Synthesis, characterization, DNA binding properties, fluorescence studies and antioxidant activity of transition metal complexes with hesperetin-2-hydroxy benzoyl hydrazone

Article abstract of DOI:10.1007/s10895-010-0635-z

A novel Schiff-base ligand (H₅L), hesperetin-2-hydroxy benzoyl hydrazone, and its copper (II), zinc (II) and nickel (II) complexes (M?H₃L) [M(II) = Cu, Zn, Ni], have been synthesized and characterized. The ligand and Zn (II) complex

Full text of DOI:10.1007/s10895-010-0635-z

J Fluoresc (2010) 20:891–905
DOI 10.1007/s10895-010-0635-z
ORIGINAL PAPER
Synthesis, Characterization, DNA Binding Properties,
Fluorescence Studies and Antioxidant Activity of Transition
Metal Complexes with Hesperetin-2-hydroxy
Benzoyl Hydrazone
Yong Li & Zheng-Yin Yang & Ming-Fang Wang
Received: 3 December 2009 /Accepted: 15 March 2010 /Published online: 30 March 2010
#
Springer Science+Business Media, LLC 2010
Abstract A novel Schiff-base ligand (H5L), hesperetin-2-
hydroxy benzoyl hydrazone, and its copper (II), zinc (II)
and nickel (II) complexes (M·H3L) [M(II) = Cu, Zn, Ni],
have been synthesized and characterized. The ligand and
Zn (II) complex exhibit green and blue fluorescence under
UV light and the fluorescent properties of the ligand and
Zn (II) complex in solid state and different solutions
were investigated. In addition, DNA binding properties
of the ligand and its metal complexes have been investi-
gated by electronic absorption spectroscopy, fluorescence
spectra, ethidium bromide displacement experiments, io-
dide quenching experiments, salt effect and viscosity
measurements. Results suggest that all the compounds bind
to DNA via an intercalation binding mode. Furthermore, the
antioxidant activity of the ligand and its metal complexes
was determined by superoxide and hydroxyl radical
scavenging methods in vitro. The metal complexes were
found to possess potent antioxidant activity and be better
than the free ligand alone and some standard antioxidants
like vitamin C and mannitol.
Introduction
DNA binding metal complexes have been extensively
investigated during the past several decades because they
can be used as potential anticancer drugs, DNA structural
probes, DNA-dependent electron transfer probes, DNA
footprinting, sequence-specific cleaving agents and so on
[1–3]. In order to develop new potential DNA targeting
antitumor drugs, the binding mechanisms of metal com-
plexes to DNA should be further studied. Basically, metal
complexes interact with double helix DNA in either non-
covalent or covalent way and the former way includes three
binding modes, i.e., intercalation, groove binding and
external static electronic effects. Among these interaction
modes, intercalation is one of the most important DNA
binding modes because numerous biological experiments
have demonstrated that DNA is the primary intracellular
target of anticancer drugs due to the intercalation between
small molecules and DNA, which can cause DNA damages
in cancer cells, blocking the division of cancer cells and
resulting in cell death [4–6]. It was reported that DNA
binding mode and affinity are affected by a number of
factors, such as planarity of ligands [7, 8], the coordination
geometry, the ligand donor atom type [9, 10], the metal ion
type and its flexible valence [11, 12].
.
Keywords Hesperetin-2-hydroxy benzoyl hydrazone
.
.
Transition metal complexes Fluorescence studies DNA
.
binding properties Antioxidant activity
Flavonoids are a broadly distributed class of naturally
occurring pigments present in vascular plants and are
responsible for most of the coloring in nature [13]. Up to
now, more than 6,000 flavonoids have already been
identified, although only a smaller number is important
from a dietary point of view [14]. Recently, flavonoids
receive considerable attention in the literature, mainly due
to their biological and physiological importance [15]. They
are potentially antioxidant, antibacterial, anticancer, anti-
:
:
Y. Li Z.-Y. Yang (*) M.-F. Wang
College of Chemistry and Chemical Engineering and State Key
Laboratory of Applied Organic Chemistry, Lanzhou University,
Lanzhou 730000, People’s Republic of China
e-mail: yangzy@lzu.edu.cn

