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Joseph et al., Afr. J. Trad. CAM (2007) 4 (4): 383 – 386
383
Research Communication
ISSN 0189-6016©2007
ISOLATION OF A STILBENE GLYCOSIDE AND OTHER CONSTITUENTS OF TERMINALIA
SERICEAE
Joseph, C. C.*1, Moshi, M.J.2, Innocent, E.2 and Nkunya, M. H. H.1
1Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania;
2Institute of Traditional Medicine, Muhimbili University College of Health Sciences
University of Dar es Salaam, P.O. Box 65005, Dar es Salaam, Tanzania
*E-mail: Cosam@chem.udsm.ac.tz
Abstract
The ethanol extract of the root bark of Terminalia sericea yielded an unreported stilbene glycoside,
3'5'-dihydroxy-4-(2-hydroxy-ethoxy) resveratrol-3-O-β-rutinoside (1) together with known compounds
resveratrol-3-β-rutinoside glycoside (2), 3',4,5'-Trihydroxystilbene (resveratrol) (3), triterpenoic acid arjungenin
and a mixture of β-sitosterol and stigmasterol. Structure determination of the isolated compounds was achieved
on the basis of spectroscopic measurements.
Key words: Terminalia sericea, stilbenes, combretastatin, resveratrol, glycosides.
Introduction
Terminalia sericea Burch. Ex. DC (Combretaceae) is widely distributed in the tropical and warm
temperate regions, especially in Africa (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). The plant
has wide spread traditional uses in eastern and southern African countries (Arnold and Gulumian, 1984;
Kokwaro, 1976; Msonthi and Magombo, 1983). Among some of its proven biological activities, include
inhibition of topoisomerase II (Wall et al., 1996), antibacterial, and antifungal activity (Eloff, 1999; Fyhrquist et
al., 2002; 2004). However, there is scanty information on compounds that are responsible for the exhibited
biological activities of the plant. The stilbene glycoside, resveratrol-3-O-β-rutinoside (2) (Bombardelli et al.,
1975), and pentacyclic triterpenes of the oleanane skeleton, together with their glucosides (Bombardelli et al.,
1974; 1975) have been isolated. This includes also a triterpene, sericoside (Maeda and Fukuda, 1996). Skin
lightening preparations containing sericoside have been patented in Japan (Maeda and Fukuda, 1996). A β-D-
glucoside (4) of resveratrol was isolated from wine, a compound that is considered to be responsible for the
protective ability of wines against coronary heart disease (Jeandet et al., 1991).
We hereby report the isolation of a an unreported stilbene glycoside 1, resveratrol-3-O-β-rutinoside (2),
resveratrol (3) and other known compounds such as sericic acid, stigmasterol, β-sitosterol and triterpenoid acid
were also isolated from the ethanol extract of the root bark of T. sericeae occurring in Tanzania.
Materials and Methods
Materials
Petroleum ether, dichloromethane, ethyl acetate (EtOAc), methanol (MeOH), chloroform (CHCl3), and
ethanol were purchased from Fisher Scientific UK Ltd (Bishop Meadow Road, Loughborough, Leicestershire,
LE 11 5RG, UK). Precoated silica gel plates (60 F254, 250 µm) were purchased from Merck and sephadex LH-
20 from Pharmacia.
Afr. J. Traditional,
Complementary and
Alternative Medicines
www.africanethnomedicines.net
Joseph et al., Afr. J. Trad. CAM (2007) 4 (4): 383 – 386
384
1: R =
2: R = H 3
13
5
3'
5' αβ
HO
H
HOH
OH
A
B
OH
1"
2"
13
5
3'
5' αβ
HO
H
HOR
O
A
B
OO
O
OH
OH
OH
OHOH
HO
CH3
OH
HO
O
O
HO
HO
OH
4
OH
Collection of Plant materials
The root barks of T. sericea were collected from Iringa region, and identified by Mr. Frank Mbago.
Voucher specimen no. IMPP 001-0144 is kept in the Herbarium of the Institute of Traditional Medicine,
Muhimbili University College of Health Sciences, University of Dar es Salaam.
