Antimicrobial efficacy of phenanthrenequinone based Schiff base complexes incorporating methionine amino acid: Structural elucidation and in vitro bio assay

Article abstract of DOI:10.1016/j.saa.2014.02.074

This work focuses the synthesis and characterization of few novel mixed ligand Schiff base metal complexes and their biological activities. For deriving the structural aspects, spectral techniques such as FT-IR, UV-Vis., ¹H NMR, Raman, EPR and

Full text of DOI:10.1016/j.saa.2014.02.074

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Antimicrobial efficacy of phenanthrenequinone based Schiff base
complexes incorporating methionine amino acid: Structural elucidation
and in vitro bio assay
⇑
Thesingu Rajan Arun, Natarajan Raman
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Few novel mixed ligand Schiff base metal complexes incorporating methionine amino acid act as efficient
DNA binding and DNA cleaving agents. They are good antimicrobial reagents. Copper complex is found to
be good amylase inhibitor.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of good DNA
metallointercalators.
Synthesis of better antimicrobial
agents of metal complexes.
Designing and synthesis of N3O type
square planar complexes.
Efficient amylase inhibition activity of
the copper complex.
Synthesis of better DNA chemical
nucleases.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 January 2014
Received in revised form 29 January 2014
Accepted 11 February 2014
Available online 27 February 2014
This work focuses the synthesis and characterization of few novel mixed ligand Schiff base metal
complexes and their biological activities. For deriving the structural aspects, spectral techniques such
as FT-IR, UV–Vis., 1H NMR, Raman, EPR and the physicochemical characterizations including elemental
analysis, molar conductance and magnetic susceptibility method have been involved. All the complexes
adopt square planar geometry. DNA binding ability of these complexes has been explored using diverse
techniques viz. UV–Vis. absorption, fluorescence spectroscopy, viscometry and cyclic voltammetry. These
studies prove that CT-DNA binding of the complexes follows the intercalation mode. Comparative DNA
oxidative cleavage ability of the complexes has been done under ultraviolet photo radiation on pUC19
DNA. In addition, the biocidal action of the complexes has been investigated against few pathogenic bac-
teria and fungi by disc diffusion method. Importantly, the amylase inhibition activity of Cu(II) complex
has been explored. The amylase inhibition property has been found to be increased upon increasing
the complex concentration.
Keywords:
Schiff base
Fluorescence
Methionine complexes
Intercalation
Amylase inhibition
Antimicrobial activity
Ó 2014 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Tel.: +91 092451 65958; fax: +91 4562 281338.
E-mail address: ramchem1964@gmail.com (N. Raman).
http://dx.doi.org/10.1016/j.saa.2014.02.074
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
293
fects [32]. With the above said natural biological importance,
attempting Cu(II) Schiff base complex as amylase inhibitor may
be the positive approach to avoid the problems associated with
the synthetic hypoglycemic agents.
Introduction
Over the years and till now, the study on Schiff base ligands is of
keen attention since they are being considered as the most privi-
leged ligands in the coordination chemistry due to their ease of
preparation through the simple condensation reaction between
primary amines and carbonyl compounds [1]. Moreover, when
such Schiff base ligands are coordinated with metal ions, their
applications are found to be enhanced. For instance, the applicabil-
ity of Schiff base complexes extends to diverse fields such as catal-
ysis [2], sensors [3], luminescent [4] and non linear optical
materials [5]. Importantly, the imine (AC@N) group of Schiff base
ligands leads to the stupendous biological activities including anti-
tumor, antibacterial, antifungal and herbicidal activities [6,7]. The
importance of Cu(II), Co(II), Ni(II) and Zn(II) ions in nature makes
them ardent interest to study the biological properties of their
new analogues. In actual fact, the role of Cu(II) in hemocyanin,
Co(II) in vitamin-B12, Ni(II) in plant urease enzyme and Zn(II) in
peptidase enzymes are the best examples for their biological
importance [8–11].
Amino acids contribute the building blocks of proteins and are
chemical species indispensable for performing a huge number of
biological functions, as exemplified by the role of enzymes
[12,13]. In recent years, they have been actively involved in the
preparation of coordination complexes due to their multiple func-
tional groups (COOH and/or NH2) which act as potential donor
sites. Importantly, such multiple functional groups present in the
amino acid side chain of the ligands can lead to the development
of unexpected and unusual structures [14,15]. The transition metal
complexes of amino acids are significant owing to their function as
model systems for the study of molecular structure, metal–metal
interactions of metalloproteins [16,17]. Among the ramp of amino
acids, methionine (met) is special case because of its coordination
behavior with diverse metal ions [18–21]. The above facts put for-
ward that incorporating amino acid into the metal complexes
structure would be the prolific attitude both in structural and bio-
logical aspects.
DNA executes an imperative function in the living process as it
contains all the hereditary information for cellular efficacy. Never-
theless, DNA molecules are prone to be smashed beneath different
setting like interactions and binding with few molecules. This
smash may lead to a variety of pathological changes in living
organisms. In this context, DNA interaction of transition metal
complexes with DNA is of particular importance for medicinal rea-
sons [22–24]. In general, the tumor cells can be destroyed by stop-
ping the replication of the affected DNA. Using Schiff base metal
complexes in particular, DNA may be damaged by either following
binding or cleavage approach. Gel electrophoresis is the best and
globally accepted technique to probe the DNA cleaving capacity
of the complexes [25,26]. In DNA binding, intercalative mode has
been established as the conventional mode when metal complexes
are under operation with DNA. Furthermore, Liu and Sadler re-
ported a review article which largely covers the list of different
complexes as DNA intercalators [27].
On the basis of above stated facts, in this present paper we re-
port the synthesis of a Schiff base from 9,10-phenanthrenequinone
and p-nitroaniline. This Schiff base ligand has been coordinated
with Cu(II), Co(II), Ni(II) and Zn(II) to form the mixed ligand
complexes using methionine as coligand. The ligand(L) and the
complexes have been characterized well by physicochemical and
various spectroscopic techniques. Significantly, their collective bio-
logical properties have been analyzed by antimicrobial, DNA cleav-
age using gel electrophoresis, DNA binding and amylase inhibition
studies.
Experimental
Materials
The chemicals involved in this work were of AnalaR grade and
were used without further purification. However, the solvents
were purified by following the standard procedure [33]. 9,10-
Phenanthrenequinone, CT-DNA, pUC19DNA, ethidium bromide
and p-nitroaniline were obtained from Sigma Aldrich. Solvents,
methionine (met) and agar were procured from Hi-media chemi-
cals. All the metal salts were received from E-Merck.
Instruments employed
Elemental analysis (C, H and N) data were obtained using a Per-
kin-Elmer 240 elemental analyzer. Electronic spectra of the com-
plexes were recorded on
a Shimadzu Model 1601 UV–Vis.
Spectrophotometer in the wavelength range of 200–1100 nm.
