, HT-29, HEP-G2, and 22RV1 cell lines treated with varying concentrations of extracts of
P. furfuracea (P.F), R. farinacea (R.F), and E. prunastri (E.P) for 72 h.

Lichen
extracts Control DMSO 12 .5 μg/mL 200 μg/mL

R. farinacea

E. prunastri

P. furfuracea

Figure 5: Morphological aspects of HT-29 cells before (Control) and after 72 h treatment with acetone extract of R. farinacea, E. prunastri,
and P. furfuracea with 12.5 µg/mL and 200 µg/mL concentrations and with DMSO at 200 µg/mL.
8 Evidence-Based Complementary and Alternative Medicine

Table 1: IC50 values of growth inhibitory effects of R. farinacea, E. prunastri, and P. furfuracea versus CHO, HT-29, Hep-G2, and 22RV1
cell lines at exposure time 72 h.
Growth inhibitory effects (IC50 (µg/mL))
Lichen species
CHO HT-29 Hep-G2 22RV1
P. furfuracea 63.60 ± 2.98a 57.10 ± 2.83 a
68.60 ± 3.77a 42.30 ± 2.55a
E. prunastri 60.80 ± 0.36a 105.52 ± 0.79b 95.71 ± 1.50ab 103.80 ± 18.40a
R. farinacea 140.24 ± 10.40b 127.677 ± 5.835c 110.15 ± 18.50b 96.42 ± 16.40a
Mytomicine 3.80 ± 0.10c 0.90 ± 0.10 d
3.21 ± 2.40c 2.56 ± 0.10b
Data are expressed in means (n � 3) ± SD. Values reported in the same column with different letters (a–d) significantly differ at p < 0.05.

Table 2: Total phenolic and flavonoids contents in R. farinacea, E. prunastri, and P. furfuracea extracts.
Lichen species TPC (μg GAE/mg of dry extract) TFC (μg CE/mg of dry extract)
R. farinacea 167.67 ± 50.20 17.63 ± 1.11∗
E. prunastri 194.33 ± 7.50 13.50 ± 2.14
P.furfuracea 328.67 ± 26.81∗∗ 12.23 ± 0.40
Data are reported as mean (n � 3) ± SD, ∗: p < 0.05, ∗∗
:p < 0.01.

0.2
Absorbance at 700 nm

Absorbance at 700 nm

0.1
1

0.0
0
0 200 400 600 800 1000 0 200 400 600 800 1000
Concentration (μg/mL) Concentration (μg/mL)

P.F Ascorbic acid

E.P Trolox
R.F
(a) (b)

Figure 6: Reducing power of the lichens R. farinacea (R.F), E. prunastri (E.P), and P. furfuracea (P.F) extracts (a) and ascorbic acid and
Trolox (b).

no inhibitory effect on Gram-negative bacteria [38]; also, diffusion of hydrophobic compounds. Without an outer
Osmana et al. reported that only Gram-positive bacteria membrane, the cell wall of Gram-positive bacteria can be
were susceptible to the acetone extract of P. furfuracea and easily permeable [40].
Evernia divaricata [39]. In contrast, other studies showed Finally, our research findings provided that the lichen
that these species have presented antibacterial effects vs. both extract tested demonstrated high antibacterial activity
Gram-positive and Gram-negative bacteria with stronger against MRSA clinical isolates from burn wounds. The ac-
inhibitory effects on Gram-positive bacteria [22, 32]. The etone extracts of P. furfuracea and E. prunastri exhibited
reason for these conflicting results may be due to variations high activity with MICs ranged from 0.039 to 0.15 mg/mL
in the genotype of the strains tested and the experimental and a bacteriostatic effect. Furthermore, R. farinacea extract
conditions. exhibited a bactericidal effect against one MRSA with MIC
The high sensibility of Gram-positive bacteria might be values ranging from 0.078 to 0.625 mg/mL for all MRSA
interpreted by the fact that the structures of the cell envelope strains. This activity could be induced by usnic acid which
are different between both Gram-positive and Gram-neg- was the major antibacterial agent in R. farinacea [37].
ative bacteria. The former has an outer membrane formed by Pompilio et al. demonstrated that usnic acid showed sig-
an inner phospholipid layer surmounted by LPS (lipo- nificantly higher activity against MRSA strains than atra-
polysaccharide) macromolecules which prevent the norin and fumarprotocetraric acid [41]. Other data indicated
Evidence-Based Complementary and Alternative Medicine 9

100 80

Scavenging of DPPH (%)

Scavenging of DPPH (%)

80
60

60
40
40

20
20

0 0
0 5 10 15 20 0 200 400 600 800 1000
Concentration (μg/mL) Concentration (μg/mL)

Ascorbic acid P.F

Trolox E.P
R.F
(a) (b)

Figure 7: Scavenging effect of standards, ascorbic acid, and Trolox (a) and P. furfuracea (P.F), E. prunastri (E.P), and R. farinacea (R.F) (b).

Table 3: IC50 values of crude extracts of R. farinacea, E. prunastri, and P. furfuracea.

