plik

Central European Journal of

Biology

* E-mail:monika.grzywacz@umlub.pl

Research Article

Department of Toxicology, Medical University of Lublin,

20-093 Lublin, Poland

Department of Pharmaceutical Botany, Medical University of Lublin,

20-093 Lublin, Poland

Department of Pharmaceutical Microbiology, Medical University of Lublin,

20-093 Lublin, Poland

Department of Virology and Immunology,

Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University,

20-033 Lublin, Poland

Department of Medical Biology, Institute of Agricultural Medicine,

20-950 Lublin, Poland

Monika Gawrońska-Grzywacz

*, Tadeusz Krzaczek

, Renata Nowak

Renata Los

, Anna Malm

, Małgorzata Cyranka

, Wojciech Rzeski

4,5

Biological activity of new flavonoid

from

Hieracium pilosella

1. Introduction

Medicinal plants are among the rich sources of beneficial
compounds exhibiting a variety of biological activities.
Polyphenolic constituents, especially flavonoids, are
still of special interest for many scientists because
they are known from antioxidant and free radical
scavenging activities. Reactive oxygen species are

involved in many processes damaging proteins, lipids
and even nucleic acids. They play an important role
in the etiology of various diseases, so the free radical
scavengers isolated from plants may be preventive
agents against cancer, inflammation, cardiovascular
and neurodegenerative disorders [

]. The search of

new antimicrobial and antiproliferative agents against
human tumor cells among the wide variety of naturally

397

Cent. Eur. J. Biol.• 6(3) • 2011 • 397-404

DOI: 10.2478/s11535-011-0017-9

Received 29 July 2010; Accepted 04 February 2011

Keywords:

Hieracium pilosella L. • Asteraceae • Isoetin 4’-O-β-D-glucopyranoside • Antiradical activity • DPPH radical • Antibacterial activity

Pseudomonas aeruginosa • Antiproliferative activity • HT-29 cell culture • MTT method

Abstract:

Hieracium pilosella L. (Asteraceae) is a well-known plant used in ethno-medicine as its inflorescences are particularly rich in

beneficial polyphenolics. This research aimed to elucidate the structure of a new flavone glycoside isolated from the inflorescences

of Hieracium pilosella and evaluate its antioxidant, antimicrobial and antiproliferative activities. The chromatographic methods were

successfully applied to isolate the new flavonoid. Its structure was determined by subsequent UV, NMR and MS experiments

and identified as isoetin 4’-O-β-D-glucopyranoside. Free radical scavenging capacity was examined by measuring the scavenging

activity of the new isoetin derivative on 2,2-diphenyl-1-picrylhydrazyl (DPPH). The compound was also screened for spectrum of

antimicrobial activity using the agar well diffusion method. Minimum inhibitory concentration (MIC) for Pseudomonas aeruginosa

ATCC 9027 was performed by the micro-dilution broth method. The antiproliferative effect of tested glycoside was assessed in two

human tumor cell lines derived from lung (A549) and colon (HT-29) carcinoma and cell proliferation was determined by means of

MTT method. The tested compound showed high antiradical activity, reducing the DPPH∙ with EC

7.9 µM (3.7 µg/ml) and exhibited

narrow antimicrobial spectrum among tested microorganisms. The compound was active against Pseudomonas aeruginosa ATCC

9027 (MIC 125 µg/ml) which is prone to causing infections that are difficult to treat due to it developing extremely rapid antibiotic

resistance. In the antiproliferative studies, cell proliferation of the colon (HT-29) carcinoma cell line was significantly decreased

after exposure to the compound. The results indicate that isoetin 4’-O-β-D-glucopyranoside possesses antioxidant capacity and

very promising antibacterial activity and could have uses as an effective antipseudomonal agent as well a antiproliferative agent.

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Biological activity of new flavonoid from

Hieracium pilosella L.

occurring substances is also a very important part of
scientific research.