Vibrational spectra were performed on FTIR-Shimadzu model IR-
Affinity-1 Spectrophotometer using KBr discs. Proton NMR spectra
of the ligand and the complex were recorded on a Bruker Avance
DRX 300 FT-NMR spectrometer using tetramethylsilane as the
internal standard. Room temperature magnetic susceptibility mea-
surements were carried out on a modified Gouy-type magnetic
balance, Hertz SG8-5HJ. The room temperature molar conductivity
of the complexes in DMSO solution (10ꢁ3 M) was measured using a
deep vision 601 model digital conductometer. The X-band EPR
spectrum was performed at LNT (77 K) using DPPH as the g-mar-
ker. Cyclic voltammetric experiments were achieved on a CHI
620C electrochemical analyzer in freshly distilled DMSO solution.
Synthesis of Schiff base ligand and its metal complexes
Synthesis of Schiff base ligand (L)
An ethanolic solution of 9,10-phenanthrenequinone (0.01 M)
was added drop wise to an ethanolic solution of p-nitroaniline
(0.01 M). Few drops of glacial acetic acid were added to the reac-
tion mixture and were refluxed for 4 h. The solid product formed
was filtered, washed, dried and recrystallized from ethanol, dried
in vacuo. Yield: 74%; yellow colour; Anal.Calc. (%): C (69.6), H
(3.6) and N (12.5); Found (%): C (69.3) H (3.2) and N (12.2); FT-IR
(KBr) (cmꢁ1): 1631(C@N), 2900–2950 (CAH) and 1400–1600
(C@C); 1H NMR (DMSO-d6) dppm: 6.9–7.4 (aromatic protons);
Amylase, one of the key enzymes in human body, deeply helps
to catalyze the hydrolysis of starch into smaller sugar products
including glucose. Though the presence of amylase is mandatory,
its amount could not exceed than 0–137 lL. Otherwise, it may fur-
ther lead to elevated post-prandial hyperglycemia or diabetes
which comes under the disorder like polyuria, polydipsia, polypha-
gia, emaciation, and weakness [28–31]. With these reasons, con-
trolling amylase activity, slowing down in specific, is very
important to manage the starch digestion. The present synthetic
hypoglycemic agents perform effectively against amylase but they
are non-specific, expensive and sometimes lead to serious side ef-
UV–Vis. in DMSO, cmꢁ1 (transition): 38,314 ( pꢂ) and 34,742
p–
(n–pꢂ).
Synthesis of mixed ligand complexes
For the synthesis of mixed ligand complexes, initially an equi-
molar ethanolic mixture of L (0.01 M) and the metal chloride salt

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T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
were found out. Finally by comparing the identified slope and inter-
cept, Kb was calculated.
(0.01 M) was heated at 60 °C under magnetic stirring for 4 h. Then,
methionine (0.01 M) in 1:1 water–ethanol was added to the above
reaction mixture and allowed to reflux for 6 h. Finally the resultant
product was washed with ethanol and then recrystallized. The ob-
tained solid product was filtered, dried in vacuo at 60 °C and kept in
desiccator.
Cyclic voltammetry
With the motive to do DNA binding studies of the complexes, a
three electrode system (working electrode (glassy carbon), auxil-
iary electrode (platinum wire) and reference electrode (Ag/AgCl))
was set in CHI620C electrochemical analyzer to carry out cyclic
voltammetric and differential pulse voltammetric studies. All the
solutions prepared for the study were degassed by purging N2.
All the electrodes were cautiously rinsed with de-ionized water
prior to the proceedings.
The degree of reversibility of one electron transfer reaction can
be received from the difference between forward and backward
peak potentials. The ratio of equilibrium constants for the binding
of oxidative and reductive ions to DNA was calculated by the
Nernst equation as given in Eq. (2).
[CuL(met)]Cl: Yield: 72%; brown colour; Anal.Calc. (%): C (53.5),
H (3.7), N (10.0), S (4.6), Cl (5.0) and Cu (9.1); Found (%): C
(53.1), H (3.5), N (9.7), S (4.8), Cl (4.9) and Cu (9.0); FT-IR
(KBr) (cmꢁ1): 3372 (NH2), 1622 (C@N), 1447 ( asy(COOꢁ)),
t
1365 (
t
sy(COOꢁ)), 536 (MAO) and 424 (MAN); Km (Oꢁ1 molꢁ1
eff (BM) 1.77; UV–Vis. in DMSO, cmꢁ1 (transition):
-
cm2) 61.12;
l
34,246 (LMCT) and 16,920 (d–d).
[CoL(met)]Cl: Yield: 68%; green colour; Anal.Calc (%): C (53.8), H
(3.7), N (10.1), S (4.6), Cl (5.1) and Co (8.5); Found (%): C (53.4),
H (3.5), N (9.8), S (4.3), Cl (5.0) and Co (8.2); FT-IR (KBr) (cmꢁ1):
3360 (NH2), 1623 (C@N), 1440 (t t
asy (COOꢁ)), 1358 ( sy(COOꢁ)),
532 (MAO) and 424 (MAN); Km (Oꢁ1 molꢁ1 cm2) 63.91; leff
(BM) 2.55; UV–Vis. in DMSO, cmꢁ1 (transition): 33,493 (LMCT)
and 16,474 (d–d).
ꢀ
ꢁ
Kred
Kox
Eob ꢁ Eof ¼ 0:0591 log
ð2Þ
[NiL(met)]Cl: Yield: 71%; yellow colour; Anal.Calc (%): C (53.9), H
(3.7), N (10.1), S (4.6), Cl (5.1) Ni (8.4); Found (%): C (53.6), H
where Eob and Eof are the potentials of the bound and free complex
forms, respectively; Kred is the binding constant for the binding of
reductive ion species with CT-DNA and Kox is the binding constant
for the binding of oxidative ion species with CT-DNA.
(3.5), N (9.8), S (4.5), Cl (5.0) and Ni (8.1); FT-IR (KBr) (cmꢁ1
)
3358 (NH2), 1625 (C@N), 1443 (tasy (COO)), 1358 (tsy(COO)),
527 (MAO) and 432 (MN); Km (Oꢁ1 molꢁ1 cm2) 69.85; leff
(BM) diamagnetic; UV–Vis. in DMSO, cmꢁ1 (transition):
33,112 (LMCT) and 23,398 (d–d).
Fluorescence spectroscopy
[ZnL(met)]Cl: Yield: 78%; greenish yellow colour; Anal.Calc (%) C
(53.3), H (3.7); N (10.0), S (4.5), Cl (5.0) and Zn (9.3); Found (%):
C (53.1), H (3.6), N (9.8), S (4.3), Cl (4.9) and Zn (9.1); FT-IR (KBr)
In DNA binding studies, fluorescence spectroscopy plays an
important part as it helps much to find out the mode of DNA bind-
ing. With such intention, a constant amount of the compound was
titrated with increasing amounts of CT-DNA. The DNA bound Cu(II)
complex was excited at 450 nm upon increasing DNA concentra-
tion. The binding constants Kb were calculated. For such calcula-
tions, Eq. (3) was employed by monitoring the changes of
fluorescence intensity with increasing concentration of DNA.
(cmꢁ1): 3361 (NH2), 1621 (C@N), 1448 ( asy(COO-)), 1304
t
(t
sy(COO-)), 534 (MAO) and 438 (MAN); 1H NMR (DMSO-d6)
dppm: 6.5–7.8 (phenyl multiplet), 3.4 (s, ANH2) 3.9–4.2 (CAH)
(met-H) d; Km (Oꢁ1 molꢁ1 cm2) 71.67; leff (BM) diamagnetic;
UV–Vis. in DMSO, cmꢁ1 (transition): 34,112 (LMCT).