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J Fluoresc (2010) 20:891–905
inflammatory and antiallergenic agents since they can
stimulate or inhibit a wide variety of enzyme systems as
pharmacological agents [16–19]. The best-known and
studied flavonoids are quercetin, morin and rutin, whereas
hesperetin is one kind of the lesser-known flavonoids.
Hesperetin (5,7,3′-trihydroxyl-4′-methoxyl-flavanone) usu-
ally occurs ubiquitously in plants, fruits, flowers and foods
of plant origin [20]. As an important bioactive Chinese
traditional medicine, hesperetin has multiple biological and
pharmacological activities, including antioxidant properties
[21–23], inhibition of cancer development [24, 25], effects
on the blood–brain barrier [26, 27] and signal transduction
pathways [28], etc.
In order to give a deep research to hesperetin ramifica-
tions and their metal complexes, in this paper, a novel
ligand, hesperetin-2-hydroxy benzoyl hydrazone (H5L,
Fig. 1) and its transition metal complexes were synthesized
and characterized. We described a comparative study of the
interaction of the complexes and ligand with calf thymus
DNA (CT DNA) using absorption spectroscopy, fluores-
cence spectra, iodide quenching experiments, salt effect and
viscosity measurements for the first time. In addition, the
antioxidant activity of the ligand and its metal complexes
was determined by superoxide and hydroxyl radical
scavenging method in vitro. Furthermore, the fluorescent
properties of the ligand and Zn (II) complex in solid state
and different solutions were investigated. Information
obtained from our work would be helpful to understand
the interaction mechanism of hesperetin hydrazones and
their complexes with DNA and useful in the development
of potential probes of DNA structure and conformation, and
new therapeutic reagents for some certain diseases.
ligand and its complexes were dissolved in a solvent
mixture of 1% DMF and 99% Tris–HCl buffer (pH 7.1)
at the concentration 1.0×10−5 M.
Carbon, hydrogen and nitrogen were determined using
an Elemental Vario EL analyzer. The metal contents of the
metal complexes were determined by titration with EDTA
(xylenol orange tetrasodium salt used as an indicator and
hexamethylidynetetraimine as buffer). The melting points
of the compounds were determined on a Beijing XT4-100X
microscopic melting point apparatus (the thermometer was
not corrected). The IR spectra were obtained in KBr discs
on a Therrno Mattson FTIR spectrophotometer in the
1
4,000–400 cm−1 region. H NMR spectra were recorded
on a Varian VR 200 MHz spectrometer in DMSO-d6 with
TMS (tetramethylsilane) as an internal standard. Mass
spectra were performed on a APEX II FT-ICR MS
instrument using DMF as mobile phase. All conductivity
measurements were performed in DMF solution with a
DDS-11C conductometer at 25.0°C. The UV-vis spectra
were recorded on a Shimadzu UV-240 spectrophotometer.
Fluorescence measurements were recorded on a Hitachi
RF-4500 spectrofluorophotometer at room temperature.
Preparation of the ligand and complexes
Synthesis of the ligand (H5L)
Scheme of the synthesis of the ligand is shown in Fig. 1.
Hesperetin (3.02 g, 10 mmol) and 2-hydroxy benzoyl
hydrazine (1.82 g, 12 mmol) were dissolved in ethanol
(50 mL). Acetic acid (1.0 mL) was added into the solution.
The solution was refluxed on an oil bath for 24 h with
stirring and a yellow precipitate formed. The yellow
precipitate was filtered, washed several times with ethanol
and recrystallized from DMF and water to give the ligand.
Yield: 83.6%. M. p.: 264–267°C. Anal Calcd for
C23H20N2O7 requires C, 63.30; H, 4.62; N, 6.42. Found:
C, 63.19; H, 4.73; N, 6.46. lmax (nm): 368, 353, 332 nm.
Experimental section
Materials and physical measurements
Ethidium bromide (EB) and CT DNA were purchased from
Sigma Chemical Co. All chemicals and solvents used were
of analytical reagent grade and used without further
purification unless otherwise noted.
Synthesis of the complexes
The ligand (43.6 mg, 0.1 mmol) was dissolved in ethanol
(20 ml) and Cu(NO3)2•3H2O (24.5 mg, 0.1 mmol) was then
added. After 5 min, triethylamine (20.2 mg, 0.2 mmol) was
added into the solution and the solution was stirred for one
day at room temperature. Then a blue precipitate, Cu (II)
complex was separated by suction filtration. It was purified
by washing several times with ethanol, and then dried for
48 h in vacuum. The Zn (II) complex and Ni (II) complex
were also synthesized as the above method. Anal Calcd for
Cu (II) complex C23H18N2O7Cu requires C, 55.48; H, 3.64;
N, 5.63; Cu, 12.76; Found: C, 55.38; H, 3.60; N, 5.68; Cu,
12.73. Yield: 73.2%. lmax (nm): 412, 387, 372 nm. Λm
All the experiments involved with the interaction of the
ligand and its transition metal complexes with CT DNA
were carried out in doubly distilled water buffer containing
5 mM Tris [Tris(hydroxymethyl)-aminomethane] and
50 mM NaCl and adjusted to pH 7.1 with hydrochloric
acid. Solution of CT DNA in the buffer gave ratios of UV
absorbance of about 1.8–1.9:1 at 260 and 280 nm,
indicating that the CT DNA was sufficiently free of protein
[21]. The CT DNA concentration per nucleotide was
determined spectrophotometrically by employing an ex-
tinction coefficient of 6,600 M−1 cm−1 at 260 nm [22]. The

J Fluoresc (2010) 20:891–905
893
Fig. 1 Scheme of the synthesis
of the ligand (H5L)
OH
3'
OH
2'
6'
8
HO
O
1'
7
HO
O
O
H+
B
OCH
3
2
4'
H
OCH
A
3
+
N
NH
2
5'
6
3
5
4
2''
Δ
3''
4''
O
H
N
OH
N
C
1''
HO
OH
5''
O
6''
HO
(S cm2 mol−1): 6.6. Anal Calcd for Zn (II) complex
C23H18N2O7Zn requires C, 55.27; H, 3.63; N, 5.61; Zn,
13.08; Found: C, 55.33; H, 3.52; N, 5.68; Zn, 13.13. Yield:
79.3%. lmax (nm): 408, 384, 372 nm. Λm (S cm2 mol−1):
7.4. Anal Calcd for Ni (II) complex C23H18N2O7Ni requires
C, 56.02; H, 3.68; N, 5.68; Ni, 11.90; Found: C, 56.07; H,
3.62; N, 5.63; Ni, 12.03. Yield: 75.4%. lmax (nm): 410,
369, 358 nm. Λm (S cm2 mol−1): 6.1.
Further support for the ligand and complexes binding to
DNA via intercalation is given through the emission quench-
ing experiment. EB is a common fluorescent probe for DNA
structure and has been employed in examinations of the mode
and process of metal complexes binding to DNA [30]. A
2.0 mL solution of 4 μM DNA and 0.32 μM EB (at
saturating binding levels) was titrated by 2.5–30 μM the
complexes and ligand. Quenching data were analyzed
according to the Stern–Volmer equation which could be
used to determine the fluorescent quenching mechanism:
DNA binding studies
F0=F ¼ 1 þ Kq½Qꢂ;
Electronic absorption spectroscopy
Where F0 and F are the fluorescence intensities in the
absence and presence of compound at [Q] concentration,
respectively; Kq is the quenching constant and [Q] is the
quencher concentration. Plots of F0/F versus [Q] appear to
be linear and Kq depends on temperature [31].
Electronic absorption titration experiments were performed
with fixed concentration drugs (10 μM), while gradually
increasing the concentration of CT DNA. When measuring
the absorption spectra, an equal amount of CT DNA was
added to both the compound solutions and the reference
solution to eliminate the absorbance of CT DNA itself. Each
sample solution was scanned in the range of 190–500 nm.
Iodide quenching experiments
The fluorescence quenching efficiency is evaluated by
Stern–Volmer equation, which varies with the experimental
conditions. Quenching plots were constructed according to
the following Stern–Volmer equation.
Fluorescence spectra
To compare quantitatively the affinity of the compounds
bound to DNA, the intrinsic binding constants (Kb) of the
compounds to DNA were obtained by the luminescence
titration method. Fixed amounts of compound were titrated
with increasing amounts of DNA, over a range of DNA
concentrations from 2.5 to 20 μM. Scan speed was 480 nm/
min, slit width was 5 nm. All experiments were conducted
at a constant room temperature in a buffer solution
containing 5 mM Tris-HCl (pH 7.1) and 50 mM NaCl
concentrations. The concentration of the bound compound
was calculated using Eq. [29].
F0=F ¼ 1 þ Ksv½Iꢁꢂ;
Where F0 and F are the fluorescence intensities in the
absence and presence of iodide at [I−] concentration,
respectively; Ksv is the quenching constant and [I−] is the
concentration of iodide. Plots of Fo/F versus [I−] appear to
be linear and Ksv was evaluated by linear least-squares
analysis of the data according to the equation [32].
Salt effect
r=Cf ¼ Kb ꢀ ðn ꢁ rÞ
Fluorescence intensities were recorded in the absence and
presence of DNA in the mixture solution of each compound
and NaCl.
Cb ¼ Ct½ðF ꢁ F0Þ=ðFmax ꢁ F0Þꢂ;
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 com-
pound. 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.
Viscosity measurements
Viscosity measurements were conducted on an Ubbelodhe
viscometer, immersed in a thermostated water-bath main-
tained to 25.0 0.1°C. Titrations were performed for the
compounds (0.5–4 μM), and each compound was intro-

