Antioxidant, Antimicrobial, and Anticancer Activity of 3 Umbilicaria Species

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DOI: 10.1111/j.1750-3841.2011.02459.x · Source: PubMed
Abstract
The aim of this study is to investigate in vitro antioxidant, antimicrobial, and anticancer activity of the acetone extracts of the lichens Umbilicaria crustulosa, U. cylindrica, and U. polyphylla. Antioxidant activity was evaluated by 5 separate methods: free radical scavenging, superoxide anion radical scavenging, reducing power, determination of total phenolic compounds, and determination of total flavonoid content. Of the lichens tested, U. polyphylla had largest free radical scavenging activity (72.79% inhibition at a concentration of 1 mg/mL), which was similar as standard antioxidants in the same concentration. Moreover, the tested extracts had effective reducing power and superoxide anion radical scavenging. Total content of phenol and flavonoid in extracts was determined as pyrocatechol equivalent, and as rutin equivalent, respectively. The strong relationships between total phenolic and flavonoid contents and the antioxidant effect of tested extracts were observed. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method. The most active was extract of U. polyphylla with minimum inhibitory concentration values ranging from 1.56 to 12.5 mg/mL. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using MTT method. All extracts were found to be strong anticancer activity toward both cell lines with IC₅₀ values ranging from 28.45 to 97.82 μg/mL. The present study shows that tested lichen extracts demonstrated a strong antioxidant, antimicrobial, and anticancer effects. That suggests that lichens may be used as possible natural antioxidant, antimicrobial, and anticancer agents.
T: Toxicolo gy &
Chemical Food Safety
Antioxidant, Antimicrobial, and Anticancer
Activity of 3 Umbilicaria Species
Marijana Kosani
´
c, Branislav Rankovi
´
c, and Tatjana Stanojkovi
´
c
Abstract: The aim of this study is to investigate in vitro antioxidant, antimicrobial, and anticancer activity of the acetone
extracts of the lichens Umbilicaria crustulosa, U. cylindrica,andU. polyphylla. Antioxidant activity was evaluated by 5 separate
methods: free radical scavenging, superoxide anion radical scavenging, reducing power, determination of total phenolic
compounds, and determination of total flavonoid content. Of the lichens tested, U. polyphylla had largest free radical
scavenging activity (72.79% inhibition at a concentration of 1 mg/mL), which was similar as standard antioxidants in the
same concentration. Moreover, the tested extracts had effective reducing power and superoxide anion radical scavenging.
Total content of phenol and flavonoid in extracts was determined as pyrocatechol equivalent, and as rutin equivalent,
respectively. The strong relationships between total phenolic and flavonoid contents and the antioxidant effect of tested
extracts were observed. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration
by the broth microdilution method. The most active was extract of U. polyphylla with minimum inhibitory concentration
values ranging from 1.56 to 12.5 mg/mL. Anticancer activity was tested against FemX (human melanoma) and LS174
(human colon carcinoma) cell lines using MTT method. All extracts were found to be strong anticancer activity toward
both cell lines with IC
50
values ranging from 28.45 to 97.82 μg/mL. The present study shows that tested lichen extracts
demonstrated a strong antioxidant, antimicrobial, and anticancer effects. That suggests that lichens may be used as possible
natural antioxidant, antimicrobial, and anticancer agents.
Keywords: anticancer activity, antimicrobial activity, antioxidant activity, lichens
Introduction
In recent years, both in practice and in science, there is great in-
terest for new preparations of natural origin in the control and pre-
vention of various human, animal, and plant diseases. It is known
that long-term use of synthetic drugs often causes numerous side
effects and sometimes resistance (Karaman and others 2003). Un-
like synthetic drugs, bioactive natural products have beneficial ef-
fect on the whole organism and without causing unwanted e ffects.
In search of new bioactive preparations of natural origin, lichens
are the subject of many research teams.
Lichens are symbiotic organisms consisting of algae and fungi
and are important constituents of many ecosystems. They usually
grow on rocks, nonfertile ground, as well as epiphytes on the trees
and leaves (Fabian and others 2005). These organisms are used for
human nutrition, animal nutrition, for getting colors, perfumes,
and alcohol. Lichens have also, for hundreds of years, been used in
many countries as a cure for diseases of humans. Namely, Lobaria
pulmonaria and Parmelia sulcata have been used in the treatment of
pulmonary and cranial diseases, respectively (Malhotra and others
2008). Similarly, Xanthoria parietina was used to cure jaundice and
Letharia vulpina in stomach diseases (Kirmizig
¨
ul and others 2003;
Malhotra and others 2008). The usage of some lichens for many
years in the traditional medicine was later justified by numerous
researches that confirmed their various biological activity.
