Oxidation and epimerization of epigallocatechin in banana fruits

Article abstract of DOI:10.1016/S0031-9422(99)00533-6

To examine the metabolism of proanthocyanidins in banana fruit, (-)- epigallocatechin was treated with the homogenate of the fruit flesh to yield (-)-gallocatechin and an oxidation product, 1-(3,4,5-trihydroxyphenyl)-3- (2,4,6-trihydroxyphenyl)-2-hydroxy-

Full text of DOI:10.1016/S0031-9422(99)00533-6

Phytochemistry 53 (2000) 311±316
www.elsevier.com/locate/phytochem
Oxidation and epimerization of epigallocatechin in banana fruits
Takashi Tanaka, Kouhei Kondou, Isao Kouno*
School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki, 852-8521, Japan
Received 8 July 1999; received in revised form 10 September 1999; accepted 5 October 1999
Abstract
To examine the metabolism of proanthocyanidins in banana fruit, ( )- epigallocatechin was treated with the homogenate of
the fruit ¯esh to yield ( )-gallocatechin and an oxidation product, 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)-2-
hydroxy-1-propanone. The latter product is a stable form of a key intermediate in the oxidative metabolism of ¯avan-3-ols, and
is also related to the biogenesis of A-type proanthocyanidins. In addition, treatment of the reaction mixture with o-
phenylenediamine a€orded monomeric and dimeric phenazine derivatives generated by condensation with the o-quinone form of
the oxidation product. # 2000 Published by Elsevier Science Ltd. All rights reserved.
Keywords: Musa acuminata; Musaceae; Proanthocyanidin; Oxidation; Epimerization; Epigallocatechin; Prodelphinidin
1. Introduction
the intermediates have not been chemically character-
ized. Hence, in order to clarify the oxidative mechan-
ism of ¯avan-3-ols and proanthocyanidins in plants,
we examined the oxidation of ( )-epigallocatechin in
banana fruits, and isolated a stable form of a key
metabolite. In addition, the presence of two unstable
metabolites having o-quinone structures was indicated
by isolation of their phenazine derivatives. This paper
describes the isolation and structure determination of
these compounds.
In the course of our chemical studies on insoluble
proanthocyanidins in fruits (Tanaka, Takahashi,
Kouno & Nonaka, 1994), prodelphinidin A-2 4'-
thioether was obtained by thiol degradation of insolu-
ble proanthocyanidin of banana fruit, together with
( )-epigallocatechin 4-thioether derived from the
major extension unit of the proanthocyanidin. The for-
mer product indicated the presence of extension units
having A-type (C-4 to C-8 and C-2 to O-7) linkages
(Weinges, Kaltenhuser, Marx, Nader & Nader, 1968;
Jacques, Haslam, Bedford & Greatbanks, 1974). The
A-type extension unit was supposed to be formed by
oxidation of proanthocyanidin with a B-type linkage
(C-4 to C-8) (Jacques, Opie, Porter & Haslam, 1977;
2. Results and discussion
Thiol degradation of insoluble proanthocyanidin,
which remained in plant debris of banana fruit after
extraction with aqueous acetone, a€orded three ¯avan-
3-ol thioethers: ( )-epigallocatechin 4-(2-hydro-
xyethyl)thio ether (1), ( )-epicatechin 4-(2-hydro-
xyethyl)thio ether (2), and ( )-epiafzerechin 4-(2-
hydroxyethyl)thio ether (3). Compounds 1 and 2 were
Nonaka, Morimoto, Kinjo, Nohara
& Nishioka,
1987). Although the oxidation mechanism of these
phenolic compounds by polyphenol oxidase has been
deduced from the structure of the stable metabolites,
1
identi®ed by direct comparison of the H-NMR spec-
tral data and speci®c rotation values with those of the
authentic samples obtained from persimmon fruits
* Corresponding author. Tel.: +81-958-47-1111; fax: +81-958-48-
4387.
E-mail address: ikouno@net.nagasaki-u.ac.jp (I. Kouno).
