Journal of Applied Botany and Food Quality 90, 330 - 338 (2017), DOI:10.5073/JABFQ.2017.090.041
1Department of Morphology and Systematic of Plants, Institute of Botany and Botanical Garden “Jevremovac”,
Faculty of Biology, University of Belgrade, Serbia
2Department of Plant Physiology, Institute for Biological Research “Siniša Stanković“, University of Belgrade, Serbia
3Institute for Medicinal Plant Research “Dr. Josif Pančić”, Belgrade, Serbia
4,5Department of Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University,
Institute for Biology and Macedonian Academy of Sciences and Arts, Skopje, Macedonia
Laserpitium ochridanum: antioxidant, antimicrobial and anti-quorum sensing activities against
Pseudomonas aeruginosa
Ksenija S. Mileski1*, Ana D. Ćirić2, Jovana D. Petrović2, Mihailo S. Ristić3, Vlado S. Matevski4,5,
Petar D. Marin1, Ana M. Džamić1
(Received June 6, 2016; Accepted August 23, 2017)
* Corresponding author
Summary
This study shows Laserpitium ochridanum essential oil composi-
tion, its antifungal potency, and antioxidant, antimicrobial and anti-
quorum sensing activities of different extracts. Monoterpene hydro-
carbons (40.9%) were the most abundant group of constituents in the
oil. Sabinene (22.8%), viridiflorol (14.7%) and α-pinene (11.40%)
were the main components of the oil. The ethanolic extract had the
highest antioxidant capacity in DPPH and ABTS assays and it was
the richest in phenolic contents. Microdilution method revealed the
strongest antibacterial activity of ethanolic extracts in comparison
to other tested extracts and streptomycin. Essential oil of L. ochri-
danum evidenced the best antifungal potential against used micro-
mycetes. Results of an anti-quorum sensing activity assay indicated
high affection of aqueous extract in reduction of PAO1 pyocyanin
production (18.07%). Used samples possessed slight reduction of
twitching and swimming motility. This study shows for the first time
anti-quorum sensing activity of L. ochridanum against Pseudomonas
aeruginosa PAO1, as well as its significant antioxidant potential.
Introduction
The genus Laserpitium L. (Apiaceae) comprises about 30 species,
mostly biennial and perennial plants, widely distributed from the
Canary Islands to Siberia and Iran (Nikolić, 1973). It is charac-
terized by ternate or several times pinnate leaves, white, yellow or
pinkish petals (TuTin, 1968). Laserpitium ochridanum Micevski
is rare and an endemic perennial plant, up to 40-60 cm with white
colour of the petals which can be found only at the National park
Mt. Galičica (FYROM), at 1600-2000 m a.s.l. (Micevski, 2005).
Different parts of some widely distributed Laserpitium species (e.g.
L. siler, L. latifolium) have been used as traditional herbal medicines
in Europe. They are usually used as tonics for strengthening and
refreshing, for treating toothache, as diuretics, for treating gastro-
intestinal disorders, heart and liver dysfunctions, pulmonary tuber-
culosis, rheumatism and topically in pruritic dermatomycoses, as
well as for sleep disorder and major depression in Taiwan (PoPović
et al., 2013; Yi-Lin chen et al., 2015). In earlier studies, sesqui-
terpene lactones were found as the main secondary metabolites in
Laserpitium extracts (Appendino et al., 1987, 1993; ĐermaNović
et al., 1996). Recently, it was published that sesquiterpene lactones
mainly belong to the class of guajanolides (PoPović et al., 2013).
However, monoterpene hydrocarbons were predominant compounds
in the essential oil (EO) of Laserpitium species (BAser and duMAn,
1997; chizzoLA et al., 1999; chizzoLA, 2007; Petrović et al., 2009;
TiriLLini et al., 2009; PoPović et al., 2010, 2013, 2014). Litarature
data showed that different Laserpitium species possessed antibac-
terial, antifungal, cytotoxic, anticancer, antinociceptive and anti-
edematous activities (Petrović et al., 2009; TiriLLini et al., 2009;
PoPović et al., 2010, 2013, 2014). Lately, the interest in studying
different pathogens is rapidly incrising, because of their resistance
towards synthetic antibiotics or antimycotics. It is known that the
pathogenic, gram-negative bacillus Pseudomonas aeruginosa is a
major cause of nosocomial infections, bronchopneumonia, septic
shock and wound infections. This opportunistic bacterium forms
populations with distinctive density-dependant behavour. By anti-
quorum sensing (anti-QS) agents, growth of P. aeruginosa can be
weaken and some of its pathologically significant virulence factors,
such as production of biofilm, swarming motility, pigment and an-
tibiotic production, can be reduced (Soković et al., 2014; sepAhi
et al., 2015). Some medical plants possess anti-QS activity and can
be considered as potential anti-quorum agents (AL-hussAini and
MAhAsneh, 2009; koh et al., 2013; sepAhi et al., 2015).
The aim of this study was to define the chemical composition of
L. ochridanum essential oil and to determined antioxidant, anti-
microbial and anti-QS activities of its extracts. To the best of our
knowledge L. ochridanum crude extracts were assayed for the first
time for their antioxidant potency combined with total phenolic and
flavonoid contents. Also, no anti-QS activity of this species has been
reported to date.
