Informed Feature-Based Molecular Networking as a Complementary Approach to Identify Bioactive Phyllobilins in...

Area of Science:

• Phytochemistry
• Natural Product Chemistry
• Analytical Chemistry

Background:

• Phyllobilins are chlorophyll metabolites with potential bioactivity but are underexplored in phytochemical research.
• Complex plant matrices often obscure the identification of these valuable compounds.
• A systematic approach is needed to uncover the diversity of phyllobilins.

Purpose of the Study:

• To develop and validate a workflow for the dereplication and identification of phyllobilins in complex plant extracts.
• To enable the annotation and prioritization of phyllobilins across various plant species.
• To address a gap in the analytical profiling of bioactive plant metabolites.

Main Methods:

• The workflow integrates Reversed-Phase Multi-Layer Perceptron (RP-MPLC) fractionation, Ultra-High-Performance Liquid Chromatography with a Vacuum Ultraviolet Detector and High-Resolution Mass Spectrometry (UHPLC-VWD-HRMS2) analysis.
• Feature-based molecular networking was employed for data analysis, utilizing public MS2 spectral libraries (GNPS).
• Antioxidant activity testing and Nuclear Magnetic Resonance (NMR) spectroscopy were used for metabolite characterization.

Main Results:

• The workflow successfully generated MS2 spectra for phylloxanthobilins from *Echinacea purpurea* and uploaded them to the GNPS database.
• Several phylloxanthobilins were annotated in senescent *Salvia officinalis* leaves, a plant with a previously unknown phyllobilin profile.
• A novel phyllobilin with a malonic acid ester moiety was isolated and structurally confirmed via 1D and 2D NMR.

Conclusions:

• The presented workflow provides a scalable and effective tool for the systematic identification and annotation of phyllobilins.
• This approach facilitates the discovery of previously overlooked bioactive natural products.
• It addresses a significant challenge in natural product research, enhancing the analytical profiling of plant extracts.