0031-9422/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00533-6

312
T. Tanaka et al. / Phytochemistry 53 (2000) 311±316
(Tanaka et al., 1994). The structure of 3 was deter-
1
probably formed by enzymatic oxidation of the B-ring
of the upper unit and subsequent addition of the C-7
hydroxyl group of the lower unit to the C-2 position
of the resulting quinonoidal structure (see Fig. 1). To
obtain the chemical evidence for this oxidative mech-
anism, the chemical conversion of ( )-epigallocatechin
(6), the major extension unit of banana proanthocyani-
din, was next examined. Thus, compound 6 was hom-
ogenized with banana fruit ¯esh, and the resulting
products were separated by Sephadex LH-20 and ODS
mined by H-NMR spectral comparison, the spectrum
was closely related to those of 1 and 2 except for the
presence of A2X2-type signals due to the p-substituted
B-ring of 3. The yields of the thioethers indicated that
the major extension unit of the insoluble proanthocya-
nidins was epigallocatechin. In persimmon fruits, the
insolubilization of proanthocyanidins was caused by
condensation with acetaldehyde, and ¯avan-3-ol
thioethers having a carbon branch originating from
acetaldehyde were isolated from the insolubilized
proanthocyanidins (Tanaka et al., 1994). However, in
this experiment, no such chemical evidence for insolu-
bilization was obtained. In addition to the monomeric
products, a thioether of the dimeric proanthocyanidin
(4) was obtained from the insoluble banana proantho-
cyanidin. The [M-H] ion peak at m/z 683 in the nega-
tive ion FABMS and two aromatic singlet signals due
to two pyrogallol-type B-rings in the 1H-NMR spec-
trum, along with signals of a hydroxyethylthio group,
suggested that 4 was a thioether of prodelphinidin
dimer. However, only one signal attributable to H-2 of
C-ring was observed at d 5.22 (H-2'), and one of the
C-2 carbons appeared at d 103.5 (C-2') in the 13C-
NMR spectrum. These ®ndings indicated that 4 is a
prodelphinidin 4-thioether having A-type linkage. This
column chromatography to a€ord compounds
(1.8%), 8 (0.2%) and 6 (48%). Most of the starting
7
material seemed to be polymerized, since the products
remained at the origin on the TLC plate. By compari-
1
son of its H-NMR data and ‰aŠ value, 7 was ident-
D
i®ed as ( )-gallocatechin, which indicated that
epimerization at C-2 position of 6 had occurred
(Kiatgrajai, Wellons, Gollob & White, 1982). The pro-
duct 8 showed an [M-H] ion peak at m/z 321 in the
negative ion FABMS spectrum, which was 16 mass
1
units larger than that of 6. The H-NMR spectrum of
8 was related to that of 6, and indicated the presence
of a pyrogallol ring ꢀd 7.27, 2H, s ), a phloroglucinol
ring ꢀd 5.98, 2H, s ), an oxygen bearing methine ꢀd
5.13, dd, J = 3, 9 Hz) and a benzylic methylene ꢀd
3.21, dd, J = 3, 14 Hz; d 2.68, dd, J = 9, 14 Hz). In
addition to signals due to the aromatic and aliphatic
parts, a conjugated carbonyl carbon signal at d 200.2
was observed in the 13C-NMR spectrum. Based on the
above evidence, the structure of 8 was concluded to be
1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)-
2-hydroxy-1-propanone. This compound can be
regarded as a stable form of an oxidation product of
6 formed by hydration of the quinone methide struc-
ture (8b) and simultaneous opening of the hemiacetal
ring (see Fig. 2). As far as we know, this is the ®rst
isolation of the intermediate generated at the ®rst
was further con®rmed by the H- and 13C-NMR spec-
1
tral comparison with those of the thioether 4a derived
from epicatechin-(4b 4 8, 2b 4 O 4 7)-epicatechin-
(4a 4 8)-epicatechin (5) (Kashiwada, Morita, Nonaka
& Nishioka, 1990). Both spectra of 4a was almost
superimposable with those of 4 except for signals aris-
ing from the B-rings.