MS 20110724 Submitted 6/13/2011, Accepted 9/16/2011. Authors Kosani
´
cand
Rankovi
´
c are with Dept. of Biology, Faculty of Science, Univ. of Kragujevac, Radoja
Domanovi
´
ca 12, Kragujevac, Serbia. Author Stanojkovi
´
c is with Inst. of Oncology and
Radiology o f Serbia, Pasterova 14, Belgrade 11000, Serbia. Direct inquiries to author
Kosani
´
c (E-mail: marijanakosanic@yahoo.com).
Lichens produce secondary metabolites the “lichen substances,”
which comprise depsides, depsidones, dibenzofurans, xanthones,
and terpene derivatives (Karagoz and others 2009). These metabo-
lites sometimes make even more than 30% of the dry mass of
thalus (Galun 1988). Lichens and their metabolites have mani-
fold biological activity: antiviral, antibiotic, antitumor, allergenic,
plant growth inhibitory, antiherbivore, ecological roles, and en-
zyme inhibitory (Kosani
´
c and Rankovi
´
c 2011). Because of that,
the present study describes the evaluation of the antioxidant, an-
timicrobial, and cytotoxic activities of the acetone extracts of the
lichens Umbilicaria crustulosa, U. cylindrica,andU. polyphylla.
Materials and Methods
Lichen samples
Lichen samples of U. crustulosa (Ach.) Frey, U.cylindrica (L.)
Delise ex Duby, and U. polyphylla (L.) Baumg. were collected
from Kopaonik, Serbia, in September of 2010. The demonstra-
tion samples are preserved in facilities of the Dept. 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 dr y ground thalli of the investigated lichens (50 g) were
extracted using acetone in a Soxchlet 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% dimethyl
sulphoxide (DMSO) for the experiments.
C
2011 Institute of Food Technologists
R
T20 Journal of Food Science
r
Vol. 71, Nr. 1, 2012 doi: 10.1111/j.1750-3841.2011.02459.x
Further reproduction without permission is prohibited
T: Toxicolo gy &
Chemical Food Safety
Antioxidant, antimicrobial, and anticancer . . .
Antioxidant activity
Scavenging 1,1-diphenyl-2-picryl-hydrazil (DPPH)
radicals. The free radical scavenging activity of lichen extracts
was measured by DPPH. The method used is similar to the
method previously used by some authors (Dorman and others
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 plant extract (1 mg/mL) were placed in cuvettes. The
mixture was shaken vigorously and allowed to s tand at room tem-
perature for 30 min. Then, the absorbance was measured at 517
nm in spectrophotometer (“Janway” Ltd., Essex, U.K.). Ascorbic
acid, butylated hydroxyanisole (BHA), and α-tocopherol, at a
concentration of 1 mg/mL, were used as positive control. The
DPPH radical concentration was calculated using the following
equation:
DPPH scavenging effect (%) = ((A0 A1)/A0) × 100,
where A0 is the absorbance of the negative control and A1 is the
absorbance of reaction mixture or standards.
Reducing power. The reducing power of extracts was deter-
mined according to the method of Oyaizu (1986). One milliliter
of extracts (1 mg/mL) was mixed with 2.5 mL of phosphate buffer
(2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (K
3
Fe(CN)
6
)
(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 mix-
ture and centrifuged. Finally, the upper layer was mixed with dis-
tilled water (2.5 mL) and FeCl
3
(0.5 mL; 0.1%). The absorbance
of the solution was measured at 700 nm in spectrophotometer
(Janway). Higher absorbance of the reaction mixture indicated
that the reducing power is increased. Ascorbic acid, BHA, and
α-tocopherol, at a concentration of 1 mg/mL, were used as
positive control.
Superoxide anion radical scavenging activity. The super-
oxide anion radical scavenging activity of lichen extracts was de-
tected according to the method of Nishimiki and others (1972).
Briefly, 0.1 mL of extracts (1 mg/mL) was mixed with 1-mL ni-
troblue tetrazolium (NBT) solution (156 μMin0.1Mphosphate
buffer, pH 7.4) and 1-mL NADH solution (468 μMin0.1M
phosphate buffer, pH 7.4). The reaction was started by adding
100 μL of phenazine methosulphate (PMS) solution (60 μMin
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 (Janway) against blank samples.
