Phytomedicine 19 (2012) 1166–1172
Contents lists available at SciVerse ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Chemical composition of three Parmelia lichens and antioxidant,
antimicrobial and cytotoxic activities of some their major metabolites
Nedeljko Manojlović a , Branislav Ranković b , Marijana Kosanić b,∗ , Perica Vasiljević c ,
Tatjana Stanojković d
a
Department of Pharmacy, Medical Faculty, University of Kragujevac, 34000 Kragujevac, Serbia
Department of Biology, Faculty of Science, University of Kragujevac, 34000 Kragujevac, Serbia
c
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, 18000 Niš, Serbia
d
Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia
b
a r t i c l e
i n f o
Keywords:
Lichens
HPLC-UV
Chemical composition
Biological activities
a b s t r a c t
The aim of this study is to investigate chemical composition of acetone extracts of the lichens Parmelia
caperata, P. saxatilis and P. sulcata and antioxidant, antimicrobial and anticancer activities of some
their major metabolites. The phytochemical analysis of acetone extracts of three Parmelia lichens were
determined by HPLC-UV method. The predominant phenolic compounds in these extracts were protocetraric and usnic acids (P. caperata) and depsidone salazinic acid (other two species). Besides these
compounds, atranorin and chloroatranorin, were also detected in some of these extracts. Antioxidant activity of their isolated metabolites was evaluated by free radical scavenging, superoxide anion
radical scavenging and reducing power. As a result of the study salazinic acid had stronger antioxidant activity than protocetraric acid. The antimicrobial activity was estimated by determination of the
minimal inhibitory concentration by the broth microdilution method. Both compounds were highly
active with minimum inhibitory concentration values ranging from 0.015 to 1 mg/ml. Anticancer
activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines
using MTT method. Salazinic acid and protocetraric acid were found to be strong anticancer activity toward both cell lines with IC50 values ranging from 35.67 to 60.18 g/ml. The present study
shows that tested lichen compounds demonstrated a strong antioxidant, antimicrobial, and anticancer
effects. That suggest that these lichens can be used as new sources of the natural antimicrobial agents,
antioxidants and anticancer compounds.
© 2012 Elsevier GmbH. All rights reserved.
Introduction
Lichens are complex symbiotic associations between a fungus (mycobiont) and photobiont which can be either an alga or
cyanobacteria (Bates et al. 2011). They are proven as the earliest
colonizers of terrestrial habitats on the earth with a worldwide
distribution from artic to tropical regions and from the plains to
the highest mountains. Their specific, even extreme, conditions
existence, slow growth and long life are the reason for producing
of numerous protective compounds against different physical and
biological influences (Mitrović et al. 2011).
Lichens synthesize a variety of secondary metabolites, mostly
from fungal metabolism. They are crystals deposited on the surface
of hiphes. They are poorly soluble in water and can usually be
∗ Corresponding author. Tel.: +381 34336223; fax: +381 34335040.
E-mail address: marijanakosanic@yahoo.com (M. Kosanić).
0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.phymed.2012.07.012
isolated from a lichen by organic dilutants (Otzurk et al. 1999). More
than one hundred secondary metabolites, mainly monoaromatics,
depsides, depsidones, pulvinates, dibenzofurans, anthraquinones
and xanthones, characteristic of lichen have been detected and
isolated (Molnar and Farkaš 2010). Chemicals structures of these
classes of compounds are similar and identification is often very
difficult.For a long time, some lichen species have been used in traditional medicine in the treatment of numerous infectious diseases
(Bown 2001). The use of lichens in medicine is based on the fact
that they contain unique and varied biologically active substances.
Lichen substances exert a wide variety of biological actions
including antibiotic, antimycotic, antiviral, anti-inflammatory,
analgesic, antipyretic, antiproliferative and cytotoxic effects
(Kosanić et al. 2012a; Manojlović et al. 2010). Thus, the aim of
the present work was to identify of secondary metabolites of P.
caperata, P. saxatilis and P. sulcata by HPLC-UV and to evaluate the
antioxidant capacity, antimicrobial and anticancer activities of the
acetone extracts from this lichen as well as their major secondary
metabolites.
