Interaction of drug based copper(II) complexes with Herring Sperm DNA and their biological activities

Article abstract of DOI:10.1016/j.saa.2012.05.037

Square pyramidal Cu(II) complexes with NS donor ligand and ciprofloxacin have been synthesized and characterized using analytical and spectral techniques. The synthesized complexes have been tested for their antimicrobial activity using double dilution te

Full text of DOI:10.1016/j.saa.2012.05.037

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Interaction of drug based copper(II) complexes with Herring Sperm DNA
and their biological activities
⇑
Mohan N. Patel , Chintan R. Patel, Hardik N. Joshi
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
The DNA binding ability of the complexes with Herring Sperm DNA has been performed using absorption
titration. With increase in DNA to complex ratio, hypochromism and red shift was observed. It suggests
that complexes bind via interaction mode.
"
Synthesis of substituted NS donor
ligand and their Cu(II) complexes.
Characterization by elemental
analysis, IR, UV–visible, LC–MS
spectrometry.
"
"
"
All Cu(II) complexes have tested for
their biological studies.
The interaction of DNA with
complexes has been performed.
a r t i c l e i n f o
a b s t r a c t
Article history:
Square pyramidal Cu(II) complexes with NS donor ligand and ciprofloxacin have been synthesized and
characterized using analytical and spectral techniques. The synthesized complexes have been tested
for their antimicrobial activity using double dilution technique in terms of minimum inhibitory concen-
tration (MIC) and colony forming unit (CFU). The DNA binding ability of the complexes with Sperm Her-
ring DNA has been performed using absorption titration and viscosity measurement. The nuclease
activity of complexes with plasmid DNA (pUC19) has been carried out using agarose gel electrophoresis
technique. Synthesized complexes have been tested for their SOD mimic activity using NBT/NADH/PMS
system. The cytotoxic properties of metal complexes have been evaluated using brine shrimp lethality
bioassay.
Received 1 March 2012
Received in revised form 7 May 2012
Accepted 17 May 2012
Available online 24 May 2012
Keywords:
NS donor ligands
Antimicrobial activity
Nuclease activity
SOD mimic activity
Cytotoxicity
Ó 2012 Elsevier B.V. All rights reserved.
Spectral techniques
Introduction
tims infected by anthrax, urinary, skin and respiratory infections
[2,3]. Quinolones are chelating agents for a variety of metal ions
Quinolones are a large group of synthetic antibacterial agents
used in a clinical practice for the treatment of variety of bacterial
infections [1]. Ciprofloxacin is the drug of choice for treating vic-
including alkaline earth and transition metal ions. Although re-
ports indicate that the coordination of quinolones to metal ions
such as Cu(II), Mg(II) and Ca(II) appear to be important for the
activity of the quinolone antibiotics [4], it has a detrimental effect
on their absorption [5]. Mixed ligand copper–quinolones com-
plexes using different NN donor ligand have been synthesized
⇑
Corresponding author. Tel.: +91 (2692) 226856x218.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.05.037

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
67
and explored for their biological activities [6–8]. The complexes of
vanadium(IV), copper(II), magnesium(II), uranium(VI), manga-
nese(II), iron(III), cobalt(II), nickel(II), molybdenum(II) and euro-
pium(III) with ciprofloxacin have been synthesized and explored
for their biological activities, because of its biological relevance
[9–16].
(Tris-Acetyl-EDTA), bromophenol blue and xylene cyanol FF were
purchased from Himedia (India). Nicotinamide adenine dinucleo-
tide reduced (NADH), Nitroblue tetrazolium (NBT) and phenazin
methosulphate (PMS) were purchased from Loba Chemie Pvt. Ltd.
Herring Sperm DNA was purchased from Sigma Chemical Co.
(India).
Metal-containing drugs have for long been of interest, but the
interest of the scientific community for medicinal aspects of metal
compounds has rapidly grown in the last few decades [17,18].
