Medicinal plants constitute an unfailing source of compounds (natural products –NPs) utilised in medicine for the prevention and treatment of various deceases. Theintroduction of new technologies and methods in the field of natural products chemistryenabled the development of high throughput methodologies for the chemical compositiondetermination of plant extracts, evaluation of their properties and the exploration of theirpotentials as drug candidates. Lately, metabolomics, an integrated approach incorporatingthe advantages of modern analytical technologies and the power of bioinformatics hasbeen proven an efficient tool in systems biology. In particular, the application ofmetabolomics for the discovery of new bioactive compounds constitutes an emerging fieldin natural products chemistry.In this context, Acronychia genus of Rutaceae family was selected based on its wellknowntraditional use as antimicrobial, antipyretic, antispasmodic and anti-inflammatorytherapeutic agent. Modern chromatographic, spectrometric and spectroscopic methodswere utilised for the exploration of their metabolite content following three basic axis: a)phytochemical investigation, identification of secondary metabolites and evaluation of theirbiological properties, b) development of analytical methods for identification ofacetophenones (chemotaxonomic markers of the genus) and dereplication strategies forthe chemical characterisation of extracts and c) application of metabolomic methodologies(LC-MS & NMR) for comparative analysis (between different species, origins, organs),chemotaxonomic studies (between species) and compound-activity correlations.Bioinformatics and sophisticated statistical tools were employed especially towards thelatter methodology. In particular:The application of various analytical and chromatographic techniques (LC-PDA, -ELSD, -HRMS, SFC-UV, HRNMR; chiral separation, FCPC) enabled the phytochemical exploration and isolation of numerous NPs (alkaloids, lignans, terpenoids). Among them,several acetophenones, an important and interesting class for the genus (Acronychia-typeacetophenones-AtA) from A. pedunculata were isolated and identified. Their particularstructural characteristics compelled their detailed and unabigious structural investigation. In addition, the evaluation of their pharmacological properties, including antimicrobial,cytotoxic (against human tumour cell lines) and anti-inflammatory activity, of all isolated AtAwas assessed in vitro.The small number of Acronychia-type acetophenones (AtA) despite their importantpharmacological profile, the limited information regarding their identification as well as thelow number of species investigated so far, led to the development of an identification anddereplication strategy for further exploration of AtA in complex mixtures. In particular, aUPLC-HRMS & HRMSn method was developed and applied for the detailed and accurateidentification thereof. Fragmentation patterns and certain ion motifs enabled theconstruction of decision trees for AtA identification and a MS nomenclature scheme wassuggested. Moreover, this methodology was utilised for the analysis of different A.pedunculata extracts enabling the dereplication of known AtA and the discovery ofpotentially new ones. This approach, having taken advantage of state of the art analyticalplatforms and concepts, could be incorporated for the investigation of other complexmixtures and plant extracts.NMR and UPLC-HRMS-based metabolomics (metabolic profiling) were used for theinvestigation of various Acronychia species and organs, from different geographical origin.Dereplication tools were also employed for the characterization of their chemical profiles.Statistical models were developed for the comparison of the different extracts revealingorgan- and species-specific biomarkers. Moreover, discrimination models allowed theidentification of certain compounds giving new insight into chemotaxonomic issues.Specific strategies were established for the identification of the revealed biomarkersincluding statistical integration of datasets obtained from both platforms. Statisticalcorrelation of the analytical and pharmacological data resulted in the development of a methodology for tracking bioactive metabolites in extracts without any prior purification.This approach provides a novel tool for the drug discovery in natural products field.Overall, the combination of these different approaches comprises a complete andcomprehensive study of Acronychia species and important knowledge was obtainedconcerning their metabolite composition and pharmacological profile. Metabolomics wereproved a promising tool in modern pharmacognosy and phytochemistry while betterinsight into the utilisation of new approaches in natural products drug discovery processwas obtained.
