Sapelenins G-J, acyclic triterpenoids with strong anti-inflammatory activities from the bark of the Cameroonian medicinal plant Entandrophragma cylindricum

Article abstract of DOI:10.1016/j.phytochem.2012.06.004

Four acyclic triterpene derivatives named sapelenins G-J (1-4), along with eight known compounds, sapelenins A-D, ekeberin D2 (5), (+)-catechin and epicatechin, and anderolide G, were isolated from the stem bark of the Cameroonian medicinal plant, Entandrophragma cylindricum Sprague, on the basis of bioassay-guided fractionation. Their structures were determined by means of high-resolution mass spectrometry and NMR spectroscopic data, as well as by comparison with the literature values of their analogs. The absolute configurations of the compounds (1-4) were assigned by the modified Mosher's method in conjunction with NOESY experiments and chemical modifications. The anti-inflammatory activities of the sapelenins were evaluated by assessing their ability to suppress or inhibit the secretion of cytokine interleukin-17 (IL-17) by human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA). The cytotoxicity of these compounds on PMBCs was further assessed for correctly interpreting their anti-inflammatory responses. The tested compounds demonstrated moderate to significant anti-inflammatory activities by suppressing the secretion of IL-17 by PHA-stimulated human PBMCs. One of them, sapelenin G (1), showed high potency in suppressing the secretion of IL-17 by PBMCs comparable to reference cyclosporine A, without causing any cytotoxic effects (negligible), and deserves further considerations towards developing an effective anti-inflammatory drug.

Full text of DOI:10.1016/j.phytochem.2012.06.004

Phytochemistry 83 (2012) 79–86
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Sapelenins G–J, acyclic triterpenoids with strong anti-inflammatory activities
from the bark of the Cameroonian medicinal plant Entandrophragma cylindricum
Simeon Fogue Kouam a,b, Souvik Kusari a,1, Marc Lamshöft a,1, Ostend Kamgue Tatuedom b,
Michael Spiteller a,
⇑
a Institute of Environmental Research (INFU) of the Faculty of Chemistry, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Str. 6,
D-44221 Dortmund, Germany
b Department of Chemistry, Higher Teachers’ Training College, University of Yaounde I, P. O. Box 47, Yaounde, Cameroon
a r t i c l e i n f o
a b s t r a c t
Article history:
Four acyclic triterpene derivatives named sapelenins G–J (1–4), along with eight known compounds, sap-
elenins A–D, ekeberin D2 (5), (+)-catechin and epicatechin, and anderolide G, were isolated from the stem
bark of the Cameroonian medicinal plant, Entandrophragma cylindricum Sprague, on the basis of bioassay-
guided fractionation. Their structures were determined by means of high-resolution mass spectrometry
and NMR spectroscopic data, as well as by comparison with the literature values of their analogs. The
absolute configurations of the compounds (1–4) were assigned by the modified Mosher’s method in con-
junction with NOESY experiments and chemical modifications. The anti-inflammatory activities of the
sapelenins were evaluated by assessing their ability to suppress or inhibit the secretion of cytokine inter-
leukin-17 (IL-17) by human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglu-
tinin (PHA). The cytotoxicity of these compounds on PMBCs was further assessed for correctly
interpreting their anti-inflammatory responses. The tested compounds demonstrated moderate to signif-
icant anti-inflammatory activities by suppressing the secretion of IL-17 by PHA-stimulated human
PBMCs. One of them, sapelenin G (1), showed high potency in suppressing the secretion of IL-17 by
PBMCs comparable to reference cyclosporine A, without causing any cytotoxic effects (negligible), and
deserves further considerations towards developing an effective anti-inflammatory drug.