Since banana fruit contains polyphenol oxidase cata-
lyzing the oxidation of catechols to o-quinones (Sojo,
Nunez-Delicado, Garcia-Carmona & Sanchez-Ferrer,
1998), the A-type linkage of the proanthocyanidin was

T. Tanaka et al. / Phytochemistry 53 (2000) 311±316
313
Fig. 1
step of the oxidation of ¯avan-3-ol by a polyphenol
oxidase.
result, two phenazine derivatives 9 and 10 were
1
obtained. The H-NMR spectrum of 9 was closely re-
Since 8 and related metabolites are metabolised con-
tinuously, it was presumed dicult to isolate the other
intermediates. However,the presence of the metabolites
having an o-diquinone structure (8c) was easily pre-
dictable from the structure of 8. To trap the unstable
o-diquinone metabolites, o-phenylenediamine was
added to the extract of the reaction mixture. As a
lated to that of 6, except for the appearance of signals
ꢀd 7.48, d, J = 1.5 Hz; d 7.93, br s; d 7.96, 2H, m; d
8.26, 2H, m ) due to a hydroxyphenazine moiety (rings
B, D and E) instead of the pyrogallol ring in 6. The
13C-NMR spectrum also supported the presence of the
monohydroxyphenazine moiety, and showed signals
due to A- and C-rings which were similar to those of
Fig. 2

314
T. Tanaka et al. / Phytochemistry 53 (2000) 311±316
6. Furthermore, the negative ion FABMS showed a
ion peak at m/z 376. On the basis of these spectral
M
data, the structure of this derivative was determined as
shown by 9, which was derived from o-quinone tauto-
mer 8c (Fig. 2).
Derivative 10 showed a M ion peak at m/z 680,
suggesting that this compound has a dimeric structure.
1
The H-NMR spectrum showed signals due to a phe-
nazine moiety ꢀd 8.28 and 7.95, each 2H, m ) and two
aromatic singlet signals ꢀd 7.03 and 8.21, each 1H).
The remaining signals due to two sets of A- and C-
ring protons, were similar to those of the theasinensins
C (11) and E (12), a pair of rotational isomers of an
epigallocatechin dimer isolated from semi-fermented
tea (Oolong tea) (Hashimoto, Nonaka & Nishioka,
1988). This observation suggested that 10 was a phena-
zine derivative of dimer formed by carbon-to-carbon
coupling between B-rings of 6 and 8c. The 13C-NMR
spectrum also showed signals arising from two sets of
A- and C-rings, the chemical shifts of which were simi-
lar to those of 11 and 12. In addition, six aromatic
carbon signals ‰d 108.1 (C-16'), 117.3 (C-12'), 130.3
(C-11'), 133.2 (C-14'), and two of the four signals
between 143.9 and 144.6 (C-13' and 15')] were related
to those of the B-ring of 11 and 12. Hence, the struc-
ture of this dimeric product was deduced to be as
shown by 10. Although the position of the biphenyl
bond in the phenazine moiety still remained undeter-
mined, comparison of the chemical shifts of 10 with
those of 9 suggested that it was located ortho to a hy-
3. Experimental
3.1. General
( )-Epigallocatechin was isolated from green tea
and recrystallized from water. The purity (>98%) and
absence of gallocatechin was con®rmed by HPLC (the
major
impurity
was
( )-epicatechin,
<2%).
Proanthocyanidin 5 was isolated from Dicranopteris
pedata (Kashiwada et al., 1990). Column chromatog-
raphy was performed with Sephadex LH-20 (25±100
mm, Pharmacia Fine Chemical), MCI-gel CHP 20P
(75±150 mm, Mitsubishi Chemical Industries),
Bondapak ODS (Waters) and Chromatorex ODS (Fuji
Silysia). Thin layer chromatography was performed on
precoated Silica gel 60 F254 plates (0.2 mm thick,
Merck) with benzene±EtOAC±HCO2H (1:7:1 or 1:7:2
v/v), as eluant, with constituents detected by ultra-
violet (UV) illumination and by spraying with 2%
ethanolic FeCl3 reagent, and by p-anisaldehyde-H2SO4
droxyl group. In the H- and 13C-NMR spectra, some
1
minor peaks arising from a closely related isomer were
observed, suggesting the presence of a rotational or
positional isomer. A possible mechanism of the for-
mation of 10 is shown in Fig. 2.
reagent. Analytical HPLC was performed on
a
Cosmosil 5C18-AR (Nacalai Tesque) column (4.6 mm
i.d. Â 250 mm) (mobile phase, CH3CN-50 mM H3PO4,
gradient elution from 10 to 50% CH3CN for 60 min;
¯ow rate, 0.8 ml/min, detection: UV absorption at 280
nm). Negative FABMS were recorded on a JEOL
JMX DX-303 spectrometer with glycerol as a matrix.
