Macrocyclic ellagitannin dimers, cuphiins D1 and D2, and accompanying tannins from Cuphea hyssopifolia

Article abstract of DOI:10.1016/S0031-9422(98)00512-3

Two new ellagitannin dimers, cuphiins D₁ and D₂, and six known compounds including oenothein B and woodfordin C have been isolated from the aerial part of Cuphea hyssopifolia (Lythraceae). Macrocyclic structures of the new tannins were elucidated based on chemical and spectral evidence.

Full text of DOI:10.1016/S0031-9422(98)00512-3

PERGAMON
Phytochemistry 50 (1999) 307±312
Macrocyclic ellagitannin dimers, cuphiins D1 and D2, and
accompanying tannins from Cuphea hyssopifolia
Lih-Geeng Chena, Kun-Ying Yena, Ling-Ling Yanga, *, Tsutomu Hatanob,
Takuo Okudab, Takashi Yoshidab
aDepartment of Pharmacognosy, Graduate Institute of Pharmaceutical Sciences, Taipei Medical College, Taipei 110, Taiwan
bFaculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
Received 20 May 1998; accepted 29 June 1998
Abstract
Two new ellagitannin dimers, cuphiins D1 and D2, and six known compounds including oenothein B and woodfordin C have
been isolated from the aerial part of Cuphea hyssopifolia (Lythraceae). Macrocyclic structures of the new tannins were elucidated
based on chemical and spectral evidence. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Cuphea hyssopifolia; Lythraceae; Ellagitannin; Cuphiin D1; Cuphiin D2; Macrocyclic structure
1. Introduction
2. Results and discussion
The aerial part of C. hyssopifolia was homogenized
in 70% aq. Me2CO. The concd aq. soln was parti-
tioned with CH2Cl2. The water layer was then sub-
jected to repeated column chromatography over
Toyopearl HW-40 and MCI-gel CHP-20P to yield
myricitrin and seven hydrolyzable tannins. Among
them, ®ve were identi®ed as 1,2,3,6-tetra-O-galloyl-b-
D-glucose (1), 1,2,3,4,6-penta-O-galloyl-b-D-glucose (2),
tellimagrandin II (3), woodfordin C (4) (Kadota et al.,
1990; Yoshida et al., 1990) and oenothein B (5)
(Hatano et al., 1990). The remaining two were found
to be new dimers and named cuphiins D1 (6) and D2
(7).
Cuphea hyssopifolia Humb. Bompl. et Kunth
(Lythraceae), a small shrub native to Mexico and
Guatemala, is cultivated in Taiwan as a horticultural
plant. Some species of the genus Cuphea have been
used for treating stomach disorders, syphilis, gonor-
rhea, cancer, as well as being used as an oral contra-
ceptive in folk medicines from South and Central
Americas (Monton, 1981; Siri & Frank, 1982; De A
Ribeiro, Fiuza de Melo, De Barrors, Gomes, & Trolin,
1986; Duke, 1986; Elisabetsky
&
Posey, 1989;
Gonzalez, Valencia, Siverio Exposito, Bermejo
Barrera, & Gupta, 1994). Flavonoid glycosides, triter-
penoids and long-chain fatty acids have been reported
as constituents of this genus (Gonzalez et al., 1994;
Santos Deborah, Salatino Maria, & Antonio, 1995;
Martins & Roque, 1995). However, their pharmaco-
logically active principles have not been described. In
the present investigation on the polyphenols of C. hys-
sopifolia, we have isolated seven hydrolyzable tannins
including two new dimeric ellagitannins with macro-
cyclic structures.