Decreased absorbance indicated increased superoxide anion radical
scavenging activity. Ascorbic acid, BHA, and α-tocopherol, at a
concentration of 1 mg/mL, were used as positive control. The per-
centage inhibition of superoxide anion generation was calculated
using the following formula:
Superoxide anion scavenging activity (%) = ((A0 A1)/A0) × 100,
where A0 is the absorbance of the negative control and A1 is the
absorbance of reaction mixture or standards.
Determination of total phenolic compounds. Total solu-
ble phenolic compounds in the lichen extracts were determined
with Folin–Ciocalteu reagent according to the method of Slinkard
and Singleton (1997) using pyrocatechol as a standard phenolic
compound. Briefly, 1 mL of the lichen extract (1 mg/mL) in a
volumetric flask diluted with distilled water (46 mL). One milliliter
of Folin–Ciocalteu reagent was added and the content of the flask
was mixed thoroughly. After 3 min, 3 mL of Na
2
CO
3
(2%) were
added and then were allowed to stand for 2 h with intermittent
shaking. The absorbance was measured at 760 nm in spectropho-
tometer (Janway). The total concentration of phenolic compounds
in the extract determined as microgram of pyrocatechol equivalent
by using an equation that was obtained from standard pyrocatechol
graph as
Absorbance = 0.0021 × total phenols (microgram pyrocate-
chol) 0.0092 (R
2
= 0.9934).
Total flavonoid content. The total flavonoid content was
determined using the Dowd method (Meda and others 2005). Two
milliliters of 2% aluminium trichloride (AlCl
3
) in methanol were
mixed with the same volume of the extract solution (1 mg/mL).
The mixture was incubated at room temperature for 10 min, and
the absorbance was measured at 415 nm in spectrophotometer
(Janway) against blank samples. The total flavonoid content was
determined as microgram of rutin equivalent by using an equation
that was obtained from standard rutin graph as
Absorbance = 0.0144 × total flavonoid (microgram rutin) +
0.0556 (R
2
= 0.9992).
Antimicrobial activity
Microorganisms and media. The following bacteria were
used as test organisms in this study: Bacillus mycoides (IPH 197),
B. subtilis (IPH 189), and Staphylococcus aureus (IPH 221) (Gram-
positive bacteria); and Enterobacter cloaceae (IPH 241), Escherichia coli
(IPH 246), and Klebsiella pneumoniae (IPH 251) (Gram-negative
bacteria). All the bacteria used were isolates of the Inst. for
Protection of Health (IPH) in Kragujevac and the Faculty of
Agriculture in Belgrade (FAB). Their identification was con-
firmed at the Microbiological Laboratory of Kragujevac, Univ. of
Kragujevac, Dept. of Biology. The fungi used as test organisms
were Aspergillus flavus (ATCC 9170), A. fumigatus (DBFS 310),
Botrytis cinerea (DBFS 133), Candida albicans (IPH 1316), Fusarium
oxysporum (DBFS 292), Mucor mucedo (ATCC 52568), Paecilomyces
variotii (ATCC 22319), Penicillium purpurescens (DBFS 418), P.
verrucosum (DBFS 262), and Trichoderma harsianum (DBFS 379).
They were from the mycological collection maintained by the My-
cological Laboratory within the Dept. of Biology of Kragujevac
Univ. Faculty of Science (DBFS). Bacterial cultures were main-
tained on M
¨
uller-Hinton agar substrates (Torlak, Belgrade, Serbia).
Fungal cultures were maintained on potato dextrose (PD) agar and
Sabourad dextrose (SD) agar (Torlak). All cultures were stored at
4
C and subcultured every 15 d.
The sensitivity of microorganisms to acetone extracts of the in-
vestigated species of lichens was tested by determining the minimal
inhibitory concentration (MIC).
Bacterial inoculi were obtained from bacterial cultures incu-
batedfor24hat37
ConM
¨
uller-Hinton agar substrate and
brought up by dilution according to the 0.5 McFarland standard
to approximately 10
8
CFU/mL. Suspensions of fungal spores were
prepared from fresh mature (3- to 7-d old) cultures that grew at
30
C on a PD agar substrate. Spores were rinsed with sterile dis-
tilled water, used to determine turbidity spectrophotometrically at
530 nm, and then further diluted to approximately 10
6
CFU/mL
according to the procedure recommended by NCCLS (1998).
Minimal inhibitory concentration. The MIC was deter-
mined by the broth microdilution method with using 96-well
microtiter plates (Sarker and others 2007). A series of dilutions
with concentrations ranging from 50 to 0.195 mg/mL for extracts
were used in the experiment against every microorganism tested.
