Free radical scavenging abilities of flavonylthiazolidine-2,4-dione compounds

Article abstract of DOI:10.1002/bio.1176

Free radical scavenging activity of flavonyl-thiazolidine-2,4-dione compounds has been evaluated using chemiluminescence, electron spin resonance spectroscopy with 5,5-dimethyl-1-pyrroline-1-oxide as spin trap and DPPH (2,2′-diphenyl-1-picrylhydrazyl) method. The examined compounds exhibited 28-50% scavenging superoxide anion radical (O₂^.-), 16.7-76.7% hydroxyl radical (HO^.) and 9-40% DPPH radical. Compounds containing carbonyl group in their structure can be considered as antioxidants with high relevance and great biological importance. Copyright

Full text of DOI:10.1002/bio.1176

Research Article
Received: 3 May 2009,
Revised: 13 July 2009,
Accepted: 14 August 2009,
Published online in Wiley Online Library: 18 November 2009
(wileyonlinelibrary.com) DOI 10.1002/bio.1176
Free radical scavenging abilities of flavonyl-
thiazolidine-2,4-dione compounds
Oya Bozdağ-Dündar,a Rahmiye Ertan,a Hassan Y. Aboul-Enein,b*
Aleksandra Kładnac and Irena Krukd
ABSTRACT: Free radical scavenging activity of flavonyl-thiazolidine-2,4-dione compounds has been evaluated using chemi-
luminescence, electron spin resonance spectroscopy with 5,5-dimethyl-1-pyrroline-1-oxide as spin trap and DPPH (2,2′-
diphenyl-1-picrylhydrazyl) method. The examined compounds exhibited 28–50% scavenging superoxide anion radical (O2•),
16.7–76.7% hydroxyl radical (HO•) and 9–40% DPPH radical. Compounds containing carbonyl group in their structure can be
considered as antioxidants with high relevance and great biological importance. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: flavone; flavonyl-thiazolidine-2,4-dione compounds; free radicals; scavenging capacity
Thiazolidinediones (TZDs) such as pioglitazone and rosigli-
tazone are a new class of antidiabetic agents that reduce plasma
Introduction
Free radical-mediated lipid peroxidation, oxidative stress and
antioxidants are widely discussed in many current research
areas. (1–3) Oxygen-centered free radicals such as hydroxyl
radical (HO•) and superoxide anion radical ( O2• ) and other reac-
tive oxygen species (ROS) are the products of normal cellular
metabolic processes and pathological stress in human body. (1)
They play dual role that is beneficial and harmful in the biology
of the cell. (2,3) Under normal conditions ROS and free radicals
are effectively eliminated by antioxidant defense systems such as
antioxidant enzymes and non-enzymatic factors. (4) However,
under pathological conditions, the balance between the genera-
tion and elimination of ROS is broken; as a result of these events,
biomacromolecules including DNA, membrane lipids and pro-
teins, are damaged by ROS-mediated oxidative stress. The term
oxidative stress in essence refers to the situation of a serious
imbalance between the production of free radicals and the anti-
oxidant defense mechanisms, leading to potential tissue damage.
