Food Sci. Technol. Res., 9 (2), 128–133, 2003 Review

Oxidation of Tea : Chemical Structures and Reaction Mechanism

Takashi TANAKA and Isao KOUNO

Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo Machi 1-14, Nagasaki 852-8521, Japan

Received January 6, 2003; Accepted February 3, 2003

Black tea accounts for almost 80% of the world’s tea production and is the most important source of polyphenol in the world. However, little has been known about the chemistry of polyphenols due to their complexity. Since most of the black tea polyphenols are produced by enzymatic oxidation of green tea catechins, in vitro model fer- mentation experiments using purified catechins are very useful, and recently structures of some novel oxidation prod- ucts of theaflavins, black tea pigments, have been elucidated. In addition, accumulation of unstable dimer quinones of epigallocatechin and its gallate during tea fermentation has been demonstrated, and the dimer quinones are converted to theasinensins, another major polyphenol characteristic of black tea, on heating. Formation and degradation of theaflavins and epigallocatechin dimer quinones are major pathways in oxidation during tea fermentation and understanding the chemical mechanism is important in clarifying black tea polyphenols.

Keywords: black tea, tea, polyphenol, catechin, theaflavin, oxidation

1. Introduction (Bryce et al., 1970; Collier et al., 1973). More recently, Nonaka Teas are produced from the leaves of Camellia sinensis (L.) O. and Nishioka applied newly developed chromatographic tech- Kuntze (Theaceae), and usually classified by the manufacturing niques to the separation of the polyphenols of fermented teas and process into three categories of fermented (black), unfermented successfully demonstrated the structures of a number of catechin (green) and semifermented (oolong). The polyphenols of fresh oxidation products (Hashimoto et al., 1987; Hashimoto et al., tea leaves are relatively simple and composed of four major cat- 1988; Hashimoto et al., 1989; Hashimoto et al., 1992). At the echins, ()-epicatechin (1), ()-epigallocatechin (2) and their same time, chemical studies on other constituents, which com- galloyl esters (3 and 4) (Fig. 1). The polyphenol composition of prise a major part of black tea polyphenols as a whole, continued green tea is similar to that of fresh tea leaves, because steaming (Brown et al., 1969; Cattel & Nursten, 1977; Robertson & Ben- or roasting at the initial stage of green tea manufacture inacti- dall, 1983; Bailey et al., 1992; Ozawa et al., 1996). However, vates enzymes involving the oxidation and hydrolysis of chemi- they were hampered by the complexity and difficulty of purifica- cal constituents of the leaves. In contrast, the enzymes play tion, and the major part of black tea polyphenols, especially important roles in black tea manufacturing, producing the char- those with larger molecular size, still remain to be clarified. Stud- acteristic color and flavor. As for polyphenols, when fresh tea ies on biological activities of black tea are also far behind those leaves are crushed at the initial stage, the four major catechins are of green tea. Black tea accounts for almost 80% of the world’s enzymatically oxidized and the resulting quinones undergo com- tea production and is a most important source of polyphenol, plex chemical changes. Since composition of the oxidation prod- which is believed to be of benefit to human health by scavenging ucts of tea catechins is extremely complex, little has been known excess reactive oxygen species (Hertog et al., 1993; Sano et al., about the chemical structures of the major part of black tea 1995; Sesso et al., 1999). Therefore, the importance of chemical polyphenols despite many studies done by a number of groups. and biological studies on black tea polyphenols is expected to in- At the end of the 1950’s, Roberts opened up the chemistry of crease. This review summarizes the chemistry of the oxidation of black tea polyphenol by applying chromatographic techniques tea catechins related to the formation of black tea polyphenols and developing the methodology of model fermentation, in based on recent results obtained primarily from model fermenta- which selected pure tea catechins were oxidized with crude tion experiments. enzymes obtained from tea leaves (Roberts & Myers, 1959; Rob- erts, 1962). After a few years, Takino and his coworkers first suc- 2. Production and Oxidation of Theaflavins ceeded in proposing the correct structure of theaflavins, which Theaflavins (5 and its galloyl esters) are most important red- are brilliant reddish-yellow pigments with a characteristic benzo- dish-orange pigments of black tea and chemically well character- tropolone moiety (Takino et al., 1964; Takino et al., 1965). Since ized. As for biological activities, the strong enzyme inhibitions of then, a number of chemical studies on theaflavins and related theaflavins, rather than their antioxidant activities, are probably pigments have been carried out, mainly by UK researchers important, because absorption from the digestive tract and blood plasma concentration of theaflavins are extremely low compared E-mail: [email protected] to those of green tea catechins (Mulder et al., 2001). It is expect- Oxidation of Tea Catechins: Chemical Structures and Reaction Mechanism 129

Fig. 1. Oxidation of tea catechins and the formation of theaflavins and epigallocatechin dimer quinones.