The starting solutions of extracts were obtained by measuring off
a certain quantity of extract and dissolving it in DMSO. Twofold
dilutions of extracts were prepared in M
¨
uller-Hinton broth for
bacterial cultures and SD broth for fungal cultures. The MIC was
determined with resazurin. Resazurin is an oxidation–reduction
indicator used for the evaluation of microbial growth. It is a blue
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Antioxidant, antimicrobial, and anticancer . . .
nonfluorescent dye that becomes pink and fluorescent when re-
duced to resorufin by oxidoreductases within viable cells. The
boundary dilution without any changing color of resazurin was
defined as the MIC for the tested microorganism at the given
concentration. As a positive control of growth inhibition, strepto-
mycin was used in the case of bacteria, ketoconazole in the case
of fungi (at a concentration of 1mg/mL). 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., U.S.A.). Both cancer cell lines
were maintained in the recommended RPMI-1640 medium sup-
plemented with 10% heat-inactivated (56
C) fetal bovine serum
(FBS), l-glutamine (3 mM), streptomycin (100 mg/mL), penicillin
(100 IU/mL), and 25 mM HEPES and adjusted to pH 7.2 by bi-
carbonate solution. Cells were grown in a humidified atmosphere
of 95% air and 5% CO
2
at 37
C.
Treatment of cell lines. Stock solutions (100 mg/mL) of ex-
tracts, made in 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, 5 different, double diluted, concentra-
tions of investigated compounds, were added to the wells. Final
concentrations applied to target cells were 200, 100, 50, 25, and
12.5 μg/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 mg/mL), and penicillin (100 IU/mL), 10% heat-inactivated
(56
C) 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
extracts on cancer cell survival was determined by MTT test (mi-
croculture 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 μLofMTT
solution (5 mg/mL PBS) were added to each well. Samples were
incubated for further 4 h at 37
Cin5%CO
2
and humidified
air atmosphere. Then, 100 μL of 10% sodium dodecyl sulphate
(SDS) were added to extract the insoluble product formazan, re-
sulting from the conversion of the MTT dye by viable cells. The
number of viable cells in each well was proportional to the in-
tensity of the absorbance of light, which was then read in an
enzyme linked immunosorbent assay (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 extracts 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. IC
50
concentration was defined as the
concentration of an agent inhibiting cell survival by 50%, com-
pared with a vehicle-treated control. As a positive control was used
cis-diamminedichloroplatinum (cis-DDP), in the same concentra-
tions as extracts. All experiments were done in triplicate.
Statistical analyses
Statistical analyses were performed with the MS EXCEL 2003
and SPSS 13.0 software packages. To determine the statistical sig-
nificance of antioxidant activity, Student’s t-test was used. Pearson’s
bivariate correlation test was carried out to calculate correlation
coefficients (r) between the content of total phenolic and flavonoid
and the DPPH radical scavenging activity, reducing power and su-
peroxide anion radical scavenging. All values are expressed as mean
± standard deviation of 3 parallel measurements.
Results and Discussion
Antioxidant activity
The DPPH free radical scavenging activities of the studied lichen
extracts are shown in Figure 1. Acetone extracts of the tested lichen
showed a relatively good scavenging activity on DPPH radical.
There was a statistically significant difference between extracts and
control (P < 0.05). The scavenging effects of all lichen extracts
were 39.41% to 72.79%. Extract from lichen U. polyphylla showed
largest DPPH radical scavenging activity (72.79%) that was similar
as standard antioxidants, ascorbic acid (86.5%), BHA (79.78%), and
α-tocopherol (63.99%) at a concentration of 1 mg/mL. The scav-
enging activity was also good for the lichen U. crustulosa (60.12%).
The lichen U. cylindrica showed a slightly weaker DPPH radical
scavenging activities (39.41%).
The results of the reducing power assay of lichen extracts are
summarized in Figure 2. High absorbance indicates high reducing
0
10
20
30
40
50
60
70
80
90
100
U.
cr
u
st
ul
o
sa
U. cylind
r
ica
U
.
pol
yp
h
yl
l
a
ascorbic ac
i
d
B
H
A
DPPH radical scavenging (%)
Figure 1–DPPH radical scavenging of the
acetone extracts of the lichens Umbilicaria
crustulosa, U. cylindrica, and U. polyphylla and
standards (at a concentration of 1 mg/mL).
