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Cytotoxic activity and antioxidative properties of Sideritis scardica extracts

  • Institute for Medicinal Plant Research "Dr. Josif Pancic"


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The promising new source of therapeutic agents
refers to plant secondary metabolites, irregularly
occurring compounds that characterize certain
plants or plant groups. There is continuously in-
creasing interest in assessing the role of the phe-
nolic compounds which show antioxidative prop-
erties and may act with beneficial health effects,
reducing the risk of chronic diseases (inflamma-
tion, cancer, osteporosis, and cardiovascular dis-
eases). Among them, flavonoids, as a large group
of plant secondary metabolites, have been pro-
duced in the plant for the purpose of protection
from photosynthetic stress, reactive oxygen spe-
cies (ROS), wounds, and herbivores. Studies of fla-
vonoids have revealed the most compelling data
for cytotoxic activities in various types of cancers,
and several flavonoids have been shown to inhibit
cancer development while exhibiting antioxidant
activities in different animal models. Further-
more, some studies suggest that the most promis-
ing use of these compounds may be as an adju-
vant to currently used therapies in antitumor
treatment [1].
Sideritis scardica Griseb. (ironwort, mountain
tea), an endemic plant of the Balkan Peninsula,
has been used in traditi onal medicine in the treat-
ment of gastrointestinal complaints, inflamma-
tion, and rheumatic disorders. This study aimed
to evaluate its gastroprotective and anti-inflam-
matory activities. Besides, continuously increas-
ing interest in assessing the role of the plant ac-
tive constituents preventing the risk of cancer
was a reason to make a detailed examination of
the investigated ethanol, diethyl ether, ethyl ace-
tate, and n-butanol extracts regarding cytotoxic-
ity. Oral administration of the investigated ex-
tracts caused a dose-dependent anti-inflamma-
tory effect in a model of carrageenan-induced rat
paw edema. Gastroprotective activity of the ex-
tracts was investigated using an ethanol-induced
acute stress ulcer in rats. The cytotoxic activity of
plant extracts was assessed on PBMC, B16, and
HL-60 cells and compared to the cytotoxicity of
phenolic compounds identified in extracts. Apo-
ptotic and necrotic cell death were analyzed by
double staining with fluoresceinisothiocyanate
(FITC)-conjugated annexin V and PI. The devel-
oped HPLC method enabled qualitative finger-
print analysis of phenolic compounds in the in-
vestigated extracts. Compared to the effect of the
positive control, the anti-inflammatory drug in-
domethacine (4 mg/kg), which produced a 50%
decrease in inflammation, diethyl ether and n-bu-
tanol extracts exhibited about the same effect in
doses of 200 and 100 mg/kg (53.6 and 48.7 %;
48.4 and 49.9%, respectively). All investigated ex-
tracts produced dose-dependent gastroprotective
activity with the efficacy comparable to that of
the reference drug ranitidine. The diethyl ether
extract showed significant dose-dependent cyto-
toxicity on B16 cells and HL-60 cells, decreasing
cell growth to 51.3% and 77.5% of control, respec-
tively, when used at 100 µg/mL. It seems that phe-
nolic compounds (apigenin, luteolin, and their
corresponding glycosides) are responsible for the
diethyl ether extract c ytotoxic effect. It also ap-
pears that induction of oxidative stress might be
involved in its cytotoxicity, since B16 and HL-60
cells increased their ROS production in response
to treatment with diethyl ether extract. Neither
of the tested extracts nor any phenolic com-
pounds showed significant cytotoxic effect to hu-
man PBMC. These results demonstrated the po-
tent anti-inflammatory and gastroprotective ac-
tivities, as well as the promising cytotoxicity.
Anti-inflammatory, Gastroprotective, and Cytotoxic
Effects of Sideritis scardica Extracts
Authors Vanja M. Tadić
, Ivica Jeremic
, Silva Dobric
, Aleksandra Isakovic
, Ivanka Markovic
, Vladimir Trajkovic
Dragica Bojovic
, Ivana Arsic
Affiliations The affiliations are listed at the end of the article
Key words
Sideritis scardica Griseb.
gastroprotective activity
received October 10, 2011
revised Dec. 12, 2011
accepted Dec. 19, 2011
Published online Januar y 24,
Planta Med 2012; 78: 415427
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 00320943
Dr Vanja Tadić, Science Advisor
Department of Pharmacy
Institute for Medicinal Plant
Research Dr Josif Pančić
Tadeusa Koscuska 1
11000 Belgrade
Phone: + 38 11 13 03 16 58
Fax: +381113031655
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
Downloaded by: Ivana Arsic. Copyrighted material.
The results of numerous preliminary investigations of plants be-
longing to the genus Sideritis L. revealed a plant-derived source of
part icular pharmacological and nutritional interest. The genus
Sideritis L. (Lamiaceae) includes approximately 150 species of an-
nual and perennial plants distributed mainly in the Mediterra-
nean region. This genus is divided into two subgenera, Sideritis
and Marrubiastrum, formed by the European and Macaronesian
species, respectively. So far, different biological activities of Side-
ritis species have been reported: anti-inflammatory, antiulcer,
analgesic, antimicrobial and antifungal [26], immunomodulat-
ing [7], macrophage NOS-2-expression inhibiting [8], and hypo-
glycemic [5]. Recently, aldose reductase inhibiting activity [9],
antiproliferative, anticholinesterase, and selective estrogen re-
ceptor modulator-like effects have been reported [1012]. The
previous studies of Sideritis species reported the presence of fla-
vonoid aglycones and glycosides, phenolic acids, di- and triterpe-
noids, fatty acids, coumarines and iridoid glycosides [3, 9, 11, 13
16], and essential oil as well [2]. Most of the studies on Sideritis
species attributed the previously cited biological activities
mainly to phenolic compounds [9,13]. Rios et al. [17] reported
that flavonoids were reducing agents able to interact with free
radical species (of relevance to autoxidation mechanism) and
could prevent generation of inflammatory mediators.
The genus Sideritis is represented in Serbia by one species only, S.
montana L. [18], but because of its pro-oxidant properties this
plant has not been used in traditional medicine [19]. S. scardica
Griseb. (ironwort, mountain tea) is an endemic plant of the Bal-
kan Peninsula belonging to the Empedoclea section. Aerial parts
of mountain tea are t raditionally known for their anti-inflamma-
tory, antimicrobial, antibacterial, antirheumatic, and gastropro-
tective properties. S. scardica is used as a loosening agent in bron-
chitis and bronchial asthma, against common cold and lung em-
physema. It has been imported in Serbia from the former Yugo-
slav Republic of Macedonia and Albania and widely used in the
treatment of inflammation, gastrointestinal disorders, and
coughs, as well as an active constituent of dietary supplements
for the prevention of anemia. In the literature, all previously cited
biological activities are mainly attributed to the phenolic content
of this plant [14].
The present study aimed to investigate anti-inflammatory and
gastroprotective activities of S. scardica extracts in order to ex-
amine the above-stated folkloric utilizations and to establish the
correlation between observed activities and phenolic constitu-
ents of the extracts based on previous studies which have recog-
nized flavonoids in S. scardica as potent biologically active sub-
stances. Besides, in the present study we investigated the in vitro
cytotoxic action of S. scardica extracts in order to establish the
connection of signif icant antitumor potential and the polyphenol
components present in the examined extracts. Qualitative and
quantitative fingerprint analyses of polyphenolic compounds in
the investigated extracts were also conducted applying the HPLC
Materials and Methods
Sodium bicarbonate (analytical grade), DPPH (1,1-diphenyl-2-
picrylhydrazyl; analytical grade), indomethacin (purity 99.0%),
carrageenan (EP grade), and t rolox (purity 99.0%) were pur-
chased from Sigma-Aldrich. Analytical grade reagents 2,6-di-
tert-butyl-4-methylphenol (BHT, purity 99.8 %), ether, petrol,
dimethyl sulfoxide (DMSO), ethyl acetate, n-butanol (BuOH), ace-
tone, and absolute ethanol (96 %, v/v) were purchased from
Merck. Acetonitrile (MeCN), water, and methanol were of HPLC
grade and also from Merck. Reference HPLC standards p-couma-
ric (purity 99.0%), protocatechuic (purity 99.0%), chlorogenic
(purity 99.0%), vanillic (purity 95.0 %), caffeic (purity
90.0%), ferulic (purity 99.0%), and syringic (purity 95.0%)
acids, luteolin-7-O-β-glucoside (purity 98.0%), apigenin-7-O-β-
glucoside (purity 99.0%), luteolin (purity 99.0%), chrysoeriol
(purity 99.0%), apigenin (purity 99.0%), hyperoside (purity
99.0%), gallic acid (purity 99.0 %), pyrogallol (purity 99.0%),
and cisplatin [cis-diamineplatinum(II) dichloride, purity
99.9%] were purchased from Sigma or from Extrasynthese.