N. Manojlović et al. / Phytomedicine 19 (2012) 1166–1172
Material and methods
to ascorbic acid and usnic acid. The DPPH radical concentration was
calculated using the following equation:
Lichen samples
DPPH scavenging effect (%) =
Lichen samples of P. caperata (L.) Ach. P. sulcata (Taylor) and
P. saxatilis (L.) Ach. were collected from Kopaonik, Serbia, in
September of 2011. The demonstration samples are preserved in
facilities of the Department of Biology and Ecology of Kragujevac,
Faculty of Science. Determination of the investigated lichens was
accomplished using standard methods.
Preparation of the lichen extracts
Finely dry ground thalli of the investigated lichens (100 g) were
extracted using acetone in a Soxhlet extractor. The extracts were
filtered and then concentrated under reduced pressure in a rotary
evaporator. The dry extracts were stored at −18 ◦ C until they were
used in the tests. The extracts were dissolved in 5% dimethylsulfoxide (DMSO) for the experiments.
High performance liquid chromatography (HPLC) analysis
Dry lichen extracts were redissolved in 500 l of acetone and
analyzed on an 1200 Series HPLC (Agilent Technologies) instrument with C18 column (C18; 25 cm × 4.6 mm, 10 m) and a UV
spectrophotometric detector with methanol–water–phosphoric
acid (75:25:0.9, v/v/v) solvent. Methanol was of HPLC grade
and was purchased from Merck (Darmstadt, Germany). Phosphoric acid was analytical-grade reagent. Deionized water used
throughout the experiments was generated by a Milli-Q academic
water purification system (Milford, MA, USA). The flow rate was
1.0 ml/min. The sample injection volume was 10 l. The standards used were obtained from the following sources: salazinic
acid (SAL, tR = 3.01 ± 0.20 min) was isolated from lichen Lobaria
pulmonaria, protocetraric acid (PRO, tR = 3.62 ± 0.20 min) isolated
from lichen Toninia candida, usnic acid (USN, tR = 10.53 ± 0.30 min),
atranorin (ATR, tR = 11.72 ± 0.10 min) and chloroatranorin (CHL,
tR = 13.23 ± 0.20 min) from lichen Evernia prunastri.
Isolation of lichen metabolites
Usnic acid (190 mg) was isolated from the acetone extract
of P. caperata (500 mg) by precipitation in benzene, and in this
study it has been used as a standard compound. Protocetraric acid
was isolated from the filtrate residue using a silica gel column
(0.149–0.074 mm; 100–200 mesh) with methanol–chloroform gradient solvent (10:1 and 5:1). Salazinic acid (300 mg) was isolated
from acetone extract of P. saxatilis (500 mg) by precipitation in
acetone. All isolated compounds were identified by their melting
points and spectroscopic data (Huneck and Yoshimura 1996).
Antioxidant activity
Scavenging DPPH radicals
The free radical scavenging activity of samples was measured
by 1,1-diphenyl-2-picryl-hydrazil (DPPH). The method used is
similar to the method previously used by some authors (Ibanez
et al. 2003; Dorman et al. 2004) but was modified in details. Two
milliliters of methanol solution of DPPH radical in the concentration of 0.05 mg/ml and 1 ml of test samples (1000, 500, 250, 125
and 62.5 g/ml) were placed in cuvettes. The mixture was shaken
vigorosly and allowed to stand at room temperature for 30 min.
Then the absorbance was measured at 517 nm in spectrophotometer (“Jenway”, UK). Free radical scavenging activity was compared
1167
A − A
1
0
A0
× 100
where A0 is the absorbance of the negative control and A1 is the
absorbance of reaction mixture or standard.
