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In vitro antioxidant, antimicrobial, and antiproliferative activities of methanol, acetone, and ethyl acetate extracts of Sedum acre L. were determined. Th eir total phenolic contents as well as the concentrations of fl avonoids were also evaluated. Th e total phenolic content was determined with Folin-Ciocalteu reagent and it ranged between 18.25 to 181.75 mg gallic acid/g. Th e concentration of fl avonoids in various extracts of S. acre was determined by using a spectrophotometric method with aluminum chloride and the obtained results varied from 8.42 to 173.42 mg rutin/g. Antioxidant activity was monitored spectrophotometrically and expressed in terms of IC 50 (μg/mL), and its values ranged from 29.57 to 987.16 μg/mL. Th e highest phenolic content and capacity to neutralize DPPH radicals were found in the acetone extract. In vitro antimicrobial activity was investigated by the microdilution method. Minimum inhibitory concentration and minimum microbicidal concentration were determined. Testing was conducted against 17 microorganisms, including 13 strains of bacteria (standard and clinical strains) and 4 species of fungi. Th e tested extracts showed signifi cant antibacterial activities against bacteria and weak to selective activities against the tested fungi. Antiproliferative activity of the methanol extract on the HCT-116 cell line was determined by MTT assay. Results showed that S. acre has medium activity on cell proliferation, with IC 50 values of 281.69 for 24 h and 126.57 for 72 h. Based on these results, S. acre is a potential source of phenols as a natural antioxidant, antibacterial, and anticancer substance of high value. Th e phenolic content of extracts depends on the solvents used for extraction. Th e results of our study showed the great potential of S. acre for use in phytotherapy, pharmaceuticals, and the food industry.
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580
Turk J Biol
36 (2012) 580-588
© TÜBİTAK
doi:10.3906/biy-1109-9
Evaluation of biological activities of goldmoss stonecrop
(Sedum acre L.)
Milan STANKOVIĆ, Ivana RADOJEVIĆ, Milena ĆURČIĆ, Sava VASIĆ,
Marina TOPUZOVIĆ, Ljiljana ČOMIĆ, Snežana MARKOVIĆ
Department of Biology and Ecology, Faculty of Science, University of Kragujevac, 34 000 Kragujevac - SERBIA
Received: 11.09.2011 Accepted: 11.05.2012
Abstract: In vitro antioxidant, antimicrobial, and antiproliferative activities of methanol, acetone, and ethyl acetate
extracts of Sedum acre L. were determined.  eir total phenolic contents as well as the concentrations of  avonoids
were also evaluated.  e total phenolic content was determined with Folin-Ciocalteu reagent and it ranged between
18.25 to 181.75 mg gallic acid/g.  e concentration of  avonoids in various extracts of S. acre was determined by
using a spectrophotometric method with aluminum chloride and the obtained results varied from 8.42 to 173.42 mg
rutin/g. Antioxidant activity was monitored spectrophotometrically and expressed in terms of IC50 (µg/mL), and its
values ranged from 29.57 to 987.16 µg/mL.  e highest phenolic content and capacity to neutralize DPPH radicals were
found in the acetone extract. In vitro antimicrobial activity was investigated by the microdilution method. Minimum
inhibitory concentration and minimum microbicidal concentration were determined. Testing was conducted against
17 microorganisms, including 13 strains of bacteria (standard and clinical strains) and 4 species of fungi.  e tested
extracts showed signi cant antibacterial activities against bacteria and weak to selective activities against the tested
fungi. Antiproliferative activity of the methanol extract on the HCT-116 cell line was determined by MTT assay. Results
showed that S. acre has medium activity on cell proliferation, with IC50 values of 281.69 for 24 h and 126.57 for 72 h.
Based on these results, S. acre is a potential source of phenols as a natural antioxidant, antibacterial, and anticancer
substance of high value.  e phenolic content of extracts depends on the solvents used for extraction.  e results of our
study showed the great potential of S. acre for use in phytotherapy, pharmaceuticals, and the food industry.