Their purity was declared as stated previously, based on the man-
ufacturerʼs internal high-precision HPLC method. Ranitidine, pu-
rity 95.0 % (Ranisan ampoules), was purchased from Zdravlje-
Actavis Company.
Plant material and the procedure for plant material
The wild growing species Sideritis scardica Griseb., Lamiaceae,
was collected on Shara Mountain (at the hill foot of the Ljuboten,
at ca. 1300 m) during the time of flowering. Plant material was air
dried, packed in paper bags and kept in a dark and cool place until
analysis. Plant material was verified, and the voucher specimen
of the plant (SS/08) was deposited at the Herbarium of the Botan-
ical Garden, Jevremovac, Belgrade, Serbia. The identification was
provided by Prof. Dmitar Lakuši ć (Institute of Botany and Botani-
cal Garden, Faculty of Biology, University of Belgrade). The shade-
dried and powdered aerial parts of S. scardica (600 g) were
coarsely extracted using 70% (V/V) ethanol. The yield of the final
extract (crude extract, 1) in terms of starting crude material was
determined to be 16.7%. The crude ethanol extract (1) was redis-
solved in distilled water, shaken vigorously and extracted with
600 mL of diethyl ether, 600 mL ethyl acetate, and 600 mL satu-
rated n-butanol in a separating funnel, successively. The obtained
extracts were: diethylether extract, 2 (2.8 g); ethyl acetate ex-
tract, 3 (1.3 g); and n-butanol extract, 4 (4.4 g). The yield of ex-
traction for extracts 2, 3, and 4 was 16.7, 7.5, and 26.3% in crude
extracts, or 0.46, 0.21, and 0.73% of dry plant, respectively.
Adult, male Wistar rats weighing 200300 g were used for esti-
mating mountain tea ethanol extract anti-inflammatory (carra-
geenan-induced paw edema test) and gastroprotective activities
(absolute ethanol-induced stress ulcer test). Experimental groups
consisted of 610 animals each. The animals were deprived of
food for 1820 h before the beginning of experiments with free
access to tap water.
This study was performed after approval from the local Institu-
tional Animal Care and Use Committee and run in accordance to
the statements of the European Union regarding handling of ex-
perimental animals (approval number 86/609/EEC, 31.01.2008).
Determination of total phenols content
The total phenolic content was determined by the Folin-Ciocalteu
method [20]. One hundred microliters of the MeOH solution of
the dry investigated extracts 1, 2, 3, and 4 (15.75, 31.5, and 63;
27.75, 55.5, and 138.75; 7.28, 14.56, and 19.13; and 7.06, 14.13,
and 28.25 µg/mL final quantity, respectively) were mixed with
0.75 mL of Folin-Ciocalteu reagent (previously diluted 10-fold
with distilled water) and allowed to stand at 22 °C for 5 min;
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
Downloaded by: Ivana Arsic. Copyrighted material.
0.75 mL of sodium bicarbonate (60 g/L) solution was added to the
mixture. After 90 min at 22 °C, absorbance was measured at
725 nm. Gallic acid (0100 mg/L) was used for calibration of a
standard curve. The calibration curve showed the linear regres-
sion at r > 0.99, and the results were expressed as milligrams of
gallic acid equivalents per gram of plant extracts dry weight (mg
GAE/g DW). Triplicate measurements were taken, and data were
presented as mean ± standard deviation (SD).
Tannins content
The percentage content of tannins was calculated using the
method described in the European Pharmacopoeia, Ph. Eur. 6.0
[21]. Shortly, decoctions prepared from the investigated extracts
were treated with phosphomolybdotungstic reagent in alkaline
medium after and without treatment with hide powder. The ab-
sorbance was measured by UVVIS spectrophotometer HP 8453
(Agilent Technologies) at λ
760 nm. From the difference in ab-
sorbance of total polyphenols and polyphenols not adsorbed by
hide powder, the percentage content of tannins expressed as py-
rogallol (%, w/w) was calculated from the expression:
where m
represents mass of the sample to be examined, in
grams; and m
is mass of pyrogallol, in grams. The results repre-
sent the mean ± SD of three determinations.
Total flavonoids content
The percentage content of flavonoids was calculated using the
method described in the European Pharmacopoeia, Ph. Eur. 6.0
[21]. Br iefly, the sample was extracted with acetone/HCl under
reflux condenser; the AlCl
complex of the flavonoid fraction ex-
tracted by ethyl acetate was measured by UVVIS spectropho-
tometer HP 8453 at 425 nm. The content of flavonoids, expressed
as hyperoside percentage, was presented as the mean ± standard
deviation of three determinations.
HPLC procedure
Chromatographic fingerprint of the extract and quantification of
identified compounds were achieved by HPLC (Agilent Technolo-
gies 1200). Detection was performed using diode array detector
(DAD), and the chromatograms were recorded at λ = 260 nm (for
protocatechin and syringic acid), 280 nm (for chlorogenic, vanilic,
p-coumaric, and caffeic acids), 325 nm (for ferulic acid), and
360 nm (for flavonoids). HPLC separation of components was
achieved using a LiChrospher 100 RP 18e (5 µm), 250 × 4 mm i.d.
column, with a flow rate of 1 mL/min and mobile phase, A
[500 mL of H
O plus 9.8 mL of 85% H3PO4 (w/w)], B (MeCN), elu-
tion, combination of gradient mode: 9075% A, 025 min; iso-
cratic 75% A, 2530 min; 7555% A, 3046 min. The sample was
prepared dissolving 118.6, 49.4, 9.4, and 53.0 mg of the extracts
1, 2, 3, and 4, respectively (obtained by the procedure previously
described) in 10 mL of MeOH, filtered through 0.2 µm PTFE filters
prior to HPLC analysis. The injected volume was 4 µL. Standard
solutions for the determination of flavonoids and polyphenolic
acid were prepared at a final concentration of 0.01 mg/mL (proto-
catechin, p-coumaric, vanilic, ferulic, and syringic acids, as well as
luteolin and chrysoeriol), 0.05 mg/mL (chlorogenic and caffeic
acids, and apigenin), or 0.12 mg/mL (apigenin-7-O-glycoside and
luteolin-7-O-glycoside) in methanol. For the purpose of the phe-
nolic compounds identification and determination in the investi-
gated extracts, three mixtures of the standards were prepared
with the already mentioned concentrations: mix 1 with caffeic
acid, apigenin-7-O-glycoside, and apigenin; mix 2 with chloro-
genic acid, luteolin-7-O-glycoside, luteolin, and chrysoer iol; mix
3 contained the rest of the investigated phenolic compounds. The
volume injected was 4 µL, the same as the investigated extract.
The identification was carried out based on retention time and
spectral matching. Once spectral matching succeeded, results
were confirmed by spiking with respective standards to achieve
a complete identification by means of the so-called peak purity
test. Those peaks not fulfilling these requirements were not
quantified. Quantification was performed by external calibration
with standards.
Determination of the free radical scavenging activity
The DPPH scavenging assay was carried out according to the pro-
cedure descr ibed by Blois, with some modifications [22]. Various
concentrations of the samples (100 µL) were mixed with 900 µL
of 0.04 mg/mL methanolic solution of DPPH. UV spect ra were re-
corded on a UVVIS spectrophotometer HP 8453. Absorbance at
517 nm was measured after 20 min. The inhibition percentage
was calculated using the following equation:
where I was the inhibition percentage, A
was the absorbance of
the negative control (contained 100 µL of MeOH instead of the
samples), and A
was the absorbance of the samples. Synthetic
antioxidants, trolox, and tert-butyl hydroxytoluene (BHT) were
used as positive controls. The inhibition percentage was plotted
against concentration of the samples, and IC
values, deter-
mined by linear regression analysis, were presented as the mean
± standard deviation of three determinations.
Carrageenan-induced rat paw edema
The carrageenan-induced rat paw edema test was used as an ex-
perimental model for screening the anti-inflammatory activity
according to the modified method of Oyanagui and Sato [23].
The extracts were administered p. o. in doses of 50, 100, and
200 mg/kg. Indomethacin, dissolved in DMSO, was used as a ref-
erence in a dose of 4 mg/kg p. o., which was a dose producing 50%
reduction of rat paw edema. The control animals were given
DMSO in a dose of 1 mL/kg p. o. Carrageenan-saline solution
(0.5% in a volume of 0.1 mL) was injected into the plantar surface
of the right hind paw 1 h after the oral administration of the ex-
tracts or indomethacin. A pure saline solution (0.9% NaCl, 0.1 mL)
was injected into the left hindpaw, which served as a control
(non-inflamed paw). The animals were killed 3 h after the carra-
geenan injection, and the paws were cut off for weighing. The dif-
ference in weight between the right and left paw, treated versus
untreated (control) rats, served as an indicator of the inflamma-
tory response intensity (i.e., anti-inflammatory activity). The per-
cent of anti-inflammatory effect was calculated from the expres-
Anti-inflammatory effect (%)
where Δk represents the difference in the paw weight in the con-
trol group; Δe is the difference in the paw weight in the treat-
ment group.