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J Fluoresc (2010) 20:891–905
duced into a DNA solution (5 μM) present in the
viscometer. Data were presented as (η/η0)1/3 versus the
ratio of the concentration of the compound and DNA,
where η is the viscosity of DNA in the presence of the
compound and η0 is the viscosity of DNA alone [33, 34].
Where Ai = the absorbance in the presence of the tested
compound; A0 = the absorbance in the absence of the
tested compound; Ac = the absorbance in the absence of
the tested compound, EDTA–Fe(II) and H2O2.
The antioxidant activity was expressed as the 50%
inhibitory concentration (IC50). IC50 values were calculated
from regression lines where: x was the tested compound
concentration in mM and y was percent inhibition of the
tested compounds.
Antioxidant activity
Superoxide radical scavenging activity
The superoxide radicals (O2•−) were produced by the
•−
system MET/VitB2/NBT [35]. The amount of O2 and
Results and discussion
•−
suppression ratio for O2 can be calculated by measuring
the absorbance at 560 nm, because NBT can be reduced
quantitatively to blue formazan by O2•−. The solution of
MET, VitB2 and NBT were prepared with 0.067 M
phosphate buffer (pH=7.8) at the condition of avoiding
light. The tested compounds were dissolved in DMF. The
reaction mixture contained MET (0.01 mol L−1), NBT
(4.6×10−5 mol L−1), VitB2 (3.3×10−6 mol L−1), phosphate
buffer solution (0.067 mol L−1) and the tested compound
(the final concentration: Ci (i=1–6)=0.4, 1.0, 2.0, 4.0, 6.0,
8.0 μM). After incubating at 30°C for 10 min and
illuminating with a fluorescent lamp for 3 min, the
absorbance (Ai) of the samples was measured at 560 nm.
The sample without the tested compound and avoiding light
was used as the control. The suppression ratio for O2•− was
calculated from the following expression:
Characterization of the complexes
The complexes were prepared by direct reaction of the
ligand with appropriate mole ratios of M (II) nitrate in
ethanol. The complexes are stable in atmospheric
conditions for extended periods and easily soluble in
DMF and DMSO; slightly soluble in ethanol, methanol
and acetone; insoluble in benzene, water and diethyl
ether. The molar conductivities of the complexes are
around 6.1–7.4 in DMF solution and in accord with
them being formulated as non-electrolytes [37]. The
elemental analyses and molar conductivities show that
formulas of the complexes conform to M·H3L [M (II) =
Cu, Zn, Ni].
IR spectra
Suppression ratioð%Þ ¼ ½ðA0 ꢁ AiÞ=A0ꢂ ꢀ 100%
The IR spectra of the ligand and its complexes are
presented in Table 1. On the basis of the very similar
spectra of the three complexes, it may be assumed that they
have the similar coordination structures. All the spectra are
characterized by vibrational bands mainly due to the O–H,
C=O, C=N, NNH, M–O and M–N groups. The ν (O–H) for
the ligand appears at 3,443 cm−1, and this peak shifts at
3,424 cm−1 or so for the complexes. The ν (C=O) vibration
of the free ligand is at 1,632 cm−1, for the complexes, this
peak shifts at 1,606 cm−1 or so; Δν (ligand-complexes) is equal
to 26 cm−1. For the complexes, the band at 570 cm−1 or so
is assigned to ν (M–O). It demonstrates that the oxygen of
the carbonyl group has formed a coordinative bond with the
metal ions [38]. δ (NNH) vibration of the free ligand
appears at 1,517 cm−1, and for the complexes the vibrations
are still at 1,519–1,524 cm−1, which indicates that the
active hydrogen of the −NNH = still exists in the
complexes. The band at 1,611 cm−1 for the free ligand is
assigned to the ν (C=N) stretch, which shifts to 1,570 cm−1
where Ai = the absorbance in the presence of the ligand or
its complexes, A0 = the absorbance in the absence of the
ligand or its complexes.
Hydroxyl radical scavenging activity
The hydroxyl radical (HO•) in aqueous media was
generated through the Fenton reaction [36]. The solution
of the tested compounds was prepared with DMF. The
reaction mixture contained 2.5 mL 0.15 M phosphate
buffer (pH=7.4), 0.5 mL 114 μM safranin, 1 mL 945 μM
EDTA–Fe(II), 1 mL 3% H2O2 and 30 μL the tested
compound solution (the final concentration: Ci (i=1−6)=1.0,
2.0, 3.0, 4.0, 5.0, 6.0 μM). 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 HO• was calculated from the
following expression:
or so for the complexes; Δν
is equal to
(ligand-complexes)
41 cm−1. Weak bands at 475 cm−1 or so are assigned to ν
(M–N) for the complexes. These further confirm that the
nitrogen of the imino-goup bonds to the metal ions [39].
Suppression ratioð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢂ ꢀ 100%

