Isoterchebulin and 4,6-O-isoterchebuloyl-D-glucose, novel hydrolyzable tannins from Terminalia macroptera

Article abstract of DOI:10.1021/np000506v

Two new hydrolyzable tannins, isoterchebulin (1) and 4,6-O-isoterchebuloyl-D-glucose (2), together with six known tannins, 3-8, were isolated from the bark of Terminalia macroptera. Their structures were elucidated by extensive 1D and 2D NMR studies, MS,

Full text of DOI:10.1021/np000506v

294
J . Nat. Prod. 2001, 64, 294-299
Isoter ch ebu lin a n d 4,6-O-Isoter ch ebu loyl-D-glu cose, Novel Hyd r olyza ble Ta n n in s
fr om Ter m in a lia m a cr opter a
J u¨rgen Conrad,† Bernhard Vogler,†,‡ Sabine Reeb,† Iris Klaiber,† Stefan Papajewski,† Gudrun Roos,†
Erlinda Vasquez,†,§ Mary C. Setzer, and Wolfgang Kraus*,†
Institut fu¨r Chemie, Universita¨t Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany, Department of Chemistry and
Department of Biological Sciences, The University of Alabama in Huntsville, Huntsville, Alabama 35899, and
Department of Plant Protection, Visayas State College of Agriculture, Baybay, Leyte, Philippines
Received October 27, 2000
Two new hydrolyzable tannins, isoterchebulin (1) and 4,6-O-isoterchebuloyl-D-glucose (2), together with
six known tannins, 3-8, were isolated from the bark of Terminalia macroptera. Their structures were
elucidated by extensive 1D and 2D NMR studies, MS, and chemical transformations. Biological activities
of all compounds were evaluated against the snail Biomphalaria glabrata, the bacteria Bacillus subtilis
and Pseudomonas fluorescens, the nematode Caenorhabditis elegans, and four cancer cell lines (Hep G2,
MCF-7/S, MDA-MB-231, and 5637 cells). All compounds except 3 showed antimicrobial activities against
B. subtilis (MIC 8-64 µg/mL), whereas only 1 was active against C. elegans (100 µg/mL) and B. glabrata
(LC100 ) 60 µg/mL). 3 and 8 were toxic against 5637 cells with LC50 ) 84.66 and 41.40 µM, respectively.
Terminalia macroptera Guill. et Perr. (Combretaceae) is
a tree widely distributed in Africa. In traditional African
folk medicine different parts of the plant, including leaves,
shoots, seeds, fruit galls, roots, and bark, are used for
treating various diseases and ailments.1 Leaf and bark
extracts showed strong antimicrobial activity toward the
Gram-positive bacteria Staphylococcus aureus and Bacillus
subtilis,2 while a root extract was active against several
strains of Neisseria gonorrheae.3 An ethanol extract of the
bark exhibited a minimum lethal dose of 250 mg/kg in
rats.4 Previous phytochemical investigations of the flowers
led to the isolation of several C- and O-glycosyl flavones.5-7
Chlorogenic acid, quercetin, isoorientin, the ellagitannins
chebulagic acid, chebulinic acid, punicalagin, and terflavin
A, gallic acid, and ellagic acid were isolated from the
leaves,8,9 while different methylated ellagic acid derivatives
and the triterpenoid terminolic acid were obtained from
the heartwood.10
We reported previously on the isolation of two galloylated
pentacyclic triterpenoids and a phenolic glucoside gallate
from the bark of T. macroptera.11,12 In the present paper
we describe the isolation and structure elucidation of two
novel hydrolyzable tannins (1, 2) having a new tetraphen-
ylic acid moiety (isoterchebulic acid), together with six
known tannins (3-8, Supporting Information). We also
report on the biological properties of these compounds
including molluscicidal, antibacterial, nematicidal, and
cytotoxic activities.
mide SC60 and Sephadex LH-20 as well as HPLC using
RP18 or CN-Phase to afford 4,6-O-(S)-isoterchebuloyl-D-
glucose (2) together with the six known tannins: 2,3-O-
(S)-hexahydroxydiphenoyl-D-glucose (3),13 punicalagin (4),13,14
terflavin A (5) and B (6),15 2-O-galloyl-punicalin (7),16 and
punicacortein C (8).17 The structures of the known tannins
were elucidated on the basis of 1D and 2D NMR (1H, 13C,
DQF-COSY, HSQC, and HMQC), ESIMS, and optical
rotation [R]D. The experimental data for 3-8 were in
agreement with values obtained from the literature.
Isoterchebulin (1) was isolated as a tan amorphous
powder. Its molecular formula of C48H28O30 was established
1
by HRFABMS. The H NMR spectrum of 1 showed dupli-
cated signals in a ratio of 4:3 for the sugar and phenolic
moieties, suggesting that the anomeric center of the sugar
moiety was not acylated. The 13C NMR spectrum displayed
12 signals due to sugar carbons including two anomeric
carbons at δ 94.2 (C-1â) and 90.8 (C-1R), which confirmed
that 1 existed as an equilibrium mixture of R and â forms
in solution (acetone-d6: D2O ) 9:1). The connectivities of
the seven sugar protons in both anomeric moieties were
determined by COSY, and their directly bonded carbons
were assigned by GHSQC (Table 1). 1H-13C long-range
correlations (GHMQC) between the anomeric protons H-1
(R: δ 5.34; â: δ 5.01) and C-5 (R: δ 70.09; â: δ 74.51) along
with H-5 (R: δ 4.12; â: δ 3.89) and C-1 (R: δ 90.81; â: δ
94.20) in each sugar unit indicated a pyranose ring with
an ether linkage between C-1 and C-5. The observed vicinal
coupling constants of J ≈ 9 Hz between the trans diaxial
oxymethine protons H-2 and H-3, H-3 and H-4, and H-4
and H-5 (each form) together with J ) 8.08 Hz for H-1â
and H-2â and J ) 3.49 Hz for H-1R and H-2R established a
glucopyranosyl moiety with 4C1 conformation for both
sugars in the case of a D configurated pyranose. Further-
more, deshielding of more than 1 ppm of the protons H-2,
H-3, H-4, and H-6 implied a tetra-acylated glucose in 1
(Table 1). Complete acid hydrolysis liberated the sugar
moiety, which was identified as D-glucose by TLC and
Resu lts a n d Discu ssion
An EtOAc extract of the dried bark was chromato-
graphed on Sephadex LH-20 followed by HPLC separation
on RP18 to give the new tannin isoterchebulin (1). The
MeOH extract was dissolved in H2O and extracted with
n-BuOH. The n-BuOH layer was concentrated in vacuo and
partitioned between CHCl3, MeOH, and H2O. The aqueous
layer was separated by column chromatography on polya-
* To whom correspondence should be addressed. Tel: 0049/7114592171.
