Secondary Metabolites of Endophytic Fungi Associated with Moringa Oleifera: Metabolite Profiling and Biological Effects on Infectious Pathogens for the Prospecting of Anti Infectives

Abstract

Endophytic fungi colonising Moringa oleifera are promising yet underexplored sources of bioactive metabolites with potential therapeutic applications. This research profiled the fungal diversity within M. oleifera tissues, optimised extraction methodologies to enhance sequential liquid-liquid extraction (LLE) recovery by adjusting cultivation parameters, and evaluated the antimicrobial properties of isolated endophytes. Additionally, the study identified anti-infective compounds through chemical characterisation and assessed their potential involvement in specific metabolic pathways through pathway mapping, thereby establishing a strong basis for the advancement of innovative anti-infective therapeutics. Leaf and twig samples from plants across various sites produced endophytic fungal communities isolated on specialised substrate media. Purified colonies were identified through combined morphological and molecular phylogenetic analyses. Optimised sequential LLE was followed by bioassay-guided screening to identify bioactive metabolite-producing organisms. Large-scale fermentation under optimised conditions enabled the recovery, fractionation, and purification of bioactive constituents using chromatographic techniques, with further identification via gas chromatography coupled with mass spectrometry, mass spectrometric analysis, and nuclear magnetic resonance spectroscopy. Twelve distinct fungal organisms were isolated, including Aspergillus flavus, Chromelosporium sp., Penicillium chrysogenum, Nodulisporium sp., Rhizopus sp., Aspergillus aculeatus, Aspergillus sp., Curvularia sp., Fusarium solani, Aspergillus niger, Penicillium notatum, and Aspergillus fumigatus, with significant variation observed across plants and collection sites. Diversity indices — including Species Richness, Simpson’s Index, Shannon Wiener Index, and Pielou’s Evenness — revealed higher fungal diversity in leaves than in twigs. During sequential LLE, semi-polar and polar solvents, such as ethyl acetate and n-butanol, produced higher extractive recoveries. However, although generally less effective at retaining bioactive metabolites, they still succeeded in extracting some active compounds when compared with a non-polar solvent like n-hexane. Organisms with shorter lag phases (< 1.5 days) exhibited higher extract recoveries, with sabouraud dextrose broth yielding optimal results compared to the GPPYSG (glucose, peptone, potassium monohydrate phosphate, yeast hydrolysate, sodium chloride, and glycerol) medium. Biological assays identified six bioactive organisms — Aspergillus flavus, Penicillium chrysogenum, Rhizopus sp., Aspergillus sp., Curvularia sp., and Aspergillus niger — with Aspergillus niger exhibiting high extract recovery and potent antibacterial and antifungal activity. Chromatographic and spectrometric analyses identified 72 volatile compounds, including hematoporphyrin, dibutyl phthalate, bis(ethylhexyl)phthalate, ginkgolide C, lycoxanthin, decanoic acid, and 3-methyl-4-propyl 2,5-furandione. Further analysis using an additional chromatography-based mass spectrometric instrument and commercial spectral libraries revealed additional variations and compounds, such as 1,3,5-trimethylbenzene, kojic acid, furfuryl alcohol, lauric acid, carvomenthone, pentyl acetate, and undecylenic acid. Identified non-volatile compounds included 4-(2-hydroxyethyl) phenol, succinic acid, 5-hydroxymethyl-2-furancarboxylic acid, fumaric acid, N-[2-(4 hydroxyphenyl) ethyl] acetamide, and 4-hydroxybenzoic acid, emphasising the metabolic adaptability of the fungal organisms and highlighting their potential as sources of bioactive metabolites. Cross-referencing these compounds with available metabolic pathway databases suggested putative biosynthetic pathways, including fatty acid biosynthesis and the biosynthesis of secondary metabolites, shedding light on their biochemical roles. The study demonstrates that fungal diversity and colonisation patterns in M. oleifera, particularly regarding the predominance of Aspergillus species, reflect distinct ecological niches within leaf and twig tissues. Notably, species such as Chromelosporium sp., Nodulisporium sp., Aspergillus aculeatus, and Curvularia sp. are relatively undocumented, presenting new avenues for research. Solvent selection, the relationship between cultivation time and recovery, and optimisation of growth conditions to enhance metabolite yield were identified as critical factors. Analysis of volatile compounds further highlighted the biochemical diversity of these fungi, revealing their potential therapeutic applications. The detection of both shared and unique compounds across different fungal extracts suggested complex biochemical interactions, which may support biotechnological and pharmacological applications. Characterisation of non-volatile metabolites from fungal extracts revealed a spectrum of bioactive compounds distinguished by their antimicrobial, antioxidant, and inflammation-modulating properties, demonstrating the metabolic versatility of Moringa associated endophytes. Significantly, this study provides the first account of 3-methyl-4 propyl-2,5-furandione, lycoxanthin, fluoxymesterone metabolite, bis(ethylhexyl)phthalate, N [2-(4-hydroxyphenyl) ethyl] acetamide, and 4-(2-hydroxyethyl) phenol from the key endophytic organism, Aspergillus niger, known for its prominent bioactive potential. This study enhances the understanding of endophytic fungi in M. oleifera, providing a robust foundation for future anti-infective drug discovery. Through fungal profiling, extraction optimisation, bioactivity screening, and metabolite analysis, Moringa-associated endophytes are accentuated as promising sources of bioactive agents for combating infectious diseases.