1H- and 13C-NMR spectra were obtained with Varian
Unity plus 500 and Varian Gemini 300 spectrometers
From the results obtained in this experiment, the
oxidation mechanism of ¯avan-3-ol in banana fruit
may be summarized as shown in Fig. 2. The outline
of the mechanism is similar to that proposed for rad-
ical oxidation of ¯avan-3-ols (Kondo, Kurihara,
Miyata, Suzuki & Toyoda, 1999). When the B-ring of
an extension unit of proanthocyanidin is oxidized in
a manner similar to the formation of 8, subsequent
addition of the C-7 hydroxyl group of the lower ¯a-
van-3-ol unit generates the A-type inter¯avan bond
(Fig. 1). Although the mechanism for the epimeriza-
tion at C-2 position is still not clear, it may be re-
lated to reduction of the quinone methide form.
Hence, the isolation of intermediate 8 and derivative
9 achieved in this experiment is important from the
viewpoint of metabolism of proanthocyanidins. In ad-
dition, isolation of derivative 10 is also of great sig-
ni®cance for understanding the mechanism of
polymerization of ¯avan-3-ols and proanthocyanidins
by polyphenol oxidase.
1
operating at 500 and 300 MHz for H and 125 and 75
MHz for 13C, respectively; chemical shifts are reported
in parts per million on the d scale with TMS as the in-
ternal standard, and coupling constants are in Hertz.
3.2. Thiol degradation of insoluble proanthocyanidin
The ripe fruit ¯esh (4.3 kg) of Musa acuminata Colla
cv. giant cavendish, imported from the Philippines,
was macerated with 1.5 l of water in a Warring blen-
der and extracted with acetone±water (4:1 v/v, 1 l).
After ®ltration, the plant debris remaining on the ®lter
paper was extracted three times with acetone±water
1.5 l, (7:3 v/v). The ®ltrate was combined and concen-

T. Tanaka et al. / Phytochemistry 53 (2000) 311±316
315
trated by rotary evaporator below 408C. The debris
3.2.2. ( )-Epiafzerechin 4-(2-hydroxyethyl)thio ether
(3)
was washed with acetone, dried in vacuo for two days,
and suspended in 10% mercaptoethanol solution (pH
2, adjusted with concentrated HCl, total volume 1 l).
The suspension was heated at 608C for one day and
kept at room temperature for three days. The mixture
was ®ltered and the ®ltrate was subjected to column
chromatography over MCI-gel CHP 20P (6.0 cm
i.d. Â 30 cm). After washing the column with water,
stepwise gradient elution with 10±70% methanol
a€orded four fractions containing FeCl3 positive com-
pounds; fr. 1 (2.86 g), fr. 2 (3.24 g), fr. 3 (3.20 g), and
fr. 4 (1.63 g). The fractions were separately subjected
to a combination of column chromatography over
Sephadex LH-20, MCI-gel CHP 20P, Bondapak ODS,
and Chromatorex ODS with water containing increas-
ing amounts of methanol to yield ( )-epigallocatechin
4-(2-hydroxyethyl)thio ether (1, 1.6 g from frs. 1 and
2), ( )-epicatechin 4-(2-hydroxyethyl)thio ether (2, 84
mg from fr. 3), prodelphinidin A-2 4-(2-hydro-
xyethyl)thio ether (4, 36 mg from fr. 3), and ( )-epiaf-
zerechin 4-(2-hydrozyethyl)thio ether (3, 4 mg from fr.
4). The acetone±water extract of the fruits was also
subjected to thiol degradation in a manner similar to
that described for the debris. MCI-gel CHP 20P col-
umn separation of the reaction mixture a€orded a
fraction containing phenolic compounds (2.4 g).
Repeated column chromatography of the fraction over
Sephadex LH-20, Chromatorex ODS, and MCI-gel
CHP 20P yielded 1 (5 mg), 2 (4 mg), and ca€eic acid
(5 mg).
Red amorphous powder, ‰aŠD+208.58 (MeOH, c
1
0.4,), H-NMR spectral data [300 MHz, (CD3)2CO +
D2O] d: 2.70±3.00 (2H, m, ±SCH2±), 3.71±4.02 (2H,
m, ±CH2OH), 4.06 (1H, br s, H-4), 4.10 (1H, br s, H-
3), 5.28 (1H, s, H-2), 5.91 (1H, d, J = 2 Hz, H-6),
6.04 (1H, d, J = 2 Hz, H-8), 6.95 (2H, d, J = 8 Hz,
H-3', 5'), 7.47 (2H, d, J = 8 Hz, H-2', 6').
