Synthesis, characterization and biological activities of Co(II), Cu(II), Zn(II), and Cd(II) metal complexes of 1,10 phenanthroline based Schiff base

A Schiff base ligand prepared from amino acid (L-Histidine) and salicylaldehyde (L1), 1,10 phenanthroline (L2) and its transition metal complexes of general formula [ML1L2] (where M = Co(II), Cu(II), Zn(II), and Cd(II)) has been synthesized and tested for in vitro antimicrobial study against harmful bacterial strains Escherichia coli (gram negative bacteria), Bacillus subtilis, Staphylococcus aureus (gram positive bacteria), and fungal organisms Aspergillus avus, Rhizophus stolonifer and Mucor ellipsoideus respectively via agar-well diffusion technique. The larvicidal activity of the complexes was tested against Culex quinquefasciatus mosquito. The synthesized complexes exhibited excellent antioxidant properties both by DPPH and Hydrogen peroxide assays. Analytical and spectroscopic techniques such as UV-Visible, FTIR, SEM, NMR, and HRMS were used to characterize the produced Schiff base metal complexes. TGA-DTA analysis was used to investigate the thermal stability of the complexes. The redox characteristics of the complexes were examined using cyclic voltammetry. Cd(II) complex shows the maximum zone of inhibition values 20 mm and 21 mm for S. aureus, and M. ellipsoideus, respectively. Among the synthesized complexes, Cd(II) complex showed highest antimicrobial, larvicidal activity and very good antioxidant property.


Introduction
Schiff bases are broad range of organic chelates widely studied in coordination chemistry. Schiff base ligands are prepared by simple condensation reaction between carbonyl group and primary amine. The researchers paid great attention towards transition metal complexes with the different Schiff base ligand because of excellent catalytic property, biological activity, industrial applications, medicinal value, and more [1][2][3][4]. Amino acids are very good biological organic chelates which can easily react with aromatic carbonyl compounds (aldehydes, ketones) to form Schiff bases [5] and interestingly with ortho hydroxy aldehydes or ketones to form a tridentate [6, 7, 2] Schiff base transition metal complex synthesized using amino acid act as effective chelating agent, antioxidant [8] and behave as a potential cytotoxic agent [9]. The interaction study between DNA and transition metal complexes has fascinated several bene ts because of their importance in molecular biology, chemotherapy and design of new kind of pharmacological molecule [10][11][12]. Transition metal complexes containing Schiff base ligands have a wide range of biological applications, including antibacterial, fungicidal, anti-in ammatory, antiradical, agrochemical, and antiviral therapy [13,14].
Mosquitoes are well known blood sucking insect that affect the human health as well as domestic animals worldwide by transmitting many dangerous diseases such as Dengue fever, encephalitis, malaria, chikungunya. C. quinquefasciatus is a common house mosquito which transmit harmful diseases. The WHO report says that every year more than one million people die due to mosquito borne diseases in worldwide [15]. So that new insecticides against mosquitoes are needed. Cu(II) complexes of amino acid derived Schiff bases as primary ligand and 2,2' bipyridyl and 1,10 phenanthroline as secondary ligand [16] was reported for larvicidal activity. Larvicidal activity was studied for amino acid based Schiff base Cu(II) complexes containing pyridine and triphenylephosphine against Anopheles and Culex mosquito [17]. In the present work, our primary intention is to synthesize the biologically active Schiff base transition metal complexes from L-histidine-N-(salicylidine) and 1,10 phenanthroline and to test antibacterial and antifungal activity against number of bacterial pathogens and fungi, further screened for in vitro antioxidant study and larvicidal activity against C. quinquefasciatus.

Materials
The essential reagents and compounds were obtained from various sources. Histidine (98.0%) and sodium hydroxide were purchased from HIMEDIA. Salicylaldehyde, DPPH (Diphenylpicrylhydrazyl), hydrogen peroxide and 1, 10 phenanthroline were purchased from sigma Aldrich. Cobalt chloride tetrahydrate, copper, zinc and cadmium acetates were purchased from SD ne.

Physical measurements
UV-Visible spectra of the metal complexes were taken using JASCO V-670 spectrophotometer, The KBr pellet FT-IR spectrometer, IRA nity-1, was used to record FTIR spectra for the solid complexes. 1 H NMR and 13 C NMR were veri ed using 400 MHz Bruker analyzer. The cyclic voltammetry of prepared complexes were recorded using 0.1M Tetrabutylammonium hexa uorophosphate (TABPF 6 ) in DMSO solvent with an Ivium vertex electrochemical workstation in a conventional three electrode system in which working electrode was gold (Au), counter electrode was platinum (Pt) and Ag/AgCl electrode as a reference electrode. Molar conductivities of synthesized complexes were analyzed in fresh solution of DMSO (1.0 x 10 − 3 M) at RT with digital conductivity TDS meter 308. The instantaneous TGA and DTA measurements were done on Thermogravimetric Analyzer, SDT Q600. Molecular weight of the complexes was calculated by JEOL GC MATE II HR Mass spectrometer, EI mass 70 eV. The powder X-Ray diffraction was veri ed using Bruker powder X-ray diffractometer, D8 Advance. Morphology of powder samples were determined by scanning electron microscope, Carl Zeiss-EVO/18.

