Biochemical relevance of Cr(III) complexes of isoniazid: synthesis, characterization, DFT, antibacterial screening, antioxidant activity and glucose-lowering effect in STZ-induced diabetic rats

Article abstract of DOI:10.1080/00958972.2019.1572885

Molecular mechanism suggests that the incorporation of an antioxidant organic moiety to chromium will be a sound strategy for the synthesis of safer and more effective hypoglycemic compounds. Two Schiff base ligands were derived by condensation of isonico

Full text of DOI:10.1080/00958972.2019.1572885

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Biochemical relevance of Cr(III) complexes of
isoniazid: Synthesis, characterization, DFT,
antibacterial screening, antioxidant activity and
glucose lowering effect in STZ-induced diabetic
rats
Satyendra N. Shukla, Pratiksha Gaur, Sangeeta Jhariya, Bhaskar Chaurasia,
Preeti Vaidya & M. Azam
To cite this article: Satyendra N. Shukla, Pratiksha Gaur, Sangeeta Jhariya, Bhaskar
Chaurasia, Preeti Vaidya & M. Azam (2019): Biochemical relevance of Cr(III) complexes of
isoniazid: Synthesis, characterization, DFT, antibacterial screening, antioxidant activity and
glucose lowering effect in STZ-induced diabetic rats, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2019.1572885
To link to this article: https://doi.org/10.1080/00958972.2019.1572885
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Biochemical relevance of Cr(III) complexes of isoniazid: Synthesis,
characterization, DFT, antibacterial screening, antioxidant activity and
glucose lowering effect in STZ-induced diabetic rats
SATYENDRA N. SHUKLA*†, PRATIKSHA GAUR†, SANGEETA JHARIYA†, BHASKAR CHAURASIA†,
PREETI VAIDYA† and M. AZAM‡
†Coordination Chemistry Research Lab, Department of Chemistry, Govt. Science College, Jabalpur (M.P.) India
‡King Saud University, Riyadh, Saudi Arabia
Molecular mechanism suggests that the incorporation of an antioxidant organic moiety to
chromium will be a sound strategy for the synthesis of safer and more effective hypoglycemic
compounds. Two Schiff base ligands were derived by condensation of isonicotinyl hydrazide
with salicylaldehyde/o-hydroxyacetophenone which further yield four novel chromium(III)
complexes of types [Cr(L)Cl2(H2O)] and [Cr(L)2]Cl. The ligands and complexes were
characterized by analytical and spectroscopic techniques. DFT study at the basic set B3LYP and
TD-SCF/6-311-G level was employed to confirm the geometry of the investigated compounds.
Ligands were tested for their antioxidant activity and exhibited good antioxidant activity.
Assessment of insulin-like activity of the complexes was initially performed in vitro by
measuring the inhibition of α-amylase. The complex with highest in vitro activity was
investigated for in vivo antidiabetic activity on the model of STZ-induced diabetic rats, which
demonstrated that complex 4 significantly lowers the blood glucose level in rats. Toxicity level
and antioxidant activity of the complex were also tested, which exhibit good tolerance level and
antioxidant activity. Histological analysis of the pancreas of animals under investigation reveals
the good condition of the pancreas treated with the complex. Ligands and complexes were also
tested for antibacterial activity against E. coli.
Keywords: α-Amylase inhibition; Schiff base; Type-2 diabetes mellitus; In vivo activity; DFT
1. Introduction
Diabetes, particularly type II diabetes, is a very severe disease affecting the health of the human
being worldwide, making it one of the most important public health challenges to all countries.
*Corresponding author. Emails: ccrl_2004@rediffmail.com; sns1963_1@rediffmail.com
1

There are reports indicating the estimation of affected people may be 349 million by 2025 [1].
Diabetes Mellitus is a disease that results in chronic hyperglycemia due to an absolute or relative
lack of insulin and insulin resistance which in turn impairs the metabolism of glucose [2, 3].
Over the years, various antidiabetic drugs with main products metformin, DPP-4 inhibitors,
GLP-1 receptor agonists, pioglitazone glucosidase inhibitors, sulfonylureas/glinides, acarbose,
and SGLT-2 inhibitors are available on the market [4]. The digestive enzyme pancreatic alpha-
amylase (EC 3.2.1.1) is a calcium metalloenzyme that catalyzes the hydrolysis of the alpha-1,4-
glycosidic linkages of the starch, amylose, amylopectin, glycogen and various maltodextrins, and
is responsible for most of the starch digestion in humans. In this context, it is important to
mention that the inhibition of carbohydrate digestive enzyme α-amylase that plays an important
role in the digestion of starch and glycogen is considered an important therapeutic strategy for
the treatment of type II diabetes and obesity as well as dental caries and periodontal disease [5].
Metal-containing compounds have been used in medicine since long [6]. Chromium is
considered an essentially important metal for normal glucose and lipid homeostasis [7].
Chromium picolinate and picolinic acid are the most extensively studied as antidiabetic and
antiobesity chromium complex [8]. Furthermore, Cr(III) ion plays a valuable role in the
regulation of insulin action, metabolic syndrome and cardiovascular disease, and facilitates the
interaction of insulin with its receptor on the cell surface [9-12].
Isonicotinyl hydrazide or isoniazid is an antibacterial compound, very active on the
bacterial strain M. tuberculosis, M. vovis and M. cansari [13]. It is highly specific and
surprisingly effective against other microorganisms and so it is now a frontline drug used in
antitubercular treatment (ATT) [14]. Schiff bases and their metal complexes are known to have
biological activities such as antifungal, antimicrobial, antitumor, antioxidant, antidiabetic and
herbicidal properties [15-23]. They have also been widely used as versatile ligands involved in
various metal chelation reactions to form metal complexes which are very interesting in many
fields, such as catalysis, enzymatic reactions and antioxidant activity.
Molecular mechanism suggests that a synergistic combination of antioxidant organic
ligands with chromium will be a sound strategy for the synthesis of safer and more effective
hypoglycemic compounds [24-26]. Thus, in view of these significant biological activities of
isoniazid as well as the biomimetic activity of chromium, we have started the work with the aim
to
synthesize
complexes
of
Schiff
bases
derived
from
isoniazid
with
2