Received 26 January 2012
Received in revised form 6 June 2012
Available online 14 July 2012
Keywords:
Entandrophragma cylindricum
Cameroonian medicinal plant
Sapelenin
Anti-inflammatory
Cytotoxic
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
pounds isolated being complex limonoids (Taylor and Wragg,
1967; Chan et al., 1970; Baxter et al., 1998; Nsiama et al., 2011)
In our search for bioactive metabolites from Cameroonian
medicinal plants, we recently encountered a plant in the Dja rain-
forest, locally known in Cameroon as ‘Sapele’. This plant is botan-
ically known as Entandrophragma cylindricum Sprague
(Meliaceae). The genus Entandrophragma contains ten species
which are generally recognized as distinct, together with some four
varieties that probably do not rank in any species (Adesida and
Taylor, 1967). E. cylindricum is one of the only five species that have
been discovered so far in Cameroon (Letouzey, 1985). It is a very
bulky and tall forest tree, up to 45 m in height and native to trop-
ical Africa. The bark of this timber has been extensively exploited
by the ‘Bantu’ and ‘Baka’ tribes in the eastern region of Cameroon,
for the treatment of rheumatism in the traditional medicine sub-
sector. Although phytochemical studies on this plant genus have
been reported earlier with the most prominent classes of com-
and highly oxygenated acyclic triterpenes (Ngnokam et al., 1993,
1995, 2005), little pharmacological investigation has so far been
performed (Huang et al., 2009).
Here, we report the isolation and the characterization of four
new acyclic triterpene derivatives named sapelenins G–J (1–4), to-
gether with eight known compounds, sapelenins A–D (Ngnokam
et al., 1993, 1995), ekeberin D2 (5) (Murata et al., 2008), (+)-cate-
chin and epicatechin (Kashiwada et al., 1990a,b), and anderolide
G (Tanaka et al., 2011). We also report the anti-inflammatory activ-
ities of the novel sapelenins by evaluating their ability to suppress/
inhibit secretion of cytokine IL-17 (interleukin-17) by human
peripheral blood mononuclear cells (PBMC) stimulated with phy-
tohemagglutinin (PHA). We further evaluated the cytotoxicity of
the tested compounds (1–4) on PMBCs in order to correctly inter-
pret their anti-inflammatory responses.
2. Results and discussion
⇑
Corresponding author. Tel.: +49 231 755 4080; fax: +49 231 755 4085.
E-mail addresses: m.spiteller@infu.tu-dortmund.de, m.spiteller@infu.
Compound 1, named sapelenin G (Fig. 1), was isolated as a col-
orless oil. Its molecular formula (C30H56O6) was determined from
uni-dortmund.de (M. Spiteller).
1
These authors contributed equally.
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.06.004

80
S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
Fig. 1. Structures of sapelenins G–J (1–4) and compounds 5–7.
the pseudomolecular ion peak at m/z = 513.4152 (calcd. for
30H57O6, 513.4155), obtained by high-resolution mass spectrom-
was observed between the protons at dH 3.32 (H-3), 1.64 (H-4a),
1.36 (H-4b), and 1.62 (H-5), respectively, indicating the oxidation
of two contiguous double bonds in 1. The relative configurations
of C-3, C-6, C-7 and C-22 could not be assigned. The compound
was assigned the structure (10E,14E,18E)-2,6,10,15,19,23-hexam-
ethyltetracosa-10,14,18-triene-2,3,6,7,22,23-hexaol and named
sapelenin G (1). Its isomer (7) has been described as one of the con-
stituents of the anti-inflammatory and anti-allergy extract of nettle
(Alberte et al., 2010).
C
etry (HR-MS) and is consistent with three degrees of unsaturation.
The IR spectrum exhibited bands for a hydroxyl functional group at
m
max = 3386 cmꢀ1. The 13C NMR (see Supplementary data, Table S1)
displays 30 carbon signals which can be sorted into eight methyls,
ten methylenes, three oxymethines, three sp2 methines and six
quaternary carbons including three sp2 carbons, and three bearing
oxygen. The 1H NMR (see Supplementary data, Table S1) spectrum
showed resonances for eight methyl groups including three vinylic
ones (dH 1.57, 2 ꢁ dH 1.59), and five attached to sp3 carbon.