Complexation with the metal protects the drug against enzymatic
degradations because of the inertness of certain metal–ligand link-
ages. The metal complex can have better hydrophobicity/hydro-
philicity properties than the free ligand and, through this; it can
improve the transport processes in the tissues. In addition, the me-
tal complex can release the active drug(s) in a specific organ, and
its activity can be reinforced by the combination of effects from
the ligands and from the metal residue. The application of these
principles has already resulted in the design of successful metal-
based drugs [19,20].
A large number of mixed ligand copper(II) complexes have been
shown to exhibit superoxide dismutase activity [21,22]. This activ-
ity depends on the Cu(II)/Cu(I) redox process, which is related to
flexibility of the geometric transformation around the metal cen-
ters [23]. Complexation with copper enhances the biological activ-
ity of a wide variety of organic ligands [24,25].
In continuation of our previous work [26], herein we synthe-
sized mixed ligand Cu(II) complexes with drug and neutral biden-
tate ligand. The in vitro antimicrobial activity of complexes was
evaluated against two Gram(+ve) and three Gram(ꢀve) microor-
ganisms. The synthesized complexes were checked for their DNA
interactions using UV spectroscopy, viscosity measurement and
gel electrophoresis. The SOD mimic behavior of the complexes
was checked under non-enzymatic condition. The cytotoxic effects
of the synthesized complexes were also checked using brine
shrimp lethality bioassay.
Physical measurements
Elemental analyses (C, H and N) of the synthesized complexes
were performed with a model 240 Perkin Elmer elemental ana-
lyzer, Massachusetts (USA). Metallic content of the complex was
determined after decomposing it under effect of acid mixture
and titrating against EDTA solution volumetrically. Room temper-
ature magnetic measurement for the complexes was made using
Gouy magnetic balance. The Gouy tube was calibrated using mer-
cury(II) tetrathiocyanatocobaltate(II) as the calibrant (
vg = 16.44 ꢁ
10ꢀ6 cgs units at 20 °C), Mumbai (India). The electronic spectra
were recorded on a UV-160A UV–visible spectrophotometer, Shi-
madzu, Kyoto (Japan). Infrared spectra were recorded on a FT-IR
ABB Bomen MB-3000 spectrophotometer (Canada) as KBr pellets
in the range 4000–400 cmꢀ1. MIC study was carried out by means
of laminar air flow cabinet, Toshiba, Delhi (India). The LC–MS spec-
tra were recorded using Thermo mass spectrophotometer (USA).
Photo quantization of the gel after electrophoresis was done using
AlphaDigiDoc™ RT. Version V.4.0.0 PC-Image software, California
(USA).
General synthesis of ligands
Ligands were prepared by modified Kronke pyridine synthesis
[27] by using halo pyridinium salt of 2-acetyl thiophene, ammo-
nium acetate and different chalcone in methanol. The mixture
was reflux for 6–7 h on sand bath. The product was obtained by
keeping the solution in ice bath. The product was purified by crys-
tallization in n-hexane. Ligands were characterized by 1H and 13C
NMR spectroscopy (Supplementary material 1: Physicochemical
parameters of ligands L1–L6). The proposed structure of ligands is
shown in Scheme 1.
Experiments
Material and reagents
All solvents, chemicals and reagents used were of analytical re-
agent grade and were used as such; double distilled water was
used throughout the experiments. Ciprofloxacin (CFLH) was pur-
chased from Bayer AG (Wuppertal, Germany). Cupric chloride
was purchased from E. Merck (India) Ltd., Mumbai. 4-Fluoro
benzaldehyde, 4-chloro benzaldehyde, 4-bromo benzaldehyde, 4-
methyl benzaldehyde, 3-bromo benzaldehyde, 3-chlorobenzalde-
hyde and 2-acetyl thiophene were purchased Spectrochem Pvt.
Ltd., Mumbai (India). Luria Broth, agarose, ethidium bromide, TAE
General synthesis of complex
Methanolic solution of CuCl2ꢂ2H2O (1 mmol) was added to a
methanolic solution of neutral bidentate ligand (Ln) (1 mmol), fol-
lowed by addition of previously prepared methanolic solution of
ciprofloxacin in presence of CH3ONa (1 mmol). The pH of reaction
mixture was adjusted at ꢃ6.8 using dilute solution of CH3ONa. The
resulting solution was refluxed for 3 h on a water bath, followed by
concentrating it to half of its volume. A fine amorphous product
Scheme 1. General synthesis of ligands and proposed structure.

