Green synthesis of biologically active transition metal nanoparticles containing novel Schiff base via catalyst free hydrothermal reaction: Structural, biological and morphology study

Article abstract of DOI:10.1002/aoc.4322

Novel cobalt, nickel and copper complexes were synthesized by regular reflux method and nano sized Co(II), Ni(II) and Cu(II) metal complexes were synthesized by a facile hydrothermal method using green solvent at various temperatures without the addition of any capping agent. The structural characterization was done by magnetic susceptibility, molar conductance, elemental analysis, thermal analysis, FT-IR, ¹H NMR, ¹³C NMR, ESI mass, UV–Visible analysis. The morphology and size of the nano metal complexes were determined using FE-Scanning electron microscopy, powder X-ray diffraction data and atomic force microscopic techniques. All the spectral and analytical results reveal 1:2 metal to ligand ratios having [ML₂(H₂O)₂] stoichiometry, here M=Co(II), Ni(II) and Cu(II), L=deprotonated ligand. The non-electrolytic nature of the compound was confirmed by molar conductance experiment. The synthesized Schiff base and its metal complexes (7, 8 and 9) were tested for their biological activity. All the tested compounds exhibit decent anticancer and DNA cleavage activity and copper complex shows better activity results than other tested compounds.

Full text of DOI:10.1002/aoc.4322

Received: 12 December 2017
DOI: 10.1002/aoc.4322
Revised: 15 January 2018
Accepted: 18 January 2018
F U L L P A P E R
Green synthesis of biologically active transition metal
nanoparticles containing novel Schiff base via catalyst free
hydrothermal reaction: Structural, biological and
morphology study
Shailaja S. Jawoor | Mahantesh Kumbar | Sangamesh A. Patil
P. G. Department of Chemistry, Karnatak
Novel cobalt, nickel and copper complexes were synthesized by regular reflux
method and nano sized Co(II), Ni(II) and Cu(II) metal complexes were synthe-
sized by a facile hydrothermal method using green solvent at various tempera-
tures without the addition of any capping agent. The structural characterization
was done by magnetic susceptibility, molar conductance, elemental analysis,
University, Dharwad 580003, Karnataka,
India
Correspondence
Sangamesh A. Patil, P. G. Department of
Chemistry, Karnatak University, Dharwad
580003, Karnataka, India.
1
13
thermal analysis, FT‐IR, H NMR, C NMR, ESI mass, UV–Visible analysis.
The morphology and size of the nano metal complexes were determined using
FE‐Scanning electron microscopy, powder X‐ray diffraction data and atomic
force microscopic techniques. All the spectral and analytical results reveal 1:2
metal to ligand ratios having [ML (H O) ] stoichiometry, here M=Co(II), Ni(II)
Email: patil1956@rediffmail.com
Funding information
University Grants Commission, Grant/
Award Number: No.KU/Sch/UGC‐UPE/
2014‐15/838
2
2
2
and Cu(II), L=deprotonated ligand. The non‐electrolytic nature of the com-
pound was confirmed by molar conductance experiment. The synthesized
Schiff base and its metal complexes (7, 8 and 9) were tested for their biological
activity. All the tested compounds exhibit decent anticancer and DNA cleavage
activity and copper complex shows better activity results than other tested
compounds.
KEYWORDS
bioactivity, FE‐SEM and AFM, green synthesis, temperature variation
1
| INTRODUCTION
new nanoparticle imaging agents can yield more sensitive
and selective imaging of cancer.
Cisplatin and other platinum based compounds are
most widely used drugs clinically. In addition, the clinical
drawbacks of cisplatin are also recognizable, including
the restricted applicability, assimilated resistance, and
severe side effects. Diagnosis of cancer disease at initial
stage is the key for effective treatment. Conversely, tradi-
tional diagnostic and imaging methods cannot detect
tumors in premature stages and have limited ability in
discriminating benign and malignant lesions. In contrast,
Transition metal compounds, whereas metal ions or
metal clusters are connected by molecular building blocks
consisting of organic molecules or organometallic com-
plexes have attracted a great attention of researchers due
to their distinct coordination geometries and useful appli-
[1]
[
2–7]
cations.
In recent years, a structural study of small
sized particles is a main fundamental interest in transition
metal compounds. On the other hand, miniaturization of
the transition metal compound's particle size to the
Appl Organometal Chem. 2018;e4322.
wileyonlinelibrary.com/journal/aoc
Copyright © 2018 John Wiley & Sons, Ltd.
1 of 12
https://doi.org/10.1002/aoc.4322

2
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JAWOOR ET AL.
[
24]
nanometer scale by functionalizing the structure inter-
faces will provide further opportunities to integrate with-
out changing the structural features. In the past,
numerous nano and micro‐sized particles has been syn-
thesized successfully from different methods viz.,
according to standard reported procedures.
