DNA binding affinity and antioxidative activity of copper(II) and zinc(II) complexes with a novel hesperetin Schiff base ligand

Article abstract of DOI:10.1016/j.ica.2009.07.008

A hesperetin Schiff base ligand (H₄L) and its complexes, [H₃CuL·OAc]·H₂O and [H₃ZnL·OAc]·2H₂O, have been synthesized and characterized on the basis of elemental analysis, molar conductivity, ¹

Full text of DOI:10.1016/j.ica.2009.07.008

Inorganica Chimica Acta 362 (2009) 4823–4831
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
DNA binding affinity and antioxidative activity of copper(II) and zinc(II)
complexes with a novel hesperetin Schiff base ligand
*
Yong Li, Zheng-yin Yang
College of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A hesperetin Schiff base ligand (H4L) and its complexes, [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O, have
been synthesized and characterized on the basis of elemental analysis, molar conductivity, 1H NMR, mass
spectra, UV–Vis spectra and IR spectra. The binding of these two complexes and the ligand to DNA has
been investigated by ultraviolet absorption spectroscopy, fluorescence spectroscopy and viscosity mea-
surements. The experiments indicate that all the compounds can bind to DNA through an intercalative
mode and the complexes intercalate into DNA more deeply than that of the ligand. In addition, the anti-
oxidative activity was also determined. The 50% inhibition obtained for the ligand and its complexes
demonstrates that, compared to the ligand, the complexes exhibit higher antioxidative activity in the
Received 17 December 2008
Received in revised form 25 April 2009
Accepted 10 July 2009
Available online 17 July 2009
Keywords:
Hesperetin Schiff base
Metal complexes
Ultraviolet absorption spectroscopy
Fluorescence spectroscopy
Viscosity measurements
DNA binding
suppression of O2 and HOÅ.
ꢁÅ
Ó 2009 Elsevier B.V. All rights reserved.
Antioxidative activity
1. Introduction
flavonoids receive considerable attention in the literature, mainly
because of their biological and physiological importance [14]. They
Since deoxyribonucleic acid (DNA) is an important cellular
receptor, many chemicals exert their antitumor effects through
binding to DNA thereby changing the republication of DNA and
inhibiting the growth of the tumor cell, which is the basis of
designing new and more efficient antitumor drugs and their effec-
tiveness depends on the mode and affinity of the binding [1–4].
Similarly, interactions of metallic antitumor agents with DNA are
also important in understanding the mechanism of their antitumor
activities [5–8]. The numerous biological experiments performed
so far suggest that DNA is the primary intracellular target of anti-
cancer drugs because the interaction between small molecules and
DNA can induce DNA damages in cancer cells, blocking the division
of cancer cells and resulting in the cell death [9–11], so exploring
the mechanism of compounds binding to DNA possesses signifi-
cant meanings, which provides certain scientific research founda-
tion and the theory basis for designing the low poisonous and
highly effective drugs.
are potentially antioxidant, antibacterial, anticancer, anti-inflam-
matory and antiallergenic agents since they stimulate or inhibit a
wide variety of enzyme systems as pharmacological agents [15–
18]. The best-known and studied flavonoids are quercetin, morin
and rutin, whereas hesperetin is one of the lesser-known flavo-
noids. Hesperetin (5,7,30-trihydroxyl-40-methoxyl-flavanone) is a
kind of flavonoid which occurs ubiquitously in plants, fruits, flow-
ers and foods of plant origin [19]. Hesperetin has multiple biolog-
ical and pharmacological activities, including antioxidant
properties [20,21], inhibition of cancer development [22,23], ef-
fects on the blood–brain barrier [24,25], signal transduction path-
ways [26], etc. However, there were rarely reports about
complexes with a hesperetin Schiff base ligand binding to DNA.
In order to study complexes with a hesperetin Schiff base ligand
binding to DNA and improve bioactivities of the complexes, we
have used hesperetin and benzoyl hydrazine to synthesize the tar-
geted Schiff base ligand.
Flavonoids are a large group of phenolic plant constituents.
They are broadly distributed in vascular plants and responsible
for most of the colors in nature [12]. To date, more than 6000 flavo-
noids have already been identified, although only a smaller num-
ber is important from a dietary point of view [13]. Recently,
In this paper, we synthesized and characterized a novel Schiff
base ligand, 5,7,30-trihydroxyl-40-methoxyl-flavanone benzoyl
hydrazone (H4L), and its complexes, [H3CuLꢀOAc]ꢀH2O and
[H3ZnLꢀOAc]ꢀ2H2O. Their DNA binding properties have been eluci-
dated using ultraviolet absorption spectroscopy, fluorescence spec-
troscopy and viscosity measurements. The results show that the
ligand and its two complexes can bind to DNA by the intercalative
mode. In addition, the antioxidative activity was also determined.
* Corresponding author. Tel.: +86 931 8913515; fax: +86 931 8912582.
E-mail address: yangzy@lzu.edu.cn (Z.-y. Yang).
0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2009.07.008

4824
Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
The 50% inhibition obtained for the ligand and its complexes dem-
onstrates that, compared to the ligand, the complexes exhibit high-
for one day with stirring. After that, a brown solution was concen-
trated and cooled, a grey solid was separated out. The grey precip-
itate was filtered, washed with massive water, and recrystallized
from DMF and water to give the ligand. Yield: 3.0 g, 72%. 1H
er antioxidative activity in the suppression of O2 and HOÅ. Our
ꢁÅ
research should be valuable for seeking and designing new antitu-
mor drugs and antioxidants, as well as for understanding the
mechanism of compounds binding to DNA and helical conforma-
tion of nucleic acids.
