carbonyl releasing schiff base complex of fe (iii
TRANSCRIPT
REGULAR ARTICLE
Carbonyl releasing Schiff base complex of Fe (III): synthesis,physicochemical characterization, antimicrobial and anticancerstudies
R V KUPWADE and V J SAWANT*
Department of Chemistry, Smt. Kasturbai Walchand College, Sangli, Maharashtra 416 416, India
E-mail: [email protected]
MS received 20 August 2019; revised 14 October 2019; accepted 15 October 2019
Abstract. The carbonyl releasing Schiff base chelate derived Fe(III) complex has been synthesized fol-
lowing the simple wet chemical method. The rhombohedral packing of ligands with the metal ion in the
complex was confirmed by its XRD pattern and FTIR spectrum. Compounds were characterized by UV-Vis.,
PL, FTIR, XRD and TEM techniques. Better biocompatibility of the complex than that of the pure Schiff base
had been elaborated on gram-positive and gram-negative bacteria by Agar well disc-diffusion antibacterial
screening. The complex also displayed pH-responsive in vitro anticancer therapy on MCF-7 breast cancer
cells as revealed by MTT assay. The release of CO and Schiff base as well as the interaction of Fe(III) with
the mitochondria inside the cell are the key factors of cell biocompatibility. This CORM complex had
exhibited higher anticancer activity on MCF-7 cells than the Schiff base and thus potential candidate for the
future biomedical applications.
Keywords. Carbonyl releasing; Schiff base; chelate; anticancer.
1. Introduction
Carbonyl releasing complexes and molecules
(CORM) are gaining more interest due to their ver-
satile properties in biomedical fields, such as antimi-
crobial, anticancer, antioxidant and anti-proliferative
agents.1 Alike the NO, carbonyl (CO) ligand is also
cell signalling species which exhibits vasodilatory,
anti-inflammatory and anti-proliferative activities.2
Carbonyl releasing species are generally
supramolecular complex agents which pass through
the cell barriers. When their uptake takes place by
lysosomes in cells at acidic pH, the release of CO
molecule takes place and ROS (Reactive Oxygen
Species) get generated by cell proton efflux. Hence,
such complexes act as an antimicrobial and anticancer
agent with better biocompatibility. Schiff bases are
generally chelating ligands containing carbimide
bonds formed after refluxing aldehydes with amines
in synthetic protocols. These ligands are the best
antimicrobial agents and form better complexes with
a variety of transition and inner transition metals with
good biocompatibility.
When such ligands combine together in a chelate of
bioactive metals like Fe(III), these complexes play
significant roles in bio signalling inside cells and give
effects of biocompatibility or cytotoxicity. Hence,
Schiff base complexes along with carbonyl ligands
result in fast endocytosis and release of ligands inside
cells to effect into ROS production at pH gradient of
cells. This ROS produced into cells leads to antimi-
crobial, anticancer or cytotoxic and biocompatible
effects on cells. Parallel effects of biocompatibility
have been shown by antibiotic ligand complexes with
special biocompatible metals.3 ROS-based antimicro-
bial activity of Pd(II) complex has been reported by W
Guerra et al.4 Recent work on heterocyclic and Schiff
base ligand complexes have been not only reported the
antimicrobial properties but also DNA cleavage
activities in cells leading to the production of ROS in
cells and biomedical interface mechanisms.5–7 Similar
research of heterocyclic and carbimide containing
Schiff base type ligands and their metal complexes
have been reported for their biomedical applica-
tions.8–12 According to recent work on such hetero-
cyclic species and Schiff bases along with their
complexes with transition metals, these complexes
have exhibited the supramolecular nature to pass from*For correspondence
J. Chem. Sci. (2020) 132:44 � Indian Academy of Sciences
https://doi.org/10.1007/s12039-020-1746-y Sadhana(0123456789().,-volV)FT3](0123456789().,-volV)
cell membranes easily and produce effects of bio-
compatibility and DNA interactions to realize poten-
tial effects on cell divisions and proliferations.13–17
In continuation of these ideas, we had synthesized
new carbonyl releasing Schiff base complex of Fe(III).
In addition to physicochemical characterization of the
simple Schiff base and its carbonyl complex with
Fe(III), the antimicrobial biocompatibility and anti-
cancer potentials have been tested by in vitro assay.