7‐
and 8‐formyl‐7‐hydroxy‐4‐
have been synthesized according to
[
25]
hydroxy‐4‐methylcoumarin
[
26]
methylcoumarin
the reported procedures, FT‐IR spectrum of compounds
were recorded in the range of 4000‐400 cm‐ using KBr
disc with Nicolet 170 SX FT‐IR spectrometer. By using
TMS as an internal reference compound, H NMR and
C NMR spectrum were recorded in CDCl solvent on
3
Bruker 400mHz spectrometer at room temperature. Fara-
day balance is used for the measurement of magnetic sus-
ceptibility of the synthesized metal complexes at room
1
[8]
[9]
microemulsion,
participation,
electro‐spinning,
solvent‐induced
[10,11]
[12]
[13]
1
solvothermal
and hydrothermal,
13
etc. These provocative studies recommend that the suit-
able design of reaction routes and selection of the metal
ions and ligands could start a novel filed to synthesize
nano‐scaled coordination compounds from the molecular
level, allowing their use in wide range of applications
temperature using Hg[Co(SCN) ] as a calibrant. Elemen-
4
[
14]
including catalysis, photochemistry,
biosensing, bio-
tal analysis was done by using Thermo Quest Elemental
Analyzer. Electronic spectra of ligand and its metal com-
plexes were recorded on a Hitachi 150‐20 Spectrophotom-
eter in the range of 1000‐200 nm. Thermal analysis of the
metal complexes was carried out with an SDT Q600 ana-
lyzer, by keeping the final temperature at 1000 °C and
the heating rate was maintained at 10 °C/min. The molar
conductivity measurements were made on ELICO‐CM‐82
Conductivity Bridge. ESI‐MS was recorded on Waters,
model‐Q‐ToF micro mass spectrophotometer equipped
with electrospray ionization (ESI) having mass range of
4000 amu in quadruple and 20,000 amu in ToF. SEM
and AFM photographs were recorded on JEOL model
JSM‐6390LV scanning electrone microscope, Multimode
nano surf easy scan atomic force microscope respectively.
Powder X‐ray diffraction spectra of the metal complexes
were recorded on Bruker AXS D8 Advance diffractometer.
[
15–20]
medical imaging, anticancer drug delivery etc.
Synthesis of transition metal compounds under hydro-
thermal conditions at relatively low temperatures of 100‐
ο
2
00 C has received much attention due to the possibility
of producing complexes with novel morphological charac-
ter. Further, Schiff base coordination complexes are still
of great importance because of their simple preparation
and wide applications in catalysis, material chemistry
[
21–
and biological modelling and as molecular magnets.
23]
However, reports on nano sized coordination com-
plexes with coumarin based ligands are limited, thus we
have concentrated on the green synthesis of nano sized
coordination compounds derived from Schiff base con-
taining coumarin and benzothiophene derivatives. The
synthesis of Schiff base described in this paper has not
been reported previously by any route. Present study
involves the two protocol for the preparation of Schiff
base metal complexes, i) hydrothermal preparation of
transition metal compounds at different temperatures in
the absence of any surfactant or capping agent and ii) reg-
ular reflux method. The synthesized compounds were
2
2
.2 | Synthesis
.2.1 | Synthesis of Ethyl 2‐amino‐5,6‐
1
13
characterized by FT‐IR, H NMR, C NMR, UV‐Vis, mass
spectroscopy, atomic force microscopy, scanning electron
microscopy, powder X‐Ray diffraction, thermal analysis,
elemental analysis, molar conductivity and magnetic
moment measurements. Further, the selected compounds
were screened for their bioassay specifically DNA cleav-
age and anticancer activity.
dihydro‐4H‐cyclopenta thiophene‐3‐
carboxylate
An
equimolar
mixture
of
cyclopentanone,
ethylcyanoacetate and sulphur in ethanol was stirred until
total dissolution of the sulphur at room temperature, to this
diethyl amine was added dropwise with continuous stirring.
After completion of the reaction as monitored by TLC,
the obtained product was purified by silica gel column chro-
matography to get pure ethyl 2‐amino‐5,6‐dihydro‐4H‐
cyclopenta thiophene‐3‐carboxylate (Scheme 1).
2
2
| EXPERIMENTAL
.1 | General considerations
Ample attention was taken to preserve the synthesized
compounds, chemicals and glassware's free from damp-
ness. Chemicals used in this work are of reagent grade.
Cyclopentanone, ethyl cyanoacetate, elemental sulphur,
pyrogallol and the metal salts were bought from SD‐Fine
chemicals and used without further purification. Com-
mercial organic solvents were distilled and dried
SCHEME 1 Synthetic route of ethyl 2‐amino‐5, 6‐dihydro‐4H‐
cyclopenta thiophene‐3‐carboxylate

JAWOOR ET AL.