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
Downloaded by: Ivana Arsic. Copyrighted material.
Absolute ethanol-induced stress ulcer in rats
To study the gastroprotective activity of the investigated extracts,
an experimental model of acute gastric mucosa damage induced
by absolute ethanol (1 mL/rat p. o.) was used. The investigated ex-
tracts, dissolved in DMSO, were administered p. o. in doses of 50
200 mg/kg 60 min prior to ethanol. Ranitidine given in doses of
520 mg/kg p. o. was used as a reference drug. The control ani-
mals were given the vehicle in a dose of 1 mL/kg p.o., also
60 min before ethanol. The animals were sacrificed 1 h after giv-
ing ethanol, and their stomachs were removed and opened along
the greater curvature. Lesions were examined under an illumi-
nated magnifier (3×). The intensity of gastric lesions was assessed
according to a modified scoring system of Adami et al. [24].
Cell lines
Mouse melanoma cell line (B16) and the human promyelocytic
leukemia cell line (HL-60) were obtained from the European Col-
lection of Cell Cultures (ECACC) while peripheral blood mononu-
clear cells (PBMC) were obtained from healthy blood donors after
written informal consent. This study was approved by the Ethical
Committee of the School of Medicine, University of Belgrade (ap-
proval number 89/101/EC, 24.02.2011). All cell lines were main-
tained at 37 °C in a humidified atmosphere with 5% CO
. B16
mouse melanoma cell lines were cultured in DMEM supple-
mented with 5% fetal calf serum-FCS while the HL-60 human leu-
kemia cell line and PBMC were cultivated in RPMI supplemented
with 10% FCS.
The adherent cells were prepared for experiments using the con-
ventional trypsinization procedure with trypsin/EDTA and incu-
bated in 96-well flat-bottom plates (2 × 10
cells/well) for viabil-
ity and LDH analyses and in 6-well flat-bottom plates for flow cy-
tometry analyses (3 × 10
cells/well). Cells were rested for 24 h
and then treated with plant extracts and different flavonoids:
apigenin, luteolin, chlorogenic acid, apigenin 7-O-glucoside, lu-
teolin 7-O-glucoside, chrysoeriol, and ferrulic acid. Suspension
cells were cultivated in 96-well flat-bottom plates (3.5 × 10
cells/well) for assessing cell viability, and in 24-well flat-bottom
plates for flow cytometry analyses (1.5 × 10
cells/well). Cells
were rested for 2 h and then treated with plant extracts or flavo-
noids. The extracts and flavonoids were dissolved in dimethyl
sulfoxide-DMSO and diluted in appropriate medium. Final con-
centration of DMSO in the incubation mixture did not exceed
0.1% and did not have any influence on cell viability. Cisplatin
(25 µM) was used as the positive control in all methods except
for lactate dehydrogenase (LDH) release assay where Triton X-
100 (3%) was used.
Determination of cell viability
Cell viability was assessed using acid-phosphatase method.
Briefly, after treatment (24 h), adherent cells were washed twice
with phosphate buffered saline, and 100 µL of reaction mixture
(0.1 M acetate buffer pH 5.5, containing para-nitrophenyl phos-
phate PNPP and 0.1% Triton-X) was added to each well. After 90
minutes, the reaction was stopped by adding 50 µL of 0.1 M
NaOH. The absorbance of the developed yellow color, which was
directly propor tional to the cells viability [25], was measured by
an automated microplate reader at 405 nm. The results were pre-
sented as percent of the control value (untreated cells), which
was arbitrarily set to 100%. For determination of suspension cell
viability, we used a modified acid-phosphatase method. After
treatment, 50 µL of reaction mixture (0.3 M acetate buffer
pH 5.5, containing PNPP and 0.2% Triton-X) was added to each
well. After 60 minutes, the reaction was stopped by the addition
of 50 µL of 0.3 M NaOH into each well, and absorbance was read
as described. At the same t ime blanks, containing cell culture me-
dium (without cells), were prepared to achieve the correction for
the absorbance caused by medium color (at 405 nm).
Analysis of apoptosis and cell cycle
Apoptotic and necrotic cell death were analyzed by double stain-
ing with fluoresceinisothiocyanate (FITC)-conjugated annexin V
and PI, in which annexin V bound to the apoptotic cells with ex-
posed phosphatidylserine, while PI labeled the necrotic cells with
membrane damage. This staining was performed according to
the manufacturerʼs instructions (BD Pharmingen).
The cell cycle was analyzed by measuring the amount of propi-
diumiodide (PI)-labeled DNA in ethanol-fixed cells, exactly as
previously described [26]. DNA fragmentation, as another marker
of apoptosis, was determined during cell cycle analysis by count-
ing the hypodiploid cells in the sub-G0/G1 cell cycle phase.
The green (FL1) and red (FL2) fluorescences of annexin/PI-
stained live cells and PI stained fixed cells were analyzed with
FACSCalibur flow cytometer (BD).
The number of viable (annexin/PI), apoptotic (annexin+/PI),
and necrotic (annexin+/PI+) cells as well as the proportion of cells
in different cell cycle phases were determined with Cell Quest Pro
software (BD). Ten thousand cells (gated to exclude cell debris)
were analyzed in each sample.
ROS measurement
Intracellular production of ROS was determined by measuring
the intensity of green fluorescence emitted by redox-sensitive
dye dihydrorhodamine 123 (DHR; Invitrogen), which was added
to cell cultures (2.5 µM) at the beginning of the treatment. At the
end of incubation, cells were detached by trypsinization, washed
in PBS, and the green fluorescence (FL1) of DHR-stained cells was
analyzed using a FACSCalibur flow cytometer. The results are ex-
pressed as mean intensity of DHR fluorescence.
Lactate dehydrogenase release assay
The release of the cytosolic enzyme lactate dehydrogenase (LDH)
reflects a loss of membrane integrity in dying cells, and it was as-
sessed by a colorimetric assay as previously described [27].
Briefly, 100 µL of cell culture supernatant after treatment (cells
grown in colorless medium) was mixed with 100 µL of solution
containing 54 mM lactic acid, 0.28 mM phenazine methosulfate,
0.66 mM p-iodonitrotetrazolium violet, and 1.3 mM NAD
. The
pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine in-
to visible hydrazone precipitate was measured using an auto-
mated microplate reader at 492 nm. The total loss of membrane
integrity resulting in complete loss of cell viability was deter-
mined by lysing the cells with 3% Triton X-100 and using this
sample as a positive control. The cytotoxicity in LDH release test
was calculated using the formula: (EC)/(TC) × 100, where E is
the experimental absorbance of cell cultures, C is the control ab-
sorbance of cell culture medium, and T is the absorbance corre-
sponding to the maximal (100 %) LDH release of Triton X-100-
lysed cells (positive control).
Statistical analysis
The statistical significance of the observed differences was ana-
lyzed by the Mann-Whitney U- test and the KruskalWallis test
(in tests for anti-inflammatory and gastroprotective activities, re-
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
Downloaded by: Ivana Arsic. Copyrighted material.
spectively) or by t-test or ANOVA followed by the Student-New-
man-Keuls test. A value of p < 0.05 was considered significant.
The current study evaluated the anti-inflammatory and gastro-
protective activities of various extracts (14) of mountain tea
and explored further perspectives in the investigation of moun-
tain tea cytotoxic activity.
