Antioxidant properties of flavone-6(4')-carboxaldehyde oxime ether derivatives.

Abstract

The in vitro antioxidant properties of some flavone-6(4)-carboxaldehyde oxime ether derivatives (Ia-f, IIa-f) were determined by their effects on the rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels by measuring the formation of 2-thiobarbituric acid reactive substances. The free radical scavenging properties of the compounds were also examined in vitro by determining their capacity to scavenge superoxide anions and interact with the stable free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The most active compounds, IIb (Flavone-4'-carboxaldehyde-O-ethyl oxime) and Id (Flavone-6-carboxaldehyde-O-[2-(1-pyrolidino) ethyl] oxime), caused 98 and 79% inhibition of superoxide anion production and DPPH stable free radical at 10(-3) M, respectively.

Full text

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Arch Pharm Res Vol 27, No 6, 610-614, 2004
610
http://apr.psk.or.kr
Antioxidant Properties of Flavone-6(4')-Carboxaldehyde Oxime
Ether Derivatives
Gülgün Ayhan-KIlcIgil, Tülay Çoban1, Meral Tunçbilek, Benay Can-Eke1, Oya Bozda -Dündar,
Rahmiye Ertan, and Mümtaz Iscan1
Department of Pharmaceutical Chemistry, Department of Toxicology1, and Faculty of Pharmacy, Ankara University,
06100 Tandoðan-Ankara, Turkey
(Received January 28, 2004)
The in vitro antioxidant properties of some flavone-6(4)-carboxaldehyde oxime ether deriva-
tives (Ia-f, IIa-f) were determined by their effects on the rat liver microsomal NADPH-depen-
dent lipid peroxidation (LP) levels by measuring the formation of 2-thiobarbituric acid reactive
substances. The free radical scavenging properties of the compounds were also examined in
vitro by determining their capacity to scavenge superoxide anions and interact with the stable
free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The most active compounds, IIb (Flavone-
4'-carboxaldehyde-O-ethyl oxime) and Id (Flavone-6-carboxaldehyde-O-[2-(1-pyrolidino) ethyl]
oxime), caused 98 and 79% inhibition of superoxide anion production and DPPH stable free
radical at 10-3 M, respectively.
Key words: Flavone, Oxime ether, Lipid peroxidation, Superoxide dismutase, DPPH, Antioxidant
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INTRODUCTION
Free radicals, including the superoxide, hydroxyl,
hydrogen peroxide and lipid peroxide radicals, have been
implicated in a number of degenerative diseases, such as
brain dysfunction, cancer, heart diseases and immune
system decline (Rice-Evans and Diplock, 1991). These
reactive oxygen species (ROS) are produced as a normal
consequence of biochemical processes in the body and
due to increased exposure to environmental and/or dietary
xenobiotics. It is an imbalance in these oxidants versus
antioxidant processes (oxidative stress) that is thought to
cause the subsequent cellular damage that leads to the
above mentioned disease processes (Griffits et al., 1998).
Neurodegenerative diseases, such as Alzheimers disease,
are also linked to damage from ROS as a result of an
imbalance between the rates of radical generation and
scavenging (Richardson, 1993). Antioxidant systems,
including superoxide dismutase, catalase and glutathione,
should keep the oxidative processes in balance. However,
deficiencies of nutritional antioxidants (flavonoids, vitamin
A, C, E, the minerals selenium and zinc, coenzyme Q10,
lipoic acid) and/or overwhelming oxidant stress can
overload these systems (Nordmann, R). The biological
activities of flavonoids have been extensively reviewed,
and some have been found to possess antibacterial (Mori
et al., 1987), anticancer (Deschner et al., 1991; Elangovan
et al., 1994), antihistaminic (Amella et al., 1985), anti-
inflammatory (Krishnaveni et al., 1997; Sala et al., 2003),
antiischemic (Rump et al., 1995), antiviral (Wleklik et al.,
1988) and hypoglycemic (Bozda et al., 2001) activities.
Flavonoids have also been found to inhibit a wide range
of enzymes involved in oxidation systems, such as phos-
pholipase A2 (Chang et al., 1994), 5-lipoxygenase, cyclo-
oxygenase (Robak et al., 1988) and xanthine oxidase
(Chang et al., 1993; Cos et al., 1998), and these activities
are related to their antioxidant properties. Flavonoids can
exert their antioxidant properties by scavenging radicals,
binding metal ions and inhibiting enzymatic systems.
In our previous studies, the synthesis and antimicrobial
evaluation of 6- or 4'-flavone carboxaldehyde oxime ether
derivatives have been described (Ayhan-KIlcIgil et al.,
1999; Tunçbilek et al., 1999). The fungustatic effect of
ketoconazole, an azole antifungal drug, associated with
its membrane stabilizing effects on Candida species is
indicated by inhibition of lipid peroxidation (Wiseman et
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Correspondence to: Gülgün Ayhan-KIlcIgil, Department of Pharma-
ceutical Chemistry, Faculty of Pharmacy, Ankara University,
06100, Tandoðan, Ankara-Turkey
E-mail: kilcigil@pharmacy.ankara.edu.tr