salicylaldehyde/o-hydroxyacetophenone in anticipation of a good antidiabetic and antioxidant
activity. In addition, mole ratios for the reactions are determined by spectral measurements.
Stability constants (K) and Gibbs free energy (ΔG) values were also calculated. We are also
interested in the antidiabetic activity of resulting complexes with in vitro and in vivo
experimental systems in order to explore their effectiveness. We have used density functional
theory (DFT) calculations to ascertain the molecular structure of the ligands and complexes.
2. Experimental
2.1. Chemicals and techniques
All the chemicals and solvents were used of A.R. grade. Chromium(III) chloride hexahydrate
was purchased from Research Lab and used without purification. Solvent methanol and ethanol
(E. Merck) were distilled before use. Chromium was determined gravimetrically [27].
Isonicotinyl
hydrazide
(Lancaster,
U.K.),
salicylaldehyde
(E.
Merck)
and
o-hydroxyacetophenone (CDH) were used as received. Enzyme alpha-amylase was purchased
from Himedia, streptozotocin (Sigma Aldrich, Germany). FTIR spectra were recorded on a
Shimadzu–8400 S FTIR spectrophotometer in the range 4000-400 cm-1. Electronic spectra were
measured on an EI-2305 UV-vis spectrophotometer in DMSO-d6. 1H-NMR and 2D-NMR spectra
(HSQC) were recorded in DMSO-d6 on an Agilent-700-vnmrs700 spectrophotometer. The ESI-
MS spectra were recorded on an Agilent 6520 (QTOF) mass spectrometer having a DART
source in methanol. Elemental analyses (C, H, and N) were performed on an Elementar Vario
EL III, Elemental Analyzer. Gouy’s method was employed for measurement of magnetic
susceptibility; Hg[Co(NCS)4] was used as a standard. Diamagnetic correction was made using
Pascal’s constants. Molar conductance was measured in DMSO with an EI-181 digital
conductivity bridge using a dipping type of cell at 25 °C [28]. Glucose levels were estimated by
Glucometer (Accu check Performa).
2.2. Synthesis of Schiff base ligands
2.2.1.
Preparation
of
(E)-N'-(2-hydroxybenzylidene)isonicotinohydrazide
(L1).
Salicylaldehyde (1.06 mL, 0.01 mol) dissolved in ethanol was added to a solution of isonicotinyl
hydrazide (1.37 g, 0.01 mol) in ethanol and the reaction mixture was stirred for 1 h. Thereafter, it
was refluxed for 12 h. Progress of the reaction was observed by TLC. After completion, the
3

reaction mixture was concentrated to one-fourth of its volume and cooled to room temperature.
After two days of slow evaporation, a cream-white precipitate was separated, which was filtered
off, washed with sodium bisulphate, vacuum dried and recrystallized from 1:1 ethanol:water
mixture (v/v). Color: cream white; Yield: 2.14 g (89.00%); m.p. = 221 °C; Found: C, 64.60; H,
4.60; N, 17.36 %; C13H11N3O2 (MW = 241.25) requires: C, 64.66; H, 4.61; N, 17.40 %. Selected
IR absorptions (KBr, cm-1): ν(O-H), 3350(w); ν(N-H), 3194(w); ν(CO)CONH, 1681(s);
ν(C=N)cyclic+ν(HC=N)imine, 1618(s). Electronic spectra (λmax, in nm (ε, in M-1cm-1)) in DMSO:
1
220(310), 250(215), 300(120). H-NMR (700 MHz; , DMSO-d6),  (ppm): 13.285 (s,
1H, -OH); 11.678 (s, 1H, -CONH); 8.512 (s, 1H, -CH=N); 8.456 (d, J = 5.4 Hz, 2H, pyridyl);
7.911 (d, J = 5.4 Hz, 2H, pyridyl); 7.316 (s, 1H, aromatic); 7.214 (d, J = 7.3 Hz, 2H, aromatic);
6.922 (dd, J = 9.5, 4.6 Hz, 1H, aromatic). ESI-mass spectra, m/z: [C6H4NO]+ = 106.03,
[C7H6NO]+ = 120.04, [C6H5N2O]+ = 121.04, [C7H7N2O]+ = 135.05, [C7H6N3O]+ = 148.05,
[C8H7N2O2]+ = 163.05, [C13H10N3O]+ = 224.08, [C13H10N3O2]+ = 240.08, [C13H11N3O2 + H+]+ =
242.04, MW = 241.09.
2.2.2. Preparation of (E)-N-(1-(2-hydroxyphenyl) ethylidene)isonicotinohydrazide (L2).
Isonicotinyl hydrazide (1.37 g, 0.01 mol) dissolved in ethanol was mixed with
o-hydroxyacetophenone (1.20 mL, 0.01 mol) in ethanol and the reaction mixture was stirred for
2 h. Thereafter, it was refluxed for 15 h. Progress of the reaction was observed by TLC. After
completion, the reaction mixture was concentrated and cooled to room temperature. After slow
evaporation, a white precipitate was obtained, which was filtered off, washed with sodium
bisulphate, vacuum dried and recrystallized from DMSO. Color: white; Yield: 2.066 g (81.00%);
m.p. = 218 °C. Found: C, 64.70; H, 5.08; N, 16.28 %; C14H13N3O2 (MW = 255.10) requires: C,
64.75; H, 5.10; N, 16.39 %; Selected IR absorptions (KBr, cm-1): ν(O-H), 3352(w); ν(N-H),
3120(w); ν(CO)CONH, 1680(s); ν(C=N)cyclic+ν(HC=N), 1610(s). Electronic spectra (λmax, in nm (ε,
1
in M-1cm-1)) in DMSO: 220(318), 250(225), 300(125). H-NMR (700 MHz; , DMSO-d6),
 (ppm): 13.188 (s, 1H, -OH); 11.589 (s, 1H, -CONH); 8.785 (d, J = 5.6 Hz, 2H, pyridyl); 7.837
(d, J = 5.5 Hz, 2H, pyridyl); 7.316 (s, 1H, aromatic); 7.646 (d, J = 7.7 Hz 2H, aromatic); 6.914
(dd, J = 9.8, 4.9 Hz, 1H, aromatic); 2.493 (s, 3H, methyl). ESI-mass spectra, m/z: [C14H13N3O2
15
+ H+]+ = 256.1; [C14H13O2N2 N + H+]+ = 257.10, MW = 255.10.
4