Extraction and isolation
Air-dried and powdered plant material (2 Kg) was cold extracted sequentially in petroleum ether (2.0 L;
40-60˚C), dichloromethane (2.0 L) and then ethanol (2.0 L). The amounts of both the petroleum ether and
dichloromethane extracts were very small to the extent that these extracts could not be worked on further. The
ethanol extract was then fractionated by vacuum liquid chromatography (VLC) eluting with a mixture of
EtOAc/petroleum ether followed by MeOH/EtOAc to give 4 bulked fractions. Thin layer chromatography (TLC)
was performed on pre-coated plates (silica gel 60 F254, 250 µm on polyester backing, Merck) eluting with
mixtures consisting of petroleum ether (b.p. 40-60˚C) and EtOAc; detection by UV and an anisaldehyde spray
reagent (Jean, 1996). Column chromatography on silica gel 60 mesh (0.063-0.2 mm, Merck) eluting with
mixtures of increasing polarity consisting of petroleum ether and EtOAc. Gel filtration was performed on
Sephadex® LH-20 (Pharmacia) eluting with MeOH or a mixture of MeOH/CHCl3. VLC was performed on
silica gel 60 (Merck), eluting with petroleum ether containing increasing amounts of EtOAc. Recrystallisation
was achieved from MeOH/CHCl3, 9:1 v/v. Flash chromatography (FC) employed Merck silica gel 60 (0.063-0.2
mm, Merck) and petroleum ether (b.p. 40-60˚C)-EtOAc mixtures. Repeated column chromatography on silica
gel of fraction 1 yielded compound 2 and 3. Compound 1 was obtained from fraction 4 after repeated column
chromatography followed by gel chromatography over Sephadex® LH-20 eluting with a mixture of methanol
and water (1:1 v/v).
Spectra
1H NMR spectra were recorded on a Bruker AM 400 spectrometer operating at 400 MHz. CD3OD, CDCl3
or d6-DMSO were used as solvents. Chemical shifts are given in δ values relative to the internal standard TMS
(δ = 0). Mass spectra were determined by direct inlet on a VG 7070 E instrument at 70 eV.
Results
The unreported stilbene glycoside, 3΄5΄-dihydroxy-4-(2 hydroxy-ethoxy) resveratrol-3-O-β-rutinoside (1), was
isolated as a reddish gum.1H-NMR (CDCl3): δ 7.49 (1H, d, J = 8.6 Hz, H-2 or H-6), 6.99 (1H, d, J = 16.3 Hz,
Joseph et al., Afr. J. Trad. CAM (2007) 4 (4): 383 – 386
385
H- α), 6.85 (1H, d, J = 16.3 Hz, H-β), 6.74 (1H, d, J = 2 Hz, H-2’), 6.66 (1H, d, J = 2 Hz, H-6΄), 6.47 (1H, d, J
= 2 Hz, H-4΄), 4.90 (1H, d, H = 1΄΄΄), 4.75 (1H, d, H = 1΄΄), 3.90 (1H, dd, J = 1.61 Hz, 3.4 Hz, H = 4΄΄), 3.71
(1H, m, H = 3΄΄΄), 3.68 (2H, m, H-1΄΄΄΄), 3.58 (2H, m, H-2΄΄΄΄), 3.56 (1H, dd, J = 3.8 Hz, 5.4 Hz, H-3΄΄), 3.46
(1H, d, J = 5.7 Hz, H-2΄΄), 3.44 (1H, m, H-2΄΄΄), 3.35 ( 1H, m, H-4΄΄΄), 1.22 (d, d, J = 6.15 Hz, Me-6΄΄΄). 13C-
NMR (CDCl3): δ 159.32 (C-3΄), 158.48 (C-5΄), 157.41 (C-4), 140.39 (C-1΄), 129.29 (C-β), 127.98 (C-2/6),
125.64 (C-C-a), 115.58 (C-3/5), 107.16 (C-C-2΄), 106.76 (C-C-6΄), 103.10 (C-C-4΄), 101.33 (C-1΄΄΄), 101.15 (C-
1΄΄), 76.93 (C-3΄΄΄), 75.85 (C-5΄΄), 73.94 (C-2΄΄), 73.08 (C-4΄΄΄), 72.49 (C-1΄΄΄΄), 71.35 (C-3΄΄΄), 71.08 (C-4΄΄),
70.33 (C-2΄΄΄), 68.82 (C-5΄΄΄), 66.60 (C-6΄΄), 61.24 (C-2΄΄΄΄), 16.93 (C-6΄΄΄). EIMS m/z (rel. int.): 580 [M]+
(C28H36O13), 564 (55), 537 (100), 391 (15), 375 (20), 229 (10). Compounds 2, 3 and 4 were also isolated.
Compound 2, resveratrol-3-O-β-rutinoside, exhibited a weak antibacterial activity against Escherichia coli at 4
mg/ml. The compound was not tested on the other organisms due to its paucity.