Compound 2 (Fig. 1) was obtained as a colorless oil. Its molec-
ular formula was determined as
C30H52O4 by HR-MS (m/z
From the above evidence and by comparing these data with
those described in the literature (Ngnokam et al., 1993, 1995; Mill-
er and Tinto, 1995; Murata et al., 2008), sapelenin G (1) was con-
firmed as a derivative of squalene with a structure similar to that
of sapelenin C (6) (Ngnokam et al., 1993). The presence of three
low field methyl groups around ꢂdH 1.5–1.8 and the lack of any
correlation peaks from two of these olefinic methyl groups in the
HMBC spectrum indicated clearly the absence of the two terminal
vinylic methyl groups and consequently, suggested the oxidation
of the terminal double bonds of the squalene skeleton assigned
to compound 1. In the HMBC experiment (Fig. 3a1), the proton sig-
nal at dH 3.28 (H-22) showed correlation peaks with carbon signals
at dC 23.4 (CH3-24), 26.7 (CH3-30) and 37.4 (CH2-20), and cross
peaks were also observed between the proton signal at dH 3.32
(H-3) and the carbon signals at dC 23.4 (CH3-1), 26.7 (CH3-25)
and 33.4 (CH2-5), respectively. Furthermore, the methyl signal at
dH 1.10 (H-26) showed cross peaks with two carbon signals at dC
33.4 (C-5) and 78.9 (C-7). In the COSY spectrum, connectivity
477.3945 [M+H]+). IR absorption bands were observed at
3430 nm (OH) and 1660 nm (C@C). The 13C NMR spectroscopic
data of 2 (see Supplementary data, Table S1) exhibited 30 typical
resonances for an acyclic triterpene skeleton including eight
methyl and ten methylene groups (Ngnokam et al., 1993, 1995;
Murata et al., 2008). A 2-hydroxypropanyl-2-methyltetrahydrof-
uranyl sub-structure was established from spectral data evidence.
An oxymethine (dC 87.6), an oxygenated tertiary carbon (dC 86.1),
two methylene carbons (dC 26.6 and 31.4) and two methyl groups
(dC 23.8 and 27.7) were observed in the 13C NMR spectrum (see
Supplementary data, Table S1). The HMBC experiment (Fig. 3a2)
showed cross peaks with proton signal at dH 3.77 (oxymethine)
and carbon signals at dC 86.1, 23.8 and 27.7. The long-range corre-
lation between the methyl protons at dH 1.15 (dC 23.8) and the car-
bon signals at dC 76.2 (oxymethine), dC 87.1 and dC 31.4 established
that the dihydrofuran moiety resided next to an oxymethine group
(dH 3.51). Furthermore, in the 1H/13C NMR spectrum, we could dis-
tinguish distinctive resonances for an epoxy function (dH/C 2.71/

S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
81
64.2, CH and dC 58.3, qC). In the HMBC experiment, important cross
peaks from H-30 (dH 1.27) to C-24 (dc 24.9); from H-30 (dH 1.27) to
C-22 (dc 64.2); H-24 (dH 1.31) to C-30 (dc 27.4) and H-24 (dH 1.31)
to C-22 (dc 64.2) were observed, indicating that the epoxy group
was located at C-22, C-23. This suggests that the structure of 2
may be a precursor of ekeberin D2 (5), isolated for the first time
from this plant (Murata et al., 2008). Compound 2, on hydrolysis
using aqueous acid (10% H2SO4) yielded ekeberin D2 (5), which
was confirmed by comparison of the spectral data with that re-
ported (Murata et al., 2008). This finding was strongly supported
by the 1H–1H COSY and HMBC results. The presence of a secondary
alcohol at C-7 of 2 allowed us to determine its absolute stereo-
chemistry by utilizing the modified Mosher’s method (Ohtani
et al., 1991; Lamshöft et al., 2003). (R)- and (S)-MTPA esters of
27.1. Resonances at dH/C 5.11/124.7 (olefinic carbon), 2.09/27.0
(methylene protons), 1.61/18.0 and 1.69/25.7 (two methyl groups)
in the 1H/13C NMR spectra were typical for those of a prenyl group
(Kouam et al., 2007). However, in the HMBC experiment (Fig. 3a3),
we observed correlations of proton resonances at dH 1.61 (CH3-30)
and 1.69 (CH3-24) with the carbon signal at dC 124.7 (C-22) which
in turn showed a cross peak with a proton signal at dH 2.01 (H-20).