2.3. Synthesis of complexes
2.3.1. Complex [Cr(L1)Cl2(H2O)] (1). To the solution of chromium(III) chloride (0.266 g,
1.0 mmol) in ethanol, recrystallized Schiff base ligand L1 (0.241 g, 1.0 mmol) dissolved in hot
ethanol was added. A few drops of sodium hydroxide were added which works as a catalyst, and
the reaction mixture was kept under stirring for 2 h in an inert atmosphere. The color of the
reaction mixture changed from dark yellow to brown. Thereafter, the reaction mixture was
refluxed for 12 h. After reduction of volume by slow evaporation, a brown solid was precipitated
in each case, which was filtered off, washed with acetone, dried under vacuum and recrystallized
from ethanol. Color: brown; Yield: 0.453 g (89%); m.p. = 250 °C. Found: C, 42.20; H, 3.80; N,
10.55; Cr, 12.95 %; C14H15Cl2CrN3O3 (MW = 396.19) requires: C, 42.44; H, 3.82; N, 10.61; Cr,
13.12 %. Selected IR absorptions (KBr, cm-1): ν(N-H), 3201(w); ν(C=O), 1642(s);
ν(C=N)cyclic+ν(C=N), 1600(s), 1570(s); (H2O), 771(s); ν(M-N), 750(m); ν(M-O), 428(m).
Electronic spectra (λmax, in nm (ε, in M-1cm-1)) in DMSO: 290(252), 320(180), 335(90), 440(20),
520(18). eff = 3.80 BM. Molar conductance in DMSO-d6, Λm at 25 °C (Ω-1cm2mol-1) = 3. ESI-
mass spectra, m/z: [Cl2CrH2O]+ = 139.89; [C14H13N3O2 + 2H]+ = 257.10; [C14H15ClCrN3O3]+ =
342.01; [C14H15Cl2CrN3O3 + H+]+ = 395.98 (MW = 396.19).
2.3.2. Complex [Cr(L1)2]Cl (2). Chromium(III) chloride (0.266 g, 1.0 mmol) dissolved in
20 mL ethanol was mixed with an ethanolic solution of recrystallized Schiff base ligand L1
(0.482 g, 2.0 mmol). A few drops of sodium hydroxide were added, and the reaction mixture was
kept under stirring for 3 h in an inert atmosphere. The color of the reaction mixture changed from
dark yellow to brown. Thereafter, the reaction mixture was refluxed for 14 h. After reduction of
volume by slow evaporation a red-brown solid was precipitated, which was filtered off, washed
with acetone, dried under vacuum and recrystallized from ethanol. Color: red-brown; Yield:
0.641 g (85%); m.p. = 240 °C; Found: C, 54.70; H, 3.50; N, 14.60; Cr, 8.95 %; C26H20Cl3CrN6O4
(MW = 567.92) requires: C, 54.99; H, 3.55; N, 14.80; Cr, 9.16 %. Selected IR absorptions (KBr,
cm-1): ν(N-H), 3053(w); ν(C=O), 1630(s); ν(C=N), 1602(s); ν(M-O), (766); ν(M-O), 464(m).
Electronic spectra (λmax, in nm (ε, in M-1 cm-1)) in DMSO: 295(320), 324(204), 330(94), 430(18),
515(22). eff = 3.65 BM. Molar conductance in DMSO-d6, Λm at 25 °C (Ω-1cm2mol-1) = 49. ESI-
mass spectra, m/z: [C13H10N3O2 + 2H+] = 242.08; [C19H14CrN3O3]+ = 384.33; [C21H16CrN5O4]+
= 454.06; [C26H20CrN6O4]+ = 532.10 (MW = 567.90).
5

2.3.3. Complex [Cr(L2)(Cl2)(H2O)] (3). Chromium(III) chloride (0.266 g, 1.0 mmol)
completely dissolved in 20 mL of ethanol was mixed with 20 mL solution of recrystallized
Schiff base ligand L2 (0.255 g, 1.0 mmol) in ethanol. A few drops of sodium hydroxide were
added, and the reaction mixture was kept under stirring for 2 h in an inert atmosphere. The color
of the reaction mixture changed from dark yellow to brown. Thereafter, the reaction mixture was
refluxed for 15 h. After reduction of volume by slow evaporation a solid was precipitated, which
was filtered off, washed with acetone, dried under vacuum and recrystallized from
methanol:carbon tetrachloride 1:2 (v/v). Color: brown; Yield: 0.501g (95%); m.p. = 250 °C;
Found: C, 43.80; H, 4.15; N, 10.20; Cr, 12.50 %; C15H15Cl3CrN3O2 (MW = 410.22) requires: C,
43.92; H, 4.18; N, 10.24; Cr, 12.68 %. Selected IR absorptions (KBr, cm-1): ν(N-H), 3155(w);
ν(C=O), 1640(s); ν(C=N), 1604(s), 1580(s); (H2O), 778(s); ν(M-N), 755(m); ν(M-O), 430(m).
Electronic spectra (λmax, in nm (ε, in M-1cm-1)) in DMSO: 290(320), 320(204), 330(94), 438(28),
525(20). eff = 3.70 BM. Molar conductance in DMSO-d6, Λm at 25 °C (Ω-1 cm2mol-1) = 5. ESI-
mass spectra, m/z: [C15H15N3O2 + 2H+] = 269.10; [C10H11Cl3CrN2O2]+ = 347.93;
[C15H15Cl2CrN3O2]+ = 390.99; [C15H15Cl3CrN3O2 + H+]+ = 409.96 (MW = 410.22).
2.3.4. Complex [Cr(L2)2]Cl (4). Chromium(III) chloride (0.266 g, 1.0 mmol) completely
dissolved in methanol and 20 mL of ethanol was mixed with a solution of recrystallized Schiff
base ligand L2 (0.51 g, 2.0 mmol) with the same solvent. A few drops of sodium hydroxide were
added, and the reaction mixture was kept under stirring for 4 h in an inert atmosphere. The color
of the reaction mixture changed from dark yellow to brown. Thereafter, the reaction mixture was
refluxed for 16 h. After reduction of volume by slow evaporation a brown solid was precipitated,
which was filtered off, washed with acetone, dried under vacuum and recrystallized from
methanol:chloroform 1:2 (v/v). Color: pale brown; Yield: 0.620g (79%); m.p. = 280 °C; Found:
C, 55.80; H, 4.01; N, 14.05; Cr, 8.40 %; C28H24ClCrN6O4 (MW = 595.98) requires: C, 56.43; H,
4.06; N, 14.10; Cr, 8.72%. Selected IR absorptions (KBr, cm-1): ν(N-H), 3066(w); ν(C=O),
1633(s); ν(C=N), 1590(s), 1573(s); ν(M-N), 576(m); ν(M-O), 426(m). Electronic spectra (λmax
,
in nm (ε, in M-1cm-1)) in DMSO: 260(320), 280(204), 340(94), 380(18), 420(26). eff = 3.73 BM.
1
Molar conductance in DMSO-d6, Λm at 25 °C (Ω-1 cm2 mol-1) = 54. H-NMR (700 MHz;
DMSO-d6), δ (ppm): 11.489 (s, 2H, -CONH); 8.710 (d, 2H, pyridyl), 7.316 (m, 8H, Ar-H); 6.002
6

(m, 4H, Ar-H); 2.487 (s, 6H, -CH3). ESI-mass spectra, m/z: [C14H12N6O4 + 2H+] = 256.10;
[C28H24CrN6O4]+ = 560.13 (MW = 595.10).
2.4. Stability parameters
Job’s method [29] was employed to determine the stoichiometric ratio and stability constant (K)
of the complexes [30]. The solutions were prepared by mixing solutions of both ligand and metal
salt with equal molar concentration (10-3 M in DMSO) in ratio varying from 1:9 to 9:1. The
absorbance was measured at 465 nm for complex 1, 475 nm for complex 2, 460 nm for
complex 3 and 455 nm for complex 4.
Preferably, only the complex species stable in solution MLn have absorbance under a
monochromatic light at the definite wavelength. The mole fraction change as increases from 0 to
1, the absorbance of solution system first increases and decreases afterwards. These changes are
plotted as absorbance versus f graph known as Job’s curve. With the help of the graph, we can
get the coordination number “n” by eq. 1:
n = 1 - f/f0
where f is molar concentration of metal ion and ligand and calculated by eq. 2.
f = [M] / ([M]+[L])
(1)
(2)
(3)
The degree of dissociation α is calculated by Job’s curve by the following formula (eq. 3).
α = (A-A0) / A
The stability constant (K) and degree of dissociation (α) of complexes are calculated by
equation 4:
(4)
where M is a concentration of metal ion, α is degree of dissociation of complex and n is
coordination number of complex.
7