The higher antimicrobial and anticancer potential of
the complex as compared to Schiff base has been
elaborated on special MCF-7 human breast cancer
cells by in vitro MTT assay. Complex exhibited
increased endocytosis inside negatively charged cell
membranes of bacteria and cancer cells and produced
higher amount of ROS at acidic pH than Schiff base,
CO, implying the effect of Fe(III) species towards
intrinsic apoptosis of cancer cells.18–20 Thus, carbonyl
releasing Schiff base complex of Fe(III) can be con-
sidered as a potential candidate for future biomedical
applications.
2. Experimental
2.1 Materials and cell cultures
All the chemicals used for the synthesis of Schiff base and
its carbonyl complex with Fe(III) and their in vitro bio-
logical screening such as Ferric nitrate, P-nitro aniline,
Benzaldehyde, Zinc powder, Calcium carbonate, Conc.
HCl, Ethanol were of A. R. grade. These chemicals were
purchased from S. D. Fine Chem. Ltd. and Merck Ltd. and
were used without further purification. The cell culture
medium such as agar growth broth and bacterial culture,
fetal bovine serum, trypsin buffer were obtained from
Himedia Ltd. and NCCS cell repository center, Pune, India.
The human breast cancer cells MCF-7 were purchased from
this cell repository. The double-distilled water was obtained
from Millipore system and used throughout the synthesis
and in vitro biological screening tests.
2.2 Synthesis of Schiff base ligand
The simple Schiff base ligand was synthesized by refluxing
P-nitroanilline and Benzaldehyde at 250 �C on the constant
flame in R.B. flask with condenser for 3 h. The yellow
product formed water-washed with ethanol to remove
unreacted organic precursors and dried in the oven below
80 �C. The Schiff base powder then subjected to physico-
chemical the complex using UV-Vis., PL, FITR spectrom-
etry. The formation of the product was confirmed on the
basis of physical constant and TLC (see protocol in
Scheme 1).
2.3 Wet chemical synthesis of CORM, carbonyl -
Schiff base complex of Fe(III)
The carbonyl-containing Schiff base metal complex of
Fe(III) was synthesized by bubbling CO into the flask
containing Schiff base and Fe(III) nitrate precursor using a
wet chemical method (Scheme 1). In detail, the Schiff base
with Fe(III) in 2:1 mM proportion in the ethanol-water
solvent system were refluxed in R. B. flask for 3 h and the
precursor was then added in the flask with double distilled
water. The gaseous CO ligand was produced in separate
flask by the reaction of conc. HCl with calcium carbonate
and zinc powder mixture. Then the CO ligands produced
separately in another flask was passed and bubbled in ligand
and metal precursor mixture to get carbonyl releasing
CORM complex of Fe(III) containing CO ligands and
Schiff base. The complex formed thus was washed with
ethanol-water solvent system and dried in the oven.
2.4 Structural and morphological
characterization of CORM-Fe(III) complex
The structure, morphology, particle diameter range and
types of bonding of functionalities in the CORM complex
of Fe(III) was determined based on physicochemical char-
acterization using UV-Vis., PL, FTIR, XRD spectrometry
OH
CHO
+ NH2 NO2
3h reflux in ethanol- H2O
CH
N NO2
OH
CO bubbling Fe(NO3)3 stirred with ligand
N
NO2
ON
NO2
OFe
CO
CO
(NO3)3
Salicyladehyde P-nitro aniline
Schiff base ligand
CORM Fe (III) Schiff base complex
Scheme 1. Synthesis protocol of Schiff base ligand andcarbonyl-schiff base Fe(III) complex.
44 Page 2 of 12 J. Chem. Sci. (2020) 132:44
techniques and TEM microscopic analysis. The spectronics
double beam UV-Vis. spectrometer with water as blank was
used to determine the absorption spectrum of complex to
compare with Schiff base. To confirm functionalities pre-
sent in complex and a with Schiff base, Perkin Elmer series
FTIR spectrometer was used with KBr pellet technique. The
PL emission spectrum of complex and Schiff base were
determined using Jasco type spectrofluorometer with exci-
tation identity of complex and Schiff base in ethanol solvent
with the same ppm. Concentrations. The X-ray diffraction
pattern of the complex was determined using X-ray spec-
trometer by powder diffraction technique to elaborate the
packing of ligands with metal in complex, hybridization and
crystal system of complex. The particle range of complex to
pass cell through membranes was determined using TEM
microscopic analysis and SAED patterns were matched with
XRD data to confirm the powder crystal system of complex.