In addition, a cross peak was observed between signal at dH 2.01
and a carbon resonating at dH 1.61 (CH3-29) suggesting a geranyl
moiety in the structure of 3. The remaining two hydroxyl groups
were located at C-10 and C-11, based on the careful examination
of the HMBC data. In fact, the methylene protons (dH 1.42 and
1.58 (CH2-8)) indicated correlations with two quaternary oxygen-
ated carbons (dC 74.6 (C-10), and 86.6 (C-6)) and the methyl group
at dH 1.15 (CH3-27) showed cross peaks with carbon signals at dC
78.9 (C-11), 74.6 (C-10) and 33.6 (C-9). Sapelenin I (3) is, thus, an
isomer of ekeberin D2 (5) (Murata et al., 2008). Because two sec-
ondary hydroxyl groups are present in the structure of sapelenin
I (3), the determinations of their absolute configurations is difficult.
However, treatment of 3 with 2,2-dimethoxypropane and p-tolu-
ene sulfonic acid in acetone afforded the corresponding acetonide
(3a). The absolute configurations of the dihydrofuran moiety were
elucidated by both NOESY and Mosher’s experiments (Figs. 2 and
3b). NOESY correlations were observed between 1-CH3, H-3 and
26-CH3, and H-7 and 26-CH3. The modified Mosher’s method
(Fig. 2) was applied to determine the absolute configuration of C-
7 to be the same as in 2 (7S) and consequently, those of C-3 and
C-6 were found to be respectively, 3S and 6R. However, it was
not possible to determine the absolute configurations at C-10
and C-11. On the basis of the above evidence, the structure of com-
pound 3 was assigned to be (1S, 8E,12E)-1-((2S,5R)-5-(2-hydroxy-
propan-2-yl)-2-methyltetrahydrofuran-2-yl)-4,9,13,17-tetra-
methyl octadeca-8,12,16-triene-1,4,5-triol.
sapelenin H (2) were prepared by treatment with (R)- and (S)-
methoxy- -trifluoromethylphenylacetyl (MTPA) chloride in DMAP
followed by drops of pyridine. Compound 2 was converted to the
corresponding MTPA esters S and R, respectively. Significant
values (
d = dS-MTPA-ester ꢀ dR-MTPA-ester) were observed for the pro-
a-
a
D
d
D
tons near the chiral center C-7 as shown in Fig. 2. According to
Mosher’s rule, the absolute configuration at C-7 was determined
as S. As a consequence, the absolute configurations of the other
two stereogenic centers in the dihydrofuran portion of 2 were de-
duced to be 3S, 6R, on the basis of the established relative stereo-
chemistry from the significant NOE effect between H-3, CH3-6 and
H-7 (Fig. 3b). The absolute configuration at C-22 could not be
determined. Thus, compound 2 is accordingly (1S,4E,8E,12E)-15-
(3,3-dimethyloxiran-2-yl)-1-((2R,5S)-5-(2-hydroxypropan-2-yl)-
2-methyltetrahydrofuran-2-yl)-4,9,13-trimethylpentadeca-4,8,12-
trien-1-ol.
Compound 3 (Fig. 1) was isolated as an optically active colorless
oil, ½a 2D0
ꢃ
¼ þ194ꢄ [c 0.01, CH3OH]. Its HR-MS mass spectrum
showed a quasi-molecular ion peak [M+H]+ at m/z = 495.4054 cor-
responding to the molecular formula C30H55O5. The IR spectrum
contained hydroxyl group absorption at 3430 cmꢀ1. Important
fragments were obtained at m/z 477 [[M+H]+ ꢀ H2O]+, 459
[[M+H]+ ꢀ 2 ꢁ H2O]+ and 441 [[M+H]+ ꢀ 3 ꢁ H2O]+ in its mass
spectrum, indicating three hydroxyl groups in the structure of 3.