2.5. Computational analysis
In order to understand the molecular structure of the ligands and complexes, we have done DFT
calculations using Gaussian 09 software package [31]. DFT calculations were performed by full-
geometry optimization of the ligands and complexes 1-4 by using DFT method of B3LYP with
6-311+G(d, 2p) and/or 3-21+G* basis set for all nonmetallic atoms and LANL2DZ basis set for
the central metal atoms in the gas phase [32]. The quantum chemical parameters such as EHOMO
,
ELUMO, Mulliken electronegativity (), dipole moment, chemical potential (Pi), global hardness
(η), global softness (S), global electrophilicity (), absolute softness (), electronic charge
(Nmax) and total energy (ETD-HF/ETDKS) were calculated after geometrical optimization of
the structure of all compounds. The optimized structural parameters such as bond lengths, bond
angles and dihedral angles of all the compounds were determined with the atom numbering
scheme of the molecule. The distribution of electronegativity/electropositivity over a large
number of atoms was presented by the molecular electrostatic potential (MEP) [33].
2.6. Biological studies
2.6.1. In vitro antioxidant assay (DPPH free radical scavenging activity) of ligands. The free
radical scavenging activity of ligands was determined with the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) method. Four solutions of different micromolar concentrations of compounds
(0.002 µM, 0.004 µM, 0.006 µM and 0.008 µM) and standard ascorbic acid were prepared in
ethanol. The volume of each test tube was adjusted to 1.0 mL by adding ethanol. DPPH was used
as a control. Thereafter, 5 mL ethanolic solution of DPPH (0.1 mM) was added. The tubes were
kept at ambient temperature for 30 min. The absorbance of test solutions and a blank solution of
DPPH (2 mL) was measured at 517 nm. The decrease in the absorbance of DPPH was calculated
comparative to the measured absorbance of the control [34]. The percentage of radical
scavenging activity was calculated using the following formula:
8

2.6.2. Antidiabetic activity
2.6.2.1. In vitro antidiabetic activity of complexes
This study was performed by a modified starch iodine protocol [35]. In short, 0.01 mL of each
compound solution (0.002 µM in DMSO) was taken in pre-labelled test tubes. The enzyme
solution was prepared by mixing 27.5 mg of alpha-amylase in 100 mL of distilled water. After
that, 0.02 mL of α-amylase was added to each test tube and incubated for 10 min at 37 °C. After
incubation, 2 mL acetate buffer was added to each test tube, thereafter, 0.2 mL of 1% starch
solution was added to each test tube and the mixture was reincubated for 1 h at 37 °C. Then
0.2 mL of 1% iodine solution was added to each test tube. The absorbance of the mixture was
taken at 565 nm. Sample, substrate and α-amylase blank were undertaken under the same
conditions. Each experiment was done in triplicate. Percent α-amylase inhibition was determined
by the following equation:
α
2.6.2.2. In vivo antidiabetic activity of complex
Complex 4 was one of the most active in the in vitro experiment, which was selected for in vivo
antidiabetic activity. Healthy male Albino wistar rats weighing 200±30 g were used for all
experiments in the present study. Rats were housed in polycarbonate cages in a group of three
animals provided with husk bedding. All animals were kept at 22±2 °C in 12:12 h, light:dark
cycle. Feeding was done with standard pellets diet supplied by Golden Feeds, New Delhi (India);
drinking allowed with purified water ad libitum. Animals were collected at random from Animal
house of PBRI, Bhopal (India). All animal experiments were approved by the Institutional
Animal
Ethics
Committee
(IAEC)
of
PBRI,
Bhopal
(CPCSEA
Reg.
No.1824/PO/ERe/S/15/CPCSEA). Protocol approval reference no. PBRI/IAEC/PN-17031.
2.6.2.2.1. Induction of diabetes. Animals (Albino wistar) were kept for overnight fasting by
depriving of food for 16 h but allowed free access to water. Streptozotocin was dissolved in
citrate buffer (pH 4.5) and nicotinamide, in normal physiological saline. Non-insulin dependent
diabetes mellitus was induced in overnight fasting rats by a single intraperitoneal injection of
60 mg/kg streptozotocin. After 15 min i.p. administration, 120 mg/kg of nicotinamide was
9

injected. Hyperglycemia confirmed by the elevated levels of blood glucose were determined at
72 h. Only rats confirmed to have permanent NIDDM (non-insulin dependent diabetes mellitus)
were used for the antidiabetic study [36]. The animals with blood glucose concentration more
than 250 mg/dL will be used for the study.
2.6.2.2.2. Acute oral toxicity. Acute oral toxicity test was performed according to OECD
guideline 423. Present study was conducted for the evaluation of acute toxicity study of the
complex in Albino wistar rat. General behavior and lethality were evaluated and toxicity was
observed through the dosing schedule of 14 days in 2000 mg/kg dose level groups.
2.6.2.2.3. Experimental design. The experimental design was modified from several previous
works [37, 38]. All animals were divided randomly into five groups with three animals in each
group. To the group first, received normal saline per oral only and served as vehicle control, the
group second, third, fourth, and fifth were made diabetic with streptozotocin (STZ) and
nicotinamide. Group second received saline, group third received glibenclamide tablet
(600 μg/kg per day per oral) at the frequency of 24 h. Group fourth and fifth diabetic animals
were administered orally 100 and 200 mg/kg chromium complex, respectively, daily for 21 days.
All dosing of test samples was done orally throughout the experimentation. Test groups and their
dosage levels are given in table S1 in the Supplementary Material.
2.6.2.2.4. Collection of blood samples. After the last day of drug treatment, the blood sample was
collected by a retro-orbital puncture at 0, 7, 14 and 21 days; the glucose levels were estimated by
Glucometer (Accu check Performa). On day 21, oxidative enzyme (SOD, GSH, LPO and
Catalase) also estimated in blood serum. Overall assessment of in vivo antidiabetic activity was
shown as % glucose lowering (% GL):
(
)
(
)
2.6.2.2.5. Statistical analyses. All experimental results are expressed as the mean±standard
deviation (SD). All statistical data were analyzed with one-way analysis of variance (ANOVA)
10

