A new Schiff base of o-phenylenediamine and m-hydroxybenzaldehyde was synthesized by condensation reaction. The Schiff base and its metal complexes of Co(II) and Ni(II) were then characterized spectroscopically. The complexes were tested for their antimicrobial properties using six selected micro-organisms, viz: (1) Staphylococcus aereus, (2) Pseudomonas aeruginosa (3) Escherichia coli (4) Enterococcus faecalis (5) Klebsiella Pneumonia and (6) Candida albicans. Results showed important antimicrobial activities which are believed to be beneficial and could be applied safely in the treatment of infections caused by any of these bacteria.
Coordination compounds also known as complexes are molecules that possess a metal centre that is bound to ligands (ions, or molecules that donate electrons to the metal)1-3. These complexes can be neutral or charged. When the complex is charged, it is stabilized by neighbouring counter-ions4-6. A coordination complex is a product of a Lewis acid-base reaction in which neutral molecules or anions (ligands) bond to a central metal atom (or ion) by coordinate covalent bonds. Coordination compounds are very useful in biological systems as they play vital role in disease treatment and control8-12.
A Schiff base complex is a coordination compound formed by a Schiff base ligand and a metal. The development in the field of bio-inorganic chemistry has increased the interest in Schiff base complexes, since it has been recognized that many of these complexes may serve as models for biologically important species13. Owing to the antimicrobial efficacy of Schiff base, it has been used over the years in the manufacture of antibiotics; however due to constant abuse of antibiotics, many strains of bacteria have grown resistance to most available brands of the complex13-16. Therefore, researches on potential metallated Schiff bases as antimicrobial agents are necessary in manufacturing novel agents that can overcome the growing menace of bacteria resistant drugs and the attendant deaths.
A schiff-base is a molecule that contains an imine moiety with an alkyl or aryl substituent attached to the imine nitrogen atom17,18; it is named after the founder Hugo Schiff. Typical conditions for imine formation require a protic solvent that is sufficiently dry to prevent subsequent hydrolysis of the newly formed imine bond. Generally, these condensation reactions proceed smoothly, although some reactants (usually as a result of electronic effects) can require forcing conditions such as heating to reflux in a high boiling solvent to remove water as the by-product. The electrophilic carbon atoms of aldehydes and ketones can be targets of nucleophilic attack by amines. The end result of this reaction is a compound in which the C=O double bond is replaced by a C=N double bond. This type of compound is known as an imine or Schiff base. A schematic of Schiff base formation is shown in figure 1. As can be seen from this figure, the orientation of the lone pair on the nitrogen atom makes it able to participate in donation into the appropriate metal ion. This donation means that a large array of transition metal coordination complexes can be prepared.
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Figure 1: Schematic of formation of a Schiff base by amine and aldehyde reactions.
The aim of this study is to investigate the antimicrobial properties of some synthesized Schiff base metal complexes using spectroscopic techniques and biochemical assay.
Synthesis of Schiff Base Ligand and Metal Complexes
0.50 g of o-phenylenediamine was dissolved in 10 ml of distilled ethanol inside a flat bottom flask and stirred on a magnetic stirrer until a homogenous solution was obtained. 1.242 g of 3- hydroxybenzaldehyde was dissolved in 10 ml of distilled ethanol and introduced dropwise into the solution in the flat bottom flask. The solution was allowed to reflux for 3 hours during which the product was obtained; thin layer chromatographic technique was used to monitor the progress of the reaction. The product was allowed to cool, then filtered, washed with ethanol and dried. The orange product weighed 1.0272 g, which equalled 70.2% yield.
Two separate solutions, each consisting of 0.1000 g of the Schiff base dissolved in 10 ml of distilled ethanol was stirred on a hot plate until a homogenous solution was obtained. A 0.0684 g of CoCl2.6H2O or 0.0683 g of NiCl2.6H2O dissolved in 10 ml of ethanol was added dropwise to the Schiff base solution. Each mixture could reflux for about 3 hours during which a colour change and precipitation was observed. The product was left to cool to room temperature, and then filtered using a moist filter paper and a funnel. The products were dried in a silica gel desiccator for 2 days and the respective weights of 0.0942 and 0.0852 g of Co(II) and Ni(II) Schiff base complexes were obtained.
