Evaluating the Antifungal Efficacy and Bioactive Metabolites of a Traditional Polyherbal Aqueous Formulation BioCC+ against Fusarium graminearum and Fusarium verticillioides

Fungal contamination of cereal crops threatens global food security by compromising grain quality and introducing harmful mycotoxins that endanger human and animal health. In West Africa, the long-standing ancestral use of native plant extracts to manage fungal contamination provides sustainable and locally adaptable solutions. Building on this knowledge, BioCC+, a polyherbal aqueous formulation containing Azadirachta indica, Cymbopogon citratus, Allium sativum, and Capsicum frutescens, was developed to control maize contamination during storage in Côte d’Ivoire. This study evaluated the antifungal activity of BioCC+ against Fusarium graminearum and F. verticillioides followed by bioactivity-guided untargeted metabolomics to explain observed effects. Crude BioCC+ extracts inhibited F. graminearum at 12.5 mg/mL and Fusarium verticillioides at 25.0 mg/mL, with the dichloromethane extract (DCMe) showing greater antifungal activity than methanol or water extracts. Based on the observed growth inhibition, DCMe was selected for fractionation using silica gel column chromatography with the following mixtures of hexane:ethyl acetate as the mobile phase: 100:0 for F1, 75:25 for F2, 50:50 for F3, 25:75 for F4, and 0:100 for F5, as well as 100% methanol for F6. Fractionation of DCMe resulted in three active fractions (F3, F4, and F6), with F4 showing the strongest inhibition (MIC = 0.4 mg/mL) against F. graminearum. Integration of antifungal activity assays with chemometric data through multivariate, univariate, and correlation-based analysis identified 6 putative antifungal candidates, two known and 4 uncharacterized metabolites. These results provide a first step for integrating bioactivity assays with metabolomics to validate ancestral knowledge and develop community-driven practices for mitigating fungal contamination in staple crops.