CT-DNA-binding studies
r=Cf ¼ Kbð1 ꢁ nrÞ
ð3Þ
All the DNA binding studies were executed in Tris–HCl buffer
[5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris–
HCl), 50 mM NaCl, pH 7.2] at room temperature.
where r = Cb/[DNA], Cf = Ct[(F ꢁ F0)/(Fmax ꢁ F0)], n is binding site size
of base pairs, Cb and Ct are the concentration of free compound and
the total compound, respectively. F is the observed fluorescence
emission intensity at a given DNA concentration, F0 is the intensity
in the absence of DNA, and Fmax is the fluorescence intensity of the
totally bound compound. The fluorescence emission intensity was
monitored for the Cu(II) complex.
Electronic absorption spectroscopy
To carry out the DNA binding experiment using absorption
spectral titrations, range of solutions was prepared in which the
concentration of the complex maintained at constant (0.1 mM)
while the concentration of stock CT-DNA increased from solution
to solution. The absorption spectra of these different metal com-
plex-DNA solutions were done in the range of 200–1100 nm. By
measuring the alterations in the LMCT absorption band upon
increasing DNA concentration, the intrinsic binding constants Kb
were calculated. For such calculations, Eq. (1) was employed by
monitoring the changes of absorption in the MLCT band with
increasing concentration of DNA.
Viscosity measurement
The room temperature viscosity measurements were carried
out on the Ostwald viscometer in a thermostatic water bath. Alter-
natively, in this experiment, DNA concentration was kept constant
and concentration of metal complexes solution increased gradu-
ally. The flow time of the solution through the capillary was deter-
mined by a stop-watch with the viscometer and it was found to
nearly 0.02 s. This flow time measurement was repeated three
times for the same complex solution to get the average flow time.
To study effect of DNA binding on the viscosity of the complex, a
½DNAꢃ
½DNAꢃ
1
¼
þ
ð1Þ
ð
ea
ꢁ
ef
Þ
ð
eb
ꢁ
ef
Þ
½Kbðeb ꢁ ef Þꢃ
plot was made between (
of the complexes to CT-DNA, where
g/go
)
1/3 and the ratio of the concentration
is the viscosity of CT-DNA in
In the above equation, [DNA] denotes the concentration of DNA,
g
absorption coefficients ea ef and eb correspond to Aobs/[complex],
free complex’s extinction coefficient and the extinction coefficient
of the complex in the totally bound form, respectively. From the
,
the presence of the compound and go is the viscosity of CT-DNA
alone. Viscosity values were calculated from the experimental flow
time of CT-DNA containing solutions corrected from the flow time
of buffer alone.
equation (Eq. (1)), slope (1/(eb
–
e
f) and intercept (1/[Kb(eb
ꢁ
ef)])

T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
295
Amylase inhibition assay
nystatin were chosen as standards for antibacterial activity and
antifungal activity, respectively. The nutrient agar and dextrose
agar in petri plates were served as the medium for the growth of
bacteria and fungi respectively. The DMSO solution of the complex
was soaked in a filter paper disc of 5 mm diameter and 1 mm
thickness. These discs were placed on the formerly seed plates
and incubated at 37 °C. After 24 h, the inhibition zone around each
disc was measured.
The inhibition assay was performed using chromogenic dinitro-
salicylic acid (DNSA) method as reported earlier [34,35]. The total
assay mixture composed of 1400
lL of 0.05 M sodium phosphate
buffer (pH = 6.9), 50 L of amylase and Cu(II) complex (at different
l
concentrations of 25, 50 and 100
10 min. After pre-incubation, 500
lg) was incubated at 37 °C for
lL of 1% starch solution in the
above buffer was added to each tube and again incubated at
37 °C for 15 min. The reaction was terminated with 1.0 mL DNSA
reagent, placed in boiling water bath for 5 min, cooled to room
temperature and the absorbance was measured at 540 nm. The
control amylase represented 100% enzyme activity and did not
contain any sample of analysis. To eliminate the absorbance pro-
duced by sample, appropriate extract controls with the extract in
the reaction mixture in which the enzyme was added after adding
DNS. The maltose liberated was determined with the help of stan-
dard maltose curve and the activities were calculated according to
the following equation, Eq. (4).
Results and discussion
All the compounds are air stable for extended periods and the
complexes are remarkably soluble in DMF and DMSO.
Elemental analysis and molar conductivity measurements
The obtained elemental analysis data of the complexes are well
comparable with the calculated values and show that the com-
plexes may have the stoichiometry of [ML(met)]Cl, wherein Schiff
base ligand (L) acts as a bidendate. All the compounds are soluble
in DMSO and their molar conductivities of their solution (10ꢁ3 M)
are measured at room temperature. The higher molar conductance
Conc: of maltose liberated ꢄ enzyme used ðmLÞ
Activity ¼
Mol: Wt of maltose ꢄ incubation time ðminÞ
ꢄ dilution factor
ð4Þ
values (61.12–71.63
X
ꢁ1 cm2 molꢁ1) of the complexes support
One unit of enzyme activity is defined as the amount of enzyme
required to release one M of maltose from starch per min under
their electrolytic nature. The formation of the Schiff base ligand
and its complexes are given in Scheme 1.
l
the assay conditions. The inhibitory property shown by the
complex was compared with that of control and expressed as
percent induction/inhibition. This was calculated according to the
following equation, Eq. (5).
FT-IR spectroscopy
As vibrational spectroscopy is the most conventional tool to
confirm the structure of the compounds, vibrational spectra of L
and the complexes have been recorded in the region 400–
4000 cmꢁ1 and are shown in Supplementary File (Fig. S1). The
bands noted between 2900–2950 cmꢁ1 in the FT-IR spectrum of L
(Fig. S1a) are allocated to the presence of CAH bonds in the aro-
matic benzene rings. In addition, the characteristic bands for aro-
Activity in presence of complex
% Inhibition ¼
ꢄ 100
ð5Þ
Control activity
Acarbose was used as a standard inhibitor and it was assayed at
above mentioned test sample concentrations. The assay method
was similar to the above mentioned procedure, instead of test sam-
ples, acarbose was added. The results were compared to that of
Cu(II) complex.
matic C@C group are observed in the range of 1400–1600 cmꢁ1
.