68
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
Scheme 2. Proposed structure and general synthesis of complexes.
obtained was washed with chloroform and dried in vacuum desic-
cators. The proposed structure of complexes is shown in Scheme 2.
calculated in order to check biocidal activity of metal complexes,
yielding 30–300 colonies.
Biological evaluation of synthesized complexes
DNA binding study by absorption titration
Binding of DNA via intercalation mode usually results in hypo-
chromism and bathochromism [29–33], because intercalation
mode involves a strong stacking interaction between an aromatic
chromophore and the DNA base pair [34]. With a selection of an
appropriate absorbance peak by performing spectrophotometric
wavelength scans of each Cu(II) complexes. After addition of equiv-
alent amount of DNA to reference cell, kept for 10 min incubation
at room temperature followed by absorption measurement. This
was specifically done to enable direct comparison between the as-
says that was required to interpret the results obtained. The intrin-
sic binding constant, Kb was determined by making it a subject in
following equation [35]
In vitro antimicrobial activity
In the present study, serial tube dilution technique was em-
ployed [28]. The tests were performed in triplicate and activities
of the compounds were tested against Escherichia coli, Pseudomonas
aeruginosa, Stapylococcus aureus, Bacillus subtilis and Serratia
marcescens. The former three are Gram(ꢀve) while later two are
Gram(+ve) organism. Selection of bacteria is of choice in prelimin-
ary screening test organism for several reasons. They are systemic
pathogens and seem to develop antibiotic resistance more readily
than any other bacteria and laboratory animals can be readily in-
fected with it. The inhibition of growth of this organism produced
by various concentrations of the test compounds was compared
under identical conditions with inhibition of growth of the same
organism in presence of several fluoroquinolone drugs, metal salt,
and ligands.
A standard volume (10 mL) of Luria Broth that would support
the growth of the test organism was added to several labeled ster-
ile stopper identical assay tubes. Solution of each test compounds
in a series of dilution was prepared. Dilution for metal salts, ligand
and standard drugs were also prepared and a control tube contain-
ing no test compound was also included. All these operations were
carefully performed under aseptic conditions. Assay tubes were
incubated at 37 1 °C for 24 h. The resultant faint turbidity was
measured. The minimum inhibitory concentration (MIC) of a
test compound is the lowest concentration showing no visible
turbidity.
½DNAꢄ=ðea
—
ef Þ ¼ ½DNAꢄ=ðeb
—
ef Þ þ 1=Kbðeb
—
ef
Þ
where, [DNA] is the concentration of DNA in base pairs,
e
a the
apparent extinction coefficient is obtained by calculating Aobs.
[complex], ef corresponds to the extinction coefficient of the com-
plex in its free form and b refers to the extinction coefficient of the
complex in the fully bound form. When each set of data, fitted to
the above equation, gave a straight line with a slope of 1/( f)
b ꢀ
and y-intercept of 1/Kb( f). The Kb value was determined from
/
e
e
e
e
b ꢀ
e
the ratio of the slope to intercept.
Hydrodynamic volume measurement
Hydrodynamic volume change was measured using Ubbelohde
viscometer immersed in thermostatic bath maintained at
a
In addition to MIC, the bactericidal action of all compounds was
evaluated against two Gram(+ve) and three Gram(-ve) bacteria in
terms of colony forming unit (CFU). For CFU, inoculum of 106 viable
bacteria/mL was prepared by diluting an overnight culture grown
in Luria Broth. Bacteria were exposed to various concentrations
of compounds. The final volume was 1 mL. Cultures were incu-
37 0.1 °C. Flow times were measured with a digital stopwatch,
each sample was measured three times, and an average flow time
was calculated. Mixing of test compound and Hearing Sperm DNA
were done by bubbling air to it. Data are presented as (
sus [complex]/[DNA], where is the viscosity of DNA in the pres-
g/g0)
1/3 ver-
g
ence of complex and g0 is the viscosity of DNA alone. Viscosity
values were calculated from the observed flow time of DNA-
containing solutions (t) corrected for that of the buffer alone (t0),
bated at 37 °C for 2 h. The 100 lL bacterial culture from above
was taken and spread over previously prepared agar plate. These
were incubated for 24 h at 37 °C and the visual colonies were
g
= (t ꢀ t0) [36].

