Salacetal, an Oleanane-type Triterpene from Salacia longipes

var. camerunensis
Brice M. Mba’ninga, Bruno N. Lentab, Silvère Ngouelaa , Didérot T. Noungouéa,
Ferdinand Tantangmoa, Ferdinand M. Talontsic , Etienne Tsamoa , and Hartmut Laatschc
a Department of Organic Chemistry, Faculty of Science, University of Yaoundé 1, P. O. Box 812,
Yaoundé, Cameroon
b Department of Chemistry, ENS, University of Yaoundé 1, P. O. Box 47, Yaoundé, Cameroon
c Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstraße 2,
37077 Göttingen, Germany
Reprint requests to Dr. Bruno Ndjakou Lenta. E-mail: [email protected]
or Prof. Etienne Tsamo. E-mail: [email protected]

Z. Naturforsch. 2011, 66b, 1270 – 1274; received September 16, 2011

Phytochemical investigation of the roots of Salacia longipes var. camerunensis led to the isola-
tion of a new triterpenoid, salacetal (1), together with the known compounds mangiferin, 2-hydr-
oxy-3-oxo-D:A-friedooleanan-29-oic acid, β -sitosterol, and stigmasterol. The structure of the new
compound as well as those of the known compounds were established by means of spectroscopic
methods and by comparison with reported data.

Key words: Salacia longipes var. camerunensis, Celastraceae, Oleanane-type Triterpene, Salacetal

Introduction quiterpenes [4, 5], stilbenes [6], and triterpenes [5]. To

the best of our knowledge, no phytochemical or phar-
Salacia longipes var. camerunensis is a liana largely
macological study has been reported on the species
distributed in Africa and commonly in Cameroon and
S. longipes var. camerunensis. In a continuing search
East of Gabon [1]. Different species of the Salacia
for biologically active compounds from Cameroonian
genus have been used over the world in the treatment
medicinal plants, we have investigated the dichloro-
of several ailments. The roots of Salacia species are
methane/methanol extract of the roots of S. longipes
extensively consumed in Japan, the United States and
var. camerunensis. We report herein on the isolation
other countries as food supplement for the prevention
and the structure elucidation of a new oleanane-type
of diabetes and obesity [2]. S. reticulata, S. oblonga
triterpene, salacetal (1), along with four known com-
and S. chinensis have long been used in India, Sri
pounds.
Lanka and China in the treatment of rheumatism and
skin diseases [3]. In Tanzania, the roots of S. madagas-
Results and Discussion
cariensis are used in folk medicine for the treatment
of fever, malaria and menorrhagia [4]. Previous phyto- The dichloromethane/methanol extract of the roots
chemical investigation of plants of the genus Salacia of Salacia longipes var. camerunensis was fraction-
resulted in the isolation of quinonemethides [3], ses- ated and purified on a silica gel column to afford five

Fig. 1. Chemical structures of com-

pounds 1 and 2.

c 2011 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com
B. M. Mba’ning et al. · An Oleanane-type Triterpene from Salacia longipes var. camerunensis 1271

Table 1. 1 H (300 MHz) and 13 C (125 MHz) NMR data in played 30 carbon signals, which account for one ke-
CDCl3 of 1, and 13 C NMR data of 2 [8]. tone carbonyl (δC = 217.0), one double bond (δC =
Attribution 1 2 152.8 and 120.5), one dioxymethine (δC = 99.1), and
one oxymethine (δC = 72.1). In addition, three me-
13 C 1H 13 C