Significantly, the expected characteristic peak of imine (C@N)
group is also presented at 1631 cmꢁ1 which confirms the forma-
tion of the Schiff base between 9,10-phenanthrenequinone and
p-nitroaniline. The collection of different peaks found at 700–
1200 cmꢁ1 may be assigned to the finger print region of L. The
FT-IR spectra of the complexes look similar to that of the ligand
(L), however shifts have been identified on some important charac-
teristic peaks. In specific, the characteristic peak of imine (C@N)
bond is shifted to the lower wave number region which might be
assigned to the coordination of L with central metal ion through
imine nitrogen [36]. This is further assured by the presence of a
band at ꢅ530 cmꢁ1 which corresponds to characteristic of MAN
bond. The appearance of a new continuing band between 3240
and 3000 cmꢁ1 could be due to the stretch of ANH2 group which
establishes the incorporation of methionine into the complex as
a co-ligand. Furthermore, the peaks appeared at ꢅ1440 and
ꢅ1350 cmꢁ1 are attributed to asymmetric and symmetric stretches
of carboxylato anion (COOꢁ) of methionine. However, the presence
of these peaks in lower wave number region than as expected may
because of the coordination of carboxyl oxygen with the metal ion.
pUC19 DNA cleavage (Gel electrophoresis)
For the solution of pUC19 DNA in the buffer (50 mM NaCl/5 mM
Tris–HCl, pH = 7.2), a ratio between the UV absorbance at 260 and
280 nm (A260/A280) was 1.8–1.9. It suggested that the chosen DNA
was sufficiently free of proteins. To accomplish cleavage reactions,
the solutions of pUC19 DNA were prepared afterward diluted with
loading dye using 1% agarose gel. To the above solution, 3
lL of
ethidium bromide (0.5 g/mL) was added and mixed well. In the
l
next step, the warm agarose was poured and instantly hold tightly
with a comb to develop sample wells. In electrophoretic tank, the
gel was placed and adequate electrophoretic buffers were added
to enclose the gel to 1 mm depth. DNA sample (20
lM), complex
(30 M) and H2O2 (50 M) in the aforesaid buffer (pH 7.2) were
l
l
mixed with a loading dye and filled into the well of the submerged
gel using a micropipette. The 50 mA electric current was passed
and the gel was taken out from the buffer. The gel was photograph-
ically captured under UV light after the completion of
electrophoresis.
This is also confirmed by the appearance of the peak at ꢅ430 cmꢁ1
,
the characteristic peak of M-O bond [37]. From the above discus-
sion, the formation of Schiff base (L) and its mixed ligand com-
plexes is confirmed and characterized.
Antimicrobial study
The in vitro potential of the complexes against bacteria and fun-
gi was tested by the disc diffusion method. The selected bacterial
strains were of Nicordia asteroide, Pseudomonas putida, Lactococcus
lactis and Escherichia coli and the fungal species were of Aspergillus
niger, Aspergillus flavus and Candida albicans. Ciprofloxacin and
UV–Vis. spectroscopy
In general, UV–Vis. spectroscopy often called as electronic
spectroscopy is used to predict the geometry of the coordination

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T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
Scheme 1. Schematic route for the synthesis of Schiff base ligand and its metal complexes.
complexes. In the UV–Vis. spectrum of ligand, notably two absorp-
tion bands are observed. The first one at 38,314 cmꢁ1 might be due
the square planar geometry to the studied Ni(II) complex. In con-
trast, Zn(II) complex does not exhibit any d–d band because of
its completely filled d10 transition.
to the p– p-electrons present in the aromatic rings (C@C),
pꢂ of the
imine group (C@N) and nitro group (N@O). The existence of second
band at 34,482 cmꢁ1 could be attributed to n–pꢂ transition due to
the presence of non-bonded electrons of C@N and NO2 groups. This
further supports the formation of L between 9,10-phenanthrene-
quinone and p-nitroaniline. These two absorption bands are also
observed in UV–Vis. spectra of all the complexes but with some
shifts either in wavelength or absorption. The shifts could be due
to the coordination of L with the metal ions. Moreover, ligand to
metal charge transfer (LMCT) transition can also be identified in
1H NMR spectra
The 1H NMR spectra of free Schiff base ligand (L) and its dia-
magnetic Zn(II) complex are recorded in d6-DMSO solution using
tetramethylsilane (TMS) as internal standard. The free ligand
shows a multiplet between 6.4 and 7.4 ppm due to aromatic pro-
tons. The NMR signals of zinc(II) complex at around 2 ppm assign
to the presence of aliphatic protons of methionine moiety. The
peak at 4.1 ppm is attributed to protons present in the ANH2
groups of methionine moiety. When compared to 1H NMR spec-
trum of L, there is no considerable change in the aromatic proton
signals in that of Zn(II) complex.
UV–Vis. spectra of all the complexes at around 35,000 cmꢁ1
.
Besides, the metal complexes exhibit an additional and much
important characteristic band which appears as the result of their
d–d transition. In detail, Cu(II) complex reveals this band at
16,920 cmꢁ1 and it can be assigned to 2B1g ? 2A1g transition, char-
acteristic of d9 square planar geometry [38]. In Co(II) complex, this
d–d band emerges at 16,474 cmꢁ1 because of 2A1g ? 2B1g transi-
tion which can be the evidence for square planar geometry of d7
Co(II) system. The Ni(II) complex reveals such d–d band at
23,398 is assigned as 1A1g ? 1B1g transition. This also allocates
Raman spectroscopy
Raman spectrum of Cu(II) complex is illustrated in Fig. 1. The
important characteristic band of C@N is observed at ꢅ1620 cmꢁ1
.
The band appeared at 1502 cmꢁ1 is assigned to the typical peak

T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
297
The EPR parameters g||, g\, gav, A|| and A were used to find out
?
the bonding parameters
measurement of covalence of the in-plane
bonding and out-of-plane -bonding respectively. The covalency
parameters of a2 and b2 have been calculated from the following
a
2, b2 and c2 which may be regarded as
-bonding, in-plane
r
p-
p
equations [42]. If
a
2 = 1.0, it indicates complete ionic character
whereas b2 = 0.5 denotes 100% covalent bonding.
a2 ¼ ꢁðAjj=0:036Þ þ ðgjj ꢁ 2:0036Þ þ ð3=7Þðg? ꢁ 2:0036Þ þ 0:04
b2 ¼ ðgjj ꢁ 2:0036ÞE= ꢁ 8ka2
c2 ¼ ðg? ꢁ 2:0036ÞE= ꢁ 2ka2
The a2 value for the present Cu(II) complex is 0.65 supporting the
covalent nature of the bonding. The b2 parameter gives an indica-
tion of the covalency of the in-plane
b2 = 0.79 which reveals the covalency of the bonding. The high value
of b2 compared to 2 indicates that the in-plane
-bonding is more
covalent than the in-plane -bonding [43]. The observed EPR data
p-bonding. The value of
Fig. 1. Raman spectrum of copper(II) complex.
a
r
p
give a supportive evidence to the conclusion obtained on the basis
of electronic spectrum and magnetic moment value. Thus the re-
sults confirm that Cu(II) complex has square planar geometry.
of aromatic C@C bonds. The asymmetric and symmetric stretches
of carboxylato anion (COOꢁ) of the methionine co-ligand are found
at ꢅ1450 and ꢅ1370 cmꢁ1 respectively. In addition, the band at
ꢅ530 cmꢁ1 is probably due to the MAO bond. The Raman spectral
assignments further support the FT-IR spectral discussions toward
the characterization of the complexes.