Material and methods
Solvents and chemical reagents
Solvents and chemicals that were provided for performing the ex-
periments were of analytical grade. Organic solvents were pro-
cured from Zorka pharma, Šabac, Serbia. Gallic acid, 3-tert-butyl-
4-hydroxyanisole (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
Folin-Ciocalteu phenol reagent, potassium acetate and aluminum
trinitrate nonahydrate were obtained from Sigma-Aldrich Co., St
Louis, MO, USA. Sodium carbonate anhydrous was purchased from
Centrohem d.o.o, Stara Pazova, Serbia. Potassium peroxide sulphate
and L(+)-ascorbic acid were obtained from Fisher Scientific UK
Ltd., Loughborough, Leicestershire, UK. 2,2’-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS) and quercetin hydrate
were purchased from TCI Europe NV, Binnenveldsweg, Belgium.
Malt-broth (MB), tryptic soy broth (TSB), Mueller-Hinton agar
(MH), Luria-Bertani medium (LB) (1% w/v NaCl, 1% w/v tryptone,
0.5% w/v yeast extract) and malt agar (MA) were obtained from the
Institute of Immunology and Virology, Torlak (Belgrade, Serbia).
Streptomycin (Sigma-Aldrich S6501 St. Louis, MQ, USA), ampi-
cillin (Sigma-Aldrich A9393 St. Louis, MQ, USA) and dimethyl sulf-
oxide (DMSO) (Sigma Aldrich, St. Louis, MQ, USA) were used in
these study. Antimicotic diflucan (containing 50 mg fluconazole) was
obtained from Pfizer PGM, Pocesur-Cisse, France.
Biological activities of Laserpitium ochridanum 331
Plant material
Plant material was collected during the flowering stage, at Mt.
Galičica, the national park in Republic of Macedonia (FYROM)
in July, 2013 (GPS: N 40°56́30 ,̋ E 20°49́34̋). It was determined
as Laserpitium ochridanum Micevski by one of the authors (prof.
Vlado S. Matevski). A voucher specimen (BU16778) is deposited
at the herbarium of the Institute of Botany and Botanical Garden
“Jevremovac”, Faculty of Biology, University of Belgrade, Serbia.
Isolation of the essential oil
The dark blue EO of L. ochridanum was obtained from 200 g of dry
aerial parts by 3 h of hydrodistilation using a Clevenger type appa-
ratus. The yield of the oil was 0.11% for herbal parts (w/w-dry bases).
The essential oil obtained was preserved in sealed vials at 4 ºC
prior to further analysis.
Preparation of plant extracts
Dried, ground plant material (10 g) was treated with 200 mL of
methanol, ethanol and distilled water to obtain different extracts. The
ultrasonic extraction procedure was performed during 24 h in the
dark; the extracts were exposed to ultrasound for the first and the last
hour of extraction and subsequently filtered through a Whatman filter
paper No 1. Methanolic and ethanolic extracts were subjected to sol-
vent evaporation under reduced pressure at maximum temperature
of 40 ºC. The frozen aqueous extracts were lyophilized, reduced to
a fine dried powder and mixed to obtain homogenous samples. The
dried and crude extracts were measured, packed in glass bottles, and
stored at 4 ºC until subjection to subsequent analysis. Obtained yields
of L. ochridanum extracts were 0.597 g for methanolic (ME), 1.323 g
for ethanolic (EE) and 0.983 g for aqueous extracts (AE).
Gas chromatography–flame ionization detector (GC–FID) and
gas chromatography–mass spectrometry (GC–MS)
Qualitative and quantitative analysis of the essential oil was per-
formed using GC and GC-MS methods. The GC analysis of the oil
was carried out on a GChP-5890 II apparatus, equipped with split-
split less injector, attached to an HP-5 column (25 m × 0.32 mm,
0.52 μm film thickness) and fitted to FID. Carrier gas flow rate (H2)
was 1 mL/min, split ratio 1:30, injector temperature was 250 °C, de-
tector temperature 300 °C, while column temperature was linearly
programmed from 40 to 240 °C (at rate of 4 °/min.). The same analy-
tical conditions were employed for GC-MS analysis, where a 1800C
Series II GCD system equipped with HP-5MS column (30 m ×
0.25 mm, 0.25 μm film thickness) was used. The transfer line was
heated at 260 °C. Mass spectra were acquired in EI mode (70 eV),
in m/z range 40-400. The identification of the individual EO com-
ponents was accomplished by comparison of retention times with
standard substances and by matching mass spectral data with those
of the Wiley 275 mass spectra library. Confirmation was performed
using AMDIS software and literature (AdAMs, 2007). Quantitative
analyses were based on area percents obtained by FID.
Analyses of total phenols and total flavonoids
Total phenolic content (TPC)
The spectrophotometric method described by singLeTon et al.
(1999) with some modifications was applied for recording total TPCs
of all tested L. ochridanum extracts, using Folin-Ciocalteu reagent
and GA as a standard. After preparing a 10% Folin-Ciocalteau re-
agent, the mixtures of 1000 μL of this solution and 200 μL of ex-
tracts solutions (1 mg/mL) were left to react for 6 min. After short
incubation, 800 μL of 7.5% sodium carbonate solution was added
and thus prepared solution was allowed to stand for 2 h at room tem-
perature in the dark. The absorbance was measured at 736 nm versus
a blank sample. Total phenols were calculated from the GA calibra-
tion curve (10-100 mg/L). Data were expressed as milligrams of GA
equivalents per gram of dry plant extract. The values were presented
as means of triplicate analysis.
