Bioassay-Guided Metabolomic Fingerprinting Analysis of Mediterranean plants using GC-MS and NMR spectroscopy

In our study, the metabolomic fingerprinting analysis of leaves and roots of eight Mediterranean plants was made by an integrated approach of GC-MS and NMR spectroscopic techniques targeted on apolar and polar metabolites respectively, following bioassay test focused on antifungal activity against two phytopathogenic fungi, Trichoderma harzianum and Aspergillus niger. The eight plant species included two perennial forbs (Dittrichia viscosa, Acanthus mollis), two grasses (Typha latifolia, Festuca drymeia), one vine (Hedera helix), one evergreen tree (Quercus ilex), and two deciduous trees (Fraxinus ornus, Fagus sylvatica), which have been used as traditional folk remedy. The research aimed at evaluating the chemical compositions of the different species both from a qualitative and a quantitative point of view, to identify the major classes of apolar and polar compounds and to integrate the spectra followed by chemometrics. The highlights of the undertaken work were: i) using an integrated approach of GC-MS and NMR spectroscopic techniques to make an intensive investigation of apolar and polar metabolites of leaves and roots of each species; ii) comparing the variation of metabolite contents in leaves and roots of eight plants simultaneously; iii) correlating internal physiologic properties (chemical profile) with the external bioactivity (antifungal activity) on some degree. The metabolic fingerprint of the Mediterranean plants showed a complex chemical composition, being specific for each species and plant tissue. Some conclusions were drew as described. Through analyzing the apolar extracts of leaf and root samples of eight species by GC-MS, combined with interpreting method of AMDIS, it was showed that apolar organic extracts were mainly composed of linear saturated fatty acids; 120 apolar metabolites, including fatty acids, n-alkanes, triterpenoids, steroids and oxygenated terpenoids were found. The exceptions were that major apolar metabolites were oxygenated terpenoids in D. viscosa leaf and unsaturated fatty acids with the richest component being linoleic acid in H. helix root, accounting for the observed antifungal activity. Triterpenoids and steroids were almost exclusively found in roots. Through analyzing the polar extracts of leaf and root samples of eight species by 1H-NMR, followd by statistical method of Principle Component Analysis (PCA), we found that extracts contained a total of 38 polar metabolites among all samples, including sugars, alkaloids, organic acids, free amino acids and aromatic compounds. Q. ilex and F. ornus contained large amounts of specific metabolites, quinic acid, quercitol and mannitol. D. viscosa and T. latifolia were characterized by a high content of aromatic compounds. The separation of A. mollis from the other species was due to the presence of betaine and sucrose in leaves and raffinose in roots. Hence, we could conclude that the research developed with the proposed approach possess the advantages of versatility and rapidity, thus making it suitable for a fast comparison among species and plant tissue types.