CT-DNA binding
For studying DNA binding behavior of metal complexes, UV–
Vis. spectroscopy is accepted as one of the most effective ways. It
is not only utilized to confirm the DNA binding as well to find
out the mode of DNA binding by calculating the binding constants
(Kb). In common, when metal complexes get bound with DNA mol-
ecules hypochromism (intercalative binding) or hyperchromism
(groove binding) has been observed in their electronic absorption
spectral bands. To study the DNA binding ability of Cu(II) complex,
its LMCT band at 402 nm has been selected and changes observed
on DNA binding are presented in Fig. 2. The observed hypochromic
shift of 13.7% in the LMCT band of Cu(II) complex proposes that the
binding mode of selected Cu(II) complex with CT-DNA molecule
might be intercalative [44]. In case of Zn(II) complex also, its LMCT
UV–Vis. spectral band (405 nm) has been chosen to explore its
DNA binding capability with CT-DNA. The noted changes in such
LMCT band upon DNA binding are given in Supplementary File
EPR spectra
The EPR spectrum of Cu(II) complex gives an enough informa-
tion about the structure of the metal ion environment in the com-
plex. i.e., the geometry, nature of the donating atoms from the
ligand and degree of covalency of the Cu(II)–ligand bonds. The
EPR spectrum of the Cu(II) complex was recorded in DMSO at li-
quid nitrogen temperature (LNT) which reveals well resolved
peaks in the low filed region. The spin Hamiltonian parameters cal-
culated for the Cu(II) complex are précised in Table 1. From this
spectral data g|| value is higher than the corresponding g\ value,
supporting the d2x ꢁ dy2 as the ground state, characteristic of square
planar geometry and axially symmetric. It has been reported that
g|| value of Cu(II) complex can be used as a measure of the covalent
character of the metal–ligand bond. If the value is more than 2.3,
the metal–ligand bond is essentially ionic and the value less than
2.3 is indicative of covalent character. The present EPR results
show that g|| is lower than 2.3 in this case suggesting that the Cu(II)
complex is covalent in nature [39]. The observed g|| value (2.14) is
in conformity with the presence of copper–nitrogen bonds in these
chelates. The present value of the g||/A|| ratio for [CuL(met)]Cl is
144 which indicates nearly square planar environments around
the Cu(II) ion with small distortions [40]. In axial symmetry the
g-values are related to the G-factor by the expression
G ¼ ðgjj ꢁ 2:0036Þ=ðg? ꢁ 2:0036Þ
which measures the exchange interaction between the Cu(II) cen-
ters in polycrystalline solid. The G value lies within the range of
4.17 for the Cu(II) complex indicating negligible exchange interac-
tion of CuACu in the complex according to Hathaway [41].
Table 1
The spin Hamiltonian parameters of copper(II) complex in DMSO solution at 300 and
77 K.
Complex
A||
A
Aiso
g||
g?
giso
a2
b2
c2
?
Fig. 2. Absorption spectrum of [CuL(met)]Cl complex in buffer pH = 7.2 at 25 °C in
the presence of increasing amount of DNA. Arrow indicates the changes in
absorbance upon increasing the DNA concentration.
[CuL(met)]Cl 132 64 85.2 2.14 2.01 2.05 0.65 0.79 0.71

298
T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
(Fig. S2). It reveals the hypochromism of 12.5%, further assigns the
intercalative mode of binding to the Zn(II) complex with CT-DNA.
Likewise, Co(II) and Ni(II) complexes involve intercalative mode
of binding with CT-DNA. The metallointercalators normally can un-
wind the DNA and their planar ligand between two intact base
pairs [45]. Here also, Cu(II) and Zn(II) complexes can insert their li-
gand L between two intact base pairs while they intercalate with
CT-DNA.
The intrinsic DNA binding constants (Kb) of complexes have
been measured using Eq. (1) to collect the information regarding
binding affinity of the complexes with CT-DNA. The calculated
values are given in Table 2. On comparison, Cu(II) exhibits the
effective intercalation than other complexes and the order of bind-
ing constants is: Cu(II) > Zn(II) > Co(II) > Ni(II). The reason for the
higher DNA binding affinity of complexes may be due to effective
release of the ligand (L) of higher planar area, extended
and aromaticity between two intact base pairs.
p-system
Cyclic voltammetry
Fig. 3. Cyclic voltammogram of [CuL(met)]Cl in buffer (pH = 7.2) at 25 °C in the
presence of increasing amount of DNA. The changes in voltammetric current upon
increasing the DNA concentration.
The consequence of the cyclic voltammetric and differential
pulse voltammetric studies is used to find out the interaction be-
tween the metal complexes and DNA which provides a beneficial
equilibrium to the formerly used spectral studies. In recent years
there is increasing curiosity in the electrochemical investigation
of interaction between complexes and DNA [46]. The cyclic
voltammograms of [CuL(met)]Cl and [ZnL(met)]Cl in buffer
(pH = 7.2) at 25 °C in the presence of increasing amount of DNA
are shown in Figs. 3 and S3. The cyclic voltammograms of the
complexes in the absence and presence of CT-DNA exhibit
important shifts in the anodic and cathodic peak potentials
followed by decrease in both peak currents, indicating the inter-
action existing between these complexes and CT-DNA [47]. The
cyclic voltammograms of the studied complexes exhibit peak cur-
rent ratio, approximately unity, which is credited that the reaction
of the complex on the glassy carbon is quasi-reversible redox
process. The data for all the complexes in DMSO upon addition of
CT-DNA and the shifts of the cathodic Epc (V) and anodic Epa (V)
potentials are shown in Table 3. The significant decrease in their
cathodic and anodic peak currents upon addition of CT-DNA to
the complexes can be attributed to the interaction of complexes
with CT-DNA. The E1/2 shifts of these complexes towards more
positive potential confirm their intercalative binding to CT-DNA [48].
The changes in the formal potential as well as the current den-
sity during the incremental addition of DNA to the complex solu-
tion confirm the intercalative interaction by using Differential
pulse voltammogram (DPV). Differential pulse voltammogram of
[CuL(met)]Cl in the absence and presence of varying amount of
[DNA] shows significant decrease of current intensity (Fig. 4). The
shift in potential is related to the ratio of binding constant by the
following equation:
in both cathodic and anodic peak potentials in the presence of
incremental addition of CT DNA. The differential pulse voltammo-
gram of the complex [ZnL(met)]Cl in the absence and presence of
varying amount of [DNA] with significant decrease of current inten-
sity is shown in Supplementary File (Fig. S4). The synthesized com-
plexes give both the anodic and cathodic peak potential shifts
which are either positive or negative. It indicates the intercalating
mode of DNA binding with methionine mixed ligand Schiff base
complexes.
Fluorescence spectrum titration
To further explore the binding mode and evaluate the similarity
of the complex bound CT-DNA the emission spectrum was studied.
Fluorescence titrations with CT-DNA are conducted by keeping the
concentration of complex invariable, and varying the CT-DNA con-
centration. Fig. 5 shows the fluorescence titration curves of the
Cu(II) complex with CT-DNA. The addition of the CT-DNA to the
Cu(II) complex leads to the obvious raise in the fluorescence inten-
sity which clearly designates that the binding of complex with
DNA can be intercalation [49]. The enrichment of fluorescence
intensity implies that the complex can insert between base pairs
of the CT-DNA. This enrichment of the fluorescence intensity is
supposed to be one of the criteria for intercalative binding
[50,51]. The intrinsic binding constants (Kb) of complexes have
been calculated using Eq. (3) to collect the information regarding
binding affinity of the complex with CT-DNA. For Cu(II) complex,
Kb value is 3.7 ꢄ 106 molꢁ1
.