Key words: Sedum acre, antimicrobial, antioxidant, antiproliferative activity
Introduction
Goldmoss stonecrop (Sedum acre L. - Crassulaceae)
is a perennial herbaceous plant, up to 10 cm high,
with smooth-margined succulent leaves 3-6 mm long
and yellow  owers. It inhabits dry sandy soil, stones,
and rocky surfaces in Europe, northern and western
Asia, Anatolia, and North Africa (1). In traditional
medicine it is widely used to treat ulcers and infected
wounds, and as a hypotensive (2).  e therapeutic
activity of S. acre is mainly caused by alkaloids
contained in this herb (3). Although it is mentioned
in some papers dealing with wild medicinal herbs
(3-7), there are no other data about the biological
activities of the S. acre plant.
Nature has long been an important source of
medicinal agents. An impressive number of modern
drugs have been isolated or derived from natural
sources, based on their use in traditional medicine
(8,9). Evaluation of plant biological e ects, such as
antioxidative, antiallergic, antibiotic, hypoglycemic,
and anticarcinogenic e ects, makes a signi cant
contribution to medicinal plant study as well as to
their pharmaceutical applications (10,11).
M. STANKOVIĆ, I. RADOJEVIĆ, M. ĆURČIĆ, S. VASIĆ, M. TOPUZOVIĆ, L. ČOMIĆ, S. MARKOVIĆ
581
Many medicinal plants have been proven
important sources of phenolic compounds that
show good antioxidant activity. Currently used
synthetic antioxidants have been suspected to cause
or promote negative health e ects; hence, stronger
restrictions have been placed on their application
and there is also a trend of substituting them with
naturally occurring antioxidants (12,13).
Plants are an abundant natural source of very
e ective antibiotic compounds. Plant antibiotics are
chemicals that kill cells or inhibit their division and
are classi ed into antibacterial, antifungal, antiviral,
and antineoplastic types according to their target.
ey a ect cell membrane permeability, and they
regulate or disable enzymatic processes and protein
translation (14,15).
A wide variety of secondary metabolites obtained
from plants are tested for their ability to treat cancer
(16).  e relation between phenolic compounds and
reduced cancer risk has been reported in previous
studies that showed a decrease in cancer risk with
consumption of vegetables and parts of plants rich in
avonoids (17).
e biological activity of S. acre is little known
up to now. e aim of this study is to investigate
the antioxidant, antimicrobial, and antiproliferative
properties of the methanol, acetone, and ethyl acetate
extracts of this plant, as well as to assess the biological
activity in relation to phenol concentration and
avonoid content in the tested extracts.
Materials and methods
Chemicals
Organic solvents and sodium hydrogen carbonate
were purchased from Zorka Pharma, Šabac, Serbia.
Gallic acid, rutin hydrate, chlorogenic acid, and
2,2-diphenyl-1-picrylhydrazyl (DPPH) were
obtained from Sigma Chemical Co., St Louis, MO,
USA. Folin-Ciocalteu phenol reagent and aluminum
chloride hexahydrate (AlCl3) were purchased from
Fluka Chemie AG, Buchs, Switzerland. Nutrient
liquid medium, a Mueller-Hinton broth, was from
Lio lchem, Roseto degli Abruzzi, Italy, while
a Sabouraud dextrose broth was from Torlak,
Belgrade, Serbia. An antibiotic, doxycycline, was
purchased from Galenika A.D., Belgrade, and an
antimycotic,  uconazole, was from P zer Inc.,
New York, NY, USA. Dulbecco’s Modi ed Eagle
Medium (DMEM) was obtained from GIBCO,
Invitrogen, Carlsbad, CA, USA. Fetal bovine
serum (FBS) and trypsin-EDTA were from PAA
Laboratories, Pasching, Austria. Dimethyl sulfoxide
(DMSO) and 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) were obtained
from SERVA, Heidelberg, Germany. All other
solvents and chemicals were of analytical grade.
Plant material
Aerial parts of S. acre were collected in June 2010
from natural populations in Trgovište in the area
surrounding the city of Vranje in southern Serbia
(42°23ʹ14.27ʺN, 22°01ʹ42.93ʺE; altitude: 574 m;
exposition: W, habitat: arid, thermophilic, rocky).
e voucher specimen of S. acre was con rmed and
deposited at the Herbarium of the Department of
Biology and Ecology, Faculty of Science, University
of Kragujevac.  e collected plant material was air-
dried in darkness at room temperature (20 °C).