Each extract was analyzed phytochemically. Total phenolic
amount ranged from 84.2 to 345.6 mg of gallic acid equivalents
per gram of plant extracts dry weight; the content of flavonoids,
expressed as hyperoside percentage, ranged from 0.4 to 1.1,
while the percentage contents of tannins were found to be from
0.5 to 5.7 (data summarized in l
Table 1). Quantitative analysis of
total phenolics, flavonoids, and tannins content, presented in
Table 1, pointed out a high amount of phenolic compounds in
the investigated extracts. The total phenolics content in crude
ethanolic extract 1 was smaller in comparison to the one of the
methanolic extract of S. condensata Boiss. & Heldr. and S. eryth-
rantha var. erythrantha Boiss. & Heldr. [28], but the extracts 3
and 4 were much richer in total phenols (l
Table 1). The total
phenolics content in S. scardica methanolic extract reported by
Tunalier et al. [29] was comparable to the results presented here
regarding the total phenolics content in the investigated extracts
3and4ofS. scardica extract 1. An HPLC method has been devel-
oped for analysis of phenolic compounds, and as analyzed by
HPLC, ferulic acid was the dominant component in all investi-
gated samples and composed 0.36, 2.34, and 2.92% of the extracts
2, 3, and 4, respect ively (l
Table 2). The identified compounds
are presented in l
Table 2 regarding their retention times. Ex-
tract 2 appeared to be more abundant in flavonoid aglycones in
comparison to the more polar extracts 3 and 4. Other identified
components were protocatechuic (1), chlorogenic acid (2), vanil-
lic (3), caffeic (4), syringic (5), p-coumaric and ferulic (6) acid, lu-
teolin-7-O-β -glucoside (8), apigenin-7-O-β-glucoside (9), luteo-
lin (10), chrysoeriol (11), and apigenin (12)(l
Figs. 1 and 2, Table
2). The percentage contents of hydroxycinnamic (2, 4, 6,and7),
4-hydroxybenzoic acid derivatives (1, 3,and5), and flavonoids
(8-12) in extracts were different (data summarized in l
Fig. 2 C).
Antioxidant activity of S. scardica extracts of different polarities
was investigated by the DPPH
free radical scavenging method.
The results demonstrated that the extracts tested possessed
DPPH free radical scavenging activity. When the extracts were
applied in the concentration range of 466.027.5 µg/mL, their
DPPH free radical scavenging activity varied approximately from
20 to 90%, respectively, with IC
values from 147.0 to 5.7 µg/mL
Table 1). Trolox and BHT, k nown as potent ant ioxidants,
ser ved as positive controls. n-Butanol extract (4) showed antiox-
idant activity comparable to positive controls (l
Table 1).
Investigated extracts applied in the doses of 50, 100, and 200 mg/
kg significantly reduced the carrageenan rat paw edema. Diethyl
ether extract, 2, possessed the strongest anti-inflammatory activ-
ity, reducing the paw edema in a dose-dependent manner. The
reduction of the edema, achieved by the doses of 100 and
200 mg/kg used, was statistically significant, and at a level com-
parable to the one of the positive control, indomethacine, applied
in a dose of 4 mg/kg producing 50% reduction (l
Fig. 3).
All tested extracts exhibited significant gastroprotective activity,
with the most effective proven to be n-butanol extract, 4, whose
effect at a dose of 100 mg/kg was even significantly better than
ranitidine, which served as a positive control (l
Fig. 4).
Table 2 Quantitative determination of flavonoids and phenolcarbonic acids in diethyl ether (2), ethyl acetate (3), and n-butanol (4) extracts.
No. Compound/extract Percentage (%) Rt
of identified compounds (nm)
1 Protocatechuic acid 0.05 0.05 5.90/6.43 218, 260, 294
2 Chlorogenic acid 0.52 1.62 1.70 8.90/8.90 218, 238, 298 sh, 324
3 Vanillic acid 0.04 –– 10.15/9.99 218, 260, 292
4 Caffeic acid 0.17 0.54 11.18/11.17 218, 238, 298 sh, 324
5 Syringic acid –– 0.16 11.22/11.25 218, 274
6 p-Coumaric acid 0.12 0.19 17.21/17.02 226, 298 sh, 366
7 Ferulic acid 0.36 2.34 2.92 22.78/22.06 218, 236, 298 sh, 324
8 Luteolin-7-O-β-glucoside 0.03 0.13 0.32 23.85/23.89 254, 266 sh, 348
9 Apigenin-7-O-β-glucoside 0.08 0.67 0.61 28.98/29.09 266, 336
10 Luteolin 0.21 –– 40.65/40.83 254, 268 sh, 348
11 Chrysoeriol –– 0.03 44.38/44.27 250, 266 sh, 292 sh, 348
12 Apigenin 0.32 –– 45.47/45.49 266, 338
The numbers refer to compounds signed on the HPLC spectrum (l
Fig. 3).
Retention times of the compounds identified in the investigated extracts.
Retention times of the
standards in the HPLC chromatogram of standards mix 1, mix 2, and mix 3
Table 1 DPPH free radical scav-
enging activity and total phenolics,
total flavonoids, and tannins con-
tents in diethyl ether (2), ethyl ace-
tate (3), and n-butanol (4) extracts
of mountain tea crude ethanol ex-
tract (1) (mean value ± SD of three
Extract DPPH activity
± SD (µg/mL)
Total phenolics ± SD
(mg GAE/g DW)
% Flavonoids ± SD % Tannins ± SD
1 (Ethanol) 31.5 ± 0.4 188.5 ± 12.9 0.4 ± 0.0 5.7 ± 0.0
2 (Diethyl ether) 147.3 ± 1.8 84.2 ± 7.3 0.4 ± 0.0 0.5 ± 0.0
3 (Ethyl acetate) 20.1 ± 0.4 345.6 ± 21.7 1.1 ± 0.0 1.5 ± 0.0
4(n-Butanol) 5.7 ± 0.4 300.3 ± 13.4 0.5 ± 0.0 3.2 ± 0.0
Trolox 5.9 ± 0.3 ––
BHT 6.0 ± 0.3 ––
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
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The cytotoxic activity of plant extracts was assessed in PBMC, B16
melanoma, and HL-60 leukemic cells and compared to the cyto-
toxic activity of the main phenolic compounds of extracts. After
24 h of incubation, only diethyl ether extract extract 2, showed
significant dose-dependent cytotoxicity in B16 cells, decreasing
cell viability to 51.3% of the control at a concentration of 100 µg/
mL (l
Fig. 5). Among the main phenolic compounds of the ex-
tracts, the most cytotoxic were luteolin, apigenin-7-O-β-glyco-
side, apigenin, and luteolin-7-O-β-glycoside, decreasing B16 cell
viability to 48.8 %, 67.3%, 77.2 %, and 82.0% of the control, respec-
tively, when used at a concentration of 100 µM (l
Fig. 5). The
other phenolic compounds, chlorogenic acid, ferrulic acid, and
chrysoeriol, did not affect cell viability. Extract 2 and extract 4 ex-
pressed signif icant c ytotoxic potential against HL-60 cells, de-
creasing their viability to 77.5 % and 81.9% of the control, respec-
tively, at a concentration of 100 µg/mL (l
Fig. 5). However, the
most toxic compounds for HL60 cells were apigenin, which de-
creased their viability to 34.4 %, luteolin (47.1%), and their glyco-
sides, luteolin-7-O-β-glucoside and apigenin-7-O-β-glucoside,
decreased viability to 66.6 % and 78.4% compared to the control,
respectively (l
Fig. 5). Neither of the tested extracts nor any phe-
nolic compounds showed a significant cytotoxic effect to human
PBMC. After 24 h treatment with cisplatin (25 µM), viability of
PBMC, B16, and HL-60 cell lines decreased to 73.6%, 56.7%, and
59.8% of the control (untreated cells), respectively.
As the most potent cytotoxic agents for further assessment of the
cell death mechanisms, we selected extract 2 (100 µg/mL) and
aglycone phenolic compounds apigenin and luteolin (both at
100 µM), because the glycosylated phenolic compounds in vivo
enter extensive deglycosylation pathways producing aglycone
components that further exhibit biological activit y [30]. The
LDH assay that reflects loss of membrane integrity in dying cells
revealed a dose-dependent increase in LDH release after 24 h
treatment with all tested compounds in both cell lines ( l
Fig. 6).
Accordingly, double staining of HL60 leukemic cells with anexin
V-FITC (Ann) and propidium iodide (PI) revealed that extract 2
and its main constituents apigenin and luteolin significantly in-
creased numbers of both early (Ann
/PI) and late apoptotic/ne-
crotic cells with membrane damage (Ann
Fig. 7). Similar
although somewhat less prominent results were obtained in B16
melanoma cells (l
Fig. 7). DNA content analysis using PI staining
showed that all three compounds caused an increase of percent-
age of HL60 cells in the subG
phase, indicating DNA fragmenta-
tion (l
Fig. 8). Cisplatin tretated cells (positive control) also
showed a significant increase in the number of early and late ap-
optotic cells (14.6% Ann
and 22.6% Ann
for B16 and
22.3% Ann
and 28.3% Ann
for HL-60 cells) as well as in-
crease in the percentage of cells in the subG
phase (27.4 % for B16
and 36.5% for HL-60 cells).
On the other hand, no increase in percentage of cells in subG
phase (hypodiploid-apoptotic cells) was observed in less sensi-
tive B16 cells treated with apigenin, luteolin, or extract 2. Howev-
er, B16 cells treated with apigenin or luteolin displayed a cell
cycle block in S/G
M phase (l
Fig. 8).