J Fluoresc (2010) 20:891–905
895
Table 1 Main IR data of the
ligand and its complexes (cm−1
)
Compounds
ν (O–H)
ν (C=O)
ν (C=N)
δ (NNH)
ν (M–O)
ν (M–N)
H5L
3,443
3,421
3,427
3,424
1,632
1,602
1,607
1,606
1,611
1,562
1,558
1,573
1,517
1,522
1,524
1,519
Cu(II) complex
Zn(II) complex
Ni(II) complex
572
570
567
479
472
475
1H NMR spectra
Fluorescence spectra
1
The H NMR spectra of the ligand and its Zn (II) complex
are assigned as follows: H5L (DMSO-d6, 200 MHz), δ
(ppm) 3.12 (2H, bs, 3-H), 3.77 (3H, s, −OCH3), 5.09 (1H,
dd, J=3.0, 12.3 Hz, 2-H), 5.88 (1H, d, J=2.2 Hz, 6-H), 5.93
(1H, d, J=2.2 Hz, 8-H), 6.91 (5H, bs, −Ar-H), 7.37 (1H, dd,
J=1.8, 7.6 Hz, 4″-H), 7.91 (1H, dd, J=1.8, 7.6 Hz, 3″-H),
9.10 (1H, s, 3′-OH), 10.01 (1H, s, 6″-OH), 11.33 (1H, s, 5-
OH), 11.65 (1H, s, −NH-C=O), 12.86 (1H, s, 7-OH). Zn
(II) complex 1H NMR (DMSO-d6, 200 MHz), δ (ppm) 2.89
(2H, bs, 3-H), 3.78 (3H, s, −OCH3), 5.00 (1H, dd, J=2.3,
10.2 Hz, 2-H), 5.55 (1H, d, J=2.0 Hz, 6-H), 5.69 (1H, d, J=
2.0 Hz, 8-H), 6.86 (5H, bs, −Ar-H), 7.23 (1H, dd, J=1.5,
7.6 Hz, 4″-H), 7.84 (1H, dd, J=1.5, 7.6 Hz, 3″-H), 9.07
(1H, s, 3′-OH), 9.30 (1H, s, −NH-C=O), 13.94 (1H, s, 7-
OH). In the complex, we can find hydrogen atoms of 5-OH
and 6″-OH were displaced by the metal ion and a new
complex was formed by coordination of zinc ion and
ligand, which is also supported by the IR spectra.
The emission spectra of the ligand and Zn (II) complex in
solid state (the excitation and emission slit widths were
1.5 nm) were recorded at room temperature. The ligand
exhibits green fluorescence and the emission is readily
observed with naked eyes under UV light. Whereas the Zn
(II) complex exhibits blue fluorescence and the emission is
also readily observed with naked eyes under UV light.
However, the fluorescence intensities of the Cu (II) and Ni
(II) complexes are much too weak in the solid state, which
are not discussed in our present work. Fluorescence spectra
of the ligand and Zn (II) complex in solid state are shown in
Fig. 2. The Zn (II) complex and ligand in the solid state at
room temperature have different emissions at 485 and
522 nm at the same excitation (350 nm), respectively.
Because Zn(NO3)2 has no fluorescence, the observed blue
fluorescence is attributed to the coordinated ligand. How-
ever, it is noteworthy that the emission wavelength of Zn
(II) complex is slightly shorter than that of the free ligand
and can probably be assigned to the intra-ligand fluorescent
emission, and can probably be the result of coordination of
the free ligand to Zn (II) that increases the ligand
conformational rigidity [40]. Thus, Zn (II) complex may
be used as an advanced material for blue light emitting
diode devices.
UV-vis spectra
The study of the electronic spectra in the ultraviolet and
visible (UV-vis) (Fig. 4) ranges for the metal complexes
and ligand was carried out in a Tris-HCl buffer solution.
The electronic spectra of the free ligand exhibit three
absorption bands at 332 (band I), 353 (band II) and 368
(band III) nm (DMF was used as the solvent). Band I is
related to ring A, band II to ring C and band III to ring B.
The complexes also yield three bands and the three bands
are shifted to 360, 380 and 410 nm or so. This shows that
there is a large conjugate system formed by the ligand in
the complexes. Therefore, the complexes should be chelates
containing both a five-membered and a six-membered ring
and the metal complex formation leads to the red shifts of
the absorption spectra. All these indicate that the metal
complexes are formed.
50
40
30
20
10
0
(a)
(b)
Mass spectra
400
450
500
550
600
650
The electrospay ionization (ESI) mass spectra of Cu(II)
complex was made. The mass spectra of Cu(II) complex
show peak at 537 (data not shown) which can be assigned
to the fragment [Cu(II) complex + K]+.
Wavelength (nm)
Fig. 2 Solid state emission spectra of the ligand (a) and Zn (II)
complex (b)