Eb ꢁ Ef ¼ 0:0591 log ðK =K½oxdꢃÞ
½redꢃ
where Eb and Ef are peak potentials of the complex in the bound and
Viscosity measurements
free form respectively. All other complexes show considerable shift
Viscosity studies of CT-DNA have been carried out with the me-
tal complexes of different concentrations for further identification
and affirmation of interaction mode between complex and DNA.
Viscosity of DNA with complex of particular concentration has
been calculated on the basis of DNA flow rate through a capillary
viscometer. From the obtained flow rates, the specific viscosity
contribution due to the DNA in the presence of metal complexes
(binding agent) was calculated. In general when the intercalative
mode of interaction plays between complex and DNA, DNA helix
gets prolonged by separating the base pairs to accommodate the
binding of ligand which further results in the enhancement of
Table 2
Electronic absorption spectral properties of Cu(II), Co(II), Ni(II) and Zn(II) complexes
with DNA.
Compound
kmax
D
k (nm)
H%
Kb ꢄ 104 (Mꢁ1
)
Free
Bound
[CuL(met)]Cl
[CoL(met)]Cl
[NiL(met)]Cl
[ZnL(met)]Cl
403.4
405.0
389.5
406.2
407.2
408.3
392.6
409.1
3.8
3.3
3.1
2.9
9.4
8.1
7.9
7.1
5.7
2.1
2.7
6.9

T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
299
Table 3
Electrochemical parameters for interaction of DNA with Cu(II), Co(II), Ni(II) and Zn(II) complexes.
Compound
Redox couple
E1/2 (V)a
bDEp (V)
Ipa/Ipc
k[red]/k[oxd]
Free
Bound
Free
Bound
[CuL(met)]Cl
Cu(III) ? Cu(II)
Cu(II) ? Cu(I)
Co(III) ? Co(II)
Ni(II) ? Ni (I)
Zn(II) ? Zn(0)
ꢁ0.403
0.430
ꢁ0.228
ꢁ0.074
ꢁ0.369
ꢁ0.439
0.410
0.242
0.104
ꢁ0.358
0.195
0.074
0.232
0.157
0.161
0.247
1.26
0.99
1.12
0.89
0.81
0.39
[CoL(met)]Cl
[NiL(met)]Cl
[ZnL(met)]Cl
0.33
0.18
0.49
ꢁ0.117
ꢁ0.208
0.218
0.205
Data from cyclic voltammetric measurements:
E1/2 is calculated as the average of anodic (EPa) and cathodic (Epc) peak potentials; Ea1=2 ¼ EPa þ Epc=2;
a
b
D
Ep = EPa ꢁ Epc
.
Fig. 6. Effect of increasing amounts of [CuL(met)]Cl (ꢄ) [CoL(met)]Cl
[NiL(met)]Cl ), [ZnL(met)]Cl ), on the relative viscosity of CT DNA vs
[complex]/[DNA] (1/R) ratio.
( ),
Fig. 4. Differential pulse voltammograms of [CuL(met)]Cl in buffer (pH = 7.2) at
25 °C in the presence of increasing amount of DNA. Arrow indicates the changes in
voltammetric currents upon increasing the DNA concentration.
(
(
a
-Amylase inhibition activity
a
-Amylase is an enzyme which is responsible for the hydrolysis
of
a-1,4-glycosidic linkages in starch and other polysaccharides
and leads to the number of smaller sugar molecules such as malt-
ose and glucose. When the amount of these small sugar molecules
obtained as the result of
hydrolysis exceeds, it ends up in diabetes. Therefore it is must to
keep the -amylase activity under control to avoid post-prandial
a-amylase catalyzed polysaccharides’
a
hyperglycemia (PPHG). A range of methods has been attempted
to inhibit the amylase activity and natural products are in particu-
lar. For example, the extracts from barley seeds in the presence of
arginine and luteolin (a flavanoid) have been reported against the
amylase activity [53,54]. However, acarbose is still used as the
standard drug for the amylase inhibition.
In an endeavor to experiment the coordination metal complex
containing biological important imine (C@N) group against the
function of amylase, Cu(II) Schiff base mixed ligand complex has
been chosen. It has been tested in three different concentrations
Fig. 5. Emission enhancement spectra of complex [CuL(met)]Cl in the absence and
presence of increasing amounts of CT-DNA. The emission intensity changes of upon
the increasing CT-DNA concentration.
(25, 50 and 100
hand, amylase inhibition activity of acarbose has also been investi-
gated in three concentrations (25, 50 and 100 g) for the compar-
lM) against the action of a-amylase. On the other
l
ison. The outcome of the amylase inhibition study is given in
Table 4. After comparison, the amylase inhibition activity of Cu(II)
complex is very much lesser than that of acarbose (standard amy-
DNA viscosity [52]. From Fig. 6, it can be noted that the viscosity of
DNA is found to be increased when it interacts with metal com-
plexes. The outcome of this study clearly proposes the intercalative
mode of binding between the complexes with CT-DNA as also evi-
denced by UV–Vis., cyclic voltammetric and fluorescence spectro-
scopic results.
lase inhibitor). The maltose liberated is 20.19
acarbose has been operated and it corresponds to 90.1% amylase
inhibition. But 25 M Cu(II) complex shows only 0.44% amylase
inhibition. Upon increasing the concentration, acarbose reaches
lg where 25 lM
l

300
T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
Table 4
Amylase inhibition assay of copper(II) complex.
Compound
OD (540 nm)
Maltose liberated (
lg)
Activity (
l
M/mL/min)
% Activity
% Inhibition
Control
2.43
2.44
2.16
1.95
0.243
0
202.19
203.02
179.69
162.19
20.19
0.00
0.056
0.056
0.050
0.045
0.0056
0.0000
0.0000
100.00
100.44
88.90
80.24
9.99
0.00
0.44
a[CuL(met)]Cl
b[CuL(met)]Cl
c[CuL(met)]Cl
aAcarbose
11.10
19.76
90.01
100.00
100.00
bAcarbose
0.00
0.00
cAcarbose
0
0.00
a, b and c are 25, 50 and 100 lM concentrations, respectively.
100% amylase inhibition with 0
concentration whereas Cu(II) complex shows 11.10% inhibition at
50 M concentration (179.69 g maltose liberation) and 19.76%
inhibition at 100 concentration with the liberation of
162.19 g maltose. The lesser amylase inhibition activity of Cu(II)
complex compared to that of acarbose may be due to the difference
in salvation behavior in the action media. Moreover, the presence
of chloride ions in the structure of the complex may also suppress
the amylase inhibition activity of Cu(II) complex. Since it has been
reported that the presence of chloride ions in the action media in-
duces the activity of amylase enzyme [55], in contrast, the reason
for the amylase inhibition activity of Cu(II) complex may be due to
its binding with the oxygen atoms of amylase.
l
g liberation of maltose at 50
l
M
comparison, Cu(II) complex stands out the best DNA cleaving agent
than other complexes. The DNA cleavage activity of metal com-
plexes may be assigned to the liberation of metal ion to the DNA
helix in the locality of stimulated oxygen or hydroxide free radi-
cals. This is further supported by the better DNA cleavage by the
metal complexes than that of corresponding ligand. The DNA
cleavage of the metal complexes may also depend on the extensive
binding of the DNA molecules to the metal complexes. The conse-
quence of this DNA cleavage indicates the important role in the iso-
lation of metal ions from the complexes. It may also be concluded
that compound which cleaves the DNA may also inhibit the growth
of the pathogenic organism by cleaving the genome.
l
l
lM
l
From this study, it is suggested that the salvation property and
preparing the metal complexes without chloride ions in their
structures are the two major phenomena to achieve the complex
with better amylase inhibition activity.
