Phenolics from Maytenus senegalensis

Article abstract of DOI:10.1016/S0031-9422(98)00571-8

Two new methylated flavan-3-ol glucosides and a methylated proanthocyanidin were isolated from the MeOH extract of the stem-bark of Maytenus senegalensis, together with five known compounds. The structures of the new compounds were determined as: (-)-4'-m

Full text of DOI:10.1016/S0031-9422(98)00571-8

PERGAMON
Phytochemistry 50 (1999) 689±694
Phenolics from Maytenus senegalensis
Ghazi Hussein, Norio Nakamura, Meselhy R. Meselhy, Masao Hattori *
Research Institute for Traditional Sino-Japanese Medicines, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194,
Japan
Received 8 May 1998; Revised 13 July 1998
Abstract
Two new methylated ¯avan-3-ol glucosides and a methylated proanthocyanidin were isolated from the MeOH extract of the
stem-bark of Maytenus senegalensis, together with ®ve known compounds. The structures of the new compounds were
0
0
0
determined as: (�)-4 -methylepigallocatechin 5-O-b-glucopyranoside, (+)-4 -methylgallocatechin 3 -O-b-glucopyranoside and
0
(
�)-epicatechin (4b 4 8) (�)-4 -methylepigallocatechin by chemical and spectroscopic means. The MeOH and H
2
O extracts
showed moderate inhibitory e€ects against HIV-1 protease. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Maytenus senegalensis; Celastraceae; Methylated ¯avan-3-ols; Proanthocyanidins
1. Introduction
MeOH and H O extracts of M. senegalensis showed
2
moderate inhibitory e€ects against HIV-1 protease
with IC50 values of 104 and 88 mg/ml respectively
(Otake, Mori, Morimoto, Ueba, Sutardjo et al., 1995),
whereas acetylpepstatin, as positive inhibitory control,
showed an IC50 of 0.24 mM.
We describe here the isolation and structure elucida-
tion of new methylated ¯avanol glucosides (2, 4) and a
methylated proanthocyanidin (6) from the MeOH
extract of the stem-bark of M. senegalensis.
The aqueous extract of the stem-bark of Maytenus
senegalensis (Celastraceae) is an important herbal
remedy in folk medicine in the Sudan. Its vernacular
name is ``Dabalab'' and it has been widely used to
treat tumors, dysentery and snake-bites. Most of the
chemical work described in the literature has been con-
®
ned to other Maytenus species (Kupchan & Smith,
1977; Bruning & Wagner, 1978; Shirota, Tamemura,
Morita, Takeya & Itokawa, 1996), with little work on
È
M. senegalensis (Tin-Wa, Farnsworth, Fong, Blomster,
2
. Results and discussion
Troja'nek et al., 1971; Abraham, Troja'nek, Mu
Fong Farnsworth, 1971; Gomez-serranillos
Zaragoza, 1979; Delle Monache, Pomponi, Marin-
È
nzing,
&
Â
Â
&
The defatted MeOH extract of the stem-bark of M.
senegalensis was partitioned between EtOAc and H O
to give EtOAc-soluble and H O-soluble fractions.
2
Repeated CC of the EtOAc-soluble fraction a€orded
®ve compounds (1, 3 and 5±7) (Fig. 1). Likewise, the
H O-soluble fraction a€orded three compounds (2, 4
Bettolo, Leoncio D'Albuquerque & Goncalves de
0
2
Lima, 1976). (�)-4 -Methylepigallocatechin, (�)-epiafz-
0
elechin (4b 4 8) (�)-4 -methylepigallocatechin (oura-
tea-proanthocyanidin) and maytenonic acid have been
ning & Wagner, 1978;
isolated from this species (Bru
È
2
0
and 8) (Fig. 1). The known compounds were (�)-4 -
Abraham et al., 1971; Delle Monache et al., 1976).