The inhibition concentration at 50% inhibition (IC50 ) was the
parameter used to compare the radical scavenging activity.
Reducing power
The reducing power of samples was determined according to
the method of Oyaizu (1986). One milliliter of test samples (1000,
500, 250, 125 and 62.5 g/ml) were mixed with 2.5 ml of phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide
(2.5 ml, 1%). The mixtures were incubated at 50 ◦ C for 20 min. Then,
trichloroacetic acid (10%, 2.5 ml) was added to the mixture and centrifuged. Finally, the upper layer was mixed with distilled water
(2.5 ml) and ferric chloride (0.5 ml; 0.1%). The absorbance of the
solution was measured at 700 nm in spectrophotometer (“Jenway”,
UK). Higher absorbance of the reaction mixture indicated that the
reducing power is increased. Reducing power was compared to
ascorbic acid and usnic acid.
Superoxide anion radical scavenging activity
The superoxide anion radical scavenging activity of samples was
detected according to the method of Nishimiki et al. (1972). Briefly,
0.1 ml of test samples (1000, 500, 250, 125 and 62.5 g/ml) was
mixed with 1 ml nitroblue tetrazolium (NBT) solution (156 M in
0.1 M phosphate buffer, pH 7.4) and 1 ml nicotinamide adenine
dinucleotide (NADH) solution (468 M in 0.1 M phosphate buffer,
pH 7.4). The reaction was started by adding 100 L of phenazine
methosulfate (PMS) solution (60 M in 0.1 M phosphate buffer, pH
7.4). The mixture was incubated at room temperature for 5 min,
and the absorbance was measured at 560 nm in spectrophotometer (“Jenway”, UK) against blank samples. Decreased absorbance
indicated increased superoxide anion radical scavenging activity.
Superoxide anion scavenging activity was compared to ascorbic
acid and usnic acid. The percentage inhibition of superoxide anion
generation was calculated using the following formula:
Superoxide anion scavenging activity (%) =
A − A
1
0
A0
× 100
where A0 is the absorbance of the negative control and A1 is the
absorbance of reaction mixture or standards.
The inhibition concentration at 50% inhibition (IC50 ) was the
parameter used to compare the radical scavenging activity.
Antimicrobial activity
Microorganisms and media
The following bacteria were used as test organisms in this study:
Bacillus mycoides (ATCC 6462), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and
Klebsiella pneumoniae (ATCC 13883). All the bacteria used were
obtained from the American Type Culture Collection (ATCC). Their
identification was confirmed at the Microbiological Laboratory of
Kragujevac, University of Kragujevac, Department of Biology. The
fungi used as test organisms were: Aspergillus flavus (ATCC 9170),
Aspergillus fumigatus (DBFS 310), Candida albicans (ATCC 10231),
Penicillium purpurescens (DBFS 418) and Penicillium verrucosum
(DBFS 262). They were from the from the American Type Culture
Collection (ATCC) and the mycological collection maintained by the
Mycological Laboratory within the Department of Biology of Kragujevac University’s Faculty of Science (DBFS). Bacterial cultures were
maintained on Müller-Hinton agar substrates (Torlak, Belgrade).
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N. Manojlović et al. / Phytomedicine 19 (2012) 1166–1172
Fungal cultures were maintained on potato dextrose (PD) agar and
Sabourad dextrose (SD) agar (Torlak, Belgrade). All cultures were
stored at 4 ◦ C and subcultured every 15 days.
The sensitivity of microorganisms to tested samples was tested
by determining the minimal inhibitory concentration (MIC).
Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 ◦ C on Müller-Hinton agar substrate and brought
up by dilution according to the 0.5 McFarland standard to approximately 108 CFU/ml. Suspensions of fungal spores were prepared
from fresh mature (3–7-day old) cultures that grew at 30 ◦ C on a
PD agar substrate. Spores were rinsed with sterile distilled water,
used to determine turbidity spectrophotometrically at 530 nm, and
then further diluted to approximately 106 CFU/ml according to the
procedure recommended by NCCLS (1998).