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J Fluoresc (2010) 20:891–905
OH
The influence of solvents on the fluorescence intensities
of the ligand and Zn (II) complex was also investigated. As
shown in Table 2, the fluorescence intensities of Zn (II)
complex in organic solvents are weaker than that of the
powder. This may be due to the quenching process of
solvent molecules in the solution. The energy transfer from
the triplet excited state of the ligand to the emitting level of
the zinc ion could not be carried out perfectly because the
oscillatory motions of the entering solvent molecules may
consume more energy. All these indicate that the polarity of
the solvents affects the fluorescence of Zn (II) complex.
Furthermore, the fluorescence intensity of Zn (II) complex,
relative to that of the free ligand, is dramatically enhanced,
which is clearly caused by the formation of coordination
bonds between the ligand and zinc ion. In addition, the
solution fluorescent spectra of the ligand and Zn (II)
complex are highly solvent-dependent.
O
OCH
3
O
N
M
NH
O
O
Fig. 3 The suggested structure of the complexes [M (II) = Cu, Zn, Ni]
Structure of the complexes
Titration with electronic absorption spectroscopy is an
effective method to investigate the binding mode of DNA
with metal complexes [41]. In the intercalative binding
mode, the π* orbital of the intercalated ligand can couple
with the π orbital of the DNA base pairs, thus, decreasing
the π→π* transition energy and resulting in the bath-
ochromism. On the other hand, the coupling π orbital is
partially filled by electrons, thus, decreasing the transition
possibilities and concomitantly resulting in the hypochrom-
ism [42]. Generally, the binding of an intercalative
molecule to DNA is accompanied by hypochromism and/
or significant red-shift (bathochromism) in the absorption
spectra due to the strong stacking interaction between the
aromatic chromophore of the ligand and DNA base pairs
with the extent of hypochromism and red-shift commonly
consistent with the strength of the intercalative interaction
[43]. Therefore, in order to obtain evidence for the binding
ability of each compound to CT DNA, spectroscopic
titration of compounds solutions with CT DNA should be
performed [44].
Since the crystal structures of the metal complexes have not
been obtained yet, we characterized the complexes and
determined its possible structure by elemental analyses,
molar conductivities, IR spectra, H NMR spectra, mass
spectra, fluorescence studies and UV-vis spectra. The likely
structure of the complexes is shown in Fig. 3.
1
DNA binding studies
Electronic absorption spectroscopy
Before reacting the ligand and its metal complexes
interacting with CT DNA, its solution behavior in Tris-
HCl buffer solution at room temperature was monitored by
UV-vis spectroscopy for 24 h. Liberation of the ligand and
its complexes was not observed under these conditions.
These suggest that the ligand and its complexes are stable
under the conditions studied.
Table 2 Fluorescence spectra
Compound
L
Solvent
lex (nm)
lem (nm)
Relative emission intensity
of the ligand and Zn (II) com-
plex in different solutions at
room temperature
Ethanol
Methanol
DMF
388
376
384
383
384
378
476
383
379
381
455
456
454
469
432
456
462
449
449
444
142.947
49.819
101.099
3.080
CH3CN
THF
2.888
Zn(II) complex
Ethanol
Methanol
DMF
57.206
34.497
201.832
29.834
10.025
CH3CN
THF
All the emission and excitation
slit widths were 3 nm

J Fluoresc (2010) 20:891–905
897
The electronic absorption spectra of the free ligand and its
metal complexes in the absence and presence of CT DNA are
given in Fig. 4. As shown, there exists three bands at 368,
353, 332 nm for the free ligand and there are also three
bands for the metal complexes. With increasing concen-
trations of CT DNA, the absorption band at 353 nm for the
free ligand exhibited hypochromism of 28.1% and was
accompanied by a small red-shift of 3 nm in λmax, from 353
to 356 nm. Upon addition of CT DNA, for the Cu(II), Zn(II)
and Ni(II) complexes, the absorption bands at 369, 387 and
384 nm also show hypochromism of 43.2%, 33.2 and
29.2%, respectively. The hypochromism observed for the
metal complexes are all accompanied by a small red shift by
about 3 nm. All these results suggest that the compounds can
interact with CT DNA quite probably by intercalating the
compounds into DNA base pairs, so we can conclude that
the free ligand and its metal complexes can interact with CT
DNA via the same mode (intercalation).
In order to further test if the complexes could bind to CT
DNA via the intercalationmod, EB (a typical indicator of
intercalation) was employed [45]. Figure 5 shows the
electronic absorption spectra when EB was employed. The
absorbance of Tris-HCl buffer solution is highest when
there is EB alone. After adding CT DNA into the buffer
solution, the absorbance decreases. However, it increases
when the ligand and metal complexes were added dropwise
into the buffer solution. Figure 5 shows that the maximal
absorption of EB at 479 nm decreased and shifted to
494 nm in the presence of DNA, which was characteristic
of intercalation mode. Curve (B) in Fig. 5 was the
absorption of a mixture solution of EB, DNA and the free
ligand and metal complexes. It was found that the
absorption at 494 nm or so increased comparing with curve
(C). It could result from two reasons: (1) EB bound to the
free ligand and metal complexes strongly, resulting in the
decrease of the amount of EB intercalated into DNA; (2)
there exists competitive intercalation between the free
ligand and metal complexes and EB with DNA, so
releasing some free EB from DNA–EB system. However,
the former reason could be precluded because there were no
new absorption peaks appearing.
Fluorescence spectra
The spectral results of the emission titrations for the metal
complexes and free ligand with CT DNA are illustrated in the
titration curves (Fig. 6). Compared to the free ligand and its
metal complexes alone, the emission intensity increases with
increasing concentrations of CT DNA. Results obtained
from fluorescence spectra suggest that all the compounds are
protected from solvent water molecules by the hydrophobic
environment inside the DNA helix and that the metal
complexes can be protected more efficiently than the free
ligand alone. This implies that all the metal complexes and
Fig. 4 Electronic absorption
a
spectra of the free ligand (a), Cu
0.25
b
0.09
(II) (b), Zn (II) (c) and Ni (II)
(d) complexes (10 μM) in the
0.08
0.07
0.06
0.05
0.04
0.03
absence (top spectrum) and
presence of increasing amounts
of CT DNA (2.5, 5.0, 7.5,
10.0, 12.5, and 15.0 μM; sub-
sequent spectra). Arrow shows
the absorbance changes upon
increasing DNA concentration
0.20
0.15
0.10
0.05
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
c
d
0.20
0.15
0.10
0.05
0.00
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)