2.5 Antimicrobial screening on gram-positive
and gram-negative bacteria by agar well disc
diffusion method
The cell-particle interactions of materials and complexes
demonstrating their reactivity and biocompatibility can be
elaborated using simple in vitro antibacterial screening in
buffer solutions to maintain physiological mimicking pH at
material cell interactions. As cell pH affects the biocom-
patibility of molecules. Here in this work 15, 20 and
25 ppm concentrations of Schiff base and CORM complex
were dosed on bacterial cell cultures grown in agar broth on
discs, inside the wells bored on plates. The gram-positive
bacteria Staph. Aurues, and gram-negative bacteria E. Coli,
Kleb. were grown on culture plates and inhibited by dosing
of Schiff base and complex solutions in buffer dispersions
with physiological pH = 7.4 by use of phosphate buffer. The
culture plates were incubated and zones of inhibition were
measured, and biocompatibility/antimicrobial property of
Schiff base and complex were compared.
2.6 In vitro MTT cell line anticancer assay
on MCF-7 human breast cancer cells
The anticancer potential of Schiff base and complex were
estimated and compared on the basis on in vitro MTT,
MCF-7cellline spectrometric assay. Cytotoxicity of Schiff
base and CORM complex were elaborated into MCF-7
(Human Breast cancer cells) with MTT assays. Cells (1 9
103/mL) were seeded in 96-well plates and then incubated
for 24 h at 37 �C and 5% CO2 atmosphere in an incubator
with 100 lL of DMEM medium supplemented with 10%
fetal bovine serum (FBS) as a growth medium and 1%
penicillin-streptomycin solution per well. This medium was
then replaced with 200 lL of the PBS, phosphate buffer
saline (pH=7.4) medium containing either Schiff bases or
complex doses (concentration 5, 10, 15, 20 and 25 lg/mL).
Cells without treatment were considered as a control as well
to evaluate its cytotoxicity. Cells were further incubated for
48 hours, and relative anticancer activity was assessed with
MTT assays. After 48 h, MTT assay was carried out. In
brief, MTT solutions (20 lL of 5 mg/mL) were added after
treatment and cell culture were again incubated for an
additional 4 h. Dimethyl sulphoxide (150 lL solution) was
added to each well to solubilize the blue formazan crystals,
and optical density at 492 nm was recorded for culture
medium. Cell viability (%) for exposure of Schiff base and
complex doses were calculated as follows,
%Cell viability ¼ Optical density at 492 nm in test cells
Optical density at 492 nm in control cells� 100
The anticancer activities of Schiff base and complex
doses were elaborated in terms of cell viability and plotted
as a function of concentrations to compare the effects.
Finally, these cultures were subjected to DAPI staining to
elaborate internalization of the complex into cancer cells,
and determine the mechanism of cell deaths or apoptosis by
interactions from the complex by the production of inter-
anions/inter-cations or release of ligands and metal ion
inside cell fluids.
2.7 DAPI staining and imaging of cancer cells
after MTT assay
After the incubation with Schiff base/complex doses and
further MTT interactions, the cells were washed with PBS
(phosphate buffer saline) solution and fixed with 4%
paraformaldehyde for 30 min, then cells were added with
20 lL of DAPI and were incubated for 20 min; finally, after
spreading on slides, the cell were examined under a fluo-
rescent microscope. The internalization of complex higher
than Schiff base was examined by the staining and mech-
anism of anticancer effects and biocompatibility/ cytotoxi-
city was demonstrated.
3. Results and Discussion
3.1 Morphological and structural characterization
of Schiff base ligand and the CORMFe(III) complex
3.1a UV-Vis. absorption and PL emission
spectrum: UV-Vis. and PL spectrum of
Schiff/complex were determined to study the
structural characteristics, metal to ligand charge
transfer transitions, bonding within the molecule and
absorption/emission property. Here UV-Vis, the
absorption spectrum of the complex (Figure 1)
explains the charge transfer transition of Fe(III)
metal ion with Schiff base and carbonyl ligand, to
elaborate the bonding interactions in the complex. The
J. Chem. Sci. (2020) 132:44 Page 3 of 12 44
spectrum exhibits the diffused broad absorption peaks
at 235 nm and 355 nm for the MLCT transitions from
Fe(III) d electrons to CO and carbimide bond of Schiff
base ligand respectively (metal to ligand charge
transfer take place probably due to non bonded and
Pi electrons of Schiff base and CO ligands and d
electrons of Fe(III) in the complex). The Schiff base
shows higher O.D. for a single peak near at same
absorption wavelength of the complex at 365 nm. (Not
shown), but dampening and peak shifting take place
for absorption maxima of Schiff base after bonding
interaction of carbimide bond with Fe(III) ions in the
complex. Hence, UV-Vis spectrum of complex
elaborates the bonding of ligands with Fe(III) and
gives the idea of probable chelate type structure of the
complex. The PL spectrum of Schiff base and complex
determined separately explain the quenching of peak
signal of Schiff base after bonding of Fe(III) with
carbimide bonds in the complex. In Figures 2A and 2B
the PL emission peaks of Schiff base and CORM
complex respectively show the quenching of signal.