Minimal inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) was determined
by the by the broth microdilution method with using 96-well
micro-titer plates (Sarker et al. 2007). A series of dilutions with
concentrations ranging from 4 to 0.00181 mg/ml for test samples
was used in the experiment against every microorganism tested.
The starting solutions of test samples were obtained by measuring
off a certain quantity of extract and dissolving it in DMSO. Two-fold
dilutions of test samples were prepared in Müller-Hinton broth for
bacterial cultures and SD broth for fungal cultures. The minimal
inhibitory concentration was determined with resazurin. Resazurin
is an oxidation–reduction indicator used for the evaluation of
microbial growth. It is a blue non-fluorescent dye that becomes
pink and fluorescent when reduced to resorufin by oxidoreductases
within viable cells. The boundary dilution without any changing
color of resazurin was defined as the minimal inhibitory concentration (MIC) for the tested microorganism at the given concentration.
As a positive control of growth inhibition, streptomycin, ketoconazole and usnic acid were used. A DMSO solution was used as a
negative control for the influence of the solvents. All experiments
were performed in triplicate.
Cytotoxic activity
Cell lines
The human melanoma FemX and human colon carcinoma LS174
cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Both cancer cell lines were maintained
in the recommended RPMI-1640 medium supplemented with 10%
heat-inactivated (56 ◦ C) fetal bovine serum, l-glutamine (3 mM),
streptomycin (100 mg = ml), penicillin (100 IU = ml), and 25 mM
HEPES and adjusted to pH 7.2 by bicarbonate solution. Cells were
grown in a humidified atmosphere of 95% air and 5% CO2 at 37 ◦ C.
Treatment of cell lines
Stock solutions (100 mg/ml) of test samples, made in dimethylsulfoxide (DMSO), were dissolved in corresponding medium to the
required working concentrations. Neoplastic FemX cells (5000 cells
per well) and neoplastic LS174 cells (7000 cells per well) were
seeded into 96-well microtiter plates, and 24 h later, after the cell
adherence, five different, double diluted, concentrations of investigated compounds, were added to the wells. Final concentrations
applied to target cells were 200, 100, 50, 25 and 12.5 g/ml, except
to the control wells, where only nutrient medium was added to
the cells. Nutrient medium was RPMI 1640 medium, supplemented
with l-glutamine (3 mM), streptomycin (100 lg/ml), and penicillin
(100 IU/ml), 10% heat inactivated (56 ◦ C) fetal bovine serum (FBS)
and 25 mM Hepes, and was adjusted to pH 7.2 by bicarbonate solution. The cultures were incubated for 72 h.
Determination of cell survival (MTT test)
The effect of test samples on cancer cell survival was determined
by MTT test (microculture tetrazolium test), according to Mosmann
(1983) with modification by Ohno and Abe (1991) 72 h upon addition of the compounds, as it was described earlier. Briefly, 20 l of
MTT solution (5 mg/ml PBS) were added to each well. Samples were
incubated for further 4 h at 37 ◦ C in 5% CO2 and humidified air atmosphere. Then, 100 l of 10% SDS were added to extract the insoluble
product formazan, resulting from the conversion of the MTT dye by
viable cells. The number of viable cells in each well was proportional to the intensity of the absorbance of light, which was then
read in an ELISA plate reader at 570 nm. Absorbance (A) at 570 nm
was measured 24 h later. To get cell survival (%), A of a sample with
cells grown in the presence of various concentrations of the investigated test samples was divided with control optical density (the A
of control cells grown only in nutrient medium), and multiplied by
100. It was implied that A of the blank was always subtracted from
A of the corresponding sample with target cells. IC50 concentration
was defined as the concentration of an agent inhibiting cell survival
by 50%, compared with a vehicle-treated control. As a positive control were used cis-diamminedichloroplatinum (Cis-DDP) and usnic
acid. All experiments were done in triplicate.