Preparation of plant extracts
Prepared plant material (10 g) was transferred to
dark-colored  asks with 200 mL of solvent (methanol,
acetone, and ethyl acetate) respectively and stored at
room temperature. A er 24 h, infusions were  ltered
through Whatman No. 1  lter paper and residue was
reextracted with an equal volume of solvents. A er 48
h, the process was repeated. Combined supernatants
were evaporated to dryness under vacuum at 40 °C
using a rotary evaporator.  e obtained extracts
were kept in sterile sample tubes and stored in a
refrigerator at 4 °C.
Determination of total phenolic contents in the
plant extracts
e total phenolic content was determined using
a spectrophotometric method (18).  e reaction
mixture was prepared by mixing 0.5 mL of methanol
solution (1 mg/mL) of extract with 2.5 mL of 10%
Folin-Ciocalteu reagent dissolved in water and 2.5
mL of 7.5% NaHCO3.  e samples were incubated at
45 °C for 15 min.  e absorbance was determined at
λmax = 765 nm.  e samples were prepared in triplicate
and the mean value of absorbance was obtained. A
blank was concomitantly prepared with methanol
instead of extract solution.  e same procedure
Evaluation of biological activities of goldmoss stonecrop (Sedum acre L.)
582
was repeated for the gallic acid and the calibration
line was construed.  e total phenolic content was
expressed in terms of gallic acid equivalent (mg of
GA/g of extract).
Determination of  avonoid concentrations in the
plant extracts
e concentrations of  avonoids were determined
using a spectrophotometric method (19).  e sample
contained 1 mL of methanol solution of the extract
in the concentration of 1 mg/mL and 1 mL of 2%
AlCl3 solution dissolved in methanol.  e samples
were incubated for 1 h at room temperature.  e
absorbance was determined at λmax = 415 nm.  e
samples were prepared in triplicate and the mean value
of absorbance was obtained.  e same procedure was
repeated for the rutin and the calibration line was
construed. Concentration of  avonoids in extracts
was expressed in terms of rutin equivalent (mg of
RU/g of extract).
Evaluation of DPPH scavenging activity
e ability of the plant extract to scavenge DPPH free
radicals was assessed using the method described by
Tekao et al. (20), adopted with suitable modi cations
from Kumarasamy et al. (21).  e stock solution
of the plant extract was prepared in methanol to
achieve a concentration of 1 mg/mL. Dilutions were
made to obtain concentrations of 500, 250, 125,
62.5, 31.25, 15.62, 7.81, 3.90, 1.99 and 0.97 µg/mL.
Diluted solutions (1 mL each) were mixed with 1 mL
of DPPH methanolic solution (80 µg/mL). A er 30
min in darkness at room temperature (23 °C), the
absorbance was recorded at 517 nm.  e control
samples contained all the reagents except the extract.
e percentage inhibition was calculated using the
following equation: % inhibition = 100 × (A control
A sample) / A control).  e IC50 values (inhibitory dose
that inhibited cell growth by 50%) were estimated
from the % inhibition versus concentration sigmoidal
curve, using a nonlinear regression analysis.  e data
are presented as mean values ± standard deviation
(SD) (n = 3).
In vitro antimicrobial assay
Test microorganisms
Antimicrobial activities of acetone, ethyl acetate,
and methanol extracts were tested against 17
microorganisms, including 13 strains of bacteria
(standard strains: Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 25923, Pseudomonas
aeruginosa ATCC 27853, Bacillus pumilus NCTC
8241; clinical strains: Escherichia coli, Staphylococcus
aureus, Proteus mirabilis, Sarcina lutea, Salmonella
enterica, Salmonella typhimurium, Sarcina lutea,
Bacillus subtilis, and Bacillus cereus) and 4 species
of fungi: Penicillium chrysogenum PMFKG-F31,
Aspergillus niger ATCC 16404, Candida albicans
(clinical isolate), and Candida albicans ATCC 10231.
All clinical isolates were a generous gi from the
Institute of Public Health in Kragujevac.  e other
microorganisms were provided from the collection
held by the Microbiology Laboratory, Faculty of
Science, University of Kragujevac.
Suspension preparation
Bacterial and yeast suspensions were prepared by
the direct colony method.  e turbidity of initial
suspension was adjusted by comparison with 0.5
McFarlands standard (22).  e initial bacterial
suspension contained about 108 colony forming
units (CFU)/mL and a suspension of yeast contained
106 CFU/mL. Additionally, 1:100 dilutions of initial
suspension were prepared into sterile 0.85% saline.
e suspensions of fungal spores were prepared by
gentle stripping of spores from slopes with growing
aspergilli.  e resulting suspensions were diluted to
1:1000 in sterile 0.85% saline.