Fax: 0049/7114592951. E-mail: kraus130@uni-hohenheim.de.
† Universita¨t Hohenheim.
optical rotation ([R]25
authentic sample.
) +59) in comparison with an
D
‡ Department of Chemistry.
A total of 10 aromatic protons for both forms resonating
as sharp singlets each were found in the chemical shift
§ Visayas State College of Agriculture.
Department of Biological Sciences.
10.1021/np000506v CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/06/2001

Novel Hydrolyzable Tannins from Terminalia
J ournal of Natural Products, 2001, Vol. 64, No. 3 295
Ta ble 1. 1H NMR and 13C NMR Spectral Data of the R- and
â-D-Glucose Residues of Compounds 1 and 2 (acetone-d6/D2O )
9:1)
1
2
pos
δ,a mult; J [Hz]
13Ca
δ,a mult; J [Hz]
13Ca
1R
2R
3R
4R
5R
6R
5.34, d; 3.5
4.93, dd; 9.4, 3.4
5.62, t; 9.6
4.78, t; 9.8
4.12, bt; 9.2
4.46, dd; 12.0, 8.6
3.14, bd; 11.9
5.01, d; 8.1
4.73, dd; 9.6, 8.1
5.34, t; 9.6
4.75, t; 9.8
3.89, bt; 9.1
90.81
74.64
75.05
69.61
70.09
64.59
5.03, d; 3.6
3.37, dd; 9.8, 3.4
4.02, t; 9.7
4.35, t; 9.5
4.03, bt; 9.4
4.31, dd; 11.7, 8.7
3.07, bd; 11.8
4.47, d; 7.8
3.19, dd; 9.4, 7.9
3.74, t; 9.2
4.36, t; 9.5
3.58, bt; 9.3
92.86
73.04
71.51
73.16
69.94
64.95
1â
2â
3â
4â
5â
6â
94.20
77.11
77.47
69.37
74.51
64.44
97.18
75.55
74.75
72.83
74.26
64.95
F igu r e 1. HHDP moiety attached to O-2 and O-3 of the central glucose
core of 1. Arrows exhibit 1H-13C long-range correlations.
carbons in the region of δ 166.6-169.8 and four carbons
at δ 157.7-160.0 in the 13C NMR spectrum suggested the
presence of four carbonyl ester functions and two aromatic
δ-lactone rings for each form similar to punicalagin13,14 and
terchebulin.18 From the remaining 72 aromatic carbons
only 63 could be seen in the 13C NMR spectrum, due to
overlapping. Ten carbons in the region δ 106-114 were
identified as hydrogen-bearing aromatic carbons by GH-
SQC. The assignment of the remaining closely resonating
quaternary carbons between δ 105 and 150 was achieved
by a series of highly optimized GHMQC experiments using
two different concentrations (60 and 300 mg/700 µL,
respectively), 4096 increments in the F1-direction for the
maximum resolution of the correlating signals, and differ-
4.42, dd; 12.0, 8.3
3.14, bd; 11.9
4.29, dd; 12.0, 7.4
3.08, bd; 11.8
a
1H NMR (500 MHz), 13C NMR 125.72 MHz (compound 1) and
75.42 MHz (compound 2). δ in ppm. Observed coupling constants
were not averaged. Observed triplet signal patterns (t, bt) in the
1H NMR spectra correspond to pseudotriplet signals. Assignments
based on COSY, DQFCOSY, GHSQC, and HMBC spectra.
n
ent J C-H optimized for 1, 2, 3, 5, and 8 Hz. Inspection of
n
the HMBC spectra revealed that a J C-H optimization of 5
and 8 Hz contained the most information about the
connectivities inside the sugar moiety. However, for the
determination of the polyphenolic substructures, optimiza-
tions for small long-range couplings on the order of 1 and
2 Hz were necessary to link as many quaternary carbons
3
4
as possible (2J , J , and J ) to the few remaining aromatic
protons. For the suggested structure only one hydrogen per
aromatic ring was proposed.
The hexahydroxydiphenoyl (HHDP) substructure (Figure
1) was represented in the 1H NMR spectrum by the
aromatic protons H-3 at δ 6.610(R)/6.615(â) and H-3′ at δ
6.35(R)/6.37(â). Carbons C-3 and C-3′ were identified by
GHSQC at δ 107.5 and δ 106.93/106.90, respectively. Each
of the protons H-3 and H-3′ of the HHDP moiety exhibited
1H-13C long-range correlations to an ester carbonyl carbon
(C-7: δ 169.05/168.95; C-7′: δ 169.82), two carbons corre-
sponding to aromatic C-C bonds (C-1, C-1′: δ 114.59,
114.49, 114.47 and 114.46; C-2: δ 126.32/126.11; C-2′: δ
126.33) and to three oxygen-bearing aromatic carbons (C-
4, C-4′: δ 145.16, 145.15, 145.26, and 145.23; C-5: δ 136.24/
136.15; C-5′: δ 136.27; C-6, C-6′: δ 144.41, 144.47, and
144.35). In agreement with the 13C NMR data obtained for
the HHDP moiety in compound 3 these results established
an HHDP substructure in 1. 1H-13C long-range correla-
tions between the low-field glucose protons H-2 (R: δ 4.93;
â: δ 4.73) and the HHDP carbonyl carbon C-7 as well as
H-3 (R: δ 5.62; â: δ 5.34) and C-7′ indicated that the HHDP
unit is attached to O-2 and O-3 of the central glucose core
by ester linkages. These findings were confirmed by partial
hydrolysis, which besides the starting material produced
ellagic acid and a hydrolysate identical to the new 4,6-O-
(S)-isoterchebuloyl-D-glucose (2). Ellagic acid19 was identi-
1
fied by EIMS and H and 13C NMR.
