SYNTHESIS, CHARACTERIZATION, ANTI-INFLAMMATORY, ANTIMICROBIAL AND DNA BINDING PROPERTIES OF Co(II). Ni(II), Cd(II) AND Cu(II) COMPLEXES WITH SCHIFF BASE

* Asha M S 1 , Othbert Pinto 1 , Nagendra Murthy V N 1 , Varadha Raj 1 and Delarasoltanesmaeili 2 . 1. St. Philomena’s College, Bannimantap, Mysore, Karnataka, India. 2. Department of Studies in Biotechnology, JSS College of Arts, Commerce and Science, Ooty road, Mysore, Karnataka, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 4 (8), 1065-1074 1066 spectrophotometer. The infrared spectra of the solid samples were recorded in the range 4000-500 cm -1 on a Perkin-Elmer 597/1650 spectrophotometer using KBr pellets. The magnetic moments were measured out using gouy balance. Purity of the compound was checked by TLC 10 .

Synthesis of Schiff base ligands and their metal complexes
General procedure for the preparation of Schiff Bases (iva,b):-A mixture of o-phenylenediamine (iii) and substituted hydroxylbenzophenone (iiab) in 1:2 molar ratio in methanol was refluxed with constant stirring. This condensation reaction was carried by using 3 drops of acetic acid for 5 hours. The formed water was removed from the reaction mixture by using sodium sulphate as dehydrating agent. After completion of the reaction, the mixture was reduced to half of its original volume and kept aside at room temperature. The white precipitate (iva,b) was formed on slow evaporation. This was filtered off and washed with ethanol. The product was recrystallized from hot ethanol and stored over anhydrous calcium chloride 11  They were also evaluated in vitro for their antifungal activity against Candida albicans. Inhibition zone diameter (IZD) in cm was used as criterion for the antimicrobial activity using agar diffusion well method. Gentamycin and Flucanazole were used as reference drugs for antibacterial and antifungal activity respectively. Microbes were grown in Nutrient Broth (NB, Merck) medium at 37°C for 24h. The bacterial number in the final inoculums was adjusted to 10 6 CFU/ml. A bacterial lawn was prepared by pouring 0.1 ml of bacterial suspension onto each plate of Nutrient Agar medium (NA, Merck), spread by a sterile cotton swab, and allowed to remain in contact for 1 min. Schiff base derivatives of different concentrations (20µg,40µg,80µg,100µg) were prepared in order to impregnate the paper discs. The sterile filter 1068 paper discs containing Schiff-base derivatives (6-mm diameter) were then placed on the bacterial lawn. The Petri dishes were subsequently incubated at 37°C for 24 h and the inhibition zone around each disc was measured in cm.

Anti inflammatory activity:-
Indirect Haemolytic assay of PLA 2 :-Protein concentration in the venom was calculated, using bovine serum albumin fraction V (0-75µg). A semi quantitative indirect haemolytic assay was employed. Briefly, packed human erythrocytes, egg yolk and phosphate buffer saline was mixed (1:1:8 V/V). 1ml of this suspension was incubated with 60 µg enzyme for 10 min at 37 0 C. The reaction was stopped by adding 9ml of cold phosphate buffer saline and centrifuged at 4 0 C for 10 min at 800Xg. The amount of haemoglobin released in the supernatant was measured at 540nm. The assay was also carried out in the presence of various concentrations 200-1000ng/ml. Enzyme and substrate without the inhibitor served as control.
DNA Binding and chelating assay using Calf thymus DNA:-For the gel electrophoresis study, calf thymus DNA (20μg) was treated with the 10 µg of Schiff base derivatives and incubated for 30 minuts in 40mM Tris-buffer (pH 8.3). The samples were electrophoresed for 3 hours at 50 V on a 1 % agarose gel in Tris-acetic acid-EDTA buffer. The gel was stained with 0.5 μg of ethidium bromide. Photographed under UV light.