To further investigate the mechanisms underlying cytotoxic ac-
tion of extract 2, apigenin, and luteolin, we investigated their
ability to induce oxidative st ress in HL60 and B16 cells. DHR
staining demonstrated that both cell types significantly increased
their ROS production in response to treatment with extract 2,
apigenin, or luteolin (l
Fig. 9). Also, cisplatin-treated cells (posi-
Fig. 1 Identified compounds in the investigated extracts: hydroxycinnamic (2, 4, 6,and7) and 4-hydroxybenzoic acid derivatives (1, 3,and5)(A); f lavonoids
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
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tive control) demonstrated the increase in ROS production of 1.8
and a 2.1-fold increase for B16 and HL-60 cells, respectively, com-
pared to untreated cells (negative control). However, the effects
of apigenin and extract 2 were more pronounced in B16 cells
Fig. 9). It therefore appears that induction of oxidative stress
might be involved in the cytotoxic activity of extract 2 and phe-
nolic compounds.
The ethanol extract (1) of S. scardica , mountain tea, and diethyl
ether (2), ethyl acetate (3), and n-butanol (4) extracts of the crude
ethanol extract were evaluated for their traditionally known but
here for the first time confirmed anti-inflammatory and gastro-
protective activities applying in vivo tests. Further, we investi-
Fig. 3 Effect of S. scardica extracts (ethanol, 1; di-
ethyl ether, 2; ethyl acetate, 3; and n-butanol, 4)
and reference substance (indomethacin IND) on
carrageen-induced rat paw edema.
Fig. 2 HPLC chromatograms of the examined mountain tea extracts (dieth-
yl ether, 2; ethyl acetate, 3; and n-butanol, 4) recorded at 360 and 280 nm,
with the spectrum of identified compounds, compared to UV spectra of ref-
erence standards and chemical structures of identified compounds (A).
Numbers refer to the following: protocatechuic acid (1), chlorogenic acid (2),
vanillic acid (3), caffeic acid (4), syringic acid (5), p-coumaric acid (6), ferulic
acid (7), luteolin-7-O-β-glycoside (8), apigenin-7-O-β-glycoside (9), luteolin
(10), chrysoeriol (11), and apigenin (12). HPLC chromatograms of the etha-
nol, diethyl ether, ethyl acetate, and n-butanol extracts (1, 2, 3, and 4, re-
spectively) of mountain tea recorded at 360 nm mirrored to each other (B).
The percentage content of hydroxycinnamic (2, 4, 6,and7), 4-hydroxyben-
zoic acid derivatives (1, 3,and5) and f lavonoids (812) in extracts (C).
* Compounds present in all investigated extracts but not identified.
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
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Fig. 4 Effect of S. scardica extracts (ethanol, 1; di-
ethyl ether, 2; ethyl acetate, 3; and n-butanol, 4)
and reference substance (ranitidine) against gastric
lesions induced by ethanol in rats. * Modified scor-
ing system of Adami et al.: 0, no lesions; 0.5, slight
hyperemia or 5 petechiae; 1, 5 erosions 5mm
in length; 1.5, 5 erosions 5 mm in length and
many petechiae; 2, 610 erosions 5 mm in length;
2.5, 15 erosions < 5 mm in length; 3, > 510 ero-
sions > 5 mm in length; 3.5, > 10 erosions > 5 mm in
length; 4, 13 erosions 5 mm in length and 0.5
1 mm in width; 4.5, 45 erosions 5 mm in length
and 0.51 mm in width; 5, 13 erosions > 5 mm in
length and 0.51 mm in width; 6, 4 or 5 grade 5 le-
sions; 7, 6 grade 5 lesions; 8, complete lesion of
the mucosa with hemorrhage. a1, a2, a3 p < 0.05;
0.01; 0.001 vs. control; b1 p < 0.05 vs. ranitidine
treated group
Fig. 5 Viability of B16 (A), HL-60 (B), and PBMC (C)
incubated with different concentrations of plant
extracts (left) and main phenolic compounds of ex-
tracts (right). The cell viability was assessed after
24 h by measurement of acidic phosphatase activ-
ity. The results are presented as means ± SD values
of triplicate observations from a representative of
three independent experiments.
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
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gated the cytotoxicity of the extracts as well, as a worldwide on-
going investigation and survey for novel natural constituents and
drugs with cytotoxic properties. In order to establish the correla-
tion of the anti-inflammatory and gastroprotective activities and
cytotoxic potential verified in this work to the polyphenol com-
ponents present in the investigated extracts, the total phenolic
content and radical scavenging capacity were determined. Be-
sides, HPLC analysis enabled the identification of 12 phenolic
compounds present in different percentages in the investigated
An antioxidant, which can quench reactive free radicals, can pre-
vent the oxidation of other molecules and may therefore have
health-promoting effects in the prevention of degenerative dis-
eases. Namely, free radicals are considered to play an important
role in numerous chronic pathologies, such as inflammation, car-
diovascular and cancer diseases among others, and are impli-
cated in the aging process. Therefore, the extracts were assessed
against DPPH radicals serving as the oxidizing substrate to deter-
mine their free radical properties. The antioxidant activity of the
tested extracts by the DPPH method was expressed by the pa-
rameter IC
, the amount of antioxidant necessary to decrease
the initial DPPH concentration by 50 %, where the lower its value,
the more efficient the antioxidant (l
Table 1). The antioxidant
capacity of the tested extracts of S. scardica aerial parts could be
attributed to their phenolic content. A good correlation between
phenolic content and antioxidant activity was established (l
ble 1). Though phenolic content served as a reasonable indicator
of an extractʼs overall antioxidant potential, activity in individual
assays depends on the quantities and properties of specific phe-
nolics in the tested extracts. The phenolic substances are capable
of scavenging reactive oxygen species (O
and OH
) and act like
hydrogen donors. Apigenin and apigenin glycosides are antioxi-
dant agents due to the acidic 4-hydroxyl group. Furthermore,
the presence of hydroxycinnamic acids enriched the antioxidant
capacity. The CH=CHCO group ensures great hydrogen-do-
nating ability and thus enforces the antioxidant capacity [31].
The investigated extracts were shown to be rich in hidroxycin-
namic derivatives, which, in respect of identified constituents,
were the most abundant group of phenolics in all investigated ex-
tracts (l
Table 2 and Fig. 3 C). The presented results were in good
accordance with the data available in the literature [29].
Anti-inflammatory activit y has been reported in different kinds
of extracts of several Sideritis species so far [4]. In the present
study, the results (l
Table 2, Figs. 3 and 4) indicated that the eth-
yl ether extract, 2, of S. scardica exhibited the highest anti-in-
flammatory, while n-butanol extract, 4, possessed the most
prominent gastroprotective activity. These results are in good ac-
cordance with the uses of this genus and another survey study
[9]. Recently, a number of flavonoids, such as apigenin, luteolin,
and quercetin, have been reported to exhibit anti-inflammatory
activity [31]. The high total phenolic content and capability of
the extrac ts tested for scavenging free radicals might partly be re-
sponsible for both their anti-inflammatory and gastroprotective
activities as demonstrated in carrageenan-induced paw edema
test and ethanol-induced acute gastric damage, respectively. It is
thought that in the early phase of the anti-inflammatory re-
sponse (within the first hour after injecting carrageenan), many
vasoactive substances (e.g. histamin, 5-hydroxyt ryptamin, bra-
dykinins, and prostaglandins) are released. On the contrary, the
second phase is related to neutrophil infiltration as well as to
the maintaining of the production of arachidonic acid metabo-
lites. In the second phase of acute inflammation, activated poly-
morphonuclear cells produce a great amount of free radical spe-
cies that additionally may damage the tissue caught by inflam-
mation. Numerous investigations have shown that flavonoids
and phenolcarbonic acids, preventing neutrophil infiltration in
the inflammed area and neutralizing free radical species, act as
anti-inflammatory agents [32]. Regarding the DPPH-scavenging
capacity of the S. scardica extracts tested and high total phenol
content, it could be hypothesized that its anti-inflammatory ef-
fect in the model of carrageenan-induced acute inflammation is
a consequence, at least partly, of their flavonols and phenolcar-
bonic acid content. Based on the mentioned investigations, our
assumption addressed phenolic compounds as the potential car-
riers of anti-inflammatory activity of investigated extracts.
The results of the present study demonstrated that the investi-
gated S. scardica extracts offered significant protection against
the ulcerogenic effect of absolute ethanol in rats, and that this ef-
fect was very close to that achieved by the current antiulcer drug
ranitidine. As known, the absolute ethanol is noxious for the
stomach and that affects the gastric mucosa topically by disrupt-
ing its barrier and thus causing hydrogen back diffusion that
leads to necrosis. As a result of the disturbed barrier function of
the gastric mucosa, a rapid and strong vasoconstriction accompa-
nied by rapid and vigorous arterial dilation occurs. As a conse-
quence, oxyradical-mediated injur y of the gastric mucosa results
Fig. 6 The effect of apigenin and luteolin as main
phenolic compounds of extracts and plant extract 2
on LDH release. B16 and HL-60 cells were incubated
with different concentrations of apigenin and lu-
teolin (µM) and plant extract 2 (µg/mL), and cyto-
toxicity was determined after 24 h by LDH test.