region of δ 6.35-7.57, indicating the presence of five
pentasubstituted aromatic rings. The low-field shift of the
proton at δ 7.56/7.57 (R/â form) was attributable to a
depside-type linkage between two aromatic rings, which
is known in the case of hydrolyzable tannins, e.g., terflavin
A, B15 and terchebulin.18 The chemical shift values of eight
The structure of the novel tetraphenylic acid moiety
(isoterchebulic acid) was elucidated as outlined in Figure
2: 1H-13C long-range correlations between the ester car-
bonyl carbon C-7 (δ 167.45/167.38) and the low-field glucose
proton H-4 (R: δ 4.78; â: δ 4.75) along with the aromatic

296 J ournal of Natural Products, 2001, Vol. 64, No. 3
Conrad et al.
m/z 1083 in FAB as well as ESIMS) could be explained by
an ether linkage between two phenolic hydroxy groups.
However, the position of the ether linkage between ring C
and D was not assignable by direct NMR measurements.
Therefore, 1 was permethylated with DMS and K2CO3 in
dry acetone to yield a mixture of the R and â forms of the
1
hexadecamethyl ether 9, which was identified by H NMR
and ESIMS ([M + H]+, [M + Na]+, [M + K]+ ions at m/z
1309, 1331, 1347, respectively). A mass difference of 224
mu equivalent to 16 introduced methyl groups and the
1
occurrence of 16 methoxy groups in the H NMR spectrum
confirmed the presence of 16 hydroxy groups in 1. Metha-
nolysis of 9 with sodium methoxide in dry methanol
afforded dimethyl-(S)-hexamethoxydiphenoate (10), which
was identified by 1H NMR, EIMS, optical rotation, and CD
together with the nonamethylisoterchebulic acid dimethyl
ester (11).
F igu r e 2. Isoterchebuloyl unit attached to O-4 and O-6 of the central
glucose core of 1. Arrows exhibit 1H-13C long-range correlations.
The ESIMS spectrum of 11 displayed its [M + Na]+ ion
at m/z 815. The 1H NMR spectrum exhibited three aromatic
protons at δ 7.46 (HA-6), 7.74 (HC-6′′), and 7.21 (HD-6′′′)
and 11 sharp singlets (3H each) in the chemical shift region
δ 3.5-4.2 which were assigned to 11 methoxy groups. An
HSQC experiment revealed the hydrogen-bearing carbons
of the three aromatic protons and the 11 methoxy groups
(Table 3). In the HMBC spectrum the protons of nine
methoxy groups correlated with one oxygen-bearing aro-
matic carbon, each indicating nine phenylmethyl ether
functions in 11. The remaining two methyl groups at δ 3.54
and 3.69 were linked to an ester carbonyl carbon each, as
proton HA-6 (δ 6.79(R)/6.76(â); C-6: δ 109.59/109.51 by
GHSQC) indicated that the aromatic ring A is linked to
O-4 of the glucose core. The remaining five ring A carbons
were identified by GHMQC at δ 145.17/144.59, 144.59,
137.72/137.63, 123.64/123.40, and 116.50/116.35. Compari-
son of these values with those of one phenolic ring of the
HHDP moiety suggested an esterified gallic acid derivative
with a similar attachment to another aromatic ring as in
the HHDP unit.
1
The aromatic ring system D was represented in the H
NMR spectrum by the proton HD-6′′′ at δ 6.535(R)/6.530-
(â) (C-6′′′: δ 107.55 by GHSQC). In the HMBC spectrum
HD-6′′′ was correlated with an ester carbonyl carbon (C-
7′′′: δ 166.73/166.67) and one carbon signal at δ 114.92/
114.77, which was assigned to the ester carbonyl bearing
carbon and, in contrast to ring A, to four oxygen-bearing
aromatic carbons at δ 142.45, 138.77/138.60, 138.06/137.99,
and 138.97/138.94. The 2,3,4,5-tetrahydroxy substitution
pattern of the tetrahydroxy benzoic acid moiety of ring D
was deduced by comparison of the observed 13C NMR data
with computer-generated carbon spectra of the three pos-
sible constitution isomers of tetrahydroxy benzoic acid.
The low-field-shifted aromatic proton HC-6′′ (δ 7.56(R)/
7.57(â); C-6′′: δ 112.55/112.52 by GHSQC) implied the
occurrence of a depside-like linkage of ring C. In the HMBC
spectra this proton correlated with an upfield-shifted
carbonyl carbon (C-7′′) at δ 160.00, which was assigned to
an aromatic δ-lactone ring. 1H-13C long-range correlations
between HC-6′′ and the five carbons at δ 150.97/150.91,
141.70/141.64, 139.03/139.00, 112.83, and 113.61 estab-
lished an additional gallic acid derivative for ring C. The
remaining 14 carbons (two of them overlapped) in the 13C
NMR spectrum showing no 1H-13C long-range correlations
as in the rings A, C, and D were attributed to another 2′,6′-
substituted gallic acid unit (aromatic ring B). Comparison
of the 13C NMR data of rings B and C with those for
punicalagin12,13 and terchebulin17 suggested that rings B
and C form an ellagic acid substructure located between
rings A and D. The orientation of the ellagic acid derivative
between rings A and D (Figure 2) was established by the
4J C-H long-range coupling from the proton HA-6 to the
quaternary carbon C-6′ of ring B at δ 124.05/124.02, which
can only be observed in the case of a C-C linkage between
C-2 of ring A and C-6′ of ring B.
1
established by H-13C long-range correlations. The attach-
ments of the phenylmethyl ether and methyl ester func-
tions to the aromatic ring systems A-D were determined
1
by H-13C long-range correlations (Figure 3).