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Antioxidant, antimicrobial, and anticancer . . .
power. Tested extracts showed a low reducing power activity. Mea-
sured values of absorbance varied from 0.035 ± 0.002 to 0.065 ±
0.004. Among the tested lichen species, U. polyphylla give highest
reducing power. The reducing power in the acetone lichen extracts
decreased in the following order: U. polyphylla > U. crustulosa >
U. cylindrica.
Results of superoxide anion scavenging activities of tested ex-
tracts are shown in Figure 3. All extracts revealed a moderate
superoxide anion scavenging activity. The superoxide anion scav-
enging activity for different lichens was within the range 33.92%
to 55.43%. There was a statistically significant difference between
extracts and control (P < 0.05). Maximum scavenging activity
(55.43%) was in the acetone extracts of the lichen U. polyphylla.
Acetone extract of lichen U. cylindrica demonstrated weakest su-
peroxide anion scavenging activity (33.92%).
Total phenolic and flavonoid constituents of tested extracts are
given in Table 1. The amount of total phenolic compounds was
determined as the pyrocatechol equivalent using an e quation ob-
tained from a standard pyrocatechol graph (y = 0.0021 ×−0.0092,
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
U. crustulosa
U. cylindrica
U. polyphylla
ascorbic acid
BHA
absorbance (700 nm)
Figure 2–Reducing power of the acetone extracts of the lichens Umbilicaria
crustulosa, U. cylindrica, and U. polyphylla and standards (at a concentra-
tion of 1 mg/mL).
0
20
40
60
80
100
120
U. crustulosa
U. cylindrica
U. polyphylla
ascorbic acid
BHA
superoxide anion scavenging (%)
Figure 3–Superoxide anion scavenging of the acetone extracts of the
lichens Umbilicaria crustulosa, U. cylindrica, and U. polyphylla and stan-
dards (at a concentration of 1 mg/mL).
Table 1–Total phenolics and flavonoid content of acetone extracts of
Umbilicaria crustulosa, U. cylindrica,andU. polyphylla.
Phenolics content Flavonoid content
microgram of microgram of
pyrocatechol equivalent/ rutin equivalent/
Lichen species milligram of extract milligram of extract
U. crustulosa 39.62 ± 1.201 28.08 ± 1.132
U. cylindrica 19.14 ± 1.318 12.95 ± 1.091
U. polyphylla 47.02 ± 1.098 30.31 ± 1.157
R
2
= 0.9934). Highest phenolic compounds were identified in
acetone extract of U. polyphylla at a 47.02 μg of pyrocatechol
equivalent while acetone extracts of U. cylindrica showed the lowest
content at 19.14 μg of pyrocatechol equivalent. The amount of
total flavonoid compounds was determined as the rutin equivalent
using an equation obtained from a standard rutin graph (y =
0.0144 ×+0.0556, R
2
= 0.9992). The total flavonoid content
for acetone extracts of U. crustulosa, U. cylindrica,andU. polyphylla
was 28.08, 12.95, and 30.31 μg of pyrocatechol equivalent,
respectively.
The tested extracts exhibited the highest radical scavenging ac-
tivity with the greatest amount of phenolic and flavonoid contents.
Correlation coefficient between phenolic and flavonoid com-
pounds of the tested extracts and free radical scavenging activity
was r = 0.992 and r = 0.964, respective ly.
Various antioxidant activities were compared to standard antiox-
idants such as ascorbic acid, BHA, and α-tocopherol. The results
showed that standard antioxidants had similar or slightly stronger
activity than tested extracts in the same concentration.
Antimicrobial activity
The antimicrobial activity of the tested lichen extracts against
the tested microorganisms was shown in the Table 2.
The maximum antimicrobial activity was found in the ace-
tone extract of the lichen U. polyphylla, which in relatively low
concentrations inhibited the tested bacteria and fungi. The MIC
for bacteria was 1.56 mg/mL against B. mycoides, B. subtilis, K.
pneumoniae and E. coli, 3.12 mg/mL against Enterobacter cloacae and
6.25 mg/mL against S. aureus. The MIC for fungi ranged from 1.56
to 12.5 mg/mL. The lowest measured MIC value (1.56 mg/mL)
was related to the C. albicans.
Extract of the lichen U. crustulosa manifested moderate antimi-
crobial activity. This lichen inhibited all of the tested bacteria,
except E. coli, which was resistant. The MIC for bacteria ranged
from 3.12 to 12.5 mg/mL. Antifungal activity was found against
6 of 10 tested fungi. MIC for fungi was 12.5 and 25 mg/mL.
The lichen U. cylindrica manifested lowest antimicrobial activity.
Antimicrobial activity was found only against C. albicans.Other
microorganisms were resistant.
The antimicrobial activity was compared with the standard an-
tibiotics, streptomycin (for bacteria) and ketoconazole (for fungi).
The results showed that standard antibiotics had stronger activity
than tested extracts as shown in Table 2. In a negative control,
DMSO had no inhibitory effect on the tested organisms.