3 of 12
2
.2.2 | Synthesis of Schiff base (SB)
ν(M‐N); 563 ν(M‐O). UV‐vis (λmax, nm, DMSO): 450,
585, 825.
To a stirred solution of ethyl 2‐amino‐5,6‐dihydro‐4H‐
cyclopenta thiophene‐3‐carboxylate (0.01 mol) in ethanol,
8
‐formyl‐7‐hydroxy‐4‐methylcoumarin (0.01 mol) in etha-
2
.2.5 | Ni(SB) Under reflux condition (2)
nol was added dropwise and stirred for 3‐4 hours. The
reaction was monitored by TLC. The precipitate obtained
after the completion of the reaction was filtered and
washed with cold ethanol and the product was dried
under vacuum for 5 h. The schematic diagram of the syn-
thesis of Schiff base is given in scheme 2.
Yield: 43%; Color: Brown; Elemental anal. calc. for
C H NiN O S : C, 56.83; H, 4.54; N, 3.16; S, 7.22; Ni,
42
40
2 12 2
6.61. Found: C, 56.41; H, 4.72; N, 3.15; S, 7.18; Ni, 6.64.
‐1
FT‐IR (KBr, cm ): 3375 ν(O–H), H O; 1580 ν(C=N);
2
452 ν(M‐N); 575 ν(M‐O). UV‐vis (λmax, nm, DMSO): 458,
610, 915.
2
.2.3 | General procedures for the synthe-
sis of metal complexes (1‐12)
2.2.6 | Cu(SB) Under reflux condition (3)
Reflux Conditions
Yield: 40%; Color: Brown; Elemental anal. calc. for
C H CuN O S : C, 56.52; H, 4.52; N, 3.14; S, 7.19; Cu,
A methanolic solution of CoCl .2H O, NiCl .6H O and
2
2
2
2
CuCl .6H O (0.1 mmol) were added dropwise to a solu-
42 40
2 12 2
2
2
6
.90. Found: C, 56.48; H, 4.50; N, 3.16; S, 7.14; Cu, 6.98.
tion of Schiff base in methanol and refluxed for 4‐5 h.
The obtained precipitate was filtered, washed with cold
methanol and dried in vacuum.
‐1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N);
2
449 ν(M‐N); 584 ν(M‐O). UV‐vis (λmax, nm, DMSO): 420,
686.
Hydrothermal Conditions
0
.1 mmol of CoCl .2H O/ NiCl .6H O/CuCl .6H O was
2 2 2 2 2 2
dissolved in 20 ml of water and then added to 0.2 mmol
of Schiff base (dissolved in 50 ml of water). The resulting
mixture was stirred for 10‐15 min and then transferred
into a Teflon‐lined stainless steel autoclave. After hydro-
2.2.7 | Nano Co(SB) Hydrothermal condi-
tion at 120 C (4)
ο
Yield: 58%; Color: Amber; Elemental anal. calc. for
C H CoN O S : C, 56.82; H, 4.54; N, 3.16; S, 7.22; Co,
42
40
2 12 2
thermal treatment (heating at different temperatures:
ο
6.64. Found: C, 56.31; H, 4.78; N, 3.12; S, 6.84; Co, 6.60.
1
20, 140 and 160 C respectively for 6 h), the autoclave
‐1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N);
2
was cooled to room temperature. The product obtained
was washed with distilled water and dried under vacuum.
The product obtained by reflux method is relatively
low in yield compared to the product obtained by hydro-
thermal method. As compared to the reflux method 10‐
470 ν(M‐N); 563 ν(M‐O). UV‐vis (λmax, nm, DMSO): 449,
575, 815.
1
5% increase in yield was observed in hydrothermal
2.2.8 | Nano Ni(SB) Hydrothermal condi-
ο
method.
tion at 120 C (5)
Yield: 55%; Color: Amber; Elemental anal. calc. for
C H NiN O S : C, 56.83; H, 4.54; N, 3.16; S, 7.22; Ni,
42
40
2 12 2
2
.2.4 | Co(SB) Under reflux condition (1)
6
.61. Found: C, 57.00; H, 4.38; N, 3.09; S, 7.16; Ni, 6.58.
‐1
Yield: 45%; Color: Brown; Elemental anal. calc. for
C H CoN O S : C, 56.82; H, 4.54; N, 3.16; S, 7.22; Co,
FT‐IR (KBr, cm ): 3375 ν(O–H), H O; 1580 ν(C=N);
2
42
40
2
12 2
452 ν(M‐N); 575 ν(M‐O). UV‐vis (λmax, nm, DMSO): 442,
6
.64. Found: C, 56.52; H, 4.52; N, 3.20; S, 7.08; Co, 6.55.
582, 928.