Species IC50 (µg/mL)
R. farinacea >1000
E. prunastri >1000
P. furfuracea 498.40 ± 44.14
Ascorbic acid 8.90 ± 0.10∗∗
Trolox 17.83 ± 0.50∗
Data are reported as mean (n � 3) ± SD, ∗: p < 0.05, ∗∗
:p < 0.01.

Table 4: In vitro antibacterial effect of crude extracts of P. furfuracea, E. prunastri, and R. farinacea.
Pseudeverina furfuracea Evernia prunastri Ramalina farinacea
Bacteria
MICa MBCa MBC/MIC MIC a
MBCa MBC/MIC MIC a
MBCa MIC/MBC
Bacillus subtilis 0.078 0.625 8.012 0.078 1.25 16.025 0.078 0.625 8.012
Listeria innocua 0.31 0.625 2.016 0.625 2.5 4 0.31 1.25 4.032
Staphylococcus aureus 0.078 0.625 8.012 0.078 0.625 8.012 0.15 1.25 8.333
MRSA N°1 0.039 0.625 16.02 0.039 0.625 16.02 0.15 0.625 4.16
MRSA N°2 0.15 0.625 4.16 0.15 1.25 8.33 0.625 1.25 2
MRSA N°3 0.078 0.625 8.01 0.15 1.25 8.33 0.625 1.25 2
MRSA N°4 0.15 0.625 4.16 0.15 0.625 4.16 0.625 1.25 2
MRSA N°5 0.078 0.625 8.012 0.15 0.625 4.16 0.625 1.25 2
Escherichia coli >25 — — >25 — — >25 — —
Pseudomonas aeruginosa >25 — — >25 — — >25 — —
Proteus mirabilis >25 — — >25 — — >25 — —
a: (mg/mL), MRSA: Methicillin-Resistant Staphylococcus aureus.

that usnic acid presented high antibacterial activity against widely investigated, and the mechanism of action of these
clinical isolates of MRSA with MIC values ranging between substances has not been sufficiently assessed [44].
25 and 50 μg/mL by disruption of the bacterial membrane
[42]. Various lichenic compounds such as lobar acid, 5. Conclusions
physodic acid, rhizocarpic acid, 3-hydroxyphysodic acid,
hybocarpone, and (R)-(+)-usnic acid isolated, respectively, The current study sheds light on the biological properties of
from Sterocaulon dactylophyllum, Hypogymnia physodes, extracts from R. farinacea, E. prunastri, and P. furfuracea
Psilolechia lucida, Hypogymnia physodes, Lecanora con- growing in Morocco. The results reported here pointed out
izaeoides, and Lecanora albescens lichen species were found that the three lichen extracts possess significant antioxidant
to be effective vs. methicillin- and multidrug-resistant and antibacterial activities. Pseudevernia furfuracea extract
Staphylococcus aureus [43]. Despite, the antibacterial activity exhibited the best antioxidant power, as well as the highest
of lichens, either as raw extracts or purified compounds, was total phenolic content. The results also demonstrated that all
10 Evidence-Based Complementary and Alternative Medicine

studied extracts have antibacterial effects against only Gram- shikimatemetabolite production, and PKS genes,” Natural
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Conflicts of Interest
prognosis and treatment,” Journal of Clinical Medicine, vol. 6,
All contributing authors declare that there are no conflicts of no. 3, p. 29, 2017.
interest. [12] N. Aoussar, R. Manzali, I. Nattah et al., “Chemical compo-
sition and antioxidant activity of two lichens species (Pseu-
devernia furfuracea L and Evernia prunastri L) collected from
Acknowledgments Morocco,” Journal of Materials and Environmental Sciences,
vol. 8, no. 6, pp. 1968–1976, 2017.
The authors extend their appreciation to the Deanship of [13] N. Aoussar, N. Rhallabi, R. Ait Mhand et al., “Seasonal
Scientific Research at King Saud University for funding this variation of antioxidant activity and phenolic content of
work through research group no. RGP-262. This research Pseudevernia furfuracea, Evernia prunastri and Ramalina
study was also supported by University Hassan II of farinacea from Morocco,” Journal of the Saudi Society of
Casablanca (Morocco) and by the Ministry of Education and Agricultural Sciences, vol. 19, no. 1, pp. 1–6, 2020.
Science of the Republic of Serbia (G. no. 172015). The au- [14] S. Huneck and I. Yoshimura, Identification of Lichen Sub-
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(University Ibn Tofail, Faculty of Sciences, Kenitra) for his [15] M. Kosanić, N. Manojlović, S. Janković, T. Stanojković, and
help in the identification of the lichen species and Professor B. Ranković, “Evernia prunastri and Pseudoevernia furfur-
K. Zerouali (University Hospital, Casablanca, Morocco) for aceae lichens and their major metabolites as antioxidant,
providing the clinical strain of Staphylococcus aureus. antimicrobial and anticancer agents,” Food and Chemical
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[16] M. Kosanić, “Extracts of five cladonia lichens as sources of
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