Hieracium pilosella L. (mouse-ear hawkweed,

hawkweed)  is a common and very invasive weed
belonging to the family Asteraceae. It is a perennial
plant with a basal rosette of leaves which undersides
are  covered  with  whitish  hairs.  The  flowering  stem  is
generally up to 30 cm long. It carries solitary pale lemon-
yellow  coloured  flowerhead  with  the  outermost  ligules
having a reddish underside. Mouse-ear hawkweed is
a colony-forming plant. It is native to Eurasia but has
been also introduced into North America and into New
Zealand. The plant favours dry, sunny, sandy and less
fertile areas [

]. In Poland mouse-ear hawkweed grows

widely on dry meadows and sunny slopes of hills.
Herba Pilosellae is well-known in European ethno-
medicine and used to treat inflammations of the urinary
tract and also skin diseases because of its diuretic,
astringent, antiseptic and antiphlogistic activity [

Hawkweed can be also employed in the phytotherapy
of hypertension, obesity and cellulitis because of its
strong diuretic properties [

]. This medicinal plant is

not reported in the European Pharmacopoeia, however
there is a monograph in the French Pharmacopoeia. The
preparations of the herb of mouse-ear hawkweed are
known and used for centuries in Polish folk medicine
to treat cystitis, nephritis, urolithiasis as well as cough
and respiratory tract inflammations and associated
bleedings. Through external application the extracts of
the plant are used to bathe septic wounds [

The antioxidant capacity of extracts of H. pilosella

has been reported by Stanojević et al. [

]. In recent

years extracts of hawkweed were also examined for
potential activities against bacteria, fungi and even
viruses, e.g. HIV [

The inflorescences of H. pilosella are especially

rich in phenolics, mainly flavonoids and also phenolic
acids [

]. Many therapeutic values e.g. diuretic and

antiseptic properties of the examined plant may be surely
attributed to them. On the other hand hawkweed contains
many phenolic compounds with phytotoxic properties
which are responsible for its strong allelopathic potential.
Until now eleven different glycosides of isoetin (hieracin,
rare 5,7,2’,4’,5’-pentahydroxyflavone) have been
identified in species mainly from the Asteraceae family,
including representatives of Hieracium genus [

Two of them appeared to be good radical scavengers
and another derivative (isoetin 5’-methyl ether) inhibited
the proliferation of tumor cell cultures – human lung
cancer cell line A549, human melanoma SK-Mel-2 and
mouse melanoma B16F1 cell lines [

Our previous work resulted in the isolation and

structure elucidation of nine flavonoid compounds from

the aerial parts of Hieracium pilosella L. [

]. In course

of the continuing studies on the phenolic constituens of
this plant, here we report the separation, identification
and bioactivity of a new glycoside of isoetin obtained
from the methanol extract of hawkweed’s inflorescences.

2. Experimental Procedures

2.1 Plant material

The inflorescences of Hieracium pilosella L., as
authenticated by Prof. T. Krzaczek, Department
of Pharmaceutical Botany of Medical University of
Lublin, were collected in Ćmiłów near Lublin (south-
eastern Poland) in June 2007. A voucher specimen
of the collected plant (No. 1976) was deposited in the
herbarium of the Department of Pharmaceutical Botany,
Faculty of Pharmacy, Medical University of Lublin,
Poland.

2.2 Extraction and isolation

The air-dried plant material (1 kg) was exhaustively
extracted with boiling 80% methanol. The obtained extract
was concentrated under reduced pressure providing a
residue (254.15 g) to preliminary column chromatography
(CC). CC was completed on polyamide with methanol
– water gradient solvent system (0-100%, v/v) and 149
fractions (100 ml each) were collected. The fraction
which showed potent antiradical effect (in DPPH method)
was fractioned by CC on polyamide with ethyl acetate
– methanol – water (18:5:2, v/v) mobile phase and 130
fractions (100 ml each) were collected. Next, fractions
1-11 were mixed after TLC control and preparative paper
chromatography on Whatman 3CHR sheets, 460 x 460
mm (upward technique development) with TBA (t-butanol
– acetic acid – water, 3:1:1 v/v) as mobile phase was
applied to yield a new glycoside (28 mg).

2.3 Instrumentation and spectroscopic

conditions

Melting point (m.p.) was determined on Boetius
heating-stage microscope (Franz Küstner Nachf.
KG, Dresden HMK, Germany) and was uncorrected.
The UV spectra were recorded on a Hitachi U-2001
UV-spectrophotometer (Hitachi Ltd., Tokyo, Japan)
using standard procedures [

]. The

H NMR,

NMR, HMBC spectra were registered in DMSO-d

spectroscope Bruker Avance DRX500 (500.13 MHz
for

H NMR and 125.75 MHz for

C NMR). Negative

and positive Liquid Secondary-Ion Mass Spectrometry
(LSI MS, Cs+, 13 keV) were performed in glycerine on
a Finnigan MAT 95 (Finnigan MAT GmbH, Bremen,
Germany) spectrometer. The obtained flavonoid was