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Antioxidant Properties of Flavone-6(4)-Carboxaldehyde Oxime Ether Derivatives611
al., 1991). Therefore, the antioxidant properties of some
flavone 6- or 4'-carboxaldehyde oxime ether derivatives,
already known to have antimicrobial activities, were
investigated (Ayhan-KIlcIgil et al., 1999; Tunçbilek et al.,
1999) by comparing with butylated hydroxytoluene (BHT)
and superoxide dismutase (SOD), a well known antioxidant
and an antioxidant enzyme, respectively.
MATERIALS AND METHODS
Chemistry
The compounds Ia-f and IIa-f (Fig. 1) were synthesized,
starting with 6- or 4'-flavone carboxaldehyde, by treatment
with the appropriate O-substituted hydroxyl amine deriva-
tives (Ayhan-KIlcIgil et al., 1999; Tunçbilek et al., 1999).
All reagents were purchased from Sigma (Taufkirchen,
Germany).
Antioxidant activity studies
Assay of lipid peroxidation
Male albino Wistar rats (200-225 g) were used in the
experiments. The animals were fed a standard laboratory
rat chow and allowed tab water ad libitum. The animals
were starved for 24 h prior to sacrifice, and then killed by
decapitation under anesthesia. The livers were removed
immediately, washed in ice-cold distilled water and micro-
somes prepared, as described previously (Iscan et al., 1984).
NADPH-dependent lipid peroxidation (LP) was deter-
mined using the optimum conditions, as previously deter-
mined and described (Iscan et al., 1984). In this assay,
the control activity has been regarded as the activity mea-
sured in the presence of the pure diluent for the chemicals
tested (Dimethylsulfoxide (DMSO) for synthesized com-
pounds and BHT). Thus, the assay has been carried out
in the presence of only solvent, as a control, or at the in-
dicated concentrations of compounds. NADPH-dependent
LP was measured spectrophotometrically by estimation
of thiobarbituric acid reactant substances (TBARS). The
amounts of TBARS were expressed in terms of nmol
malondialdehyde (MDA)/mg protein. The assay was
essentially derived from the methods of Wills (Wills,
1966; Wills, 1969), as modified by Bishayee (Bishayee
and Balasubramanian, 1971). A typical optimized assay
mixture contained 0.2 nM Fe++, 90 mM KCl and 62.5 mM
potassium-phosphate buffer, pH 7.4, and the NADPH
generating system (0.25 mM NADP+, 2.5 mM MgCl2, 2.5
mM glucose-6-phosphate, 1.0 U glucose-6-phosphate
dehydrogenase and 14.2 mM potassium phosphate buffer
pH 7.8), and 0.2 mg microsomal protein, in a final volume
of 1.0 mL. The reaction was initiated by the addition of the
NADPH generating system to the microsomal mixtures.
The reaction was carried out at 37oC for 30 minutes, and
trichloroacetic acid added to stop the reaction. The
denatured proteins were then removed by centrifugation.
Finally, the supernatant was mixed with thiobarbituric acid
(TBA) and boiled for 15 minutes. The absorbance was
measured spectrophotometrically at 532 nm. Each experi-
ment was performed in triplicate. The protein contents of
liver microsomes were determined by the method of
Lowry et al. (Lowry et al., 1951), using bovine serum
albumin as a standard.
Superoxide radical scavenging activity
The capacity of compounds to scavenge superoxide
anions was determined spectrophotometrically on the
basis of the inhibition of cytochrome c reduction, according
to the modified method of McCord et al (McCord and
Fridowich, 1969).
Superoxide anions were generated in the xanthine/
xanthine oxidase system. The reaction mixture contained
0.05 M phosphate buffer, pH 7.8, 0.32 U xanthine oxidase,
50 µM xanthine, 60 mM cytochrome c and 100 µL of
different concentrations of the synthesized compounds in
a final volume of 1 mL. DMSO and BHT were used as the
control solution and reference compound, respectively.
The absorbance was measured spectrophotometrically at
550 nm to determine the cytochrome c reduction. Each
experiment was performed in triplicate, and the results
expressed as a percentage of the control.
Fig. 1. General form ula of the com pounds