NMR (DMSO-d6, 200 MHz),
d (ppm) 2.51 (1H, dd, J = 12.3,
17.0 Hz, 3(a)-H), 2.88 (1H, dd, J = 3.0, 17.0 Hz, 3(e)-H), 5.06 (1H,
dd, J = 3.0, 12.3 Hz, 2-H), 5.87 (1H, d, J = 2.2 Hz, 6-H), 5.93 (1H, d,
J = 2.2 Hz, 8-H), 6.93 (3H, bs, 20, 50, 60H), 7.52 (2H, dd, J = 1.8,
7.4 Hz, 300, 500H), 7.56 (2H, dd, J = 7.4, 11.6 Hz, 400H), 7.88 (2H, dd,
J = 1.8, 11.6 Hz, 200, 600H), 9.09 (1H, s, 30-OH), 9.99 (1H, s, 7-OH),
11.12 (1H, s, –NH–C@O), 13.06 (1H, s, 5-OH). M.p. 264ꢁ266 °C.
Anal. Calc. for C23H20N2O6: C, 65.71; H, 4.79; N, 6.66. Found: C,
2. Experimental
2.1. Materials and methods
65.51; H, 4.92; N, 6.76%. IR
1605. kmax(nm): 238, 327 nm.
m m(C@O): 1642, m(C@N):
max(cmꢁ1):
2.1.1. Materials
Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2), ethidium bromide (EB) and calf thymus DNA (CT DNA)
were purchased from Sigma Chemical Co. Ethylenediaminetetra-
aceticacid (EDTA), safranin, benzoyl hydrazine, acetic acid, hes-
peretin, M(OAc)2ꢀnH2O [M = Cu(II), n = 1; Zn(II), n = 2] were
produced in China. All chemicals used were of analytical grade.
EDTA–Fe(II) and KH2PO4–K2HPO4 buffers were prepared with dou-
bly distilled water.
All the experiments involved with the interaction of the com-
plexes with DNA were carried out in doubly distilled water buffer.
The water buffer was produced by mixing 5 mM Tris
[Tris(hydroxymethyl)-aminomethane] and 50 mM NaCl with dou-
bly distilled water and adjusted to pH 7.2 with hydrochloric acid.
The solution of DNA in the buffer gave a ratio of UV absorbance
of about 1.8–1.9:1 at 260 and 280 nm, which indicated that the
DNA was sufficiently free of protein [27]. DNA concentration per
nucleotide was determined spectrophotometrically by employing
an extinction coefficient of 6600 Mꢁ1 cmꢁ1 at 260 nm [28]. The
complexes were dissolved in a solvent mixture of 1% DMF and
99% Tris–HCl buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.2) at the
concentration 1.0 ꢂ 10ꢁ5 M.
2.2.2. Preparation of the complexes
The ligand (84 mg, 0.20 mmol) was dissolved in acetone
(20 mL). After 5 min, Cu(OAc)2ꢀH2O (48 mg, 0.24 mmol) was added
into the ligand solution quickly and the solution was refluxed on a
water-bath for 10 h with stirring.
A
green precipitate,
[H3CuLꢀOAc]ꢀH2O, was separated from the solution by suction fil-
tration, purified by washing several times with methanol and dried
for 24 h in vacuo. [H3ZnLꢀOAc]ꢀ2H2O, a yellow solid, was prepared
by the same way as above. Anal. Calc. for [H3CuLꢀOAc]ꢀH2O: C,
53.62; H, 4.32; N, 5.00; Cu, 11.35. Found: C, 53.78; H, 4.23; N,
5.16; Cu, 11.63%. IR
m
max(cmꢁ1):
m
(C@O): 1616, (C@N): 1590,
m
m
as(COOꢁ): 1564, s(COOꢁ): 1369,
m
m
(M–O): 481, m(M–N): 410.
kmax(nm): 240, 347 nm. KM (S cm2 molꢁ1) 10ꢁ3 DMF solution,
10.8. EI-ESI: m/z = 561 [M+H]+. Anal. Calc. for [H3ZnLꢀOAc]ꢀ2H2O:
C, 51.78; H, 4.52; N, 4.83; Zn, 11.28. Found: C, 51.85; H, 4.63; N,
4.69; Zn, 11.48%. IR
m
max(cmꢁ1):
m(C@O): 1616,
m(C@N): 1591,
m
as(COOꢁ): 1562,
m
s(COOꢁ): 1364,
m(M–O): 493, m(M-N): 409.
kmax(nm): 240, 329 nm. KM (S cm2 molꢁ1) 10ꢁ3 DMF solution, 24.3.
2.2.3. Electronic absorption titration
Absorption titration experiments were performed with fixed
concentration drugs (10 lM), while gradually increasing the con-
centration of DNA. When measuring the absorption spectra, an
equal amount of DNA was added to both the complex solutions
and the reference solution to eliminate the absorbance of DNA it-
self. Each sample solution was scanned in the range 190–500 nm.
2.1.2. Methods
Elemental analysis (C, H, N) was carried out on an Elemental
Vario EL analyzer. The metal contents of the complexes were deter-
mined by titration with ethylenediaminetetraacetic acid (EDTA).
