Abstract: Owing to the biological importance of metalloelements in many biological processes, especially metabolic processes, magnesium(II) complexes were synthesized and examined for their antimicrobial and cytotoxic activities. Among the two synthesized organometalic complexes [Mg(2-ap)2, A] and [Mg(2-ab)2, C], the maximum antibacterial and antifungal activities were shown by the compound A. Among the ligands, 2-aminophenol (B) showed more antibacterial activity than 2-aminobenzoic acid (D). The minimum inhibitory concentration for the complex A against five pathogenic bacteria Streptococcus-haemolyticus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli were found in the range of 32-64 μg ml-1. The complexes were also tested for their cytotoxicity using brine shrimp lethality bioassay method and the LC50 values of the complexes A and C were found to be 5.7 and 24.3 µg ml-1 respectively. Antibacterial, antifungal and cytotoxic studies undertaken for the above compounds indicated structure-activity relationships. These metalloderivatives were more active than the parent compounds (ligands).
INTRODUCTION
Metal coordination complexes have been reported in cancer therapy for their cytotoxic activities. Platinum coordination complexes form a new class of active anticancer agents in animals and man (Rosenberg, 1977). Various tumor cell lines are now growing resistance to conventional antitumor metal coordination complexes e.g acquired cisplatin (platinum complex) resistance in some (mainly murine) preclinical tumor models (Kelland, 1993). Thinking of tumor resistance and its circumvention there remains scope for substantial improvement in the clinical utility of metal coordination complexes through the discovery of additional platinum-based complexes (or possibly alternative metals). In the continuation of this strategy of new drug discovery we have studied two new coordination complexes for their antimicrobial and cytotoxic activities.
MATERIALS AND METHODS
Sources of compounds: In the present study the compounds were synthesized according to the following procedure:
Preparation of [Mg(2-ap)2, A] about 0.3764 gm of magnesium chloride (MgCl) was dissolved in 5 ml methanol in a 50 ml beaker. A solution of 1.080 gm of 2-aminobenzoic acid was made in 5 ml methanol in a 50 ml beaker. A solution of 0.5342 gm of ethylene diamine was also made in 5 ml methanol in an another 50 ml beaker. Three solutions were mixed, stirred for half an hour at room temperature and allowed to stand for half an hour. A gray white colored precipitate was observed. The precipitate was filtered and dried in vacuum desiccators over anhydrous CaCl2.
Preparation of [Mg(2-ab)2, C] About 0.3764 gm of magnesium chloride (MgCl) was dissolved in 5 ml methanol in a 50 ml beaker. A solution of 0.87304 gm of 2-amino phenol was made in 5 ml methanol in a 50 ml beaker. A solution of 0.5342 gm of ethylene diamine was also made in 5 ml methanol in an another 50 ml beaker. Three solutions were mixed, stirred for half an hour at room temperature and allowed to stand for half an hour. A light yellow colored precipitate was observed. The precipitate was filtered and dried in vacuum desiccators over anhydrous CaCl2. The prepared complexes were characterized by IR, UV, magnetic moment, melting point, conductivity measurements and literature review.
Antimicrobial and Cytotoxicity Activity Studies: In vitro Antimicrobial screening is generally performed by disc diffusion method (Bauer et al., 1966; Rios et al., 1988) for primary selection of the compounds as therapeutic agent. Disc diffusion method is highly effective for rapidly growing microorganisms and the activities of the test compounds are expressed by measuring the diameter of the zone of inhibition. Generally, the more susceptible the organism the bigger is the zone of inhibition. The method is essentially a qualitative or semi quantitative test indicating sensitivity or resistance of microorganisms to the test materials as well as bacteriostatic or bactericidal activity of a compound (Reiner, 1982). The standard test microorganisms were collected from the Microbiology Laboratory of the Institute of Nutrition and Food Sciences (INFS), University of Dhaka, Bangladesh. The diameters of zones of inhibition produced by the compounds were compared with the standard antibiotic (Kanamycin 30 μg disc–1) and antifungal (Nystatin 50 μg disc–1) agents for their antibacterial and antifungal activities. The experiment was performed in duplicate to minimize errors.
Minimum inhibitory concentration (MIC) of a compound is defined as the lowest concentration of that compound in a medium without visible growth of the test organisms. The basic principle is the dilution tests which comprises the serial dilution of the antimicrobial agent inoculated with the organism. For the test, standard serial dilution technique (Reiner, 1982) was employed.