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
69
DNA cleavage study
of concentration of samples were plotted against percent of mor-
tality of nauplii [40].
Gel electrophoresis of plasmid DNA (pUC19 DNA) was carried
out in TAE buffer (0.04 M Tris–Acetate, pH 8, 0.001 M EDTA). Fif-
teen microliters of reaction mixture containing plasmid DNA
Result and discussion
(150
200
the reactions were quenched by addition of 5
l
l
g/mL) in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and
M complex were allowed to proceed for 24 h at 37 °C. All
L loading buffer
Characterization of complexes
l
(40% sucrose, 0.25% bromophenol blue, 0.25% xylene cyanole FF,
200 mM EDTA). The aliquots were loaded directly onto 1% agarose
gel and electrophoresed at 50 V in 1ꢁ TAE buffer. Gel was stained
FT-IR, UV–visible spectroscopy, magnetic measurement and LC–
MS techniques were used to evaluate the structure of the com-
plexes. Physico-chemical parameters and microanalysis data (Sup-
plementary material 2: Physico-chemical parameters and
microanalysis data of complexes) are in good agreement with pro-
posed structure.
with 0.5 lg/mL ethidium bromide and was photographed on a UV
trans-illuminator. The percentage of each form of DNA was quan-
tified based on illumination using AlphaDigiDoc™ RT. The degree
of DNA cleavage activity was expressed in terms of the percentage
of cleavage of the SC-DNA according to the following equation [37]
Spectrophotometric titration
The amount of copper was determined by spectrophotometric
titration technique [41,42]. The calculated results from the equiv-
alent endpoint (Fig. 1, Supplementary material 3: Spectrophoto-
metric titration data for the complexes) reveal the metallic
content of the complex 1 (Supplementary material 4: Spectropho-
tometric titration curves for equivalent endpoint determination of
the complexes).
Percent of DNA cleavage ¼ðPercent of SC-DNAÞcontrol
ꢀ ðPercent of SC-DNAÞsample
=
ðPercent of SC-DNAÞcontrol ꢁ 100
Enzymatic behavior
NBT/NADH/PMS system was used to study SOD-like behavior of
the complexes. The superoxide radial produced by 79 M NADH,
M PMS in phosphate buffer (pH = 7.8) was responsible for
M NBT in system, and 0.25–3.0 M tested compound are
l
IR spectroscopy
30
75
l
l
Major changes in IR spectra of ligands on complexation are
comprised in Table 1 and some important points are as follows:
l
responsible for retardation in the reduction rate of NBT. Retarda-
tion in the reduction rate of NBT was determined spectrophoto-
metrically by monitoring the concentration of blue formazan
form which absorbs at 560 nm. All measurements were carried
ꢅ Bands at 1780 and 1624 cmꢀ1 in case of ciprofloxacin corre-
sponds to m(C@O)carb and m(C@O)p, respectively, which on com-
plexation with metal ion shift to about 1580 and 1350 cmꢀ1
respectively [43].
,
out at room temperature. The percent inhibition (
g) of NBT reduc-
tion was calculated using following equation [38],
ꢅ Unidentate nature for the carboxylato group of ciprofloxacin
is proved by frequency separation of about 220 cmꢀ1
g
ðpercent inhibition of NBT reductionÞ ¼ ð1 ꢀ k0=kÞ ꢁ 100
(Dm@COOas–mCOOs) [44].