‐1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N); 470
2
2
.2.9 | Nano Cu(SB) Hydrothermal condi-
ο
tion at 120 C (6)
Yield: 61%; Color: Amber; Elemental anal. calc. for
C H CuN O S : C, 56.52; H, 4.52; N, 3.14; S, 7.19; Cu,
42
40
2 12 2
6
.90. Found: C, 56.55; H, 4.28; N, 3.12; S, 7.12; Cu, 6.83.
‐
1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N); 449
2
SCHEME 2 Synthetic route of the Schiff Base
ν(M‐N); 584 ν(M‐O). UV‐vis (λmax, nm, DMSO): 428, 698.

4
of 12
JAWOOR ET AL.
2
.2.10 | Nano Co(SB) Hydrothermal con-
2.2.15 | Nano Cu(SB) Hydrothermal con-
dition at 160 C (12)
ο
ο
dition at 140 C (7)
Yield: 54%; Color: Amber; Elemental anal. calc. for
C H CoN O S : C, 56.82; H, 4.54; N, 3.16; S, 7.22; Co,
Yield: 46%; Color: Amber; Elemental anal. calc. for
C H CuN O S : C, 56.52; H, 4.52; N, 3.14; S, 7.19;
42
40
2
12 2
42 40
2 12 2
6
.64. Found: C, 56.78; H, 4.42; N, 3.09; S, 6.99; Co, 6.62.
Cu, 6.90. Found: C, 56.54; H, 4.65; N, 3.36; S, 7.13; Cu,
‐
1
‐1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N);
6.78. FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578
2
2
4
70 ν(M‐N); 563 ν(M‐O). UV‐vis (λmax, nm, DMSO): 442,
ν(C=N); 449 ν(M‐N); 584 ν(M‐O). UV‐vis (λmax, nm,
5
63, 816.
DMSO): 430, 685.
2
.3 | Pharmacology
2
.2.11 | Nano Ni(SB) Hydrothermal condi-
ο
From the literature it is well known fact that, as the size of
the particle decreases the penetration of the compounds
through membrane will be easy. Therefore we have car-
ried out the bioactivity for compounds 7, 8 and 9. The fol-
lowing cytotoxicity and DNA cleavage studies were
undertaken to evaluate the primary examination of the
synthesized nano metal complexes (7, 8 and 9).
tion at 140 C (8)
Yield: 51%; Color: Amber; Elemental anal. calc. for
C H NiN O S : C, 56.83; H, 4.54; N, 3.16; S, 7.22; Ni,
42
40
2 12 2
6
.61. Found: C, 56.72; H, 4.62; N, 3.25; S, 7.15; Ni, 6.58.
‐1
FT‐IR (KBr, cm ): 3375 ν(O–H), H O; 1580 ν(C=N); 452
ν(M‐N); 575 ν(M‐O). UV‐vis (λmax, nm, DMSO): 448,
2
5
85, 914.
2.3.1 | Cytotoxic activity in in vitro assay
The in vitro cytotoxicity assay of the ligand and its
metal complexes 7, 8 and 9 were tested against human
ovarian cancer cell line (PA‐1) by MTT (Standard 3,4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide)
2
.2.12 | Nano Cu(SB) Hydrothermal con-
ο
dition at 140 C (9)
Yield: 58%; Color: Amber; Elemental anal. calc. for
C H CuN O S : C, 56.52; H, 4.52; N, 3.14; S, 7.19; Cu,
[
27,28]
42
40
2 12 2
method
and cisplatin was used as a standard refer-
6
.90. Found: C, 56.48; H, 4.60; N, 3.18; S, 7.10; Cu, 6.94.
ence compound. The cell lines were obtained from
National Centre for Cell Sciences, Pune, India. The cells
were cultured in DMEM medium supplemented with
‐
1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N);
2
4
49 ν(M‐N); 584 ν(M‐O). UV‐vis (λmax, nm, DMSO):
4
30, 694.
10% (v/v) heat inactivated fetal bovine serum (FBS) and
ο
0.1 % antibiotic solution for 24 h at 37 C and for adher-
ence 5% CO were used. Cells were incubated with the
2
tested compounds to achieve final concentrations of 10,
20, 25, 30 and 50 μL and control wells were prepared by
the addition of culture medium (100 μL). After 48 hours
of incubation, 20 μL of stock solution of MTT was added
2
.2.13 | Nano Co(SB) Hydrothermal con-
ο
dition at 160 C (10)
Yield: 50%; Color: Amber; Elemental anal. calc. for
C H CoN O S : C, 56.82; H, 4.54; N, 3.16; S, 7.22; Co,
ο
to each well. After incubation at 37 C for 4 h, the superna-
42
40
2 12 2
6
.64. Found: C, 56.41; H, 4.72; N, 3.02; S, 7.18; Co, 6.58.
tant was carefully aspirated and the separated formazan
product was dissolved by addition of 100 μL of buffer con-
taining dimethylformamide (50%) and sodium dodecyl
sulfate (20 %). Absorbance was measured at 570 nm using
an ELISA plate reader and the optical density of each well
was then measured for three times to obtain the mean
values. Further, percentage inhibition was calculated
from the below equation:
‐
1
FT‐IR (KBr, cm ): 3456 ν(O–H), H O; 1578 ν(C=N);
2
4
70 ν(M‐N); 563 ν(M‐O). UV‐vis (λmax, nm, DMSO): 452,
5
91, 828.