Its 1H/13C NMR spectra showed resonances at dH/C 3.79/88.1,
3.56/77.7 and 3.44/78.9, corresponding to three oxymethine
groups (see Supplementary data, Table S1). Furthermore, the 1H
NMR spectrum indicated resonances for eight methyl groups [dH
1.17, 1.22, 1.65, 1.69 (each 3H), 1.15 (6H), 1.61 (6H)] and reso-
nances of three olefinic protons at dH 5.18 (J = 6.9), 5.11 (J = 6.5),
5.12 (J = 6.8). In the 13C NMR spectrum, a total of 30 resonances
were observed. Compound 3 is, thus, an acyclic triterpene contain-
ing at least three hydroxyl groups. In the 13C NMR spectrum, one
can distinguish resonances for a 2-hydroxypropanyl-2-methyl-tet-
rahydrofuran-2-yl moiety at dC 86.6, 31.5, 27.0, 88.1, 71.1, 23.9, and
Compound 4 (Fig. 1) was obtained as an optically active color-
less oil {½a 2D0
þ 36 (c 0.01, CH3OH)} with the molecular formula
ꢃ
C
32H56O6, established by HR-MS in conjunction with the NMR
spectra. The IR spectrum of 4 indicated the presence of hydroxyl
(3430 and 3360 cmꢀ1) and ester carbonyl (1730 cmꢀ1) groups.
The chemical shift values of the 1H and 13C NMR spectra of 4 in
CDCl3 (see Supplementary data, Table S1) were almost identical
with those of compound 3. However, in the 1H NMR spectrum of
compound 4, a resonance was observed for an acetyl group at dH
2.09 and the signal at dH 3.56 (H-7) in 3 shifted downfield to dH
4.95, confirming the presence of an acetyl group. This was corrob-
orated by the resonances at dC 171.1 in the 13C NMR spectrum.
From this evidence, it was clear that the hydroxy moiety was re-
placed in 3 by an acetoxyl group. Based on the formation of the
sapelenins G–I (1–3) in the same plant extract, we speculate that
they possess the same biogenetic origin. In addition to the consis-
Fig. 2.
D
d(dS ꢀ dR) values (in ppm) for the MTPA esters of 2 and 3.

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S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
Fig. 3. (a) 1H–1H COSY (bold bonds) and selected HMBC (curved arrows) correlations for 1–4. (b) Key NOE correlation (curved arrows) observed for the dihydrofuran portion
for 2–5 NOESY spectrum (in CDCl3, 500 MHz).
Fig. 4. Schematic representation of the chemical conversion of sapelenin derivatives. (a) 2,2-dimethoxypropane, p-toluenesulfonic acid, acetone at rt, 30 mn, 98%; (b) (+) or
(ꢀ)- MTPA chloride, DMAP, at RT, overnight, 74%.
+
tent chemical correlations (Fig. 4), we assume identical absolute
configurations of the three stereogenic centers C-3, C-6 and C-7
of the derivatives presented in Fig. 4. The absolute configurations
at C-10 and C-11 could not be determined. The structure of 4
was, thus, determined as a (1S,8E,12E)-4,5-dihydroxy-1-((2S,5R)-
5-(2-hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl)-4,9,13,
17-tetramethyloctadeca-8,12,16-trienyl acetate.
helper 17 (Th17) cell subset of CD4 T cells, and is associated with
several immune regulatory functions, including the inflammatory
process during infection and in autoimmune diseases (Iwakura
et al., 2011; Angkasekwinai and Dong, 2011). In fact, the IL-17 fam-
ily of cytokines is involved in the development of inflammation
and host defense against infection by inducing the expression of
genes encoding proinflammatory cytokines (such as TNF, IL-1, IL-
6, G-CSF, and GM-CSF), chemokines (such as CXCL1,CXCL5, IL-8,
CCL2, and CCL7), antimicrobial peptides (such as defensins and
The IL-17 (or IL-17A), originally called cytotoxic T-lymphocyte
antigen-8, is the signature cytokine of the recently identified T

S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
83
S100 proteins), and matrix metalloproteinases (such as MMP1,
MMP3, and MMP13) from fibroblasts, endothelial cells, and epithe-
lial cells (Kolls and Linden, 2004; Iwakura et al., 2008, 2011;
Reynolds et al., 2010). Furthermore, IL-17 also promotes SCF- and
G-CSF-mediated granulopoiesis and recruits neutrophils to the
inflammatory sites (Iwakura et al., 2011). Therefore, we employed
the 96 well-plate format of measurement of IL-17 secretion by hu-
man PBMCs stimulated with PHA using the human IL-17 ELISA kit
(Ray Biotech, Inc.). Sapelenins G–J (1–4) demonstrated moderate to
significant anti-inflammatory activities by suppressing the secre-
tion of IL-17 by human PBMCs stimulated with PHA (Fig. 5). We
used cyclosporine A (CsA) as an additional control (reference). Sap-
elenins G (1), I (3), and J (4) significantly suppressed IL-17 secretion
gradient to 0% A over 2 min, after 100% B isocratic for 7 min. After-
wards, the system returned to its initial condition (70% A) within
1 min, and was equilibrated for 5 min.