ed that their strong inhibition activities against amylase (Hara & yellow pigment was established by two-dimensional NMR spec- Honda, 1990) and sucrase (Honda & Hara, 1993) in the digestive troscopic analysis and the chemical derivatization (Tanaka et al., tract reduce the rapid increase of blood sugar level after meals. 2000), and the oxidation mechanism of the formation of 7 from 5 These pigments are catechin dimers having a characteristic ben- was deduced as shown in Fig 3. In this mechanism, the epicat- zotropolone moiety produced by the condensation of a pair of echin quinone (1a) was responsible for the initial oxidation of the quinones (1a–4a) derived from dihydroxy and trihydroxy B- benzotropolone ring of 5 (Tanaka et al., 2002a). In addition, two rings of catechins (1–4) (Fig. 1). Haslam proposed an alternative new oxidation products of 5, bistheaflavin A (8) (Tanaka et al., pathway for formation of theaflavin, in which initial intermolecu- 2001) and dehydrotheaflavin (9) (Tanaka et al., 2002b) were iso- lar C-C bond formation occurred between 1 (or 3) and 2a (or lated in similar model fermentation experiments using banana 4a), based on the intermolecular - complex formation be- and tea leaf homogenates, respectively. The mechanism for the tween a catechol ring of 1 (electron rich) and a hydroxy o-quino- formation of these yellow pigments is proposed as shown in Fig. ne (electron poor) of 2a prior to the C-C bond formation 2 and 3. (Haslam, 1998). As shown in Fig. 1, the initial step producing the o-quinone 1a Besides tea, many including loquat, Japanese pear, is most important in the oxidation cascade of tea catechins. It is apple and banana are capable of synthesizing theaflavins when thought that this initial oxidation was catalyzed by polyphenol epicatechin and epigallocatechin are added, and it was suggested oxidase, which transfers two electrons to an oxygen molecule that their mechanism of theaflavin production was similar to that from the catechol B-ring. There is another oxidation enzyme, in tea fermentation (Tanaka et al., 2002a). Especially, the homo- peroxidase, which catalyzes the oxidation of polyphenols in the genates of banana and Japanese pear can be used as excellent presence of hydrogen peroxide. The importance of peroxidase in enzyme sources for a model fermentation system applicable to tea fermentation is pointed out because hydrogen peroxide is large-scale experiments, because these fruits show strong en- generated during catechin oxidation (Subramanian et al., 1999; zyme activities, and practically no oxidation products of their Nakayama et al., 2002). In our experiments, the initial oxidation own were detected on HPLC analysis. Thus, we examined pro- was probably catalyzed by , because the pres- duction of theaflavin (5) from a mixture of purified epicatechin ence of excess amounts of catalase, which decompose hydrogen (1) and epigallocatechin (2) by model fermentation mainly with peroxide, did not affect the reaction in an experiment using Japa- banana fruits. Our results were consistent with the coupled oxi- nese pears. Theoretically, peroxidase withdraws one electron dation mechanism for theaflavin formation (Fig. 1), in which the from a to initiate a radical coupling reaction. However, it enzyme preferentially oxidizes epicatechin (1) into its is known that one-electron oxidation of catechins also results in quinone 1a and in turn, the quinone 1a oxidizes epigallocatechin the production of B-ring o-quinones in various reaction condi- (2) into the quinone 2a (Robertson, 1983). In this mechanism, 1 tions (Sawai & Sakata, 1998; Kondo et al., 1999; Sawai & appeared to be oxidized very slowly compared to 2 because 1 Moon, 2000). Actually, Sang et al. (2002) reported that theafla- was regenerated by reduction of 1a. Its rapid oxidation-reduction vin monogallate was produced on treatment of 1 and 4 with per- turnover was chemically proved by a similar experiment in the oxidase and hydrogen peroxide along with a related new presence of glutathione, in which 1 was completely converted to oxidation product. The results suggested that peroxidase and glutathione adducts of 1a and, in contrast, about 70% of 2 was polyphenol oxidase result in similar products to the oxidation of unchanged (Tanaka et al., 2002a). This coupled oxidation mech- a mixture of 1–4. The following condensation reactions of o- anism can be explained by higher enzyme specificity to 1 and the quinones 1a and 2a are probably non-enzymatic, because lower oxidation-reduction potential of 2 (Roberts, 1957a). In a theaflavin can be synthesized from 1 and 2 by treatment with series of experiments, we isolated an oxidation product of 5 and inorganic oxidants, such as potassium ferricyanide (Takino et al., named it theanaphthoquinone (7) (Fig. 2). The structure of this 1964). In addition, autoxidation of 5 in a phosphate buffer of pH 130 T. TANAKA et al.