Flow cytometry analysis
Cellular DNA content and cell distribution were quantified by flow cytometry using propidium iodide (PI). Cells
(3 × 105 cells/well) were seeded in 6-well plates and incubated with
or without IC50 concentration of investigated compounds for 24 h.
After treatment, the cells were collected by trypsinization, and fixed
in ice-cold 70% ethanol at −20 ◦ C overnight. After fixation, the cells
were washed in PBS and pellets obtained by centrifugation was
treated with RNase (100 lg/ml) at 37 ◦ C temperature for 30 min
and then incubated with propidium iodide (PI) (40 lg/ml) for at
least 30 min. DNA content and cell cycle distribution were analyzed
using a Becton Dickinson FAC-Scan flow cytometer. Flow cytometry
analysis was performed using a CellQuestR (Becton Dickinson, San
Jose, CA, USA), on a minimum of 10,000 cells per sample (Clothier
1995).
Statistical analyses
Statistical analyses were performed with the EXCEL and SPSS
softwares package. To determine the statistical significance of
antioxidant activity, Student’s t-test was used. All values are
expressed as mean ± SD of three parallel measurements.
Results and discussion
Chromatograms for standards and P. caperata, P. sulcata, and
P. saxatilis acetone extracts eluted by HPLC are represented in
Figs. 1 and 2. As it is evidenced in the chromatograms, there
were the presence of depsidones, depsides and dibenzofuran as
the most abundant substance classes in the extracts examined.
The HPLC chromatogram of the acetone extracts of P. sulcata and
P. saxatilis showed that the highest peaks belong to the depsidone salazinic acid (SAL) (tR = 3.01 ± 0.20 min). On the other
hand, protocetraric acid (PRO) (tR = 3.62 ± 0.20 min) and usnic acid
(USN) (tR = 10.53 ± 0.30 min) were detected as the major secondary
metabolites in the acetone extract of P. caperata. Identification of
these compounds was achieved by comparison of their tR values
with the standard substances previously isolated from lichens. The
UV absorbance spectral data (200–400 nm) also corresponded with
those of standards and found in Yoshimura et al. (1994). Table 1
shows the retention time of the detected lichen substances and
their absorbance maxima (nm). The structures of the detected
compounds are shown in Fig. 3. Some of the compounds are identified for the first time in this lichen. After detection of the present
N. Manojlović et al. / Phytomedicine 19 (2012) 1166–1172
1169
Fig. 1. Chromatogram of the standards used for identification of the compounds present in Parmelia species.
compounds in lichens, major lichen metabolites in these species
(salazinic acid, protocetraric acid and usnic acid – which was used
as a standard compound) were isolated and used for antioxidant,
antimicrobial and anticancer investigations.
The scavenging of DPPH radicals and superoxide anion radicals
by the tested compounds is shown in Table 2. There was a statistically significant difference between samples and control (p < 0.05).
Salazinic acid showed stronger DPPH radicals and superoxide anion
radicals scavenging activity than protocetraric acid. The IC50 values were 91.57 and 138.23 g/ml for salazinic acid and 119.10 and
177.60 g/ml for protocetraric acid (for DPPH radicals and superoxide anion radicals scavenging activity, respectively). As a shown in
Table 3, salazinic acid also demonstrated greater reducing power.
Various antioxidant activities were compared to ascorbic acid
and usnic acid. The results showed that standards have stronger
activity than tested samples.
The tested lichen compounds have a strong antioxidant activity
against various oxidative systems in vitro. The isolated components
belong phenols which indicate an important role of phenol in the
antioxidant activity for lichens. In fact, number of previous studies
Fig. 2. HPLC chromatograms acquired at 254 nm of the acetone extracts of P. caperata, P. sulcata and P. saxatilis. Chromatographic peaks identities are reported in Table 1.