Cytotoxic activity
The cytotoxic activity of the studied lichen extracts related to
tested cell lines was shown in the Table 3.
The tested lichen extracts manifested a strong cytotoxic activ-
ity against target cells in vitro. The inhibition concentration at
50% inhibition (IC
50
) was the parameter used to compare the
cytotoxic activity. A lower IC
50
meant better cytotoxic activity.
Umbilicaria cylindrica exhibited highest cytotoxic activity. The
IC
50
against FemX and LS174 cell lines was 28.45 and
40.55 μg/mL, respectively.
The extract of U. polyphylla also showed a good cytotoxic activity
against both cell lines. The IC
50
value was 35.05 μg/mL against
FemX cell and 39.45 μg/mL against LS174 cell.
The U. crustulosa manifested a slightly weaker cytotoxic activ-
ity. The IC
50
value was 69.43 μg/mL related to FemX cell and
97.82 μg/mL related to LS174 cell.
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Antioxidant, antimicrobial, and anticancer . . .
As shown in table, positive control (cis-DDP) had better cyto-
toxic activity than tested lichen extracts in the same concentrations.
In the present study, in vitro antioxidant, antimicrobial, and cy-
totoxic activities of acetone extract from the lichens U. crustulosa,
U. cylindrica,andU. polyphylla were examined.
The tested lichen extracts have a strong antioxidant activity
against var ious oxidative systems in vitro. Strong antioxidant activ-
ity of tested lichen extracts is correlated with a high content of total
phenols. In fact, it was observed that the examined lichen extracts
where found the higher content of phenols and flavonoids exert
stronger DPPH radical scave nging effect, suggesting that pheno-
lics are the main agents for their antioxidant activity. These results
mostly agree with the literature, where we can find a number
of report for the antioxidant activity of extracts with high con-
tent of phenolic compounds (Behera and other s 2009). However,
some authors believe that the antioxidant activity of extracts may
not be necessarily correlated with the content of polyphenolics
(Rankovi
´
c and others 2010). It is necessary understand that ex-
tracts are mixtures of natural compounds, and their antioxidant
activity is not only a result of the different activities of individ-
ual polyphenolics but may be the result of their antagonistic or
synergistic interactions with other phenolics or other nonphe-
nolic components. Same authors showed that nonphenolics play
an important role in the antioxidant activity of lichen extracts
(Odabasoglu and others 2004). This is corroborated by our results
showing the strong DPPH radical activity, but weaker reducing
power of the tested extracts. This suggests that nonphenolics play
important role in antioxidant activities of lichen extracts. An-
tioxidant effect of some other lichens was also studied by other
researchers. For example, Behera and others (2005) for different
extracts from the lichen Usnea ghattensis. Kekuda and others (2009)
find an antioxidant activity for the extracts of the lichen Parmotrema
pseudotinctorum and Ramalina hossei. Manojlovi
´
c and others (2010)
explored antioxidant properties of Laurera benguelensis.
In our experiments, the tested lichen extracts show a relatively
strong antimicrobial activity. The intensity of the antimicrobial
effect depended on the species of lichen, its concentration, and
the tested organism. The extract of U. polyphylla had the strongest
antimicrobial activity among the tested species in this study, in-
hibiting the tested bacteria and fungi at low concentrations, while
the lowest activity showed U. cylindrica. Differences in antimicro-
bial activity of different species of lichens are probably a conse-
quence of the presence of different components with antimicrobial
activity (Adedapo and others 2008), among which the main agents
for antimicrobial activity emphasize the phenols. This is confirmed
by our results showing that extracts of lichen U. polyphylla and
U. crustulosa, where it found a higher content of phenols and
flavonoids, show a stronger antimicrobial effect, while the lichen
U. cylindrica, which had the weakest activity, also contains small
amounts of phenolic components. Otherwise, U. crustulosa con-
tains gyrophoric acid, U. cylindrica contains norstictic acid, while U.
polyphylla contains gyrophoric acid, umbilicaric acid, and lecanoric
acid (Brodo and others 2001).
The extracts used in this study had a stronger antibacterial than
antifungal activity. This observation is in accordance with other
studies (Yang and Anderson 1999), focused on the antimicrobial
activity that has 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 cell wall of
Gram-positive bacteria is made of peptidoglucanes and teichoic
acids, while the cell wall of Gram-negative bacteria is made of
peptidoglucanes, lipopolysacharides, and lipoproteins (Heijenoort
2001). The cell wall of fungi is poorly permeable and it consists
of polysaccharides such as chitin and glucan (Farka
ˇ
s 2003).