followed by Bonferroni test. Differences resulting in P values < 0.05 were considered as the level
of significance.
2.6.2.2.6. Histopathological examination of the pancreas. For dissection of the pancreas, a
laparotomy was performed and the pancreas was immediately isolated and fixed overnight in 4%
paraformaldehyde solution [39]. Cross sections of 4-μm were cut and processed for routine
staining with hematoxylin and eosin (H&E) and were used for histopathological examination
under a light microscope (OPTICA). Photographs were then captured and diameters of ten
random islets of Langerhans in each section were determined using GIMP 2.8.14 software.
Diameters were then averaged for each animal and compared to determine the effect of drugs on
the diseased islets.
2.6.3. Determination of oxidative enzyme levels
2.6.3.1. Preparation of tissue for assay
The organ was rinsed with ice-cold normal saline followed by 0.15 M HCl (pH 7.4). Thereafter,
for LPO 10% w/v tissue homogenate in 0.15 M HCl (pH 7.4) and for GSH 10% w/v in 0.1 M
phosphate buffer (pH 7.4) was taken as sample. However, for Catalase and SOD 10% w/v in
0.15 M HCl / 0.1 M phosphate buffer (pH 7.4) was used. The sample was centrifuged at 15,000
rpm for 15 min at 4 °C and supernatant were taken as a sample.
2.6.3.2. Determination of LPO level
To 0.2 mL of prepared tissue homogenate, 0.2 mL of 4% sodium dodecyl sulphate, 1.5 mL of
20% acetic acid and 1.5 mL of 0.5% thiobarbituric acid was added. The mixture was heated for
60 min at 95 °C in a temperature controlled water bath to give a pink color. The mixture was
centrifuged at 3500 rpm for 10 min [40]. The absorbance of the supernatant was read
spectrophotometrically at 532 nm.
2.6.3.3. Determination of GSH level
To 1 mL of tissue homogenate, 1 mL of 10% TCA was added. The precipitated fraction was
centrifuged and to 0.5 mL supernatant, 2 mL of DTNB reagent was added. The final volume was
made up to 3 mL with phosphate buffer. The color developed was read at 412 nm. A standard
11

curve for GSH was prepared using glutathione. Results were expressed as micromole of GSH/mg
tissue [41].
2.6.3.4. Determination of CAT level
CAT level was estimated by the continuous spectrophotometric rate determination method [42].
A 50 μL supernatant was added to the buffered substrate (50 mM phosphate buffer, pH 7
containing 10 mM H2O2) to make total volume 3 mL. The decrease in the absorbance was read at
240 nm for 2.5 min at an interval of 15 sec. The activity was calculated using extinction
coefficient of H2O2 0.041/moles/cm2. Results were expressed as moles of H2O2 utilized/min/mg
protein.
2.6.3.5. Determination of SOD level
The assay involves the production of superoxide from O2 using reduced b-nicotinamide adenine
dinucleotide (NADH) as a reductant and phenazine methosulphate (PMS) as a catalyst in the
presence of an indicator, nitro-blue tetrazolium (NBT), which turns blue when reduced by
superoxide. The color change was monitored spectrophotometrically in the visible range at
560 nm. When aliquots of common beverages are added to the reaction, superoxide scavengers
(i.e. antioxidants) compete with NBT to react with superoxide. The percent inhibition of NBT
reduction can be used to quantify superoxide-scavenging [43, 44].
2.6.4. Antibacterial screening. Complexes 1-4 and ligands L1 and L2 were screened for
antibacterial activity against gram-negative bacteria E. coli at different concentrations. Agar well
diffusion method was used for antibacterial screening [45].
3. Results and discussion
3.1. Characterization of ligands
Empirical formulas of Schiff base ligands L1 and L2 were in agreement with elemental analysis.
ESI-mass spectra of L1 and L2 exhibit several peaks depending on the fragmentation pattern and
a molecular ion/pseudo-molecular ion peak [46]. L1 shows peaks at m/z: 106.03, 120.04, 135.05,
224.08 and 242.04 attributed for [C6H4NO]+, [C7H6NO]+, [C7H7N2O]+ and [C13H11N3O2+H+]+,
respectively. A peak at 242.04 was attributed to the pseudo-molecular ion. ESI-MS spectra of L2
12

(figure S1 in the Supplementary Material) shows peaks at m/z: 256.10, 257.10 attributed for
14
15
pseudo-molecular ion [C14H13 N3O2 + H+]+ and its isotope [C14H13 O2 N2 N+H+]+.
FT-IR spectra of L1 and L2 show absorption bands at 1618 cm-1 and 1610 cm-1,
respectively, which may be assigned for the combination of ν(C=N)cyclic and ν(C=N)imine [47].
However, both ligands exhibit a weak band at about 3350 cm-1 assigned for ν(O-H) vibration. A
signal at ~1681 cm-1 was assigned to ν(C=O) of CONH moiety. For ligands, theoretical IR
spectra were elucidated with the help of DFT, which show a close resemblance with
experimental FT-IR data. Theoretical IR spectrum of L2 is shown in figure S2. However, FT-IR
spectra of ligand L2 and complex 4 are shown in figure S3.
The electronic spectra of Schiff base ligands exhibit three bands at ~220 nm, 260 nm and
300 nm. The first band was assigned to the π-π* transition of the C=C chromophore, however,
the other two bands are assigned to n-* transition associated with C=N and C=O groups,
respectively. The UV-vis spectrum of L2 is given in figure S4.
1H-NMR spectrum of L1 exhibits a signal at 8.512 ppm assigned for one azomethine
proton. Both L1 and L2 displayed a signal at 13.285 ppm and 13.188 ppm for one proton, each
attributed to phenolic-OH. The signals observed at 11.678 ppm in L1 and 11.589 ppm in L2 for
one proton each were assigned to (CO-NH) proton. L2 shows a signal centered at 3.366 ppm for
3H was assigned for –CH3 proton. In L1, two doublets centered at 8.456 ppm and 7.919 ppm for
two protons were assigned to pyridyl protons. A singlet observed at 7.316 ppm for one proton,
another doublet at 7.214 ppm for two protons and a triplet centered at 6.922 ppm were assigned
to aromatic protons of the phenolic ring.
However, L2 exhibits two doublets centered at 8.785 ppm and 7.845 ppm for two protons
each assigned to pyridyl proton. A doublet centered at 7.316 ppm for one proton, another doublet
at 7.646 ppm for one proton and a double doublet centered at 6.914 ppm for 2H were assigned to
aromatic protons of the phenolic ring. 1H-NMR spectrum of L2 is shown in figure S5.
L2 was also studied by 2D-HSQC spectrum (figure S6) which actually shows the
connectivity of protons with different carbon atoms [48]. The two contours observed at
6.914 ppm were connected to phenolic carbon at 118 ppm and 119 ppm. The contours observed
at 7.314 ppm and 7.646 ppm were connected with carbons at 132 ppm and 128 ppm. The
contours observed at 7.845 and 8.780 ppm were connected with pyridyl carbons at 122.0 ppm
and 149.0 ppm.
13