2
.2.14 | Nano Ni(SB) Hydrothermal condi-
ο
tion at 160 C (11)
%
inhibition = mean OD of untreated cells (control)/
mean OD of treated cells X 100
Yield: 48%; Color: Amber; Elemental anal. calc. for
C H NiN O S : C, 56.83; H, 4.54; N, 3.16; S, 7.22; Ni,
42
40
2 12 2
6
.61. Found: C, 56.72; H, 4.48; N, 3.20; S, 7.04; Ni, 6.55.
2.3.2 | DNA Cleavage Studies
‐
1
FT‐IR (KBr, cm ): 3375 ν(O–H), H O; 1580 ν(C=N);
2
4
52 ν(M‐N); 575 ν(M‐O). UV‐vis (λmax, nm, DMSO): 460,
The cleavages of ligands with DNA interaction ability are
5
95, 910.
always eye‐catching due to their ability to cause several

JAWOOR ET AL.
5 of 12
effects like gene expression modification, cell cycle arrest
and DNA cleavage activity. DNA cleavage activity of
ligand and its metal complexes (7, 8 and 9) were studied
3.1 | FT‐Infrared Spectral analysis
FT‐Infrared spectra of all the synthesized metal complexes
exhibited a weak band around 2900 and 3100 cm‐ indi-
cates the aromatic and aliphatic C‐H stretching frequen-
cies. On comparison with the FT‐infrared spectra of the
metal complexes with Schiff base, the azomethine group
is shifted to lower wavelengths in all the metal complexes
i.e. in the range of 1565‐1580 cm‐ showed the coordina-
tion through nitrogen of azomethine group to the metal
1
[29,30]
by using agarose gel electrophoresis method.
In
brief, 250 mg of agarose was dissolved in 25 ml of TAE
buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) by
boiling. The warmed agarose was poured into the gel
cassette fitted with comb. Later comb was removed
carefully after the gel solidified and gel placed in electro-
phoresis chamber flooded with TAE buffer. 20 μL of
sample mixture and calf‐thymus DNA (mixed with
bromophenol blue dye @ 1:1 ratio), were loaded care-
fully into individual wells along with standard DNA
marker and gel was run in electrophoresis chamber with
1
[
31]
‐1
ion.
The broad band around 3400 cm and two weak
‐1
‐1
bands around 700‐725 cm and 750‐800 cm indicate the
coordinated water molecules due to rocking and wagging
[
32,33]
modes of vibrations respectively.
The complexes
‐1
showed a band around 1298 cm due to phenolic ν(C‐O)
5
0 V of electricity for 45 min. Then gel was immersed in
‐
1
whereas the band is shifted to 1364 cm in the Schiff base
is attributed to the high mesomeric interaction in the com-
plexes. The supportive evidence of bonding is also shown
by the presence of new bands in all the spectra of the com-
ETBR solution for about 15 min to stain and then bands
were observed under UV transilluminator and the
photographs of the gel bands were recorded. The cleav-
age ability of samples was compared with CT‐DNA and
DNA marker.
‐
1
plexes, which appear in the regions of 450‐470 cm and
1
5
60‐590 cm‐ are the characteristic bands of ν(M‐N) and
ν(M‐O) stretching vibrations respectively, these bands are
[
34]
missing in the spectrum of Schiff base.
Therefore, the
FT‐IR spectral results provide the confirmation of the
bonding mode of Schiff base to metal complexes, the
representative spectra of Schiff base and metal complex
3
| RESULTS AND DISCUSSION
All the metal complexes were prepared in the ratio of
9
are presented in Figure 1 and Figure 2. The FT‐IR
spectra of compound 7 and 8 are given in supplementary
S1 and S2).
1
1
:2 (metal: ligand) by regular reflux method (compounds
, 2 and 3) and hydrothermal process at relatively
ο
ο
(
low temperatures (compounds 4‐12) viz., 120 C, 140 C
and 160 C respectively. Structural and morphological
ο
characterization of the compounds was done by various
spectro‐analytical techniques. All the spectral data of the
compounds which are synthesized by hydrothermal route
at different temperatures were in good agreement with
those of the compounds which are synthesized under
reflux condition.