Ethanolic extracts of the stems of M. senegalensis
demonstrated cytotoxic e€ects against carcinoma in
cell cultures and leukemia in mice (Tin-Wa et al.,
methylepigallocatechin (1) (Drewes
1
& Mashibaye,
993), (�)-epigallocatechin (3), epicatechin (4b 4 8)
epigallocatechin (5) and epicatechin (4b 4 8) epicate-
chin (procyanidin B-2) (7) (Nonaka, Kawahara &
Nishioka, 1983; Nonaka, Hsu & Nishioka, 1981;
Porter, Newman, Foo, Wong & Hemingway, 1982;
Hashimoto, Nonaka & Nishioka, 1989), and phloro-
glucinol 1-O-b-D-glucopyranoside (8), and identi®-
1971). Upon antiviral screening of medicinal plants,
* Corresponding author. Fax: +81-764-34-5060; E-mail: sai-
bo421@ms.toyama-mpu.ac.jp
0031-9422/98 $ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00571-8

6
90
G. Hussein et al. / Phytochemistry 50 (1999) 689±694
Fig. 1. Compounds 1±9.
cation of the three new compounds (2, 4 and 6) are
described below.
The atmospheric pressure ionization (API) mass
tern for ring B (two protons as a singlet at d 6.60), a
sugar moiety (anomeric proton signal at d 4.87), and a
methoxy group (singlet at d 3.80, 3H). The singlet at d
4.84 (H±2) (C±2 at d 79.3) and the multiplet at d 4.20
(H±3) (C±3 at d 66.7) are characteristic for the 2,3-cis
con®guration of the ¯avan-3-ol unit (Shen, Chang &
Ho, 1993; Markham & Ternai, 1976). The fragment
spectrum (positive ion mode) of 2 showed a molecular
+
ion peak at m/z 483 [M +H]
molecular formula C H O . The H NMR spectrum
consistent with the
1
22
26 12
showed signals characteristic for a ¯avan-3-ol skeleton
with a phloroglucinol pattern for ring A (two doublets
at d 6.07 and 6.29, J= 2.2 Hz) and a pyrogallol pat-
+
ion peak at m/z 321 [M +H� 162] suggested a hex-
ose unit which was identi®ed as glucose by comparing

G. Hussein et al. / Phytochemistry 50 (1999) 689±694
691
Table 1
13
C NMR spectral data of compounds 1±4 and 8
a
1
b
2
a
3
a
4
a
8
Position
1
2
3
4
160.8
96.7
79.6
67.3
29.1
79.3
66.7
28.8
79.8
67.4
29.1
82.1
68.4
27.6
160.0
98.0
4
5
6
7
8
8
1
2
3
4
5
6
1
2
3
4
5
6
a
100.0
157.9
96.3
102.2
158.1
97.8
100.0
157.9
96.3
100.6
157.7
96.4
160.0
96.7
157.5
95.8
157.6
96.5
157.6
95.8
157.5
95.5
a
0
157.1
135.9
107.1
151.3
136.5
151.3
107.1
156.6
136.1
107.0
150.9
136.1
150.9
107.0
102.0
74.6
157.2
131.4
106.9
146.6
133.5
146.6
106.9
156.5
136.7
107.4
152.0
137.9
151.6
110.0
102.6
74.8
0
0
0
0
0
�
�
�
�
�
�
102.0
74.7
77.9
71.3
77.9
62.5
78.6
71.3
77.0
71.0
79.0
62.7
77.9
61.5
OCH
3
60.7
60.6
62.3
a
b
3 3 3 2
Measured at 125 MHz (in CD OD and CD COCD ±D O [2:1] )
1
3
0
the C NMR spectral data of the sugar carbons with
those reported for methyl O-glucosides (Agrawal &
Bansal, 1989). These ®ndings suggested that 2 was the
glucoside of 1 (Drewes & Mashibaye, 1993) (Table 1).
