Antioxidant capacities of Gundelia tournefortii L. extracts and
inhibition on glutathione-S-transferase activity
N. C ¸oruha,*, A.G. Sag ˇdıc ¸og ˇlu Celepb, F. O¨zgo ¨kc ¸ec, M._Is ?canb
aDepartment of Chemistry, Middle East Technical University, 06531 Ankara, Turkey
bDepartment of Biochemistry, Middle East Technical University, 06531 Ankara, Turkey
cDepartment of Biology, Yu ¨zu ¨ncu ¨ Yil U¨niversity, Van, Turkey
Received 22 July 2005; received in revised form 21 November 2005; accepted 2 December 2005
Gundelia tournefortii L. is an important food source and a well-known medicinal plant in Eastern Anatolia. Therapeutic effects of
medicinal plants are known to be closely related to their antioxidant capacities. Antioxidant activities of G. tournefortii, both for the
aerial parts and seeds, were investigated by using both 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and lipid peroxidation
inhibition methods. The seeds were found to have higher antioxidant potential than the aerial, with IC50values of 0.073 mg/mL for
DPPH scavenging and 0.146 mg/mL for lipid peroxidation inhibition capacities. In addition, total phenolic contents of the Gundelia
tournefortii L. extracts, especially the seed extracts correlates to its high antioxidant activity with 105.1 ± 8.7 lg gallic acid equivalents
(GAEs) per mg of seed extract. Plant extracts with high phenolics content are known to have important effects on various enzymes, as
well as glutathione-S-transferases, which are important detoxification enzymes in phase II systems with an important role in developing
multi-drug resistance to chemotherapy in tumour cells. Consequently, the effects of G. tournefortii extracts on crude cytosolic glutathi-
one-S-transferase was also studied and the seed extracts have shown effective inhibition of cytosolic GST activity, with an IC50of
? 2005 Elsevier Ltd. All rights reserved.
Keywords: Gundelia tournefortii L.; Antioxidant; DPPH; Lipid peroxidation; Total phenolics; Glutathione-S-transferase
Gundelia tournefortii L. var. armata Freyn. and Sint.
from the Asteraceae (Compositae) family, is a medicinal
plant, native to Asian-temperate zones of Western Asia,
namely Cyprus, Egypt, Iran, Israel, Jordan, Turkey, Azer-
baijan and Turkmenistan. It is recorded that the flowers,
leaves, seeds and stems of G. tournefortii are used as food
sources (Ertug ˇ, 2000). In the Middle East, the young and
still undeveloped flower buds are sold in the local markets
just like artichoke hearts;itis ahighly sought item (Hedrick,
1972; Kunkel, 1984). Dry seeds of G. tournefortii are also
known to be effective for the treatment of vitiligo disease,
in Eastern Anatolia folk medicine. Fresh seeds of G. tourne-
fortii are used in pickles and also are effective diuretics.
In this study, the antioxidant activities of G. tournefortii
extracts were evaluated for their DPPH radical scavenging
and lipid peroxidation inhibition capacities and for their
total phenolics contents. The effects of G. tournefortii
extracts on glutathione-S-transferase (GST) activities were
also investigated along with their antioxidant capacities.
GST enzymes are important for the detoxification of by-
products of lipid peroxidation or DNA hydroperoxides in
biological systems (Hayes & Pulford, 1995; Nordberg &
Arn’er, 2001). Resistance developed, against antitumor
drugs used in chemotherapy, is due to the ability of tumor
cells to promote GSH conjugation catalyzed by glutathi-
one-S-transferase (Guadiano et al., 2000; Zanden et al.,
0308-8146/$ - see front matter ? 2005 Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: +90 312 210 51 39; fax: +90 312 210 12
E-mail address: email@example.com (N. C ¸oruh).
Food Chemistry 100 (2007) 1249–1253
2004). Therefore, investigation of novel antioxidants with
inhibitory effects on GST activity is another important rea-
son for this research (Gyamfi, Ohtani, Shinno, & Aniya,
2. Materials and methods
Preparative chromatography grade methanol used in the
preparation of extracts was purchased from Merck
(Darmstadt, Germany). Potassium chloride (KCl), ethyl-
enediaminetetraacetic acid (EDTA), glycerol, bovine serum
albumin (BSA), copper sulphate (CuSO4), sodium potas-
sium tartarate, sodium hydroxide (NaOH), sodium carbon-
ate (Na2CO3), Tris–HCL, ferrous sulphate (FeSO4Æ 7H2O),
thiobarbituric acid (TBA), ortho-phosphoric acid (H3PO4),
(DPPH), gallic acid, 1-chloro-2,4-dinitrobenzene (CDNB),
and reduced glutathione (GSH), were purchased from
Sigma Chemical Company, (St. Louis, MO, USA).
