Tetrahedron Letters
Synthesis of theaflavins with Camellia sinensis cell culture and
inhibition of increase in blood sugar values in high-fat diet mice
subjected to sucrose or glucose loading
b
c
Masumi Takemoto a, , Hiroaki Takemoto , Asuka Sakurada
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a School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi-Aza Misumido, Koriyama 963-8611, Japan
b School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
c School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Theaflavin and its galloyl esters are major polyphenolic pigments of black tea. We compared the
efficiency of a variety of oxidizing enzyme systems to synthesize theaflavin and its galloyl esters. Camellia
sinensis cell culture efficiently synthesized theaflavin from epicatechin and epigallocatechin with 70%
yield and 100% conversion in 4 min. In an administration experiment performed in mice, theaflavin
inhibited the increase blood glucose levels in mice that were fed a high-fat diet and subjected to glucose
or sucrose loading in mice.
Received 6 May 2014
Revised 17 July 2014
Accepted 23 July 2014
Available online 30 July 2014
Keywords:
Theaflavin
Ó 2014 Elsevier Ltd. All rights reserved.
Theaflavin galloyl esters
Camellia sinensis cell cultures
Attenuation of blood sugar level increase
Theaflavin (TF) and its galloyl esters are the main red pigments
in black tea, and have recently been shown to exhibit various
anti-obesity,1 glucose-lowering,2 and anticancer3 effects. There-
fore, it has become recognized as potentially useful not only as a
natural coloring agent but also as a bioactive substance. However,
further research on the compound is required. TF has no galloyl
esters. Theaflavin’s galloyl esters have three different galloyl
esters: theaflavin-3-O-gallate (TF3G), theaflavin-30-O-gallate
(TF30G), and theaflavin-3,30-di-O-gallate (TFDG). Their chemical
peroxidase (HRP) is a commercially available metalloporphyrin
enzyme. We observed that Camellia (C.) sinensis cell culture is a rich
source of peroxidase (POD) enzymes.7 Therefore, we applied them to
the oxidative coupling of 2-naphthol derivatives8 and production of a
variety of biologically active compounds.7 In this Letter, we report a
method using C. sinensis cell culture for the efficient synthesis of TF
from EC and EGC with high yield.9
To investigate the synthesis of TF by oxidation of EC and EGC,
we surveyed a variety of oxidizing agents [commercial polyphenol
oxidase (PPO) (Funakoshi Co., Ltd), commercial HRP10 (Wako Co.,
Ltd), C. sinensis cell culture, Nicotiana (N.) tabacum cell culture,7
and Daucus (D.) carota cell culture7] to optimize conditions as
shown in Table 1. Synthesis of TF from EC (20 mg) and EGC
(21 mg) was performed with a stirrer. (A) 10 mg PPO was added
to a mixture of EC and EGC in 0.1 M H3PO4 buffer (pH 6, 20 ml),
(B) 10 mg HRP was added to a mixture of EC and EGC in 0.1 M
H3PO4 buffer (pH 6, 20 ml), acetone (2 ml), and 3% H2O2 (0.5 ml),
(C) 10 ml of plant cell culture was added to a mixture of EC and
EGC in 0.1 M H3PO4 buffer (pH 6, 20 ml), acetone (2 ml), and 3%
H2O2 (0.5 ml).
structures are shown below in Scheme 1.
TF and its galloyl esters (TF3G, TF30G, TFDG) are produced from
their parent catechins [epicatechin (EC), epigallocatechin (EGC),
epicatechin gallate (ECG), and epigallocatechin-3-O-gallate
(EGCG)] by polyphenol oxidase or peroxidase in tea leaves during
production of black tea leaves or in tea fermentation, and are
obtained by extraction from black tea.4,5 However, the content in
black tea is extremely low as shown in Scheme 1.6 Therefore,
it has been difficult to obtain sufficiently usable amounts of TF
and its galloyl esters simply by extraction from black tea leaves.
TF is biosynthesized from EC and EGC. TF3G is biosynthesized
from EC and EGCG. TF30G is biosynthesized from ECG and EGC and
TFDG is biosynthesized from ECG and EGCG (Scheme 1).4,5 Develop-
ment of the use of enzymes for oxidative reactions, which is a green
chemistry approach, has been increasing in recent years. Horseradish
As shown in Table 1 as oxidizing agent entry 3, EC and EGC were
converted into TF in 15 min with 48% yield as the sole product, and
the residual starting materials (EC, EGC) were absent. For the other
oxidizing agents (entries 1, 2, 4, and 5), yields of TF were low, and
the starting materials (EC, EGC) were recovered. For C. sinensis cell
culture (entry 6), a shortened reaction time led to the highest
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Corresponding author.
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.