2
3
4
As J C-H, J C-H, and J C-H long-range couplings cannot
be distinguished in HMBC experiments, the exact position
of each phenylmethyl ether group in 11 was established
by NOE experiments. In the NOESY spectrum each of the
aromatic protons showed cross-peaks to the protons of the
adjacent methyl ether group and additionally in the case
of HA-6 and HD-6′′′ to the methyl ester group of rings A
and D. NOESY correlations between neighboring methyl
groups clearly indicated the position and sequence of each
phenylmethyl ether function in the aromatic ring systems.
The ether linkage between C-4′′ of ring C and C-2′′′ of ring
D was deduced from the following considerations: The
proton HD-6′′′ showed 1H-13C long-range correlations to
three methyl-bearing phenolic carbons and to one without
a methyl group. The NOESY experiment clearly estab-
lished the 3′′′-5′′′ positions of the phenylmethyl ether
groups in ring D, indicating that the phenolic carbon
C-2′′′was chemically modified before methylation.
In the HMBC spectrum HC-6′′ correlated with three
phenolic carbons of which only the adjacent one was linked
to a methoxy group. This observation led to the conclusion
that one of the non-methoxylated phenolic carbons (C-3′′)
had to be assigned to the aromatic δ-lactone and the other
(C-4′′), in agreement with the molecular weight, to an ether
bridge between rings C and D. The atropisomerism of the
phenyl-phenyl bond of 11 ([R]26D ) -33.9) was established
to be in the S-series by comparison of its CD curve with
similar structures of known tannins.13,15,18
4,6-O-(S)-Isoterchebuloyl-D-glucose (2) was isolated as a
tan amorphous powder. Its molecular formula of C34H22O22
was established by HRFABMS. The 1H and 13C NMR
spectra of 2 showed duplicated signals for the sugar and
polyphenolic moieties, indicating an equilibrium mixture
of R and â anomeric forms in solution (ratio: 4:3 of the R/â
Considering the 13 carbons of the isoterchebuloyl moiety
that were assigned as phenolic carbons including the two
associated with the aromatic δ-lactone rings, a molecular
weight of 1102 mu should be expected for 1. The difference
of 18 mu from the observed molecular mass ([M - H]- at

Novel Hydrolyzable Tannins from Terminalia
J ournal of Natural Products, 2001, Vol. 64, No. 3 297
Ta ble 2. 1H NMR and 13C NMR Spectral Data of the HHDP and Isoterchebuloyl Units of 1 and 2 (acetone-d6/D2O ) 9:1)
1
2
isoterchebuloyl
HHDP
isoterchebuloyl
1HR,â
13Ca
pos
1HR,â
13Ca
pos
1HR,â
13Ca
a
a
a
pos
1
2
3
4
5
6
7
1′
2′
3′
4′
5′
6′
7′
1′′
2′′
3′′
4′′
5′′
6′′
7′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
123.63,b 123.40b
116.50, 116.35
144.59
137.72, 137.63
145.21,c 145.17d
109.59, 109.51
167.45, 167.38
107.63,e 107.47e
113.19, 113.14
136.66
139.58, 139.57
147.86, 147.82
124.05,b 124.02b
157.79, 157.75
113.61
1
2
3
4
5
6
7
1′
2′
3′
4′
5′
6′
7′
114.59,g 114.46g
126.32, 126.11
107.42,e 107.36e
145.16,c 145.15c
136.24, 136.15
144.41h
169.05, 168.95
114.49,g 114.47g
126.33
1
2
3
4
5
6
7
1′
2′
3′
4′
5′
6′
7′
1′′
2′′
3′′
4′′
5′′
6′′
7′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
124.19, 123.96
116.12, 116.01
144.34
137.82, 137.78
145.15
110.47, 110.40
167.75
107.59, 107.53
113.51, 113.20
137.43, 137.33
136.53
147.69, 147.66
124.56, 124.52
157.78, 157.71
112.38
113.14, 112.85
139.45, 139.44
141.79, 141.72
150.97, 150.91
112.34
6.610, 6.615
6.35, 6.37
6.79, 6.76
7.09
106.93, 106.90
145.26, 145.23c
136.27
144.35,h 144.47h
169.82
112.83
139.03,f 139.00f
141.70, 141.64
150.97, 150.91
112.55, 112.52
160.0
7.56, 7.57
7.540, 7.544
6.476, 6.482
159.93
114.92, 114.77
142.45
115.12, 115.04
142.40
138.77,f 138.60f
138.06,f 137.99f
138.97f, 138.94f
107.55e
138.90,b 138.89b
138.60,b 138.44b
138.92,b 138.91b
107.39, 107.30
166.82, 166.73
6.535, 6.530
166.73, 166.67
a
1H NMR (500 MHz),13C NMR 125.72 MHz (compound 1) and 75.42 MHz (compound 2). δ in ppm. Assignments based on GHSQC and
HMBC spectra. 13C NMR values without differentiation between R/â forms because of similar intensities of the 13C NMR resonances.
-h13C NMR values with same letters as well as values of positons 3′/4′, 3′′/4′′, 1′/2′, and 1′′/2′′ of the isoterchebuloyl unit may be
b
interchanged.
Ta ble 3. 1H NMR (300 MHz) Spectral Data of 11 in
Acetone-d6 and MeOH-d4 and 13C NMR (75.42 MHz) Data of 11
in Acetone-d6
pos
1H (Me)a: acetone/MeOH
13C (Me)a
1
2
3
4
5
126.26b
124.95b
(3.58/3.62)
(3.88/3.948)
(3.95/4.00)
7.46/7.54
151.85 (60.98)
146.64 (60.88)
153.66 (56.33)
110.17
166.75 (51.99)
112.28
6 (HA)
7
1′
(3.54/3.63)
2′
3′
116.19
142.35
4′
5′
6′
(4.21/4.27)
(3.66/3.71)
146.24 (62.04)
153.02 (61.40)
133.50
7′
1′′
2′′
3′′
157.28
113.57c
114.18c
140.54d
4′′
140.00d
5′′
6′′ (HC)
7′′
(3.94/3.945)
7.74/7.83
153.61 (57.45)
108.72
159.01
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′ (HD)
7′′′
118.73
145.56
146.54 (61.60)
147.34 (61.15)
150.53 (56.54)
109.15
F igu r e 3. Nonamethylisoterchebulic acid dimethyl ester (11). Arrows
show NOESY (T) and HMBC correlations (f). 1H-13C long-range
correlations between the methyl groups and their corresponding
methoxy-bearing carbons are not shown.