(1,3) Uncontrolled generation of free radicals that attack mem-
brane lipids, protein and DNA is believed to be involved in many
health disorders such as diabetes mellitus, cancer and neurode-
generative and inflammatory diseases. (5–9)
glucose and glucose production and also increase glucose clear-
ance in patients with type 2 diabetes, thus reducing insulin resis-
tance. (10,14) Besides their anti-diabetic potency, these TZDs
have been shown to exert antioxidant activity. Pioglitazone
inhibits O−2 radical production in endothelial cells. Rosiglitazone
ameliorates the impaired coronary arteriolar dilation in mice with
type 2 diabetes by reducing oxidative stress via a mechanism
unrelated to its effect on hyperglycaemia. (14)
Flavonoids are a large family of compounds with common
chemical structure that have recently attracted considerable
interest due to their broad pharmacological and therapeutical
effects. They exist extensively in all parts of plants, including
fruits, vegetables, seeds, nuts, herbs, flowers and bark. (15) They
have been described as health-promoting, disease-preventing
dietary supplements which are extremely safe and associated
with low toxicity. (16) In addition, traditional herbal medicines
having flavones have been recommended for the treatment of
diabetes and the antidiabetic activity of several plant extracts has
been correlated with their antioxidant properties. As antioxi-
* Correspondence to: H. Y. Aboul-Enein, Pharmaceutical and Medicinal
Chemistry Department, The Pharmacetical and Drug Industries Research
Division, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail:
enein@gawab.com
Diabetes mellitus, a state of chronic hyperglycemia, is a major
cause of serious micro- and macrovascular diseases, affecting,
therefore, nearly every system in the body. Growing evidence
indicates that oxidative stress is increased in diabetes due to
overproduction of ROS and decreased efficiency of antioxidant
defenses, a process that starts very early and worsens over the
course of the disease. (5,6,8,10,11)
a
Ankara University, Faculty of Pharmacy, Department of Pharmaceutical
Chemistry, 06100 Tandoğan, Ankara, Turkey
b
Pharmaceutical and Medicinal Chemistry Department, The Pharmacetical
Hyperglycemia is reported to induce oxidative stress through
multiple pathways such as redox imbalances secondary to
enhanced aldose reductase activity, increased advanced glyca-
tion end products, altered protein kinase C activity, especially
β-isoforms, prostanoid imbalances and mitochondrial over-
production of superoxide. (12) All these pathways converge in
the production of oxidative stress. (8,12,13)
and Drug Industries Research Division, National Research Centre, Dokki,
Cairo 12311, Egypt
c
Department of Medical History and Ethics, Pomeranian Medical Academy,
ul. Rybacka 1, 70-204 Szczecin, Poland
d
Institute of Physics, West Pomeranian University of Technology, Szczecin, Al.
Piastów 48/49, 70-311 Szczecin, Poland
Luminescence 2011; 26: 10–16
Copyright © 2009 John Wiley & Sons, Ltd.

Free radical scavenging of flavonyl-thiazolidine-2,4-dione
dants, these compounds may prevent the progressive impair-
ment of pancreatic β-cell function due to oxidative stress, thus
reducing the occurrence of type 2 diabetes. (17) As free radical
scavengers, flavonoids (15,16,18,19) can act as powerful antioxi-
dants, even more so than the traditional vitamins. (20,21)
In our previous studies, we reported the synthesis and the in
vitro antidiabetic and aldose reductase inhibitory activity results
of the flavonyl-2,4-thiazolidinediones (FTDs) 1–6. (22) In this
paper, the in vitro effect of FTDs (Fig. 1) having antidiabetic and
aldose reductase inhibitory activity was examined on the reac-
tions generating O−2 , HO and DPPH radicals.
Materials and methods
The compounds 1–3 were synthesized by Knoevenagel reaction
of ethyl 2,4-dioxothiazolidine-3-ylacetate with flavone-3′/4′/6-
carboxaldehydes using acetic acid–sodium acetate mixture.
The compounds 4–6 were obtained with acidic hydrolysis of
compounds 1–3. (22)
Chemicals
Ammonium ferrous sulphate hexahydrate was from Fluka
(Buchs, Switzerland); catalase (19,900 U mg−1) from bovine liver
and p-nitroblue-tetrazolium chloride (NBT) were from Sigma
(St Louis, MO, USA). All remaining reagents were purchased from
E. Merck (Darmstadt, Germany) and Aldrich (Milwaukee, MI, USA).
The reagents were propared fresh daily; dimethylsulfoxide
(DMSO) was prepared as a 0.1 mol L−1 aqueous solution immedi-
ately before use and stored in darkness at −15°C under nitrogen
to avoid any oxidation of the spin trap.
Code
Formula
O
1
O
O
N
S
O
O
O
Superoxide anion radical scavenging activity
Superoxide anion radical was generated in
a KO2–DMSO
system. (23) 18-Crown-6 (1,4,7,10,13,16-hexaoxacyclo-octadec-
ane) (60 mg) in 10 mL anhydrous dimethylsulfoxide (DMSO) was
mixed quickly to avoid contact with air humidity with 7 mg of
KO2. The mixture was stirred for 1 h to give a pale yellow solution
of O2• , which was stable at room temperature for at least 1 h. The
O
2
O
O
N
O
O
O2• concentration was determined using a UV spectrum (λmax
=
S
251 nm, ε = 2686 29 mol cm−1).