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et al.

analyzed by RP-HPLC to verify the purity. The HPLC
system (Knauer, Berlin, Germany) consisting of a HPLC
Pump K-1001, Solvent Organizer K-1500, UV-VIS
Detector Fast Scanning Spectrophotometer K-2600,
Degasser K-5004, Column Thermostat, 20 ml sample
injector (Rheodyne, Cotati, CA, USA), column Hypersil
ODS (particle size 5 µm, 200 mm x 4.6 mm I.D., Merck,
Germany) and gradient elution with acetonitrile - water
(5:45, v/v, 30 min), the flow rate 1ml/min and detection
at 254 nm and 360 nm were used.

Analytical TLC (co-chromatography with authentic

standard, LGC Promochem, ChromaDex, Inc. USA) of
a sugar liberated after acid hydrolysis of a new flavonoid
was performed on cellulose plates (Merck, Germany)
using pyridine – ethyl acetate – acetic acid – water
(5:5:1:3, v/v/v/v) as mobile phase and also on silica
gel 60 F

254

plates (Merck, Germany) using n-propanol

– ethyl acetate – water (7:2:1, v/v/v). Aniline phthalate
spray reagent revealed the nature of the sugar.

2.4 Antioxidant assay

The free radical scavenging activity of the isolated
compound was estimated according to the procedure
described by Brand-Williams, Cuvelier & Berset (1995)
with some modifications [

]. Ethanol of analytical

grade (POCH, Poland), 1,1-diphenyl-2-picrylhydrazyl
radical - DPPH

(Aldrich, Steinheim, Germany), quercetin

and trolox (both from Sigma, Steinheim, Germany)
were used in the assay. Briefly, the tested compound,
at concentrations ranging from 25 to 250 µg/ml (50 µl
in ethanol), was mixed with 1.95 ml DPPH

solution

(6 x 10

-5

M in ethanol). The absorbance was measured at

517 nm after incubating the mixture at room temperature
for 30 min in the dark. The antiradical activity was
expressed as EC

(antiradical dose required to cause

a 50% inhibition), which was calculated by the following
formula: [(A

blank

− A

sample

)/A

sample

] × 100, where A

blank

the absorbance of the DPPH radical solution and A

sample

is the absorbance of the DPPH radical solution after
the addition of the sample [

]. EC

was obtained by

linear regression analysis of the dose-response curve,
plotted between % inhibition and concentration (µg/
ml). A Hitachi U-2001 UV-spectrophotometer (Hitachi
Ltd., Tokyo, Japan) was used. Trolox and quercetin -
standards known for strong antioxidant activity, were
used as positive control. A minimum of five different
concentrations for compound were tested in triplicate
analyses.

2.5 Antimicrobial assay

Agar well diffusion method was used to screen for
spectrum of antimicrobial activity of a new flavone
glycoside. Twelve reference microbial strains were used,

six Gram-positive bacteria (Staphylococcus epidermidis
ATCC 12228, Staphylococcus aureus ATCC 25923,
Staphylococcus aureus ATCC 6538, Bacillus cereus
ATCC 10876, Bacillus subtilis ATCC 6633, Micrococcus
luteus ATCC 10240), four Gram-negative bacteria
(Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 13883, Pseudomonas aeruginosa ATCC 9027,
Proteus mirabilis ATCC 12453) and two yeasts (Candida
albicans ATCC 10231, Candida parapsilosis ATCC
22091). The surface of Mueller-Hinton agar and Mueller-
Hinton agar supplemented with 2% glucose were
inoculated with bacterial or yeast inocula (0.5 McFarland
standard scale - approximately 150 x 10

CFU/ml),

respectively. Next, 40 µl of tested compound (dissolved
in DMSO - 1 mg/ml) was dropped into wells (d=6 mm)
on the agar media. The agar plates were preincubated
at room temperature for 1 h, next they were incubated
at 37

C for 24 h and 25

C for 48 h for bacteria and

yeasts, respectively. After the incubation period, the
zones of growth inhibition were measured and recorded
(subtracting the diameter of the well) and average values
were calculated. The well containing DMSO without test
compound was used as negative control. Gentamicin
and fluconazole were used in order to control the
sensitivity of the bacteria and fungi tested, respectively.