(3.61/3.67)
(3.81/3.86)
(3.87/3.91)
7.21/7.28
(3.69/3.78)
165.83 (52.21)
The structure of the isoterchebuloyl moiety and its 4-O
and 6-O linkage to the sugar core was elucidated by
GHMQC experiments in a manner similar to that described
for compound 1 (Table 2). The atropisomerism of the
phenyl-phenyl bond of the isoterchebulic acid residue of
2 was deduced to be in the S-series from comparison of the
a
NMR data of methyl groups (Me) in parentheses. δ in ppm.
Assignments based on NOESY, ROESY, NOE-difference, GHSQC,
and HMBC spectra. b -d13C NMR values with same letters may
be interchanged.
forms in acetone-d6/D2O ) 9:1). The sequence of the seven
sugar protons in both anomeric sugar moieties and their
directly bonded carbons was established by DQFCOSY and
optical rotation ([R]25 ) -204) and CD curve of 2 with
D
corresponding data of the 4,6-O-(S)-isoterchebuloyl-D-
glucose obtained by partial hydrolysis of 1.
4
GHSQC, respectively (Table 1). Again, a C1 conformation
for the sugar parts of both isomers was established by the
vicinal coupling constants of J ≈ 9 Hz between the sugar
protons H-2 and H-3, H-3 and H-4, and H-4 and H-5.
Compounds 1-8 were tested for their activities against
the bacteria Bacillus subtilis and Pseudomonas fluorescens,
the nematode Caenorhabditis elegans, and Biomphalaria

298 J ournal of Natural Products, 2001, Vol. 64, No. 3
Conrad et al.
separations. Thin-layer chromatography was carried out on
precoated Si gel 60 F254 plates (Merck), and spots were detected
by UV illumination.
P la n t Ma ter ia l. The bark of T. macroptera (Combretaceae)
was collected at Wakwa, Cameroon, in 1995 and identified by
Dr. S. Yonkeu, Chief of the herbarium at the Institute de
Recherche Zoo Veterinaire, Wakwa, Cameroon. A voucher
specimen (no. 47542 HNC) is located at the Cameroon National
Herbarium, Ngaoundere, Cameroon.
Extr a ction a n d Isola tion . The air-dried powdered bark
of T. macroptera was extracted and treated as previously
described.11 Sephadex LH-20 chromatography of the EtOAc
extract (40 g) with MeOH as eluant afforded seven fractions,
A1-A7. HPLC purification [5 mL/min, 250 × 25 mm, CH3CN-
H2O (32:68) + 0.01% TFA, UV 280 nm] of an aliquot (1.4 g) of
fraction A6 (8.1 g) on RP 18 afforded compound 1 (1.2 g). The
n-BuOH extract (106 g) was partitioned between CHCl3,
MeOH, and H2O (1.5:1:1). The aqueous layer was extracted
six times with the organic phase and concentrated under
reduced pressure to give 70 g of a brown crystalline residue.
A part of this residue (36 g) was subjected to a polyamide SC60
column (200 g) and eluted with MeOH-H2O (2:8, 5:5, 10:0) f
MeOH-EtOH (5:5, 0:10) f EtOH-acetone-DMF-n-PrOH (5:
1:1:3). Fractions were monitored by analytical HPLC (1 mL/
min, RP 18, CH3CN-H2O (10:90) and (15:85) + 0.07% TFA,
UV 254 nm), and fractions with the same HPLC pattern were
combined to yield 11 fractions, B1-B11, altogether. Comparison
of the retention times and 1H NMR spectra of fractions B10
and B11 (15.7 g in total) revealed that 1 was the only compound
in these fractions.
glabrata, one of the snail vectors of schistosomiasis, as well
as the cancer cell lines Hep G2 (human hepatocellular
carcinoma), MCF-7/S (N.I.H. human breast adenocarci-
noma, pleural effusion), MDA-MB-231 (human breast
adenocarcinoma), and 5637 cells (human primary bladder
carcinoma). All compounds except 3 showed antimicrobial
activities against B. subtilis in the microdilution assays:20
minimal inhibition concentrations (MICs) of 8 µg/mL for
compounds 1, 2, and 7, 16 µg/mL for 8, 32 µg/mL for 4,
and 64 µg/mL for 5 and 6, compared to 2 µg/mL for the
â-lactam antibiotic Cefotaxim. However, the observed
activities originated from a bacteriostatic and not a bac-
teriocidal mode of action, as revealed by regrowth of
bacteria treated with the MIC value of each compound on
drug-free HNB agar plates. None of the compounds were
active against P. fluorescens even in the highest concentra-
tion (1024 µg/mL). Only 1 exhibited moderate activity (++)
at 100 µg/mL against the nematode C. elegans21 and was
molluscicidal against B. glabrata (LC100 ) 60 µg/mL).22 To
determine the piscicidal property of 1, this compound was
tested against tilapia Oreochromis mossambicus at a
concentration that was toxic to the snails (50 and 75 µg/
mL).23 At both concentrations compound 1 caused only 10%
mortality. In the screening against the above-mentioned
cancer cell lines only the compounds 3 and 8 were toxic
against 5637 cells (human primary bladder carcinoma)
with LC50 values of 84.66 ( 2.66 and 41.40 ( 3.34 µM,
respectively.