898
J Fluoresc (2010) 20:891–905
Fig. 5 The ultraviolet visible
absorption spectra of 1×10−5
M
a
b
0.015
0.010
0.005
0.015
0.010
0.005
A
B
C
EB (A); (A) +2.5×10−5 M DNA
(C); (C) +2.5×10−5 M free
ligand (a), Cu (II) (b), Zn (II) (c)
and Ni (II) (d) complexes
(B) in Tris-HCl buffer (5 mM
tris–HCl, 50 mM NaCl, pH 7.1)
solution
A
C
B
400
450
500
550
600
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
c
d
0.015
0.010
0.005
0.000
0.015
0.010
0.005
A
B
A
B
C
C
400
450
500
550
600
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
free ligand can insert between DNA base pairs deeply and
the metal complexes can bind to DNA more strongly than
the free ligand alone. Since the hydrophobic environment
inside the DNA helix reduces the accessibility of solvent
water molecules to the compound and the compound
mobility is restricted at certain binding sites, a decrease of
the vibrational modes of relaxation results. The binding of
the compounds to DNA leading to a significantly increase in
emission intensity also agrees with those observed for other
intercalators [46]. According to the Scatchard equation, a
plot of r/Cf versus r gave Kb values of (3.02 0.25) ×105,
(1.35 0.05) ×106, (3.58 0.38) ×106 and (2.07 0.09)
×106 M−1 from the fluorescence data for the ligand, Cu(II),
Zn(II) and Ni(II) complexes, respectively. The higher
binding affinities of the metal complexes are probably
attributed to the extension of the π system of the intercalated
ligand due to the coordination of transition metal ions, which
also leads to a planar area greater than that of the free ligand
and the coordinated ligand penetrating more deeply into and
stacking more strongly with the base pairs of DNA.
intensity gives a measure of DNA binding propensity of the
compounds and stacking interaction (intercalation) between
the adjacent DNA base pairs [48, 49]. Steady-state emission
quenching experiments are usually used to observe the
binding mode of the metal complexes and free ligand to CT
DNA. Figure 7 shows the emission spectra of the DNA–EB
system with increasing amounts of the metal complexes and
free ligand. The addition of the compounds to the DNA-
bound EB solutions caused obvious reduction in emission
intensities. The quenching plots illustrated that the quench-
ing of EB bound to DNA by the complexes and free ligand
is in good agreement with the linear Stern–Volmer equation.
In the plots of F0/F versus [Q], Kq is given by the ratio of the
slope to the intercept and Kq values for the ligand, Cu (II),
Zn (II) and Ni (II) complexes are (1.57 0.12) ×105, (1.79
0.06) ×105, (2.26 0.07) ×105 and (2.10 0.08) ×105 M−1,
respectively. The data show that the interaction of the metal
complexes with DNA is stronger than that of the free ligand,
which is consistent with the electronic absorption spectral
results. Since these changes indicate only one kind of
quenching process, it may be concluded that the metal
complexes and free ligand bind with DNA via the same
mode (intercalation mode).
Binding modes of the compounds with DNA were further
monitored by a fluorescent EB displacement assay. EB is one
of the most sensitive fluorescence probes that can bind to
DNA. The fluorescence of EB increases after intercalating
into DNA base pairs. If the compounds intercalates into DNA,
it leads to a decrease in the binding sites of DNA available for
EB and resulting in the decrease in the fluorescence intensity
of DNA-EB system [47]. The reduction of the emission
Iodide quenching experiments
The results of the emission titrations for the Zn (II) complex
and potassium iodide with absence and presence of CT

J Fluoresc (2010) 20:891–905
899
Fig. 6 The emission enhance-
ment spectra of free ligand (a),
Cu (II) (b), Zn (II) (c) and Ni
(II) (d) complexes (10 μM) in
the presence of 0, 2.5, 5.0, 7.5,
10.0, 12.5, 15.0, 17.5 and
20.0 μM DNA. Arrows show
the emission intensity changes
upon increasing DNA concen-
tration. Inset: Scatchard plot of
the fluorescence titration data of
ligand and metal complexes, Kb
(a) = (3.02 0.25) ×105 M−1; Kb
(b) = (1.35 0.05) ×106 M−1; Kb
(c) = (3.58 0.38) ×106 M−1; Kb
(d) = (2.07 0.09) ×106 M−1
a
600
500
400
300
200
100
0
2.0
1.5
1.0
0.5
0.0
Kb = (3.02 0.25)×10 5 M-1
Kb = (3.02 0.25)×105 M-1
0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
350
400
450
500
550
600
Wavelength (nm)
r
b
14
12
10
8
700
600
500
400
300
200
100
0
Kb = (1.35 0.05)×106 M-1
Kb = (1.35 0.05)×106 M-1
6
4
2
0
350
400
450
500
550
600
0.4
0.6
0.8
1.0
1.2
1.4
Wavelength (nm)
r
c1000
10
8
900
800
700
600
500
400
300
200
100
0
Kb = (3.58 0.38)×106 M-1
Kb = (3.58 0.38)×106 M-1
6
4
2
0
350
400
450
500
550
600
0.75
0.80
0.85
0.90
0.95
1.00
Wavelength (nm)
r
d
900
800
700
600
500
400
300
200
100
0
9
8
7
6
5
4
3
2
1
6
Kb = (2.07 0.09)×10 M-1
Kb = (2.07 0.09)×106 M-1
350
400
450
500
550
600
0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95
Wavelength (nm)
r