Microdilution method
Antimicrobial activity was tested by determining
the minimum inhibitory concentration (MIC) and
minimum microbicidal concentration (MMC)
using the microdilution method with resazurin, an
indicator of microbial growth (23).  e 96-well plates
were prepared by dispensing 100 µL of nutrient broth,
Mueller-Hinton broth for bacteria and Sabouraud
dextrose broth for fungi, into each well. A volume
of 100 µL from the stock solution of tested extracts
(concentration: 80 mg/mL) was added into the  rst
row of the plate. Serial dilutions were then performed
2-fold by using a multichannel pipette.  e obtained
concentration range was from 0.0195 mg/mL to 40
mg/mL. MIC was de ned as the lowest concentration
of tested extracts that prevented a resazurin color
change from blue to pink.  e method was described
in detail in a previous paper (24).
M. STANKOVIĆ, I. RADOJEVIĆ, M. ĆURČIĆ, S. VASIĆ, M. TOPUZOVIĆ, L. ČOMIĆ, S. MARKOVIĆ
583
MMC was determined by plating 10 µL of samples
from wells, where no indicator color change was
recorded, on nutrient agar medium. At the end of the
incubation period, the lowest concentration with no
growth (no colony) was de ned as the MMC.
Doxycycline and  uconazole, dissolved in nutrient
liquid medium, were used as a positive control.  e
tested compounds were dissolved in DMSO and then
diluted into nutrient liquid medium to achieve a
concentration of 10% DMSO. A solvent control test
was performed to study the e ect of 10% DMSO on
the growth of microorganisms. In the experiment,
the concentration of DMSO was additionally
decreased because of the 2-fold serial dilution assay
(the working concentration was 5% and lower). Each
test included growth control and sterility control. All
tests were performed in duplicate and MICs were
constant.
Antiproliferative assay
Cell preparation and culturing
e HCT-116 cell line was obtained from the
American Type Culture Collection. Cells were
maintained in DMEM supplemented with 10%
FBS, with 100 units/mL penicillin and 100 µg/mL
streptomycin. Cells were cultured in a humidi ed
atmosphere with 5% CO2 at 37 °C. Cells were grown
in 75-cm2 culture bottles supplied with 15 mL of
DMEM, and a er a few passages cells were seeded in
96-well plate. All studies were done with cells at 70%
to 80% con uence.
Cell viability assay (MTT assay)
HCT-116 cells were seeded in a 96-well plate
(10,000 cells per well). A er 24 h of cell incubation,
the medium was replaced with 100 µL of medium
containing various doses of methanol extracts at
di erent concentrations (1, 10, 50, 100, 250, and 500
µg/mL) for 24 h and 72 h. Untreated cells served as
the control. A er 24 h and 72 h of treatment, the cell
viability was determined by MTT assay (25).  e
proliferation test was based on the color reaction
of mitochondrial dehydrogenase in living cells by
MTT. At the end of the treatment period, MTT ( nal
concentration: 5 mg/mL phosphate bu er solution)
was added to each well, which were then incubated
at 37 °C in 5% CO2 for 2-4 h.  e colored crystals
of produced formazan were dissolved in 150 µL of
DMSO.  e absorbance was measured at 570 nm on
a microplate reader. Cell proliferation was calculated
as the ratio of absorbance of treated group divided
by the absorbance of control group and multiplied by
100 to give percentage proliferation.
Statistical analysis
e data are expressed as the means ± SD. All
statistical analyses were performed using SPSS 17
for Windows (SPSS Inc., Chicago, IL, USA). Mean
di erences were established by Students t-test. Data
were analyzed using one-way analysis of variance
(ANOVA). In all cases, P < 0.05 was considered
statistically signi cant.
Results and discussion
Total phenolic content,  avonoid concentrations,
and in vitro antioxidant, antibacterial, and antifungal
activities were determined with methanol, acetone,
and ethyl acetate extracts of S. acre. Various solvents
were used to achieve extraction of active substances
with diversity in their polarity.  is choice of solvents
was proven to be very e ective in earlier studies (26).