Total flavonoid content (TFC)
Measurements of TFCs of L. ochridanum extracts were based on
the method described by pArk et al. (1997) with slight modification.
An aliquot of each extract solution (1 mL) was mixed with 80% etha-
nol, 10% aluminium nitrate nonahydrate and 1 M potassium acetate.
Absorption readings at 415 nm using a spectrophotometer were taken
after 40 min. against a blank sample consisting of a 0.5 mL 96%
ethanol instead of the tested sample. The TFCs were determined
from the QE standard curve (10-100 mg/L). Results were expressed
as mg of QE equivalents/g of dry extract. Generally, All measure-
ments were done in triplicates.
Antioxidant capacity
DPPH assay
Series of EO and extracts solutions in appropriate solvents, with con-
centrations of 0.25-2 μL/mL for EO and 0.025-0.2 mg/mL for ex-
tracts were subjected for examination of free radical scavenging ac-
tivity by DPPH assay. This spectrophotometric procedure described
by BLois (1958), was performed to evaluate the quantity of tested
solutions needed to reduce 50% of the initial DPPH radical concen-
tration. 0.2 mL of each dilution was mixed with 1.8 mL of DPPH
methanol solution (0.04 mg/mL). The absorbance was recorded at
517 nm after 30 min. of dark incubation at room temperature. BHA
and ascorbic acid were used as reference standards and methanol as
a blank. The corresponding percentage of inhibitions of each sample
was calculated from obtained absorbance values by using following
equation:
Percentage (%) of inhibition = (Ac-As)/Ac × 100
Tested concentrations of EO and extracts which decrease absorption
of DPPH solution for 50% (IC50) were obtained from the curve de-
pendence of absorption of DPPH solution on 517 nm from concentra-
tion for each tested solution and used standards.
ABTS assay
The procedure of MiLLer and rice-evAns (1997) with slightly mo-
difications was followed for determination of in vitro ABTS radical-
scavenging potency. Before usage, 5 mL of the mixture of 2.46 mM
potassium persulphate and 19.2 mg of ABTS was allowed to react in
the dark for 12-16 h at room temperature to obtain ABTS+ solution.
100-110 mL of distilled water was added to 1 mL of ABTS+ solution
to adjust an absorbance of 0.7 ± 0.02 units at 734 nm. The mixtures
of 2 mL of diluted ABTS·+ solution and 50 μL of each tested extract
solution were incubated for 30 min. at 30 ºC and the absorbance
was determined spectrophotometrically at 734 nm, using water as a
blank. For every experiment a fresh ABTS+ solution was prepared.
The results were expressed from an ascorbic acid calibration curve
(0-2 mg/L) in mg of ascorbic acid equivalents/g of dry extract. Tests
were carried out in triplicate and all measurements were expressed as
average of three analyses ± standard deviation.
Evaluation of antimicrobial properties
Microorganisms and culture conditions
The antimicrobial activity of all investigated samples was tested
using pure control strains obtained from the mycological laboratory,
Department of Plant Physiology, Institute for Biologycal Research
332 K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić
“Siniša Stanković”, Belgrade, Serbia. The microorganisms included
following bacterial strains: Bacillus cereus (food isolate), Listeria
monocytogenes (NCTC 7973), Micrococcus flavus (ATCC 10240)
and Staphylococcus aureus (ATCC 6538), Enterobacter cloacae
(human isolate), Escherichia coli (ATCC 35210), Pseudomonas
aeruginosa (ATCC 27853), and Salmonella typhimurium (ATCC
13311). The following micromicetes were used: Aspergillus fumi-
gatus (ATCC 9197), Aspergillus niger (ATCC6275) Aspergillus
ochraceus (ATCC 12066), Aspergillus versicolor (ATCC 11730),
Candida albicans (ATCC 10231), Penicillium funiculosum (ATCC
10509), Penicillium ochrochloron (ATCC 9112) and Trichoderma
viride (IAM 5061). Dilutions of bacterial inocula were cultured on
solid MH medium, while micromycetes were maintained on solid
MA medium. The cultures were subcultured once a month and stored
at +4 °C for further usage (BooTh, 1971).
Microdilution method
For determination of antimicrobial activity of L. ochridanum oil
and extracts, the modified microdilution technique described by
hAneL and rAeTher (1998) was applied. The assay was performed
by sterile 96-well microtiter plates, by adding pure EO or dilutions of
tested extracts (in 5% DMSO) into corresponding medium - TSB and
MA for bacteria and fungi, respectively. To achieve the concentration
of 1.0 × 108 colony forming units (CFU)/mL for bacterial strains,
100 μL of overnight cultures were mixed with 900 μL of medium
in eppendorf. Fungal inocula were prepared by washing spores with
sterile 0.85% saline solution (containing 0.1% Tween 80 (v/v)). The
microbial cell suspensions were adjusted with sterile saline to a
concentration of approximately 1.0 × 106 CFU/mL for bacteria and
1.0 × 105 CFU/mL for fungi in a final volume of 100 μL per well.