Cell viability assay:-
Tryphan blue exclusion test is based on the principle that living cell membrane has the ability to prevent the entry of the dye, hence they remain unstained and can be easily distinguished from the dead cell which take up the dye. The percentage of viable cells was determined by diluting the isolated lymphocytes with 0.4% tryphan blue, mixed and loaded into haemocytometer. The viable cells were counted under microscope and % of viability was calculated.

Isolation of Human Peripheral Lymphocytes:-
10ml of blood and 2ml of ACD were mixed, it is incubated with 4 volumes of haemolysing buffer at 4 0 C for 30 minutes centrifuged at 1500 rpm for 12min at 4 0 C, pellet was subjected to10ml lysing buffer, centrifuged at 1500 rpm for 12 min, pellet was washed with HBSS solution, suspended in 2ml of same solution.
Cell Viability by dye exclusion method:-Lymphocytes (10×10 6 ) suspended in 1ml of HBSS were treated with Schiff base derivatives(5µl -500ƞg) dissolved in DMF and incubated for 60 minutes in a shaking water bath at 37 0 C.At the end of incubation, aliquot of cells was taken for viability assay by tryphan blue exclusion method. Stained and stainless cells were counted. % of viable cell calculated by using the following formula % of viable cells = number of living cells/Total number of cells × 100 Result and discussion:-Chemistry:-Benzoylation of phenols 12 , Fries rearrangement used to convert a phenyl ester to an ortho-and para-hydroxy aryl ketone iia,b.
Based on these information ligands (iva,b) were achieved by condensation reaction of substituted hydroxybenzophenone (ii) with o-phenylenediamine in presence of methanol by adopting the above method. Benzoyl phenol based cobalt(II), nickel(II), cadmium(II) and copper(II) complexes were synthesised using respective salts and organic ligands. All the complexes vM 1 -M 8 are colored, stable at room temperature, soluble in DMSO and melt with decomposition above 250°C. Unfortunately, our efforts to obtain single crystals of complexes were not successful. Therefore the ligands and their complexes were characterized on the basis of elemental analysis, 1 H NMR, IR, magnetic susceptibility measurement, electronic spectra data, colours, melting point, partial elemental analyses and molar conductivities.
The elemental analysis of the compounds is in good agreement with their proposed formulae and the molar conductance of all the complexes in DMSO shows that they are non-electrolytes 13 . Thin layer chromatographic analysis was monitored for the progress of the reactions. The yield, elemental analysis and molar conductance data of metal complexes are presented in Table 1  In the spectra of the Schiff base ligands the absorption band observed at 275-293 nm were assigned to a π→π* transition and the band at 355-398 nm were assigned due to n→π* transition associated with the azomethine chromophore (-C=N). The new bands in complexes near 457-515 nm can be assigned to L→M charge transfer band.
The magnetic moment of the complexes were measured at room temperature. The magnetic moment of the cobalt (II) lay in the range 4.50 and 4.55 BM (ivM 1 and ivM 5 ) which corresponds to 3 unpaired electrons. The solution spectra of the cobalt (II) complexes exhibited absorption in the region 290, 320, 440 (ivM 1 ) and 270, 340, 450nm (ivM 5 ). The spectra resemble that reported for octahedral complex 20 . Thus the various bands can be assigned to: 4 T 1 g → 4 T 2 g, 4 T 1 g → 4 A 2 g, 4 T 1 g→ 4 T 1 g.