7.3 yielded a yellow pigment bistheaflavin B (10) along with the major part of these metabolites probably derive from epigal- theanaphthoquinone (7) (Fig. 2) (Tanaka et al., 2001). Bistheafla- locatechin (2) and its gallate (4), because these two catechins vin B (10) is a dimeric product of theaflavin and deduced to be together account for over 70% of the total tea catechins in fresh formed by coupling of the reduced form (7a) of theanaphtho- tea leaves. There have been a number of reports on enzymatic quinone with theaflavin-quinone (5b). The results showed that 7 and non-enzymatic oxidation of epigallocatechin and its gallate, was also formed non-enzymatically from 5. and most of them have suggested that oxidation and subsequent intermolecular condensation between two pyrogallol B-rings 3. Formation of Epigallocatechin Dimer Quinones dominantly occurred (Hashimoto et al., 1988; Tanaka et al., The concentration of theaflavins in black tea is not high. The 2001; Tanaka et al., 2002b; Valcic et al., 1999; Zhu et al., 2000). major components of the color of black tea infusions are the so- In black tea, the most important metabolites derived from 2 and 4 called , which are heterogeneous mixtures of cat- are theasinensins (11), in which pyrogallol rings of two mole- echin oxidation products and have not yet been chemically char- cules of epigallocatechin or its gallate are linked through the C-C acterized. In addition, there are many uncharacterized colorless bond. Recent publications have revealed the importance of polyphenolic compounds derived from green tea catechins. theasinensins by demonstrating some biological activities, such These chemically unknown substances may be partly produced as the induction of apoptosis (Pan et al., 2000) and the inhibition by further oxidation of theaflavins as described above. However, of squalene epoxidase (Abe et al., 2000). Originally, production

Fig. 2. Oxidation of theaflavin.

Fig. 3. Proposed mechanism for the formation of theanaphthoquinone (7) and dehyfrotheaflavin (9). Oxidation of Tea Catechins: Chemical Structures and Reaction Mechanism 131 of these dimeric compounds during tea fermentation was pre- mentation experiment yielded another known dimer (15) (Tanaka sumed by Roberts (Roberts & Myers, 1959), and after almost ten et al., 2002b), which has recently been reported as an oxidation years, their presence in black tea was confirmed chemically (Fer- product obtained by treatment of 4 with a radical initiator in an retti et al., 1968). The complete structures of theasinensins A-E organic solvent (Valcic et al., 1999) and with peroxidase-H2O2 including the absolute configuration of biphenyl bonds were (Zhu et al., 2000). Besides production of theaflavins, formation established by Hashimoto et al. (1988). They also demonstrated of these dimeric products from epigallocatechin and its gallate is that oolongtheanins (12) was derived from epigallocatechins (2 an alternative major metabolic pathway of tea catechins. Very and/or 4) via theasinensins by enzymatic oxidation. More recent- recently, we proposed a mechanism for the production of ly, we reported the structures of new dimers of epigallocatechin theasinensins, in which theasinensins are produced by the reduc- and its gallate, 13 (Tanaka et al., 2001) and 14 (Tanaka et al., tion of corresponding o-quinones at the final stage of black tea 2002b), respectively, which were produced by model fermenta- manufacture (Tanaka et al., 2002c). The experimental evidence is tion with fresh tea homogenate (Fig. 4). The same model fer- as follows: HPLC analysis of crushed tea leaves showed peaks

Fig. 4. Structures of phenazine derivative of epigallocatechin dimer quinone (6a) and oxidation products (11–15) of epigallocatechin and its gallate.

Fig. 5. HPLC profiles of fermented tea leaves. a, extract of fermented tea leaves; b, ethanol extract of fermented tea leaves after treatment with o- phenylenediamine; c, ethanol extract of fermented tea leaves after heating (90˚C, 10 min); 1, epicatechin; 2; epigallocatechin; 3, epicatechin-3-O-galate; 4, epi- gallocatechin-3-O-gallate; Q, dimer quinones; P, phenazine derivatives of dimer quinones; T, theasinensins. 132 T. TANAKA et al. arising from theaflavins along with several broad peaks attribut- their importance in the biological activities of black tea polyphe- able to quinones (peaks designated as Q in Fig. 5a), while no nols. Ozawa et al. (1996) proposed a partial structure of thearub- peaks corresponding to theasinensins were observed. After treat- igins based on chemical degradation, and their results showed ment of the crushed leaves with o-phenylenediamine in order to how complex the thearubigins are. Our present results are still far trap the o-quinones, several large peaks attributable to phenazine from establishing the chemical constituents of the most impor- derivatives of o-quinones appeared (peaks designated as P in Fig. tant polyphenol supply for humans. 5b). Chromatographic separation and subsequent spectral analy- sis revealed the structures of these phenazine derivatives as repre- sented by formula 6a (Fig. 4). The production of these deriva- References tives indicates that theasinensin quinones (6) are accumulated Abe, I., Seki, T., Umehara, K., Miyase, T., Noguchi, H., Sakakibara, J. and Ono, T. (2000). Green tea polyphenols: Novel and potent inhib- during tea fermentation (Fig. 1). The phenazine derivatives 6a itors of squalene epoxidase. Biochem. Biophys. Res. Commun., 268, were not produced when commercial black tea was treated with 767–771. the same reagent, suggesting that the dimer quinones were de- Bailey, R.G., Nursten, H.E. and McDowell, I. (1992). 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