Total phenolic content
e content of total phenolic compounds in the
examined plant extracts, which was determined by
using Folin-Ciocalteu reagent, is expressed in terms
of gallic acid equivalent (standard curve equation:
y = 7.026x – 0.0191, r2 = 0.999) as mg GA/g extract
(Figure 1).  e concentrations of total phenols in
the examined extracts ranged from 18.25 mg/g to
181.75 mg/g.  e high concentration of phenols was
measured in the acetone and ethyl acetate extracts.
Methanol extract had a considerably smaller
concentration of phenolic compounds.  e extracts
obtained by using moderately polar solvents had
higher concentrations of phenolic compounds.  e
content of phenols in extracts of S. acre depended on
the polarity of the solvent used for extraction. High
dissolubility of phenols in polar solvents provided
a high concentration of these compounds in the
extracts obtained by using polar solvents for the
extraction (27,28).
Concentration of  avonoids
e concentration of  avonoids in various extracts of
S. acre was determined by using a spectrophotometric
Evaluation of biological activities of goldmoss stonecrop (Sedum acre L.)
584
method using AlCl3.  e values for concentrations of
avonoids are expressed in terms of rutin equivalent
(standard curve equation: y = 17.231x – 0.0591,
r2 = 0.999) as mg RU/g extract. A summary of the
identi ed quantities of  avonoids in the tested
extracts is shown in Figure 1.  e concentration of
avonoids in plant extracts ranged from 8.42 mg/g to
173.42 mg/g. A high concentration of  avonoids was
measured in the acetone and ethyl acetate extracts.
e lowest  avonoid concentration was measured in
the methanol extract.  e concentration of  avonoids
in plant extracts depended on the polarity of solvents
used in the extract preparation (29). Based on the
obtained values of the concentration of  avonoids in
the examined extracts from S. acre, it was found that
the highest concentration of these compounds was in
the extracts obtained by using solvents of moderate
polarity.
Antioxidant activity
e antioxidant activity of di erent plant extracts
from S. acre was determined by using a methanolic
solution of DPPH reagent. Unlike free radicals
generated in vitro such as the hydroxyl radical and
superoxide anion, DPPH has the advantage of being
una ected by certain side reactions, such as metal ion
chelation and enzyme inhibition, brought about by
various additives. A freshly prepared DPPH solution
exhibits a deep purple color with an absorption
maximum at 517 nm.  is purple color generally
fades when antioxidant molecules quench DPPH
free radicals (i.e., by providing hydrogen atoms or
by electron donation, conceivably via a free-radical
attack on the DPPH molecule) and convert them into
colorless products (i.e., 2,2-diphenyl-1-hydrazine or
a substituted analogous hydrazine), resulting in a
decrease in absorbance at 517 nm (30).
e antioxidant activity of 3 di erent extracts from
the whole plant of S. acre is expressed in terms of IC50
(µg/mL) values. A summary display of obtained IC50
values of the antioxidant activity of tested extracts
is given in Table 1. Parallel to examination of the
antioxidant activity of plant extracts, the values for
3 standard compounds (Table 2) were obtained and
compared to the values of the antioxidant activity.
e standard substances were rutin, chlorogenic
acid, and 3-tert-butyl-4-hydroxyanisole (BHA).  e
obtained values of antioxidant activity examined
with DPPH
radical were in the range of 29.57 µg/mL
to 987.16 µg/mL.  e largest capacity to neutralize
DPPH radicals was found for the acetone extract,
which neutralized 50% of free radicals at the
0
20
40
60
80
100
120
140
160
180
200
Methanolc Acetone Ethyl acetate
Concentratons mg/ml
Total phenolc
content
Flavonod
cocncentratons
Figure 1. Total phenolic content expressed in terms of gallic
acid equivalent (mg of GA/g of extract) and  avonoid
concentrations expressed in terms of rutin equivalent
(mg of RU/g of extract) in di erent S. acre extracts.
Table 1. Values1 for antioxidant (DPPH scavenging) activity of
S. acre extracts.
Extracts IC50 (μg/mL)
Methanol 987.16 ± 1.69
Acetone 29.57 ± 1.21
Ethyl acetate 40.42 ± 1.05
1Average of 3 analyses ± SD.
Table 2. Values1 of antioxidant (DPPH scavenging) activity of
standard substances obtained for comparison with the
values of S. acre.