3.2. Plant material
The stem bark of E. cylindricum was collected from the Dja rain-
forest, east region, Cameroon, in June 2010. The botanical identifi-
cation was made at the National Herbarium of Cameroon in
Yaoundé by Mr. Victor Nana (botanist) and a voucher specimen
(No. 54965/SFR/CAM) was deposited.
3.3. Extraction and isolation
at the concentration of 11.1 lM, which became pronounced in a
concentration-dependent manner. Their activity was comparable
to that of the reference (CsA) at higher concentrations. Sapelenin
H (2) was not potent in suppressing the secretion of IL-17 even
The air-dried stem bark of E. cylindricum (7 kg) was powdered
and extracted twice with MeOH at room temperature for 48 and
8 h, respectively. The organic solvent was concentrated to afford
a crude extract (890 g) which was re-extracted with ethyl ace-
tate/methanol (90/10) to obtain a 390 g extract, which was further
subjected to flash silica gel column chromatography, using a gradi-
ent of ethyl acetate in hexane, to give seven fractions named fr0
(pure hexane); fr1 (hexane/ethyl acetate, 9/1); fr2 (hexane/ethyl
acetate, 4/1); fr3 (hexane/ethyl acetate, 7/3); fr4 (hexane/ethyl ace-
tate, 1/1); fr5 (pure ethyl acetate) and fr6 (ethyl acetate/methanol,
4/1). Fr4 and fr5 were combined based on their LC-MS profiles.
Fraction fr0 was found to contain mainly mixtures of hydrocarbons
and phytosterols. Fractions fr1 (38 g), fr2 (41 g), fr3 (36 g), fr4 (20 g)
and fr5 (30 g) which showed strong anti-inflammatory activities,
were again subjected separately to flash silica gel column chroma-
tography, using the same gradient of solvent, to afford four series
of six (A1–A6); eight (B1–B8), seven (C1–C7) and seven (D1–D7) sub-
fractions. Each subfraction was submitted to the semipreparative
HPLC at wavelength 205 nm with the solvent system H2O (B)-
MeOH (A) with gradient program as described above. Fractions
A6 and B4 gave, respectively, sapelenins H (2, 22 mg) and J (4,
7 mg); fractions C4 and C6 yielded ekeberin D2 (5, 76 mg) and sap-
elenins B (80 mg), D (49 mg) and I (3, 50 mg); Fractions D5 gave
sapelenins A (37 mg) and C (92 mg); and Fraction D6 afforded
(+)-catechin (68 mg), epicatechin (20 mg) and sapelenin G (1,
21 mg).
at the highest concentration tested (100.0 lM). However, to con-
firm that the data obtained for IL-17 secretion was not affected
by the cytotoxicity of the sapelenins towards PBMCs, we evaluated
the viability of the PBMCs treated with the respective compounds
by a cell viability assay (CellTiter-BlueÒ, Promega) in parallel
(Fig. 6).