The microplates were incubated for 24 h at 37 °C for bacteria and for
72 h at 28 °C for fungi. The lowest concentrations of tested samples
completely inhibiting the growth of used pathogens were defined as
minimum inhibitory concentrations (MICs). The minimum bacte-
ricidal/fungicidal concentrations (MBCs, MFCs) were determined
as the lowest concentrations with no visible growth after serial sub-
cultivation, indicating 99.5% killing of the original inoculums
(hAneL and rAeTher, 1998). In addition, bacterial growth was de-
termined by a colorimetric microbial viability assay, based on reduc-
tion of an 0.2% p-iodonitrotetrazolium violet color (INT) aqueous
solution (I 8377-Sigma Aldrich, St. Louis, MQ, USA) and com-
pared with positive control for each bacterial strain (cLsi, 2009;
TsukATAni et al., 2012). Two replicates were done for each sample.
The solution of synthetic standard streptomycin with concentration
of 1 mg/mL 5% DMSO was used as positive control for bacteria,
while the fluconazole solution (antimicotic diflucan containing 50 mg
fluconazole) at concentration of 2 mg/mL 5% DMSO was included
for fungi. Sterilized distilled water containing 0.02% Tween 80 and
5% DMSO was used as negative control.
Preparation of stock solutions of plant extracts for antimicrobial
tests
Different quantities of stock solutions of L. ochridanum extracts, dis-
solved in 5% DMSO (20 mg/mL) were tested against various patho-
genic microorganisms.
Anti-quorum sensing activity of extracts
Bacterial strains, growth media and culture conditions
In this study, Pseudomonas aeruginosa PAO1 from the Institute for
Biological Research “Siniša Stanković”, Belgrade, Serbia, was used.
Bacteria were routinely grown in Luria-Bertani (LB) medium with
shaking (220 rpm) and cultured at 37 °C.
Biofilm formation
Considering the results obtained in antimicrobial assay and low
yields of isolated EO and ME, further anti-QS analyzes were con-
tinued with ethanolic and aqueous extracts of L. ochridanum. The
samples (0.5, 0.25, 0.125 of MICs, respectively) were tested on bio-
film forming ability on polystyrene flat-bottomed microtitre 96 well
plates as described by spoering and Lewis (2001); drenkArd
and AusuBeL (2002), with some modifications. In brief, 100 μL of
overnight culture of P. aeruginosa (1.0 × 108 CFU/mL) was added
to each well of the plates in the presence of 100 μL subinhibitory
concentrations (subMIC) of L. ochridanum samples (0.5, 0.25 and
0.125 MIC) or 100 mL medium (control). After incubation for 24 h at
37 ºC, each well was washed twice with sterile PBS (pH 7.4), dried,
stained for 10 min with 0.1% crystal violet in order to determine the
biofilm mass. After drying, 200 μL of 95% ethanol (v/v) was added
to solubilise the dye that had stained the biofilm cells. The excess
stain was washed off with distilled water. After 10 min, the content
of the wells was homogenized and the absorbance at λ = 620 nm was
read on a Sunrise™ - TecanELISA reader. The experiment was done
in triplicate and repeated two times and values were presented as a
mean values ± SE.
Twitching and flagella motility
After growth in the presence or absence of subMICs of L. ochri-
danum ethanolic and aqueous extracts, streptomycin and ampicil-
lin, the cells of P. aeruginosa PAO1 were washed twice with sterile
PBS and re-suspended in PBS at 1.0 × 108 CFU/mL (OD of 0.1 at
660 nm). In brief, the cells were stabbed into a nutrient agar plate
with a sterile toothpick and incubated overnight at 37 °C. The plates
were then removed from the incubator and incubated at room tem-
perature for two more days. Colony edges and the zone of moti-
lity were measured with a light microscope (o’TooLe and koLTer,
1998a, b). SubMICs of extracts (0.5 MICs) were mixed into 10 mL
of molten LB medium and poured immediately over the surface of
a solidified LB plate as an overlay. The plate was point inoculated
with an overnight culture of PAO1 once the overlaid agar had solidi-
fied and incubated at 37 °C for 3 days. The extent of swimming was
determined by measuring the area of the colony (sAndY and Foong-
Yee, 2012). The experiment was done in triplicate and repeated two
times. The colony diameters were measured three times in different
direction and values were presented as a mean values ± SE.
Inhibition of synthesis of P. aeruginosa PAO1 pyocyanin
The flask assay was used to quantify the inhibitory activity of the
L. ochridanum against P. aeruginosa pyocyanin production. Over-
night culture of the bacillus PAO1 was diluted to OD600 nm 0.2. Then,
0.5 MICs of tested extracts dissolved in 5% of DMSO (1.25 mg/mL
for EE and 12.50 mg/mL for AE), were added to the bacteria (5 mL)
and incubated at 37 °C for 24 h. The treated culture was extracted
with chloroform (3 mL), followed by mixing the chloroform layer
with 0.2 M HCl (1 mL). Absorbance of the extracted organic layer
was measured at 520 nm using a Shimadzu UV1601 spectrophoto-
meter (Kyoto, Japan) (sAndY and Foong-Yee 2012). The experiment
was done in triplicate and repeated two times. The values were ex-
pressed as ratio (OD520/OD600) × 100.