3.2.3. Preparation of thioether 4a from trimer 5
A solution of 5 (38 mg) and mercaptoethanol (0.3
ml) in EtOH±0.1 M HCl (2:1 v/v 3 ml) was heated at
50 8C for 30 h. The mixture was applied to a column
of MCI-gel CHP 20P with water and eluted with 20±
40 % MeOH (stepwise gradient) to a€ord 4a (13 mg);
white amorphous powder, ‰aŠ
64.18 (MeOH, c 0.9).
D
negative ion FABMS m/z: 651 (M-H) , 1H-NMR
spectral data [300 MHz, (CD3)2CO] d: 2.70±3.01 (2 H,
m, ±SCH2±), 3.74±4.00 (2H, m, ±CH2OH), 4.11 (1H,
d, J = 2 Hz, H-3'), 4.14 (1H, d, J = 3 Hz, H-3), 4.20
(1H, d, J = 2 Hz, H-4'), 4.34 (1H, d, J = 3 Hz, H-4),
5.30 (1H, s, H-2'), 5.98, 6.08 (each 1H, d, J = 2 Hz,
H-6, 8), 6.16 (1H, s, H-6'), 6.85, 6.91 (each 1H, d, J =
8 Hz, H-15, 15'), 7.05, 7.14 (each 1H, dd, J = 2, 8 Hz,
H-16, 16'), 7.19, 7.37 (each 1H, d, J = 2 Hz, B ring
H-12, 12'); 13C-NMR spectral data [75 MHz,
(CD3)2CO] d: 28.7 (C-4), 35.3 (±SCH2±), 44.4 (C-4'),
62.6 (±CH2OH), 67.2 (C-3'), 71.0 (C-3), 77.2 (C-2),
96.1, 97.1, 97.9 (C-6, 8, 6), 99.7, 102.3 (C-10, 10),
103.6 (C-2), 106.7 (C- 8'), 115.1, 115.3, 115.6, 116.3
(C-12, 15, 12', 15'), 119.5, 120.8 (C-16, 16'), 130.2,
132.0 (C-11, 11'), 145.4, 145.6, 145.8, 146.1 (C-13, 14,
13', 14'), 151.3, 153.2, 153.8, 156.6, 157.0, 157.9 (C-5,
7, 9, 5', 7', 9').
3.2.1. Prodelphinidin A-2 4'-(2-hydroxyethyl)thio ether
(4)
Red amorphous powder, ‰aŠ
18.68 (MeOH, c
3.3. Isolation of metabolites of epigallocatechin
D
1
0.2), negative ion FABMS m/z: 683 (M-H) , H-NMR
spectral data [300 MHz, (CD3)2CO + D2O] d: 2.70±
3.01 (2 H, m, ±SCH2±), 3.71±4.00 (2H, m, ±CH2OH),
4.09 (1H, d, J = 2 Hz, H-3'), 4.12 (1H, d, J = 3 Hz,
H-3), 4.18 (1H, d, J = 2 Hz, H-4'), 4.34 (1H, d, J = 3
Hz, H-4), 5.22 (1H, s, H-2'), 5.98 (1H, d, J = 2 Hz,
H-6), 6.07 (1H, J = 2 Hz, H-8), 6.14 (1H, s, H-6'),
6.76, 6.88 (each 2H, s, H-12, 16; H-12', 16'); 13C-
NMR spectral data [75 MHz, (CD3)2CO] d: 28.7 (C-4),
35.3 (±SCH2±), 44.4 (C-4'), 62.6 (±CH2OH), 67.3 (C-
3'), 71.0 (C-3'), 77.2 (C-2'), 96.2, 97.1, 97.9 (C-6, 8,
6'), 99.7, 102.3 (C-10, 10'), 103.5 (C-2), 106.7 (C-8'),
107.2 (2C), 108.2 (2C) (C-12, 16; C-12', 16'), 129.6,
131.3 (C-11, 11'), 133.7, 133.8 (C-14, 14'), 145.7 (2C),
146.2 (2C) (C-13, 15, 13', 15'), 151.3, 153.3, 153.7,
156.5, 157.0, 157.9 (C-5, 7, 9, 5', 7', 9'). Assignments
were made by comparison with the data for the related
thioether previously described (Kashiwada et al.,
1990).