Minimum inhibitory concentration (MIC) of the new

flavonoid was examined for P. aeruginosa ATCC 9027 by
the micro-dilution broth method, using two-fold dilutions
of compound in Mueller-Hinton broth (MHB) prepared
in 96-well polystyrene plates. Final concentrations of
the compound ranged from 15.6 to 500 µg/ml. Bacterial
inoculum was added to all wells to obtain final optical
density corresponding to 5 x 10

CFU/ml. After incubation

at 35

C for 24 h, the MIC was assessed visually as

the lowest flavonoid concentration showing complete
bacterial growth inhibition. Appropriate DMSO, growth
and sterile controls were carried out.

2.6 Antiproliferative assay

The cell lines: human caucasian lung carcinoma (A549)
and human colon adenocarcinoma (HT 29) were obtained
from the European Collection of Cell Cultures (Centre
for Applied Microbiology and Research, Salisbury, UK).
The following culture media, purchased from Sigma
(Sigma Chemicals, St. Louis, MO, USA), were applied:
1:1 mixture of DMEM and Nutrient mixture F-12 Ham
(HT-29), 3:1 mixture of DEMEM and Nutrient mixture
Ham’s F-12 (A549). All media were supplemented
with 10% foetal bovine serum (FBS, Sigma), penicillin
(100 U/ml) (Sigma) and streptomycin (100 µg/ml)
(Sigma). The cultures were kept at 37

C in a humidified

atmosphere of 95% air and 5% CO

. Cell proliferation

was assessed by means of MTT method as previously

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Biological activity of new flavonoid from

Hieracium pilosella L.

described [

]. Tumor cells were placed on 96-well

microplates (Nunc, Roskilde, Denmark) at a density of
1 x 10

(A549) and 3 x 10

(HT-29). Next day, the culture

medium was removed and the cells were exposed to
serial dilutions of tested compound in a fresh medium.
Cell proliferation was assessed after 96 h by using
the MTT method (Cell proliferation kit I, Boehringer
Mannheim, Germany) in which the yellow tetrazolium salt
(MTT) is metabolized by viable cells to purple formazan
crystals. Tumor cells were incubated for 3 h with MTT
solution (5 mg/ml). Formazan crystals were solubilised
overnight in SDS buffer (10% SDS in 0.01 N HCl), and
the product was quantified spectrophotometrically by
measuring absorbance at 570 nm wavelength using
an E-max Microplate Reader (Molecular Devices
Corporation, Menlo Park, CA, USA).

2.7 Statistical analysis

The reported value for each sample was calculated
as the mean of three measurements. Correlation
coefficients (R) and coefficients of determination (R

)

were calculated using Microsoft Excel 2000. Results of
antiproliferative studies are expressed as percentage of
control and mean values ± SEM of 5-6 trials.

3. Results and Discussion

3.1 Structure elucidation

The structure of the isolated compound was established
by physical, chemical and spectroscopic methods. The
compound had a melting point of 260-261ºC (yellow-
orange powder, crystallized from methanol). Mass
spectra showed quasimolecular ion peaks [M + H]

m/z 465 and [M – H]

at m/z 463 and also other important

ions at m/z 303 [aglycone + H]

and 301 [aglycone – H]

Acid hydrolysis (2M HCl at 100°C for 1 h) yielded glucose
identified by TLC and aglycone - isoetin, determined by
comparing the obtained UV and

H NMR data with that

in the literature [

The following data for isolated compound

were obtained: UV λ

max

MeOH

nm: 262, 368;

(+ NaOMe): 267, 336, 424; (+ AlCl

): 271, 293, 324, 404;

(+ AlCl

+ HCl): 270, 292, 323, 402; (+ NaOAc): 263, 371;

(+ NaOAc + H

): 268, 309, 368. The comparison of

the neutral spectra of isoetin glycosides [

] showed

the glycosylation in the B-ring led to a hypsochromic shift
to shorter wavelengths in band I in relation to aglycone
spectrum suggesting a flavone which is glycosylated in
the B-ring. In the spectrum after the addition of NaOAc,
the hypsochromic shift in relation to aglycone spectrum
was observed and pointed out the B-ring glycosylation
as well

[

]. There was neither a bathochromic effect

in the NaOAc/H

spectrum nor a hypsochromic effect

in the AlCl

/HCl spectrum (towards the AlCl

spectrum)

because free 4’,5’ (ortho)-dihydroxy groups were lacking
[

]. These above-mentioned effects were obviously

present in aglycone spectra. Addition of NaOMe gave
a bathochromic shift due to a free 2’-OH group [

