Characterization and Structural Elucidation of the Complex Mixture Thearubigins in Black Tea using Advanced Analytical Mass Spectrometry
- Thearubigins are the most abundant pigments found in black tea, contributing to a wide range of heterogeneous fractions of polyphenolic oxidation products. The term thearubigins has been introduced fifty years ago but up till now their chemical nature remained unresolved and a challenge for the scientists despite many attempts and efforts made to understand their composition. Detailed structural knowledge of thearubigins is of high interest to understand their sensory properties in order to assist the manufacturing and quality of black tea as an economically important beverage. By employing a strategy in combining a series of advanced powerful mass spectrometry techniques, the chemical composition and structural elucidation of these compounds were achieved. A series of analytical mass spectrometry experiments including ESI-LC tandem mass and ESI-LC-TOF mass spectrometry, MALDI-TOF-TOF mass spectrometry, ESI-UPLC ion mobility mass spectrometry, MALDI imaging mass spectrometry were employed in combination with experimental potentiostatic studies and theoretical computational analysis. In this work, two classes of compounds were identified and a new structural hypothesis in thearubigin formation was further substantiated. The data revealed the presence of polyhydroxylated dimers of the theanaphthaquinone and theasinensin C structures, which were consistent with the polyhydroxylation hypothesis previously formulated. Furthermore, new class of peroxo-/ epoxi- compounds in the series of theasinensin A were identified, thus indicating the presence of H2O2 and its important contribution in the tea fermentation process. A series of novel trimeric and tetrameric catechins were identified. The results showed that the structures of these compounds provide for the first time experimental evidence on oxidative oligomerisation of flavan-3-ols through an oxidative coupling pathway, most notably between theaflavin/ theasinensin and oxidized flavan-3-ols. Furthermore, the regiochemistry of the new trimeric structures were suggested based on computational studies. Isomeric structures were also resolved based on the energy dimension in direct infusion MSn experiments.
An electrochemical model oxidation system was established. Theasinensins, theaflavins and their gallate esters were identified and shown as the main reaction products obtained from the electrochemical oxidation process performed, thus acting as the main precursors for further oxidation processes in thearubigin formation. Since thearubigins are one of the most complex mixtures present in nature, being composed of sets of multiple isomers, the differentiation between isomeric structure of theasinensins, proanthocyanidins B-type, and rutin (Quercetin-3-O-rutinoside) were studied according to their mobility drift times acquired and the assignment of the individual isomeric structures were based on comparison between their experimental drift times and theoretical collisional cross sections. Furthermore, polyphenolic components present in the tea leaves and their spatial distribution were located allowing a molecular mapping of the green tea constituents to be achieved. A new free matrix MS imaging method was developed for the first time, where the biochemical process occurring in a young green tea leaf has been determined. Finally, the importance of thearubigins on the health benefits by studying its inhibitory activity on nutrient cultures containing both gram positive and gram negative bacteria was highlighted.
This study characterizes the thearubigin as a complex mixture, reveals the important reaction mechanism occurring and identifies novel components, shows the main polyphenolic occurring components as main precursors in the oxidation process, demonstrates the isomerism present in thearubigin fractions, reveals the initial biochemical processing in the leaf matrix, and shows its important biological activity. In a word, this study serves a step forward for unraveling the mystery of black tea.