900
J Fluoresc (2010) 20:891–905
Fig. 7 The emission spectra of
DNA-EB system, in the pres-
ence of 0, 2.5, 5.0, 7.5, 10.0,
12.5, 15.0, 17.5, 20.0, 22.5,
25.0, 27.5 and 30.0 μM free
ligand (a), Cu (II) (b), Zn (II) (c)
and Ni (II) (d) complexes.
Arrows show the emission in-
tensity changes upon increasing
ligand and complexes. Inset:
Stern–Volmer plot of the fluo-
rescence titration data of ligand
and complexes. Kq (a) = (1.57
0.12) ×105 M−1; Kq (b) = (1.79
0.06) ×105 M−1; Kq (c) = (2.26
0.07) ×105 M−1; Kq (d) = (2.10
0.08) ×105 M−1
a
6
5
4
3
2
1
700
600
500
400
300
200
100
0
Kq=(1.57 0.12)×105 M-1
-1
Kq=(1.57 0.12)×105 M
0
5
10
15
20
25
30
550
600
650
700
750
[Compound] × 106M
Wavelength (nm)
b
c
d
7
6
5
4
3
2
1
600
500
400
300
200
100
0
Kq=(1.79 0.06)×105 M-1
Kq=(1.79 0.06)×105 M-1
550
600
650
Wavelength (nm)
700
750
0
5
10
15
20
25
30
[Compound] × 106M
600
500
400
300
200
100
0
8
7
6
5
4
3
2
1
Kq=(2.25 0.07)×105 M-1
-1
Kq=(2.25 0.07)×105 M
550
600
650
700
750
0
5
10
15
20
25
30
[Compound] × 106M
Wavelength (nm)
600
500
400
300
200
100
0
7
6
5
4
3
2
1
Kq=(2.10 0.08)×105 M-1
Kq=(2.10 0.08)×105 M
-1
0
5
10
15
20
25
30
550
600
650
Wavelength (nm)
700
750
[Compound] × 106M

J Fluoresc (2010) 20:891–905
901
DNA are illustrated in the titration curves (Fig. 8).
Compared to the Zn (II) complex alone, the emission
intensity decreases with increasing concentrations of CT
DNA. The quenching plots illustrated that the quenching
studies of the Zn (II) complex are in good agreement with
the linear Stern–Volmer equation.
conclude that the metal complexes are intercalated into the
DNA helix and they should be protected from the anionic
quencher, owning to the base pairs above and below the
intercalators [50].
Salt effect
In the aqueous solution, iodide and ferrocyanide anions
quench the fluorescence of Zn (II) complex very efficiently,
so we used potassium iodide as the quencher to determine
the relative accessibilities of the free and bound Zn (II)
complex. From Fig. 8, we can find that the addition of Zn
(II) complex and CT DNA results in extensive quenching
of the fluorescence intensity. The quenching data were
plotted according to the Stern–Volmer equation and the
slopes were calculated by the linear least-squares method.
The observed quenching constants were 4.59 0.22 and
10.5 0.63 M−1 with and without CT DNA, respectively.
The quenching of the Zn (II) complex fluorescence was in
fact enhanced by a factor of about 3 when the Zn(II)
complex was bound to the DNA helix. Ksv values were
used to deduce the interaction mode of the fluorescence
probe with DNA. Higher binding constants should corre-
spond to the better protection by the DNA and a stronger
inhibition of quenching by anionic species. Thus, we can
The effects of the ionic strength on the Zn (II) complex
fluorescence intensity was tested by the addition of a strong
electrolyte, such as NaCl, instead of potassium iodide.
Cations of the salts can neutralize the negatively charged
phosphate. If the compound binds to DNA through an
electrostatic interaction mode, the surface of DNA will be
surrounded by the sodium ions with the increasing ionic
strength. Then the compound has difficulty to approach
DNA molecules and the strength of interaction with DNA
decreases, and then the degree of fluorescence quenching
also decreases [29]. As seen from Fig. 9, addition of NaCl
to the Zn (II) complex in the absence and presence of CT
DNA has few or no effects on the fluorescence intensity,
showing that the interaction of the Zn (II) complex with CT
DNA is not electrostatic interaction. Thus, we can come to
a conclusion that the compounds interact with DNA is not
via an electrostatic mode.
Fig. 8 a Fluorescence spectra
of Zn (II) complex (10 μM) with
increasing concentration of KI
a
b
900
900
(5, 10, 15, 20, 25, 30 and
800
700
600
500
400
300
200
100
0
800
35 mM). b Fluorescence spectra
of Zn (II) complex (10 μM) and
DNA (20 μM) system with
increasing concentration of KI
(5, 10, 15, 20, 25, 30 and
35 mM). c Inset: Stern–Volmer
plot of the fluorescence titration
data of Zn (II) complex. Effect
of KI concentration (1: Zn(II)
complex + KI; 2: Zn (II) com-
plex + KI + CT DNA). Ksv (1) =
10.5 0.63 M−1, Ksv (2) = 4.59
0.22 M−1
Ksv = 10.5 0.63 M-1
700
600
500
400
300
200
100
0
K
sv = 4.59 0.22 M-1
650
400
450
500
550
600
650
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
c
1.40
K
sv (1) = 10.5 0.63 M-1
sv (2) = 4.59 0.22 M-1
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
K
1
2
0.005 0.010 0.015 0.020 0.025 0.030 0.035
[KI]