Fraction B3 was dissolved in 10 mL of MeOH-H2O (1:1) and
centrifuged. The evaporated supernatant (300 mg) was purified
by HPLC on RP18 [5 mL/min, 250 × 10 mm, H2O + 0.05%
TFA, UV 254 nm] to afford compound 3 (216 mg). An aliquot
(500 mg) of fraction B4 (1 g) was applied to HPLC on RP18 [5
mL/min, 250 × 10 mm, CH3CN-H2O (2:98 f 7:93) + 0.05%
TFA, UV 254 nm] to give compound 6 (35 mg). An aliquot (500
mg) of fraction B9 (5.6 g) was purified by HPLC on RP18 [5
mL/min, 250 × 10 mm, CH3CN-H2O (10:90 f 13:87) + 0.05%
TFA, UV 254 nm] to yield compounds 4 (58 mg) and 5 (23 mg).
Compounds 2, 7, and 8 found in fraction B9 (4 g) could not
be separated by HPLC on RP18. Therefore, B9 was subjected
to Sephadex LH-20 chromatography with MeOH-H2O (9:1)
as eluant to afford nine fractions, C1-C9. HPLC purification
[5 mL/min, 250 × 10 mm, CH3CN-H2O (7.5:92.5) + 0.05%
TFA, UV 254 nm] of fraction C4 (347 mg) gave compound 2
(84 mg) and a mixture of compounds 7 and 8. HPLC on CN [5
mL/min, 250 × 25 mm, CH3CN-H2O (0.4:99.6) + 0.075% TFA,
UV 254 nm] of this mixture yielded pure compounds 7 (28 mg)
and 8 (40 mg).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Bu¨chi SMP-20 apparatus and are uncorrected.
UV and CD spectra were obtained on a Hewlett-Packard
8452A diode array spectrometer and on
a J asco J -500A
spectropolarimeter at 25 °C, respectively. Optical rotations
were recorded on a Perkin-Elmer 241 polarimeter. IR spectra
(KBr disks) were measured on a Perkin-Elmer FT-IR Paragon
1000 spectrometer.
NMR spectra were recorded on a Varian Unity Inova 500
or 300 MHz instrument. The H and 13C chemical shifts were
1
referenced to solvent signals at δH/C 2.04/29.8 (acetone-d6), 3.35/
49.0 (methanol-d4), 8.71/149.8 (pyridine-d5), and 7.27/77.0
(CDCl3) relative to TMS. All 1D (1H, 13C, NOE difference) and
2D NMR (COSY, DQFCOSY, NOESY, ROESY, GHSQC,
GHMQC, G ) gradient enhanced) measurements were per-
formed using standard Varian pulse sequences. 1H-13C cor-
relation spectra were recorded by GHSQC (J C-H ) 140 Hz)
for the determination of proton-bearing carbons and GHMQC
(nJ C-H optimized for 1, 2, 3, 5, and 8 Hz; 4096 increments in
F1) for multibond correlations (HMBC). NOESY and ROESY
experiments were carried out with 0.5 and 1.0 s mixing time.
13C NMR spectra were recorded at 125.72 and 75. 42 MHz with
∼0.003 and ∼0.004 ppm digital resolution (FT-size 128 K) to
assign carbon shifts with two decimal places. Program for
computer-generated carbon spectra: ACD-labs, version 2.0,
1996. Mass spectra were obtained on a Finnigan MAT 95
(FABMS, HRFABMS with NBA as matrix), a Finnigan MAT
TSQ 700 (ESIMS), and a Varian MAT 311A (EIMS; 70 eV).
Open and low-pressure column chromatography was per-
formed with polyamide SC60 (0.05-0.16 mm;Macherey&Nagel),
Si gel G60 (40-63 µm; Merck), and Sephadex LH-20 (Fluka
Biochemica). Analytical and semipreparative HPLC were
performed on a Merck Hitachi 655A-12 liquid chromatograph
equipped with a Shimadzu SPD-2A UV/vis detector and a
Knauer HPLC Pump 64 equipped with an ABI Applied
Biosystems 785A detector, respectively. Merck LiChroCART
125 × 4 mm cartridges (LiCrospher 100 RP18 and LiCrospher
100; 5 µm) were used for analytical and a Merck LiChroCART
250 × 10 mm cartridge (LiCrospher 100 RP18; 10 µm) as well
as Bischoff HPLC 250 × 25 mm columns (LiCrospher 100
RP18, 10 µm and Spherisorb CN, 5 µm) for semipreparative
Isoter ch ebu lin (1): tan amorphous powder; mp (H2O)
240-245 °C (decomp); [R]25 -169°(c 0.465, MeOH); UV
D
(MeOH) λmax (log ꢀ) 220 (4.84), 258 (4.82), 378 (4.13) nm; CD
(c 2.5 × 10-5, MeOH) θ (nm) -4.16 × 104 (215), +5.68 × 104
(237), -2.24 × 104 (295), -0.56 × 104 (372); IR (KBr) νmax 3416,
1733, 1610, 1517, 1458, 1360, 1312, 1180, 1059, 965, 877, 836,
770, 746 cm-1; 1H and 13C NMR, see Tables 1 and 2; negative
ESIMS and FABMS m/z 1083 [M - H]-; HRFABMS m/z
1083.0588 [M - H]- (calcd for C48H27O30 requires 1083.0576).
P a r tia l Hyd r olysis of 1. A solution of 1 (80 mg) in 1 N
H2SO4 (5 mL) was heated under reflux for 6 h. After cooling,
the reaction mixture was neutralized with Na2CO3 and the
solvent removed in vacuo. The residue was dissolved in
MeOH-H2O (1:1; 20 mL) and filtered. The filtrate was
concentrated under reduced pressure and applied to HPLC on
RP18 [5 mL/min, 250 × 10 mm, MeOH-H2O (5:95 f 100:0 in
30 min) + 0.1% TFA, UV 254 nm] to give the starting material
(17 mg), ellagic acid (8 mg), and a hydrolysate identical to
compound 2: [R]25 -196° (c 0.5, MeOH); 1H and 13C NMR
D
identical to 2 (Tables 1 and 2), negative ESIMS m/z 781 [M -
H]-. Ellagic acid: 1H NMR (pyridine-d5, 300 MHz) δ 8.2; 13C
NMR (pyridine-d5, 75.42 MHz) δ 160.9, 142.1, 137.8, 113.8,
111.8, 108.7; EIMS m/z 302 [M]+ (61), 44 (100), 28 (55).