This was further shown by enzymatic hydrolysis of 2,
which gave compound 1. Signi®cant HMBC corre-
lations between C±5 (d 158.1) and the proton signals
at d 4.87 (H±1�) and 6.29 (H±6), linked the glucose
unit to C±5, whereas C±7 was correlated with both H±
whereas enzymatic hydrolysis of 4 a€orded (+)-4 -
methylgallocatechin (Plazzo De Mello, Petereit &
0
Nahrstedt, 1996). The down®eld shift of C±3 (d
152.0) and the up®eld shift of C±2 (107.4), when com-
0
0
pared with that of C±6 (110.0), suggested glycosyla-
0
tion at C±3 (Table 1). This was further con®rmed by
0
the HMBC correlations C±3 (d 152.0) with H±1� (d
0
0
4.68) and H±2 (6.61). Long range correlations C±4 (d
0
137.9) with the methoxy protons (d 3.84) and H±2 ,
connected the methoxy group to C±4 . From these
0
6
and H±8. Correlations between the carbon signal at
0
0
d 136.1 (C±4 ) and the proton signals at d 3.80 (3H, s)
and 6.60 (2H, s), placed the methoxyl group at C±4 .
data, compound 4 was determined to be (+)-4 -
0
0
methylgallocatechin 3 -O-b-glucopyranoside.
The positive ion API-mass spectrum of 6 revealed a
The coupling constant (J =7.2 Hz, H±1�) was indica-
tive of a b-anomeric con®guration of the glucose unit.
+
molecular ion peak at m/z 631 [M + Na] consistent
0
Thus, compound 2 was determined to be (�)-4 -methy-
with the molecular formula C H O . The NMR
28 13
31
lepigallocatechin 5-O-b glucopyranoside.
Compound 4 was assigned the molecular formula
data of 6 showed features characteristic for ¯avan-ols
of proanthocyanidin dimers similar to that of 5
(Table 2) (Nonaka et al., 1981; Porter et al., 1982;
Hashimoto et al., 1989), and a methoxyl group (dH
3.76/dC 60.7). A pair of singlets at d 4.58 and 5.10 of
H±2 and that of multiplets at d 3.91 and 4.29 of H±3,
were characteristic for two ¯avan-3-ol units with epica-
techin stereochemistry (C2, C3: cis) (Nonaka et al.,
1981; Nonaka, Kawahara & Nishioka, 1982). The
broad singlets at d 5.98 and 6.02 were assigned for H±
8 (upper = u) and H±6u, respectively, and the singlet
at d 5.91 was assigned for H±6 (lower = l). The singlet
at d 6.71 (2H) suggested a pyrogallol hydroxylation of
C H O on the basis of the API-mass spectrum (m/z
26 12
2
2
+
at 483 [M +H] ). The doublet (J= 7.5 Hz) at d 4.82
H±2) and carbon signals at d 68.4 (C±3) and 82.1 (C±
) (Table 1) suggested a ¯avan-3-ol skeleton with 2,3-
trans con®guration (Porter et al., 1982). Signals
characteristic for a hexose moiety (anomeric proton at
(
2
+
d 4.68, fragment ion at m/z 321 [M +H� 162] ) and
a methoxyl group (d 3.84, 3H, s) were also observed in
the H NMR spectrum (see experimental). The hexose
1
moiety was identi®ed as glucose with the b-anomeric
con®guration (J =6.8 Hz) (Agrawal & Bansal, 1989),

6
92
G. Hussein et al. / Phytochemistry 50 (1999) 689±694
Table 2
3
. Experimental
13
a
C NMR spectral data of compounds 5±7
Position
5
6
7
3
.1. General
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
(u)
(u)
(u)
a(u)
(u)
(u)
(u)
(u)
a(u)
77.0
73.4
37.1
76.9
73.1
36.9
77.1
73.4
37.1
Optical rotations were taken with a JASCO DIP-360
automatic polarimeter. UV: MeOH (UV-2200,
SHIMADZU, Japan). IR: KBr (FT/IR-230, JASCO,
Japan). NMR spectra were measured with a VARIAN
100.5
158.2
97.3
100.4
158.1
96.5
