J. Agric. Food Chem. 1998, 46, 111−114
111
NMR An a lytica l Ap p r oa ch To Cla r ify th e An tioxid a tive Molecu la r
Mech a n ism of Ca tech in s Usin g 1,1-Dip h en yl-2-p icr ylh yd r a zyl
Yusuke Sawai*,† and Kanzo Sakata‡
National Research Institute of Vegetables, Ornamental Plants and Tea, Kanaya, Shizuoka 428, J apan, and
Research Laboratory of Marine Biological Science, Faculty of Agriculture, Shizuoka University,
Mochimune, Shizuoka 421-01, J apan
Each tea catechin was reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the reaction mixture
was subjected to NMR analysis. The antioxidation mechanism of (+)-catechin [(+)-C] is considered
to be due to the change of the B-ring to an o-quinone structure at first because of the appearance
of two carbonyl signals. This is substantiated by trapping the compound as an adduct of a 1,2-
phenylenediamine to an o-quinone. (-)-Epicatechin [(-)-EC] was also confirmed to give a similar
result, but in the case of (-)-epigallocatechin [(-)-EGC] and ethyl gallate (EG) no carbonyl signals
were observed. The antioxidation mechanisms of (-)-EGC and EG are different from those of (+)-C
and (-)-EC. This may be one of the reasons for the differences of the antioxidative activities between
the two types of catechins.
Keyw or d s: Antioxidation mechanism; NMR; DPPH; (+)-catechin; tea catechins
INTRODUCTION
Isola tion of Com p ou n d A (1). (+)-C (43.5 mg, 0.15 mmol
in acetone) was reacted with DPPH (118.2 mg, 0.30 mmol),
and after the color faded, the mixture was further reacted with
1,2-phenylenediamine (16.2 mg, 0.15 mmol). The reaction
mixture was chromatographed on a silica gel column (CHCl3/
MeOH). Each product showing Rf value higher than that of
(+)-C was subjected to NMR analysis to find a phenylenedi-
amine adduct: compound A (1) [4.0 mg; Rf ) 0.4, silica gel
GF254 (Merck), CHCl3/MeOH ) 9:1] EI-MS, m/z (%) 360 (M+,
100), 222 (95), 193 (44), 169 (53), 139 (25); HR-EIMS, m/z
360.1090 (M+, -2.0 mmu for C21H16N2O4); 13C NMR (acetone-
d6, 67.5 MHz) δ 158.0, 157.3, 156.5 (C-5,-7,-8a), 127.6-131.5
(phenazyl), 96.6 (C-6), 95.5 (C-8), 82.4 (C-2), 68.3 (C-3) (Figure
3); 1H NMR (acetone-d6, 270 MHz) δ 7.80-8.50 (phenazyl), 6.12
(H-8), 6.05 (H-6), 5.09 (H-2), 4.35 (H-3).
Tea catechins (flavan-3-ol derivatives) are known to
possess potent antioxidative activities. Several inves-
tigations on the activities of tea catechins have been
attempted (Matsuzaki and Hara, 1985; Hirose et al.,
1990). The antioxidation mechanism is considered to
be due to their radical scavenging ability. Oxidation
products of (+)-catechin [(+)-C] have been identified by
Nakayama and Hirose (1994) and Kobayashi (1994).
Recently Nanjo et al. (1996) reported that the B-ring is
important in the antioxidative activity of (+)-C. We
tried to clarify the antioxidation mechanism using a free
radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), on a
molecular basis.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
1H- a n d 13C-NMR Sp ectr a l Mea su r em en t. 1H- and 13C-
NMR spectra of the reaction mixtures and some authentic
specimens were measured with a J EOL J SX-270 FT-NMR
spectrometer. Chemical shifts are expressed as δ values using
TMS as an internal standard.
Sa m p le P r ep a r a tion for NMR Mea su r em en t. (1) Reac-
tion of (+)-C, (-)-Epicatechin [(-)-EC], and (-)-Epigallocat-
echin [(-)-EGC]. After the 1H- and 13C-NMR spectral mea-
surement of (+)-C (4.35 mg, 0.015 mmol) in acetone-d6 (0.7
mL), DPPH (11.82 mg, 0.030 mmol) was added into the NMR
cell. The purple color of DPPH faded gradually. The reaction
mixture was then subjected to 1H- and 13C-NMR analyses. (-)-
EC and (-)-EGC were treated in the same way.
First of all, (+)-C (0.015 mmol) was reacted with
DPPH (0.030 mmol) in acetone-d6 (0.7 mL) as a model
reaction of catechins. The purple color of DPPH faded
1
gradually. The reaction mixture was subjected to H-
and 13C-NMR analyses. The spectra were compared
with those of (+)-C (Porter et al., 1982). The charac-
teristic signals due to H-2′, -5′, and -6′ (δ 6.75-6.96) (B-
ring) decreased, although those signals ascribable to H-6
(δ 5.86) and H-8 (δ 6.02) (A-ring) remained unchanged.
The characteristic signals due to C-2′ (δ 115.3) and C-5′
(δ 115.7) (B-ring) decreased, although the signals as-
cribable to C-6 (δ 96.0) and C-8 (δ 95.5) (A-ring)
remained unchanged (Figure 1). These strongly suggest
that the two hydroxyl groups of the B-ring are more
important as radical scavengers among the four phenolic
hydroxyl groups of (+)-C, in good accordance with the
conclusion by Nanjo et al. (1996). They obtained
peracetylated (+)-C and its glycoside and measured
their radical scavenging ability using ESR. The impor-
tance of the B-ring was also mentioned in their paper.
(2) Reaction of Ethyl Gallate (EG). EG (2.97 mg, 0.015
mmol) in acetone-d6 (0.7 mL) was reacted with DPPH (5.91
mg, 0.015 mmol). The same treatment was repeated three
times. Total amount of added DPPH was 0.045 mmol.
* Author to whom correspondence should be addressed
(telephone +81-547-45-4101; fax +81-547-46-2169; e-mail
ysawai@tea.affrc.go.jp).
†National Research Institute of Vegetables, Ornamental
Plants and Tea.
The B-ring has two hydroxyl groups. A 2-fold amount
of DPPH (0.030 mmol) completely reacted with 0.015
‡Shizuoka University.
S0021-8561(97)00342-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/19/1998