ꢅ Deprotonation of the hydroxyl group of ciprofloxacin is con-
firmed by deduction of band at 3519 cmꢀ1 from spectra due
to hydrogen bonding [43].
where k0 and k present the slopes of the straight line of absorbance
values as a function of time in presence and absence of SOD mimic
or a model compound, respectively. IC50 value of the complex was
determined by plotting the graph of percentage inhibition of NBT
reduction against increase in concentration of the complex. Concen-
tration of the complex which causes 50% inhibition of NBT reduc-
ꢅ The band at 1624 cmꢀ1 responsible for
m(C@O)p in ciprofloxacin
is observed between 1622 and 1632 cmꢀ1 in case of complexes
[12], these data are further supported by m(M–O) appears at
ꢃ570–540 cmꢀ1 [45],
m
(M–N) appears at ꢃ490–530 cmꢀ1 and
tion is reported as IC50
.
m
(M–S) appears at ꢃ440–470 cmꢀ1 [46].
Cytotoxic activity – Brine shrimp lethality bioassay
Electronic and magnetic behavior
Brine shrimp (Artemia cysts) lethality bioassay technique was
applied for the determination of general toxic property of com-
plexes. The in vitro lethality test has been carried out using brine
shrimp eggs i.e. Artemia cysts. Brine shrimp eggs were hatched in
a shallow rectangular plastic dish (22 ꢁ 32 cm), filled with artificial
seawater, which was prepared with commercial salt mixture and
double distilled water. An unequal partition was made in the plas-
tic dish with the help of a perforated device. Approximately 50 mg
of eggs were sprinkled into the large compartment, which was
darkened while the minor compartment was opened to ordinary
light. After 2 days nauplii were collected by a pipette from the
lighter side. A stock solution of the test complex was prepared in
DMSO. From this stock solution, solutions were transferred to the
Copper(II) complexes, i.e. d9 system with a simple ligand at low
temperature exhibit an absorption band with a large width make
them very difficult to interpret. Complexes of copper(II) with dif-
vials to make final concentration 2, 4, 6, 8, 10, 12 lM, etc. (three
for each dilutions were used for each test sample and LC50 is the
mean of three values) and three vial was kept as control having
of DMSO only. After 2 days, when the of nauplii were ready, 1 mL
of seawater and 10 of nauplii were added to each vial and the vol-
ume was adjusted with seawater to 2.5 mL per vial [39]. After 24 h
each vial was observed using a magnifying glass and the number of
survivors in each vial was counted and noted. Data were analyzed
by simple logit method to determine the LC50 values, in which log
Fig. 1. The spectrophotometric titration curves for equivalent endpoint determi-
nation of the complex 1.

70
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
Table 1
Characteristic absorptions bands of IR spectra of the complexes and CPFH (cmꢀ1).
Compound
m(C@O) pyridone
m
(COO)as
m
(COO)s
D
m
m
(M–O)
m
(M–N)
m(M–S)
CPFH
1624
1625
1629
1627
1632
1622
1630
1708a
1575
1574
1578
1577
1580
1576
–
–
–
–
–
[Cu(CPF)L1Cl] (1)
[Cu(CPF)L2Cl] (2)
[Cu(CPF)L3Cl] (3)
[Cu(CPF)L4Cl] (4)
[Cu(CPF)L5Cl] (5)
[Cu(CPF)L6Cl] (6)
1352
1349
1358
1354
1355
1355
223
225
220
223
228
221
565
570
540
560
575
545
510
505
525
520
490
515
455
465
440
485
470
450
a
As m(COOH).
Fig. 2. LC–MS spectrum of complex [Cu(CPF)L1Cl].
ferent coordination numbers resulting in different geometry. The
Biological evaluation of synthesized complexes
In vitro antimicrobial activity
The complexes were screened for in vitro antimicrobial activity
against two Gram(+ve) i.e. S. aureus, B. subtilis and three
Gram(ꢀve), i.e., S. marcescens, P. aeruginosa and E. coli microorgan-
isms using serial tube dilution technique. The antimicrobial activ-
ity data are shown in Table 2, indicate that:
copper(II) complexes exhibit
a
broad band at ꢃ15,300 cmꢀ1
[47,48] corresponding to a characteristic d–d transition in tetrago-
nal field, suggesting distorted square pyramidal geometry for cop-
per(II) complexes.