100.5
158.3
96.3
1
13
UNITY 500 ( H� , 500 MHz; C� , 125 MHz) or a
157.8
96.1
157.6
96.1
157.8
96.2
1
13
JEOL JNA-LA 400WB-FT ( H� , 400 MHz; C� ,
1
00 MHz) spectrometers. The atmospheric pressure
156.3
131.1
115.2
145.5
145.8
115.9
119.2
78.8
156.3
132.2
115.1
145.4
145.7
115.8
119.2
79.2
156.4
132.0
115.2
145.6
145.8
115.9
119.4
78.8
0
(u)
(u)
ionization (API) mass spectra were measured with a
PE SCIEX API III biomolecular mass analyzer. TLC:
precoated silica gel 60 F254 and RP-18 F254S plates
(0.25 mm, MERCK) developed with solvent system, A
(benzene±ethyl formate±formic acid, 1:7:1), and B
0
0
0
0
(u)
(u)
(u)
(u)
0
(l)
(l)
(l)
a(l)
(l)
(l)
(l)
(l)
a(l)
(acetonitrile±0.1% tri¯uoroacetic acid/H O, 3:7), re-
2
66.7
29.8
66.6
29.7
66.9
29.7
spectively. Detection of the spots: by UV and anisalde-
hyde/H SO4 and ferric chloride reagents. CC:
2
100.5
158.2
97.5
100.4
158.1
97.3
100.5
158.3
96.5
Sephadex LH-20 (PHARMACIA, Sweden), Diaion
HP-20 and MCI gel CHP-20P (MITSUBISHI,
Tokyo, Japan) and Silica gel 60 (70±230 mesh,
MERCK). MPLC: LiChroprep RP-18 (size A,
MERCK).
157.8
106.8
156.3
132.5
106.8
146.6
133.4
146.4
106.8
157.6
106.8
156.3
135.8
106.8
151.1
136.1
151.1
106.8
60.7
157.8
107.3
156.4
132.5
115.9
145.6
145.8
115.2
119.4
0
0
0
0
0
0
(l)
(l)
(l)
(l)
3
.2. Isolation of 1±8
(l)
(l)
The powdered stem-bark (2.8 kg) of M. senegalensis,
±
OCH
3
collected in December 1995 from Arkowit district,
East of Sudan, was extracted with MeOH (2lÂ2) at
room temp. and then by re¯ux (1.5lÂ2). The MeOH
was concentrated to 300 ml and shaken with hexane
(600 ml). The MeOH layer was then evaporated under
a
Measured in CD
3
OD at 100 MHz.
ring B, similar to that of 2, while an ABX-like spin
system at d 6.66, 6.73 and 6.89 suggested a B-ring of
catechol pattern. Twin carbon signals at d 29.7 and
red. press., suspended in H O (500 ml) and shaken
2
with an equal volume of EtOAc. The EtOAc layer was
evaporated to dryness to give the EtOAc-soluble frac-
tion (10.5 g) which was further fractionated by CC/
Sephadex LH-20 (EtOH, H O to MeOH) to give 6
2
fractions. CC/silica gel (CHCl ±MeOH, 8:2) of fr. 2
3
a€orded 1 (1.87 g). CC/MCI gel (30% aq. MeOH) of
fr. 3 followed by silica gel CC (eluted with CHCl3±
MeOH±H O, 6:4:1) gave 5 (80 mg) and 7 (29 mg).
2
Similarly, fr. 4 a€orded 3 (24 mg) and 6 (186 mg).
The aqueous layer was concentrated and passed
through a column of Diaion HP-20. Elution was
3
6.9 assigned for the lower (l) and upper (u) C±4, re-
spectively. From these ®ndings, 6 was determined to
be composed of epicatechin, epigallocatechin units and
a methoxy group. The position of a carbon±carbon
linkage between the two ¯avan units was determined
by comparison of the NMR data of 6 with those of 5.
A singlet at d 4.65 ascribed for H±4u and a carbon
shift at d 76.9 (C±2u) are characteristic for the b-link-
age of the two ¯avan units (Nonaka et al., 1982;
Morimoto, Nonaka, Chen & Nishioka, 1988). The
relatively up®eld shifts of C±4l (at d 29.7), C±8l (d
started with H O, 50% MeOH and then MeOH. CC
2
1
2
06.8) and H±2l (d 4.58) and the down®eld shift of H±
u (d 5.1) suggested a 4b 4 8 linkage, as the most fre-
of the 50% MeOH eluate (30 g) over Sephadex LH-20
(EtOH, H O to MeOH) gave ®ve fractions, fr. I±V.