O
O
Chemiluminescence (CL) measurements were made using an
EMI9553Q photomultiplier with an S20 cathode sensitive in the
range of 200–800 nm, interfaced with a computer for data acqui-
sition and handling. Reagents were introduced to a thermostated
glass cuvette placed in a light-tight chamber. The cuvette was
exhausted and washed using a B-169 vacuum system (Büchi,
Flawill, Switzerland). The quenching activity was calculated as
follows:
3
4
O
S
N
O
O
O
O
O
Quenching ratio Q(%) = [(ΣI0 − ΣIx)/ΣI0] × 100%, where ΣI0 is
the integrated light intensity (CL sum) measured during
t=3
H
O
O
N
S
⎛
⎝
⎞
⎠
O
3 min
in the absence of a inhibitor (an FTD
O
ΣI =
( )
I t dt
∫
⎜
⎟
t=0
compound), ΣIx is the light sum measured in the presence of the
inhibitor. Because FTDs were dissolved in DMSO and added to
the reaction mixture caused a short-lasting small ‘flash’ followed
by a very small increase in the light emission (data not shown);
this reaction was considered as the control reaction.
O
O
O
5
H
O
O
N
O
Hydroxyl radical scavenging activity
S
Electron spin resonance (ESR) and the spin trap agent
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used to deter-
mine the scavenging hydroxyl radical generated in the Fenton
reaction: (24)
O
O
6
O
O
S
( ) ( )
H2O2 +Fe II III
→HO• +Fe +HO−
(1)
N
O
H
O
This reaction is accompanied by two other reactions
+HO• →Fe +HO−
H2O2 +HO• →HOO• +H2O
O
Fe
II
( )
( )
III
(2)
(3)
Figure 1. Chemical structures of flavonyl-thiazolidine-2,4-dione compounds
(FTDs).
Luminescence 2011; 26: 10–16
Copyright © 2009 John Wiley & Sons, Ltd.
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O. Bozdağ-Dündar et al.
In the presence of the DMPO trap, HO• is rapidly trapped by this
Superoxide anion radical in DMSO elicits strong light emission
according to the following reactions: (29–31)
spin trap at high rate constant of 3.4 × 109 mol−1 s−1: (25)
•
DMPO +HO• →DMPO − OH
(4)
O2• → 1O2 + electron
(5)
(6)
(7)
The rate constant for reaction of the HOO• with this nitrone spin
trap is very low (∼10 mol−1 s−1).
−
2O2• + 2Me2SO → 1O2 +Me2SO2 + OH− +MeSO
( )
CH2
The Fenton reaction contained 200 μL of 0.1 mol−1 DMPO
(aqueous solution), 50 μL of 4 mmol L−1 H2O2, 50 μL of
80 mmol L−1 sodium trifluoroacetate (pH 6.15) and 50 μL of
ammonium ferrous sulfate hexahydrate solution at concentra-
tion of 0.5 mmol L−1. The reaction mixture was placed in the ESR
21O → 1O
→
3 O2 2 +light emission
(
)
2
2
(
)
2
Addition of the O−2 radical inhibitors leads to a decrease in the light
intensity, allowing the evaluation of their antioxidant activity. This
reaction has high sensivity and selectivity and has been applied
successfully for evaluation of an antioxidative property of the bio-
logically reactive compounds in nonaqueous media. (32)
Typical chemiluminescence intensity–time diagrams in the
absence (blank) and in the presence of a antioxidant (sample)
were givn in our previous paper. (33)
cavity using a quartz flat cell with an optical pathlength of
•
DMPO−OH
0.25 mm. The spin-trapped
signal was analyzed
5 min after the addition of ammonium ferrous sulfate hexahy-
drate on a standard ESR spectrometer operating at 9.3 GHz with
100 kHz modulation of the steady magnetic field.