Statistical analysis
Three samples were used and all the assays were carried out in trip-
licates. The results are expressed as mean values and standard de-
viation (SD). The results were analyzed using one-way analysis of
variance (ANOVA) followed by Tukey’s HSD Test with a = 0.05. This
analysis was carried out using SPSS v. 18.0 program.
Biological activities of Laserpitium ochridanum 333
Results
Essential oil composition
Referring to the results presented in Tab. 1, fifty nine components
were identified in L. ochridanum EO. Monoterpene hydrocarbons
were the most abundant group of compounds (40.87%), followed
by oxygenated sesquiterpenes (24.14%), oxygenated monoterpenes
(15.27%) and sesquiterpene hydrocarbons (13.17%). The dominant
compounds of the EO were sabinene (22.8%), viridiflorol (14.7%) and
α-pinene (11.4%) (Tab. 1).
Total phenolic contents
According to the results obtained for TPC in L. ochridanum extracts
(Tab. 2), phenols were present from 111.28 to 141.30 mg GA/g of dry
extract for methanolic and ethanolic extract, respectively. In tested
extracts, TFC ranged from 21.38 to 67.69 mg QE/of dry extract for
aqueous and ethanolic extract, respectively. In general, greater varia-
tion in tested extracts was recorded in flavonoid contents. The high-
est phenolic and flavonoid concentrations were measured in EE of
L. ochridanum (Tab. 2).
Antioxidant activity
The results of obtained antioxidant activity for L. ochridanim are
listed in Tab. 2. In DPPH test, used extracts exhibited similar anti-
oxidant activity, stronger than BHA, but lower activity compared to
ascorbic acid. Still, the strongest radical scavenging activity was re-
corded for EE (0.113 ± 0.002 mg/mL), which was in accordance with
the highest measured total phenolic and flavonoid contents. EO of
L. ochridanum showed the lowest antioxidant potency compared to
all other samples. Results obtained by the ABTS test showed that
the AE was the most effective agent in concentration of 2.172 ±
0.005 mg ascorbic acid/g of dry extract. According to the obtained
results this sample had slightly lower antioxidant capacity than stan-
dard QE (2.749 ± 0.004 mg ascorbic acid/g of dry extract).
Antimicrobial properties
Antibacterial activity
The results presented in Tab. 3 indicate that L. ochridanum extracts
exhibited moderate antibacterial activity. The EE was the strongest
in bactericidal activity (MBCs = 1.00-5.00 mg/mL), while the lowest
potency had AE (MBCs = 11.00-14.00 mg/mL). Both, methanolic
and ethanolic extracts were more effective compared to streptomy-
cin, but all extracts, including aqueous, showed stronger inhibitory
activity on L. monocytogenes and E. cloacae than used antibiotic.
Tab. 1: Chemical composition of EO of L. ochridanum aerial parts.
Compounds KIE KIL %
α-Thujene 919.1 924 0.32
α-Pinene 924.8 932 11.36
Thuja-2,4(10)-diene 944.9 953 0.17
Sabinene 965.6 969 22.76
β-Pinene 974.7 974 0.80
Myrcene 985.7 988 0.69
n-Octanal 997.2 998 0.39
α-Terpinene 1009.4 1014 1.02
p-Cymene 1018.0 1020 0.35
β-Phellandrene 1020.9 1025 0.96
γ-Terpinene 1051.3 1054 1.93
cis-Sabinene hydrate 1057.3 1065 0.56
n-Octanol 1069.3 1063 3.86
Terpinolene 1080.7 1086 0.51
6-Camphenone 1088.1 1095 0.59
trans-Sabinene hydrate 1097.0 1098 0.29
6-Camphenol 1113.5 1111 0.43
α-Campholenal 1119.3 1122 0.56
trans-Pinocarveol 1132.4 1135 0.85
trans-Sabinol 1135.5 1137 0.40
trans-Verbenol 1139.6 1140 1.79
Terpinen-4-ol 1171.0 1174 3.86
Thuj-3-en-10-al 1178.9 1181 0.31
α-Terpineol 1186.8 1186 0.28
Myrtenal 1188.8 1193 0.37
Myrtenol 1192.1 1194 0.54
Verbenone 1204.0 1204 0.19
Octanyl acetate 1207.5 1211 1.26
Isobornyl acetate 1277.8 1283 0.50
α-Terpinyl acetate 1343.0 1346 0.21
Cyclosativene 1366.2 1369 0.27
α-Copaene 1370.2 1374 1.12
Daucene 1376.8 1380 0.69
β-Cubebene 1381.1 1387 0.23
β-Elemene 1383.4 1389 0.43
(E)-Caryophyllene 1409.0 1417 1.62
trans-α-Bergamotene 1427.0 1432 0.18
α-humulene 1443.5 1452 0.66
(E)-β-Farnesene 1450.2 1454 0.19
cis-Muurola-4(14),5-diene 1460.6 1465 0.20
γ-Himachalene 1467.9 1468 0.81
Dauca-5,8-diene 1471.4 1471 1.96
ar-Curcumene 1475.5 1479 0.19
β-Selinene 1487.0 1489 1.73
cis-Eudesma-6,11-diene 1488.0 1489 0.83
Bicyclogermacrene 1494.9 1500 0.44
β-Bisabolene 1500.6 1505 0.12
δ-Cadinene 1514.3 1522 0.70
α-Calacorene 1546.1 1544 0.80
Spathulenol 1570.3 1577 2.65
Caryophyllene oxide 1573.4 1582 1.16
Viridiflorol 1588.9 1592 14.71
1-epi-Cubenol 1627.5 1627 0.54
β-Cedren-9-one 1631.6 1630 2.44
β-Eudesmol 1649.4 1649 0.44
α-Bisabolol 1679.3 1685 2.20
Chamazulene 1721.9 1730 3.04
Neophytadiene (isomer II) 1829.5 1830 1.02
Incensole acetate 2181.9 2184 0.52
Total 100.00
Number of constituents 59
Monoterpene hydrocarbons 40.87%
Oxygenated monoterpenes 15.27%
Sesquiterpene hydrocarbons 13.17%
Oxygenated sesquiterpenes 24.14%
Others 6.55%
KIE = Kovats (retention) index experimentally determined (AMDIS)
KIL = Kovats (retention) index - literature data (ADAMS, 2007)
334 K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić
B. cereus and S. aureus (MBCs = 1.00-11.00 mg/mL) were the most
sensitive bacteria, while E. coli and M. flavus (MBCs = 5.00->14.00
mg/mL) proved to be the most resistant strains.