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612G. Ayhan-KIlcIgil et al.
DPPH free radical scavenging activity
The free radical scavenging activities of these com-
pounds were tested by their ability to bleach the stable
radical DPPH, as described by Blois (Blois, 1958). This
assay has often been used to estimate the antiradical
activity of antioxidants. Because of its odd electronic
structure DPPH gives a strong absorption band at 517 nm
in its visible spectrum. DPPH was dissolved in methanol
to give a 100 µM solution. 0.1 mL of the test compounds
and BHT, dissolved in DMSO, was added to 1.0 mL of the
methanolic DPPH solution. The absorbance at 517 nm
was determined after 30 min at room temperature, and
the scavenging activities calculated as a percentage of
the radical reduction. Each experiment was performed in
triplicate. DMSO was used as a control solution and BHT
as a reference compound. The radical scavenging activity
Table I. Effects of the com pounds on liver superoxide anion production and reduction of DPPH radical a
Com poundR
Concentration in
incubation m edium (M)
Superoxide anion (O2
production percent
of control
•-)
LP Percent
of control
DPPH free radical
scavenger activity
(percent of control)
Controlb
100100100
Ia CH3
10-4
10-3
106±4
131±6
180 103±3
IbC2H5
10-4
10-3
178±9
114±1
--
IcCH2CH2N(CH3)2
10-4
10-3
197±2
131±2
--
Id
10-4
10-3
146±2
142±2
131 121±1
Ie
10-4
10-3
188±6
118±4
221191±1
If
s
10-4
10-3
103±3
137±4
124 102±1
IIaCH3
10-4
10-3
231±8
131±6
144 105±2
IIbC2H5
10-4
10-3
197±2
112±2
158198±3
IIc CH2CH2N(CH3)2
10-4
10-3
199±4
101±6
138182±3
IId
10-4
10-3
199±4
106±3
141155±1
IIe
10-4
10-3
178±4
164±2
128183±4
IIf
10-4
10-3
114±2
162±3
102 184±3
BHT
10-4
10-3
103±3
152±6
165119±1
Controlc
Water100±2
SOD
30 IU
45 IU
124±2
111±1
aEach value represents the mean ± S.D. of 2-4 independent experim ents
bDim ethylsulfoxide only, control for com pounds and BHT
cDistilled water, control for SOD
− : not tested