The melting point of the ligand was determined on a Beijing
XT4-100X microscopic melting point apparatus. The IR spectra
were obtained in KBr discs on a Therrno Mattson FTIR spectropho-
tometer in the 4000–400 cmꢁ1 region. 1H NMR spectra were re-
2.2.4. Fluorescence spectra
To compare quantitatively the affinity of the compounds bound
to DNA, the intrinsic binding constants Kb of all the compounds to
DNA were obtained by the luminescence titration method. Fixed
amounts of compound were titrated with increasing amounts of
corded on
a Varian VR 200-MHz spectrometer in DMSO
(dimethyl sulfoxide)-d6 with TMS (tetramethylsilane) as internal
standard. Mass spectra were performed on a APEX II FT-ICR MS
instrument using methanol as mobile phase. Conductivity mea-
surements were performed in DMF (N,N-dimethylformamide) with
a DDS-11C conductometer at 25.0 °C. The UV–Vis spectra of the
compounds (DMF solution) were recorded on a Varian Cary 100
Cone spectrophotometer. Fluorescence measurements were re-
corded on a Hitachi RF-4500 spectrofluorophotometer. Quantum
yields of the ligand and its complexes were determined by an abso-
lute method using an integrating sphere on FLS920 of Edinburgh
Instrument. The absorbance was performed in DMF solution with
a 721-E spectrophotometer in measuring the antioxidative activity
of all the compounds.
DNA, over a range of DNA concentrations from 2.5 to 20 lM. An
excitation wave length of 329 nm ([H3CuLꢀOAc]ꢀH2O) and 322 nm
([H3ZnLꢀOAc]ꢀ2H2O) was used, and fluorescence emission intensity
was monitored at 454 and 453 nm, respectively. Kb values of all the
compounds bound to DNA were calculated using equation [29]
r=Cf ¼ Kbðn ꢁ rÞ;
Cb ¼ Ct½ðF ꢁ F0Þ=ðFmax ꢁ F0Þꢃ;
Cf ¼ Ct ꢁ Cb;
where Ct is the total compound concentration, F is the observed
fluorescence emission intensity at given DNA concentration, F0 is
the intensity in the absence of DNA, and Fmax is the fluorescence
of the totally bound compound. Binding data were cast into the
form of a Scatchard plot of r/Cf versus r, where r is the binding ratio
Cb/[DNA]t, Cf is the free compound concentration and n is the
binding site number. All experiments were conducted at 20 °C in
a buffer containing 5 mM Tris–HCl (pH 7.2) and 50 mM NaCl
concentrations.
2.2. Synthesis of ligand (H4L) and complexes
2.2.1. Preparation of ligand (H4L)
Scheme of the synthesis of the ligand is shown in Fig. 1. Hes-
peretin (3.02 g, 10 mmol) and benzoyl hydrazine (1.50 g, 11 mmol)
were dissolved in methanol (40 mL). Acetic acid (1.0 mL) was
added to this solution. The solution was refluxed on a water-bath

Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
4825
Fig. 1. Scheme of the synthesis of the ligand (H4L).
Further support for the ligand and complexes binding to DNA
via intercalation is given through the emission quenching experi-
ment. EB is a common fluorescent probe for DNA structure and
has been employed in examinations of the mode and process of
was prepared with DMF. The reaction mixture contained 2.5 mL
0.15 M phosphate buffer (pH 7.4), 0.5 mL 114 M safranin, 1 mL
945 M EDTA–Fe(II), 1 mL 3% H2O2 and 30 L the tested compound
solution (the final concentration: Ci (i=1–6) = 1.0, 2.0, 3.0, 4.0, 5.0,
6.0 M). The sample without the tested compound was used as
the control. The reaction mixtures were incubated at 37 °C for
60 min in a water-bath. Absorbances (Ai, A0, Ac) at 520 nm were
measured. The suppression ratio for HOÅ was calculated from the
following expression:
l
l
l
metal complexes binding to DNA [7]. A 2.5 mL solution of 4
DNA and 0.32 M EB (at saturating binding levels) was titrated
by the 5–50 M complexes and ligand. Quenching data were ana-
lM
l
l
l
lyzed according to the SternꢁVolmer equation, which could be
used to determine the fluorescent quenching mechanism:
F0=F ¼ 1 þ Kq½Qꢃ;
Suppression ratio ð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢃ ꢂ 100%
where F0 and F are the fluorescence intensity in the absence and in
the presence of drug at [Q] concentration respectively; Kq is the
quenching constant and [Q] is the quencher concentration. Plots
of Fo/F versus [Q] appear to be linear and Kq depends on tempera-
ture [30].
where Ai is the absorbance in the presence of the tested compound;
A0 is the absorbance in the absence of the tested compound; Ac is
the absorbance in the absence of the tested compound, EDTA–Fe(II)
and H2O2.
3. Results and discussion
2.2.5. Viscosity measurements
Viscosity experiments were conducted on an Ubbelodhe vis-
cometer, immersed in a thermostated water-bath maintained to
3.1. Characterization of complexes
25.0 °C. Titrations were performed for the complexes (0.5–3
l
l
M),
M)
versus
The likely structures of complexes are shown in Fig. 2, which is
in accord with elemental analysis, IR spectra, UV–Vis spectra and
molar conductivity. The results suggest that the compositions of
the two complexes is [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O,
respectively. The two complexes are air stable for extended periods
and easily soluble in DMF and DMSO; slightly soluble in acetone,
chloroform, methanol, ethanol and water; insoluble in benzene
and diethyl ether. The molar conductance values of the complexes
is 10.8 and 24.3 Scm2 molꢁ1 in 10ꢁ3 DMF solution respectively,
which show that the two complexes are non-electrolytes in DMF
solution [35].
and each compound was introduced into a DNA solution (5
1/3
present in the viscometer. Data were presented as (g/g0)
the ratio of the concentration of the compound and DNA, where
is the viscosity of DNA in the presence of the compound and g0 is
the viscosity of DNA alone [31,32].