The cytotoxicity assay (Fig. 6) revealed that sapelenin G (1) had
only negligible cytotoxic effects against the PBMCs even at higher
concentrations. Sapelenin H (2) did not show any cytotoxic effect
at any tested concentration. Sapelenins I (3) and J (4) started dem-
onstrating cytotoxicity against the PBMCs at 33.3
respectively, that increased in a concentration-dependent expo-
nential manner at higher concentrations; that is, at 100 M for
lM and 11.1 lM,
l
both the sapelenins. This assay, thus, allowed us to discriminate
the real anti-inflammatory responses of the tested compounds
from the false positives.
Taken together, the anti-inflammatory and cytotoxicity assays
revealed that sapelenin G (1) is highly potent in suppressing the
secretion of IL-17 by PBMCs without causing any cytotoxic effects,
and deserves further consideration for development as an anti-
inflammatory drug. Sapelenin H (2) neither possessed the potency
to suppress IL-17 secretion by the PBMCs nor had any cytotoxic
activity towards them. However, since sapelenins I (3) and J (4)
showed a concentration-dependent cytotoxic effect on the PBMCs,
their IL-17 suppression data were considered invalid at the higher
concentrations.
3.4. Accurate mass measurement
The high-resolution mass spectra were obtained with an LTQ-
Orbitrap Spectrometer (Thermo Fisher, USA) equipped with a
HESI-II source. The spectrometer was operated in positive mode
(1 spectrum sꢀ1; mass range: 200–1000) with nominal mass
resolving power of 60000 at m/z 400 with a scan rate of 1 Hz with
automatic gain control to provide high-accuracy mass measure-
ments within 2 ppm deviation using an internal standard; Bis(2-
ethylhexyl)phthalate: m/z = 391.284286. The spectrometer was
attached with an Agilent 1200 HPLC system (Santa Clara, USA)
consisting of LC-pump, PDA detector (k = 205 nm), auto sampler
3. Experimental
3.1. General experimental procedures
3.1.1. NMR
The NMR spectra were recorded on a Bruker DRX-500 NMR.
Chemical shifts (d) were quoted in parts per million (ppm) from
internal standard tetramethylsilane (TMS). The JC,H couplings
were measured by means of pulsed field gradient HMBC spectra re-
corded by varying the J-refocusing time between t = 0.04 and
0.14 s.
3
(injection volume 10 ll) and column oven (30 °C). MS/MS experi-
ments were performed by CID (collision induced decay, 35 eV)
mode. Following parameters were used for experiments: spray
voltage 5 kV, capillary temperature 260 °C, tube lens 70 V. Nitro-
gen was used as sheath gas (50 arbitrary units) and auxiliary gas
(five arbitrary units). Helium served as the collision gas. The sepa-
rations were performed by using a Nucleodur Gravity column
3.1.2. Column chromatography and preparative HPLC
Flash column chromatography was performed using silica-gel
60 (Merck, 0.040–0.063 mm). Preparative reversed-phase HPLC
was carried out with a Gilson system consisting of pump 322 with
a UV detector 152 (k = 205 nm) using a Nucleodur Gravity column
(50 ꢁ 2 mm, 1.8
Germany) with a H2O (+0.1% HCOOH, +10 mM NH4Ac) (A)/acetoni-
trile (+0.1% HCOOH) (B) gradient (flow rate 300
l minꢀ1). Samples
lm particle size) from Macherey–Nagel (Düren,
from Macherey–Nagel (Düren, Germany) (250 ꢁ 16 mm, 5
lm par-
ticle size). Separation was achieved by using a H2O (A)-MeOH (B)
gradient program as follows (flow rate 4 mL minꢀ1): 70% A linear
to 75% B for 15 min, linear gradient to 20% A over 13 min, linear
l
were analyzed by using a gradient program as follows: 90% A iso-
cratic for 2 min, linear gradient to 100% B over 13 min, after 100% B

84
S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
Fig. 5. Effect of the tested compounds on the secretion of IL-17 by PHA-stimulated PBMCs. (a) Sapelenin G (1); (b) Sapelenin H (2); (c) Sapelenin I (3); (d) Sapelenin J (4). As
negative control, IL-17 secretion in unstimulated PBMCs was determined. As positive control, IL-17 secretion in PHA-stimulated PBMCs without adding any tested compound
was determined. An additional control (reference) of CsA was used to determine the comparative efficacy of the tested drugs. (Concentrations used: 0.41, 1.23, 3.70, 11.11,
33.33, and 100 lM).