1 34.8 2.56, m 40.3

thines, nine methylenes, seven methyls and six qua-
2 33.0 1.50 (m), 2.62 (m) 34.2
3 217.0 – 217.8 ternary carbons were observed. Based on these NMR
4 47.2 – 47.7 data, compound 1 was assumed to be an olean-12-ene-
5 48.1 2.02 (m) 55.3 type triterpenoid with one hydroxy group and one ke-
6 19.7 1.64 (m) 19.7 tone carbonyl. In fact, signals at δC = 120.5 and 152.8
7 30.4 1.42 (m) 32.6
8 46.6 – 43.2 are characteristic of an olean-12-ene triterpenoid skele-
9 53.7 1.96 (d, J = 4.5 Hz) 48.5 ton [7].
10 37.8 – 37.5 The NMR data of 1 were similar to those of 3-
11 72.1 4.45 (dd, J = 4.5; 4.2 Hz) 82.0
12 120.5 5.46 (d, J = 4.2 Hz) 121.2 oxo-11β -hydroxyolean-12-ene (2) [8]. The differences
13 152.8 – 152.8 were the disappearance of the 26-methyl and the 11-
14 41.7 – 42.3 hydroxy groups in compound 1 and the appearance
15 26.7 0.86 (m), 2.04 (td, J = 13.4, 4.6 Hz) 27.9 of a dioxymethine moiety and an additional double
16 26.6 1.06 (m), 1.84 (td, J =13.4, 4.6 Hz ) 26.6
17 32.8 – 33.1 bond equivalent. The additional double bond equiva-
18 48.0 2.02 (m) 47.0 lent in compound 1 may be derived from the formation
19 46.1 0.96 (m), 1.58 (dm, J = 13.7 Hz) 46.7 of an additional ring between the 11-hydroxy and the
20 31.1 – 31.1 26-CH3 groups (δC = 16.2) of compound 2. In fact,
21 34.6 1.10 (m), 1.32 (m) 34.6
22 36.9 1.22 (m), 1.42 (m) 36.9 couplings were observed in the 1 H-1 H COSY spec-
23 27.9 1.12 (s) 26.9 trum between the oxymethine (δH = 4.45, dd, J = 4.5
24 19.4 1.10 (s) 21.5 and 4.2 Hz) and the olefinic proton (δH = 5.46, d, J =
25 19.1 1.20 (s) 18.0 4.2 Hz) and the methine proton at δH = 1.96 (1H, d,
26 99.1 5.80 (d, J = 4.2 Hz) 16.6
27 23.6 1.08 (s) 24.7
J = 4.5 Hz). A further diagnostic coupling was seen be-
28 28.7 0.83 (s) 28.5 tween the dioxymethine (δH = 5.80, d, J = 4.2 Hz) and
29 33.3 0.86 (s) 33.2 the hydroxy group (δH = 3.07, d, 4.2 Hz). In the HMBC
30 23.7 0.87 (s) 23.6 spectrum (Fig. 2) the dioxymethine signal showed cor-
OH – 3.07 (d, J = 4.2 Hz) –
relations with C-11 (δC = 72.1, oxymethine) and C-8
(δC = 47.2), confirming the existence of an additional
compounds including salacetal (1), mangiferin [21], ring in 1 (formed between the 11β -hydroxy and 26-
2-hydroxy-3-oxo-D:A-friedooleanan-29-oic acid [22], methyl groups present in 2). Additional correlations
β -sitosterol [23], and stigmasterol [24]. observed between the oxymethine proton H-11 and
Compound 1 (Fig. 1) was obtained as a color- C-13 (δC = 152.8), C-12 (δC = 120.5), C-9 (δC = 53.7)
less powder. It gave a positive response with the
Liebermann-Buchard test for triterpenes. Its molecu-
lar formula, C30 H46 O3 , with eight double bond equiv-
alents, was determined by HR-ESIMS, which showed
the quasi-molecular ion peak [M+Na]+ at m/z =
477.3342 (calcd. m/z = 477.3339 for C30 H46 O3 Na).
The 1 H NMR spectrum (Table 1) revealed the presence
of singlet signals due to seven angular methyls (δH =
0.83, 0.86, 0.87, 1.08, 1.10, 1.12 and 1.20), a doublet
of one methine proton (δH = 1.96, J = 4.5 Hz), and
a double doublet due to an oxymethine proton (δH =
4.45, J = 4.5 and 4.2 Hz). The spectrum also exhibited
three doublets due to an olefinic proton (δH = 5.46,
J = 4.2 Hz), a dioxymethine proton (δH = 5.80, J =
4.2 Hz) and a hydroxy group (δH = 3.07, J = 4.2 Hz).
The 13 C NMR (Table 1) and APT spectra of 1 dis- Fig. 2. Selected HMBC correlations of compound 1.
1272 B. M. Mba’ning et al. · An Oleanane-type Triterpene from Salacia longipes var. camerunensis