2
quently encountered C±4/C±8 bonds in proanthocyani-
dins (Morimoto et al., 1988). On thiolytic degradation
using benzylmercaptan and acetic acid, 6 produced 1
and compound 9 (Nonaka, Nishioka, Nagasawa &
Oura, 1981). Accordingly, the structure of 6 was deter-
Repeated CC of fr. II over Sephadex LH-20 (70%
EtOH), silica gel (CHCl ±MeOH±H O) a€orded 8
3
2
(36 mg). Likewise, repeated CC of fr. III over
Sephadex LH-20, silica gel and then MPLC
(system B) gave 2 (35 mg) and 4 (18 mg). CC/
Sephadex LH-20 of fr. IV gave additional amount of 1
(115 mg).
0
mined to be (�)-epicatechin (4b 4 8) (�)-4 -methylepi-
gallocatechin.

G. Hussein et al. / Phytochemistry 50 (1999) 689±694
693
0
3
.3. (�)-4 -Methylepigallocatechin 5-O-b-
tral data were consistent with reported values (Plazzo
De Mello et al., 1996).
glucopyranoside (2)
0
Light brown amorphous powder, (R 0.53, in solvent
f
3.7. (�)-Epicatechin (4b 4 8) (�)-4 -
system B). [a]D �13.18 (MeOH, c 0.15). UV lmax nm
methylepigallocatechin (6)
(
n
log E): 210 (4.59), 230 sh (4.14), 270 (3.41). IR
1 1
�
cm : 3400 (OH), 1610 (arom. C.C). H-NMR
Light brown amorphous powder, (R 0.29, in solvent
f
system A). [a] +22.78 (MeOH, c 0.72). UV l
(log E): 220 (4.3), 280 (3.39). IR n
(OH), 1610 (arom. C.C). H-NMR (400 MHz,
CD OD) d: 2.82 (2H, m, H±4l), 3.76 (3H, s, ±OCH ),
3.91 (1H, br s, H±3u), 4.29 (1H, br s, H±3l), 4.58 (1H,
br s, H±2l), 4.65 (1H, br s, H±4u), 5.10 (1H, br s, H±
2u), 5.91 (1H, br s, H±6l), 5.98 (1H, br s, H±6u), 6.02
max
(
J= 16.4, 4.6 Hz, H±4ax, as in CD OD), 2.90 (1H, dd,
400 MHz, CD COCD /D O, [2:1]) d: 2.94 (1H, dd,
nm
max
3
3
2
D
�
cm : 3360
max
1
3
1
J= 16.4, 4.1 Hz, H±4eq, as in CD OD), 3.34±3.84
3
6H, H±2�� H±6�), 3.80 (3H, s, OMe), 4.20 (1H, m,
H±3), 4.84 (1H, s, H±2), 4.87 (1H, d, J =7.2 Hz, H±
(
3
3
1
�), 6.07 (1H, d, J= 2.2 Hz, H±8), 6.29 (1H, d,
13
0
0
J= 2.2 Hz, H±6), 6.60 (2H, s, H±2 and H±6 ). C-
NMR (Table 1). API-MS (positive ion mode) m/z (rel.
0
(1H, br s, H±8u), 6.66 (1H, br s, H±6 u), 6.71 (2H, s,
0 0 0
+
int.%): 505 [M + Na] (49), 483 [M +H] (17) and
+
H±2 l, H±6 l), 6.73 (1H, br s, H±5 u), 6.89 (1H, br s,
+
0
13
H±2 u). C-NMR (Table 2). API-MS (positive ion
3
21 [M + H� Glc] (44).