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Antioxidant Properties of Flavone-6(4)-Carboxaldehyde Oxime Ether Derivatives613
was obtained from the equation: Radical scavenging
activity % = {(ODcontrol − ODsample) / ODcontrol } × 100
RESULTS AND DISCUSSION
The inhibitory effects of the compounds on the NADPH-
dependent lipid peroxidation levels were determined using
rat liver microsomes by measuring the formation of 2-
thiobarbituric acid reactive substances. Only two of the
compounds (IIa and IIb), at a concentration of 10-3 M,
showed marked inhibition (56 and 42%, respectively) of
the lipid peroxidation. These inhibitions were close to
those obtained with 10-3 M BHT (65%), a well known
antioxidant, but the others had no effects on the level of
lipid peroxidation (Table I).
The superoxide anion radical scavenging activities of
the compounds were investigated using the xanthine/
xanthine oxidase system, and the results are presented in
Table I. The most active compounds were found: IIb
(98%), Ib (96%), and Ie (92%) at a concentration of 10-3 M.
The -N-OEt derivatives especially were more potent than
the others. The compounds appeared to have stronger
inhibitory effects on superoxide anion formation than that
of the BHT (48% at 10-3 M) that was used as the positive
control. In addition, they had similar inhibitory effects on
superoxide anion formation to that of SOD (89% inhibition
at 45 IU), the antioxidant enzyme used for comparison.
Compounds IIc and IId had no effect on superoxide anion
formation. The scavenging rates of the rest of the com-
pounds were in the range of 36-69%. In general, substitu-
tions on the A ring of the flavone ring system results in an
improvement of the superoxide radical scavenging pro-
perties, with the exception of compound IIb. The structure-
activity correlation within the 6- and 4'- substituted
derivatives is not straightforward; there is a difference in
activity between the aminoalkyl substituted member of
each series and its counterpart. Compounds Ib, IIa, and IIf
showed biphasic effects on superoxide anion formation.
These compounds increased and decreased the superoxide
anion formation at concentrations of 10-4 and 10-3 M,
respectively. This pattern of differing effects can be seen
in biological assays, where increases may occur at lower
concentrations, whereas inhibition results at higher con-
centrations (Iscan, 1984; Al-Assadi et al., 1992). Biphasic
effects of other chemicals, such as thiazolidinedione/
imidazolidinedione (Dündar et al., 2002) and hydroxy-
chalcones (McCord and Fridowich, 1969; Kornbrust and
Maris, 1980; Parke et al., 1991; Dix and Aikens, 1993) on
the formation of superoxide anions have been well
established in various in vitro systems.
The free radical scavenging properties of the compounds
were also examined by the interaction with the stable free
radical, DPPH. As seen in Table I, compounds Id and IId,
which bear a pyrolidino moiety, as an R substituent, were
found to have some marginal DPPH scavenger activities
(79 and 45% respectively) at concentrations of 10-3 com-
pared with BHT (91%). The compounds Ie, IIc, IIe, and IIf
showed slight inhibitions of the DPPH stable free radical,
whereas the others were ineffective.
There are two mechanisms by which an antioxidant can
scavenge DPPH. First, a direct H-atom abstraction process
(eq 1), and second, a proton concerted electron-transfer
process (eq 2) (Litwinienko and Ingold, 2003).
DPPH• + RXH → DPPHH + RX•
(1)
DPPH• + RXH → DPPH− + RXH•+
DPPH• + RXH → DPPHH + RX•
(2)
Since some theoretical methods, especially density func-
tional theory, have been successfully used to investigate
and elucidate the structure-activity relationship (SAR) of
antioxidants, this would also be helpful in clarifying our
compounds, and thus in the design of novel antioxidants
with better pharmacological effects.
The activity patterns of compounds on LP, superoxide
anion formation and DPPH radical scavenging activity
were dissimilar. Distinct antioxidant effects of chemicals
have also been previously noted in different in vitro assay
systems (Dündar et al., 2002; Ölgen and Coban, 2003).
Thus, herein, observation of the distinct effects of some
compounds on the assay systems utilized was not
surprising, as the mechanisms of production of oxidative
stress in these assays are different (Kornbrust and Mavis,
1980; Parke et al., 1991; Dix and Aikens, 1993).
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