Scheme 1. Characteristic fragment

peaks in the EI-MS spectrum of
compound 1.

and C-10 (δC = 37.8) confirmed the position of the

double bond. Cross peaks were also observed between
Me-23 (δH = 1.10) and C-3 (δC = 217.0), indicating
the location of the carbonyl group at C-3. This was fur-
ther confirmed by a characteristic fragment ion peak at
m/z = 191 (Scheme 1) obtained by a retro Diels-Alder
fragmentation in the EIMS spectrum of 1. In addition,
the base peak observed at m/z = 408 [M–HCOOH]+
due to loss of formic acid confirmed the presence of a
hemi-acetal group in compound 1. Fig. 3. Orientation of the hemi-acetal group in compound 1.
Studies relative to the orientation of the oxygenated
group at C-11 in several 11-hydroxy and -methoxy 12- ried out on the crude extract showed moderate effects
oleane- and ursane-type triterpenes have been reported against Bacillus subtilis and Escherichia coli with in-
from different plant families (Labiatae [9 – 13], Burs- hibition diameters of 11 and 12 mm at 40 µ g per
eraceae [14, 8] and Celastraceae [15 – 17]). In most platelet, respectively. The crude extract was also tested
cases, the hydroxy group was in the less hindered equa- in vitro for its preliminary cytotoxicity assay using
torial 11α -position, based on the large axial-axial J9,11 Artemia salina. It exhibited a 40 % mortality of larvae
values (8 – 11 Hz) of the coupling constant between at 10 µ g/mL.
H-9 and H-11. Two 11β -hydroxy-oleane-type triter-
penes, 3-oxo-11β -hydroxy-olean-12-ene (2) [8] and Experimental Section
11β ,21β -dihydroxy-olean-12-en-3-one [18], have also General
been reported. With respect to the B and C rings with
the trans junction and chair conformation in oleanane Optical rotation was determined on a Perkin-Elmer po-
triterpenes [19 – 20], the value of the coupling con- larimeter (model 241). NMR spectra were recorded in CDCl3
stant (J11 ,9 = 4.5 Hz) observed between H-11 and H-9 on Mercury 300 and Inova 600 spectrometers. The chemi-
clearly indicated that the oxygen at C-11 is in the ax- cal shifts are given in ppm relative to TMS as internal ref-
ial position (β -oriented) in compound 1 (Fig. 3). Thus, erence. HR-ESIMS was performed on a microTOF 10237
spectrometer (Bruker). UV spectra were recorded in CHCl3
compound 1 is 11β ,26-epoxy-26-hydroxy-olean-12-
on a Carry 300 spectrophotometer. Pre-coated TLC Poly-
en-3-one, named salacetal.
gram SilG/UV254 plastic sheets from Macherey-Nagel were
The isolated compounds were tested for their an- used for thin-layer chromatography (0.2 mm layer thickness)
tibacterial potency against Bacillus subtilis, Staphy- and visualized under UV light (254 and 365 nm) or sul-
lococcus aureus and Escherichia coli, and their an- furic anisaldehyde reagent (85 mL MeOH, 14 mL EtOAc,
tifungal activity against Candida albicans and Mu- 1 mL H2 SO4 and 1 mL 4-methoxybenzaldehyde). Col-
cor miehei. No significant effects were detected in umn chromatography and flash chromatography were con-
these bioassays. However, the preliminary tests car- ducted using silica gel (Merck 230 – 400 and 70 – 230 mesh)
B. M. Mba’ning et al. · An Oleanane-type Triterpene from Salacia longipes var. camerunensis 1273