Brine shrimp lethality bioassay (Persoone et al., 1980; Meyer, 1982; Mclaughlin et al., 1988; Mclaughlin, 1990) is a recent development in the assay procedure of bioactive compounds which indicates cytotoxicity as well as a wide range of pharmacological activities (e.g. anticancer, antiviral, insecticidal, pesticidal, AIDS, etc.) of the compounds. Here in vivo lethality, a simple zoological organism (Artemia salina) was used as a convenient monitor for the screening.
RESULTS AND DISCUSSION
Antibacterial activity: The complexes A [Mg (2-ap)2] and C [Mg(2-ab)2] and the ligands 2-aminobenzoic acid (2-ab) and 2-amino phenol (2-ap) at a concentration of 30 μg disc–1 and 100 μg disc–1 showed antibacterial activity against four Gram positive (Sarcina lutea, Bacillus subtilis, Streptococcus-β-haemolyticus and Staphylococcus aureus ) and five Gram negative (Salmonella typhi, Shigella dysenteriae, Pseudomonas aeruginosa, Escherichia coli and Shigella flexneri) bacteria. The results are given in Table 1. From this present investigation we can conclude that when ligands bind with metals then the formed complexes show more activity than the ligands themselves. 2-amino phenol shows more activity than 2-aminobenzoic acid and this is probably due to phenolic hydroxyl group. To investigate the exact mechanism of higher antibacterial activity of 2-amino phenol than 2-aminobenzoic acid further study will be needed.
| Table 1: | In vitro antibacterial activity of the compounds A, B, C, D and Standard Kanamycin |
| Table 2: | Minimum Inhibitory Concentration (MIC) values of the compounds A, B, C and D |
| Table 3: | In vitro antifungal activity of the compounds A, B, C, D and standard Nystatin |
| Table 4: | The results of cytotoxic effect of the compounds A, B, C and D |
Minimum Inhibitory Concentration (MIC): The minimum inhibitory concentrations (MIC) of the compounds A, B, C and D were determined against four pathogenic bacteria by serial dilution technique (Reiner,1982) and the values were given in the Table 2. The MIC values of the compound A against Bacillus subtilis, Streptococcus-β-haemolyticus, Salmonella typhi and Escherichia coli were found to be 32, 32, 64 and 32 μg ml–1 respectively, for compound B 64, 64, 64 and 128 μg ml–1 respectively, for compound C 64, 32, 64 and 64 μg ml–1 respectively, for compound D 128, 64, 64 and 64 μg ml–1 respectively. From the MIC values it was found that the complex A [Mg(2-ap)2] was more potent than the complex C [Mg(2-ab)2].
Antifungal activity: The compound A, B, C and D were tested against the pathogenic fungi Candida albicans, Aspergillus niger and Aspergillus flavus at a concentration of 50 μg disc–1 for each and the result was compared with standard Nystatin 50 μg disc–1. The activity was determined after 72 h of incubation at room temperature (30°C) and the obtained result are cited in Table 3. It was observed that only the complex A [Mg(2-ap)2] is little active against the tested fungi in comparison with the standard Nystatin. Complex C [Mg(2-ab)2] and both the ligands 2-amino phenol and 2-aminobenzoic acid were inactive in the concentration of 50 μg disc–1 against the three pathogenic fungi.
These results (antibacterial and antifungal) suggest that the complex [Mg(2-ap)2] is more active than others compounds (C, B and D) which is an interesting finding.
Cytotoxicity activity: The mortality rate of brine shrimp napulii was found to increase with concentration of sample and a plot concentration versus percent mortality on graph paper gave an almost linear correlation. The median lethal concentration (LC50) of the complexes A and C was found to be 5.7 μg ml–1 and 24.3 μg ml–1 respectively (Table 4). The lowest LC50 found for the complex A, indicating that the cytotoxicity of the magnesium chelate with 2-amino phenol [Mg(2-ap)2, A] is greater than the magnesium chelate with 2-aminobenzoic acid [Mg(2-ab)2, C]. The cytotoxicity activity for the ligands 2-amino phenol (2-ap, B) and 2-aminobenzoic acid (2-ab, D) were little with high LC50 values 56μg ml–1 and 63μg ml–1 respectively but comparatively 2-amino phenol showed more cytotoxicity than 2-aminobenzoic acid. In the present investigation we can conclude that the complex [Mg(2-ap)2] may have anticancer activity as it showed more cytotoxic activity and further investigations are needed to establish it as an anticancer agent.