Discussion
Compound 1 was obtained as a red gum. Its structure was established on the basis of extensive
spectroscopic analysis, in particular H/H-COSY, HMQC, HMBC and MS measurements, as well as from
comparison of the observed spectral data with those reported in the literature for the main skeleton (Bombardelli
et al., 1975., Breitmaier and Voelter, 1989., Dorman et al., 1970). Thus, the aromatic region of the 1H and 13C
NMR spectra exhibited features which were virtually the same as those observed for the stilbene moiety in
resveratrol-3-O-β-rutinoside (2) and this indicated the presence of a resveratrolyl unit in structure 1, as was
further also confirmed by the MS which consisted of a peak at m/z 228 due to the fragment ion corresponding to
3. Furthermore, the 1H NMR spectrum displayed two doublet at δ 4.94 and 4.75 which were assigned to
protons at anomeric carbon atoms, thereby indicating the presence of a disaccharide unit that consisted of a
rhamnoside and a glucoside unit, whereas the methyl carbon signal for the rhamnose unit was observed at δ
16.93 (Bombardelli et al., 1975., Breitmaier and Voelter, 1989., Dorman et al., 1970). The α-L-O-glycosidic
linkage between the two sugar units was indicated from the observed J values as well as the 13C NMR
resonances of the anomeric carbon atoms, which appeared at δ 101.18 and 101.33, respectively (Kasai et al.,
1977; 1979). The 1H and 13C NMR spectral features for the disaccharide unit were virtually the same as those
observed in the spectra of 2 and this indicated that, like in 2, glycoside 1 contained a rutinoside residue
(Bombardelli et al., 1975., Breitmaier and Voelter, 1989., Dorman et al., 1970). The 13C NMR spectrum with
DEPT 135 measurements of compound 1 indicated signals due to 14 carbon atoms in the region δ 16-101. Of
these signals, 12 were characteristic resonances observed for the rutinoside sugar residue in compound 2 as
reported in the preceeding discussion, also corresponding to literature data for the rutinoside 13C NMR
resonances for compound 2 (Bombardelli et al., 1975., Breitmaier and Voelter, 1989. Dorman et al., 1970). The
remaining two 13C NMR signals at δ 61.24 and 72.49 were attributed to a dioxymethylene group. These
spectral features, as well as the positions of the 13C NMR resonances in the high field region being comparable
to those observed for the resveratrolyl unit in compound 2 but having only slight differences led to the
conclusion that compound 1 must be having structural features similar to those of 2, the former compound
however bearing an additional hydroxyethane group. Addition of a two carbon atom in compound 1 as
compared to compound 2 was also indicated by the MS which exhibited a molecular ion peak at m/z 580, which
is 44 amu higher than the M+ peak for glycoside 2 (m/z 536). The additional 44 amu could be accounted for by
considering the presence of an additional CH2CH2O unit in compound 1.
As for compound 2, the position of the rutinoside sugar residue in glycoside 1 was deduced based on
the upfield shift of the 13C NMR resonances for C-3' and C-5' in ring A as compared to the corresponding
signals in the spectrum of 3. The position of the rutinoside sugar residue was further corroborated by long range
H/C interactions involving the anomeric proton at C-1'' as revealed in the HMBC spectrum.
Establishment of the position of the hydroxyethane group at C-4 was facilitated by considering the slight upfield
shift of the 13C NMR resonance for C-4 in compound 3 as compared to the corresponding signals in the
spectrum of glycoside 2. Apparently, the substitution could also be at C-5' in ring B which makes structure 1 to
be only tentative.
The MS of 1 consisted of the molecular ion peak at m/z 580 and a fragment ion peak at m/z 536 which
corresponds to the M+ peak for compound 2, being formed through rearrangement of 1 followed by extrusion of
Joseph et al., Afr. J. Trad. CAM (2007) 4 (4): 383 – 386
386
acetaldehyde. Cleavage of the sugar residue yielded a fragment ion at m/z 229 due to the resveratrolyl unit. The
MS fragmentation pattern of compound 1 is consistent with the proposed structure.
Acknowledgements
Financial support by Swedish International Development Agency (SIDA)/Department of Research
Cooperation (SAREC) within an overall research support to the Faculty of Science at the University of Dar es
Salaam is gratefully acknowledged. We also thank Prof. Berhanu M. Abegaz of the University of Botswana for
availing spectral analytical facilities to us. One of the authors (IE) thanks the Vlaamse Interuniversitaire Raad-
Flemish Interuniversity Council (VLIR) for fellowship.
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