PL spectra of Schiff base and complex support for
the bonding, structure and nature of complex con-
taining Schiff base and CO ligand simultaneously
linked with Fe(III) ions to give more stability for the
chelate. According to PL spectra, the PL intensity of
Schiff base at 815 nm. due to non bonded and Pi
electrons was quenched by Fe(III) bonding in complex
proving MLCT transition and coordinate/covalent
linkages of carbimide bonds with a metal ion. Overall
these spectra elaborate chelate nature of complex
containing CO ligand liked with planer Schiff base
ligand with Fe(III) ion in the complex. So the spectra
support for CO releasing nature of the complex, as
Schiff base is firmly linked by coordinate bonds and
CO electrons are representing different absorption
maxima in UV-Vis spectrum of the complex at
235 nm representing more non-bonding free electrons
over it. This elaborated the free site of CO ligands for
easy release from complex to behave as CORM
complex for better cell biocompatibility. Table 1
supports for MLCT transition identities in Schiff base
and CORM complex determined from UV-Vis and PL
spectrum.
The composition of the complex for its elements and
ions C, H, O, N, Fe (III) along with functionalities of
carbimide, carbonyl bonds to metal are confirmed by
using UV-Vis and PL electron transitions and peaks in
respective spectra. These evidence are supported by
FTIR signals as per spectral studies in works of B. Sinha
et al.,21 hence these analyses of Schiff base and CORM
complex not only prove the composition but also
probable structure and functionalities in the complex.
3.1b FTIR spectrum of Schiff base and CORM
complex: FTIR spectrum of Schiff base and
complex were determined to estimate the
functionalities present inside the molecules and to
confirm the formation of CORM complex on the basis
of signals represented by functional groups in
fingerprint and functional regions of spectra.
Figure 3A represents the FTIR spectrum of Schiff
base, in this spectrum presence of carbimide bond –
C=N- was elaborated due to signal at 1918 and
2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0
0
1
2
3
4
5
d 5 F e ( II I) M L C T w ith C O a n d c a rb im id e
Abso
rban
ce
W a v e le n g th n m
U V -V IS a b s o rp t io n s p e c tra o f s c h if f b a s e c o m p le x
Figure 1. UV-Visible absorption spectrum of CORM Schiff base complex of Fe(III).
44 Page 4 of 12 J. Chem. Sci. (2020) 132:44
Figure 2. (A) PL emmission spectrum of schiff base. (B) PL emmission spectrum of complex.
Table 1. MLCT transitions of non bonded/Pi electrons of ligands and d electrons of Fe(III).
Absorption or emissionpeak shown in UV-Vis. OrPL spectrum Schiff base transition Metal CORM complex charge transfer transition
UV-Vis. Absorption at 355nm.
n to Pi absorption maxima A1g to T2g and A1g to A2g from d5 state of Fe(III) for splittingfrom bonding of carbimide and CO species
PL quenching at 815 nm. n to Pi relaxation-no quenching
Pi to Pi relaxation – quenching due to Fe(III) and carbimidelinkage
J. Chem. Sci. (2020) 132:44 Page 5 of 12 44
1971 cm-1. The signals at 531, 665 and 805 cm-1
represent the fingerprint identity of aromatic groups
from benzaldehyde and nitroaniline. Signals at 2555
and 2673 cm-1 are attributed towards the aromatic
nitro and nitrile entity of Schiff base. Signals from
3034 to 3379 cm-1 are raised in the spectrum due to
the presence of aromatic –OH and carbimide hydroxyl
side interactions in Schiff base. In Figure 3B FTIR
spectrum proves functionalities present in CORM
Fe(III) complex elaborates the presence of carbimide
metal bond and side CO ligands over the metal ion.
Signals at 1760 and 1822 cm-1 elaborates the
presence CO groups over Fe(III) metal ion in the
complex. The signals at 1936 and 1969 cm-1 are
attributed to Fe(III) bonded with carbimide linkage in
the complex. The broadening of signals in complex
FTIR spectrum at 2585 and 2884 cm-1 is attributed to
carbimide and CO linkages at Fe(III) ion and covalent
aromatic-O linkage to Fe(III) from Schiff base near to
carbimide metal bond. Overall all these observations
lead to prove the chelate nature of the complex and
octahedral site of Schiff base with metal ion and free
carbonyl groups over it. Table 2 throws light on
specific signals of the FTIR spectrum for the
functionalities in the Schiff base and the complex.
