Unraveling the structure of the black tea thearubigins

Abstract

Thearubigins are the most abundant group of phenolic pigments found in black tea accounting for an estimated 60% of the solids in a typical black tea infusion. Fifty years ago the term thearubigins was first introduced and up to now the chemical nature of the thearubigins remains largely unresolved if not mysterious despite many efforts clarifying their structure. This paper summarizes some of our attempts to clarify and elucidate the chemical nature of the thearubigins, presenting for 15 commercially representative teas data obtained using combustion analysis, IR spectroscopy, NMR spectroscopy, Diffusion NMR spectroscopy, UV–vis spectroscopy, Circular Dichroism spectroscopy and atomic force microscopy, MALDI-TOF-MS and ESI-FT-ICR-MS. The thearubigin fractions from these 15 teas are remarkably similar with respect to their spectroscopic fingerprint. The data obtained are consistent with the thearubigins being structures of not more than 2000 Da with more than 5000 individual chemical entities detected that are susceptible to concentration-driven aggregation in aqueous solution, and that retain the chiral properties of the flavanols and theaflavins. By applying petrolomics-style data interpretation strategies and by developing novel data interpretation strategies a structural model for the thearubigins was developed.

Introduction

Black tea is, second only to water, the most consumed beverage on this planet with an average per head consumption of around 500 ml per day. Annual production of tea leaves reached a record high in 2008 with a global harvest of 3.749.775 Mt [1]. Production of dried tea comprises 20% green, 2% oolong and the remainder black. In January 2008, tea prices were US$1.56 kg⁻¹ and had increased to a record high in June 2008 of US$3.40 kg⁻¹[2], making black tea one of the most economically important agricultural products. Despite its importance, the majority of black tea’s chemical composition remains unresolved if not mysterious.

Black tea is produced from the young green shoots of the tea plant (Camellia sinensis), which are converted to black tea by a so-called fermentation. There are two major processes, the ‘orthodox’ and the ‘cut–tear–curl’. In both, the objective is to achieve efficient disruption of cellular compartmentation bringing phenolic compounds into contact with polyphenol oxidases and activating many other enzymes. A detailed account of the operation is beyond the scope of this paper [3] but oxidation for 60–120 min at about 40 °C before drying is representative.

The chemical composition of black tea brew can be divided into (i) a series of well characterized small molecules including alkaloids (e.g. theobromine and caffeine), carbohydrates and amino acids (including theanine), and a series of glycosylated flavonoids together accounting for 30–40% of the dry mass of a typical black tea infusion and (ii) the heterogeneous and poorly characterized polyphenolic fermentation products accounting for the remaining 60–70%. This material was originally referred to as oxytheotannin [4].

Polyphenol oxidase is the most important enzyme and the main substrates are the flavan-3-ols or catechins 1–6, especially epigallocatechin 3 and its gallate ester 1. These account for 10–25% of the dry weight of a fresh green tea leaf and are sequestered in the vacuoles of the leaf cells (representative structures shown in Chart 1). The substrates are oxidized and extensively transformed into novel dimeric, oligomeric and polymeric compounds, few of which have been fully characterized. Oxytheotannin was subsequently divided into the reddish-orange, ethyl acetate-soluble theaflavins, and the brownish water-soluble (or ethyl acetate-insoluble) thearubigins. Although first observed in 1959 [5], the term thearubigins was not introduced until 1962 [6], but even 50 years later these oligomeric and polymeric transformation products remain poorly characterized. Better progress has been made with the dimeric products, and five classes are known, the theacitrins 7, [7] the theaflavins 8[8], the theasinensins 9[9], theanaphthoquinones 10[10] and oolongintheanins 11[11] (see Chart 2).

Mechanistically the accepted hypothesis of oligomerisation of green tea polyphenols involves an initial two electron oxidation of the 1,2-diol unit of a catechin B-ring to give an ortho-quinone 12 (see Scheme 1). For this rapid oxidation step even a quantum tunneling pathway was postulated [12]. This highly electrophilic ortho-quinone 12 can react with an appropriate nucleophile, e.g. another catechin and undergo an electrophilic aromatic substitution reaction furnishing initially carbocation 13. Here the chemistry branches out and further loss of a proton leads to a theasinensin, whereas ring contraction or rearrangements leads to 7, 8, 10 or 11. In theory, the products of this first dimerisation can again act either as a substrate or co-substrate for oxidation, or as a nucleophile, to furnish more complex oligomeric polyphenolic compounds.

Reviews by Harbowy and Balentine [13] and Haslam [14] have summarized the state of knowledge on the chemical structure of the TRs, but their conclusions are somewhat speculative. It is commercially important to elucidate the chemical structure of the TRs for a variety of reasons. Structural characterization will improve the understanding of the components contributing to taste, color and shelf-life of black tea-based products, thus assisting process and product control, while facilitating the manufacture of distinctive products. Furthermore, structure elucidation of the TR fraction will facilitate the identification of chemical compounds responsible for any of the various and wide ranging beneficial biological activities and health effects, associated with the consumption of black tea and black tea-based products [15].

The TRs were originally defined by Roberts as a fraction of acidic brown phenolic pigments that form a broad streak on two-dimensional paper chromatography [16], and what is probably the same material elutes as a broad Gaussian hump from reversed phase HPLC packings [17]. Several theories have been put forward to account for the Gaussian hump including (i) a too large number of compounds to be chromatographically resolved, (ii) the presence of chelating multivalent metal cations and (iii) non-covalent interactions such as hydrogen bonding and π–π-interactions between the individual thearubigin components that collectively result in peak broadening and chromatographic problems such as partial separation followed by rapid equilibration and re-equilibration [18].

However, information about the numbers of individual compounds present in a typical thearubigin fraction is rare, with the most reliable data originating from MALDI-TOF mass spectrometric investigations counting around 200 individual mass peaks [19]. Further mass spectrometrical investigations of the thearubigins were carried out by Chen [20], Engelhardt [21], Clifford [22] and Nursten [23]. Similarly, experimental information about the content of metal cations in the thearubigin fraction are scarce and little clear experimental evidence on the extent of non-covalent interactions between individual tea polyphenols exist, although there are data suggesting hyperchromic interactions between small mass flavonoids and polymeric material during chromatography [24]. Data regarding the molecular weight distribution of the components in a typical thearubigin fraction are contradictory and controversial. While some authors suggested that the thearubigins are polymeric materials of molecular weights of up to 40,000 g/mol [25] Clifford et al. using size exclusion HPLC suggested that the molecular weight of the constituents are in the range 700–2000 g/mol with no larger compounds being present [26].

This paper summarizes our findings on the structure of the thearubigins, which are mainly based on mass spectrometry. This paper represents a written version of a presentation on the structure of the thearubigins given at the ICPH 2009 in Harrogate. For this reason many experimental detail and detailed in depth discussions on the findings are absent and beyond the scope of this article but are in the process to be published elsewhere.