Antibacterial and antifungal screening
From Figs. 8 and 9, it is observed that the ligand functions feebly
against the bacteria and fungi involved. But after metallation, the
same ligand shows higher activity in the form of complexes. The
presence of imine group may be one of the reasons for the biocidal
action of both the ligand and metal complexes. In addition, it has
been already proved that the electron-withdrawing substituent
such as nitro group on the benzene ring exhibited a comparable
growth-inhibitory activity against microorganisms. The presence
of nitro group in our complexes may also be the reasonable for
the better antimicrobial activity of metal complexes.
The above figures reveal the antibacterial and antifungal activ-
ity of metal complexes. Among the reported complexes, Cu(II)
complex shows the highest biocidal activity than other complexes.
Furthermore, it is necessary to be indicated that Zn(II) complex re-
veals good antibacterial and antifungal activity, followed by copper
species. This may be due to the inhibition by several structural en-
zymes. These enzymes play a vital role in important metabolic
activities of the microbes. On comparison, metal complexes show
better biocidal action against E. coli and L. lactis (Gram-negative
bacteria) than that of N. asteroides and P. putida (Gram-positive
bacteria). This difference might be due to their difference in Gram
status. In fact, Gram-negative bacteria are surrounded by an outer
membrane having lipopolysaccharides. The Schiff base can com-
bine with the lipophilic layer to improve the membrane permeabil-
ity of Gram-negative bacteria. The cell with the lipid membrane
Cleavage of plasmid pUC19 DNA
For the existence of any genomic disease like cancer in the hu-
man body DNA is accountable. Therefore, it is always better to
damage the diseased DNA to destroy the affected cells. With this
motive enormous amount of work has been reported in the past
and it is still being the matter of concern. In the continuation, we
are also reporting here the cleavage of pUC19 DNA with Cu(II),
Co(II), Ni(II) and Zn(II) complexes. Their cleavage efficacy can be
identified by using the gel electrophoresis diagram as shown in
Fig. 7.
When circular plasmid DNA is conducted by electrophoresis,
the fastest migration will be observed for the super coiled form-I
(SC). If one strand is cleaved, the SC form will relax to produce a
slow-moving open circular form-II (OC). If both strands are
cleaved, a nicked form-III (NC) will be generated that migrates in
between the electrodes [56]. As seen from Fig. 7, there is no cleav-
age observed in the case of control (DNA itself). However, all the
metal complexes reveal some good DNA cleaving properties. In de-
tail, Cu(II) and Ni(II) complexes have shown complete cleavage
DNA as evidenced by diminishing of lanes intensity while Zn(II)
and Co(II) complexes have shown partial DNA cleavage. On
Fig. 7. The gel electrophoretic separation of plasmid pUC19 DNA treated with [M(L)(met)]Cl complexes. Lane 1; DNA control ; Lane 2: DNA + ligand + H2O2; Lane 3:
DNA + [CuL(met)]Cl + H2O2; Lane 4: DNA + [CoL(met)]Cl + H2O2; Lane 5: DNA + [NiL(met)]Cl + H2O2; Lane 6: DNA + [ZnL(met)]Cl + H2O2.

T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
301
the growth of bacteria and fungi to a greater extent compared to
the standard drugs.
Conclusion
In the present study, we have attempted to synthesize few no-
vel mixed ligand Schiff base metal complexes of Cu(II), Co(II), Ni(II)
and Zn(II) in the motive to reach new bioinorganic agents with bet-
ter biological properties. The complexes have been also employed
to bind CT-DNA and such binding ability has been explored using
diverse techniques. It is concluded that all these studies proved
the CT-DNA binding of the complexes through the intercalation
mode. Amongst all of them, Cu(II) complex, which reveals the
strongest DNA binding affinity and effective DNA cleavage effi-
ciency. The antimicrobial screening data reveal that the complexes
exhibit higher activity than the free ligand. As copper complex has
highest biocidal activity, the amylase inhibition activity of Cu(II)
complex has been studied. The amylase inhibition property has
been found to be increased upon increasing the complex concen-
tration. The collective biological activities of the complexes may
be helpful for the designing of metal based drugs.
Fig. 8. The antibacterial activity of Schiff base ligand (L) and its mixed ligand metal
complexes as compared to the standard drug. (1) Free Schiff base ligand, (2)
CuL(met)]Cl, (3) [CoL(met)]Cl, (4) [NiL(met)]Cl, (5) [ZnL(met)]Cl and (6) Ciproflox-
acin (standard).
Acknowledgments
The authors express their heartfelt thanks to the College
Managing Board, Principal, and Head of the Department of
Chemistry, VHNSN College for providing necessary research facili-
ties. IIT Bombay and CDRI, Lucknow, are gratefully acknowledged
for providing instrumental facilities.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.074.
References
Fig. 9. The antifungal activity of Schiff base ligand (L) and its mixed ligand metal
complexes as compared to the standard drug. (1) Free Schiff base ligand, (2)
[CuL(met)]Cl, (3) [CoL(met)]Cl, (4) [NiL(met)]Cl, (5) [ZnL(met)]Cl and (6) Nystatin
(standard).
[1] P.G. Cozzi, Chem. Soc. Rev. 33 (2004) 410–421.
[2] R. Antony, S.T. David, K. Saravanan, K. Karuppasamy, S. Balakumar,
Spectrochim. Acta A 103 (2013) 423–430.
[3] M.R. Ganjali, M.R. Pourjavid, M. Rezapour, T. Poursaberi, A. Daftari, M.S. Niasari,
Electroanalysis 16 (2004) 922–927.
[4] Q. Hou, L. Zhao, H. Zhang, Y. Wang, S. Jiang, J. Lumin. 126 (2007) 447–451.
[5] P.G. Lacroix, Eur. J. Inorg. Chem. 2001 (2001) 339–348.
[6] N. Raman, A. Selvan, S. Sudharsan, Spectrochim. Acta A 79 (2011) 873–883.
[7] R. Antony, S.T.D. Manickam, K. Saravanan, K. Karuppasamy, S. Balakumar, J.
Mol. Struct. 1050 (2013) 53–60.
[8] C. Gerdemann, C. Eicken, B. Krebs, Acc. Chem. Res. 35 (2002) 183–191.
[9] I. Sagi, M.D. Wirt, E. Chen, S. Frisbie, M.R. Chance, J. Am. Chem. Soc. 112 (1990)
8639–8644.
[10] D. Volkmer, B. Hommerich, K. Griesar, W. Haase, B. Krebs, Inorg. Chem. 35
(1996) 3792–3803.
[11] B.L. Vallee, D.S. Auld, Biochemistry 29 (1990) 5647–5659.