3
.2 | Molar conductance, Magnetic
moment measurements and Thermal
analysis of metal complexes
The molar conductivity values observed in the range of
17.05 ‐ 25.20 Ω cm mol indicated that, the complexes
‐
1
2
‐1
FIGURE 1 FT‐IR Spectrum of Schiff base

6
of 12
JAWOOR ET AL.
FIGURE 2 FT‐IR spectrum of compound 9
are non‐electrolytic in nature. Electronic structures of the
metal ions ligated with organic ligand are focused by the
3.4 | Solution electronic spectral studies of
Schiff base and its metal complexes (1‐12)
[
35]
magnetic susceptibility of the compound.
The mag-
[
36]
The solution electronic spectral studies of the Schiff base
and its metal complexes were collected in DMSO solution.
The Schiff base exhibits absorption bands at 260 nm and
netic moment value of all the cobalt (4.53‐4.75 BM),
[37,38]
nickel (3.13‐3.28 BM)
and copper (1.71‐1.79
complexes are paramagnetic in nature and
[
39,40]
BM)
[41]
358 nm for π→π* and n→π* transitions respectively.
belonging to octahedral geometry.
All the Co(II) complexes of Schiff base shows bands
The thermal stability and decomposition pattern of
all the synthesized complexes have been studied by
thermogravimetric analysis in the temperature range
4
4
around 815‐828 nm and 563‐591 nm of T (F)→ T (F)
(
1g
2g
4
1 1g 1g 3
4
ν ) and T (F)→ T (P)(ν ) transitions. The electronic
ο
ο
spectrum of Ni(II) complexes showed d‐d transition band
around 910‐928 nm and 582‐610 nm corresponds to
of 25‐1000 C with heating rate of 10 C/min and the
decomposition details are given in Table (S3). All
3
3
3
3 2
3
[42]
ο
A
2g(F)→ T1g(P)(ν
), A2g(F)→ T1g(F)(ν ) transitions.
the complexes exhibited a weight loss around 200 C
In the Cu(II) complexes, broad band around the region
indicating the loss of coordinated water molecules.
2
2
ο
of 685‐700 nm is assigned to E → T2g transition and fur-
g
The weight loss of around 250‐650 C temperature is
ther the high intensity band around 420 nm is due to sym-
due to the ligand moieties and the plateau obtained
after the loss of ligand corresponds to the formation of
metal residue.
metry forbidden ligand to metal charge transfer
[
43]
transition. This nature of absorption spectra shows that
all the Co(II) and Ni(II) complexes of Schiff bases are in
octahedral geometry and all the Cu(II) complexes are in
[
44]
distorted octahedral geometry.
1
13
3
.3 | H and C NMR Spectral studies
The nuclear magnetic resonance spectrum of the Schiff
3
.5 | Powder X‐ray diffraction studies
1
13
base was screened for both H NMR and C NMR
(PWXRD)
1
spectroscopy experiment. H NMR spectrum of Schiff
base (Figure 3) shows a singlet at 9.22 ppm is ascribed
to azomethine (‐CH=N) proton of the Schiff base. A
singlet at 14.27 ppm is due to the phenolic –OH of the
Schiff base. A characteristic triplet and quartet proton
X‐ray diffraction studies considered to be very useful prac-
tice that can afford realistic structural knowledge about
[
45]
the compounds under attention.
However the synthe-
sized nano‐metal complexes (7‐9) were soluble in some
organic solvents like DMSO and DMF, crystals that are
suitable for single crystal analysis are not obtained. So,
the degree of crystallinity of the synthesized metal com-
plexes (7‐9) was tested by powder X‐ray diffraction pattern
studies. The powder X‐ray diffraction pattern of com-
signals at 2.41 and 4.4 ppm are assigned to –CH and
3
–
CH respectively. In addition to this, the multiplet sig-
2
nals observed between 6.1–7.5 ppm are due to aromatic
13
protons. The C NMR spectrum of Schiff base (S4),
signals at 164.70, 160.30, 60.69 and 18.94 ppm are due
to C=N, C=O, ‐CH2 and ‐CH3 respectively. Signals
appeared in the region 107.03–157.54 ppm are assigned
to aromatic carbons.
ο
plexes 7‐9 were scanned in the range of 2‐80 (θ) at wave-
length 1.54 Å. The diffractogram and associated data
depict the 2θ value, relative intensity and inter‐planar

JAWOOR ET AL.
7 of 12
1
FIGURE 3 H NMR spectrum of the schiff base
spacing (d‐values). In complexes 7‐9, the trend of the
Similarly PWXRD for compounds 8 and 9 were done
and the results were depicted in Table and the patterns
were given in figure (S5, S6, S7 and S8). Also, for com-
pounds 1‐6 and 10‐12 PWXRD analysis were done and
the representative patterns were given in supplementary
file (S9, S10, S11). All the complexes showed presence of
forbidden numbers like 7, 15, 23, 71 etc., indicates the
complexes are belonging to hexagonal or tetragonal
symmetry.
curves decreased from maximum to minimum intensity
[
46]
indicating the amorphous nature of the complexes.