Each value on the graph represents means ± SD val-
ues from at least three independent experiments
(* p < 0.05 denotes significant difference in com-
parison with control).
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
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from ischemia followed by reperfusion. The oxygen-derived rad-
icals are directly implicated in that mechanism, and their remo-
tion stimulates the healing of ethanol-induced gastric lesions
[33]. Many studies have demonstrated that flavonoids and phe-
nolic acids, known substances with antioxidant properties, may
protect against gastric damaging effects of absolute ethanol and
possess antiulcerogenic activity. Since the extracts of S. scardica
tested in this study contained the phenolic components (l
1 and 2, Fig. 2) and showed antioxidant activity, it could be sug-
gested that the significant gastroprotective effect of the extracts,
similarly to their anti-inflammatory effect, might at least in part
be consequences of the presence of phenolic compounds in this
Today, cancer is becoming one of the most important causes of
mortality worldwide. Cancer screening and cancer prevention
are a main challenge for the health care system in the developed
world. Many studies showed that flavonoids widespread phyto-
chemicals have strong cytotoxic properties against different
forms of cancer [34,35]. According to this, we further assessed
the in vitro cytotoxic activity of the extracts and their phenolic
compounds. We chose the adherent melanoma B16 cell line
based on previous results showing that some flavonoids (apigen-
in, quercetin) inhibit melanoma cell growth and metastatic po-
tential in vivo [35]. In parallel, experiments on leukemia suspen-
sion cell line HL60 and on human normal PBMC were performed.
The main constituents of plant extracts were also assessed for
their cytotoxic activity in order to explain anticancer activity of
the extracts. None of the tested extracts showed toxicity to hu-
man PBMC, but diethyl ether extract (extract 2), as well as its
main constituents, flavonoids apigenin and luteolin, showed sig-
nificant cytotoxicity to both tumor cell lines. The mechanisms of
their anticancer activity apparently included induction of apo-
ptotic cell death characterized by phosphatidylserine exposure
and DNA fragmentation, which correlated with the induction of
ROS generation. Overproduction of ROS and ensuing oxidative
stress are well-known factors able to trigger cell death [36] and
might be crucial for the cytotoxic activity of extract 2 and its fla-
vonoid ingredients.
Apigenin and luteolin have been shown to inhibit proteasome ac-
tivity and induce apoptosis in human leukemia cells [37]. Protea-
some inhibitors induce accumulation of proteasome target pro-
teins and subsequent activation of caspases as well as cleavage
of poly-ADP ribose polymerase finally leading to apoptosis in
transformed but not in normal cells. Decreasing viability of
HL60 cells could also be attributed to cell dif ferentiation, as api-
genin and luteolin induce morphological differentiation of HL60
cells into granulocytes. It could be expected that extract 2 has
even greater potential to induce differentiation because of high
apigenin-7-O-β-glucosyde content which previously has been
shown to have very high ability to induce differentiation in
HL60 cells [38]. Moreover, it has been shown that flavonoids can
directly bind to some protein kinases including Akt, Fyn, Janus ki-
nase 1, mitogen-activated protein kinase (MAPK) kinase 1, MAPK
kinase 4, phosphoinositide 3-kinase, and Raf1, and then alter
their phosphorylation state to regulate multiple cell signaling
pathways in carcinogenic processes [39].
The type of tumor cell death induced by some agents could influ-
ence therapy response. Even though the majority of anticancer
drugs promote apoptotic cell death, the resistance to chemother-
apy-induced apoptosis seems to be a hallmark of most common
cancers. Necrotic tumor cells potentiate macrophage-mediated
antitumor response in vitro, while apoptosis has the opposite ef-
fect. Necrotic cell death acts as an important stimulus for the in-
duction and maintenance of an efficient immune response medi-
ated by dendritic cells [40]. Numerous data describing immuno-
stimulatory properties of necrotic cell death have fostered a hy-
pothesis that necrosis might be more efficient than apoptosis in
inducing tumor regression. Thus, the ability of a plant extract to
induce necrotic cell death could be an advantage to classical anti-
tumor agents and potentially a very useful adjunctive therapy in
Fig. 7 The effects of apigenin and luteolin as main phenolic compounds
of extracts and plant extract 2 on the induction of apoptosis/necrosis in
mouse melanoma B16 and human leukemia HL-60 cells. B16/HL-60 cells
(Ctrl as control representing untreated cells) were incubated with 100 µM
of apigenin (Apig) and luteolin (Lut) as well as with 100 µg/mL of extract 2
(Extr 2), and the induction of apoptosis/necrosis was investigated after 24 h
by flow cytometry. The representative dot blots are presented, while the
cell numbers (%) in each graph represent means ± SD values from at least
three independent experiments (* p < 0.05 denotes significant difference
in comparison with control).
Tadić VM et al. Anti-inflammatory, Gastroprotective, and Planta Med 2012; 78: 415427
Original Papers
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cancer treatment. While we have observed an increase in cell
membrane permeability of cancer cells treated with extract 2,
apigenin and luteolin, the possibility that necrotic cell death was
secondary to apoptosis induction could not be completely ex-
cluded. We are currently investigating the contribution of necro-
sis to the anticancer activity of extract 2 and its flavonoid constit-
A multitude of cytotoxic effects of flavonoids deserve attention
and further investigation as adjuvant anticancer drugs, as well
as very potent chemicals for cancer chemoprevention. Plant-
based diet, rich in phytochemicals, has been long considered to
have an important role in cancer prevention. Many studies
showed that flavonoids as widespread phytochemicals have
strong cytotoxic properties against different forms of cancer
[35]. Plants rich in flavonoids, used for centuries in traditional
medicine, could be a very good source for providing appropriate
daily intake of flavonoids components as a simple and cheap tool
in cancer prevention. The data presented here support further
exploration of flavonoids as chemotherapeutic and chemopre-
ventive agents.
The authors wish to thank the Serbian Ministry of Science and
Technological Development for financial support, projects III
45017 and III 41025.
Conflict of Interest
There are no conflicts of interest among all authors.
Institute for Medicinal Plant Research Dr Josif Pančić, Belgrade, Serbia
Institute of Rheumatology, University of Belgrade, Belgrade, Serbia
Institute of Biochemistry, School of Medicine, University of Belgrade,
Belgrade, Serbia
Institute for Scientific Information, Military Medical Academy, Belgrade,
Institute of Microbiology and Immunology, School of Medicine, University
of Belgrade, Belgrade, Serbia
ICN Montenegro, Podgorica, Montenegro
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Fig. 9 The effects of apigenin and luteolin as main
phenolic compounds of extracts and plant extract 2
on the induction of ROS production in B16 and HL-
60 cells. Cells were incubated with 100 µM of api-
genin (A), luteolin (B), and 100 µg/mL of extract 2
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difference in comparison with control).
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... 94,5 %). Diese entsprach einem EC 50 -Wert von 5,7 µg/mL (Koleva et al., 2003;Tadic et al., 2008). Ethanol-Extrakte hingegen zeigten Werte von bis zu 8,9 mg/mL (Karapandzova et al., 2013). ...
... Es wurden dosisabhängige, zytotoxische in vitro Wirkungen gegen die Zelllinien HeLa, B16 und FemX und HL-60 beschrieben; vor allem Diethylether-und Ethylacetat-Extrakte waren aktiv (Tadic et al., 2008;2012). Zudem konnte nachgewiesen werden, dass S. scardica-Auszüge die Produktion von reaktiven Sauerstoffspezies in Gliomazellen und primären Astrozyten von Ratten erhöhen; auch Zellzyklus-Arrest und Autophagie gehören zu den Wirkmechanismen. ...