Tested FTDs were dissolved in DMSO, which is suitable for dis-
solving water-insoluble samples. (26)
Four of the six examined FTDs compounds dissolved in DMSO
reduced the light sum at least by 28% (range 28–50%) at a con-
centration of 0.5 mmol L−1 (Fig. 2). One of these compounds
(denoted as 4) exhibited the O−2 radical scavenging activity com-
parable to that for specific this radical scavenger NBT (1 mmol L−1)
and a little lower than (1 mmol L−1) Tiron (1,2-dihydroxy benzene-
3,5-disulphonic acid) (56%) at the same concentration. The CL
sums for these two inhibitors were accounted for by integrating
the area under the kinetic curve I = f(t) after 20 min. The sums
were compared with those of the control (without examined
compound), considered as 90%. In contrast, under the conditions
examined, comounds 1 and 2 increased light emission by about
99 and 207%, respectively.
The ability to scavenge the HO radicals was calculated using
the following formula:
Hydroxyl radical scavenging ratio R(%) = [H0 − H) / H0] × 100%
where Ho and H represent the relative height of the second peak
in the ESR spectrum of the spin-adduct from the blank (DMSO)
and a sample dissolved in DMSO, respectively.
The conditions of ESR measurement were as follows: micro-
wave power 0.63 mW, modulation amplitude 0.2 mT, time con-
stant 0.1 s, receiver gain 4 × 104, and temperature 293 K.
The enhanced chemiluminescence from the O−2 radical–DMSO
system in the presence of compounds 1 and 2 indicates that both
compounds were able to stimulate the O−2 radical dismutation as
follows:
DPPH radical scavenging activity
Free radical scavenging activity was evaluated according to
the method reported by Shimada et al. (27) Two milliliters of
0.1 mmol L−1 DPPH radical in ethanol was mixed with 2 mL of
sample solution (2.5 mmol L−1) in DMSO and incubated at 24°C
for 30 min in the dark. The decrease in absorbance at 517 nm was
measured against ethanol using a UV–vis Zeiss M-40 spectro-
photometer (Zeiss, Germany). Scavenging capacity A (%) was
calculated using the following equation:
2O2• + 2H+ → 1O2 +H2O2
(8)
This means that these compounds might deliver H+ for reaction
(8) that is a source of singlet oxygen. (34) To check if H2O2 and 1O2
are generated in reaction (8) the effect of catalase (an enzyme
responsible for destruction of H2O2 (28,35)) and histidine (a
1
quencher of O2 (28)) was examined. An addition of the enzyme
( )
A % = 1−(A − A ) : A ×100%
[ ]
1
2
o
at a concentration of 200 μg/mL caused a decrease in the CL sum
of about 40%. Also the aqueous solution of histidine (1 mmol L−1)
inhibited the light emission by 46%, although water alone at the
same volume (5%) as these tested antioxidant H2O solutions
quenched the CL by about 10%. In contrast, an addition of SOD
(100 μg mL−1), enzyme which catalyzes the dismutation of O−2
radical into hydrogen peroxide, (35) to the O2• –DMSO solution
increased the light sum by about 45%. Direct addition of H2O2
(0.1 mmol L−1) elicited a time-dependent increment of light emis-
sion by about 20% (data not shown).
where A1 is the absorbance of 2 mL DPPH radical solution and
2 mL sample of a FTD, solution, A2 is the absorbance of 2 mL
sample of a FTD solution and 2 mL ethanol, Ao is the absorbance
of 2 mL DPPH radical solution and 2 mL DMSO.
Results and discussion
The antioxidant activities of the individual compound may
depend on the structure of the free-radical inhibitors, the sub-
stituents present on the rings, e.g. free carboxylic group and the
degree of polymerization.
Compounds containing the –CH2COOH group exhibited the
strong direct activity towards the O−2 radical. This is consistent
with a previous finding of Sawyer and Gibian, (36) who reported
that weakly acidic O−2 radical as a weak base can promote the H+
transfer from organic compounds.
