Discovery and characterization of bioactive compounds from Limnophila aromatica: nevadensin and related flavonoids as potent antimicrobial agents.

Limnophila aromatica, a traditional medicinal plant, has been previously reported to possess notable antimicrobial properties. However, the specific bioactive constituents responsible for this activity remain largely unidentified. This study aimed to isolate, identify, and evaluate the antibacterial potential of compounds from a 100% ethanolic extract of L. aromatica. The crude ethanolic extract exhibited the highest antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and was subsequently subjected to Diaion HP-20 column chromatography, followed by preparative HPLC. Seven major peaks were identified using UV-Vis spectra, LC-QTOF-MS, and NMR analyses. The antibacterial efficacy of these isolated compounds was assessed using disc diffusion and broth microdilution assays against Bacillus subtilis, S. aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa. The isolated compounds were identified as norethindrone acetate, isothymusin, nevadensin, gardenin B, 5,3'-dihydroxy-7,8,2'-trimethoxyisoflavone, jasmolin II, and oleanolic acid. Nevadensin, the predominant compound (79.55%), demonstrated potent bactericidal activity against B. subtilis, S. aureus, and MRSA. Jasmolin II and gardenin B also exhibited promising antibacterial effects. Although the disc diffusion assay was limited by compound diffusion, the broth microdilution method confirmed significant MIC values, ranging from 0.59 to 2.86 mg/ml while the MBC values ranged from 0.59 to 12.72 mg/ml. This study highlights nevadensin and other flavonoids from L. aromatica as potential candidates for the development of alternative antibacterial therapies, particularly against drug-resistant Gram-positive bacteria.