Antifungal activity
The results obtained for the antifungal activity of investigated
samples are presented in Tab. 4. The EO of this species had the
strongest activity in inhibition of micromycetes growth (MFCs =
0.55-2.20 mg/mL) and it was similar to the activity of applied fluco-
nazole (MFCs = 0.03-1.50 mg/mL). Among the investigated extracts,
the EE showed the highest activity (MFCs = 5.00 mg/mL) on all used
fungal strains, exept for A. niger (MFCs =18.00 mg/mL) (Tab. 4). The
most resistant micromycetes were A. niger and A. fumigatus, while
the most sensitive strains were A. versicolor, P. ochrochloron and
P. funiculosum. Fungi T. viride and P. ochrocloron were more sensi-
tive to L. ochridanum oil (MFC = 1.10 mg/mL), than to fluconazole
(MFC = 1.50 mg/mL) (Tab. 4).
Standards / / QE 2.75 ± 0.004a
Tab. 2: TPC, TFC and antioxidant activity of L. ochridanum extracts and EO (means ± SD).
L. ochridanum extracts/ Total phenolic contents Antioxidant activity
TPC 1 mg/mL TFC 1 mg/mL DPPH ABTS 1 mg/mL
(mg GA/g of DE) (mg QE/g of DE) (IC50 = mg/mL) (mg ascorbic acid/g of DE)
ME 111.28 ± 0.005c 31.31 ± 0.010b 0.12 ± 0.011b 1.63 ± 0.009b
EE 141.30 ± 0.013a 67.69 ± 0.018a 0.11 ± 0.002b 1.56 ± 0.004b
AE 125.30 ± 0.010b 21.38 ± 0.004c 0.12 ± 0.000b 2.17 ± 0.005a
EO / / 1.88 ± 0.009c /
BHA 0.13 ±0.012b
ascorbic acid 0.03 ± 0.008a
Indicated letters mean significant difference (p < 0.05)
EO
Tab. 3: Antibacterial activity of L. ochridanum extracts in mg/mL (means ± SD).
ME EE AE Streptomycin
MIC MBC MIC MBC MIC MBC MIC MBC
B. cereus 0.40 ± 0.06a 1.00 ± 0.03a 0.50 ± 0.02a 1.00 ± 0.00a 6.00 ± 0.02a 11.00 ± 0.01a 1.50 ± 0.01a 2.50 ± 0.06a
M. flavus 4.00 ± 0.02c 5.00 ± 0.02b 2.00 ± 0.01ab 5.00 ± 0.00b 10.00 ± 0.01b >14.00 ± 0.03b 2.50 ± 0.00a 5.00 ± 0.00b
L. monocytogenes 3.00 ± 0.04c 4.00 ± 0.07b 0.50 ± 0.00a 2.00 ± 0.10a 5.00 ± 0.02a 11.00 ± 0.01a 15.00 ± 0.10c 20.00 ± 0.02c
P. aeruginosa 3.00 ± 0.00c 4.00 ± 0.05b 0.50 ± 0.02a 2.00 ± 0.01a 5.00 ± 0.00a 11.00 ± 0.02a 2.50 ± 0.07a 5.00 ± 0.01b
E. coli 4.00 ± 0.01c 8.00 ± 0.03c 4.00 ± 0.02b 5.00 ± 0.01b 10.00 ± 0.00b >14.00 ± 0.01b 2.50 ± 0.04a 5.00 ± 0.03b
E. cloacae 2.00 ± 0.01b 4.00 ± 0.01b 1.00 ± 0.10a 4.00 ± 0.02b 5.00 ± 0.10a 11.00 ± 0.01a 10.00 ± 0.02b 20.00 ± 0.00c
S. typhymurium 2.00 ± 0.00b 3.00 ± 0.05b 1.00 ± 0.06a 2.00 ± 0·05a 10.00 ± 0.01b >14.00 ± 0.02b 2.50 ± 0.01a 5.00 ± 0.01b
S. aureus 0.40 ± 0.10a 1.00 ± 0.07b 1.00 ± 0.05a 2.00 ± 0.03a 5.00 ± 0.07a 11.00 ± 0.00a 2.50 ± 0.05a 5.00 ± 0.00b
Indicated letters mean significant difference (p < 0.05)
L. ochridanum/
Bacteria
L. ochridanum/
Fungi
Tab. 4: Antifungal activity of L. ochridanum extracts and EO in mg/mL (means ± SD).
