¹H-NMR-Based Chemometrics and ¹³C-NMR Dereplication Analysis Applied to the Bioprospecting of the Yucatecan Flora: Identification of 3-O-Acetyl-Ceanotic Acid as an Inhibitor of Bacterial Virulence Factors from Colubrina yucatanensis

The emergence and reappearance of a growing number of diseases, together with the loss of biodiversity, have restructured bioprospecting methodologies when studying secondary metabolites, which continue to play a key role in the discovery of natural products with pharmacological activity. In this investigation, a combined approach using chemometric analyses of ¹H-NMR and HPLC metabolic and chromatographic profiles, respectively, and ¹³C-NMR dereplication analysis has been used to identify inhibitors of virulence factors in bacteria as part of a new approach in the fight against antimicrobial resistance. Multivariate statistical analysis was used to correlate the ¹H-NMR metabolic and HPLC chromatographic profiles of the root crude extract and semipurified fractions from Colubrina yucatanensis (M.C.Johnst.) G.L.Nesom, Rhamnaceae, with the results from their inhibition of the production of pyocyanin, proteases, and biofilm formation in a model of Pseudomonas aeruginosa. The methodology used allowed the identification of activity-associated signals and components in the ¹H-NMR metabolic and HPLC chromatographic profiles of the bioactive extract and semipurified fractions, respectively. ¹³C-NMR dereplication analysis of the semipurified bioactive fractions using the MixONat software confirmed that one of the activity-associated signals detected in the ¹H-NMR profile belonged to 3-O-acetyl-ceanotic acid. Purification and evaluation of the pure ceanotane confirmed its identification as one of the bioactive metabolites produced by C. yucatanensis. This is the first report of 3-O-acetyl-ceanotic acid as a natural inhibitor of bacterial virulence factors.