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Antitumor , Cytotoxic and Antimicrobial Studies of A Novel Schiff Base , Ortho-Vanillin-( 1 , 2-Ethylenediimine ) Ortho-Hydroxyacetophenone and its Transition Metal Complexes
| Content Provider | Semantic Scholar |
|---|---|
| Author | Kumar, S. P. Mohan |
| Copyright Year | 2017 |
| Abstract | The Schiff base, o-vanillin-(1,2-ethylenediimine)o-hydroxyacetophenone(VEH), and its six metal complexes were synthesized, characterised and tested for their cytotoxic activities. The copper complex was found to have high IC50 value, around 48 μ/ml. Daltons Lymphoma Ascites cell (DLA cell) induced solid and Ehrliche`s Ascites Carcinoma cell (EAC cell) induced ascites tumour models were used for antitumor studies of the compounds. Copper complex administrated at different concentrations in mice inhibited the solid tumour development and increased the mean survival rate and the life span of Ascites tumour enduring mice in a concentration dependent manner. The ligand and its metal complexes were screened against C. albicans, C. Tropicalis and A. Flavus fungi and Pseudomonas aeruginosa, (PTCC 1074) Staphylococcus aureus (PTCC 1112), Escherichia coli (PTCC 1330), and Bacillus cereus (PTCC 1015) bacteria to assess their potential antimicrobial activities. The results are quite promising. It is clear from the antifungal screening data that the metal complexes are more fungitoxic than the chelating agent itself. The bacterial screening results revealed that the free ligand has more sensitivity for gram-positive than gram-negative bacteria. Keyword: o-vanillin-(1,2-ethylenediimine)o-hydroxyacetophenone (VEH); Trypan blue exclusion metod; survival rate; Micro-both dilution method; cytotoxicity; anticancer; antimicrobial and antifungal Subin Kumar K & Aravindakshan KK 272 J Pharm Chem Biol Sci, September-November 2017; 5(3):271-281 base metal complexes, a gigantic study has been made on the field of pharmacology of these types of compounds[21]. Literature review reported that the metal complexes especially transition metal metals compounds of Schiff bases have better antitumour properties, when compared to free ligand [22]. They act in mammalian cells by encumber the enzyme, ribonucleotide reductase, is an essential in the preparation of DNA precursors[23]. Wang M and Wang L F et al [24] reported that the anticancer studies of metal compounds of thiosemicarbazone derived from 3acetylumbelliferon. Among these complexes, Co(II) and Cu(II) exhibit better inhibitory effect compared to others. The present work is an extension of our previous studies and is devoted to the preparation and identification of metal complexes with a Schiff base ligand synthesized from o-vanillin, ohydroxyacetophenone and 1,2-ethylenediamine. The characterization was done based on elemental analysis, magnetic measurements, molar conductance, IR, 1H NMR and electronic spectral data. The ligand, o-vanillin-(1,2ethylenediimine)o-hydroxyacetophenone(VEH), and its copper complex were tested for their cytotoxic and antitumour activities. The ligand and its metal complexes were screened against C. albicans, C. tropicalis, A. flavus, and A. niger fungi and Pseudomonas aeruginosa, (PTCC 1074) Staphylococcus aureus (PTCC 1112), Escherichia coli (PTCC 1330), and Bacillus cereus (PTCC 1015) bacteria to evaluate their efficacious antifungal and antibacterial activities respectively. MATERIALS AND METHODS The metal salts used in this study were BDH AnalaR quality. For the preparation of the HEH, o-Vanillin, o-hydroxyacetophenone and 1,2ethylenediamine were used. Mainly chlorides, sulphates, nitrates and acetate of Cr(III), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) were selected for the preparation of metal compounds. The commercially available solvents, ethanol, methanol, chloroform, DMF, DMSO etc. were taken for the preparation, extraction and recrystalization. All the solvents except E. Merk reagent grade were purified by standard method [25]. Synthesis of the Ligand, O-vanillin-(1,2ethylenediimine)o-hydroxyacetophenone: (VEH)): A methanolic solution of o-Vanillin (0.025 mol in 25 ml) and o-hydroxyacetophenone (0.025 mol in 25 ml) were added to 1,2-ethylenediamine (0.025 mol) in minimum amount of methanol in a 250 ml round bottom flask with stirring[13]. The mixture was kept under reflux for two hours, and then the resultant yellow product was cooled in an ice bath. The product formed was filtered, washed a number of times with ethanol and finally with petroleum benzene and allowed to dried over anhydrous CaCl2. Yellowish crystalline solid; yield 80 %; m. p: 1420C; Solubility: DMSO, DMF; UV-Vis λmax: 280 nm, 420 nm; IR: ν = 1612 cm-1 (C=O), ν = 1684 cm-1 (C=N)azomethine, ν = 1518 cm-1 (C=N)azomethine, ν = 3345 cm-1 (-OH), ν = 3043 cm-1 (-NH), ν = 3115 cm-1 (Ar). (Fig.1) Fig. 1: Synthesis of Ligand *IUPAC name of the novel VEH Schiff base ligand : 2-((E)-((2-((E)-(1-(2hydroxyphenyl)ethylidene)amino)ethyl)imino)methyl)-6-methoxyphenol Subin Kumar K & Aravindakshan KK 273 J Pharm Chem Biol Sci, September-November 2017; 5(3):271-281 Formation of metal complexes: The 0.005 mol of metal salts (Cr (III), Fe (III), Co (II), Ni (II), Cu (II) and Zn (II) ) in minimum amount of ethanol was added to DMSO solution of the ligand (0.005 mol in 20 ml) in dimethyl sulphoxide (DMSO) in 1:1 molar ratio and it was kept under reflux for about 4 h. It was then cooled and allowed to evaporate. After filtering, the solid complex obtained was washed with petroleum benzene and finally with ethanol and dried over anhydrous CaCl2. (Yield: 70–80%, m.p = 250-3500C) (Fig.2). Fig.2: Synthesis of metal complexes Characterization of the Ligand, VEH The ligand (Fig.3a) and the complexes (Fig.3b) were characterized based on their elemental analysis, magnetic moment data, and IR, UV/Vis and 1H NMR spectral techniques. Fig. 3: Structures of (a). VEH and (b). Cu(II) complex of VEH Assessment of anticancer potential: The Cancer Institute (WIA) at Adayar, Chennai provided the essential EAC cell lines and Dalton’s Lymphoma Ascites (DLA) and disseminated as transplantable tumours in the peritoneal cavity of BALB/C mice. The NCCS, is a national level biotechnology, tissue engineering and tissue banking research centre, Pune, India was supplied L929 (mouse lung fibro blast) cell line for our investigation. The Swiss albino laboratory mice (20-26 g) were acquired from the Mannuthy small Animal Breeding Station, Thrissur, Kerala. They were stored in Amala Cancer Research Centre animal house, Trissur Kerala and provided standard condition of humidity and temperature with standard food (mouse chow) and water ad libitum. Institutional Animal Ethics Committee (IAEC) were given the prior permission for the all animal experiments in the present investigation and carried out strictly according to the strategy of CPCSEA established by the Animal Welfare Division, Government of India. Mouse lung fibroblasts (L929 cells) were allowed to culture in DMEM medium supplemented with FBS (10% v/v), streptomycin (100 μg/ml) and penicillin (100 μ/ml) and stored at 370C in an incubator with 5% carbon dioxide. DLA and EAC cells were perpetuated in mouse (intraperitoneal cavity) were treated for the experiment. The synthesis of the drug: To 1 ml of the dimethylsulphoxide (DMSO) added 50 milligrams of the sample (Ligand and metal complexes) and dissolve. Using the solution in vitro studies was conducted. For conducting in vivo studies, 50 mg of the sample was first dissolved in 1 ml DMSO and it was further diluted with distilled water to the desired concentration. Subin Kumar K & Aravindakshan KK 274 J Pharm Chem Biol Sci, September-November 2017; 5(3):271-281 Trypan blue exclusion method: Using DLA cells, the compounds, to be tested, were investigated for short-term in vitro cytotoxicity studies. The cancer affected tumour cells for conducting the study aspirated from the peritoneal cavity of tumour bolstering mice where washed three times using PBS (phosphate buffered saline). Trypan blue exclusion method was used for the determination of Cell viability. The suspension of viable cell (1×106 cells in 0.1 ml) was added to test tubes enclosing various concentrations of the test samples and the volume was made up to 1 ml with PBS. The test tubes labelled as control consist of the cell suspension only. These samples and control to be analysed were allowed to incubate at 370C for 3 hours. 0.1 ml of 1% Trypan blue was added to the cell suspension and placed for 2 to 4 minutes and loaded on a haemocytometer. It was observed that, the dead cells appeared in blue colour, due to the absorption blue stain of Trypan blue, while live cells did not absorb the dye. The number of each stained and unstained cells were estimated separately. Toxicity analysis Schiff base and its metal complexes: 30 laboratory Swiss albino mice were categorized into 5 groups (6 mice/group). Group 1, 2, 3 and 4 were treated with 5mg/kg, 10 mg/kg, 15 mg/kg and 20 mg/kg respectively. The group 5 was treated as control. The drug was given to the mice once in a day by intraperitoneal injection and continued for 6 weeks. The mortality rate of the animals was noted[24]. Effect of o-vanillin-(1,2-ethylenediimine)ohydroxyacetophenone copper complex Survival rate of ascites tumour enduring Swiss albino mice; Mice (female, 40 – 56 days old) weighted 24–30 g were spitted into 5 groups having 6 animals per group. Viable EAC cells 106 in 0.1 ml of PBS were administrated by injecting on the peritoneal cavity of the animal. Group 1 and 5 were treated as control and standard (cyclophosphamide) respectively and group 2, 3 and 4 treated with 5mg/kg, 10 mg/kg and 15 mg/kg respectively. Drug and standard drug cyclophosphamide were administrated by intraperitoneal injection to the animal from the first day of tumour induction [24]. The mortal rate of mice due to tumour encumbrance was recorded and percentage of increase in life span (ILS) was determined as, % ILS= (T-C/C) ×100, where T and C are mean survival of treated and control mice, respectively. On solid tumour development: Swiss albino mice (35 – 56 days old) weighing 24–28 g were splitted into five groups, each group composed of 6 animals for the above studies. DLA cells (0.1 ml of 106 cells per mouse) were administrated by in |
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