Numerous lichens were screened for antimicrobial activity in
search of the new antimicrobial agents. Rankovi
´
c and others
(2010) find an antimicrobial activity for the methanol extract of the
lichens Parmelia centrifuga
. Similar results were reported by Candan
and others (2007) for different extracts extracted from the lichen
P. sulcata.
In the present study, the results clearly demonstrate that acetone
extracts of studied lichens induced significant cytotoxic effect on
the tested cancer cell lines. Until now, only few reports show
that lichen have anticancer activity. Bezivin and others (2003)
reported significant anticancer effect for Parmelia caperata, Cladonia
Table 3– Growth inhibitory effects of acetone extracts of Umbilicaria
crustulosa, U. cylindr ica,andU. polyphylla on FemX and LS 174 cell
lines.
FemX LS 174
Lichen species IC
50
(μg/mL)
U. crustulosa 69.43 ± 2.85 97.82 ± 3.04
U. cylindrica 28.45 ± 4.99 40.55 ± 2.95
U. polyphylla 35.05 ± 2.56 39.45 ± 3.92
cis-DDP 0.94 ± 0.35 2.3 ± 0.31
Table 2–Minimum inhibitory concentration (MIC) of acetone extracts of Umbilicaria crustulosa, U. cylindrica,andU. polyphylla.
Lichen species U. crustulosa U. cylindrica U. polyphylla S–K
B. mycoides 3.12
a
–1.56 7.81
B. subtilis 6.25 1.56 7.81
E. cloacae 3.12 3.12 1.95
E. coli ––1.56 31.25
K. pneumoniae 12.5–1.56 1.95
S. aureus 6.25 6.25 31.25
A. flavus 25 12.5–3.9
A. fumigatus 12.5–12.5–3.9
B. cinerea ––6.25 1.95
C. albicans 12.5 1.56 1.95
F. oxysporum 25 12.5–3.9
M. mucedo 12.5–12.5–31.25
P. v a r i o t i i ––6.25 1.95
P. purpurescens ––12.5–3.9
P. verrucosum 12.5–12.5–3.9
T. harsianum 12.5–12.5–7.81
a
Minimum inhibitory concentration (MIC); values given as milligram per milliliter for lichen extract and as microgram per milliliter for antibiotics. Values are the mean of 3 replicate.
Antibiotics: K = ketoconazole; S = streptomycin.
T24 Journal of Food Science
r
Vol. 71, Nr. 1, 2012
T: Toxicolo gy &
Chemical Food Safety
Antioxidant, antimicrobial, and anticancer . . .
convoluta, C. rangiformis, Platisma glauca,andRamalina cuspidata.
Also, Manojlovi
´
c and others (2010) explored anticancer properties
of Thamnolia vermicularis.
Some literature data reported that lichen components are re-
sponsible for anticancer activities of lichens. Anticancer activity of
various lichens components is known such as usnic acid, lecanoric
acid, gyrophoric acid, salazinic acid, lobaric acid, evernic acid,
vulpinic acid, and protolichesterinic acid (Bucar and others 2004;
Burlando and others 2009). However, it is difficult to determine
the contribution of individual components for the overall anti-
cancer effect. Often, the activity of the extracts may be the result
of an synergistic effect of several compounds (Manojlovi
´
cand
others 2010). Due to the fact that phenols are to most numerous
group of lichen components, it is evident that they have an im-
portant contribution to anticancer activity. This is confirmed by
our results showing a high content of phenolic components of the
tested lichen species and their significant anticancer activity.
Conclusions
In conclusion, it can be stated that tested lichen extracts have
a strong antioxidant, antimicrobial, and anticancer activity in vitro.
On the basis of these results, lichen appears to be good and safe
natural antioxidant, antimicrobial, and anticancer. Further studies
should be done to search new compounds from lichens that exhibit
strong antioxidant, antimicrobial, and anticancer activity.
Acknowledgments
This work was financed in part by the Ministry of Science, Tech-
nology, and Development of the Republic of Serbia and was car-
ried out within the framework of project nrs 173032 and 175011.