Dimethyl sulphoxide (DMSO) and Folin-Ciocalteu’s phe-
nol reagent were purchased from Merck (Darmstadt, Ger-
many). All other chemicals were analytical grade and were
obtained from commercial sources at the highest purity
2.2. Plant materials
Plant samples of Gundelia tournefortii L. var. armata
Freyn. & Sint. were collected at various periods in 2003,
from Van in Turkey. The plant specimens with their local-
ities and the necessary field records were written and enu-
merated as voucher specimen number, F 11174. They
were press-dried according to herbarium techniques and
identified by ‘‘Flora of Turkey’’ (Davis, 1975), and stored
in the herbarium at Yu ¨zu ¨ncu ¨ Yil University (VANF).
2.3. Preparation of plant extracts
Plant samples were ground using a Waring type stainless
steel commercial blender. Chemical extraction was carried
out in a Heidolph Laborota 4000 rotary evaporator, over-
night at 50 ?C. For each extraction, 10 g of the sample were
extracted in methanol with a sample to solvent ratio of 1:10
(w/v). Extracts were then filtered through a double cheese-
cloth and dried under vacuum using a rotary evaporator.
2.4. Isolation of sheep liver microsomes
Sheep livers, bought from the slaughterhouse of Sincan,
Ankara Turkey, were homogenized using a glass-teflon
homogenizer and centrifuged by a Sorvall RC 5C Plus with
SS-34 rotor and Sorvall Combi Plus Ultracentrifuge with
10,800g for 25 min. The microsomes in the suspension,
containing 25–30 mg protein/mL, were stored in an Sanyo
ultra-low freezer (?84 ?C) for up to 3 months (Iscan et al.,
1998) and their protein content was measured by the
Lowry’s Method (Lowry, Rosebrough, Farr, & Randall,
2.5. Lipid peroxidation inhibition evaluated by thiobarbituric
Lipid peroxidation of the microsomes was induced using
a 100 lM ferrous sulphate solution. The end products of
lipid peroxidation were quantified by determining the
formed malondialdehyde (MDA), and related products.
In acidic medium, MDA reacts with thiobarbituric acid
(TBA) upon boiling and the resultant MDA-TBA adduct
as other TBA reactive substances (TBARS) absorbs at
532 nm. Formation of MDA-TBA adduct was monitored
by a Cary 50 Bio UV–vis spectrophotometer in both the
presence and absence of the extracts, and was expressed
as nmoles/mg of protein/minute. The TBARS values were
calculated using the extinction coefficient of 1.56 · 10?5
M?1cm?1(Heath & Packer, 1968). Percent inhibition in
lipid peroxidation was calculated using the equation
% Inhibition ¼ ½ðA0? A1Þ=A0? 100?;
where, A0is the absorbance of control with methanol and
A1is the absorbance with the extract samples. Fifty percent
inhibitory concentration (IC50) values were calculated after
constructing the percent inhibition versus log (extract con-
2.6. Free radical scavenging activity by DPPH method
The free radical scavenging activities were determined
by the DPPH method of Blois (1958) with some modifica-
tions. A 0.05 mg/mL of DPPH ethanol solution which
absorbs at 517 nm, produces approximately 1.3 unit of
absorbance. A series of extract solutions with varying con-
centrations were prepared by dissolving the dried extracts
in ethanol. A solution of each extract (0.1 mL) was added
to 1.4 mL of DPPH solution. The absorbance at 517 nm
was recorded after 5 min of incubation at room tempera-
ture. The radical scavenging capacity of each extract was
calculated as the percent DPPH radical scavenging affect
DPPH Scavenging Effect ð%Þ ¼ ½ðA0? A1Þ=A0? 100?;
where, A0is the absorbance of the control with ethanol and
A1is the absorbance of the sample in the presence of the
extracts. IC50concentrations were also calculated as ex-
2.7. Determination of total phenolics content
The total concentration of phenolic compounds in the
solutions (0.05–0.3 mg/mL) as described by Singleton and
tion (0.1 mL) was mixed with 2 mL of a 2% (w/v) sodium
carbonate solution and vortexed vigorously. The same pro-
N. C ¸ oruh et al. / Food Chemistry 100 (2007) 1249–1253
cedure was also applied to the standard solutions of gallic
acid. After 3 min, 0.1 mL of 50% Folin-Ciocalteu’s phenol
reagent was added and each mixture was vortexed again.