1
On the basis of the elemental analysis, ESI-MS, electronic, FT-IR and H-NMR spectra,
the following structures of L1 and L2 were proposed (figure 1).
3.2. Characterization of the complexes
The results of the elemental analysis of the metal complexes were in close agreement with the
proposed chemical formula for the complexes. Molar conductance (Λm) of all complexes in
10-3 M in DMSO at 25 °C was also helpful in proposing their formula. Molar conductance of
complexes 1 and 3 is between 3-5 Ω-1cm2mol-1 indicating their non-ionic nature, however, 2 and
4 exhibit molar conductance of 49 and 54 Ω-1cm2mol-1 indicating 1:1 electrolytic nature in
DMSO solution [49]. These complexes have shown the presence of Cl- outside the coordination
sphere by positive qualitative silver nitrate test. The structure of these complexes was also
supported by ESI-MS.
ESI-mass spectra of the four complexes exhibit several peaks depending upon the
fragmentation pattern including a molecular ion, M+ or pseudo-molecular ion, [M + H+] peak
[50]. Complex 1 shows peaks at m/z 139.89, 257.10, 342.01, 342.01, and 395.98 attributed to
[C14H13N3O2 + 2H]+, [C14H15ClCrN3O3]+, [C14H15ClCrN3O3]+, and [C14H15Cl2CrN3O3 + H+]+,
respectively. The peak at m/z 395.98 was a pseudo-molecular ion peak. In complex 2, peaks
appeared at m/z 242.08, 384.33, 454.06 and 532.10 attributed to [C13H10N3O2 + 2H+],
[C19H14CrN3O3]+, [C21H16CrN5O4]+ and [C26H20CrN6O4]+, respectively. The peak observed at
m/z 532.10 was attributed to molecular ion, [C26H20CrN6O4]+. Complex 3 exhibits peaks at m/z
269.10, 347.93, 390.99 and 409.96 attributed to [C15H15N3O2 + 2H+], [C10H11Cl3CrN2O2]+,,
[C15H15Cl2CrN3O2]+, and [C15H15Cl3CrN3O2 + H+]+, respectively. The peak at m/z 409.96 was
attributed for pseudo-molecular ion, [C15H15Cl3CrN3O2 + H+]+. However, complex 4 exhibits
peaks at m/z = 256.10 and 560.13 attributed to [C14H12N6O4 + 2H+] and [C28H24CrN6O4]+,
respectively. The peak at m/z 560.13 was due to molecular ion [C28H24CrN6O4]+, indicating the
ionic nature of complex. Since naturally occurring chromium is composed of four stable isotopes
(50Cr, 52Cr, 53Cr, and 54Cr with 52Cr being the most abundant (83.789% natural abundance)), ESI-
MS spectrum of 4 (figure S7) clearly shows four peaks at m/z 558.13, 560.12, 561.13 and 562.13
assigned for [M+] ion peaks of the four isotopes.
FT-IR spectra of ligands display characteristic one/two peaks between 1610-1618 cm-1
assigned to a combination of ν(C=N) + ν(HC=N). This peak was shifted in complexes 1-4 and
14

appeared in the range 1604-1570 cm-1, clearly indicating coordination to metal through imine-N
[51]. Ligands L1 and L2 exhibit a weak peak at 3350 cm-1 and 3352 cm-1, respectively, assigned
for ν(-OH), which vanished in complexes 1, 2, 3 and 4 and new peaks appeared at 428, 464, 430
and 426 cm-1, respectively, attributed for new ν(M-O) bond indicating the bonding of metal to
the oxygen. A signal exhibited in 1 and 3 at 771 cm-1 and 778 cm-1, respectively, was attributed
to wagging and rocking mode of coordinated water [52]. FT-IR spectrum of 4 is given in
figure S3.
Electronic spectra of complexes (10-3 M) were measured in DMSO. Complexes 1-4 are
paramagnetic with magnetic susceptibility in between 3.65-3.80 BM. The electronic spectra of
L1 and L2 exhibit three bands at 220 nm, 260 nm and 300 nm. The first band was assigned to the
π-π* transition of the C=C chromophore, however, the other two bands are assigned to the n-*
transition associated with C=N and C=O groups, respectively. The first band assigned to C=C
chromophore and auxochrome of benzene ring was red-shifted in the complexes and appeared at
260 nm in the complexes. The other two bands assigned to n-* transition of (C=N)imine and C=O
groups are also red-shifted and appeared with decreased intensity at 280 nm and 340 nm in the
complexes, indicating an interaction of the ligand with metal ion due to the transfer of a non-
bonding electron pair from donor to metal. In the electronic spectra of the complexes, two low-
4
4
intensity broad bands appeared at 420 and 460 nm, attributed to the d-d transitions A2g  T2g
4
4
(v1) and A2g  T1g (v2) of the Cr(III) ion. These bands are of low molar absorptivity (ε) being
‘Laporte forbidden’ transitions. However, in chromium complexes, two transitions bands are
4
observed in the region as expected, but the third transition, A2g (F) 4T1g (P), could not be
identified; it may lie in the ligand field. The electronic spectral transition suggests an octahedral
environment around Cr(III) ion [53]. Electronic spectrum of 4 is shown in figure S8.
Since all complexes were paramagnetic, NMR spectra of the complexes were
complicated. The theory of the unpaired electron-nucleus interaction and its consequences for
NMR spectroscopy has been developed over the past decades and has been summarized to some
extent. In diamagnetic molecules, the orbital shift (dorb) provides the principal contribution to the
observed chemical shifts. In paramagnetic samples, the hyperfine shift (dhf) adds to the orbital
shift, leading to the observed chemical shift:
dobs = dorb + dhf
15

In view of common evidence and inevitable paramagnetic interference, we have explained
1H-NMR spectra of 4 (figure S9). A comparison of signals of the ligand with complexes reveals
that there are four signals between 2.452 ppm and 11.489 ppm. The two very weak and broad
signals between 6.80 ppm to 7.80 ppm can be attributed to phenolic protons. However, a signal
observed at 8.70 ppm was assigned to pyridyl protons. Signals expected for methyl proton not
observed in the normal scale may be due to paramagnetic interference. A signal at 11.489 ppm
was assigned to CONH proton. Signal observed at 13.188 ppm for one phenolic-OH proton in L2
vanished in 4, indicating removal of O-H proton and formation of M-O bond [54, 55]. 2D-HSQC
of 4 was not appropriate.
3.3. Stability constants of the complexes
The stoichiometries of reactions and complexing behavior of Schiff base ligands with chromium
Job’s method of continuous variation [56] was used in the investigation of composition and
stability constant of complexes during the reaction. The stability constants (K) for 1, 2, 3 and 4
were 1.919103, 2.473105, 1.266103 and 2.011105 M-1, respectively. From the close
observation of the data it was clearly observed that stability constants of 2 and 4 are quite high,
which may be ascribed to their 1:2 metal ratio. Job’s plot of 4 is shown in figure 2 and Job’s
plots of 1-3 are shown in figures S10-S12.
The Gibb’s free energy of formation of complexes is also calculated from the
relationship:
G = -RTlnK
where R is the gas constant (8.314 J/mole K), T is absolute temperature (273.16 + 25 =
298.16 K) and K is stability constant. The Gibbs free energies of 2 and 4 are also highly
negative, which indicate more feasibility of formation of these complexes in solution.
Thermodynamic and coordinating parameters of all complexes are presented in table S2.
3.4. DFT calculations
3.4.1. Molecular geometry, bond length and bond angles. In order to achieve better insight
into the molecular structure of ligands L1 and L2 and complexes 1-4, computational analysis has
16