The magnetic moments measurement for any geometry in cop-
per(II) complexes generally results in 1.8 BM, which is very close to
spin-only value, i.e. 1.73 BM. The observed values in our case are
very close to the spin-only values (Supplementary material 2:
Physico-chemical parameters and microanalysis data of com-
plexes) expected for S = ½ system (1.73 BM) which lead to a path
of conclusion that metal center in synthesized complexes posses
five coordination number with one unpaired electron responsible
for S = ½ system [49,50].
ꢅ Except complex 4, all the complexes are more active against S.
aureus.
ꢅ In case of B. subtilis the complexes 1–3 are more active than the
complexes 4–6.
ꢅ All the complexes are more active in case of S. marcescens.
ꢅ Complexes 1 and 2 are more active, 3 is comparatively active
while complexes 4–6 are not active against P. aeruginosa.
ꢅ Complexes 1, 2, 3 and 6 are more active while 4 and 5 are not
active against E. coli.
LC–MS spectra analysis
LC–MS spectrum of complex [Cu(CPF)(L1)Cl] is shown in Fig. 2.
Mass spectrum of the complex show molecular ion [M+] at 760.74
m/z and [M+2] at 762.24 m/z. Peaks at 430.40 and 432.40 m/z are
due to ligand attached with copper and one chlorine atom. The peak
at 429.33 and 431.33 m/z corresponds to ciprofloxacin attached to
copper(II) including coordinated chlorine atom. The peak at
394.95 m/z is due to removal of chlorine atom from neutral biden-
tate ligand. The peak at 331.41 m/z is due to the loss of copper from
neutral bidentate ligand. The peak at 331.15 m/z corresponds to cip-
rofloxacin moiety. The peak at 248.28 m/z is due to fragment of neu-
tral bidentate ligand. Peak at 247.14 m/z corresponds to loss of
piperidine moiety from ciprofloxacin. Peak at 203.21 m/z is due to
loss of COOH group from fragment of ciprofloxacin (Supplementary
material 5: Proposed mass fragmentation pattern of complex 1).
This increase in antimicrobial activity may be due to Overtone’s
concept [51], chelation theory [52,53] or the following factors [54]
such as: (i) the chelate effect of the ligands; (ii) the nature of the
NS-donor ligands; (iii) the total charge of the complex; (iv) the
existence and the nature of the ion neutralizing the ionic complex;
(v) the nuclearity of the metal center in the complex.
The first two of the five above-mentioned factors may be
responsible for higher antimicrobial activity; that is the chelate ef-
fect provided by both the ciprofloxacin ligand and the NS-donor li-
gand and the nature of the ligands. This is probably one of the main
reasons for the diverse antibacterial activities shown by the com-

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
71
Table 2
Antimicrobial activities of CPFH, copper(II) salt and complexes in terms of minimum inhibitory concentration (MIC) (
lM).
Compound
Gram positive
Gram negative
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl2ꢂ2H2O
2698.0
1.52
0.75
0.80
0.92
1.55
1.30
1.23
2815.0
1.04
0.30
0.40
0.45
1.40
1.22
1.10
2756.0
1.52
0.45
0.75
0.80
1.35
1.28
0.95
2404.0
1.32
0.95
1.15
1.30
1.73
1.65
1.60
3402.0
1.36
0.80
0.90
1.12
1.52
1.40
1.25
CPFH
[Cu(CPF)(L1)Cl] (1)
[Cu(CPF)(L2)Cl] (2)
[Cu(CPF)(L3)Cl] (3)
[Cu(CPF)(L1)Cl] (4)
[Cu(CPF)(L5)Cl] (5)
[Cu(CPF)(L6)Cl] (6)
Fig. 3. Electronic absorption spectra of [Cu(CPF)L1Cl] in phosphate buffer
Fig. 4. Effect of increasing amount of EtBr, CPFH and complexes on the relative
viscosity of Herring Sperm DNA at 37 0.1 °C.