The absorbance at 750 nm of each mixture was measured,
after incubation for 30 min at room temperature. Results
were expressed as milligrams of total phenolics content per
milligrams of extract as gallic acid equivalents (GAE).
2.8. Determination of sheep liver glutathione-S-transferase
activity toward CDNB
GST activities were determined spectrophotometrically
by monitoring the thioether formation at 340 nm using
1-chloro-2,4-dinitrobenzene (CDNB) as the substrate
(Habig, Pabst, & Jakoby, 1974). Sheep liver cytosolic frac-
tions were prepared and used as the enzyme source to mea-
sure GST activity toward CDNB. Potassium phosphate
buffer (0.2 M) and final 1 mM GSH were used throughout
the experiments. Enzyme activity determinations were car-
ried out at 25 ?C. Sheep liver microsomes (21 mg/mL) were
used after 1/100 dilution with 10 mM potassium phosphate
buffer (pH 6.0). Concentrations ranging between 0.2 and
2 mM CDNB were used as the substrates. The reactions
started by the addition of the enzyme. Incubation mixtures
without an enzyme source were used as the blanks (non-
enzymatic reactions). Enzyme activities with 10 lL of
DMSO were regarded as control. The enzyme activities
were expressed in terms of percent specific activity. One
unit of GSTs activity is defined as the amount of enzyme
producing 1 lM thioether per min. The enzyme activities
were expressed in terms of percent specific activity. The
extinction coefficient for the CDNB conjugate at 340 nm
is 0.0096 lM?1cm?1(Habig et al., 1974).
Each data point was obtained by making at least 6 inde-
pendent measurements. The results were expressed as
mean ± SD and levels of significance were assessed using
ANOVA test (Analysis of Variance).
3. Results and discussion
Each extract was prepared by dissolving 10 g of dry
samples in 100 mL methanol. The methanol extraction of
the aerial parts and the seeds of G. tournefortii yielded
6.3% and 2.18% of dry weight, respectively. In order to
identify the antioxidant capacity of plants extracts, DPPH
and lipid peroxidation inhibition tests were selected as the
methods of choice. The DPPH method, is the direct test
used to evaluate the real-time free radical scavenging. On
the other hand, lipid peroxidation inhibition test was used
to mimic a biomembrane medium.
3.1. Lipid peroxidation inhibition by thiobarbituric acid test
Iron induced microsomal lipid peroxidation inhibition
was used as one of the methods to evaluate antioxidant
capacity. a-Tocopherol, a well-known antioxidant, was
used as the standard in this study. During the measure-
ments, the absorbance of each sample was corrected for
the methanol background. The lipid peroxidation inhibi-
tion capacities of each methanol extract were calculated
as percent inhibiton as shown in Fig. 1. Results are also
listed in Table 1 as the concentration of each extract neces-
sary to inhibit microsomal lipid peroxidation by 50 per-
cent, (IC50). As a result IC50values for inhibition of lipid
peroxidation were found to be 0.255 and 0.146 mg/mL,
for aerial parts and seeds extracts of Gundelia tournefortii
L., respectively. The IC50 value of a-tocopherol was
0 0.10.20.3 0.4 0.5
G. tournefortii aerialG. tournefortii seeds alpha-tocopherol
Fig. 1. Inhibition of iron induced lipid peroxidation (%) by Gundelia
tournefortii L. aerial and seed extracts compared with a-tocopherol.
Comparison of 50% inhibitory concentration for lipid peroxidation, % DPPH radical scavenging activity, and total phenolic contents of Gundelia
tournefortii L. extracts and their effects on GST activity
TP (lg GAE/mg extract)GST inhibition
Gundelia tournefortii aerial parts
Gundelia tournefortii seeds
64.4 ± 4.8
105.1 ± 8.7
DPPH scavenging IC50: concentration of plant extracts for 50 percent of DPPH scavenging. LP inhibition IC50: concentrations of plant extracts for 50
percent inhibition of lipid peroxidation. TP: total phenolics content lg equivalents of Gallic acid/mg of plant extract. GST inhibition IC50: concentration
of plant extracts for 50 percent inhibition of GST activity. ND: not determined. NA: not applicable.
N. C ¸ oruh et al. / Food Chemistry 100 (2007) 1249–1253
3.2. Free radical scavenging activity by DPPH method
Free radical scavenging activity of the extracts was eval-
uated by using the DPPH radical scavenging method. Dry
extracts were dissolved in ethanol in concentrations rang-
ing between 0.1 and 0.8 mg/mL. DPPH radical scavenging
activity of the extracts are expressed as % radical scaveng-
ing, by measuring the absorbance decrease at 517 nm due
to disappearance of the DPPH radical. Results are pre-
sented as % DPPH radical scavenging activity of the Gund-
elia extracts in mg/mL concentrations and shown in Fig. 2.