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Sideritis scardica Griseb., also known as Greek mountain tea, is a plant that belongs to the family of Lamiaceae. It is endemic to the Balkan region, where the herb is traditionally used against a broad range of afflictions, mostly as an infusion. Over the past few years, S. scardica has been scientifically investigated for the pharmacological properties it is said to have, especially focusing on the effects in the central nervous system, such as an improved cognitive performance and the reduction of β-amyloid plaques which are typical in Alzheimer's disease. Following this issue, the main goal of the present study was to examine whether extracts of Greek mountain tea counteract further pathomechanisms of this and other neurodegenerative disorders which are a rising health concern in our ageing society. For this purpose six hydroalcoholic S. scardica extracts of different lipophilicity, that were produced and phytochemically analysed by Finzelberg GmbH & Co. KG, were investigated for their neuroprotective activity in the model organism Caenorhabditis elegans. Additionally, six fractions of one of the most potent extracts, seven isolated pure compounds and extracts of some well-known medicinal plants were tested. The mid-polar extracts of S. scardica in particular, as well as the more lipophilic fractions and the phenylethanoid glycosides, were counteracting the aggregation or toxicity of β-amyloid peptides, α-synuclein, 6-hydroxydopamine, tau- and polyglutamine proteins in vivo, whereas they did not reveal any antioxidant properties in the nematodes. The spectrum of activities of S. scardica was especially comparable with that of Bacopa monnieri (Brahmi), a plant used in ayurvedic medicine. The alleviation of the neurotoxicity of the proteins and compounds make S. scardica extracts interesting for the preventive or therapeutic treatment of Alzheimer's, Parkinson's, Huntington's and other neurodegenerative diseases. The phenylethanoids could be identified as active constituents, although synergistic effects between extractives of different polarity remain probable and have to be taken into consideration. Also, a hypothetical mode of action could be postulated: the bonding of polyphenols to the peptides, directly inhibiting their aggregation to toxic oligomers or plaques and fibrils. In the present study, the activity of hydroalcoholic Sideritis scardica extracts against hallmarks of Alzheimer's disease could be affirmed and for the first time, positive effects on further neurotoxic substances could be shown, expanding the neuropharmacological profile of the Greek mountain tea. As some studies on vertebrates and humans assessing the influence on cognitive performance and proving the harmlessness have already been conducted, high-quality clinical studies investigating the preventive or therapeutic efficacy against Alzheimer's etc. should be performed.
... Sideritis leptoclada O. Schwarz et P. H. Davis kızlançayı bitkisinin fitoterapik özelliği üzerine çok az sayıda bilimsel çalışma vardır. Bu bağlamda bu çalışmada, Göktepe'ye endemik olan S. leptoclada (kızlançayı) bitkisinin antioksidan aktivite değerini açığa çıkarılması amaçlanmaktadır [4]- [10]. ...
... The activity was close to that of the rosmarinic acid (94.5%). The same method was applied to extracts of different concentrations and the results varied from 20 to 90% [104,108,151]. Relatively high antioxidant activity was found by Ivancheva et al. [106] using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid extraction method and regarding the standard 0.86 Trolox equivalent antioxidant capacity. ...
Background: Over the last two decades there has been a substantial increase of the number of studies on the species of genus Sideritis. Species of section Empedoclia, occurring in the Eastern Mediterranean region and in part of Western Asia possess some remarkable characteristics and are known as valuable medicinal plants used by local people in the traditional medicine and for herbal tea. The objective of the review is to make a survey on the recent studies on the ethnopharmacology and biological activity of the species in Southeastern Europe and in Turkey, which is the center of distribution and their main occurrence. Main body: The review focuses to the ethnopharmacological studies and on biological activities of the species of interest. The numerous phytochemical studies were not considered for this review, except in the cases when combined with the target issues. The survey revealed that total 47 species belonging to section Empedoclia have been studied either in ethnopharmacological aspect, or in relation to their biological activities, or both. Most species have been used traditionally by the local people as herbal tea or for treatment various health problems, most frequently flu, cold and respiratory diseases. The species demonstrate numerous biological activities and are promising for use in the therapy of many diseases and health disorders. Antioxidant activity was found in 40 species, antimicrobial and antibacterial activity – in 27 species, anti-inflammatory – in 14 species, antifungal – in 8 species, cytotoxic – in 7 species. There were also some other, more specific biological activities, found in a few species, but considered promising for further studies and application. Short conclusion: The species of genus Sideritis, section Empedoclia have been used by local people as herbal tea and in traditional medicine since long time ago. People are taking advantage of the high species diversity are aware of their useful properties. Much more information is available about the biological activities of the target species than about their traditional uses. Most species demonstrate various biological activities and are of substantial interest for further studies on their pharmacological properties and their potential for pharmacy and medicine.
... The results are similar to those reported in the literature [14][15][16]. From the available data it is clear that the mean values for total phenolics and total flavonoids content in S. scardica and S. raeseri are not significantly different (p<0.05), although the mean values for S. raeseri are slightly higher. ...
... The results are similar to those reported in the literature [14][15][16]. From the available data it is clear that the mean values for total phenolics and total flavonoids content in S. scardica and S. raeseri are not significantly different (p<0.05), although the mean values for S. raeseri are slightly higher. ...
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The essential-oil composition of six native populations of Sideritis scardica from Bulgaria was studied by GC-FID and GC/MS analyses. Altogether, 37 components, representing 73.1 to 79.2% of the total oil content were identified. Among them, a-pinene (4.4 –25.1%), b-pinene (2.8 –18.0%), oct-1-en-3-ol (2.3 –8.0%), phenylacetaldehyde (0.5 –9.5%), b-bisabolene (1.3–11.0%), benzyl benzoate (1.1 –14.3%), and m-camphorene (1; 0.3 –12.4%) were the main compounds. All samples were characterized by low contents of oxygenated mono- and sesquiterpenes ( �1.6 and 2.3%, resp. ). Principal component analysis (PCA) and cluster analysis (CA) showed a significant variability in the chemical composition of the studied samples as well as a correlation between the oil profiles and the ecological conditions of the natural habitats of S. scardica.
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Medicinal plants are widely used in the treatment of numerous diseases. These plants synthetize a wide variety of bioactive metabolites that exhibit countless biological and pharmacological activities. Nepeta nepetella and Sideritis arborescens are aromatic and medicinal plants belonging to the Lamiaceae family. These species are used in folk medicine in the treatment of several diseases. The present investigation aims to carry out a phytochemical screening of the aqueous extracts of N. nepetella and S. arborescens to reveal the different groups of phytochemicals present in the polar extract of these species, to estimate the amounts of phenolic compounds including total polyphenols, total flavonoids, flavonols, tannins, anthocyanins, and hydroxycinnamic acids, and to evaluate their antioxidant capacity using radical scavenging activity, molybdenum and ferric reduction potentials, and lipid peroxidation inhibition effects. The results of this study showed that these two Lamiaceae species contain a wide variety of metabolites including terpenoids, cardiac glycosides, phenols, proteins, flavonoids, tannins, quinons, coumarins, saponins, essential oils, and reducing sugars. Moreover, the estimation of the amounts of different phenolic compounds showed that both species have important phenolic compounds contents including flavonoids, tannins, and hydroxycinnamic acids. The antioxidant tests showed that both species display good antioxidant activity in which S. arborescens exhibit the highest antioxidant potential. In conclusion, the findings of this study suggest that Nepeta nepetella and Sideritis arborescens have important amounts of antioxidant phenolic compounds that can be used in the treatment of several diseases.
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Sideritis species have been known as medicinal plants since ancient times, and used as tea in Mediterranean countries such as Turkey, Greece, and Spain. They are also used for the treatment of several ailments such as a cough, common cold, and gastrointestinal disorders. The aim of the present study was to perform the chemical composition, antioxidant, anticholinesterase, and anti-tyrosinase activities of the essential oils of Sideritis albiflora and S. leptoclada. β-caryophyllene (21.2%) and Germacrene D (17.9%) were identified as the major compounds in S. albiflora and S. leptoclada essential oils, respectively. The essential oil of S. albiflora showed the highest lipid peroxidation inhibitory (IC50: 73.8 ± 0.8 µg/mL), DPPH free radical scavenging (28.3±0.1%), ABTS cation radical scavenging (IC50: 50.6 ± 1.0 µg/mL), reducing power (A0.05: 181.7 ± 0.6 µg/mL), acetylcholinesterase (22.1 ± 0.4%), butyrylcholinesterase (IC50: 157.2 ± 0.9 µg/mL) and tyrosinase (15.2 ± 0.4%) inhibitory activities. Moreover, S. albiflora essential oil had rich total phenolic and flavonoid contents indicating 41.5 ± 0.8 µg PEs/mg and 21.4 ± 1.0 µg QEs/mg respectively. This study suggests that consumption of Sideritis species as tea may protect one against melanogenesis, amnesia, and oxidative stress without any observable side effect.