Cuphiin D1 (6) was obtained as a white amorphous
powder and showed a retention time close to that of
woodfordin C (4) in normal-phase HPLC, which
suggested a dimeric character (Okuda, Yoshida, &
Hatano, 1989). The molecular formula C82H56O52 was
indicated by its negative-ion FAB mass spectrum (m/z
1
1871 [M H] ) and elemental analysis. The H NMR
spectrum of 6 recorded at ambient temperature was of
limited usefulness due to broadening of signals for
some of the aromatic and sugar protons, presumably
arising from restricted conformational changes in the
molecule such as that observed for 5 (Hatano et al.,
* Corresponding author. Tel.: +886-2-23773554; Fax: +886-2-
27388351; E-mail: llyang@mail.tmc.edu.tw.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00512-3

308
L.-G. Chen et al. / Phytochemistry 50 (1999) 307±312
4
1990). However the spectrum recorded at an elevated
temperature (388C) indicated four 2H-singlets at d
7.31, 7.23, 7.20 and 7.06 due to four galloyl groups
and six aromatic 1H-singlets attributable to two valo-
neoyl groups. The sugar proton signals which were
assigned with the aid of the 1H±1H shift correlation
spectrum were characteristic of fully acylated C1 glu-
copyranose. The anomeric proton signals at d 7.24 (d,
J =3.0 Hz) and 5.44 (d, J=7.2 Hz), suggest that
anomeric centres of two glucose cores have an a- and
b-oriented acyloxy group, respectively. The 13C reson-
ances of the glucose moieties of 6, whose assignments

L.-G. Chen et al. / Phytochemistry 50 (1999) 307±312
309
C-7)±dH 4.83, 3.94 (glucose H-60)) clearly indicate that
two carboxy groups of the HHDP moiety in each valo-
neoyl group are on O-4/O-6 of each glucose core
(Yoshida, Hatano, Kuwajima, & Okuda, 1992), and
that the orientation of the valoneoyl group on O-4/O-6
of glucose I (the lower glucose core in the formula) is
the same as that in the rugosins (rugosin type), while
that of glucose II (the upper glucose core) is of the iso-
rugosin type (Yoshida et al., 1992). Four galloyl
groups were also allocated at O-1, O-10, O-3 and O-30
by long-range correlations as depicted by arrows in the
formula 6a. These spectral features indicated a macro-
cyclic structure (6) for cuphiin D1 (Yoshida et al.,
1990).
The atropisomerism at each biphenyl moiety of
these two valoneoyl groups was determined as S by a
positive Cotton e€ect at 221 nm ([y] +4.8  105) in the
CD spectrum of 6 (Okuda et al., 1982), which was
similar to that of woodfordin C (4) ([y]218 +4.1 Â105).
Cuphiin D1 (6) was thus characterized as a gallate
of woodfordin C (4).
Cuphiin D2 (7), an o€-white amorphous powder,
exhibited an [M + NH4] + ion peak at m/z 1738 in the
ESI-MS, corresponding to the molecular formula
C75H52O48. The 1H NMR spectrum of 7 recorded at
ambient temperature showed broad signals like 4±6.
The spectrum of 7 recorded at 408C showed close
resemblance to that of 6 except for lack of one galloyl
group. The presence of three galloyl groups (d 7.25,
7.20 and 7.06) and two valoneoyl groups (d 7.22, 6.71,
6.62, 6.57, 6.51 and 6.30) was supported by methanoly-
sis of a methylated derivative of 7 which a€orded 9
and 10 in the molar ratio of 3:2.
Another distinguishable feature of 7 from 6 was an
up®eld shift of one of the anomeric proton signals (d
7.24 (d, J =3 Hz) in 6 to d 6.18 (d, J =3 Hz) in 7).
This shift suggests that the hydroxyl group at an
anomeric center of a glucose core (glucose I) is free,
and the other anomeric center has an b-oriented acy-
loxy group. However, the absence of duplication of
any proton signal and also the observation of a single
peak in the reversed-phase HPLC indicate that 7 exists
only an anomeric form (a-anomer) for glucose I.
The absence of an acyl group at the anomeric center
of glucose I was further con®rmed by reduction of 7
with sodium borohydride, which yielded dihydrocu-
phiin D2 (8), ESI-MS m/z 1740 [M + NH4] + . The
1H±1H COSY of 8 revealed the presence of one gluci-
tol core in the molecule. The structure of cuphiin D2
was thus represented by the formula 7 which is a
regioisomer of woodfordin C (4) concerning a galloyl
group. The structural relationship of cuphiin D1 (6)
and D2 (7) was substantiated by enzymatic degalloyla-
tion of 6 with tannase yielding 4, 5, 7 and gallic acid.
Cuphiin D2 (7) also produced oenothein B (5) and gal-
lic acid upon enzymatic hydrolysis with tannase.
were con®rmed by the 1H±13C shift correlation spec-
trum, also showed the presence of a- (d 90.8, C-1) and
b- (d 92.8, C-10) glucosidic linkages (Yoshida et al.,
1984).
