PhD: Polyphenolic profile and antioxidant activity of mountain tea of genus Sideritis from the Balkans and characterization of metabolites of its polyphenols in urine

Jasmina Petreska Stanoeva
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia
July, 2012


Twenty one sample of Sideritis species (S. scardica, S. raeseri, S. taurica, S. syriaca and S. perfoliata ) from various sites on the Balkan Peninsula were analysed with regards to the nature and abundance of secondary metabolites, particularly phenolic compounds, which have several roles in the plant physiological processes and health beneficial effects. Also, the phenolic profile of different parts (leaf, flower and stem) of wild and cultivated grown Sideritis species from Macedonia has been analysed using water and methanol extracts to give an insight in the nature and content of various polyphenols.

A systematic method for phenolic compounds identification, using tandem mass spectrometry coupled to high performance liquid chromatography with diode array detection was developed. The characterization of each phenolic compound was performed using MS/MS product-ion analysis and common–neutral-loss analysis. The structures of flavonoid glycosides were analysed before and after alkaline hydrolysis and the effect of sugar substitution and acetylation on the flavonoids and ESI ionization and fragmentation discussed.

Three new phenylethanoid glycosides, alyssonoside, echinacoside, and forsythoside were detected for the first time. The occurrence of the hydroxycinnamic acids, phenyletahnoid glycosides and flavonoids has been investigated in taxonomically related taxa of the genus Sideritis. All taxa analysed produced very similar phenolic patterns characterized by the presence of 5-caffeoylquinic acid, forsythoside B, verbascoside, hypolaetin 7-O-[6′′′-O-acetyl]-allosyl(1→2)gluco¬side, apigenin, 7-(4"-p-coumaroylglucoside) and 4'-O-methylisoscutellarein 7-O-[6′′′-O-acetyl]-allosyl(1→2)glucoside and minor amounts of isoverbascoside, apigenin 7-O-allosyl(1→2)glucoside, isoscutellarein 7-O-allosyl-(1→2)-[6′′-O-acetyl]-glucoside hypolaetin 7-O-allosyl-(1→2)-[6′′-O-acetyl]-glucoside and 4'-O-methylhypolaetin 7-O-[6′′′-O-acetyl]-allosyl-(1→2)-[6′′-O-acetyl]-glucoside. These results show that these species are systematically very closely related. Phenylethanoid glucosides and flavonoid acetylglucosides are dominant and they present 90 % of total phenolic compounds. Principal component analysis (PCA) was performed for the nature and content of the different compounds to be correlated to the particular species of the genus Sideritis and also to the locations. The antioxidant capacity was measured with three methods (DPPH·, ABTS·+ and FRAP assays) and was linearly correlated with phenolic content. Concerning the phenolic content in the different aerial parts, leaf was the richest plant organ in phenolics followed by flower and stem with the lowest amount.

A nutrition experiment was performed for studying the bioavailability of the polyphenols from Sideritis with healthy human subjects, who consumed S. scardica decoction after which urine was collected and analysed. 63 metabolities were identified using HPLC/MSn, which were mainly glucuronide, sulphated and methylated metabolities. The identification and structure elucidation of these metabolities provided essential data for further pharmacological and clinical studies of Sideritis polyphenols bioavailability.
The application of an ion trap mass spectrometer, usually employed for identification, was systematically evaluated for quantitative analysis of various conjugated forms of flavonoids and compared to UV quantification using nine referent standards of flavonoids with six different aglycones. The analytical characteristics of the tested MS methods were shown to be comparable to UV with regards to precision and accuracy and superior for selectivity and sensitivity. Moreover, it was shown that in MS detection one flavone derivative can be quantified using other available derivatives with similar substitution pattern with regards to sugars attached and acetylated sugars whereas the nature of the aglycone is not essential.