PWXRD pattern of compound 7, 8 and 9 showed
seven, nine and six reflections with maxima at 2θ =
ο
ο
4
0
5.51 , 45.44 and 43.30ο corresponding to d values
.1957, 0.1960 and 0.1965 respectively. The inter‐planar
distance (d) has been calculated using Bragg's equation,
nλ=2dsinθ. For the complex 9 unit cell calculations have
been calculated for cubic symmetry using entire signifi-
2
2
2
cant peaks and h + k + l vales were determined in
Table 1 and the PWXRD pattern were represented in the
3
.6 | Scanning electron microscope and
2
2
2
Figure 4. The h + k + l vales of compound 9 are 1, 3,
, 9, 46 and 93. The calculated lattice parameters for the
Atomic force microscope
7
above compound is a = b = c = 18.582 Å. The presence
of forbidden number indicates the complex may belong
to hexagonal or tetragonal symmetry.
Scanning electron microscope images of the compounds
1‐12 which are prepared under regular reflux and hydro-
thermal method are shown in Figure 5 (S12 and S13).
TABLE 1 Powder x‐ray diffraction results of compound 9
2
2
2
2
2
2
Peak
2θ
θ
Sin θ
Sin θ
1000 Sin θ
1000 Sin θ/CF (h +k +l )
(hkl)
d
a in A
1
2
3
4
5
6
4.63
2.315
4.165
6.245
6.94
0.0403
0.0726
0.1087
0.1208
0.2727
0.3851
0.0016
0.0052
0.0118
0.0145
0.0743
0.1483
1.60
1.00 (1)
(1 0 0)
(1 1 1)
‐‐‐
18.784
10.426
6.9640
6.2660
2.7750
1.9650
18.784
18.058
18.425
18.798
18.481
18.949
8.33
5.20
3.25 (3)
12.49
13.88
31.67
45.30
11.00
14.50
74.30
148.3
6.875 (7)
9.062 (9)
46.43 (46)
92.68 (93)
(2 2 1)
‐‐‐
15.83
22.65
‐‐‐

8
of 12
JAWOOR ET AL.
further the particles were noticed to be fragmented when
ο
the temperature is increased to 160 C, this may be due to
rise in temperature [Figure 5 (11)]. Therefore these results
suggest the changing temperature can affect the morphol-
ogy of the nanomaterials.
In addition, surface topography of the prepared com-
plexes was characterized by using atomic force micro-
scopic technique. As shown in Figure 6 (S14 and S15),
the compounds are having the morphology similar to that
of SEM images, where the thickness is observed to be
7
3
7
3.8 nm, 45.2 nm, 72.8 nm, 49.9 nm, 39.5 nm, 41.3 nm,
2.5 nm, 17.6 nm, 32.9 nm, 69.3 nm, 26.0 nm and
8.1 nm for synthesized compounds via reflux and hydro-
thermal method at 120 °C. When the temperature is
increased to 140 °C, thickness seen to be reduced and
clear image of nano surface is observed in all three nano
metal complexes. Further increasing the temperature
up‐to 160 °C resulted in the agglomerated morphology
of the complex. This indicates that at 140 °C well defined
nano structured morphology of complex is formed.
FIGURE 4 Powder X‐ray pattern of compound 9
From the figure it can be seen that the compounds 1‐3 do
not have a much distinctive morphology. Further, tem-
perature dependent morphology study was carried out at
ο
ο
ο
different temperatures i.e. 120 C, 140 C and 160 C. As
ο
can be seen from Figure 5 (5), at a temperature of 120 C
nano Co (II) complex formed are not in clear morphology.
On increasing the temperature from 120 to 140 C, irregu-
lar rod like morphology were noticed [Figure 5 (8)],
3.7 | Cytotoxicity
ο
When dealing with the biological activity, cytotoxicity to
human cells is one factor which is very important. In
FIGURE 5 Scanning electron microscope images of nickel complexes of schiff base at reflux and hydrothermal method (different
temperatures)

JAWOOR ET AL.
9 of 12
FIGURE 6 AFM Images of nickel complexes of schiff base at reflux and hydrothermal method (different temperatures)
order to evaluate and compare the cytotoxic activities of
Schiff base and their complexes (7‐9), the in‐vitro anti-
cancer activities of the compounds against PA‐1 (human
ovarian cancer) cell line by MTT assay were carried out,
keeping the cisplatin as a standard positive control to
evaluate the results of the Schiff base and their com-
plexes (7‐9). The results were represented by means of
IC50 values and % cell survival and illustrated in
Figure 7. The IC50 values obtained from the experiment
showed that the tested nano‐metal complexes were
more active than the free ligand, these results indicate
the enhancement of anticancer activity upon coordina-
tion to the metal ions, and this may be due to the pres-
[47]
ence of stronger hydrogen bonds.