Methylation of 6 with dimethyl sulfate and potass-
ium carbonate gave an octacosamethyl derivative (6a).
Although some 1H signals of 6 showed considerable
broadening even in the spectrum measured at 388C,
the spectrum of 6a measured at ambient temperature
exhibited sharp signals attributable to four galloyl
groups and two valoneoyl groups. The presence of
these acyl groups in 6a was further supported by ten
ester carbonyl carbon resonances (d 164.4±168.3) in
the 13C NMR spectrum, and production of methyl tri-
O-methylgallate (9) and trimethyl octa-O-methylva-
loneate (10) in the molar ratio of 2:1 upon methylation
of 6 followed by methanolysis. Both of the galloyl
moieties of the valoneoyl groups in 6a were determined
to be at O-2 of each glucose core, based on the long-
range 1H±13C COSY (dH 7.30 (valoneoyl-I HC)±dC
167.2 (valoneoyl-I C-7 )±dH 5.97 (glucose H-2) and dH
7.08 (valoneoyl-II HC)±dC 166.6 (valoneoyl-II C-7 )±
dH 5.92 (glucose H-20)). The other four sets of the
long-range correlations among valoneoyl proton±ester
carbonyl carbon±glucose proton (dH 6.68 (valoneoyl-I
HA)±dC 168.3 (valoneoyl-I C-7)±dH 6.12 (glucose H-4);
dH 6.42 (valoneoyl-I HB)±dC 167.4 (valoneoyl-I C-70)±
dH 5.26, 3.76 (glucose H-6); dH 6.35 (valoneoyl-II
HB )±dC 167.1 (valoneoyl-II C-70)±dH 5.11 (glucose H-
0
40); dH 6.85 (valoneoyl-II HA )±dC 167.9 (valoneoyl-II
0

310
L.-G. Chen et al. / Phytochemistry 50 (1999) 307±312
3. Experimental
3.1. General
MCI-gel CHP-20P with aq. MeOH to yield cuphiin D1
(6) (189 mg).
The 20% MeOH eluate (7 g) from column chroma-
tography of Dia-ion HP-20 was also rechromato-
graphed over Toyopearl HW-40 (F) (2.5 cm i.d. Â 45
cm) developing with H2O 4 60% MeOH 4 70%
MeOH 4 MeOH±H2O±Me2CO (7:2:1). The 70%
MeOH eluate gave an additional crop of woodfordin
C (4) (120 mg). The 60% MeOH eluate was rechroma-
tographed with MCI-gel CHP-20P to give oenothein B
(5) (118 mg) and cuphiin D2 (7) (100 mg).
The 60% MeOH eluate (8.5 g) of Dia-ion HP-20
column chromatography was rechromatographed over
Toyopearl HW-40(F) (2.5 cm i.d. Â 52 cm) developing
with H2O 4 40% MeOH 4 60% MeOH 4 70%
MeOH 4 MeOH±H2O±Me2CO (7:2:1). The 60%
MeOH eluate gave myricitrin (494 mg), and the
MeOH±H2O±Me2CO (7:2:1) eluate gave 1,2,3,4,6-
penta-O-galloyl-b-D-glucose (2) (101 mg), tellimagran-
din II (3) (53 mg) and 1,2,3,6-tetra-O-galloyl-b-D-glu-
cose (1) (15 mg).
1H (500 MHz) and 13C NMR (126 MHz) spectra
were measured on a Varian VXR 500 or Bruker DRX
500 instrument and chemical shifts are given in d
(ppm) values. FAB-MS were taken on a VG 70-SE
mass spectrometer using 3-nitrobenzyl alcohol contain-
ing NaCl as the matrix agent, and ESI-MS on a
Micromass Autospec OA-TOF mass spectrometer with
direct injection of 50% aq. MeOH soln containing
small amount of CH3COONH4. Normal-phase HPLC
was conducted on YMC-pack SIL-A003 (4.6
mm  250 mm) using the following solvent systems:
(NP1) n-hexane±MeOH±THF±HCOOH (60:45:15:1)
contained oxalic acid (500 mg/1.2 l), (NP2) n-hexane±
EtOAc (2:1) (¯ow rate, 1.5 ml/min; detection 280 nm)
at room temp. Reversed-phase HPLC was performed
on a YMC-pack J'sphere ODS H-80 (4.6 mm  250
mm) column, using the following solvent systems:
(RP1) 0.1 M H3PO4±0.1 M KH2PO4±EtOH±EtOAc
(42.5:42.5:10:5), (RP2) 0.1 M H3PO4±0.1 M KH2PO4±
EtOH±EtOAc (44:44:7:5) (¯ow rate, 1.0 ml/min; detec-
tion 280 nm) at 408C. Column chromatography was
carried out on Toyopearl HW-40 (®ne grade) (Tosoh),
Dia-ion HP-20 and MCI-gel CHP-20P (Mitsubishi
Chemical Industry). TLC was conducted on Kieselgel
PF254 (Merck) plates with n-hexane±CHCl3±Me2CO
(5:4:1), and visualized under UV irradiation.