+
mode) m/z (rel. int.%): 631 [M + Na] (10) and 609
+
M +1] (27). [u =upper; l = lower]
[
3
.4. Enzymatic hydrolysis of 2
3
.8. Thiolysis of compound 6
Glucoside 2 (5 mg) was dissolved in 50 mM NaOAc
bu€er (pH 5.0) and incubated with crude naringinase
Sigma Chemical Co.) at 408C for 48 h. The ethyl acet-
ate-soluble portion of the mixture was puri®ed over
A mixture of 6 (24 mg) in ethanol (3 ml), benzylmer-
(
captan (0.5 ml) and acetic acid (0.5 ml) was stirred
under argon at 50±608C for 48 h. The mixture was
evaporated under red. pres. and chromatographed
over Sephadex LH-20 (benzene±EtOH) to a€ord 1
0
Sephadex LH-20 (EtOH) to a€ord (�)-4 -methylepigal-
locatechin 1 (2 mg), [a]D �23.28 (EtOH, c 0.1). Lit.
(
Delle Monache et al., 1976): [a] �60.08 (EtOH, c
D
1
.0). The H-NMR spectral data were consistent with
and (�)-epicatechin-4b-benzylthioether 9, [a] �28.98
D
1
reported values (Drewes & Mashibaye, 1993).
(
Me CO, c 0.15). Lit. (Nonaka et al., 1981): [a]
2
D
�
288(Me CO, c 1.0). API-MS (positive ion mode) m/z
2
+ 1
(
rel. int.%): 413 [M +1] (64). The H NMR spectral
data were in agreement with lit. values (Nonaka et al.,
981).
0
0
3
.5. (+)-4 -Methylgallocatechin 3 -O-b-
glucopyranoside (4)
1
Yellowish amorphous powder, (R 0.71, in solvent
f
system B). [a]D �15.28 (MeOH, c 0.15). UV lmax nm
Acknowledgements
(
log E): 210 (4.53), 230 sh (3.98), 280 (2.9). IR
1 1
�
n
cm : 3400 (OH), 1610 (arom. C.C). H-NMR
max
The authors would like to thank Prof. Sami Khalid,
Faculty of Pharmacy, University of Khartoum, Sudan,
for providing the plant samples. They are also
indebted to Dr. Gamal El-Ghazali, Medicinal and
Aromatic Plants Research Institute, National Centre
for Research, Khartoum, for the botanical identi®-
cation of the samples.
(
500 MHz, CD OD) d: 2.51 (1H, dd, J=16.4, 7.2 Hz,
3
H±4ax), 2.72 (1H, dd, J= 16.4, 5.3 Hz, H±4eq), 3.23
1H, m, H±5�), 3.42 (1H, m, H±4�), 3.44 (1H, m, H±
�), 3.74 (1H, m, H±2�), 3.70±3.74 (2H, m, H±6�), 3.84
3H, s, OMe), 3.99 (1H, m, H±3), 4.68 (1H, d,
(
3
(
J= 6.8 Hz, H±1�), 4.82 (1H, d, J =7.5 Hz, H±2), 5.91
(
1H, d, J =2.2 Hz, H±8), 5.94 (1H, d, J =2.2 Hz, H±
0
6), 6.61 (1H, d, J=1.9 Hz, H±2 ), 6.71 (1H, d,
J= 1.9 Hz, H±6 ). C-NMR (Table 1). API-MS (posi-
+
tive ion mode) m/z (rel. int.%): 505 [M + Na] (73),
0
13
References
+
+
4
83 [M + H] (12) and 321 [M + H� Glc] (10).
È
Abraham, D. J., Troja'nek, J., Munzing, H. P., Fong, H. H. S., &
Farnsworth, N. R. (1971). Journal of Pharmaceutical Society, 60,
1085±1087.
3.6. Enzymatic hydrolysis of 4
Agrawal, P. K., & Bansal, M. C. (1989). In: P. K. Agrawal, Carbon-13
NMR of ¯avonoids (pp. 283±364). Amsterdam: Elsevier.
Glucoside 4 (5 mg) was treated as for 2 to a€ord
0
Bru
Delle Monache, F., Pomponi, M., Marin-Bettolo, G. B., Leoncio
D'Albuquerque, I., Goncalves de Lima, O. (1976).
Phytochemistry, 15, 573±574.
È
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