with different mixtures of n-hexane-ethyl acetate, and combined on the basis of TLC analysis. Further purifica-
dichloromethane-methanol as eluents. tion of sub-fractions 71 – 76 afforded 2-hydroxy-3-oxo-D:A-
friedooleanan-29-oic acid (10.2 mg). Purification of frac-
Plant material tion D with mixtures CH2 Cl2 -MeOH of increasing polarity
as eluent afforded mangiferin (71.3 mg). Fraction C was a
Salacia longipes var. camerunensis was collected in De- complex mixture and was not further studied.
cember 2008 at Mount Kala (Yaoundé), in the Centre Re-
gion of Cameroon. The plant was identified by Mr. V. Nana, Evaluation of antimicrobial activity
botanist at the National Herbarium of Cameroon, where
a voucher specimen (No 28963 / SRF/Cam) has been de- Three bacterial species, including Bacillus subtilus,
posited. Staphylococcus aureus and Escherichia coli, the yeast Can-
dida albicans and the fungus Mucor miehei were used. In
Extraction and separation vitro assays were performed according to the methods de-
scribed in previous reports by Latha et al. [24].
The roots of S. longipes var. camerunensis (2.4 kg) were
crushed and extracted at r. t. with a mixture of CH2 Cl2 - Salacetal [11β ,26-epoxy-26-hydroxy-olean-12-en-3-one] (1)
MeOH (1 : 1), (2 × 7 L, 48 h each). The extract was concen-
trated under vacuum to afford 70.6 g of a residue. 60.0 g of Colorless powder. – [α ]20
D = +37 (c = 0.1, MeOH). –
the extract was chromatographed on silica gel (Merck, 230 – UV/Vis (MeOH): λmax (log ε ) = +246 (3.2), 332 (3.1) nm. –
1 H NMR (CDCl , 300 MHz) and 13 C NMR (CDCl ,
400 mesh) using mixtures CH2 Cl2 -MeOH of increasing po- 3 3
larity as eluent. 91 fractions of 300 mL each were collected 125 MHz) spectroscopic data: see Table 1. – MS (EI, 70 eV):
and combined on the basis of TLC analysis to yield four m/z (%) = 454 (40) [M]+ , 439 (19), 409 (33), 408 (100),
main fractions labelled A (11.2 g), B (9.5 g), C (13.1 g), and 393 (30), 322 (38), 287 (13), 255 (25), 203 (12), 191 (20). –
D (18.0 g). HRMS ((+)-ESI): m/z = 477.3342 (calcd. m/z = 477.3339
Fraction A (11.2 g) was subjected to column chromatog- for C30 H46 O3 Na, [M+Na]+ ).
raphy on silica gel (Merck, 70 – 230 mesh). Elution with
Acknowledgements
n-hexane-EtOAc gradient mixtures resulted in the collec-
tion of 549 fractions of 25 mL each, which were com- The authors wish to acknowledge the German Academic
bined on the basis of TLC analysis. Further purification of Exchange Service (DAAD) for awarding a grant to B. M.
sub-fractions 16 – 32 afforded the mixture of β -sitosterol Mba’ning, and the European Commission for awarding a
and stigmasterol (17.8 mg) while that of sub-fractions 183 – Marie Curie fellowship to B. N. Lenta, contract MIF2-CT-
189 gave salacetal (1, 6.2 mg). Chromatography of frac- 2006-021591, Nr. 980033. They also acknowledge the Third
tion B on silica gel (Merck, 70 – 230 mesh) and elut- World Academy of Science (TWAS) for the research grant
ing with n-hexane-EtOAc gradient mixtures resulted in the Nr. 07-141 LDC/CHE/AF/AC-UNESCO FR: 3240171776 to
collection of 107 fractions of 25 mL each, which were our TWAS Research Unit.

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