3.4. Cuphiin D1 (6)
A white amorphous powder. [a]D +2758 (MeOH; c
1.0). Found: C, 46.6; H, 3.7. Calc. for
C82H56O52Á13H2O: C, 46.7; H, 3.9%. FAB-MS m/z:
1871 [M H] . ESI-MS: m/z 1890 [M + NH4] + . UV
lmax (MeOH) (nm): 219, 275. CD (MeOH) [y] (nm):
+4.8 Â105 (221), +2.2 Â 105 (239), 4.4 Â 104 (263),
5
1
+1.2 Â10 (284). H NMR (500 MHz, 388C, Me2CO-
d6) d: 7.31, 7.23, 7.20, 7.06 (each 2H, s, galloyl(Gal)),
7.12, 6.75, 6.61, 6.54, 6.49, 6.32 (each 1H, s, valoneoyl
(Val)), 7.24 (d, J=3 Hz, Glc H-1), 6.09 (dd, J= 3, 10
Hz, Glc H-2), 6.21 (t, J= 10 Hz, Glc H-3), 6.01 (brt,
J =10 Hz, Glc H-4), 4.67 (ddd, J=10, 7, 1 Hz, Glc
H-5), 5.34 (dd, J =7, 13.3 Hz, Glc H-6), 3.72 (dd,
J =13.3, 1 Hz, Glc H-6), 5.44 (d, J =7.2 Hz, Glc H-
10), 5.65 (m, Glc H-20), 5.68 (m, Glc H-30), 5.00 (m,
Glc H-40 and H-60), 4.30 (dd, J=5, 10 Hz, Glc H-50),
3.86 (brd, J= 13.3 Hz, Glc H-60). 13C NMR (126
MHz, 388C, Me2CO-d6) d: 90.8 (Glc C-1), 72.6 (Glc C-
2), 70.9 (Glc C-3), 69.2 (Glc C-4), 71.6 (Glc C-5), 62.3
(Glc C-6), 92.8 (Glc C-10), 72.4 (Glc C-20 and Glc C-
30), 73.3 (Glc C-40), 72.6 (Glc C-50), 64.5 (Glc C-60),
106.0, 107.1, 107.9, 108.2, 109.0, 114.3 (Val C-2, C-2
and C-2 ), 109.9, 110.1, 110.2, 110.6 (Gal C-2 and C-
6), 164.6, 165.2, 165.6, 166.3, 167.2, 167.3, 167.9, 168.9
(ester carbonyl).
3.2. Plant materials
The aerial part of C. hyssopifolia was collected in
Taipei, Taiwan, in April 1996. A voucher specimen is
deposited in the Graduate Institute of Pharmacognosy
Science, Taipei Medical College.
3.3. Isolation of tannins
The dried aerial part of C. hyssopifolia (1.7 kg) was
homogenized in 70% aq. Me2CO (20 l Â3) and ®l-
tered. The concd ®ltrate was extracted with CH2Cl2 to
remove nonpolar constituents, and the aq. layer was
evapd. A part (118 g) of the aq. extract was chromato-
graphed over Dia-ion HP-20 (9.5 cm i.d. Â 45 cm)
with H2O, and H2O±MeOH (10% MeOH 4 20% 4
40% 4 60% 4 80%). A part (6 g) of the 40%
MeOH eluate was chromatographed over Toyopearl
HW-40(F) (2.5 cm i.d. Â 50 cm) developing with
H2O 4 50% MeOH 4 60% MeOH 4 70% MeOH 4
MeOH±H2O±Me2CO (7:2:1) 4 MeOH±H2O±Me2CO
(8:1:1). The 70% MeOH eluate was rechromato-
graphed over MCI-gel CHP-20P with aq. MeOH to
yield woodfordin C (4) (243 mg). The MeOH±H2O±
Me2CO (7:2:1) eluate was rechromatographed over
3.5. Methylation of cuphiin D1 (6)
A mixture of 6 (21 mg), Me2SO4 (0.1 ml) and
K2CO3 (200 mg) in dry Me2CO (5 ml) was stirred
overnight at room temp, and then re¯uxed for 2.7 h.