Substances IC50 (μg/mL)
BHA 5.39 ± 0.31
Rutin 9.28 ± 0.27
Chlorogenic acid 11.65 ± 0.52
1Average of 3 analyses ± SD.
M. STANKOVIĆ, I. RADOJEVIĆ, M. ĆURČIĆ, S. VASIĆ, M. TOPUZOVIĆ, L. ČOMIĆ, S. MARKOVIĆ
585
concentration of 29.57 µg/mL.
A similar activity was
found for the ethyl acetate extract.  e lowest capacity
to inhibit DPPH radicals was determined for the
methanol extract.
In comparison to the IC50 values of BHA, rutin,
and chlorogenic acid (Table 2), the acetone and
ethyl acetate extracts of S. acre manifested a stronger
capacity for neutralization of DPPH radicals.
Antimicrobial activity
e results of in vitro testing of the antimicrobial
activities of acetone, ethyl acetate, and methanol extracts
of S. acre are shown in Table 3. For comparison, the
MIC and MMC values of doxycycline and  uconazole
are also listed in Table 3.  e solvent (10% DMSO) did
not inhibit the growth of the tested microorganisms.
Antimicrobial activity of the tested extracts
was evaluated by determining MICs and MMCs in
relation to the 17 species of microorganisms. MIC
and MMC values were in the range of 0.039 mg/mL
to 40 mg/mL. Extracts showed selective antimicrobial
properties while the activities depended both on
the species of microorganism and on the type and
concentration of extract. In general, the tested
extracts demonstrated high antimicrobial activity,
showing more potent inhibitory e ects on the growth
of bacteria than on fungi.
e weakest antimicrobial activity was shown by
the methanol extract (P < 0.05). When the activity
is observed according to groups of microorganisms,
this extract was the only one with strong activity on
gram-positive bacteria, but had the weakest activity
on fungi (P < 0.05).
e tested extracts showed high antibacterial
activity against gram-positive bacteria. MIC values
were in the range of 0.039 mg/mL to 2.5 mg/mL.
e extracts showed a signi cant e ect in relation
to Bacillus cereus (MIC: 0.039 mg/mL; MMC: 0.625
mg/mL) and Sarcina lutea (MIC: 0.039 mg/mL;
MMC: 2.5 mg/mL).  e extracts also showed good
antibacterial e ect on gram-negative bacteria.  e
best e ect was with acetone and ethyl acetate extracts
Table 3. Antimicrobial activities of acetone, ethyl acetate, and methanol extracts of S. acre.
Species Methanol extract Acetone extract Ethyl acetate extract Doxycycline / Fluconazole
MIC*MMC*MIC MMC MIC MMC MIC MMC
Escherichia coli ATCC 25922 2.5 20 2.5 5 2.5 10 15.625 31.25
Escherichia coli 5 40 2.5 10 20 20 7.81 15.625
Pseud. aeruginosa ATCC 27853 1.25 20 0.313 5 0.313 2.5 62.5 125
Proteus mirabilis 2.5 2.5 0.625 1.25 0.625 1.25 250 >250
Salmonella typhimurium 2.5 10 1.25 2.5 1.25 2.5 15.625 125
Salmonella enterica 2.5 5 1.25 2.5 15.625 31.25
Staphylococcus aureus 2.5 2.5 1.25 2.5 0.448 7.81
Staphylococcus aureus ATCC 25923 0.625 2.5 0.313 2.5 0.625 10 0.224 3.75
Sarcina lutea 0.039 2.5 0.039 2.5 <0.448 3.75
Bacillus subtilis 1.25 2.5 1.25 1.25 0.313 0.625 0.112 1.953
Bacillus cereus 0.039 0.625 0.039 0.625 0.977 7.81
Bacillus pumilus NCTC 8241 1.25 1.25 1.25 2.5 0.112 7.81
Candida albicans ATCC 10231 5 20 2.5 2.5 0.313 0.313 31.25 1000
Candida albicans 10 10 1.25 1.25 2.5 2.5 62.5 1000
Penicillium chrysogenum 10 10 10 10 10 10 62.5 500
Aspergillus niger ATCC 16404 10 20 10 20 20 20 62.5 62.5
*Minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) values are given as mg/mL for plant
extract and g/mL for antibiotics; –, not tested.
Evaluation of biological activities of goldmoss stonecrop (Sedum acre L.)