Fourier Transform Infrared (FTIR) and UV/VIS Spectroscopy
FTIR was performed with Buck scientific M530 USA. The instrument is equipped with a detector of deuterated triglycine sulphate and beam splitter of potassium bromide. The software of the Gram A1 was used to obtain the spectra and to manipulate them. An approximately 1.0 g of each sample was used. During measurement, FTIR spectra was obtained at frequency regions of 4,000 – 600 cm-1 and co-added at 32 scans and at 4 cm-1 resolution. UV-visible spectrophotometric analysis was conducted on the sample using a UV-visible spectrophotometer (APEL 3000UV) with a slit width of 2 nm, using a 10 mm cell at room temperature. The extract was examined under visible and UV light in the wavelength ranging from 300 to 800 nm.
The broth dilution method was used to test for the antimicrobial activities of the Schiff base complexes against six microorganisms: staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa, escherichia coli, aspergillus niger and candida albicans. 23 g of the broth was dissolved in 1000 ml of distilled water and placed in multiple microtiter plates. Varying concentrations of the Schiff base complexes and the microorganisms to be tested were then added to the plate. The plate was then heated at 37 oC for 24 h (bacteria) and at 25 oC for 48h (fungi); the pH of the media was maintained between 7.2 to 7.4. Following the allotted time, the plate was removed and checked for bacterial growth.
RESULTS AND DISCUSSION
Infrared and UV/VIS Spectroscopy
As can be seen in figure 2a, the band at 3300 cm-1 was assigned to ʋO-H in the Schiff base ligand. The band was absent in the spectra of Ni and Co complexes, indicating the participation of the phenol oxygen in the chelation. The ʋC=N of azomethine in the free ligand was observed at 1650 cm-1 which is characteristic of azomethine absorption. This band was shifted to lower frequencies (550-680 cm-1) in the metal complexes, indicating coordination of the nitrogen atom of Schiff base to the metal in the complexes via M-N bonds. The ʋC-N stretching vibration, which appeared at 1312 cm-1 in the Schiff base underwent a shift towards higher frequency (1332-1335 cm-1) in the complexes, confirming the involvement of oxygen in coordination to the metal ions. The C-N band at 1275 cm-1 was observed for the Schiff base while for the complexes it occurred in the range 1281-1289 cm-1.
In Figure 2b, the absorption of the metal complexes showed only one distinct peak, however for the Schiff base, the bands at 332 and 395 nm in the Uv/Vis spectra was assigned to the π-π* of phenyl ring and n- π* of (H-C=N) transitions respectively.
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Figure 2 (a) FTIR spectra of the synthesized Schiff base and its meal complexes, and (b) UV/Vis spectra of synthesized the Schiff base and its meal complexes
Zones of Inhibition and Minimum Inhibitory Concentration (MIC)
The zone of inhibition test (Kirby-Bauer test) is a qualitative method used to measure antibiotic resistance and to test the ability of antimicrobial agents to inhibit bacterial growth. The zone of inhibition is a circular area around the spot of the antibiotic in which the bacteria colonies do not grow. The zones of inhibition of the samples and the control were measured and recorded in the table 1. As can be seen from this table a larger zone of inhibition of the metal complexes means that they are more potent antimicrobial agents than the uncomplexed Schiff base.
The minimum inhibitory concentration (MIC) which represents the lowest concentration of an antimicrobial agent that will inhibit the visible growth of microorganisms after overnight incubation is shown in table 2. Lower MIC value indicates that less of the drug is required in order to inhibit growth of the organism; therefore, drugs with lover MIC scores are more effective antimicrobial agents19-20. Antimicrobial agents with MIC values of 8000μg/l and less are considered active. As can be seen in table 2, Synthesized nickel metal complex [Ni(HL)] exhibited better bactericidal activity against E. coli, S . auerus and A. niger. On the other hand [Co(HL)] and the uncomplexed Schiff base [HL] showed similar MIC scores, although their zones of inhibition differ.