Furthermore, the

H NMR spectrum showed doublets at

δ 6.17 (H-6) with J=1.7 Hz and 6.43 (H-8) with J=1.8 Hz,
singlets at δ 7.05 (H-3), 6.79 (H-3’) and 7.32 (H-6’). By
means of

H-

C HMBC three further singlet signals at δ

12.99, 10.81, 10.35 and 8.48 were assigned to protons
from the following hydroxyl groups: 5-OH, 7-OH, 2’-OH
and 5’-OH. The compound showed the pyranose form
and β-configuration of glucose (the signal at δ 4.78, d,
J=6.5 Hz, H-1”). In addition, only the H-3’ signal was
apparently shifted downfield compared with one in the
aglycone spectrum suggesting this proton located at
the ortho position of the glycosylated hydroxyl group
and the HMBC analysis proved that 2’-OH group was
surely free. These observations were further supported
by HMBC correlation between δ 4.78 (H-1”) and δ 148.9
(C-4’). All the NMR data are given in Table

In the results of obtained data, the isolated compound

was identified as isoetin 4’-O-β-D-glucopyranoside, a
new derivative of this rare flavone (Figure

). This is

the first report of finding this compound in the nature
and in the examined species. Therefore, the presence
of flavonoids in this plant is in accord with its traditional
uses as a diuretic and anti-inflammatory drug [

3.2 Biological activities

The free radical scavengers in plants have received a
great amount of attention as being primary preventive
ingredients against various diseases such as cancer,
inflammation or cardiovascular disorders [

]. Recent

studies have proved that the antioxidant properties (to
remove free radicals)

of medicinal plants are due to

their phenolic compounds, such as flavonoids, phenolic
acids, and tannins [

]. The DPPH (1,1-diphenyl-2-

picrylhydrazyl) method is one of the most frequently
used for testing the antiradical activity of plant extracts
and compounds [

Figure 1.

The structure of isoetin 4’-O-β-D-glucopyranoside.

5’

2’

4’

1’’

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et al.

There is only one report in the literature about the

high radical scavenging effect of methanol extract
of H. pilosella [

]. In our preliminary TLC DPPH

experiments of flavonoids isolated from H. pilosella
(not shown), the tested compound exhibited a
high activity. Thus, antioxidant capacity of isoetin
4’-O-β-D-glucopyranoside was evaluated through
colorimetric scavenging activity assay against DPPH
radical with trolox and quercetin as positive controls
(EC

3.2 µg/ml, 1.34 µg/ml and 12.9 µM, 3.98 µM,

respectively). This new isoetin glycoside showed
high scavenging activity with an EC

value of 7.9 µM

(3.7 µg/ml) and exhibited a much stronger scavenging
activity than the recently identified

and so far the

only tested 2’,7-substituted isoetin glycosides from
Taraxacum mongolicum [

]. Its anti-oxidative activity

is comparable to trolox and only three times weaker
than quercetin, a strong antioxidant (Table

). Other

investigators reported similar results for quercetin and
trolox and weaker for the above mentioned derivatives

Table 1.

The

H and

C NMR data of isoetin 4’-O-β-D-glucopyranoside in DMSO-d

a,b

δ values in ppm, J values in Hz

Assignments were established by HMBC experiment

Compound

Correlation equation

[µg/ml]

[µM]

Isoetin 4’-O-β-D-glucopyranoside (IG)

464

y=5925.7x+28.17

0.9986

3.7±0.35

7.9±0.68

Trolox (T)

250.3

y=14792x+ 2.3168

0.9968

3.2±0.2

12.9±0.76

Quercetin (Q)

338.3

y=30641.2x+8.7886

0.9987

1.34±0.05

3.98±0.32

Table 2.

The scavenging effect of isoetin 4’-O-β-D-glucopyranoside on DPPH radical

Mean value ± SD, n=3; EC

IG/EC

T=1.16; EC

IG/ EC

Q=2.74

Position

δ C

δ H

H-

C correlations (HMBC)

161.4

H-3, H-6’

107.8

7.05 (1H, s)

181.8

H-3

161.1

5-OH, H-6

98.6

6.17 (1H, d, J=1.7)

5-OH, H-8

164.1

H-6, H-8

93.7

6.43 (1H, d, J=1.8)

H-6

157.3

H-8

103.6

H-3, H-6, H-8, 5-OH

1’

110.0

H-3, H-3’, H-6’, 2’-OH

2’