been performed, which is designed for the realization of large-scale DFT calculations. It was
clearly observed from the fully-optimized structure of ligands that in both ligands (figure 3)
benzene ring is not in the plane of pyridyl ring, but actually situated at ~45 angles. The
molecular structure of complexes shows almost octahedral geometry around Cr(III) ion as it was
revealed from the bond distances and bond angles (table 1). Some bond length becomes slightly
longer than ligands. Since ligand was coordinated through azomethine-N and formation of an
M-N bond takes place, elongation in azomethine C=N was observed due to a partial shift in
electron density towards M-N bond. Similarly, coordination was also observed from the
deprotonated oxygen of hydroxybenzylidene moiety and the carbonyl oxygen of isonicotinyl
hydrazide. It was interestingly observed that pyridyl and benzene ring which were not in the
same plane in ligands were rotated and moved closer to each other in order to facilitate
coordination with metal. However, small angular distortions were observed in N-Cr-O, Cl-Cr-Cl,
O-Cr-O, O-Cr-Cl and N-Cr-N bond angles.
3.4.2. Quantum chemical parameters. The quantum chemical parameters; highest occupied
molecular orbital energy (EHOMO), lowest unoccupied molecular orbital energy (ELUMO), the
difference between HOMO and LUMO energy levels (ΔE), Mulliken electronegativity (),
dipole moment chemical potential (Pi), global hardness (η), global softness (S), global
electrophilicity (), absolute softness () and electronic charge (ΔNmax) have been estimated and
listed in table 2.
On the basis of the above data, we can conclude that:
1. The value of energy gap (ΔE) is higher in L1 than L2, which indicates the hard nature
as well as low reactivity of L1.
2. Complexes 2 and 4 exhibit lower energy gap than other complexes, which indicates
their soft nature, high reactivity and high flexibility in the transfer of electrons from
a donor (ligand) to acceptors (metal ions).
3. The ELUMO value was low in L2 which is an indicator of high acceptability for
electrons. The ELUMO values were almost the same in complexes.
4. The high value of EHOMO was observed in L2 which may be due to its powerful
donation behavior. Global hardness (η) and absolute softness () parameters are the
17

indicators of molecular stability and reactivity. The negative data of both ELUMO and
EHOMO were attributed to the stability of the complexes.
3.4.3. Mulliken atomic charge analysis. The Mulliken population analyses of ligands and
complexes 1-4 were done by using method B3LYP/6-311++G(d, p) with LANL2DZ basis set in
the gas phase. The net atomic charges of ligands and their complexes were obtained by means of
Mulliken population analysis. Mulliken charge is directly related to the vibrational properties of
the molecule. Distribution of positive and negative charges are the major cause to increase or
decrease bond length. The result shows that L1 has more positive charges on carbon atom C13
(+0.469029) due to the highly electronegative imine N11 (-488796) and O25 (-415137) attached
to this carbon atom. One positively charged carbon C15 (+0.202289) and one hydrogen H29
(+0.374521) are connected to more electronegative phenolic oxygen O26 (-0.625646) of the
benzene ring. However, in L2 more positive charges appear on carbon atom C13 (+0.411532)
because this atom is connected to more electronegative nitrogen N11 (-0.463831) and oxygen
O25 (-0.596106). Another positively charged carbon atom C15 (+0.169524) and hydrogen atom
H27 (+0.320235) is connected with the more electronegative phenolic O26 (–0.596106) of the
benzene ring. This is due to highly shielded atoms such as oxygen and nitrogen attached to less
electronegative or positively charged atoms such as carbon and hydrogen, acquiring a highly
negative charge. However, in 1-4, chromium(III) ion carries higher positive charge, +1.095398,
+1.245587, +1.712005 and +1.456922, respectively. Therefore, in 1 and 3, three electronegative
atoms, an oxygen atom of CONH moiety, another oxygen atom of dehydrogenated hydroxyl
moiety and an imine nitrogen of ligands are points of coordination with the electropositive metal
centered. Of the remaining three coordinating sites, two coordinating points were occupied by
chloride ion and one coordinating point was occupied by the oxygen atom of a neutral water
molecule. However, in 2 and 4, six electronegative atoms which are two phenolic oxygen atoms,
two imine nitrogen atoms and two oxygen of CONH moiety of ligands are coordinating points
with an electropositive metal center. Mulliken charge values of electropositive carbon and
electronegative oxygen/nitrogen atoms are also higher than ligand, probably due to an
occurrence of back-bonding of π-electrons during complex formation. This result is consistent
with the molecular electrostatic potential (MEP) [57-59]. The illustration of atomic charges for
L2 and 4 are shown as bar diagrams in figure S13.
18

3.4.4. Molecular electrostatic potential analysis (MEP) and contour maps. The MEP is
related to electron density and provides information about electron rich and electron deficient
sites in the molecules, which is a very useful description to understand metal-ligand interactions
as well as hydrogen bonding. The negative regions in MEP are related to the electronegative site,
while the positive regions are related to the electropositive site. The MEP of the ligand molecule
shows several regions of positive and negative potential. The negative regions are mainly over
the oxygen and nitrogen atoms (deep red/yellow) on -CONH groups of INH moiety and nitrogen
atoms of imine groups of ligand. Most positive regions (blue/green) are observed around the
hydrogen atoms of C–H groups as well as carbon atoms of imine groups. The color codes of this
map are in the ranges -0.0723 a.u. (deep red) to +0.0723 a.u. (deep blue) in L1 and -0.0755 a.u.
(deep red) to +0.0755 a.u. (deep blue) in L2. In 1 and 3, MEP is between -0.0012 a.u. (deep red)
to +0.0012 a.u. (deep blue) and -0.0010 a.u. to +0.0010 a.u., respectively. However, in 2 and 4
MEP is between -0.0752 a.u to +0.0752 a.u. and -0.0775 a.u. to +0.0775 a.u., respectively. The
potential increases in the order: red < orange < yellow < green < blue. The different colors
represent the different values of the electrostatic potential at the different atomic surface.
Molecular electrostatic potentials of L2 and 4 are shown in figure 4.
On the basis of the elemental analysis, molar conductance measurements, magnetic
susceptibility, ESI-MS, electronic, FT-IR, 1H-NMR, 2D-NMR spectra and DFT studies, the most
plausible ChemDraw structure and DFT optimized structure of complex 4 is shown in figure 5
and of complexes 1-3 in figures S14-S16.
3.5. Biological activity
3.5.1. In vitro antioxidant activity of ligands (free radical scavenging activity). The
antioxidant activity of the ligands was examined by measuring the scavenging effect of DPPH
radicals. The results of the free radical scavenging activity of the compounds at different
micromolar concentrations are shown in table 3. Ascorbic acid was used as a control to estimate
the effectiveness of the tested compounds under the same conditions. It was observed that the
free radical scavenging activity of these compounds was concentration-dependent. The examined
compounds have exhibited significant scavenging activity but lower than ascorbic acid. It has
19