N. Manojlović et al. / Phytomedicine 19 (2012) 1166–1172
1170
Fig. 3. Structures of the identified compounds.
found that the lichens where found the higher content of phenols
exert stronger antioxidant activity (Behera et al. 2009; Kosanić et al.
2012a), which means that phenols are important antioxidants.
Antioxidant effect of some other lichen compounds was also
studied by other researchers. For example, antioxidant activity was
found for lecanoric acid, salazinic acid, stictic acid, usnic acid (Luo
et al. 2009; Amo de Paz et al. 2010).
The antimicrobial activity of the lichen components against
the test microorganisms are shown in Table 4. Salazinic acid
and protocetraric acid showed similar antimicrobial activity, but
antibacterial activity was stronger than antifungal activity for both
Table 1
Retention time of the examined lichen compounds and their absorbance maxima
(nm).
Peaks
Compound
Retention time
(tR ± SD)a (min)
Absorbance maxima (nm)
UV spectrum
SAL
PRO
USN
ATR
CHL
Salazinic acid
Protocetraric acid
Usnic acid
Atranorin
Chloroatranorin
3.01
3.62
10.53
11.72
13.23
213, 238, 312
212, 240, 320m
234, 282
210, 252, 321m
213, 252, 315m , 350
±
±
±
±
±
0.20
0.20
0.30
0.10
0.20
m, minor absorbance maximum.
a
Values are the means of three determinations ± SD.
compounds. The MIC for components relative to the tested microorganisms ranged from 0.015 to 1 mg/ml.
The antimicrobial activity was compared with the standard
antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi)
and usnic acid. As shown in Table 2, standards have stronger activity
than tested samples. In a negative control, DMSO had no inhibitory
effect on the tested organisms.
Previously, numerous lichen compounds were screened for
antimicrobial activity in search of the new antimicrobial agents
(Candan et al. 2006; Kosanić and Ranković 2011; Turk et al. 2006).
In our experiments, the tested lichen compounds show very strong
antimicrobial activity. Antibacterial activity was stronger than antifungal. This observation is in accordance with other studies (Yang
Table 2
DPPH radical scavenging activity and superoxide anion scavenging activity of isolated compounds.
Lichen compounds
DPPH radical
scavenging IC50
(g/ml)
Superoxide anion
scavenging IC50
(g/ml)
Salazinic acid
Protocetraric acid
Usnic acid
Ascorbic acid
91.57
138.23
60.73
6.42
119.10
177.60
97.28
115.61
Ascorbic acid and usnic acid were used as a standards.
N. Manojlović et al. / Phytomedicine 19 (2012) 1166–1172
1171
Table 3
Reducing power of isolated compounds.
Lichen compounds
Salazinic acid
Protocetraric acid
Usnic acid
Ascorbic acid
Absorbance (700 nm)
1000 g/ml
500 g/ml
250 g/ml
125 g/ml
62.5 g/ml
0.2722
0.1209
0.6723
2.113
0.1842
0.0903
0.5468
1.654
0.0758
0.0721
0.2692
0.9571
0.0534
0.0502
0.1249
0.4783
0.0321
0.0312
0.0901
0.2472
Ascorbic acid and usnic acid were used as a standards.
Table 4
Minimum inhibitory concentration (MIC) of isolated compounds.
Lichen compounds
Salazinic acid
Protocetraric acid
Usnic acid
S
K
B. mycoides
B. subtilis
E. coli
K. pneumoniae
S. aureus
A. flavus
A. fumigatus
C. albicans
P. purpurescens
P. verrucosum
0.015
0.0312
1
0.5
0.125
1
1
0.25
1
0.5
0.015
0.015
1
0.5
0.015
1
0.25
0.25
1
0.5
0.0008
0.0008
0.25
0.0625
0.125
0.5
0.25
0.125
0.5
0.5
7.81
7.81
31.25
31.25
15.72
–
–
–
–
–
–
–
–
–
–
3.9
3.9
1.95
3.9
3.9
Values given as mg/ml for lichen compounds and as g/ml for antibiotics. Values are the mean of three replicate. S, streptomycin; K, ketoconazole and usnic acid were used
as a standards.
and Anderson 1999; Kosanić et al. 2012b), focused on the antimicrobial activity which have demonstrated that bacteria are more
sensitive to the antimicrobial activity than the fungi due to differences in the composition and permeability of the cell wall.
