Unveiling the functional components and antivirulence activity of mustard leaves using an LC-MS/MS, molecular networking, and multivariate data analysis integrated approach

Abstract

Plant extracts have recently received increased attention as alternative sources of antimicrobial agents in thefight against multidrug-resistant bacteria. Non-targeted metabolomics liquid chromatography-quadrupole timeof-flight tandem mass spectrometry, molecular networking, and chemometrics were used to evaluate themetabolic profiles of red and green leaves of two Brassica juncea (L.) varieties, var. integrifolia (IR and IG) and var.rugosa (RR and RG), as well as to establish a relationship between the elucidated chemical profiles and antivirulenceactivity. In total, 171 metabolites from different classes were annotated and principal componentanalysis revealed higher levels of phenolics and glucosinolates in var. integrifolia leaves and color discrimination,whereas fatty acids were enriched in var. rugosa, particularly trihydroxy octadecadienoic acid. All extractsdemonstrated significant antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, presentingthe IR leaves the highest antihemolytic activity against S. aureus (99 % inhibition), followed by RR (84 %), IG(82 %), and RG (37 %) leaves. Antivirulence of IR leaves was further validated by reduction in alpha-hemolysingene transcription (~4-fold). Using various multivariate data analyses, compounds positively correlated tobioactivity, primarily phenolic compounds, glucosinolates, and isothiocyanates, were also identified.