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Vol. 71, Nr. 1, 2012
r
Journal of Food Science T25
    • "Numerous lichens were screened for antimicrobial activity in search of the new antimicrobial agents. Kosanić et al. (2012) find an antimicrobial activity for the acetone extract of the lichens Umbilicaria crustulosa, U. cylindrica, and U. polyphylla. Similar results were reported by Karthikai Devi et al. (2011) for different extracts extracted from the lichen Roccella belangeriana. "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract: Here we investigate antioxidant and antimicrobial activities of the acetone extract of the Cladonia rangiferina and their fumarprotocetraric acid. Antioxidant activit y was evaluated by free radical scavenging, superoxide anion radical scavenging, reducing power and determination of total phenolics. As a result, fumarprotocetraric acid had larger free radical scavenging activity with (IC 50 = 228.46 μg/mL). Tested sample s also had effective reducing power and superoxide anion radical scavenging. Total phenolic content was determined as pyrocatechol equivalent. The antimicrobial activity was estimated by determination of the MIC, where most active was fumarprotocetraric ac id with MIC values ranging from 0.03 to 0.25 μ g/mL.
    Full-text · Conference Paper · Mar 2016 · EXCLI Journal
    • "In our study, we have for the first time investigated the cytotoxic, anti-proliferative and apoptotic effects of methanolic extracts from C. rangiformis and C. convoluta species on MCF-7 cells. only the MTT method by Kosanić et al. (2012b). The present study indicated that the methanolic extracts of C. rangiformis and C. convolute can show cytotoxic effects on MCF-7 cells. "
    [Show abstract] [Hide abstract] ABSTRACT: This study tries to elucidate the anti-proliferative and apoptotic effects of methanolic lichen extracts from Cladonia rangiformis and Cladonia convolute in MCF-7 human breast cancer cells. Lichen extracts (0-2 mg/ml) were added to MCF-7 cells for 24 h. Cell viability was tested using 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Cell proliferation was observed using bromodeoxyuridine (BrdU) labelling and proliferating cell nuclear antigen (PCNA) by immunocytochemistry. The TUNEL method was used for cell death detection. The effective dose (ED50) values of methanolic extracts from C. rangiformis and C. convolute were found to be 0.905 and 0.977 mg/ml, respectively. Treatment with C. rangiformis methanolic extract (0.2-0.8 mg/ml) dose-dependently inhibited proliferation of MCF-7 cells as detected by BrdU incorporation. The inhibition was started in 0.2 mg/ml concentration of C. convoluta methanolic extract. The percent of PCNA immunopositive cells showed a decrease in MCF-7 cells treated with two lichen extracts compared to control MCF-7. Both methanolic extracts showed a significant increase in percentage of apoptosis-positive cells. These results indicate that methanolic lichen extracts from C. rangiformis and C. convolute inhibited proliferation of MCF-7 cells and caused apoptosis in MCF-7 cells. The lichens may be novel natural agents for treating breast cancer disease.
    Full-text · Article · Jul 2015
    • "This observation is in accordance with other studies (Yang and Anderson, 1999; Kosanić et al., 2012b; Ličina et al., 2013) focused on 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 cell wall of Gram-positive bacteria is made of peptidoglucanes and teichoic acids, while the cell wall of Gram-negative bacteria is made of peptidoglucanes, lipopolysaccharides and lipoproteins (Heijenoort, 2001; Kosanić et al., 2012b). The cell wall of fungi is poorly permeable and consists of polysaccharides such as hitchin and glucan (Farkaš, 2003). "
    [Show abstract] [Hide abstract] ABSTRACT: The aim of this study is to investigate chemical composition of acetone extracts of the lichens Parmelia arseneana and Acarospora fuscata and in vitro antioxidant, antimicrobial, and anticancer activities of these extracts and gyrophoric acid isolated from A. fuscata. The HPLC-UV method was used for the identification of secondary metabolites. Stictic acid, norstictic acid, gyrophoric acid, usnic acid, atranorin and chloroatranorin were identified in the A. fuscata. In P. arseneana, we detected stictic acid, norstictic acid, usnic acid and atranorin, while gyrophoric acid was not identified. Antioxidant activity was evaluated by measuring the scavenging capacity of tested samples on DPPH and superoxide anion radicals, reducing the power of samples and determination of total phenolic compounds in extracts. As a result of the study, gyrophoric acid was found to have the largest DPPH radical scavenging activity with an IC50 value of 105.75 mu g/ml. Moreover, the tested samples had an effective superoxide anion radical scavenging and reducing power. The total content of phenol in extracts was determined as pyrocatechol equivalent. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method. The most active was also gyrophoric acid, with minimum inhibitory concentration values ranging from 0.019 to 1.25 mg/ml. Anticancer activity was tested against LS174 (human colon carcinoma cell line), A549 (human lung carcinoma cell line), Fem-x (malignant melanoma cell line), and a chronic myelogeneous leukaemia K562 cell line using the MTT method. Extract of P. arseneana expressed the strongest anticancer activity against all cell lines with IC50 values ranging from 11.61 to 47.06 mu g/ml.
    Full-text · Article · Nov 2014
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