After removal of inorganic materials by centrifugation,

L.-G. Chen et al. / Phytochemistry 50 (1999) 307±312
311
4
1
the supernatant was concd and puri®ed by prep. TLC
(Kieselgel PF254) with toluene±Me2CO (3:1) to yield
the octacosamethylate (6a) (12 mg).
+9.3 Â10 (284). H NMR (500 MHz, 408C, Me2CO-
d6) d: 7.25, 7.20, 7.06 (each 2H, s, Gal), 7.22, 6.71,
6.62, 6.57, 6.51, 6.30 (each 1H, s, Val), 6.18 (d, J= 3
Hz, Glc H-1), 6.13 (brt, J= 10 Hz, Glc H-2), 5.76 (m,
Glc H-3), 5.79 (m, Glc H-4), 4.58 (dd, J=7, 9.5 Hz,
Glc H-5), 5.27 (dd, J=7, 12.5 Hz, Glc H-6), 3.65 (d,
J =12.5 Hz, Glc H-6), 5.47 (brd, J= 9 Hz, Glc H-10),
5.66 (t, J =9 Hz, Glc H-20), 5.65 (t, J= 9 Hz, Glc H-
30), 4.98 (m, Glc H-40 and H-60), 4.27 (dd, J= 5, 10
Hz, Glc H-50), 3.82 (d, J =14 Hz, Glc H-60). 13C
NMR (126 MHz, 408C, Me2CO-d6) d: 91.3 (Glc C-1),
71.4 (Glc C-2), 74.7 (Glc C-3), 70.4 (Glc C-4), 68.7
(Glc C-5), 63.2 (Glc C-6), 93.3 (Glc C-10), 72.0 (Glc C-
20), 72.7 (Glc C-30), 73.6 (Glc C-40), 73.0 (Glc C-50),
64.9 (Glc C-60), 106.2, 107.6, 108.2, 109.1, 109.4, 110.7
(Val C-3, C-30, C-6 ), 110.5, 111.0, 114.9 (Gal C-2, C-
6), 165.61, 165.64, 166.8, 167.7, 167.9, 168.0, 168.3,
169.3 (ester carbonyl).
3.6. Compound 6a
A white amorphous powder. ESI-MS: m/z 2282
[M + NH4] + . H NMR (500 MHz, 278C, Me2CO-d6)
1
d: 7.50, 7.42, 7.36, 7.32 (each 2H, s, Gal), 7.30 (1H, s,
0
Val-I HC), 7.08 (1H, s, Val-II HC ), 6.85 (1H, s, Val-II
0
HA ), 6.68 (1H, s, Val-I HA), 6.42 (1H, s, Val-I HB),
0
6.35 (1H, s, Val-II HB ), 7.14 (d, J =3.7 Hz, Glc H-1),
5.97 (dd, J =3.7, 10 Hz, Glc H-2), 6.18 (t, J=10 Hz,
Glc H-3), 6.12 (t, J =10 Hz, Glc H-4), 4.78 (dd,
J= 7, 9 Hz, Glc H-5), 5.26 (dd, J =7, 13 Hz, Glc H-
6), 3.76 (Glc H-6, overlapped with OMe), 5.53 (d,
J= 9 Hz, Glc H-10), 5.92 (t, J= 9 Hz, Glc H-20), 5.60
(t, J= 10 Hz, Glc H-30), 5.11 (t, J =10 Hz, Glc H-40),
4.30 (dd, J=6, 10 Hz, Glc H-50), 4.83 (dd, J= 6, 13
Hz, Glc H-60), 3.94 (Glc H-60, overlapped with OMe),
4.00±3.38 (28 Â OMe). 13C NMR (126 MHz, 388C,
Me2CO-d6) d: 91.5 (Glc C-1), 73.4 (Glc C-2), 72.1 (Glc
C-3), 69.5 (Glc C-4), 71.2 (Glc C-5), 63.5 (Glc C-6),
94.0 (Glc C-10), 71.5 (Glc C-20), 74.0 (Glc C-30), 73.6
(Glc C-40), 73.2 (Glc C-50), 64.5 (Glc C-60), 108.25,
108.28, 108.34, 109.4 (Gal, C-2 and C-6), 106.0 (Val-II
C-3), 106.2 (Val-I C-3), 106.8 (Val-II C-30), 109.0 (Val-
I C-30), 109.1 (Val-I C-6 ), 110.0 (Val-II C-6 ), 164.4
(Gal C-7), 165.3 (Gal C-7), 166.0 (Gal C-7), 166.6
(Val-II C-7 ), 166.9 (Gal C-7), 167.1 (Val-II C-70),
167.2 (Val-I C-7 ), 167.4 (Val-I C-70), 167.9 (Val-II C-
7), 168.3 (Val-I C-7).
