Untargeted and targeted chemical profiling for efficacy-directed discrimination of Hedera helix L. subspecies using HPTLC- image analysis and HPTLC/MS

Abstract

A strategy combining untargeted and targeted chemical profiling for discrimination of the different subspecies of Hedera helix L (Ivy) based on HPTLC fingerprints and antioxidant and antimicrobial activity testing followed by chemometric analysis has been implemented for the first time for the holistic profiling of bioactive secondary metabolites in the different subspecies. Principal component analysis (PCA) based on HPTLC-image analysis fingerprints of secondary metabolites loading plots were utilized for determination of chemical markers responsible for the classification which were then identified using HPTLC/MS technique as the six phenolics; caffeic acid, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, rutin and chlorogenic acid and the three saponins; alpha-hederin, hederasaponin B and hederacoside C. The identified markers were then quantified using a HPTLC-high resolution plate imaging technique for targeted profiling. Biomarkers were determined from the coefficients plot of orthogonal projection to latent structures (OPLS) model based on the secondary metabolites quantitative data together with the biological activity data which revealed that the phenolic metabolites were strongly correlated to the antioxidant and antimicrobial activity data points compared to saponins. According to targeted hierarchical clustering analysis (HCA)-heat-map, samples could be divided into three main clusters; H. helix subsp. helix cluster, H. helix subsp. rhizomatifera and H. helix subsp. hiberinica sub-clusters and H. helix subsp. poetrum and H. helix subsp. canariensis sub-clusters, and such clustering pattern was directly related to the variation in the content of bioactive phenolics among the different subspecies rather than their saponins content, providing an insight into the unsettled taxonomy of the ivy subspecies and successful precise efficacy-directed discrimination of the different samples.