g
2.3. Antioxidative activity
2.3.1. Superoxide radical scavenging activity
The superoxide radicals (O2ꢁÅ) were produced by the system
ꢁÅ
MET/VitB2/NBT [33]. The amount of O2 and suppression ratio
IR spectra provide a lot of information on the structure of com-
ꢁÅ
plexes. m
(C@O) vibration of the free ligand is at 1642 cmꢁ1, for the
for O2
can be calculated by measuring the absorbance at
two complexes the peaks shift to 1590 and 1591 cmꢁ1, respec-
tively. The bands, which appear at 481 and 493 cmꢁ1 respectively
560 nm, because NBT can be reduced quantitatively to blue forma-
zan by O2ꢁÅ. The solution of MET, VitB2 and NBT were prepared
with 0.067 M phosphate buffer (pH 7.8) at the condition of avoid-
ing light. The tested compounds were dissolved in DMF. The reac-
after reaction with the metal ions are assigned to m(M–O). This
means that the oxygen of the carbonyl has likely formed coordina-
(0.01 mol Lꢁ1),
NBT
tion bonds with the metal ions [36]. The band at 1604 cmꢁ1 for the
tion
mixture
contained
MET
(4.6 ꢂ 10ꢁ5 mol Lꢁ1), VitB2 (3.3 ꢂ 10ꢁ6 mol Lꢁ1), phosphate buffer
free ligand is assigned to
shift to 1616 and 1562 cmꢁ1. Bands appearing at 410 and 409 cmꢁ1
respectively are assigned to (M–N). These data indicate that the
m(C@N); for the two complexes, the peaks
solution (0.067 mol Lꢁ1) and the tested compound (the final con-
centration: Ci (i=1–6) = 0.4, 1.0, 2.0, 4.0, 6.0, 8.0 lM). After incubating
m
at 30 °C for 10 min and illuminating with a fluorescent lamp for
3 min, the absorbance (Ai) of the samples were measured at
560 nm. The sample without the tested compound and avoiding
nitrogen of the imino group also bonds to the metal ions [37].
[H3CuLꢀOAc]ꢀH2O displays both asymmetric and symmetric
stretching vibration of COOꢁ at 1564 and 1369 cmꢁ1 and
ꢁÅ
[H3ZnLꢀOAc]ꢀ2H2O is at 1562 and 1364 cmꢁ1. The
Dm = 195 and
light was used as the control. The suppression ratio for O2 was
198 cmꢁ1 are bigger than that of CH3COONa (ca. 150 cmꢁ1), which
suggests the acetate group is monodentate coordination with
Cu(II) and Zn(II) ions.
calculated from the following expression:
Suppression ratio ð%Þ ¼ ½ðA0 ꢁ AiÞ=A0ꢃ ꢂ 100%
where Ai is the absorbance in the presence of the ligand or its com-
plexes, A0 is the absorbance in the absence of the ligand or its
complexes.
The studies of the electronic spectra in the ultraviolet and visi-
ble ranges for the ligand and complexes were carried out in the
buffer solution. The ligand has an intensive band at 238 nm and
a less intensive band at 327 nm. There are two bands at 240 and
347 nm for [H3CuLꢀOAc]ꢀH2O, two bands at 240 and 329 nm for
[H3ZnLꢀOAc]ꢀ2H2O. These changes indicate that the complexes
are formed.
2.3.2. Hydroxy radical scavenging activity
The hydroxyl radical in aqueous media was generated through
the Fenton reaction [34]. The solution of the tested compounds

4826
Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
(a)
(b)
OH
OH
O
O
HO
O
HO
O
H2O
2H2O
O
N
O
O
N
O
NH
Cu
NH
Zn
AcO
AcO
,
Fig. 2. (a) Conjectural structure of [H3CuLꢀOAc]ꢀH2O. (b) Conjectural structure of [H3ZnLꢀOAc]ꢀ2H2O.
3.2. DNA binding interaction
was employed since EB interacts with DNA as a typical indicator
of intercalation [39]. Fig. 4 shows that the maximal absorption of
EB at 479 nm decreased and shifted to 492 nm in the presence of
DNA, which was characteristic of interaction. Curve (B) in Fig. 4a
The interaction of the free ligand, [H3CuLꢀOAc]ꢀH2O and
[H3ZnLꢀOAc]ꢀ2H2O with DNA was investigated by means of elec-
tronic absorption, spectrofluorimetric titration and viscosity mea-
surements to evaluate their binding affinities and mechanisms.
Electronic absorption spectroscopy was an effective method in
examing the binding mode of DNA with metal complexes. Interca-
lative mode of binding usually results in hypochromism and red
shift because of the strong stacking interaction between an aro-
matic chromophore and the base pairs of DNA. The extent of the
spectral change is related to the strength of binding [38]. The elec-
tronic absorption titration of the ligand and complexes is shown in
Fig. 3. The absorption bands of the ligand at 327 nm exhibited hyp-
ochromism of 40% and bathochromism of 2 nm. Otherwise, the
absorption band of [H3CuLꢀOAc]ꢀH2O at 347 nm exhibited hypo-
chromism of 98%, and bathochromism of 3 nm and [H3ZnLꢀOAc]ꢀ
2H2O at 340 nm exhibited hypochromism of 50% and bathochro-
mism of 4 nm. These results suggest an association of these com-
pounds with DNA and we primarily speculate that the ligand and
its complexes interacting with DNA have the same mechanism
(intercalation mode) of electrostatic effect or interaction.
was the absorption of
a mixture solution of EB, DNA and
[H3CuLꢀOAc]ꢀH2O. Curve (B) in Fig. 4b was the absorption of a
mixture solution of EB, DNA and [H3ZnLꢀOAc]ꢀ2H2O. It was found
that the absorption at 492 nm increased comparing with curve
(C) in Fig. 4. It could result from two reasons: (1) EB bound to
the complexes strongly, resulting in the decreased amount of
EB intercalated into DNA; (2) there exists competitive intercala-
tion between the complexes and EB with DNA and then releases
some free EB from DNA–EB system. However, the former reason
could be precluded because there were no new absorption peaks
appearing.