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Hagers monograph of Sideritis scardica
Full-text available
Hagers monograph of Sideritidis scardicae herba
The present thesis aims at biological and phytochemical study of the Sideritis species with conservation status in Bulgaria. The study includes broad range of topics related to the analysis of flora in the localities of target species, evaluation of their population status, morphometric variation of the perennial species, reproduction biology of Sideritis syriaca. The quantitative and qualitative composition of the phenolic compounds of the species was studied in detail both in their natural localities and in cultivation. Additionally, the anti-tumor activity of S. scardica and S. syriaca was studied in laboratory test. As a result of the floristic analysis total 358 plant species were recorded in the localities of Sideritis scardica, 155 species in the localities of S. syriaca and 98 species in the localities of S. lanata. In the case of S. scardica the highest plant diversity was found in the localities in the Rhodopes and Slavyanka, where the percentage of endemic species was very high. Predominant geolements in the localities of all three species were these having Mediterranean component. The species of conservation importance and medicinal plants in the localities of the three species studied were identified and analyzed. The natural habitats of all studied localities of the three species are of high nature conservation importance and are among the priority habitats for the EU. The population status of the three species studied indicates necessity of measures for their conservation. The most endangered species is S. syriaca, whose populations are characterized by the lowest census number and occupy the smallest area. The morphometric variation of S. scardica and S. syriaca underlined the differences between the two taxa, but also distinguished the population of S. scardica from Chervenata stena as having an intermediate position and, therefore, needing further investigation. The stem, foliar, and calyx’s epidermis was similar in S. scardica and S. syriaca. The embryological study of S. syriaca revealed a high reproductive capacity and confirmed the sexual reproduction as main type of reproduction for this species, thus limiting at some extent its adaptive mechanisms. The phytochemical studies have shown that the quantity of total phenolics in S. scardica and S. syriaca has similar values but the quantity of flavonoids in the second species was lower. The content of the two studied groups of compounds was significantly lower in S. lanata. The accumulation of phenolic compounds was higher in the leaves, as compared to flowers. The comparison between wild and cultivated plants showed that in some cases the quantity of biologically active substances in the cultivated plants is not inferior to that in wild plants and can be even higher, which fact allows to recommend the cultivation of the two species – S. scardica and S. syriaca. The qualitative HPLC analysis included two species more (S. raeseri and S. taurica), and the qualitative composition was quite similar, thus underlining the genetic similarity of the species studied. The phenolic composition allowed to distinguish the species belonging to the two sections Hesiodia and Empedoclia. Some differences were found between S. syriaca from Bulgaria (Strandzha) and from Crete Island, and the Bulgarian sample was closer to S. taurica from Turkey, which indicates unresolved taxonomic relationships and necessity of further studies in this respect. High phytotoxic activity of methanol extract of S. scardica was recorded regarding the tumor cell line HCT-116, which offers new perspectives for studying the medicinal properties of the species and its application. Numerous activities were performed within the framework of scientific and applied project, aimed at conservation of S. scardica. These include meeting with local people in the regions close to the natural localities, providing information about the species status (as presentation and printed materials), about the measures for its conservation and possibilities for conservation. An experimental field for observations and investigations was established and planting material was produced, which was put at disposal of people willing to cultivate the species. An initiative for change of the conservation status of the species was launched, consisting of well-grounded proposal for its protection. As a result of these efforts, an order signed by the Minister was issued by the Ministry of Environment and Waters, fully prohibiting the collecting of S. scardica from its natural localities.
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The antioxidant properties and phenolic composition of 27 Sideritis species were studied. Plant samples were extracted with petroleum ether using a Soxhlet apparatus. The defatted plant materials were extracted with 70% methanol. Antioxidant activities of the extracts were measured using Fe+2 induced linoleic acid peroxidation, as indicated by thiobarbituric acid reactive substance (TBARS) production. Free radical scavenging activities were determined based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Results were compared with standard BHT. Total phenol concentration of the extracts was estimated with Folin-Ciocalteu reagent using gallic acid as standard, and phenolic components were quantified by HPLC-DAD.
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The antioxidant activities and total phenolics of 28 plant products, including sunflower seeds, flaxseeds, wheat germ, buckwheat, and several fruits, vegetables, and medicinal plants were determined. The total phenolic content, determined according to the Folin−Ciocalteu method, varied from 169 to 10548 mg/100 g of dry product. Antioxidant activity of methanolic extract evaluated according to the β-carotene bleaching method expressed as AOX (Δ log A470/min), AA (percent inhibition relative to control), ORR (oxidation rate ratio), and AAC (antioxidant activity coefficient) ranged from 0.05, 53.7, 0.009, and 51.7 to 0.26, 99.1, 0.46, and 969.3, respectively. The correlation coefficient between total phenolics and antioxidative activities was statistically significant. Keywords: Antioxidant activity; phenolics; medicinal plants; oilseeds; buckwheat; vegetables; fruits; wheat products
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To prevent bone loss that occurs with increasing age, nutritional and pharmacological factors are needed. Traditional therapeutic agents (selective estrogen receptor modulators or SERMs, biphosphonates, calcitonin) may have serious side effects or contraindications. In an attempt to find food components potentially acting as SERMs, we submitted four plant aqueous extracts derived from Greek flora (Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum) in a series of in vitro biological assays reflective of SERM profile. We examined their ability (a) to stimulate the differentiation and mineralization of osteoblastic cell culture by histochemical staining for alkaline phosphatase and Alizarin Red-S staining, (b) to induce, like antiestrogens, the insulin growth factor binding protein 3 (IGFBP3) in MCF-7 breast cancer cells, and (c) to proliferate cervical adenocarcinoma (HeLa) cells by use of MTT assay. Our data reveal that all the plant extracts studied at a concentration range 10-100 microg/mL stimulate osteoblastic cell differentiation and exhibit antiestrogenic effect on breast cancer cells without proliferative effects on cervical adenocarcinoma cells. The presence of estradiol inhibited the antiestrogenic effect induced by the extracts on MCF-7 cells, suggesting an estrogen receptor-related mechanism. In conclusion, the aqueous extracts derived from Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum may form the basis to design "functional foods" for the prevention of osteoporosis.
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Apigenin, a naturally occurring plant flavone, abundantly present in common fruits and vegetables, is recognized as a bioactive flavonoid shown to possess anti-inflammatory, antioxidant and anticancer properties. Epidemiologic studies suggest that a diet rich in flavones is related to a decreased risk of certain cancers, particularly cancers of the breast, digestive tract, skin, prostate and certain hematological malignancies. It has been suggested that apigenin may be protective in other diseases that are affected by oxidative process, such as cardiovascular and neurological disorders, although more research needs to be conducted in this regard. Human clinical trials examining the effect of supplementation of apigenin on disease prevention have not been conducted, although there is considerable potential for apigenin to be developed as a cancer chemopreventive agent.
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Protein kinases play crucial roles in the regulation of multiple cell signaling pathways and cellular functions. Deregulation of protein kinase function has been implicated in carcinogenesis. The inhibition of protein kinases has emerged as an important target for cancer chemoprevention and therapy. Accumulated data revealed that flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoids can bind directly to some protein kinases, including Akt/protein kinase B (Akt/PKB), Fyn, Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase 1 (MEK1), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase kinase 4 (MKK4), Raf1, and zeta chain-associated 70-kDa protein (ZAP-70) kinase, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes. In this review, we report recent results on the interactions of flavonoids and protein kinases, especially their direct binding and molecular modeling. The data suggest that flavonoids act as protein kinase inhibitors for cancer chemoprevention that were thought previously as conventional hydrogen-donating antioxidant. Moreover, the molecular modeling data show some hints for creating natural compound-based protein kinase inhibitors for cancer chemoprevention and therapy.
METHODS for measuring antioxidants and appraising antioxidant activity appear to be of two general types. If the chemical nature of the antioxidant is known, one may strive for a test specific for the compound or group of interest; for example, the nitroprusside test for sulphydryl groups. Alternatively one may observe the inhibition of some natural oxidative process such as the β-oxidation of fats, as a function of the added antioxidant.
Cell death is an essential phenomenon in normal development and homeostasis, but also plays a crucial role in various pathologies. Our understanding of the molecular mechanisms involved has increased exponentially, although it is still far from complete. The morphological features of a cell dying either by apoptosis or by necrosis are remarkably conserved for quite different cell types derived from lower or higher organisms. At the molecular level, several gene products play a similar, crucial role in a major cell death pathway in a worm and in man. However, one should not oversimplify. It is now evident that there are multiple pathways leading to cell death, and some cells may have the required components for one pathway, but not for another, or contain endogenous inhibitors which preclude a particular pathway. Furthermore, different pathways can co-exist in the same cell and are switched on by specific stimuli. Apoptotic cell death, reported to be non-inflammatory, and necrotic cell death, which may be inflammatory, are two extremes, while the real situation is usually more complex. We here review the distinguishing features of the various cell death pathways: caspases (cysteine proteases cleaving after particular aspartate residues), mitochondria and/or reactive oxygen species are often, but not always, key components. As these various caspase-dependent and caspase-independent cell death pathways are becoming better characterized, we may learn to differentiate them, fill in the many gaps in our understanding, and perhaps exploit the knowledge acquired for clinical benefit.