3.1c XRD (X-ray diffraction) pattern
of complex: As per XRD pattern of the complex in
Figure 4, the complex evidenced to rhombohedra
crystalline packing for metal ion and ligands, it
elaborates the structure and morphology for CORM
complex. The diffraction pattern gives signals for
metal ion planes in an octahedral environment of
Schiff base and carbonyl ligands, Fe(III) of the
complex are in octahedral sites of packing with
ligands in complex, hence phase purity of complex
crystal have been proved by XRD patterns. The XRD
data is determined using miller indices of pattern,
FTIR spectra of Schiff base
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 40088.90
89.2
89.4
89.6
89.8
90.0
90.2
90.4
90.6
90.8
91.0
91.2
91.4
91.6
91.8
92.00
Frequency cm-1
%T
FTIR spectra of CORM complex
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 40033.034
36
38
40
42
44
46
48
50.0
Frequency cm-1
%T
A
B
Figure 3. (A) FTIR spectrum of Schiff base ligand. (B) FTIR spectrum of CORM complex.
44 Page 6 of 12 J. Chem. Sci. (2020) 132:44
Scherer’s formula and packing of planes and it was
matched with octahedral Fe(III) data in JCPDS card
no. 85-3854. As per Table 3, the crystal parameters of
rhombohedral packing of complex clearly represented
the octahedral packing of the metal ion with ligands
producing rhombohedral crystal. Hence, XRD data
proved the phase purity, crystal packing, geometry,
chelate octahedral nature of CORM complex.
3.1d TEM image and SAED pattern
of Complex: The TEM image of the complex in
Figure 5A throws light on spherical elongated
morphology and some aggregation of complex
molecules due to supramolecular neighbouring
bonding interactions. The particle sizes of complex
crystals ranged from 60 nm to 75 nm. But most of the
particles of complex molecules exhibited average size
08060402
0
2000
4000
6000
8000
10000
12000
[Fe(III) in rhom bohedra ]
[111 ]
[002 ]
[121 ]
[001 ]
[020 ]F e(III) ion in carb im ide and C O ligand env ironm ent O ctahedra l rhom bohedra l crysta l sym m etry
Inte
nsity
2 T he ta
X R D spectra o f com plex
Figure 4. XRD pattern of the Complex.
Table 2. Matching of FTIR signals elaborating the functionalities of Schiff base and CORM complex.
Signal in FTIR spectrum Schiff base functionality CORM complex functionality
1969 cm-1 Carbimide bond -C=N- Carbimide linked to Fe(III)1760 cm-1 Absence of CO Presence of linked CO ligands over Fe(III)3034 cm-1 Aromatic -OH Ether aromatic-O- linkage with Fe(III)1790 and 1824 cm-1 Presence of an aromatic nitro group Out of plane nitro groups2555 and 2585 cm-1 Presence of nitrile entity Presence of linked nitrile entity
Table 3. Crystal parameters of CORM Fe(III) complex matched with standard Fe(III) octahedral JCPDS card.
Crystallite planes(Miller Indices)(h,k,l)Rhombohedra ofoctahedral chelate phase
d Calculated A0
d = a/H(h2?k2?l2)or
2dSinh = nk
d Standard A0
JCPDS card no.- 89-3854Fe(III)
Lattice Constanta and b A0 from main XRD peak
at theta = 37.5 of CORM complex crystalfrom rhombohedra
001 5.088 5.089 a standard = 3.249b standard = 5.266121 4.032 4.045
020 7.039 7.043002 3.089 3.083 a calculated = 3.251 and
b calculated = 5.272111 6.334 6.329
J. Chem. Sci. (2020) 132:44 Page 7 of 12 44
range 60 nm which can pass through cell membranes
by supramolecular interactions easily. The TEM
results were matched with XRD data and proved the
chelate octahedral nature of complex with the
crystalline state for good water solubility and further
biocompatibility. The SAED patterns greatly matched
with XRD data with crystalline dot patterns as per
Figure 5B. Overall TEM image and SAED pattern of
the complex had proved the morphology of the
complex for further water-loving nature and
biomedical applications.
3.2 Antimicrobial properties Schiff base
and complex for better biocompatibility
As per the images of bacterial cultures and zones of
inhibition exhibited by Schiff base and complex on
Figure 5. (A) TEM image of complex. (B) SAED pattern of complex.
