Antibacterial activity guided isolation of constituents from leaves of Croton gratissimus Burch. var. gratissimus and their chemophenetic significance

Antibacterial-guided investigation of Croton gratissimus Burch. var. gratissimus (Euphorbiaceae) leaves led to the isolation and identification of fifteen compounds including one amino acid, (2R,4S)-4-hydroxy-1-methyl-pyrrolidine-2-carboxylic acid (1), two clerodane diterpenoids, crotocorylifuran (2) and 7,8-dehydrocrotocoryli-furan (3), one halimane diterpenoid, crassifolin B (15), three flavonoids, ayanin (4), vitexin (5) and isovitexin (6), two pentacyclic triterpenoids, 3(1-acetyl-12 alpha-hydroxy-oleanane-13(1,28-olide (7) and lupeol (8), one disaccharide, sucrose (9), two sterols, (1-sitosterol (10) and 3-O-(1-D-sitosterol glucoside (11), and three fatty derivatives, henicosan-1-ol (12), methyl n-pentadecanoate (13) and methyl 9-octadecenoate (14). Their structures were established by means of spectrometric and spectroscopic data and comparison with literature data. The zwitterionic form (1a, (2R,4S)-4-hydroxy-1-methylpyrrolidinium-2-carboxylate) of compound 1 was also determined by X-ray crystallography, therefore confirming the absolute configuration of 1. Esterification of ayanin (4) led to a new flavonoid derivative, 3'-(3 ''-amino-6 ''-bromobenzoyl)-5-hydroxy-3,4 ',7-trimethoxyflavone (4a). The antibacterial activity of extract, fractions and compounds was assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Gram-positive and Gramnegative bacteria. The study identifies crotocorylifuran (2) and henicosan-1-ol (12) as particularly potent bactericidal agents, and flavonoids such as ayanin (4) and vitexin (5) as effective antibacterial constituents. Mechanistically, the MeOH extract and ayanin (4) disrupt proton-pumping activity and respiratory chain dehydrogenase function, thereby impairing energy metabolism and viability. The chemophenetic significance of these compounds was also discussed.