Bioassay-guided isolation and structural characterization of antimicrobial phytoconstituents from Limonia acidissima L. leaves and evaluation of antimicrobial, anthelmintic, and cytotoxic effects: an in vitro and in silico approach.

INTRODUCTION: Rising antimicrobial and antiparasitic resistance drives the need for novel bioactive compounds from medicinal plants, a key source of therapeutic natural products. Limonia acidissima is widely used in traditional medicine, but its leaves are underexplored despite the reported pharmacological properties of other plant parts. This study evaluated the antimicrobial potential of L. acidissima leaves using a bioassay-guided approach and assessed the antimicrobial, anthelmintic, and cytotoxic properties of the isolated compounds, supported by molecular docking and in silico analyses. METHODS: Leaf extracts of L. acidissima were prepared using acetone, ethanol, and water and screened against representative microbes. The most active acetone extract was selected for bioassay-guided fractionation using silica gel chromatography and HPLC. Structural characterization was performed using UHPLC-MS/MS and NMR spectroscopy. The isolated compounds were subsequently evaluated in relevant biological assays and in silico assessment. RESULTS AND DISCUSSION: Bioassay-guided isolation yielded four compounds, tentatively identified as 2,2'-dihydroxy-4,7,4',7'-tetramethoxy-1,1'-biphenanthrene (C1), kaempferol (C2), luteolin (C3), and kaempferol-3-O-α-L-rhamnopyranoside or afzelin (C4). All isolated compounds exhibited significant antimicrobial activity against Gram-positive bacteria, with comparatively limited activity against Gram-negative bacteria and yeast. They also showed moderate anthelmintic activity and selective cytotoxicity. Docking analysis revealed that C4 showed the strongest and most consistent binding across most targets, while C3 also exhibited high affinity, particularly against microbial receptors. C2 demonstrated moderate to strong activity, especially against S. aureus, whereas C1 showed generally weak binding. Overall, kaempferol-3-O-α-L-rhamnoside (C4) and luteolin (C3) emerged as the most promising candidates for further biological evaluation. CONCLUSION: This study underscores that L. acidissima leaves are a potential source of bioactive compounds with multifunctional biological activities. Notably, C1, C3, and C4 may be reported for the first time from this plant, and several biological activities, together with molecular docking analyses, are described for this species. These findings support the traditional medicinal use of plants and reinforce the importance of a bioassay-guided approach and in silico studies in the discovery of potential therapeutic agents.