Figure 6. (A) to (C) Anti microbial effects of schiff base ligand at 25 ppm. on (A) E-Coli.-Gram negative bacteria,(B) Kleb.- Gram negative bacteria, (C) Staph. aureus- Gram positive bacteria.
44 Page 8 of 12 J. Chem. Sci. (2020) 132:44
gram-positive and gram-negative bacteria in Fig-
ures 6A to 6C and 7A to 7C, it clearly indicated that
the Schiff base and complex show better antimicrobial
activities. The CORM complex shows higher activity
than Schiff base and as per Table 4, especially on
gram-positive bacteria, higher activity was estimated
than gram-negative bacteria. So the CORM complex
exhibit higher biocompatibility than Schiff base, due
to release of CO, Schiff base ligand and Fe(III) metal
ion inside bacterial cells causing interactions with
DNA. So, the complex show higher cell compatibility
and internalization due to its chelate supramolecular
nature proving its applicability in biomedical fields.
3.3 Anticancer potential of Schiff base
and complex by in vitro MTT assay on MCF-7 cells
As per Figure 8, pH stimulated higher anticancer
activity had been shown by complex than Schiff base
compared with free control cells on the basis of
in vitro MTT assay on MCF-7 breast cancer cells. The
cell viabilities determined are related to cytotoxicity
and anticancer effects on these cancer cells at 5, 10,
15, 20, 25 ppm. Concentrations of doses of Schiff base
and CORM complex. Higher cell line apoptosis was
observed at 25 ppm. Giving dose-dependent and pH
triggered cytotoxicity of CORM complex. About 77%
Table 4. Anti-microbial activities of Schiff base and complex compared for gram-positive and gram-negative bacteria.
Type/name of bacterial culture in Agar broth[as per Figures 6A to 6C and 7A to 7C]
Zones of inhibition for gram-positive bacteria/gram-negative bacteria aszone diameter in mm. for concentrations of drug/dose of complex or Schiff
base 25 lg/mL (ppm.) in vitro on bacterial cells
For Schiff base For CORM complex
E. Coli (gram -ve) 10 mm. 35 mm.Kleb.(gram -ve) 5 mm. 15 mm.Staph aureus(gram ?ve) 8 mm. 48 mm.
Figure 7. (A) to (C) Anti microbial effects of CORM complex at 25 ppm. on (A) E-Coli.-Gram negative bacteria,(B) Kleb.-Gram negative bacteria, (C) Staph. aureus- Gram positive bacteria.
J. Chem. Sci. (2020) 132:44 Page 9 of 12 44
apoptosis was given by CORM complex at 25 ppm
dose, hence, the complex show good cytotoxicity on
cancer cells and proved its potential application in the
biomedical area. Supramolecular chelate nature to
pass from cell barriers, presence key signaling CO
ligand and Schiff base with electron providing Fe(III)
ion and pH triggered action on cancer cell endocytosis
and apoptosis are the main entities which play role in
anticancer effects of this new CORM complex. These
all observations are supported and proved by DAPI
staining image of MCF-7 cells after the action of
CORM complex by MTT assay. In Figure 9, it was
observed that the complex after endocytosis shrinks
cancer cells fastly after incubation and show DAPI
fluorescence at center of cells under a microscope.
This observation indicates the action of released spe-
cies with DNA producing pH-sensitive ROS flux
inside cancer cells from CORM complex resulting in
intrinsic apoptosis of MCF-7 cells.
3.4 Mechanism for antimicrobial activity
and anticancer potentials of the CORM complex
As per Scheme 2, when CORM complex enters into
bacterial or cancer cells with production of ROS (re-
active oxygen species) like O2, OH, OOH and H2O2
after endocytosis and lysosomal digestion at acidic pH,
it causes prominent biocompatible effects on these
cells. CORM complex release CO ligand, Schiff base
and dissociate Fe(III) inside acidic pH of cells, so by
pH trigger it releases proton flux, CO, intercations
inside cell organelles. The ROS produced inside bac-
terial cells by these species on the basis of cell
molecule interaction enters into rough organelles and
mitochondria of cells disturbing membrane potentials
of cell organelles and damage DNA or mutated DNA.
The free electrons from Fe(III) ions, CO ligand, Schiff
base activity, inter cation and proton flux are key
factors disturbing the cancer cell mitochondria and
mutated DNA. Overall these intrinsic pathways of
apoptosis by CORM complex cause higher anticancer
effects than Schiff base on MCF-7 cells with probable
ROS-lipid peroxidation mechanism which are pH-re-
sponsive and CO signaling affecting biomedical
activities.19,22,23 Hence, this new CORM complex
5 10 15 20 250
10
20
30
40
50
60
70%
cel
l via
bilit
y fo
r MC
F-7
cells
afte
r MTT
ass
ay
Concentrations of dosing for complex and schiff base in ppm.
action of CORM complex action of Schiff base
Figure 8. Anticancer activities of schiff base and CORMcomplex by MTT assay on MCF-7 cells.