586
on P. aeruginosa (MIC: 0.313 mg/mL; MMC: 2.5
and 5 mg/mL) and P. m i ra bi li s (MIC: 0.625 mg/mL;
MMC: 1.25 mg/mL).
e tested extracts showed low to moderate
antifungal activity. MIC and MMC values were in
the range of 1.25 mg/mL to 20 mg/mL.  e exception
was the ethyl acetate extract on the species C.
albicans ATCC 10231, where the MIC and MMC
were 0.313 mg/mL.  ese results are in accordance
with the preliminary results of Tosun et al. (4) on
the antifungal activity of the methanol extract of S.
acre on C. albicans ATCC 10231 and C. krusei ATCC
6258.
While the acetate extract had good antifungal
activity on Aspergillus restrictus as well as on A.
fumigatus (6), the other extracts of Sedum acre had
no signi cant activity on other  lamentous fungi.
e activity of the acetone extract was in relation
with its total phenol and  avonoid content. In some
previous studies it was claimed that the phenolic
compounds from the plant might play a major role in
their antimicrobial activities (31).
With the exception of the assay of anti-Aspergillus
activities, this is the  rst study on the antimicrobial
activity of the acetone and ethyl acetate extracts of
S. acre.  e results of our research indicated a good
antimicrobial potential of S. acre, based on which
it could be considered as a source of potential
antimicrobial substances.
Antiproliferative activity
Secondary metabolites derived from natural sources
have been receiving increasing attention in recent
years since they were reported to have a remarkable
spectrum of biological activities including
antioxidant, antiin ammatory, antibacterial, and
anticarcinogenic activities.  ey may have many
health bene ts and can be considered as possible
chemopreventive agents against cancer (32,33).
In order to provide complete screening of
biological activities, cell proliferation ability was also
examined. To explore the antiproliferative activity
of methanol extracts of S. acre on the HCT-116
cell line, the MTT cell viability assay was used. We
treated HCT-116 cells with di erent concentrations
of extract (in a concentration range from 1 µg/mL to
500 µg/mL) and determined cell viability a er 24 h
and 72 h.
e shape of dose-response curves indicated a
signi cant inhibition of cell growth in a dose- and
time-dependent manner (Figure 2). Cell growth was
signi cantly lower (P < 0.05) when extract-treated
cells were compared to control cells.  e extract
exhibited higher cytotoxic e ects a er a longer
exposure.  e e ects of extracts were expressed by
IC50 values.  e IC50 value was used as a parameter
for cytotoxicity.  e IC50 value was 281.69 a er 24
h and 126.57 a er 72 h of exposure.  e methanol
extract of S. acre a ects cell proliferation and inhibits
cell growth, but according to the IC50 values, we can
conclude that it has an intermediate e ect on HCT-
116 cell proliferation.
Results reported here contributed to the
knowledge on the biological activity of S. acre. It
could be concluded that S. acre is a source of phenolic
compounds that demonstrate e ective antioxidative,
antibacterial, and antiproliferative activities. Acetone
is a very e ective solvent for extraction of phenolic
compounds from this plant.  e determined
cytotoxic and antimicrobial activities of S. acre are
of great importance in preservation of potential
antimicrobial substances and they also present
important information for further studies and
applications in phytotherapy, pharmacy, and food
industry.
0
20
40
60
80
100
120
1 10 50 100 250 500
% of vable cells
Concentratons (µg/ml)
aer 24 h
aer 72 h
Figure 2. e dose-response curves of the e ects of S. acre on
cell growth in HCT-116 cells.  e cells were treated
with various concentrations of drugs a er 24 h and
72 h of exposure.  e antiproliferative e ects were
measured by MTT assay. Results were expressed
as means ± standard error for 3 independent
determinations.
M. STANKOVIĆ, I. RADOJEVIĆ, M. ĆURČIĆ, S. VASIĆ, M. TOPUZOVIĆ, L. ČOMIĆ, S. MARKOVIĆ
587
Acknowledgments
is investigation was supported by the Ministry
of Education and Science of the Republic of Serbia,
grants No. III41010 and No. OI173032.
Corresponding author:
Milan STANKOVIĆ
Department for Biology and Ecology,
Faculty of Science,
str. Radoja Domanovića No. 12,
34000 Kragujevac, P.O. Box 60 - SERBIA
E-mail: mstankovic@kg.ac.rs
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