ME EE AE EO Fluconazole
MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC
C. albicans 8.00 ± 0.02c 12.00 ± 0.01b 6.00 ± 0.10c 12.00 ± 0.05b 12.00 ± 0.01b 14.00 ± 0.03a 0.55 ± 0.00b 1.10 ± 0.02b 0.02 ± 0.05a 0.03 ± 0.03a
T. viride 3.00 ± 0.00a 8.00 ± 0.02a 5.00 ± 0.05b 14.00 ± 0.07b 10.00 ± 0.01a 12.00 ± 0.02a 0.55 ± 0.07b 1.10± 0.03b 1.00 ± 0.02d 1.50± 0.02d
P. ochrochloron 6.00 ± 0.03b 10.00 ±0.10a 4.00 ± 0.02a 5.00 ± 0.05a 10.00 ± 0.00a 14.00 ± 0.00a 0.28 ± 0.05a 1.10 ± 0.01b 1.00 ± 0.00d 1.50 ± 0.02d
P. funiculosum 3.00 ± 0.01a 8.00 ± 0.05a 4.00 ± 0.00a 5.00 ± 0.00a 12.00 ± 0.07b 14.00 ± 0.02a 0.55 ± 0.05b 2.20 ± 0.01c 0.25 ± 0.01b 0.50 ± 0.00b
A. fumigatus 6.00 ± 0.00b 14.00 ± 0.01b 6.00 ± 0.10c 14.00 ± 0.20b 12.00 ± 0.10b 14.00 ± 0.02a 0.55 ± 0.10b 2.20 ± 0.07c 0.50 ± 0.02c 1.00 ± 0.00c
A. versicolor 6.00 ± 0.03b 8.00 ± 0.20a 4.00 ± 0.01a 5.00 ± 0.03a 10.00 ± 0.20a 12.00 ± 0.07a 0.28 ± 0.02a 0.55± 0.00a 0.13 ± 0.00a 0.50 ± 0.01b
A. ochraceus 6.00 ± 0.02b 19.00 ± 0.03c 4.00 ± 0.03a 5.00 ± 0.02a 10.00 ± 0.00a 14.00 ± 0.05a 0.55 ± 0.10b 1.10 ± 0.02b 0.50 ± 0.05c 1.00 ± 0.03c
A. niger 8·00 ± 0.01c 19.00 ± 0.05c 6.00 ± 0.00c 18.00 ± 0.05c 12.00 ± 0.01b 18.00 ±0.10b 1.10 ±0.07c 2.20 ± 0.00c 0.25 ± 0.03b 1.00 ± 0.10c
Indicated letters mean significant difference (p < 0.05)
Biological activities of Laserpitium ochridanum 335
Anti-QS activity evaluation
Biofilm formation
Tab. 5 presents the effects of L. ochridanum extracts on P. aerugi-
nosa PAO1 biofilm formation. The samples were tested at 0.5, 0.25
and 0.125 of MIC values. L. ochridanum extracts showed significant
difference in terms of anti-biofilm formation activity. L. ochrida-
num EE showed dose dependant inhibitory activity, reducing from
8.63% to 63.88% of biofilm formation, where the best result was ob-
tained in the presence of 0.5 MIC of the extract. Results revealed
that L. ochridanum EE reduced biofilm formation more effectively
than both antibiotics allowing formation of PAO1 in the range from
36.12% to 91.37%. Contrary, the tested subMIC concentrations of
AE did not show any suppression of P. aeruginosa biofilm forma-
tion (Tab. 5). In the presence of commercial antibiotics streptomycin
and ampicillin, biofilm formation occurred in narrower range, with
slightly stronger biofilm inhibition recorded for streptomycin.
Twitching and flagella motility
In addition, the ethanolic and aqueous extracts of L. ochridanum
reduced the twitching and flagella motility activity of P. aeruginosa
(Tab. 6 and Fig. 1). As presented in Tab. 6, the color of the colo-
ny ranged from white, through light green to green. Used extracts
changed the color and diameter of treated colonies to a certain ex-
tent. In the presence of extracts, colonies where white and larger
(14.00 mm and 17.67 mm for EE and AE, respectively) in compari-
son to colonies treated with antibiotics (Tab. 6). The green colony of
P. aeruginosa with streptomycin, had minimal growth (11.00 mm)
and completely reduces protrusions. Also, the most reduced flagella
in size, shape and number were in colony with streptomycin (Fig. 1).
Inhibition of synthesis of P. aeruginosa PAO1 pyocyanin
SubMICs of L. ochridanum samples were tested for inhibition of
P. aeruginosa pigment production and both extracts showed sub-
stantial activity in pigment synthesis inhibition. The affection was
observed by the reduction of the green pigmentation of the samples,
Tab. 5: Effects of L. ochridanum extracts on biofilm formation of P. aeru-
ginosa PAO1 (%).
Biofilm formation*
0.5 MIC 0.25 MIC 0.125 MIC
(% ± SE) (%± SE) (% ± SE)
EE 36.12 ± 1.73 60.82 ± 1.05 91.37 ± 0.42
AE n.d. n.d. n.d.