Within the framework of this paper, the superoxide radical scav-
enging effect of six novel synthesized compounds was estimated
employing the CL from O−2 radicals generated in DMSO. Superox-
ide anion radical is a reduced form of molecular oxygen formed
by receiving one electron. (28) The radical is known as a precursor
of very harmful species to cellular components such as HO
radicals as well as singlet oxygen (1O2) and H2O2. In addition,
this radical is a one-electron reductant of transition metal
ions. (28)
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Free radical scavenging of flavonyl-thiazolidine-2,4-dione
50
50
41
5
44
6
56
28
50
4
0
1
2
3
NBT
Tiron
-50
-100
-150
-200
-250
-99
-207
Compound
Figure 2. Scavenging effect of the examined flavonyl-thiazolidine-2,4-dione compounds (0.5 mol L−1) on the chemilumines-
cence from 1 mol L−1 O−2 radicals generated in DMSO. The examined scavengers were dissolved in 0.5 mL DMSO. Temperature
294 K. All data represent means SD of three different experiments. Numbers given at the bottom of the figure refer to com-
pounds listed in Fig. 1. The remaining condictions are reported under Materials and Methods.
7.2 mmol L−1 led to an 80% inhibition. The specificity of the
Fenton probe for H2O2 and further HO radicals generation was
checked by adding catalase (180 μg mL−1). We observed a near-
complete prevence of appearance of any ESR signal, whereas in
the presence of a heat-denaturateed enzyme the signal was
decreased by 5%, indicating that the origin of the DMPO–OH
signal was dependent on HO radical generation. This conclusion
Among the oxygen-derived free radicals HO radical is the most
reactive unstable oxidizing species and can be produced from O−2
radical and H2O2 in the presence of transition metal ions, such as
cooper or iron. (28) This species reacts with a variety of organic
compounds, including biomolecules such as lipids, proteins,
polypeptides and DNA. When a hydroxyl radical reacts with aro-
matic compounds, its addition across a double bond is reported,
and reaction products such as hydroxycyclo-hexadienyl radical
and peroxyl radical are thought to be formed. (37) Except for
addition reactions, the HO radical may react by hydrogen abstrac-
tion and electron transfer reactions. Indeed the reactions of HO
radicals with biomolecules result in generation of other radicals,
but they are usually of lower toxicity.
The ability of the tested compounds to scavenge the HO
radical was monitored using the ESR spin-trapping assay, the
technique routinely used for the sensitive and specific detection
of short-lived hydroxyl radicals. The Fenton reaction, a well-
documented HO radical generator, was utilized to test the ability
of tested compounds to scavenge this radical. Hydroxyl radicals
trapped by DMPO form spin-adducts, for which a spectrum is
shown in Fig. 3A(a). The spectrum exhibits four splitting lines
with an intensity ratio of 1:2:2:1 and hyperfine splitting
finds confirmation in a fact that superoxide dismutase (SOD) even
•
DMPO−OH
at a high concentration did not prevent
formation.
The scavenging activities of the tested compounds dissolved
in DMSO to this Fenton reaction system with DMPO on HO radi-
cals are shown in Fig. 3B. The scavenging abilities were, in
descending order, 6 > 5 > 4 > 3 > 1 > 2 > DMSO, and the ampli-
tude decrease ranged from 16.7 3.5 to 76.7 4.6%. The scav-
enging effect of 0.37 mmol L−1 compound 6 (76.7%) was nearly
equal to that of the salicylate at concentrartion of 7.2 mmol L−1.
The ability of tested compounds to exhaust their H-donating
capacity was examined using 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH•). The compound is a stable free radical that has a
proton free radical with an absorption maximum band around
515–528 nm. The characteristic absorption decreases signifi-
cantly on exposure due to the proton-donating ability of the
antioxidants proportional to the concentration and antioxidant
activity of the antioxidant. (39) The DMPPH radical can be used
to the primary characterization of the scavenging activity of
compounds. (40–43)
constant of aN = aH = 14.9 G. The parameters are consistent with
•
those values for the
adduct reported by other
DMPO−OH
authors. (25,38) No ESR signal was measured from the Fenton–
DMPO reaction, when one substrate was omitted.
According to the conclusion of Sanchez-Moreno, (44)
compounds able to scavenge the DPPH radical in vitro may also
scavenge polyaromatic hydrocarbon cations in vivo by donating
hydrogen ions or electrons.
The height of the second peak of the spectrum determined the
•
relative amounts of DMPO−OH adduct formed in the reaction.
When hydroxyl radical scavenger was added, a decrease in the
•
DMPO−OH
amount of
adduct was observed.