Figure 9. DAPI staining image of internalization ofcomplex for apoptosis of MCF-7 cells.
pH trigger CORM Complex (Excitation at 385 nm.) emission at 815 nm.
[Release of H+ / Fe3+/ CO and Schiff base at acidic pH in lysosome]
Fe(III) to Schiff base CT transition VB+ + e- to CB in Fe(III) - (free electrons)
Dissociation-mitochondrial disruption
[CO] and Schiff base releasing at lysosomes pH change --- ROS in cells- O2., OH., .OOH
Mitochondrial cyt.-c destruction and caspase activation Apoptosis of cancer cell
Scheme 2. Mechanism for antimicrobial and anticancer activities of complex.
44 Page 10 of 12 J. Chem. Sci. (2020) 132:44
clearly indicates potential biomedical applications for
antimicrobial and anticancer activities.
4. Conclusions
The new carbonyl-Schiff base CORM complex of
Fe(III) exhibited rhombohedral packing and chelate
nature with an octahedral site of ligands. The complex
had exhibited 60 nm mean particle diameter and
crystalline oval morphology as confirmed by XRD
pattern and TEM analysis. This chelate with CO
releasing ability has the potential to pass cell barrier
with supramolecular nature and hence show better
biocompatibility than Schiff base. The quenching of
signal in PL spectrum elaborated the bonding of
Fe(III) ion with Schiff base and CO ligand. Further-
more, the complex exhibits ROS in bacterial and
cancer cells after the release of the Schiff base, Fe(III)
and CO by pH trigger. The CORM complex shows
better biocompatibility by in vitro than Schiff base.
The synergic effects of the release of CO, Schiff base
and interaction of Fe(III) with mitochondria inside the
cells are the key factors for cell biocompatibility.
Hence, this CORM complex of Fe(III) exhibits an
intrinsic pathway for apoptosis of cancer cells. Overall
this CORM complex has potential application in
biomedical fields for cytotoxicity and
biocompatibility.
Acknowledgements
The author is thankful to analytical instrumentation labo-
ratory, Jaysingpur College, Jaysingpur, India for providing
some spectroscopic characterizations of samples, and to the
Biotechnology laboratory of Smt. K. W. College, Sangli for
providing cell cultures and in vitro testing facilities.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
References
1. Wang P, Liu H, Zhao Q, Chen Y, Liu B, Zhang B andZheng Q 2014 Synthesis and Evaluation of drug-likeproperties of CO-releasing molecules containing ruthe-nium and group 6 metal Eur. J. Med. Chem. 74 199
2. Orlowska E, Babak M V, Domotor O, Enyedy E A,Rapta P, Zalibera M, Bucinsky L, Malcek M, Govind C,Karunakaran V, Farid Y C S, McDonell T E, Luneau D,Schaniel D, Ang W H and Arion V B 2018 NOreleasing and anticancer properties of octahedral
Ruthenium-Nitrosyl complexes with equatorial 1H-inadazole ligands Inorg. Chem. 57 10702
3. Shaikh A R, Giridhar R and Yadav M R 2007 Bismuthnorfloxacin complex: Synthesis, physicochemical andantimicrobial evaluation Int. J. Pharm. 6 24
4. Guerra W, de Andrade Azevedo E, de Souza MonteiroA R, Bucciarelli-Rodriguez M, Chartone-Souza E,Nascimento A M, Fontes A P, Le Moyec L andPereira-Maia E C 2005 Synthesis, characterization, andantibacterial activity of three palladium(II) complexesof tetracyclines J. Inorg. Biochem. 99 2348
5. Ejidike I and Ajibade P 2016 Ruthenium (III) Com-plexes of Heterocyclic Tridentate (ONN) Schiff Base:Synthesis, Characterization and its Biological Propertiesas an Antiradical and Antiproliferative Agent Int.J. Mol. Sci. 17 60
6. Li M X, Chen C L, Zhang D, Niu J Y and Ji B S 2010Mn(II), Co(II) and Zn(II) complexes with heterocyclicsubstituted thiosemicarbazones: Synthesis, characteri-zation, X-ray crystal structures and antitumor compar-ison Eur. J. Med. Chem. 45 3169