Synthesis
An ethanolic solution of salicylaldehyde (0.122 g, 1 mmol) was added to aqueous solution of L-Histidine (0.208 g, 1 mmol) with NaOH (0.08 g, 2 mmol). The reaction mixture was stirred at 60 o C for an hour. To this reaction mixture suitable metal salts such as 1 mmol of cobalt (II) chloride tetrahydrate (0.238 g)/copper (II) acetate monohydrate (0.200 g)/zinc (II) acetate dihydrate (0.22 g)/cadmium (II) acetate dihydrate (0.266 g) were added and this mixture was continuously stirred then 1,10 phenanthroline (0.181g, 1 mmol) was added after 1 h. Stirring was continued for 2 more hours. The formed precipitate was separated by ltration and then washed in cold ethanol and dried under vacuum. TLC was used to observe the reaction progress.

Antimicrobial activity
Antimicrobial activity of the synthesized Schiff base metal complexes was studied against bacterial strains S. aureus, B. subtilis, E. coli and three fungal strains A. avus, R. stolonifer and M. ellipsoideus by WD (well diffusion) method [18]. Microorganisms were seeded and spreaded uniformly in sterilized petri plates with Muller Hinton Agar medium. Three wells were cut onto a Muller Hinton agar spread plate with 6 mm diameter using sterile well cutter. 100 µL of freshly prepared sample solution in DMSO was transferred into the wells, DMSO was concede as -ve control and streptomycin as + ve control. Finally, the sample plates were left undisturbed for an appropriate span to allow the sample to disperse prior to the development of microorganisms, following which the plates were kept in an incubator for organism growth at 37° C for one day for bacteria and 2-3 days for fungi [11]. The results of study were determined by measuring the zone of inhibition and compared with zone of positive control.

Antioxidant activity
Antioxidant activity was assessed using two methods: the DPPH assay and the H 2 O 2 assay.
Synthesized complexes (2 mg/mL of the complex in DMSO) were taken to test the free radical scavenging ability using 2 mL of DPPH in methanol (0.05M). The prepared solution was incubated for 30 minutes at room temperature in a dark place before the absorbance measurement. A spectrometer was used to detect the absorbance at 517 nm using methanol as a blank and a methanolic solution of DPPH as a control. The radical scavenging effects of compounds were determined by recording the absorbance at same wavelength. Decrease in absorption of reaction mixture indicated an increase in antioxidant property [22].
The following equation was used to calculate the obtained results in this study.

% of inhibition =
Were, A c -absorbance of the control: A s -absorbance of reaction mixture

H 2 O 2 scavenging activity
For free radical scavenging activity using hydrogen peroxide, 2 mg/mL of sample in DMSO was taken and added to 40 mM of hydrogen peroxide in phosphate buffer (pH 7.4) [13,14]. Phosphate solution without hydrogen peroxide was used as the blank and the absorbance was measured at 230 nm. Percentage of radical scavenging effects were calculated using Eq. (1) given in the DPPH method.

Larvicidal assay
The larvicidal activity of the samples were carried out using Culex quinquefasciatus mosquito larvae collected from Zonal Entomological Research facility, Vellore, Tamil Nadu. The larvicidal bioactivity was evaluated as per procedure reported by World Health Organization [23] with slight modi cation [24]. 10 fourth instar larvae of C. quinquefasciatus were situated in 100 mL of sterile deionized water containing various concentrations (1.0, 2.0, 3.0, 4.0, 5.0 mg) of prepared Schiff base metal complexes. Sterile double distilled water without complex is negative control and commercial C. quinquefasciatus larvicide with test sample is positive control. Dead larvae in each set were calculated for each hour and continued for twentyfour hours exposure time. The % of mortality was assessed as an average of triplicates after 24 h of exposure period.

Synthesis
The scheme of synthesis of Schiff base ligand and metal complex are shown in Fig. 1. In the rst step Schiff base ligand 1 was obtained by condensation of salicylaldehyde with L-histidine. The needed metal salts are added to the Schiff base ligand solution. Finally, ligand 1, 10 phenanthroline was added. The resultant product 2 was puri ed by recrystallization. Different spectroscopic techniques were used to con rm the structure of the synthesized compounds. All the complexes were soluble in DMF and DMSO and were non-hygroscopic. The CHNS analysis data were in good agreement with calculated values which revealed that the formula of the complexes are as expected. The molar conductivity of the complexes was determined using DMSO solvent (10 − 3 M) and summarized in Table 1. The non-electrolytic nature of the complexes were indicated by very low values.