The IC50 values for DPPH percent scavenging were
determined from the percent inhibition versus log (extract
concentration) curve, and for the aerial parts and the seeds
of G. tournefortii extracts, IC50values were found to be
0.442 and 0.073 mg/mL, respectively. IC50for quercetin,
which was used as a standard was 0.0061 mg/mL, as listed
in Table 1.
3.3. Total phenolics content of G. tournefortii extracts
The total phenolics content of the G. tournefortii
extracts was determined by using the method of Singleton
and Rossi (1965) and the results are expressed in Table 1
as lg per mg of gallic acid equivalents (GAE).
The amount of total phenolic compounds found in the
extract from aerial parts of G. tournefortii was 64.4 ±
4.8 lg in 1 mg of methanolic crude extract. The total phen-
olics content in the seeds, however, was 105.1 ± 8.7 lg in
1 mg of methanolic crude extract, gallic acid equivalents.
The results are consistent with the respective antioxidant
capacities of each extract.
Phenolic constituents have been studied extensively as
important contributers to the antioxidant activity in plants
(Aruoma, Bahorun, & Jen, 2003; Lo ´pez, Martı´nez, Del
Valle, Ferrit, & Luque, 2003; Rice-Evans, Miller, &
Paganga, 1997; Skerget et al., 2005). There are reports in
the literature which correlate the total phenolics content
of a plant extract with its antioxidant activity (Cai, Luo,
Sun, & Corke, 2004; Kaur & Kapoor, 2002; Velioglu,
Mazza, Gao, & Oomah, 1998; Yen & Duh, 1993) and this
was also the case in the present study, as shown in Table 1.
The seed extracts with high phenolics content, also has high
DPPH radical scavenging and lipid peroxidation inhibition
3.4. Effects of G. tournefortii extracts on glutathione-S-
GSTs activity was determined spectroscopically by mon-
itoring the thioether formation at 340 nm using CDNB as
the substrate (Habig et al., 1974). The extracts were used
within 0.01–0.15 mg/mL final concentrations in order to
calculate the percent inhibition of GST activity and respec-
tive IC50values. The inhibitory effect of extracts (100 lg/
mL concentration) on GST activity was compared with
their phenolics content in lg equivalent of GAE/mg of
extract (Fig. 3). Concentrations of the extacts for 50% inhi-
bition of GST activity were also listed in Table 1.
Phenolic content of the seed extracts of G. tournefortii
was higher (105.1 lg/mg extract) than the aerial parts of
the plant (64.4 lg/mg extract). Plant extracts with high
polyphenols are known to have important inhibitory
effects on glutathione-S-transferases in the literature. Con-
sequently, G. tournefortii seed extracts which have higher
phenolics content, are also expected to be more effective
in GST inhibition, as given in Fig. 3 and Table 1, coincid-
ing with literature (Gyamfi et al., 2004). Furthermore,
GST activity of cancer cells was inhibited by polyphenols
(Zhang & Wong, 1997). Hence, the search for novel anti-
oxidants with GST inhibitory activity has become an
important issue because of their role in multi-drug
0 0.20.4 0.60.8
G. tournefortii aerial G. tournefortii seedsQuercetin
Fig. 2. DPPH radical scavenging activity (%) of Gundelia tournefortii L.
aerial and seed extracts compared with quercetin.
G. tournefortii aerial G. tournefortii seeds
GST Inhibition (%)
Total Phenolics ( μg GAE equivalent)
Fig. 3. Comparison of the GST inhibition of Gundelia tournefortii L.
extracts (100 lg/mL), with their phenolics content in lg equivalent of
GAE/mg of each extract from aerial parts and the seeds.
N. C ¸ oruh et al. / Food Chemistry 100 (2007) 1249–1253
4. Conclusions Download full-text
This is the first study in the literature, on the antioxi-
dant capacity, content of total phenolics and the GST
inhibitory activities of G. tournefortii plant. Methanol
extracts of G. tournefortii, especially the seed extracts, have
considerable antioxidant capacity compared with a-
tocopherol. The seed extracts, having higher phenolic con-
tent, were also more effective GST inhibitors, with an IC50
of 97.5 lg/mL.
This study was supported by the grant from The State
Planning Organization awarded to N. C ¸oruh (Grant No:
BAP-07-02-DPT-2003-K120920-16) and the grant from
The Scientific and Technological Research Council of Tur-
key awarded to F. O¨zgo ¨kc ¸e (Grant No: TUBITAK-TBAG
2049 (01 T 054)).
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