7. ManjunathM, Kulkarni A D, Bagihalli G B,Malladi S andPatil S A 2017 Bio-important antipyrine derived SchiffBases and their transition metal complexes: Synthesis,spectroscopic characterization, antimicrobial, anthelminticand DNA cleavage investigation J. Mol. Struct. 1127 314
8. Hranjec M, Starcevic K, Pavelic S K, Lucin P, PavelicK and Zamola G K 2011 Synthesis, spectroscopiccharacterization and antiproliferative evaluation in vitroof novel Schiff Bases related to benzimidazoles Eur.J. Med. Chem. 46 2274
9. Huang H, Chen Q, Ku X, Meng L, Lin L, Wang X, ZhuC, Wang Y, Chen Z, Li M, Jiang H, Chen K, Ding J andLiu H 2010 A Series of a-Heterocyclic CarboxaldehydeThiosemicarbazones Inhibit Topoisomerase IIa Cat-alytic Activity J. Med. Chem. 53 3048
10. Badea M, Calu L, Chifiriuc M C, Bleotu C, Marin A,Ion S, Ionita G, Stanica N, Marutescu L, Lazar V,Marinescu D and Olar R 2014 Thermal behaviour ofsome novel antimicrobials based on complexes with aSchiff Base bearing 1,2,4-triazole pharmacophore J.Therm. Anal. Calorim. 118 1145
11. Sinha D, Tiwari A K, Singh S, Shukla G, Mishra P,Chandra H and Mishra A K 2008 Synthesis, character-ization and biological activity of Schiff Base analoguesof indole-3-carboxaldehyde Eur. J. Med. Chem. 43 160
12. Sonmez M and Sekerci M 2010 A New HeterocyclicSchiff Bases and Its Metal Complexes Synth. React.Inorg. Met.-Org. Chem. 5714 489
13. Barot K P, Manna K S and Ghate M D 2013 Design,synthesis and antimicrobial activities of some novel1,3,4-thiadiazole, 1,2,4-triazole-5-thione and 1,3-thia-zolan-4-one derivatives of benzimidazole J. SaudiChem. Soc. 21 S35
14. Abd-El-Aziz A S, Agatemor C and Etkin N 2017Antimicrobial resistance challenged with metal-basedantimicrobial macromolecules Biomaterials 118 27
15. Dhanaraj C J and Johnson J 2016 Transition metalcomplexes of a novel quinoxaline-based tridentate ONOdonor ligand: synthesis, spectral characterization, ther-mal, in vitro pharmacological and molecular modelingstudies Appl. Organomet. Chem. 30 860
J. Chem. Sci. (2020) 132:44 Page 11 of 12 44
16. Krishnamoorthy P, Sathyadevi P, Cowley A H, ButoracR R and Dharmaraj N 2011 Evaluation of DNA binding,DNA cleavage, protein binding and in vitro cytotoxicactivities of bivalent transition metal hydrazone com-plexes Eur. J. Med. Chem. 46 3376
17. Arjmand F, Jamsheera A and Mohapatra D K 2013Synthesis, characterization and in vitro DNA bindingand cleavage studies of Cu(II)/Zn(II) dipeptide com-plexes J. Photochem. Photobiol. B 121 75
18. Bharti S K and Singh S K 2009 Metal Based Drugs:Current Use and Future Potential Pharm. Lett. 1 39
19. Uivarosi V 2013 Metal Complexes of QuinoloneAntibiotics and Their Applications: An Update Mole-cules 18 11153
20. Alpaslan E, Geilich B M, Yazici H and Webster T J2017 pH-controlled cerium oxide nanoparticle
inhibition of both gram-positive and gram-negativebacterial growth Sci. Rep. 7
21. Sinha B, Bhattacharya M and Saha S 2019 Transitionmetal complexes obtained from an ionic liquid-sup-ported Schiff base: synthesis, physicochemical charac-terization and exploration of antimicrobial activities J.Chem. Sci. 131 1
22. Sawant V J and Bamane S R 2018 PEG-beta-cyclodex-trin functionalized zinc oxide nanoparticles show cellimaging with high drug payload and sustained pHresponsive delivery of curcumin in to MCF-7 cells J.Drug. Del. Sci. Tech. 43 397
23. Bamane S R and Sawant V J 2019 Folate tetheredGd2O3 nanoparticles exhibit photoactive antimicrobialeffects and pH responsive delivery of 5-fluorouracil intoMCF-7 cells Drug Del. Lett. 9 58
44 Page 12 of 12 J. Chem. Sci. (2020) 132:44