150.9

H-3’, H-6’

3’

104.6

6.79 (1H, s)

4’

148.9

H-3’, H-6’, 5’-OH, H-1”

5’

139.6

H-3’, H-6’, 5’-OH

6’

113.7

7.32 (1H, s)

5’-OH

1”

101.2

4.78 (1H, d, J=6.5)

2”

73.1

3”

75.9

3.26-3.40 (1H, m)

4”

69.2

5”

77.1

6”

60.3

3.72 (1H, dd, J=11.4, 4.8, H-6a”)

3.57 (1H, m, H-6b”)

5-OH

12.99 (1H, s)

7-OH

10.81 (1H, br s)

2’-OH

10.35 (1H, br s)

5’-OH

8.48 (1H, s)

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of isoetin [

]. The potency of a molecule for scavenging

free radicals

is due to the number of hydrogens available

for donation by the hydroxyl groups. Flavonoids bearing
free hydroxyl groups are known to be good radical
scavengers, an assumption that is particularly true for
the flavonoid B-ring. The presence of a catechol moiety
in this ring is the main factor [

]. However, stronger

antioxidant activity of isoetin 4’-O-β-D-glucopyranoside
than 2’,7-substituted isoetin glycosides isolated from
T. mongolicum may indicate the significance of free
2’-hydroxyl group in antioxidant capacity of isoetin
derivatives. The glycosyl groups also do not contribute
effectively to radical scavenging [

]. The differences

in the structure may explain the stronger antioxidant
activity of this new isoetin glycoside than other recently
described isoetin derivatives [

A variety of techniques have been reported for

determining the antimicrobial activity of plant materials.
The most frequently used techniques include the
agar well diffusion method and the dilution method
based on incorporation of plant samples in the media
prior to inoculation. These methods and the panel
of standard bacterial and yeast strains are routinely
used as a preliminary check and help to select
efficient plant materials. In the current study, isoetin
4’-O-β-D-glucopyranoside was first screened for
spectrum of antimicrobial activity using the agar well
diffusion method. At the compound concentration used
(1 mg/ml) no activity against yeasts was found, while
out of 10 bacterial strains tested only P. aeruginosa
was sensitive to the compound with an inhibition zone

of 7 mm diameter. The minimum concentration capable
of preventing microbial growth (MIC) was examined in
more details using the micro-dilution broth method [

P. aeruginosa growth was inhibited at MIC=125 µg/ml.
According to our results the tested new flavonoid
exhibited narrow antibacterial spectrum, including only
P. aeruginosa ATCC 9027, among used microorganisms.
However, it is interesting to note that non-fermentative,
Gram-negative rods belonging to this species are
important nosocomial pathogens with an ability to
spread on medical devices, hospital environment and
even in disinfectants. These microorganisms cause
high morbidity and mortality in the services of oncology,
haematology, surgery or burn and intensive care units
[

]. The infections due to P. aeruginosa are often

difficult to treat because of its intrinsic resistance to
many conventional antimicrobial agents. The bacteria
from this group also have the ability to develop antibiotic
resistance extremely rapidly, especially among clinical
isolates [

]. Isoetin 4’-O-β-D-glucopyranoside inhibits

growth of this microorganism with MIC=125 µg/ml.
Further research is needed in order to determine the
activity of this new flavone glycoside against a panel
of strains, including clinical isolates of P. aeruginosa.
In addition, on the basis of our investigations it may be
concluded that this new isoetin glycoside is the main
constituent responsible for the antipseudomonal activity
of plant extracts examined in earlier studies [

The antiproliferative effect of isoetin 4’-O-β-D-

glucopyranoside was assessed in two human tumor
cell lines derived from lung (A549) and colon (HT-29)

Figure 2.

Effect of isoetin 4’-O-β-D-glucopyranoside on proliferation of (A) human lung carcinoma cells (A549), (B) human colon adenocarcinoma

cells (HT-29) measured by means of MTT assay. *at least P<0.05 vs. control (c), one-way ANOVA,

post test: Tukey.

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et al.

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carcinomas. Cells were exposed to either culture medium
(control) or tested flavonoid compound (1-100 µM) for 96
hours. Proliferation of A549 cells was not affected by up
to 25 µM, however at the highest concentrations (50 and

100 µM) a significant stimulatory effect was observed
(Figure

A). In the case of HT-29 cell culture, the

proliferation was significantly decreased (10-100 µM)
in a non-dose dependent manner (Figure

B).

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