Biosci. Biotechnol. Biochem., 74 (4), 875–877, 2010
Note
Epimerization of Tea Catechins under Weakly Acidic and Alkaline Conditions
y
Nobuyoshi ISHINO, Emiko YANASE, and Shin-ichi NAKATSUKA
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Received December 1, 2009; Accepted January 18, 2010; Online Publication, April 7, 2010
Tea catechins in a buffer at pH 7 with N2 replacing
O2 epimerized rapidly at 80 ꢀC with less than 10% of
oxidative side reactions and gave catechin epimers in a
50–63% yield. The epimerization of catechins with three
hydroxyl groups was faster than with two groups, and of
galloyl-free catechins was faster than catechins with a
galloyl ester.
An epimerization experiment was conducted in which
each tea catechin 1a–4a with >98% respective purity
was heated in a buffer, and the epimer of each in the
reaction mixture was identified against an authentic
sample by a reversed-phase HPLC analysis.
(ꢀ)-EC 1a in a buffer at pH 8 was heated at 80 ꢁC in a
normal atmosphere to confirm how much catechin was
degraded under alkaline conditions. During the reaction,
the amounts of 1a and 1b were decreased rapidly, and
the reaction mixture was turned brownish by catechin
oxidants. N2 gas was then introduced into the pure 1a
solution for 5 min to remove oxygen dissolved in the
buffer. The 1a buffer solution was then heated at 80 ꢁC
in the dark and was hardly discoloured by the reaction.
1H-NMR data for the reaction mixture showed that it
contained few oxidative by-products.
1a was then epimerized in buffers of various pH
value. The relative epimerization rates of 1a at 80 ꢁC for
20 min in the various buffers, based on the rate in a pH 5
buffer solution, are shown in Fig. 2A. These rates show
that the aqueous solutions of higher pH resulted in faster
epimerization, the epimerization being about 50 times
faster at pH 8 than at pH 5. Other tea catechins 2a–4a
were similarly epimerized, further validating the alka-
line-promoted reaction mechanism.
Key words: tea catechin; epimerization; catechin
epimer; introduction N2 gas
Green tea catechins like (ꢀ)-EC 1a, (ꢀ)-EGC 2a, (ꢀ)-
ECg 3a, and (ꢀ)-EGCg 4a, as shown Fig. 1, possess a
variety of biologically relevant properties which include
anti-oxidative1) and anti-viral2) activities. Freudenberg
et al.3,4) found the epimerization of catechin in 1922 and
obtained a catechin epimer in a low yield under alkaline
conditions in 1924. Many studies about catechin epime-
rization have since been carried out. It has recently been
suggested that green tea catechins in tea drinks were
epimerized during brewing, sterilisation and long-term
heating in tea shops and dispensers.5,6) Recent studies
have revealed that catechin epimers displayed more
biologically relevant activities (inhibiting cholesterol
absorption7) and exhibiting antiallergic8) and antioxida-
tive9) properties) than the original catechins. The changes
in these biological activities only depend on changes in
the configuration at C2. Investigating this epimerization
would provide better insight into the mechanism govern-
ing these biological activities; it is therefore necessary to
obtain catechin epimers in a good yield.
The epimerization mechanism shown in Fig. 1 for
catechins under alkaline conditions was proposed by
Metha and Whalley10) in 1963. However, Seto et al.11)
have developed a useful epimerization method under
weakly acidic conditions (pH 5) at 120 ꢁC for 30 min
and yielded catechin C2 epimers (ꢀ)-C 1b, (ꢀ)-GC 2b,
(ꢀ)-Cg 3b, and (ꢀ)-GCg 4b from respective tea
catechins 1a–4a in a 19–36% yield. Although catechins
are prone to oxidative degradation and polymerisation
under high-pH conditions, their epimerization should be
faster and more efficient under higher pH conditions if
oxidative side reactions can be inhibited. In addition, tea
infusions brewed with hard water (pH 7.2) have been
found to contain more catechin epimers than with
purified water (pH 5.9).12) We therefore examined an
efficient epimerization method for catechins that inhib-
ited oxidative side reactions.
Inhibiting most of the side reactions therefore enabled
the rapid epimerization of catechins in the pH 8 buffer.
Each tea catechin 1a–4a was then favourably epimer-
ized in the pH 8 buffer at 70 ꢁC. This was conducted
three times for catechins 1a–4a, the average epimeriza-
tion rate being shown in Fig. 2B. The three main factors
relating the catechin structure to the epimerization
reaction were therefore found. First, the catechins with
three electron-donating hydroxyl groups in the B ring
were epimerized faster than those with two groups
(2a > 1a and 4a > 3a). Second, the galloyl-free cat-
echins were epimerized faster than the catechins with a
galloyl ester at the 3 position that contained more acidic
Hb at the 400 position than that of Ha at the 40 position in
the B ring (Fig. 2C, 1a > 3a and 2a > 4a). Third, the
epimer ratio at equilibrium of the catechins with a
galloyl ester at the 3 position was 10% lower than that of
the galloyl-free catechins (1ab and 2ab > 3ab and
*
4ab). These factors suggest the possibility of some sort
of interaction between the B ring and galloyl group that
reduced the stability difference between tea catechins 3a
and 4a and their epimers 3b and 4b.
*
This trend was also observed in the data of Reference 11.
y
To whom correspondence should be addressed. Tel/Fax: +81-58-293-2919; E-mail: nakatsu@gifu-u.ac.jp
Abbreviations: (ꢀ)-EC, (ꢀ)-epicatechin; (ꢀ)-EGC, (ꢀ)-epigallocatechin; (ꢀ)-ECg, (ꢀ)-epicatechin gallate; (ꢀ)-EGCg, (ꢀ)-epigallocatechin
gallate; (ꢀ)-C, (ꢀ)-catechin; (ꢀ)-GC, (ꢀ)-gallocatechin; (ꢀ)-Cg, (ꢀ)-catechin gallate; (ꢀ)-GCg, (ꢀ)-gallocatechin gallate
Ishino, Nobuyoshi