The ¯esh (81 g) of banana fruit was homogenized in
Waring blender. TLC analysis of the homogenate did
not show any components corresponding to ¯avan-3-
ols and related compounds, which were positive to ani-
saldehyde±H2SO4 reagent. An aqueous solution (5 ml)
of ( )-epigallocatechin (6, 1.0 g) was mixed with the
homogenate and stirred for 30 min at room tempera-
ture. The mixture was extracted with acetone±water
(4:1 v/v 200 ml) three times. After removal of acetone
by rotary evaporator, the extract was partitioned with
EtOAC (4Â). The EtOAC layer was concentrated and
applied to a column of Sephadex LH-20 (3.0 cm
i.d. Â 40 cm) eluting with 60% MeoH to give two frac-
tions positive to FeCl3 reagent. The ®rst fraction con-
tained polymeric products and was not separated
further. The second fraction was chromatographed on
Chromatorex ODS with water containing increasing
amounts of methanol to give ( )-gallocatechin (7, 18

316
T. Tanaka et al. / Phytochemistry 53 (2000) 311±316
mg) and a metabolite 8 (2.0 mg), along with 6 (482
mg).
4'', 5'', 6''), 135.8 (C-1'), 142.0, 144.4, 144.5, 145.2 (C-
4', 5', 1'', 2''), 152.9 (C-3'), 156.3, 157.6 (2C) (C-5, 7,
8a).
3.3.1. ( )-Gallocatechin (7)
Red amorphous powder, ‰aŠ
5.48 (MeOH, c 0.5),
3.4.2. Phenazine derivative (10)
Brown amorphous powder, ‰aŠ + 43.38(MeOH, c
D
1H-NMR spectral data [300 MHz, (CD3)2CO + D2O]
d: 2.50 (1H, dd, J = 9, 16 Hz, H-4a), 2.89 (1H, dd, J
= 6, 16 Hz, H-4b), 3.97 (1H, m, H-3), 4.47 (1H, d, J
= 8 Hz, H-2), 5.86, 6.02 (each 1H, d, J = 2 Hz, H-6,
H-8), 6.47 (2H, s, H-2', H-6').
D
0.1), negative ion FABMS m/z: 680 (M ), 1H-NMR
spectral data [300 MHz, (CD3)2CO + D2O] d: 2.12
(1H, dd, J = 4, 16 Hz, H-4 or 4'), 2.42 (1H, dd, J =
4, 16 Hz, H-4 or 4'), 2.54 (1H, d, J = 16 Hz, H-4 or
4'), 2.76 (1H, d, J = 16 Hz, H-4 or 4'), 4.11, 4.30
(each 1H, br s, H-3, 3'), 4.55, 4.98 (each 1H, s, H-2,
2'), 5.83, 5.88, 6.00, 6.02 (each 1H, br s, H-6, 8, 6', 8'),
7.04 (1H, s, H-16), 7.92±7.99 (2H, m, H-4'', 5''), 8.21
(1H, s, H-16), 8.25±8.31 (2H, m, H-3'', 6''); 13C-NMR
spectral data [75 MHz, (CD3)2CO + D2O] d: 28.9±
30.5 (C-4, 4', overlapped with solvent signal), 63.8,
64.9 (C-3, 3'), 77.3, 77.7 (C-2, 2'), 95.5, 95.6, 96.0, 96.3
(C-6, 8, 6', 8'), 99.0, 99.1 (C-10, 10'), 108.1 (C-16'),
117.3 (C-12'), 119.3 (C-16), 129.2 (C-12), 130.1 (2C),
131.1, 131.6 (C-19, 20, 21, 22), 130.3 (C-11'), 133.3 (C-
14'), 135.5 (C-11), 142.0, 143.9, 144.1, 144.5, 144.6,
146.0 (C-14, 15, 13', 15', 1'', 2''), 151.3 (C-13), 156.9,
157.0, 157.1, 157.3, 157.4, 157.6 (C-5, 5', 7, 7', 9, 9').