3.9. Reduction of 7 with NaBH4
To a soln of 7 (10 mg) in MeOH (2.0 ml) was added
NaBH4 (40 mg). After 25 min, the reaction was termi-
nated by addition of HOAc and the solvent was
evapd. The residue was dissolved in H2O and applied
to a Sep-Pak C18 cartridge. Elution was conducted
with H2O and then with aq. MeOH (5%
MeOH 4 10% 4 20% 4 30% 4 40% 4 50%). The
30% MeOH eluate gave dihydrocuphiin D2 (8) (8.3
mg).
3.10. Dihydrocuphiin D2 (8)
3.7. Hydrolysis of 6 with tannase
A light brown amorphous powder. [a]D +158
(MeOH; c 1.0). ESI-MS: m/z 1740 [M + NH4] + . UV
lmax (MeOH) (nm): 218, 274. CD (MeOH) [y] (nm):
+2.0 Â105 (223), +1.4 Â 105 (238), 5.0 Â 104 (264),
+4.3 Â104 (287), 1.4 Â 104 (317). 1H NMR (500
MHz, 408C, Me2CO-d6 + D2O) d: 7.17, 7.15, 6.96
(each 2H, s, Gal), 6.91, 6.83, 6.71, 6.66, 6.60, 6.52
(each 1H, s, Val), 3.86 (brd, J =12 Hz, glucitol H-1
and H-6), 4.02 (dd, J= 5, 11 Hz, glucitol H-1), 5.13
(brs, glucitol H-2), 5.88 (dd, J= 5, 7 Hz, glucitol H-3),
5.58 (t, J =7 Hz, glucitol H-4), 4.17 (d, J=7 Hz, glu-
citol H-5), 4.67 (d, J =12 Hz, glucitol H-6), 5.92 (d,
J =7.5 Hz, Glc H-10), 5.36 (dd, J= 7.5, 9 Hz, Glc H-
20), 5.77 (t, J= 9 Hz, Glc H-30), 5.20 (t, J =9 Hz, Glc
H-40), 4.29 (dd, J =4.5, 9 Hz, Glc H-50), 5.24 (m, Glc
H-60), 3.92 (d, J =13.5 Hz, Glc H-60).
A soln of 6 (40 mg) in H2O (20 ml) was incubated
with tannase at 378C for 46 h. The reaction was
terminated by acidi®cation with 1% HCl, and the
reaction mixture was applied to CC over MCI-gel
CHP-20P (1.2 cm i.d. Â14.5 cm). Elution was con-
ducted with H2O and then with aq. MeOH (5%
MeOH 4 10% 4 20% 4 30% 4 40% 4 50%). The
5% MeOH eluate a€orded gallic acid (1.4 mg), 20%
MeOH eluate gave oenothein B (5) (5.6 mg) and 30%
MeOH eluate gave 7 (15 mg). The 40% MeOH eluate
was further puri®ed with prep. HPLC with 0.1 M
H3PO4±0.1 M KH2PO4±CH3CN (44:44:12) to give
woodfordin C (4) (2.1 mg) and the starting material 6
(1.0 mg).