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The magnetic moment of the nickel (II) lay in the range 3.11 and 3.02 BM (vM 2 and vM 6 ) which corresponds to 2 unpaired electrons. The solution spectra of the nickel (II) complexes exhibited absorption in the region 330, 380, 420, 470 (ivM 2 ) and 350, 375, 480, 490 nm (ivM 6 ). The spectra resemble that reported for octahedral complex. Thus the various bands can be assigned to: 4 T 1 g → 4 T 2 g, 4 T 1 g → 4 A 2 g, 4 T 1 g→ 4 T 1 g and L→M charge transfer respectively.
The observed magnetic moment of the Cu(II) complexes are 1.92 and 1.90 BM (vM 4 and vM 8 ) which confirms the octahedral structure of this complex [21][22][23] . For octahedral Cu(II) complexes, the expected transition is assigned to 2 Eg → 2 T 2 g transition. Due to Jahn-Teller distortions, Cu(II) complexes give a broad absorption at 615 and 695 nm (vM 4 and vM 8 ).
Cd(II) ion with d 10 electronic configuration permits a wide range of symmetries and coordination numbers. Since d 10 configuration affords no crystal field stabilization, the stereochemistry of a particular compound depends on the size and polarizing power of the M(II) cation and the steric requirement of the ligands and have no d-d transition and is diamagnetic.
Analytical, conductance and magnetic moment data of compound.     3 have showed the maximum inhibition with IC50 value of 531 µg /ml, 539 µg ml and 547 µg /ml respectively which is followed by compounds vM 4 , iva, vM 5 , vM 6 , vM 7 , vM 8 and ivb Showed inhibition with IC50 value of 563 µg /ml, 578 µg /ml, 612 µg /ml, 638 µg /ml, 655 µg /ml, 671 µg /ml and 684 µg /ml respectively. All the compounds show almost complete inhibition at less than 1µg/ml.  Antimicrobial activity:-Compounds (iva, vM 1 to vM 4 and ivb, vM 5 to vM 8 ) were tested in vitro for their antimicrobial activity against two Gram-positive and two Gram-negative bacterial strains. Commercial antibiotics such as gentamycin and flucanazole were used as standard drugs. The results were compared with standard drugs and depicted in Table 5. Among the Schiff-base derivatives (vM 1 to vM 4 and vM 5 to vM 8 ) compound vM 3 and vM 6 were showing maximum antibacterial activity than the other Schiff-base derivatives. vM 2 is potent against both Gram+ve and Gram -ve bacteria, only vM 1 and vM 2 of showed antifungal activity with the MIC of 9.9µg and 18.63µg/ml respectively. vM 4 did not show any microbial activity.

Cell viability assay:-
The cell viability assay reveals that the compounds vM 1 to M 3 and vM 5 to M 7 are found to be cyto protective in nature as the percent viability is greater than the control. The compound M 4 and ivb appears to be cytotoxic as the percent viability was lesser than the control. iva was found to be neither cytotoxic nor cytoprotective as the percent viability is similar to that of control.  The compound iva and α of ivb not only bound to the DNA but also degraded the DNA as it can be confirmed by the less intensity of the DNA. The remaining compounds are not involved in the degradation of the DNA, the same thing is also confirmed by cell viability assay.

Conclusion:-
The author has synthesized and characterized two new schiff-base ligands (iva, ivb) and eight metal complexes (vM 1 -M 8 ). These complexes have been characterized by utilizing the various physico-chemical methods. The molecular formulae of the complexes were calculated from the elemental analyses data and are in good agreement with theoretical values. The observed molar conductance of all the complexes in DMSO, magnetic susceptibility and spectral data confirm octahedral environment around the M(II) ions with N, O as donor atoms and shows that they are non-electrolytes. The presence of coordinated water molecules in all the complexes has been further confirmed from IR. The mass spectrum of the complex confirms the proposed structure. Based on these evidences the following structures have been proposed for the complexes. A series of Schiff-base derivatives, iva, M 1 -M 4 and M 5 -M 8 of were screened for their DNA binding and chelating, anti-inflammatory and antimicrobial activity. Of the ten compounds tested M 1 and M 2 found to be potent antimicrobial compound with broad specificity, M 6 and M 7 showed only antibacterial activity. The compound iva and ivb were found to have potent DNA binding and chelating ability the remaining compounds were found to be cyto protective in nature but all the compounds proven to be potent