(Na2HPO4/NaH2PO4, pH 7.2) in the absence and presence of increasing amount of
DNA. The [Cu] complex = 10
lM; [DNA] = 0–150
lM. The incubation period is
10 min. at room temperature, Inset: Plot of [DNA]/(ea
shows the absorbance change upon increasing DNA concentrations.
ꢀ
e
f) versus [DNA]. Arrow
Table 3
Binding constant (Kb), IC50 and LC50 values of synthesized complexes.
Complex
Kb (Mꢀ1
)
IC50 value (
lM)
LC50
8.07
11.18
13.01
25.67
21.64
15.89
(lM)
[Cu(CPF)(L1)Cl] (1)
[Cu(CPF)(L2)Cl] (2)
[Cu(CPF)(L3)Cl] (3)
[Cu(CPF)(L4)Cl] (4)
[Cu(CPF)(L5)Cl] (5)
[Cu(CPF)(L6)Cl] (6)
2.45 ꢁ 105
2.04 ꢁ 105
1.41 ꢁ 105
1.02 ꢁ 105
1.29 ꢁ 105
1.37 ꢁ 105
0.55
0.75
0.89
1.25
1.16
0.95
Fig. 5. Photogenic view of cleavage of pUC19 DNA (300
copper(II) complexes (200 M) using 1% agarose gel containing 0.5
bromide. All reactions were incubated in TE buffer (pH 8) in a final volume of 15
l
g/mL) with series of
g/mL ethidium
L,
l
l
l
for 24 h at 37 °C. Lane 1, DNA control; Lane 2, CuCl2ꢂ2H2O; Lane 3, ciprofloxacin;
Lane 4, [Cu(CPF)L1Cl]; Lane 5, [Cu(CPF)L2Cl]; Lane 6, [Cu(CPF)L3Cl]; Lane 7,
[Cu(CPF)L4Cl]; Lane 8, [Cu(CPF)L5Cl]; Lane 9, [Cu(CPF)L6Cl].
plexes. The significant improvement of the activity of ciprofloxacin
when coordinated to the copper complex is simply an evidence of
the role of the coordinated metal ion.
binding constant of complex 1 is due to electron withdrawing
group present on the ancillary ligand [60].
In addition, our study regarding bactericidal activity in terms of
CFU/mL of above metal complexes against same microorganisms
reveals that, decrease in number of colonies with increasing the
concentration of complexes. The CFU/mL for different microorgan-
ism against complexes is shown in Supplementary material 6.
Hydrodynamic volume measurement
Binding modes of the complexes were further investigated by
viscosity measurements. Though photophysical experiments give
necessary information about binding modes of metal complexes
with DNA, but not provide conclusive evidences for the exact mode
of binding. Viscosity of DNA is increased in the case of classical
intercalator due to increase in the length of DNA helix, as base
pairs are separated to accommodate the intercalator.
In absence of crystallographic study, it is found that relative vis-
cosity measurement study is the most critical tests for exploding
interaction properties between complexes and DNA, in solution
state [61]. Fig. 4 shows that the binding ability of classical interca-
lator ethidium bromide is more compare to all complexes. In our
case increase in viscosity was observed, hence complexes bind to
DNA via intercalation mode [62] and the magnitude of increase
in relative viscosity is same as shown by Haq et al. [63], and out
of all, complex 1 interact more strongly compare to other.
DNA binding study by absorption titration
Basic principle of absorption titration is change in spectral tran-
sition of coordination compounds on interaction with DNA. With
increase in DNA to complex ratio, hypochromism and red shift
was observed (Fig. 3). The extent of binding strength of complexes
was quantitatively determined by measuring an intrinsic binding
constants Kb (Table 3). This is lower than Kb value of classical inter-
calator ethidium bromide but higher than reported [CuII(phen)(bn-
p)H2O] [55], [CuL1(ClO4)2] [56], [Zn(erx)2(H2O)2] [57],
[Ni(sf)2(bipyam)] [58] and [Ni(oxo)2(H2O)2] complexes [59]. Thus,
there is a possibility of intercalation of complexes. The highest

72
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
Table 4
Complex mediated DNA cleavage data by gel electrophoresis.