FT-IR
FT-IR spectra of the synthesized complexes are shown in gure S1, and their corresponding data displayed in Table 2. An intense band corresponding to imine group coordinate with metal ions appeared in the range of 1597 − 1586 cm − 1 [25]. Bands appeared in the range of 1519-1592 cm − 1, [26] assigned to symmetric stretching and bands in the range of 1321-1342 cm − 1 assigned to asymmetric stretching of carboxylate anion which are coordinated to the central metal ion. The disparity between the asymmetric and symmetric stretching frequencies Δυ = [υ as COO − − υ s COO − ] was found the higher than the free COO − anion (185 cm − 1 ). From this the monodentate coordination of the COO − anion to the metal ion was established [27]. IR data of the complexes revealed that the Schiff base ligand coordinated to the metal ion through the imine nitrogen, phenolic oxygen and oxygen atom exist in the carboxylate anion and acted as a tridentate ligand.

Electronic spectral studies
The optical properties of the Schiff base metal complexes were investigated by UV-Visible absorption spectroscopy in 10 − 3 M DMSO solution ( gure S8). All the prepared Schiff base metal complexes possess two discrete absorption bands. The high intensity peak appear in the region of 285-320 nm were allocated to π-π * and n-π * transitions due to azomethine group and aromatic chromophores [28]. The low intensity/absorption minima of the complex observed in the region of 340-420 nm corresponds to Metalto-Ligand Charge Transfer (MLCT) transitions [29]. in the complex. There was no d-d absorption in the diamagnetic Zinc (II) complex [15]. Electron transfer from metal d-orbital to lled π-orbital of the ligand has been designated as M(dπ)-to-L(π * ). These important strong absorption bands of both charge transfer and M-L transition support the pioneering idea of a square pyramidal habitat for the ligand and ve coordination with the metal.

PXRD analysis
In order to check the crystalline nature of the synthesized complex, powder XRD was recorded between 2 theta from 10 o to 90 o . Powder X-ray diffraction analysis of Schiff base metal complexes shown in gure S9, Co(II) complex displayed well-de ned sharp peaks while other complexes are not. Co(II) complexes are highly crystalline in nature, while Cu(II) and Zn(II) complexes exhibited broad peak and no identi able peak is observed for Cd(II) complex. The average crystallite size of CoL1L2, CuL1L2, and ZnL1L2 complexes are135 nm, and 145 nm respectively.

Morphology Study
The surface morphology and particle size of the synthesized complexes are investigated using a scanning electron microscope (SEM). shows small sized grains with agglomeration and appeared as coral-rag-like structure. The layers in each complex micrograph demonstrate that the system contains atoms in a well-de ned pattern, indicating that the reactants have completely reacted to form a distinct homogenous product. The particles in the complexes are only a few microns in size. Particles smaller than 100 nm were also found, which clumped together to form bigger agglomerates.

Electrochemical studies
The cyclic voltammetry (CV) measurement is a primary characterization technique to identify the redox behavior of synthesized complexes. Cyclic voltammetry analysis was performed in dimethyl sulfoxide solution (DMSO) using 0.1 M TABPF 6 as supporting electrolyte, glassy carbon, Pt and Ag/AgCl as working electrode, counter electrode, and reference electrode respectively with the voltage scan rate of 50 mV/s. The resultant cyclic voltammograms of the complexes 1-4 are displayed in gure S10 and the electrochemical data are tabulated in Table 3.  occur at the cathodic half cycle in the scan rate of 50 mV/s. Each reduction is associated to quasi-reversible one electron transfer process at room temperature [30]. The rst redox couple peak separation (Ep) value is higher than the second redox couple of all the complexes. From this higher Ep value of the complexes, it is observed that there is a difference between the original complex and the reduced species [31].

Thermal properties
Thermal stability of synthesized Cu(II), Co(II) and Zn(II) complexes was carried out by Thermogravimetric (TGA) and Differential Thermo analysis (DTA) in nitrogen atmosphere with heating rate from 25 o C to 800 o C. Figure S11 and Table 4 [32]. When compared to other transition metal complexes, the thermal stability of Co (II) complex was found to be higher (large proportion of residue).

Stability Study
The synthesized complexes 1-4 were investigated for their photophysical stability in the presence of UV light in DMSO solution (1X10 − 3 M) before being tested for biological activity. The solutions were exposed to UV light (254 nm, 16 watts) for 150 minutes and the spectra were measured for every 30 minutes in the range 200-800 nm to detect the colour change and stability [33]. Figure S12 depicts the UV-Visible spectrum over time and the impacts of UV radiation exposure. When the samples were exposed to UV light irradiation for 150 minutes, there were no signi cant alterations in the colour of the samples and ignorable changes in the absorption spectra which revealed that the complexes 1-4 possess good stability.

Antioxidant activity
In vitro radical scavenging activity of prepared Schiff base metal complexes were studied by DPPH and H 2 O 2 method. The values are shown in Table 5. The DPPH has a free radical when dissolved in methanol at room temperature that can take up either electrons or hydrogen radicals, transformed into stable molecule.
Owing to the existence of unpaired electron, DPPH showed a characteristic intense absorption band at 517 nm. The absorption decreases stoichiometrically, when this electron becomes paired off with respect to the number of hydrogen atom or number of an electron taken up. The notable change in the absorbance by this kind of reaction could be extensively adopted to study the free radical scavenger's capability of several molecules [35].

Conclusion
In Antifungal assay of Schiff base metal complexes 1-4 Figure 5