Ampicillin 69.16 ± 0.65 56.46 ± 0.46 92.16 ± 0.37
Streptomycin 49.40 ± 0.46 70.97 ± 0.36 88.36 ± 0.42
*Biofilm formation values were calculated as: ((mean A620 control well-
mean A620 treated well)/mean A620 control well) × 100.
− Values are expressed as means ± SE.
− n.d. not determinate
L. ochri-
danum/
Standards
Tab. 6: Effects of L. ochridanum extracts on twitching and flagella motility of P. aeruginosa (PAO1).
L. ochridanum extracts/ Colony diameter Flagella diameter Colony colour Colony edge
Standards (mm ± SE) (μm)
EE 14.00 ± 2.65 40-160 White Rare flagella
AE 17.67 ± 5.51 16-80 White Tiny flagella
Streptomycin 11.00 ± 1.00 24-56 Green Tiny flagella
Ampicillin 13.33 ± 5.03 16-56 Green Regular flagella
P. aeruginosa (PAO1) 12.00 ± 1.00 56-80 Light green Regular flagella
Fig. 1: Light microscopy of colony edges of P. aeruginosa in twitching motility, grown in the presence or absence of L. ochridanum extracts and commercial
antibiotics. The colonies from the bacteria grown with extracts in concentration of 0.5 MIC (A-B). The colony with EE was with moderately reduced
protrusions (A); In the presence of AE colony formed almost regular protrusions (B); P. aeruginosa colony in the presence of streptomycin (0.5 MIC)
with reduced protrusion (C); P. aeruginosa colony in the presence of ampicillin with regularly formed protrusions (D); P. aeruginosa produced a flat,
widely spread, irregularly shaped colony in the absence of extracts and commercial antibiotics (E); Magnification: (A-D) × 100.
336 K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić
compared to the coloration of the control PAO1 sample (Fig. 2). The
strongest inhibition of pigment’s production was detected for L.
ochridanum AE. In the presence of tested concentrations of extracts,
pyocyanin was less produced (23.46% and 18.07% for ethanolic and
aqueous extracts, respectively), than by control strain (141.55%). All
extracts were better in prevention of pigment production regarding to
applied antibiotics (Fig. 2).
screening results indicated that EE and AE samples showed some
potential of anti-QS activity. L. ochridanum EE demonstrated an
inhibition at the initial stage of biofilm formation in the manner of
different tested concentrations. That is of great importance since
bacteria form biofilms as a protection against host’s immune sys-
tem and as a factor of antibiotic resistance (Soković et al., 2014). It
was clearly indicated that tested concentrations of L. ochridanum
extracts were more effective on P. aeruginosa pigment production
than those of applied antibiotics. This green, toxic pigment acts as a
virulence factor in bacteria, so reduction of its production is crucial
for increasing the effectiveness of host defense. While AE notably
demonstrated the best activity in suppression of pyocyanin synthe-
sis, it did not show any reduction of colony formation at the tested
subMICs. Opposite results obtained for AE in these two assays could
be associated with possible different mechanisms responsible for its
activity. At this point, there is no sufficient data to highlight the ex-
act method of QS inhibition. A few potential modes of action have
been proposed, many of them to interfere with the QS system such as
inhibition of biosynthesis of auto-inducer molecules, inactivation or
degradation of the auto-inducer, interference with the signal receptor
and inhibition of the genetic regulation system. Due to complex phy-
tochemistry of plant extracts, different compounds could be associ-
ated with specific effects linked to the QS system, so the difference
in the activities of L. ochridanum aqueous sample suggest less polar
nature of L. ochridanum active compounds in reduction of P. aerugi-
nosa biofilm formation. The importance of plant extracts preparation
should be also considered (Adonizio et al., 2006; Adonizio, 2008:
Glamočlija et al., 2015). In the research of Adonizio et al. (2008),
where aqueous extract of Schefflera actinophylla, with no anti-QS
activity, was used as a negative control, it was concluded that plant
extracts differentially affect biofilm formation. Inhibition of swar-
ming and twitching motility of PAO1 by L. ochridanum samples was
achieved in moderate extent with better results obtained for EE. Both
types of motilities are important in the initial stages of biofilm for-
mation of P. aeruginosa (o’TooLe and koLTer, 1998b).
Conclusions
This study established the chemical characterization of L. ochrida-
num EO and provided new data concerning antioxidant and anti-QS
activities of crude extracts/EO. The presence of phenolic compounds
and previously reported sesquiterpene lactones mostly contribute to
biological potency of L. ochridanum extracts. Among all extracts,
EE possessed the best potency in evaluated antioxidant and anti-
bacterial activities. The EO revealed strong antifungal potential.
L. ochridanum showed promising anti-QS effectiveness that was
sufficient for the reduction of biofilm formation and pyocyanin
production. To establish the application of this species in various
pharmaceutical, dietary or alternative medicine branches, further
research is needed especially concerning that the exact mechanistic
interactions with the QS system should be resolved.
Acknowledgements
The authors are grateful to the Ministry of Education, Science and
Technological Development of the Republic of Serbia for financial
support Grants No. 173029 and 173032.
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0
20
40
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Address of corresponding author:
Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology,
University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
E-mail: ksenija.mileski@bio.bg.ac.rs