Fig. 5 exemplifies the spectrofluorimetric titration of the ligand
and complexes with DNA. It is observed that the fluorescence
intensity increases with the addition of DNA into the buffer solu-
tion of the free ligand and its complexes, which agrees with those
observed for other intercalators and indicate their intercalation
with DNA. The results of the emission titrations suggest that all
the compounds are protected from solvent water molecules by
the hydrophobic environment inside the DNA helix and that the
complexes can be protected more effectively than the free ligand.
This indicates that all the compounds can insert between DNA base
In order to test whether the complexes can bind to DNA by
the mode of intercalation, competitive EB binding studies were
undertaken to gain supports for the above spectral result. EB
(c)
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
-0.04
0.18
(b)
(a)
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
270 300 330 360 390 420 450
Wavelength (nm)
250
300
350
400
450
250 300 350 400 450
Wavelength (nm)
500
Wavelength (nm)
Fig. 3. (a) Electronic spectra of ligand in the presence of increasing amounts of DNA; [DNA] = 0–25
amounts of DNA; [DNA] = 0–25
M. (c) Electronic spectra of [H3ZnLꢀOAc]ꢀ2H2O in the presence of increasing amounts of DNA; [DNA] = 0–25
changes upon increasing DNA concentration.
l
M. (b) Electronic spectra of [H3CuLꢀOAc]ꢀH2O in the presence of increasing
l
l
M. Arrows show the absorbance

Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
4827
0.05
A
0.05
0.04
0.03
0.02
0.01
0.00
(a)
(b)
A
B
B
C
0.04
0.03
0.02
0.01
0.00
C
400
500
600
400
500
600
Wavelength (nm)
Wavelength (nm)
Fig. 4. (a) The visible absorption spectra of 1 ꢂ 10ꢁ5 M EB (A); (A) + 2.5 ꢂ 10ꢁ5 M DNA (C); (C) + 2.5 ꢂ 10ꢁ5 M [H3CuLꢀOAc]ꢀH2O (B) in Tris–HCl buffer (5 mM Tris–HCl, 50 mM
NaCl, pH 7.2) solution. (b) The visible absorption spectra of 1 ꢂ 10ꢁ5 M EB (A); (A) + 2.5 ꢂ 10ꢁ5 M DNA (C); (C) + 2.5 ꢂ 10ꢁ5 M [H3ZnLꢀOAc]ꢀ2H2O (B) in Tris–HCl buffer (5 mM
Tris–HCl, 50 mM NaCl, pH 7.2) solution.
pairs and the complexes can bind to DNA more strongly than that
of the free ligand. Since the hydrophobic environment inside the
DNA helix reduces the accessibility of solvent water molecules to
the compounds and the compounds mobility is restricted at the
binding site, a decrease of the vibrational modes of relaxation re-
sults. And then the compounds binding to DNA leads to a marked
increase in the fluorescence emission intensity, which accords with
other intercalators [27]. According to the Scatchard equation, Kb of
the free ligand, [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O are
decrease upon the addition of the compounds, which shows that
all the compounds can bind to DNA via the same mode (intercala-
tion mode). According to linear Stern–Volmer equation, in the plots
of F0/F versus [Q], Kq is given by the ratio of the slope to the inter-
cept. Kq values of the free ligand, [H3CuLꢀOAc]ꢀH2O and
[H3ZnLꢀOAc]ꢀ2H2O are (4.73 0.05) ꢂ 104, (8.91 0.04) ꢂ 104 and
(6.36 0.06) ꢂ 104 Mꢁ1, respectively. These data show that the
strength of complexes binding to DNA is stronger than that of
the free ligand, which is consistent with the results obtained from
electronic absorption titration and spectrofluorimetric titration.
In order to further clarify the interactions between the com-
pounds and DNA, viscosity measurements were carried out.
Hydrodynamic measurements that are sensitive to length change
(i.e. viscosity and sedimentation) are regarded as the least ambig-
uous and the most critical tests of binding in solution in the ab-
sence of crystallographic structural data [44]. The effects of the
free ligand, [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O on the vis-
cosity of DNA at 25 °C are shown in Fig. 7. Viscosity experimental
results clearly show that all the compounds can intercalate be-
tween adjacent DNA base pairs, causing an extension in the helix,
and thus increase the viscosity of DNA. The complexes can inter-
calate more strongly than the free ligand and [H3CuLꢀOAc]ꢀH2O
can intercalate more deeply than [H3ZnLꢀOAc]ꢀ2H2O, leading to
a greater increase in viscosity of the DNA with an increasing con-
centration of complexes. The results obtained from viscosity mea-
surements substantiate those obtained from spectroscopic
studies.