The DPPH radical scavenging capacities of tested FTDs are
showed in Fig. 4A. For compounds 2, 4, 5 and 6 the reaction was
biphasic, with a faster decay in the absorbance in the first dozen
minutes, followed by a slower step. The DPPH radical scavenging
activity of the examined compounds was evident (p < 0.05), but
Figure 3A(b and c) shows the ESR spectrum measured in the
Fenton–DMPO reaction in the presence of salicylic acid and
compound 5, the OH radical scavenger, respectively. Salicylic acid
can remove this radical from the reaction at the reaction rate
1.2 × 101- L mol−1 s−1 (39) The use of salicylic acid at concentration
Luminescence 2011; 26: 10–16
Copyright © 2009 John Wiley & Sons, Ltd.
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O. Bozdağ-Dündar et al.
A
90
80
70
60
50
40
30
20
10
0
B
76.7
60.1
38.3
32.5
28.5
16.7
2
1
3
4
5
6
Compound
Figure 3. (A) The ESR spectra of the DMPO–OH spin adduct arising in the Fenton reaction: (a) ESR spectrum pro-
duced by the reaction mixture containing 50 mol L−1 DMPO, 0.5 mol L−1 H2O2, 1.0 mol L−1 sodium trifluoracetate, pH
6.15, 0.5 mL DMSO, and 50 μmol L−1 FeSO4 (NH4)2 SO4. The reaction was started by an addition of the Fe(II) ion. (b) The
signal of DMPO−OH radical quenched by the addition of 7.2 mol L−1 salicylic acid dissolved in 0.5 mL DMSO. (c)
The DMPO−OH radical signal quenched by the presence of 0.37 mol L−1 compound 5 dissolved in 0.5 mL of DMSO.
(B) The inhibitory effect of FTD compounds exerted on the DMPO−OH radical formation. The composition of the
reaction mixtures was the same as in (A, part c). Data are expressed as means SD of three measurements. The
numbers in figure and on the x-axis refer to the compounds given in Fig. 1. Measurement conditions are reported
under the Materials and Methods section.
was lower than ascorbic acid and Trolox at concentrations similar
free radical scavenging power. The –COOH group is capable of
to that used during the compounds examination. The com-
pounds’ hierarchy of reducing capacity was 6 > 4 > 5 > 2 > 3 > 1,
ranging from 9 5 to 40 3% (Fig. 4B).
It can be observed that FTDs possessing a free carboxylic
group, denoted as 4–6, exhibited the highest effectiveness in the
readily donating hydrogen electron.
The inhibitory effect examined FTDs containing the –CH2–
COOH group on DPPH, O−2 and HO radicals may represent the
direct scavenging activity. Compounds 1–3 could not scavenge
free radical as effectively as 4–6. Other structural properties
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Luminescence 2011; 26: 10–16

Free radical scavenging of flavonyl-thiazolidine-2,4-dione
A
0.0
1
3
2
-0.2
-0.4
-0.6
-0.8
5
4
6
trolox
ascorbic acid
0
10
20
30
40
50
60
Time [s]
120
100
80
60
40
20
0
B
97
93
40
35
32
14
2
11
3
9
1
4
5
6
Trolox
Ascorbic acid
Compounds
Figure 4. DPPH radical scavenging activity of the FTDs compounds, ascorbic acid 1 mol L−1 and Trolox. (A) Kinetic curves of
the reaction between DPPH radical and solutions of the examined compounds. (B) The maximal scavenging effect exerted by
examined compounds on DPPH•. Results are means SD of triplicate measurements. Numbers given in the figure refer to
compounds listed in Fig. 1. Reaction conditions are described in Materials and Methods.
important for antioxidant nature of the examined compounds
found that among tested compounds those containing the car-
boxylic group exerted stronger antioxidant effect than FTDs
without this group. These findings suggest that there is promise
for FTDs to be used successfully in treating diseases that involve
free radicals.
include the presence of a C2–C3 double bond in conjugation
with 4-oxo function in the C ring. (45) It has been reported that
the degree of unsaturation of the C2–C3 bond is an important
determinant of high bioactivity of flavonoids. (46) The presence
of nitrogen atom in the thiazole ring may also be an active center
of FTDs, due to its lone pair electrons. In addition, all examined
compounds contain the benzene ring; this also may be valid
due to HO and O−2 radicals abilities for substitution on the
ring. (47)
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