3.3.2. 1-(3,4,5-Trihydroxyphenyl)-3-(2,4,6-
trihydroxyphenyl)-2-hydroxy-1-propanone (8)
Red amorphous powder, ‰aŠ + 234.68(MeOH, c
D
0.8,), negative ion FABMS m/z: 321 (M-H) , 1H-
NMR spectral data [300 MHz, (CD3)2CO + D2O] d:
2.68 (1H, dd, J = 14, 9 Hz, H-3a), 3.21 (1H, dd, J =
14, 3 Hz, H-3b), 5.13 (1H, dd, J = 9, 3 Hz, H-2), 5.98
(2H, s, H-3', 5'), 7.27 (2H, s, H-2', 6'); 13C-NMR spec-
tral data [75 MHz, (CD3)2CO + D2O] d: 29.5±30.6
(C-3, overlapped with solvent signal), 74.5 (C- 2), 95.8
(C-3'', 5''), 103.9 (C-1''), 109.4 (C-2', 6'), 126.0 (C-1'),
133.9 (C-4'), 146.1 (C-3', 5'), 157.8 (C-2'', 4'', 6''),
200.2 (C-1).
3.4. Isolation of phenazine derivatives of metabolites
Acknowledgements
( )-Epigallocatechin, 6 (1.0 g) was treated with a
homogenate of fruit ¯esh (86 g) for 30 min as
described above, and the mixture was extracted with
acetone±water (4:1 v/v 200 ml) three times. The extract
was concentrated until acetone was completely
removed, and the aqueous solution (50 ml) was mixed
with a solution of o-phenylenediamine (1 g) in 20%
acetic acid±ethanol (20 ml). After stirring for 30 min,
the mixture was ®ltered and the ®ltrate was partitioned
with EtOAC (4Â). The EtOAC layer was concentrated
and separated by column chromatography as described
above to a€ord derivatives 9 (5 mg) and 10 (7 mg)
together with 6 (571 mg).
The authors are grateful to Mr. K. Inada and Mr.
N. Yamaguchi (Nagasaki University) for NMR and
MS measurements.
References
Hashimoto, F., Nonaka, G., & Nishioka, I. (1988). Chem. Pharm.
Bull, 36, 1676.
Jacques, D., Haslam, E., Bedford, G. R., & Greatbanks, D. (1974).
J. Chem. Soc. Perkin Trans, 1, 2263.
Jacques, D., Opie, C. T., Porter, L. J., & Haslam, E. (1977). J.
Chem. Soc. Perkin Trans, 1, 1637.
Kashiwada, Y., Morita, M., Nonaka, G., & Nishioka, I. (1990).
Chem. Pharm. Bull, 38, 856.
3.4.1. Phenazine derivative (9)
Red amorphous powder, ‰aŠ
61.38 (MeOH, c
Kiatgrajai, P., Wellons, J. D., Gollob, L., & White, J. D. (1982). J.
Org. Chem, 47, 2910.
D
0.5,), negative ion FABMS m/z: 376 (M ), 1H-NMR
spectral data [300 MHz, (CD3)2CO + D2O] d: 2.80
(1H, dd, J = 4, 7 Hz, H-4), 2.99 (1H, dd, J = 4, 7
Hz, H-4), 4.52 (1H, br s, H-3), 5.31 (1H, br s, H-2),
6.08 (2H, br s, H-6, 8), 7.48 (1H, d, J = 1.5 Hz, H-2'),
7.93 (1H, br s, H-6') 7.90±8.3 (2H, m, H-4'' 5''), 8.23±
8.29 (2H, m, H-3'', 6''); 13C-NMR spectral data [75
MHz, (CD3)2CO + D2O] d: 29.8 (C-4), 66.5 (C-3),
79.3 (C-2), 95.4 (C-8), 96.3 (C-6), 99.5 (C-4a), 110.1
(C-2'), 117.8 (C-6'), 130.2 (2C), 131.0, 131.6 (C-3'',
Kondo, K., Kurihara, M., Miyata, N., Suzuki, T., & Toyoda, M.
(1999). Arch. Biochem. Biophys, 362, 79.
Nonaka, G., Morimoto, S., Kinjo, J., Nohara, T., & Nishioka, I.
(1987). Chem. Pharm. Bull, 35, 149.
Sojo, M. M., Nunez-Delicado, E., Garcia-Carmona, F., & Sanchez-
Ferrer, A. (1998). J. Agric. Food Chem, 46, 4924.
Tanaka, T., Takahashi, R., Kouno, I., & Nonaka, G. (1994). J.
Chem. Soc. Perkin Trans, 1, 3013.
Weinges, K., Kaltenhuser, W., Marx, H., Nader, E., Nader, F.,
Perner, J., & Seiler, D. (1968). Justus Liebigs Ann. Chem, 711,
184.