902
J Fluoresc (2010) 20:891–905
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
H5L
Cu(II) Complex
Zn(II) Complex
Ni(II) Complex
2
1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.0
0.1
0.2
0.3
0.4
0.5
[M]/[DNA]
[NaCl] (mol· L-1)
Fig. 10 Effects of increasing amounts of the ligand and its transition
metal complexes on the relative viscosity of CT DNA at 25.0 0.1°C
Fig. 9 Effect of NaCl concentration (1: Zn (II) complex + NaCl; 2:
Zn(II) complex + NaCl + CT DNA)
On the basis of all the spectroscopic studies together
with the viscosity measurements, we find that the transition
metal complexes and free ligand can bind to CT DNA via
an intercalative mode (Fig. 11) and the transition metal
complexes bind to CT DNA more strongly and deeply than
the free ligand.
Viscosity measurements
In order to further clarify the interaction nature between the
compounds and DNA, viscosity measurements were carried
out. Optical photophysical probes provide necessary but not
sufficient evidence to support a binding model. Hydrody-
namic measurements that are sensitive to length change (i.e.
viscosity and sedimentation) are regarded as the least
ambiguous and the most critical tests of a binding model
in solution in the absence of crystallographic structural data
[51]. A classical intercalation model demands that the DNA
helix must lengthen as base pairs are separated to
accommodate the binding complexes, leading to the
increase of DNA viscosity, as for the behaviors of the
known DNA intercalators [52]. In contrast, a partial and/or
non-classical intercalation of the complex could bend (or
kink) the DNA helix, reducing its viscosity concomitantly
[51]. In addition, some complexes such as [Ru(bpy)3]2+,
which interacts with DNA by an electrostatic binding
mode, have no influence on DNA viscosity [53]. The
effects of all the compounds on the viscosity of CT DNA
are shown in Fig. 10. The viscosities of DNA increase
steadily with increasing concentrations of ligand and
complexes and the extent of the increase observed for the
ligand is smaller than those for the complexes. Viscosity
measurements clearly show that all the compounds can
intercalate between adjacent DNA base pairs, causing an
extension in the DNA helix and thus increasing the
viscosity of DNA and that the complexes can intercalate
more strongly and deeply than the free ligand, leading to
the greater increase in viscosity of the DNA with an
increasing concentration of the complexes. The results
obtained from the viscosity experiments validate those
obtained from the spectroscopic studies.
Antioxidant activity
Due to the ligand and its transition metal complexes
exhibiting good DNA binding affinity, it is considered
worthwhile to investigate their antioxidant activity. Reac-
•−
tive oxygen species (ROS), such as superoxide anion (O2
)
and hydroxyl radical (HO•), are generated by all aerobic
cells during normal oxygen metabolism, and cumulative
information obtained has proved that the oxidation induced
by ROS is involved in the pathogenesis of various diseases
through direct effects on DNA directly and by acting as a
tumor promoter [54–56]. In this paper, the antioxidant
+
OH
O
OCH3
O
N
M
O
O
M =Cu, Zn and Ni
Fig. 11 Molecular mode for the transition metal complexes (via an
intercalative mode)

J Fluoresc (2010) 20:891–905
903
Fig. 12 Scavenging effect of
b
a
the ligand and transition metal
•−
60
50
40
100
H L
complexes on O2 (a) and
5
H5L
HO• (b)
Cu(II) Complex
Zn(II) Complex
N(II) Complexi
Cu(II) Complex
Zn(II) Complex
Ni(II) Complex
80
60
40
20
0
30
20
10
0
0
1
2
3
4
5
6
1
2
3
4
5
Concentration (μM)
Concentration (μM)
scavenging abilities for HO• and O2 radicals. It was
believed that the information obtained form present work
would be useful to develop new potential antioxidants and
therapeutic agents for some diseases.
•−
activity of the ligand and its transition metal complexes was
studied by comparing their scavenging effect on superoxide
radical and hydroxyl radical.
Figure 12 depicts the inhibitory effects of the ligand and
•−
transition metal complexes on O2 (a) and HO• (b). The
antioxidant activity of the compounds is expressed as 50%
inhibitory concentration (IC50 in μM). From Fig. 12(a), the
inhibitory effect of the compounds studied on O2 is
Conclusion
•−
concentration dependent and the suppression ratio increase
with increasing sample concentrations in the range tested.
IC50 values of the ligand, Cu (II), Zn (II) and Ni (II)
complexes are 118.06, 0.42, 1.93 and 4.46 μM, respective-
In summary, a novel hesperetin Schiff-base ligand and three
transition metal complexes have been synthesized and
characterized. The ligand and Zn (II) complex can emit
fluorescence in the solid state and their emission spectra are
highly solvent-dependent. Kb values obtained from the
fluorescence titration data of the ligand, Cu (II), Zn (II) and
Ni (II) complexes are (3.02 0.25) ×105, (1.35 0.05) ×106,
(3.58 0.38) ×106 and (2.07 0.09) ×106 M−1, respectively,
indicating that all the compounds can bind to DNA via the
intercalation mode and metal complexes can bind to DNA
more strongly than the free ligand. This is due to the
chelating effect (metal ions to free ligand) which can
enhance the planar functionality of the metal complexes
and the metal complexes can insert and stack between the
DNA base pairs more easily and deeply than the free ligand
alone. It is noteworthy that Zn (II) complex can not only
emit blue fluorescence but also possess better DNA binding
ability, and it should be further studied in the future.
Antioxidant activity was also studied by the scavenging
superoxide and hydroxyl radical methods and experimental
results indicate the metal complexes possess better antiox-
idant activity than the free ligand. Our present work would
be helpful to understand the interaction mechanism of small
molecule compounds binding to DNA and useful in the
development of potential probes of DNA structure and
conformation, new therapeutic reagents and antioxidants.
•−
ly. It is clear that the scavenger effect on O2 can be
enhanced by the formation of metal-ligand coordination
compounds and the nature of metal ions also affects the
ability. As seen from Fig. 12(b), IC50 values of the ligand,
Cu (II), Zn (II) and Ni (II) complexes are 85.63, 4.88, 8.36
and 39.46 μM, respectively. The metal complexes are better
antioxidants than the ligand. Obviously, the metal com-
plexes exhibit considerable scavenging activity due to the
chelation of organic molecules to the metal ions and the
metal ions also exert differential and selective effects on
scavenging radicals of the biological system. Furthermore,
we find that the metal complexes show scavenging HO•
effects than mannitol which is usually used as special
scavenger for HO• and it is IC50 value is 10.19 μM [57],
indicating that the synthesized complexes are better
potential antioxidants. In addition, it is reported that IC50
value of ascorbic acid (Vc, a standard agent for non-
enzymatic reaction) for HO• is 1.537 mg mL−1
•−
(8.727 mmol), and the scavenging effect for O2 is only
25% at 1.75 mg mL−1 (9.94 mmol) [58]. In addition, the
magnetic properties influence on the reactivity of the metal
complexes with superoxide anion and hydroxyl radicals,
and we find complexes which are paramagnetic can
scavenge the superoxide and hydroxyl radicals more greatly
than complexes which are diamagnetic. Notably, the
investigated ligand and complexes have much stronger
Acknowledgements This work is supported by the National Natural
Science Foundation of China (20975046) and Gansu NSF
(0710RJZA012).

904
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