been observed that L2 exhibited higher antioxidant activity of about 5-8% at every concentration
than L1.
3.5.2. In vitro antidiabetic activity. In vitro antidiabetic activity results are displayed in table 4.
A close observation of data reveals that:
1. Complexes exhibit better inhibition efficiency than Schiff base ligands.
2. Complexes 3 and 4 exhibit high inhibition efficiency. However, inhibition efficiency of 4
was almost the same as acarbose, used as a standard for comparison. It may be due to a
synergistic combination of L2 to chromium(III) ion, in particular steric environment results in
better absorption at the surface of the enzyme leading to better inhibition.
3. However, complexes 1 and 2 exhibited less inhibiting activity than expected. These
complexes show inhibition efficiency just above the ligand, probably due to the fact that
congregation of some function present in ligand in combination with chromium(III) in a
particular steric environment may not work in a synergistic way for the improvement of
antidiabetic activity.
3.5.3. In vivo antidiabetic activity. To assess the in vivo antidiabetic activity of chromium
complexes, the animal study was carried out on Albino wistar rats. The glucose level and body
weight were monitored before and after the treatment with the complex. The standard drug
glibenclamide was used for the comparison. Antioxidant activity and histology of pancreas were
studied on the twenty-first day in order to observe the effect of the drug on the animal.
3.5.3.1. Body mass of rats. The body weight of each group of rats before and after treatment with
the complex is shown in figure S17. The body weight of diabetic rats (Group 2) reduced with
time as compared to the initial weight. In the case of non-diabetic control rats (Group 1), weight
gain was observed. However, the body weight of complex-treated groups (Group 4 and Group 5)
and standard drug glibenclamide-treated group (Group 3) were significantly lowered initially in
the first week because of lower food and water consumption (first three days after drug
administration) and thereafter, considerably increases throughout the experiment.
20

3.5.3.2. Blood glucose level. The blood glucose levels before and after treatment are shown in
table 5 and figure S18. There is a significant gradual decrease in the glucose level observed after
the treatment with the complex. The percentage of decrease in glucose level was slightly higher
in Group 5, indicating the effect of a high dose of 4 to mice. However, in standard mice group
treated with antidiabetic drug glibenclamide, a sudden decrease in glucose level was observed in
the first seven days and thereafter decrease in blood glucose level was steady. The glucose level
of diabetic rats (Group 2) increases throughout the experiment.
3.5.3.3. Histological analysis. Histology of pancreas was analyzed on the basis of an effect on
the islet of Langerhans (figure 6). In animals treated with the standard drug, normal intercalated
ducts were clearly observed in histological slides 1 and 2, interlobular duct, acini, blood vessels
and pancreatic islets were distinguished from surrounding exocrine tissue on the basis of
continuous connective tissue capsule and extensive vascularity. Similarly, in the complex-treated
group, histology is almost the same. In the diabetic group at some location, alteration in the
structural morphology of islets was observed. Interestingly, it can be easily observed that in
Group 5 animals, which were treated with higher dose of the complex (200 mg/kg), histology of
pancreas is exactly similar to normal group animal.
3.5.4. In vivo antioxidant activity of complex. Complex 4 was also tested for antioxidant
activity for lipid peroxides (LPO), superoxide dismutase (SOD), glutathione (GSH) and catalase
(CAT) on the twenty-first day. It was observed that complex-treated groups (Group 4 and 5)
have higher levels of antioxidative parameters (catalase, superoxide dismutase and glutathione)
and decreased levels of lipid peroxidation, indicating its efficacy to reduce the LDL-c oxidation.
In the complex-treated groups, a significant increase in the liver CAT, SOD and reduced GSH
levels were observed as compared to the STZ control animals, which suggests good antioxidant
activity of this complex. In vivo antioxidant activity is shown in table 6.
3.5.5. Antibacterial screening of ligands and complex. The results of in vitro antibacterial
activity of the Schiff base ligands and their complexes 1-4 are presented in table 7. Amoxicillin
was used as positive standards and DMSO was used as negative control for this antibacterial
activity. Metal complexes have been more effective than the free ligands. Complex 4 displayed
21

the highest bactericidal activity against E. coli. However, other complexes exhibit lower
antibacterial activity [60]. The activity order of the synthesized compounds was as follows: 4 > 3
> 2 > 1 > L2 > L1. It is postulated that improved activity arises from the delocalization of
positive charge between the organic moiety and the metal, which favors the drug entering into
normal cellular processes of the bacteria. Probably the enhanced lipophilicity of the complexes
leads to breakdown of the permeability barrier of the cell and thus retards the normal cell process
in bacteria. The minimum inhibitory concentration (MIC) of the complexes is also displayed in
the table 7 since the MIC is the concentration of the highest dilution tube, in which bacterial
growth was absent. It was observed that 1-4 has exhibited MIC in between 31-37 μg/mL.
4. Conclusion
Two Schiff bases and their four chromium(III) complexes were synthesized and characterized by
spectroscopic techniques. Higher stability constant values of 2 and 4 suggested high stability of
1:2 metal chelate complexes. Gibbs free energy (G) of these complexes is also high, which
suggests high feasibility of their formation reaction. DFT was used to achieve better insight into
molecular structure. These compounds were initially studied for their in vitro antidiabetic and
antioxidant activity. Complexes 3 and 4 have exhibited a very promising in vitro antidiabetic
activity. Complex 4 shows in vitro antidiabetic activity almost the same as acarbose and was
selected for in vivo antidiabetic and antioxidant activity in Albino wistar rats. A significant
gradual decrease in the blood glucose level was observed in complex-treated groups. It was also
observed that complex-treated groups have higher levels of antioxidative parameters (catalase,
superoxide dismutase and glutathione) and decreased levels of lipid peroxidation, indicating its
efficacy to reduce the LDL-c oxidation. In the complex-treated groups, a significant increase in
the liver CAT, SOD and reduced GSH levels were observed as compared to the STZ control
animals, which suggests the good antioxidant activity of this complex. Interestingly, it was also
obvious from the histological slides that the group treated with a high dose of the complex has
the same histology as normal. It means higher doses of complex helps in maintaining the health
of the pancreas. In view of such promising results, we can say that this therapeutic approach may
be used as an important strategy in the management of blood glucose. Thus, the study of these
compounds is quite interesting not only in terms of their reactivity but also due to their varied
range of activity.
22

Acknowledgements
The authors are grateful to Principal, Government Science College, Jabalpur and Head,
Chemistry Department, for providing laboratory facilities. We sincerely thank SAIF, CDRI,
1
Lucknow, for recording ESI-MS, H-NMR spectra. We are also thankful to Daksh laboratory,
Jabalpur and PBRI, Bhopal for their valuable help rendered in in-vitro and in-vivo activity. One
of us (Sangeeta Jhariya) is also grateful to the UGC-New Delhi for financial support through
RGNF (Award letter no. F1-17.1/2015-16/RGNF-2015-17-SC-MAD-19472/ (SA-III/Website)).
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26

Graphical abstract
27

(a)
(b)
Figure 1. Structure of (a) (E)-N'-(2-hydroxybenzylidene) isonicotinohydrazide (L1) and (b) (E)-
N-(1-(2-hydroxyphenyl) ethylidene)isonicotinohydrazide (L2).
28

Figure 2. Job’s plot of complex 4.
29