[12] M.A. Neelakantan, R. Rusalraj, J. Dharmaraja, S. Johnsonraja, T. Jeyakumar, M.S.
Pillai, Spectrochim. Acta A 71 (2008) 1599–1609.
favours the passage of lipid soluble materials only. Therefore, to
design the Schiff base complexes as antimicrobial drugs, the lipo-
philicity is an important factor. If lipophilicity gets enhanced then
Schiff bases get more chances to penetrate into the lipid mem-
branes and much strongly restrict growth of the organism. Schiff
base mixed ligand complexes have very high activity against fungi.
This may also be evidenced by the charge transfer interaction
between the Schiff base molecules and the lipopolysaccharide mol-
ecules which leads to the loss of permeability barrier activity of the
membrane [57].
[13] K.H. Zimmermann, An Introduction to Protein Informatics, Kluwer Academic
Publishers, London, 2003.
[14] Z.H. Chohan, M. Arif, M. Sarfraz, Appl. Organomet. Chem. 21 (2007) 294–302.
[15] B.K. Singh, H. Rajour, A. Prakash, Spectrochim. Acta A 94 (2012) 143–151.
[16] P. Deschamps, P.P. Kulkarni, B. Sarkar, Inorg. Chem. 42 (2003) 7366–7368.
[17] R.H. Holm, P. Kennepohl, E.I. Solomon, Chem. Rev. 96 (1996) 2239–2314.
[18] P. Bauwens, M. Mertens, J. Knight, H. Alberto, J. Am. Chem. Soc. 128 (2006)
15996–15997.
[19] M.S. Masoud, M.F. Amira, A.M. Ramadan, G.M.E. Ashry, Spectrochim. Acta A 69
(2008) 230–238.
[20] B.B. Ivanova, M.I. Mitewa, J. Coord. Chem. 57 (2004) 217–221.
[21] T. Grochowski, K. Samochocka, J. Chem. Soc. Dalton Trans. (1992) 1145–1149.
[22] L.R. Kelland, Eur. J. Cancer 41 (2005) 971–979.
[23] Y. Song, X. Lu, M. Yang, X. Zheng, Transit. Met. Chem. 30 (2005) 499–513.
[24] Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, J. Inorg. Biochem. 83 (2001) 49–
55.
[25] R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B.U. Nair, Biochim.
Biophys. Acta 1475 (2000) 157–162.
As previously mentioned, the metal complexes show improved
antimicrobial activity than their corresponding ligand. This can be
explained by Overtone’s concept and Tweedy’s chelation theory.
When chelation operates, the polarity of the metal ion gets sup-
pressed to a greater extent. Due to this, the delocalization of
p-
electrons may increase on the whole chelation ring for the
improvement towards better lipophilicity of complexes. This could
definitely lead to the much better penetration of the complexes
into lipid membranes than that of ligand. This can further block
the metal binding sites on the enzymes of the microorganisms.
Thus, in general, the complexes show better activity than their
Schiff base ligand. It may be concluded that our complexes inhibit

302
T.R. Arun, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 292–302
[26] V.C.D. Silveira, J.S. Luz, C.C. Oliveira, I. Graziani, M.R. Ciriolo, A.M.C. Ferreira, J.
Inorg. Biochem. 102 (2008) 1090–1103.
[27] H.K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349–359.
[28] F. Akhter, A. Hashim, M.S. Khan, S. Ahmad, D. Iqbal, A.K. Srivastava, M.H.
Siddiqui, South African J. Bot. 88 (2013) 265–272.
[29] D.J. Newman, G.M. Cragg, K.M. Snader, Nat. Prod. Rep. 17 (2000) 215–234.
[30] R. Gupta, P. Gigras, H. Mohapatra, V.K. Goswami, B. Chauhan, Biochemistry 38
(2003) 1599–1616.
[31] G. Muralikrishna, M. Nirmala, Carbohydr. Polym. 60 (2005) 163–1735.
[32] A.Y.Y. Cheng, I.G. Fantus, Can. Med. Assoc. J. 172 (2005) 213–226.
[33] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of Laboratory Chemicals,
Oxford, U.K., 1980.
[34] G.L. Miller, Anal. Chem. 31 (1959) 426–428.
[35] P. Sudha, S.S. Zinjarde, S.Y. Bhargava, A.R. Kumar, BMC Complem. Altern. Med.
11 (2011) 11–15.
[36] M. Dolaz, V. McKee, S. Urus, N. Demir, A.E. Sabik, A. Golcu, M. Tumer,
Spectrochim. Acta A 76 (2010) 174–181.
[37] B.S. Creaven, M. Devereux, A. Foltyn, S. McClean, G. Rosair, V.R. Thangella, M.
Walsh, Polyhedron 29 (2010) 813–822.
[41] B.J. Hathaway, D.E. Billing, Coord. Chem. Rev. 5 (1970) 143–207.
[42] R.K. Ray, G.B. Kauffman, Inorg. Chim. Acta 173 (1990) 207–214.
[43] B.J. Hathaway, Struct. Bonding 14 (1973) 60.
[44] S. Zhang, J. Zhou, J. Coord. Chem. 61 (2008) 2488–2498.
[45] G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251–262.
[46] A.M.O. Breet, T.R.A. Macedo, D. Raimundo, M.H. Marques, S.H.P. Serrano,
Biosens. Bioelect. 13 (1998) 861–867.
[47] M.T. Carter, A.J. Bard, J. Am. Chem. Soc. 109 (1987) 7528–7530.
[48] N. Raman, A. Selvan, P. Manisankar, Spectrochim. Acta A 76 (2010) 161–173.
[49] A. Nohara, T. Umetani, Y. Sanno, Tetrahedron Lett. 22 (1973) 1995–1998.
[50] B.D. Wang, Z.Y. Yang, T.R. Li, Bioorg. Med. Chem. 14 (2006) 6012–6021.
[51] Z.Y. Yang, B.D. Wang, Y.H. Li, J. Org. Met. Chem. 691 (2006) 4159–4166.
[52] M.C. Prabahara, H.S.B. Naik, Biometals 21 (2008) 675–684.
[53] J. Abe, U. Sidenius, B. Svensson, Biochem. J. 293 (1993) 151–155.
[54] J.S. Kim, C.S. Kwon, K.H. Son, Biosci. Biotechnol. Biochem. 64 (2000) 2458–
2461.
[55] M. Qian, V. Nahoum, J. Bonicel, H. Bischoff, B. Henrissat, F. Payan, Biochemistry
40 (2001) 7700–7709.
[56] J.K. Barton, A.T. Danishefsky, J.M. Goldberg, J. Am. Chem. Soc. 106 (1984) 2172–
2176.
[38] H. Unver, Z. Hayavali, Spectrochim. Acta A 75 (2010) 782–788.
[39] P. Kamalakannan, D. Venkappayya, Russ. J. Coord. Chem. 28 (2002) 423–433.
[40] S.M.M. Romanowski, F. Tormena, V.A. Santos, M.F. Hermann, A.S. Mangrich, J.
Braz. Chem. Soc. 15 (2004) 897–903.
[57] M. Imran, J. Iqbal, S. Iqbal, N. Ijaz, Turk. J. Biol. 31 (2007) 67–72.