Su ga r An a lysis of 1. A solution of 1 (150 mg) in 3 N TFA
(8 mL) was heated under reflux for 24 h. After cooling, the

Novel Hydrolyzable Tannins from Terminalia
J ournal of Natural Products, 2001, Vol. 64, No. 3 299
precipitate was removed by filtration and the filtrate evapo-
rated to dryness. The residue was subjected to a polyamide
SC60 column (5 g, 0.1% TFA) followed by chromatography on
Si gel [10 g, CHCl3-MeOH-H2O (60:35:5)]. Final purification
was achieved by chromatography on NH2 phase (solid-phase
extraction cartridge Adsorbex, Merck) using CH3CN as eluant.
The resulting D-glucose (11 mg) was identified by TLC and
compounds (n ) 6, in three replicates for each concentration).
Bayluscid was used as positive control (LC100 5 µg/mL).
P iscicid a l Activity. Compound 1 was tested at 50 and 75
µg/mL (n ) 30, in three replicates for each concentration)
against O. mossambicus using the standard protocol according
to Balza.23
determination of its optical rotation, [R]25 +59 (c 0.5, H2O),
Cytotoxic Assa y. Compounds 1-8 were screened against
Hep G2 (human hepatocellular carcinoma), MCF-7/S24 (N.I.H.
human breast adenocarcinoma, pleural effusion), MDA-MB-
231 (human breast adenocarcinoma), and 5637 cells (human
primary bladder carcinoma) using the method according to
Setzer.25
D
in comparison with an authentic sample (Merck).
Meth yla tion of 1. A mixture of 1 (500 mg), dimethyl sulfate
(2 mL), and anhydrous K2CO3 (2.8 g) in dry acetone (25 mL)
was heated under reflux for 4 h. After cooling, the precipitate
was removed by filtration. The resulting filtrate was acidified
with formic acid (pH 4) and concentrated under reduced
pressure. The residue was chromatographed on Si gel (35 g)
with petroleum ether-EtOAc (60:40 f 35:65) to afford the
hexadecamethyl ether 9 (279 mg) as a yellow crystalline
powder; positive ESIMS m/z 1309 [M + H]+, 1331 [M + Na]+,
Ack n ow led gm en t. We thank Norbert Nieth, Organisch
Chemisches Institut, Universita¨t Heidelberg, for recording the
HRFAB MS spectra.
1
1347 [M + K]+; H NMR (acetone-d6, 500 MHz) δ 7.71, 7.02
(2H in total, each s, aromatic H), 6.85, 6.82, 6.80, 6.79, 6.77,
6.67 (3H in total, each s, aromatic H), 4.30, 4.29, 4.00, 3.98,
3.94, 3.93, 3.89, 3.88 (×2), 3.87, 3.86, 3.84, 3.83, 3.82, 3.79,
3.78, 3.77 (×2), 3.73, 3.71, 3.70, 3.66, 3.65, 3.61, 3.60, 3.42,
3.32 (each s, total 16 CH3), 5.54 (t, J ) 9.5 Hz), 5.40 (m), 5.04
(dd, J ) 3.7, 9.7 Hz), 4.99 (d, J ) 3.7 Hz), 4.89 (t, J ) 9.8 Hz),
4.81 (m), 4.60 (dd, J ) 9.0, 12.0 Hz), 4.59 (dd, J ) 8.8, 12.0
Hz), 3.02 (bd, J ) 12.0 Hz), 3.00 (bd, J ) 12.0 Hz) (12H in
total, R/â sugar protons).
Su p p or tin g In for m a tion Ava ila ble: Structures of 1-8, part of
the GHMBC spectrum of 1, derivatization and degradation scheme of
1 and CD spectrum of 11. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) von Maydell, H.-J . Trees and Shrubs of the Sahel; Verlag J oseph
Margraf: Weikersheim, 1990; pp 63-76 and 393.
(2) le Grand, A.; Wondergem, P. A.; Verpoorte, R.; Pousset, J . L. J .
Ethnopharmacol. 1988, 22, 25-31.
(3) Silva, O.; Ferreira, E.; Vaz Pato, M.; Gomes, E. Int. J . Pharmacogn.
1997, 35, 323-325.
(4) Sandberg, F.; Cronlund, A. J . Ethnopharmacol. 1982, 5, 187-204.
(5) Nongonierma, R.; Proliac, A.; Raynaud, J . Pharmazie 1987, 42, 871-
872.
Alk a lin e Meth a n olysis of 9. A solution of 9 (110 mg) in
dry methanol (50 mL) was treated dropwise with 5% sodium
methoxide to a final sodium methoxide concentration of 0.04%
in the reaction mixture. After 16 h at room temperature the
reaction mixture was neutralized with acetic acid, filtered, and
concentrated under reduced pressure. Chromatography of the
residue on Si gel (20 g) with petroleum ether-EtOAc (50:50
f 35:65) yielded the colorless oily dimethyl-(S)-hexamethoxy-
diphenoate (10) (17 mg) and nonamethylisoterchebulic acid
dimethyl ester (11) (14 mg) as a pale yellow powder. Compound
(6) Nongonierma, R.; Proliac, A.; Raynaud, J . Pharmazie 1988, 43, 293.
(7) Nongonierma, R.; Proliac, A.; Raynaud, J . Pharm. Acta Helv. 1990,
65 (8), 233-235.
(8) Noguetra, L.; de Almeida e Silva, L.; Correira Alves, A. Garcia Orta
1962, 10, 501-509; Chem. Abstr. 1964, 61, 12326.
(9) Silva, O.; Ferreira, E.; Romao, N.; Franco, S.; Porem, L.; Esmeraldo,
N.; Vaz Pato, M.; Gomes, E. T. In Book of Abstracts of the J oint
Meeting of the American Society of Pharmacognosy; GA, Phytochemi-
cal Society of Europe and AFERP: Amsterdam, 1999; abstract 429.
(10) Idemudia, O. G. Phytochemistry 1970, 9, 2401-2402.