The IC50 value of
standard cisplatin for PA‐I cell line is 1.92 μM. Schiff
base showed lower cytotoxic effect with IC50 value
FIGURE 7 Cytotoxicity evaluation of Schiff base (SB) and 7–9
against PA‐I cell line
7
9.41 μM followed by nickel complex (8) 8.33 μM,
cobalt complex (7) 8.06 μM and copper complex (9)
.46 μM. Cytotoxicity of the copper complex is higher
7
[
48]
than the remaining tested compounds because, ability
to produce reactive oxygen species is high in copper
complexes as compared to the other first row transition
metal complexes,
and we found that the synthesized
compounds showed reduction in the rate of cell prolifer-
[
49]
ation.
The other important feature is the particle size

10 of 12
JAWOOR ET AL.
[
51]
Mahendra Raj and Mruthyunjayaswamy
have studied
Schiff base metal complexes derived from coumarin, for
the DNA cleavage assay and obtained the similar
kind of good results. The presence of heteroatoms like
nitrogen and oxygen in the metal complexes and the
size of the complexes played very important role in the
cleavage activity.
4
| CONCLUSIONS
In conclusion, novel Schiff base has been prepared suc-
cessfully by condensation reaction; the metal complexes
were prepared by one step hydrothermal method and
regular reflux method. The structure of the compound
in both methods is same. AFM and SEM results reveal
that the size of the nano metal complexes is controlla-
ble. It is found that the change in temperature in the
hydrothermal method have variations in their size and
FIGURE 8 DNA Cleavage Activity of Schiff base (SB) and its
metal complexes (7‐9). Photograph of gel electrophoresis
experiment of above series on CT‐DNA. Lane M, DNA marker; lane
C, treated DNA
[
52]
morphology of the nano metal complexes.
The evi-
of the metal complexes, nano‐sized metal complexes
were used for the activity, and this nano feature of
the tested metal complexes increased the anticancer
activity via stress‐free dispersion of compounds to
tumor cells.
dences obtained from the analytical and spectroscopic
data can support the octahedral geometry of the metal
complexes as shown in Figure 9. The biological investi-
gation of the Schiff base and metal complexes 7‐9 have
cleaved the DNA completely and the anticancer activity
showed the tested compounds were cytotoxic to the
human ovarian cancer line (PA‐1). It is known that
the inorganic nanomaterials were widely used in the
medicinal field for various reasons: as their size is
smaller, surface area to volume ratio increases gradu-
ally. Thus, the existing surface area of the active compo-
nent of the metal complexes is significant, which
increases contact between the biomolecules and nano
compounds helps to achieve a better reaction rate and
high productivity.
Finally, the present research work confirmed the
size of the particle may influence the bio‐activity of
the metal complexes and change in the temperature
can affect the morphology of the compounds. All these
interesting properties of the nano‐metal complexes
motivated the researcher to work more in inorganic
nanomaterial filed to contribute in the area of
nanomedicine.
3.8 | DNA cleavage studies by gel
electrophoresis method
Agarose gel has been used for the DNA cleavage studies
of ligand and its metal complexes (7, 8 and 9). The metal
ions play significant role in the cleavage of DNA because
of nature of metal complexes can be attributed to π‐π
[50]
interaction through the aromatic phenyl rings.
The
DNA interactions with Schiff base and treated metal
complexes were shown in Figure 8. The experiment
works on migration of DNA with the influence of electric
potential. The photograph of experiment shows different
band widths than their control DNA; all the tested com-
pounds cleaved the DNA completely, so the compounds
have high ability to cleave the DNA completely. K.
ACKNOWLEDGEMENT
The authors are thankful to the USIC, Karnatak Univer-
sity, Dharwad, IISC‐ Bangalore, IIT‐Bombay for providing
the spectral facilities. We are thankful to UGC for the
financial assistance through UGC‐UPE Fellowship.
Authors are thankful to Shirajahammad M. Hunagund
and Vani R. Desai for their help in synthesis of nano
metal complexes.
FIGURE 9 Tentative structures of the metal complexes

JAWOOR ET AL.
11 of 12
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How to cite this article: Jawoor SS, Kumbar M,
52] X. C. Jiang, W. M. Chen, C. Y. Chen, S. X. Xiong, A. B. Yu,
Patil SA. Green synthesis of biologically active
transition metal nanoparticles containing novel
Schiff base via catalyst free hydrothermal reaction:
Structural, biological and morphology study. Appl
Organometal Chem. 2018;e4322. https://doi.org/
10.1002/aoc.4322
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