Antimicrobial investigation

Antimicrobial investigation
Standardized disc-agar was applied with the intention of conducting antimicrobial investigation, focusing on it as a tool for in vitro assessment. In this case, it was used to evaluate the activity of selected organisms by looking at synthesized compounds from selected microorganisms. The organisms comprised of fungi, gram negative, and gram positive bacteria to include Aspergillus fumigatus (RCMB 02568), Salmonella typhimurium (RCMB 010315), Mycobactrium tuberculosis (RCMB010120), Klebsiella pneumoniae (RCMB 0010093), Escherichia coli (RCMB 010052), Bacillis subtilis (RCMB 010067), Streptococcus pneumoniae (RCMB 010010), and Staphylococcus aureus (RCMB 010027). For the gram negative bacteria, antibiotic penicillin was applied as the standard reference while gentamicin was applied for gram positive bacteria. Amphoteric B was applied for all bacteria samples as the standard antifungal control. The synthesized compounds were then collected by dissolving in DMF at concentrations of 1 mg mL-1 and 5 mg mL-1 before commencing testing.
Evaluation of the anticancer activity
The synthesized compounds (dissolved in DMF) were assessed for cytotoxic activity against colon carcinoma (HCT-116) and breast carcinoma (MCF-7) cell lines as the experimental groups. The experiment used Baby Hamster Kidney (BHK) cells as the controls since they are a normal cell line. The evaluation for the experimental and control cells focused on the ratio for inhibition of cell growth (IC50) by applying the formula: (C-T / C) x 100 = IC50.

Within the IC50 formula, the C implies mean absorbance for the untreated BHK cells as controls while the T implies the mean absorbance for the cells in the experimental group. In addition, the cell cytotoxic effect (CC50) was also evaluated as a percentage using the formula: [1-(ODt / ODc)] x 100 = CC50. Within the CC50 formula, ODt and ODc imply the absorbance values for the treated and control cells respectively. The test results revealed that minute amounts of copper can cause cytotoxicity in HCT-116 cells with higher amounts required to cause the same effect in the control cells. The significant differences noted for the cytotoxicity of the synthesized compounds in both the experiment and control cells was supported by the ratio for the concentration that caused half of the BHK cells to die against the concentration that caused a similar proportion of death in the HCT-116 cells.
Antimicrobial studies
Disc-agar diffusion approach was used to evaluate the antifungal and antibacterial activities for novel compounds. In this case, the novel compounds were collected from selected fungi, gram negative, and gram positive bacteria to include Candida albicans (RCMB 05036), Aspergillus fumigatus (RCMB 02568), Salmonella typhimurium (RCMB 010315), Klebsiella pneumoniae (RCMB 0010093), Pseudomonas aeruginosa (RCMB 010043), Escherichia coli (RCMB 010052), Bacillis subtilis (RCMB 010067), Streptococcus pneumoniae (RCMB 010010), and Staphylococcus aureus (RCMB 010027). Standard references were also used whereby amphoteric B was applied for the case of standard antifungal reference control sample, gentamicin applied was applied for gram negative bacteria sample, and antibiotic ampicillin applied for gram positive bacteria sample.
Evaluation was conducted for Mycobactrium tuberculosis (RCMB 010126) strain by focusing on the anti-mycobacterial activity of its synthesized compounds. The strain was acquired from Al-Azhar University’s Regional Center for Mycology and Biotechnology located in Cairo, Egypt. In addition, the reference drugs used were pyrazinamide and isoniazide. The evaluation applied microplate Alamar Blue solution, carried out in clear-bottomed, black microplates with 96 wells. Using clear-bottomed black mucroplates was based on a need to minimize background effects since any changes to the plate contents would easily be seen and contrasted. The wells on the perimeter were packed with sterile water to preclude the experimental wells from being dehydrated. The dilution was first conducted using dimethyl sulfoxide before being subjected to twofold dilution within the wells. M. tuberculosis inoculum in the quantity of 0.1 ml of 105 CFU/mL was then added to the experimental wells. The control wells were set up to contain only the bacteria. Next, incubation for the microplates was conducted at 37°C. On the fourth day of incubation, 12.5 µL of 20% Tween 80 and 20 µL of Alamar Blue assay were added to the microplates as reagents before being incubated for an additional 24 hours at 37°C. Finally, the contents of the plates were subjected to absorbance readings at 590 nm and the results recorded. The absorbance readings were evaluated for percentage inhibition defined as the average of experiment wells divided by the average of the control wells multiplied by 100. Additional evaluation of the results was conducted by presenting the visual MICs, defined as the lowest concentration of the drugs capable of preventing a change in color.
Antitumour activity and Cytotoxicity assay
Cytotoxic and anticancer activities testing for the synthesized compounds was conducted against HEPG-2, MCF-7, and HCT-116 cell lines. This included the determination of ratio for inhibition of cell growth (IC50) by applying the formula (C-T/C)x100=IC50 whereby the C implied mean absorbance for the untreated BHK cells as controls while the T implied the mean absorbance for the cells in the experimental group. In addition, the cell cytotoxic effect (CC50) was also evaluated as a percentage using the formula [1-(ODt/ODc)]x100=CC50 whereby ODt and ODc implied the absorbance values for the treated and control cells respectively.
Antimicrobial activity
Antimicrobial activity evaluations was also conducted for the metal ion complexes produced by fungi, gram negative, and gram positive bacteria to include Candida albicans (RCMB 05036), Aspergillus fumigatus (RCMB 02568), Pseudomonas aeruginosa (RCMB 010043), Klebsiella pneumoniae (RCMB 0010093), Escherichia coli (RCMB 010052), Bacillis subtilis (RCMB 010067), Streptococcus pneumoniae (RCMB 010010), and Staphylococcus aureus (RCMB 010027). Some of the compounds showed high antibacterial activity against selected fungi and bacteria. Firstly, compounds 22 and 25 showed high antibacterial activity against C. albicans, A. fumigatus, B. subtilis, K. pneumoniae, S. pneumoniae, E. coli, S. aureus, and S. Typhimurium. Secondly, compound 21 showed high activity against K. pneumonia. Finally, compound 24 exhibited high activity against Mycobacterium tuberculosis.
In vitro anticancer studies
New metal complexes exhibited some activity against anti-human liver cancer (HEPG-2), anti-human colon cancer (HCT-116), and anti-human breast cancer (MCF-7) cell lines. In this case, copper metal complex displayed significant activity against HEPG-2 cell line at SI value of 5.30 when compared to MTX that produced SI value of 4.46 as a reference drug. In addition, copper and silver metal complexes displayed significant activity against HCT-116 at SI value of 28.33 and 36.20 respectively when compared to MTX that produced SI value of 4.46 as a reference drug. Also, copper and silver metal complexes displayed significant activity against MCF-7 at SI value of 30.55 and 39.78 respectively when compared to MTX that produced SI value of 5.37 as a reference drug. As such, copper and silver cell lines showed activity against cancer cell lines.