(2.70 0.19) ꢂ 105,
(2.87 0.05) ꢂ 106
and
(1.43 0.06) ꢂ
106 Mꢁ1, respectively. The values of these compounds are some-
what larger than some known DNA intercalators, such as
Kb = 4.8 ꢂ 104 Mꢁ1 for [Ru(bpy)2(phi)]Cl2 (phi = 9,10-phenanthren-
equinonediimine) [40], 2.1 ꢂ 104 Mꢁ1 for [Ru(bpy)2(ddt)]2+
(ddt = 3-(pyrazin-yl)-5,6-diphenyl-as-triazine) [41], 6.3 ꢂ 104 Mꢁ1
for [Ru(bpy)2(dpt)]2+ (dpt = 3-(pyrazin-yl)-as-triazino[5,6-f]phen-
anthrene) [41], and also larger than that observed for [Ru(b-
py)2(dppz)]2+ (>106) [42]. These data show that all the
compounds can bind to DNA and the binding strength of com-
plexes is stronger than that of the free ligand. The large Kb value
is the direct evidence that the three compounds have good abilities
to bind to DNA. In addition, the binding site numbers of the ligand,
[H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O were 3.25, 0.98 and 0.97,
respectively. Generally, the binding site numbers of other com-
pounds are 1 binding site per 4 nucleotides. The noticeable binding
site numbers may be due to the multi-hydroxyls of theses
compounds.
Quantum yields of the ligand and its complexes were deter-
mined by an absolute method using an integrating sphere on
FLS920 of Edinburgh Instrument. The quantum yields of the ligand
and [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O were 0.0504, 0.0459
and 0.0354, respectively, indicating that the fluorescence of all
the compounds is not very strong.
3.3. Antioxidative activity
3.3.1. Superoxide radical scavenging activity
Fig. 8 and Table 1 depict the inhibitory effect of the compounds
on O2ꢁÅ. The average suppression ratio for O2 increases with the
ꢁÅ
The DNA binding mode of all the compounds was further mon-
itored by a fluorescent EB displacement assay. It is well known that
EB can intercalate nonspecifically into DNA which causes it to fluo-
resce strongly. Competitive binding of other drugs to DNA and EB
will result in displacement of DNA and a decrease of the fluores-
cence intensity. The fluorescence-based competition technique
can provide indirect evidence for the DNA binding mode. The emis-
sion band of the DNA–EB system decreased in intensity with an in-
crease of the compounds concentrations, which indicated that the
compounds can displace EB from DNA–EB system [43]. Such char-
acteristic change is usually observed in DNA intercalative interac-
tions. Fig. 6 shows the emission spectra of DNA–EB system with
the increase of the free ligand, [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ
2H2O. Obviously, the fluorescence intensities of DNA–EB system
increasing amount of the compound concentration in the range
of tested concentration. The average suppression ratio of the ligand
(IC50 = 31.287
lM) is the least in all the compounds and
[H3CuLꢀOAc]ꢀH2O (IC50 = 0.401
l
M) is the more effective inhibitor
than [H3ZnLꢀOAc]ꢀ2H2O (IC50 = 4.098
lM). It is clear that the scav-
ꢁÅ
enger effect on O2 can be enhanced by the formation of metal-li-
gand complexes and the metal ions also affect the ability. The
transition metal ions, such as Cu(II) and Zn(II) may have different
and selective nature for scavenging O2ꢁÅ. Due to the lower IC50 val-
ues, they are potential drugs to eliminate the radicals.
3.3.2. Hydroxy radical scavenging activity
The data of suppression ratio for HOÅ are shown in Fig. 9 and
Table 2. The inhibitory effect of all the compounds is marked and

4828
Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
8
200
150
100
50
(a)
K = (2.70 0.19)X10 M
K = (2.70 0.19)X10 M
6
4
2
0
0.5
1.0
1.5
2.0
2.5
400
450
500
550
r
Wavelength λ/nm
7
6
5
4
3
2
1
(b) 600
500
400
300
200
100
0
K = (2.87 0.52)X10 M
K = (2.87 0.52)X10 M
400
450
500
550
600
0.75
0.80
0.85
r
0.90
0.95
Wavelength λ/nm
6
5
4
3
2
1
600
(c)
500
400
300
200
100
0
K = (1.43 0.63)X10 M
K = (1.43 0.63)X10 M
400
450
500
550
600
0.55
0.60
0.65
0.70
0.75
r
0.80
0.85
0.90
0.95
Wavelength λ/nm
Fig. 5. (a) The emission enhancement spectra of ligand (10
l
M) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5
l
M DNA. Inset: Scatchard plot of the fluorescence
titration data of ligand, Kb = (2.70 0.19) ꢂ 105 Mꢁ1. (b) The emission enhancement spectra of [H3CuLꢀOAc]ꢀH2O (10
lM) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15 and
17.5
(10
l
M DNA. Inset: Scatchard plot of the fluorescence titration data of [H3CuLꢀOAc]ꢀH2O, Kb = (2.87 0.05) ꢂ 106 Mꢁ1. (c) The emission enhancement spectra of ligand
l
M) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5
lM
DNA. Inset: Scatchard plot of the fluorescence titration data of [H3ZnLꢀOAc]ꢀ2H2O,
Kb = (1.43 0.06) ꢂ 106 Mꢁ1. Arrows show the emission intensity changes upon increasing DNA concentration.