(11) Conrad, J .; Vogler, B.; Klaiber, I.; Roos, G.; Walter, U.; Kraus, W.
Phytochemistry 1998, 48 (4), 647-650.
10: [R]25 -25° (c 1.69, CHCl3); CD (c 8.89 × 10-5, CH3CN) θ
D
(nm) -4.33 × 104 (248), +0.79 × 104 (308); H NMR (CDCl3,
1
300 MHz) δ 7.37 (2H, s, aromatic H), 3.94, 3.93, 3.60 (×2),
(each s, 8 CH3); positive ESIMS m/z 451 [M + H]+. Compound
11: [R]26D -33.9° (c 0.36, CHCl3); UV (CH3CN) λmax (log ꢀ) 214
(4.72), 254 (4.79), 370 (4.21) nm; CD (c 5.13 × 10-5, CH3CN) θ
(nm) -4.37 × 104 (242), +0.21 × 104 (257), -1.57 × 104 (270),
+1.33 × 104 (312); 1H and 13C NMR, see Table 3; positive
ESIMS m/z 815 [M + Na]+.
(12) Conrad, J .; Vogler, B.; Klaiber, I.; Reeb, S.; Guse, J .-H.; Roos, G.;
Kraus, W. Nat. Prod. Lett. 2000, 15, 35-43.
(13) J ossang, A.; Pousset, J . L.; Bodo, B. J . Nat. Prod. 1994, 57, 732-
737, and references therein.
4,6-O-(S)-Isoter ch ebu loyl-D-glu cose (2): tan amorphous
(14) Doig, A. J .; Williams, D. H.; Oelrichs, P. B.; Bacynskyi, L. J . Chem.
Soc., Perkin Trans. 1 1990, 2317-2321, and references therein.
(15) Tanaka, T.; Nonaka, G. I.; Nishioka, I. Chem. Pharm. Bull. 1986,
34, 1039-1049.
(16) Tanaka, T.; Nonaka, G. I.; Nishioka, I. Chem. Pharm. Bull. 1986,
34, 650-655.
(17) Tanaka, T.; Nonaka, G. I.; Nishioka, I. Chem. Pharm. Bull. 1986,
34, 656-663.
(18) Lin, T. C.; Nonaka, G. I.; Nishioka, I.; Ho, F. C. Chem. Pharm. Bull.
1990, 38, 3004-3008.
powder; mp (H2O) 212-215 °C (decomp); [R]25 -204° (c 0.5,
D
MeOH); UV (MeOH) λmax (log ꢀ) 220 (4.70), 262 (4.70), 378
(4.10) nm; CD (c 2.5 × 10-5, MeOH) θ (nm) -7.66 × 104 (218),
+5.83 × 104 (250), -0.80 × 104 (298), -1.20 × 104 (372); IR
(KBr) νmax 3413, 1723, 1608, 1521, 1459, 1435, 1362, 1178,
1
1059, 1017, 963, 881, 837, 771, 723 cm-1; H and 13C NMR,
see Tables 1 and 2; negative ESIMS and FABMS m/z 781 [M
- H]-; HRFABMS m/z 781.0554 [M - H]- (calcd for C34H21O22
requires 781.0516).
(19) Li, X.-C.; Elsohly, H. N.; Hufford, C. D.; Clark, A. M. Magn. Reson.
Chem. 1999, 37, 856-859.
An tim icr obia l Activity. Samples in serial dilutions [start-
ing concentration 1024 µg/mL, dilution factor 1/2n, two repli-
cates for each concentration] were tested in microdilution
assays according to established protocols20 using B. subtilis
and P. fluorescens as test organisms. The â-lactam antibiotic
Cefotaxim was used as a positive control (MIC 2 µg/mL).
An th elm in th ic Activity. The bioassay against C. elegans
was performed according to Simpkin and Coles.21 Compounds
were tested at 50 and 100 µg/mL (n ) 60, in two replicates for
each concentration), and biotests were evaluated by assessing
the increase in number of worms and their movement.11 The
anthelminthic Pyrantelembonate was used as a positive
control with strong activity (+++) at 50 µg/mL.
(20) (a) Vanden Berge, D. A.; Vlietinck, A. J . In Methods in Plant
Biochemistry; Dey, P. M., Harborne, J ., Eds.; Academic Press:
London, 1991; Vol. 6, pp 52-57. (b) ‘‘Arbeitsausschuss chemothera-
peutischer Untersuchungsmethoden‘‘ des Normenausschusses Mediz-
in (NAMed); DIN 58940, part 8. In Medizinische Mikrobiologie und
Immunologie; DIN pocket book 222; Beuth Verlag: Berlin, Ko¨ln, 1991.
(21) Simpkin, K. G.; Coles, G. C. J . Chem. Technol. Biotechnol. 1981, 31,
66-69.
(22) (a) Moser, D.; Klaiber, I.; Vogler, B.; Kraus, W. Pestic. Sci. 1999, 55,
336-339. (b) Nakanishi, K.; Kubo, I. Isr. J . Chem. 1977, 16, 28-31.
(c) Marston, K.; Hostettmann, K. In Methods in Plant Biochemistry;
Dey, P. M., Harborne, J ., Eds.; Academic Press: London, 1991; Vol.
6, pp 153-178.
(23) Balza, F.; Abranowski, Z.; Towers, G. H. N.; Wiryachitra, P. Phy-
tochemistry 1989, 23, 1827-1830.
(24) Osborne, C. K.; Hobbs, K.; Trent, J . M. Breast Cancer Res. Treat.
1987, 9 (2), 111-121.
(25) Setzer, M. C.; Setzer, W. N.; J ackes, B. R.; Gentry, G. A.; Moriarity,
D. M. Pharm. Biol. 2000, in press.
Mollu scicid a l Activity. Molluscicidal properties of the
compounds against B. glabrata were examined using a rapid
screening method.22 Samples were tested at 100 µg/mL (n )
6, in three replicates). In case of toxicity LC100 were determined
by serial dilutions (80, 60, 40, 20, 10 µg/mL) of the active
NP000506V