The cytotoxic activity of the tested compounds against the
tested cell lines is shown in Table 5. The tested samples exhibited high cytotoxic activity against the target cells in vitro. The IC50
value for both compounds relative to the tested cells ranged from
35.67 to 60.18 g/ml. The best cytotoxic activity was exhibited the
salazinic acid. As shown in this table, Cis-DDP and usnic acid have
slightly better cytotoxic activity than tested samples.
The effect of protocetraric acid and salazinic acid on cell cycle
progression was investigated in FemX and LS174 cells. Cell cycle
was assessed by cytofluorimetric analysis, using propidium iodide
to label DNA. Results are given in Table 6. Cells treated for 24 h
with IC50 (g/ml) concentrations of both compounds showed a significant increase in the number of cells in sub-G1 phase, and an
reduction in the number of cells in S phase. Specially, incubation
of the cell line FemX with salazinic acid induced G0-G1 and G2-M
cell cycle blockade. In addition, treatment LS174 cells of protocetraric acid and salazinic acid leads to a somewhat more pronounced
increase in sub-G1 phase and concomitant decrease in G2/M were
observed, supporting a G1 phase arrest. An increase in cells containing sub-G1 amounts of DNA was observed, indicating that the
compounds was inducing cell death.
Some literature data reported that lichen components are
responsible for anticancer activities of lichens (Bucar et al. 2004;
Burlando et al. 2009). However, it is difficult to determine the contribution of individual components for the overall anticancer effect.
Table 5
Growth inhibitory effects of isolated compounds on FemX and LS174 cell lines CisDDP and usnic acid were used as a standards.
Lichen compounds
Apoptotic cells sub-G1
G1
S
G2/M
FemX
Control
Salazinic acid
Protocetraric acid
Usnic acid
0.15
16.23
16.20
10.18
62.65
43.74
43.81
54.38
16.69
13.64
15.30
14.64
16.64
24.93
18.62
20.37
LS174
Control
Salazinic acid
Protocetraric acid
Usnic acid
3.68
20.88
22.90
12.89
51.68
41.37
39.71
52.77
12.61
14.27
16.96
15.06
30.19
15.90
23.92
19.28
Effect of compounds on cell cycle phase distribution. FemX and LS174 cell lines were
exposed to compounds (IC50 g/ml) for 24 h and then collected for analysis of cell
cycle phase distribution using flow cytometry. Percentage of cells under different
stages of cell cycles (sub-G1, G1, S, G2/M) is shown. Usnic acid was used as a standard
compound.
Often, the activity of lichens may be the result of synergistic or
antagonistic effect of several compounds.
In conclusion, it can be stated that tested lichen compounds
have a strong antioxidant, antimicrobial and anticancer activity in
vitro, which suggest that lichens could be good natural antioxidant,
antimicrobial and anticancer agents.
Acknowledgement
This work was financed in part by the Ministry of Science, Technology, and Development of the Republic of Serbia and was carried
out within the framework of projects no. 173032, 175011 and
172015.
References
IC50 (g/ml)
FemX
Salazinic acid
Protocetraric acid
Usnic acid
Cis-DDP
Table 6
Effect of isolated compounds on cell cycle progression.
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58.68
12.72
0.94
LS174
±
±
±
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0.56
2.11
0.35
0.35
35.67
60.18
15.66
2.3
±
±
±
±
1.89
0.59
1.45
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