the average suppression ratio for HOÅ increases with the increasing
compounds concentration. The average suppression ratio of the
ligand is the poorest in all the compounds, [H3CuLꢀOAc]ꢀH2O
(IC50 = 6.065 M) are
M) and [H3ZnLꢀOAc]ꢀ2H2O (IC50 = 10.709
l
l

Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
4829
100
80
60
40
20
0
2.0
1.9
1.8
(a)
K = (4.73 0.05) X 10 M
K = (4.73 0.05) X 10 M
1.7
1.6
1.5
1.4
1.3
1.2
1.1
0
2
4
6
8
10
12
14
16
18
20
550
600
650
700
Wavelength (nm)
[Compound] X 10
M
120
100
80
60
40
20
0
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
(b)
K = (8.91 0.04) X 10 M
K = (8.91 0.04) X 10 M
0
2
4
6
8
10
12
14
16
18
20
550
600
650
700
[Compound] X 10 M
Wavelength (nm)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
100
80
60
40
20
0
(c)
K = (6.36 0.06) X 10 M
K = (6.36 0.06) X 10 M
0
2
4
6
8
10
12
14
16
18
20
550
600
650
700
[Compound] X 10
M
Wavelength (nm)
Fig. 6. (a) The emission spectra of DNA–EB system, kex = 525 nm, kem = 550–600 nm, in the presence of 0, 2, 4, 6, 8, 10, 12, 14, 16,18 and 20 lM ligand. Inset: Stern–Volmer
plot of the fluorescence titration data of ligand. Kq = (4.73 0.05) ꢂ 104 Mꢁ1. (b) The emission spectra of DNA–EB system, kex = 525 nm, kem = 550–600 nm, in the presence of
0, 2, 4, 6, 8, 10, 12, 14, 16,18 and 20
l
M [H3CuLꢀOAc]ꢀH2O. Inset: Stern–Volmer plot of the fluorescence titration data of [H3CuLꢀOAc]ꢀH2O. Kq = (8.91 0.04) ꢂ 104 Mꢁ1. (c) The
emission spectra of DNA–EB system, kex = 525 nm, kem = 550–600 nm, in the presence of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20
lM [H3ZnLꢀOAc]ꢀ2H2O. Inset: Stern–Volmer plot
of the fluorescence titration data of [H3ZnLꢀOAc]ꢀ2H2O. Kq = (6.36 0.06) ꢂ 104 Mꢁ1. Arrows show the emission intensity changes upon increasing concentration.

4830
Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
60
1.5
1.4
1.3
1.2
1.1
1.0
b
c
50
a
40
30
20
10
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0
1
2
3
4
5
6
[M]/[DNA]
Concentration (μM)
Fig. 7. Effect of increasing amounts of the ligand (a), [H3CuLꢀOAc]ꢀH2O (b) and
[H3ZnLꢀOAc]ꢀ2H2O (c) on the relative viscosity of DNA at 25.0 °C.
Fig. 9. Scavenging effect of the ligand and its complexes on OHÅ; j, experimental
data points of the ligand; d, experimental data points of [H3CuLꢀOAc]ꢀH2O; .,
experimental data points of [H3ZnLꢀOAc]ꢀ2H2O.
100
80
the more effective inhibitors than the ligand (IC50 = 50.429 lM),
which illuminates that because of the formation of coordination
compounds, the scavenger effect can be enhanced and the transi-
tion metal ions, Cu(II) and Zn(II) also affect the scavenger ability.
60
4. Conclusions
Taken together, a novel hesperetin Schiff base ligand, 5,7,30-tri-
hydroxyl-40-methoxyl-flavanone benzoyl hydrazone (H4L), and its
complexes, [H3CuLꢀOAc]ꢀH2O and [H3ZnLꢀOAc]ꢀ2H2O have been
prepared and characterized by elemental analysis, molar conduc-
tivity, 1H NMR, mass spectra, IR spectra and UV–Vis spectra. DNA
binding properties of the free ligand and its complexes were inves-
tigated by ultraviolet absorption spectroscopy, fluorescence spec-
troscopy and viscosity measurements. The results support the
fact that all the compounds can bind to DNA via intercalation
mode. Kb values show that the complexes can bind to DNA more
strongly than that of the free ligand. In addition, the complexes
40
20
0
3
6
9
Concentration (μM)
Fig. 8. Scavenging effect of the ligand and its complexes on O2ꢁÅ; j, experimental
data points of the ligand; d, experimental data points of [H3CuLꢀOAc]ꢀH2O; .,
experimental data points of [H3ZnLꢀOAc]ꢀ2H2O.
have active scavenging effects on O2 and HOÅ. Our research
ꢁÅ
should be valuable for seeking and designing new antitumor drugs
Table 1
The influence of investigated compounds for O2ꢁÅ. IC50 values were calculated from regression lines where: x was log of the tested compound concentration and Y was percent
inhibition of the tested compounds. When the percent inhibition of the tested compounds was 50%, the tested compound concentration was IC50. R2 = correlation coefficient.
ꢁÅ
Compound
Average inhibition (%) for O2
0.4 1.0
S.41
49.67
10.66
Equation
IC50
(
l
M)
R2
lM
l
M
2.0
l
M
4.0
l
M
6.0
l
M
8.0 lM
H4L
15.11
66.59
25.53
22.06
73.43
38.78
28.54
85.52
48.84
34.17
95.61
57.90
37.89
97.23
60.25
Y = 22.67x + 16.10
Y = 36.80x + 64.60
Y = 38.95x + 26.14
31.287
0.401
4.098
0.989
0.987
0.997
[H3CuLꢀO Ac]ꢀH2O
[H3ZnLꢀOAc]ꢀH2O
Table 2
The influence of investigated compounds for HOÅ. IC50 values were calculated from regression lines where: x was log of the tested compound concentration and Y was percent
inhibition of the tested compounds. When the percent inhibition of the tested compounds was 50%, the tested compound concentration was IC50. R2 = correlation coefficient.