Lane No.
Compound
Form I (SC)
Form II (OC)
Form III (LC)
%Cleavage
1
2
3
4
5
6
7
8
9
DNA
81
76
63
10
20
25
26
28
31
19
24
23
50
50
41
40
37
33
–
–
–
6.17
CuCl2ꢂ2H2O
Ciprofloxacin
14
40
30
34
34
35
36
22.22
87.65
75.31
69.14
61.72
65.43
67.90
[Cu(CPF)(L1)Cl] (1)
[Cu(CPF)(L2)Cl] (2)
[Cu(CPF)(L3)Cl] (3)
[Cu(CPF)(L1)Cl] (4)
[Cu(CPF)(L5)Cl] (5)
[Cu(CPF)(L6)Cl] (6)
Fig. 7. The plot of percentage of inhibiting NBT reduction with an increase in the
concentration of complex 1.
Fig. 6. Plot of absorbance (Abs560) as a function of time (t) to determine% inhibition
of formazan formation at various concentration of complex
function of time.
1 (0.5–3 lm) as
scavenging activity toward superoxide radicals, which may be
accredited to the redox potential of Cu(II) complex, geometry at
metal center, the nature of coordinating ligand and acidic anion
on apical position. Distorted geometry of complexes may favor
the geometrical change, which is essential for the catalysis of cop-
per in the Cu–Zn SOD that also changes distorted square pyramidal
geometry [67,68].
DNA cleavage study
Transition metal complex mediated DNA cleavage is the center
of interest [64,65]. When plasmid DNA was subjected to electro-
phoresis after interaction, upon illumination of gel (Fig. 5) the fast-
est migration was observed for super coiled (SC) Form I, where as
the slowest moving was open circular (OC) Form II and the inter-
mediate moving is the linear (LC) Form III, generated on cleavage
of open circular. The data of plasmid cleavage are presented
in Table 4. Here the complex 1 shows the maximum cleavage
ability compared to all synthesized complexes. The different
DNA-cleavage efficiency of the complexes, metal salt and drugs is
due to the difference in binding affinity of the complexes to DNA
and the structural dissimilarities of ligands.
Cytotoxic activity – Brine shrimp lethality bioassay
LC50 values of test complexes observed after 24 h are shown in
Table 3. From the results it was found that the complexes showed
considerable cytotoxicity in the brine shrimp (Artemia cysts) lethal-
ity bioassay, but complex 1 showed maximum activity than rest of
the complexes. The increasing order of cytotoxic assay of com-
plexes are as follow 1 > 2 > 3 > 6 > 5 > 4.
Conclusion
Enzymatic behavior
Here in this work, we have synthesized six Cu(II) metallointer-
calators with different neutral bidentate ligands and ciprofloxacin.
The antibacterial activity of ciprofloxacin is changed upon coordi-
nation with metal ion. Hypochromism and bathochromism of band
in absorption titration and increase in relative viscosity of DNA
suggest that all complexes bind with DNA via classical intercalative
mode. Complexation of drug with copper(II) ion enhances their
DNA cleavage ability. Ligand which can facilitate the stabilization
of bonding between metal center and oxygen radical anion favors
enhancement in enzymatic behavior. These results suggest that
the synthesized complexes can be kept forward for their in vivo
nuclease, antibacterial and enzymatic behavior.
NBT/NADH/PMS system was used to check SOD like activity of
the synthesized complexes. The percentage inhibition of formazan
formation at various concentrations of complexes as a function of
time was measured by measuring the absorbance at 560 nm and
plotted to have a straight line (Fig. 6). With increase in concentra-
tion of tested compounds decrease in slope (m) was observed. Per-
centage inhibition of the reduction of NBT plotted against
concentration of the complex (Fig. 7). The SOD activity of the pres-
ent synthesized complexes is given in Table 3. All the complexes
show better radical scavenging ability than complexes reported
by Chao et al. [66]. It is clear that the complexes exhibit greater

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 66–73
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