3.8. Cuphiin D2 (7)
3.11. Hydrolysis of 7 with tannase
An o€-white amorphous powder. [a]D +1078
(MeOH; c 1.0). ESI-MS: m/z 1738 [M +NH4] + . UV
lmax (MeOH) (nm): 218, 271. CD (MeOH) [y] (nm):
+4.0 Â 105 (222), +2.1 Â 105 (238), 4.5 Â 104 (262),
A soln of 7 (0.5 mg) in H2O (0.5 ml) was incubated
with 3 drops of tannase at 378C for 46 h. Production
of oenothein B (5) and gallic acid was con®rmed by
comparison of retention times on reversed-phase

312
L.-G. Chen et al. / Phytochemistry 50 (1999) 307±312
HPLC (RP1) and 1H NMR of the reaction mixture
with authentic samples.
References
De A Ribeiro, R., Fiuza de Melo, M. M. R., De Barrors, F.,
Gomes, C., & Trolin, G. (1986). Journal of Ethnopharmacology,
15, 262.
3.12. Quantitative analysis of constituent units of 6 and
7
Duke, J. A. (1986). Economic Botany, 39, 278.
Elisabetsky, E., & Posey, A. D. (1989). Journal of Ethnopharmacology,
26, 299.
A soln of tannin (1.0 mg) in EtOH (0.5 ml) was
treated with etheral CH2N2 (1.5 ml) at room temp
overnight. The residue obtained after removal of the
solvent was methanolyzed with 1% NaOMe (0.2 ml)
in MeOH (0.5 ml) at room temp overnight. The reac-
tion mixture was acidi®ed with HOAc and the solvent
was evapd. The residue was partitioned between H2O
(1.0 ml) and EtOAc (1.0 ml). The EtOAc layer was
evapd and re-dissolved in EtOAc (1.0 ml), and then
analyzed by normal phase HPLC (NP2). The ratio of
the amounts of the products was estimated from their
peak area, referring to a known ratio of the methano-
lysate, methyl tri-O-methylgallate (9) and trimethyl
octa-O-methylvaloneate (10), obtained by similar
methylation of woodfordin C (3) followed by metha-
nolysis.
Gonzalez, A. G., Valencia, E., Siverio Exposito, T., Bermejo Barrera,
J., & Gupta, M. P. (1994). Planta Medica, 60, 592.
Hatano, T., Yasuhara, T., Matsuda, M., Yazaki, K., Yoshida, T., &
Okuda, T. (1990). Journal of the Chemical Society, Perkin
Transactions 1, 2735.
Kadota, S., Takamori, Y., Nyein, K. N., Kikuchi, T., Tanaka, K., &
Ekimoto, H. (1990). Chemical and Pharmaceutical Bulletin, 38,
2687.
Martins, D., & Roque, N. F. (1995). Fitoterapia, 66, 187.
Monton, J. F. (1981). In C. C. Thomas (Ed.), Atlas of Medicinal Plants
of Middle America±Bahamas to Yucatan (p. 612). Spring®eld,
IL.
Okuda, T., Yoshida, T., & Hatano, T. (1989). Journal of Natural
Products, 52, 1.
Okuda, T., Yoshida, T., Hatano, T., Koga, T., Toh, N., & Kuriyama,
K. (1982). Tetrahedron Letters, 23, 3937.
Santos Deborah, Y. A. C., Salatino Maria, L. F., & Antonio, S.
(1995). Biochemical Systematics and Ecology, 23, 99.
Siri, V. R. & Frank, J. L. (1982). In New Plant Sources for Drugs and
Foods from The New York Botanical Garden Herbarium (p. 204).
London: Harvard University Press.
Acknowledgements
Yoshida, T., Chou, T., Nitta, A., Miyamoto, K., Koshiura, R., &
Okuda, T. (1990). Chemical and Pharmaceutical Bulletin, 38,
1211.
The authors thank Ms. Shwu-Huey Wang for
the NMR measurements with the Bruker DRX 500
NMR spectrometer. We are also grateful to the
Instrumentation Center of Taipei Medical College and
the SC-NMR Laboratory of Okayama University for
the NMR experiments.
Yoshida, T., Hatano, T., Kuwajima, T.,
Heterocycles, 33, 463.
& Okuda, T. (1992).
Yoshida, T., Hatano, T., Okuda, T., Memon, M. U., Shingu, T., &
Inoue, K. (1984). Chemical and Pharmaceutical Bulletin, 32,
1790.