Compound
Average inhibition (%) for OHÅ
1.0 2.0
1.17 8.61
Equation
IC50
(
l
M)
R2
l
M
lM
3.0
l
M
4.0
l
M
5.0
l
M
6.0 lM
H4L
14.03
39.51
30.83
17.54
43.82
33.92
21.05
46.84
40.94
23.79
51.46
42.76
Y = 29.06x + 0.52
Y = 32.88x + 24.26
Y = 32.96x + 16.06
50.429
6.065
10.709
0.995
0.980
0.971
[H3CuLꢀOAc]ꢀH20
25.73
17.54
32.23
24.59
[H3ZnLꢀOAc]ꢀH20

Y. Li, Z.-y. Yang / Inorganica Chimica Acta 362 (2009) 4823–4831
4831
[18] A. Nkengfack, T. Vouffo, Z. Fomun, M. Meyer, S.O. Bergendorff, Phytochemistry
36 (1994) 1047.
and antioxidants, as well as for understanding the mechanism of
compounds binding to DNA and helical conformation of nucleic
acids.
[19] J.V. Formica, W. Regelson, Food Chem. Toxicol. 33 (1995) 1061.
[20] Y.Z. Cai, Q. Luo, M. Sun, H. Corke, Life Sci. 74 (2004) 2157.
[21] K.E. Heim, A.R. Tagliaferro, D.J. Bobilya, J. Nutr. Biochem. 13 (2002) 572.
[22] H.C. Cooray, T. Janvilisri, H.W. Veen, S.B. Hladky, M.A. Barrand, Biochem.
Biophys. Res. Commun. 317 (2004) 269.
Acknowledgements
[23] F.V. So, N. Guthrie, A.F. Chambers, K.K. Carroll, Cancer Lett. 112 (1997) 127.
[24] Y. Mitsunaga, H. Takanaga, H. Matsuo, M. Naito, T. Tsuruo, H. Ohtani, Y.
Sawada, Eur. J. Pharmacol. 395 (2000) 193.
[25] K.A. Youdim, M.S. Dobbie, G. Kuhnle, A.R. Proteggente, N.J. Abbott, C. Rice-
Evans, J. Neurochem. 85 (2003) 180.
This work is supported by the National Natural Science Founda-
tion of China (20475023) and Gansu NSF (0710RJZA012).
[26] J.O. Jim, J. Brown, J. Fleming, P.R. Harrison, Biochem. Pharmacol. 66 (2003)
2075.
References
[27] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319.
[28] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8547.
[29] G.M. Howe, K.C. Wu, W.R. Bauer, Biochemistry 19 (1976) 4339.
[30] M.R. Efink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199.
[31] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Gradsland, Biochemistry 33
(1994) 5031.
[32] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, R.B. Yu, J.
Chem. Soc., Dalton Trans. 1 (1999) 19.
[33] C.C. Winterbourn, Biochem. J. 182 (1979) 625.
[1] A.E. Friedman, C.V. Kumar, N.J. Turro, J.K. Barton, Nucleic Acids Res. 19 (1991)
2595.
[2] A.M. Pyle, T. Morri, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 9432.
[3] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986)
2081.
[4] P.H. Proctor, E.S. Reynolds, Physiol. Chem. Phys. Med. NMR 16 (1984) 175.
[5] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaircs, Biochemistry 31 (1992) 931.
[6] C. Hiort, P. Lincoln, B. Norden, J. Am. Chem. Soc. 115 (1993) 3448.
[7] C.V. Kumar, J.K. Barton, N.J. Turro, J. Am. Chem. Soc. 107 (1985) 5518.
[8] J.K. Barton, A.T. Danishefsky, J.M. Goldberg, J. Am. Chem. Soc. 106 (1984) 2172.
[9] V.S. Li, D. Choi, Z. Wang, L.S. Jimenez, M.S. Tang, H. Kohn, J. Am. Chem. Soc. 118
(1996) 2326.
[10] G. Zuber, J.C. Quada Jr., S.M. Hecht, J. Am. Chem. Soc. 120 (1998) 9368.
[11] S.M. Hecht, J. Nat. Prod. 63 (2000) 158.
[12] P. Knekt, R. Jarvinen, R. Seppanen, M. Heliovaara, L.T. Pukkala, A. Aromaa, Am. J.
Epidemiol. 146 (1997) 223.
[13] I. Erlund, Nutr. Res. 24 (2004) 851.
[14] Y. Sakaguchi, Y. Maehara, H. Baba, T. Kusumoto, K. Sugimachi, R. Newmanv,
Adv. Cancer Res. 52 (1992) 3306.
[34] C.C. Winterbourn, Biochem. J. 198 (1981) 125.
[35] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81.
[36] F. Maarchetti, C. Pettinari, R. Pettinari, D. Leonesi, A. Lorenzotti, Polyhedron 18
(1999) 3041.
[37] K. Narang, V.P. Singh, Transition Met. Chem. 18 (1993) 287.
[38] Z.D. Xu, H. Liu, S.L. Xiao, M. Yang, X.H. Bu, J. Inorg. Biochem. 90 (2002) 79.
[39] W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski, D. Boykin, Biochemistry 32
(1993) 4098.
[40] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3051.
[41] H. Deng, J.W. Cai, H. Xu, H. Zhang, L.N. Ji, Dalton Trans. (2003) 325.
[42] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem.
Soc. 112 (1990) 4960.
[15] S.J. Habtemariam, Nat. Prod. 60 (1997) 775.
[16] I. Aljancic, V. Vajs, N. Menkovic, I. Karadzic, N. Juranic, S. Milosavljevic, S.
Macura, J. Nat. Prod. 62 (1999) 909.
[43] Y.B. Zeng, N. Yang, W.S. Liu, N. Tang, J. Inorg. Biochem. 97 (2003) 258.
[44] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573.
[17] F. Ko, C. Chu, C. Lin, C. Chang, C. Teng, Biochim. Biophys. Acta 1389 (1998) 81.