Determination of total flavonoid content and antibacterial activity of solvent fractions from ‘Cacao’ (Theobroma cacao L.) leaves

Cacao is renowned for its rich phytochemical composition, especially in its pods, which exhibit significant antibacterial properties. However, the leaves remain underexplored for such potential. This study seeks to examine the antibacterial properties of cacao leaves by fractionating its ethanolic extract, identifying bioactive compounds, and evaluating their activity against common pathogens, Escherichia coli and Staphylococcus aureus, in light of the growing challenge of antimicrobial resistance. Dried and pulverized cacao leaves were macerated in 80% ethanol to extract flavonoids. The crude extract was partitioned using water, hexane, and ethyl acetate. Each fraction was subjected to qualitative phytochemical screening and quantification of flavonoid content (TFC). The fraction with the highest TFC was further tested for antibacterial activity using the disk diffusion assay at concentrations of 50, 75, and 100 µg/mL. The positive control was cefoxitin (30 µg/disc), while the negative control was 1% DMSO. All three fractions tested positive for flavonoids, glycosides, tannins, and phenols. The ethyl acetate fraction (EAF) exhibited the highest TFC at 581.67 mg QE/g. Despite its high flavonoid content, the EAF exhibited minimal antibacterial activity, with maximum zones of inhibition of 6.83 ± 0.00 mm for E. coli and 6.84 ± 0.00 mm for S. aureus at 100 µg/mL. Cefoxitin showed significantly larger zones: 20.46 ± 0.08 mm and 31.45 ± 0.03 mm, respectively. The findings underscore the need for optimized extraction and compound characterization methods. Further research is recommended to explore synergistic effects with antibiotics and investigate additional biological activities of cacao leaf extracts.