S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
85
Sapelenin H (2): oil, ½a D20
ꢃ
þ 58 (c 0.01, MeOH); IR (KBr); mmax
3430 (OH), 1660 (C@C), 1447, 1378, 1173, 1078, 1026 cmꢀ1 1H
;
NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) are shown
in Table S1 (Supplementary data); HR-MS [M+H]+m/z 477.3945
(calcd. for C30H53O4, 477.3944).
Sapelenin I (3): oil, ½a D20
ꢃ
þ 194 (c 0.01, MeOH); IR (KBr); mmax
3430 (OH), 1660 (C@C), 1447, 1378, 1173, 1078, 1026 cmꢀ1
;
1H
NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) are shown
in Table S1 (Supplementary data); HR-MS [M+H]+m/z 495.4054
(calcd. for C30H55O5, 495.4049).
Sapelenin J (4): oil, ½a D20
þ 36 (c 0.01, MeOH); IR (KBr); mmax
ꢃ
3430 (OH), 3360 (OH), 1730 (C@O), 1660 (C@C), 1443, 1373,
1243, 1082, 1021 cmꢀ1 1H NMR (500 MHz, CDCl3) and 13C NMR
;
(125 MHz, CDCl3) are shown in Table S1 (Supplementary data);
HR-MS [M+H]+m/z 537.4150 (calcd. for C32H57O6, 537.4155).
3.6. Chemical conversion of sapelenin derivatives
3.6.1. Acetonidation of sapelenin I (3)
To a solution of sapelenin I (3) (20.0 mg, 0.040 mmol) in acetone
(5 ml) was added 1 ml of 2,2-dimethoxypropane and 4 mg of p-tol-
uene sulfonic acid. After 30 min at 20 °C, the solution was
quenched with water and the reaction mixture was extracted with
hexane and washed with NaHCO3, and then dried over Na2CO3. The
solvent was evaporated at reduced pressure and the extract was
subjected to the HPLC to afford the acetonide (3a) (21.4 mg, 99%)
as a colorless oil. HR-MS [M+Na]+ m/z 557.4166 (calcd. for
C33H58O5Na, 557.4182).
3.6.2. Acetonidation of ekeberin D2 (5)
The same reaction was performed with 5 (7.0 mg, 0.014 mmol)
to afford the expected acetonide (5a) (7.5 mg, 99.1%) as a colorless
oil. HR-MS [M+Na]+ m/z 557.4171 (calcd. for C33H58O5Na,
557.4182).
3.6.3. Acetylation of sapelenin I (3)
Dry pyridine (0.5 ml) and Ac2O (1.0 ml) were added to sapelenin
I (3, 10 mg, 0.020 mmol) and stirred for 12 h. After the usual work-
up, the extract was subjected to the HPLC to afford sapelenin I diac-
etate (3d) (5.7 mg, 48.7%) as
a
major colorless oil;
diacetylsapelenin I,
½
a 2D0
ꢃ
¼ þ192ꢄ (c 0.005, MeOH); 1H NMR
(500 MHz, CDCl3): 5.11 (1H, t, J = 5.5, H-22), 5.12 (2H, t, J = 6.5,
H-14,18), 4.85 (1H, dd, J = 2.8, 9.7), 4.72 (1H, dd, J = 2.1, 10.2),
3.78 (1H, t, J = 5.7), 2.23 (1H, m, H-13), 2.12 (1H, m, H-5), 2.11
(3H, s, H3CCO), 2.09 (3H, s, H3CCO), 2.07 (4H, m, H-17,H-19), 2.00
(m, H-20), 1.85 (2H, m, H-4), 1.69 (3H, s, CH3-24), 1.65 (2H, m,
H-9), 1.61 (3H, s, CH3-30), 1.58 (6H, s, CH3-28,29), 1.54 (1H, m,),
1.53 (3H, m), 1.38 (1H, m), 1.23 (3H, s, CH3-25), 1.17 (3H, s, CH3-
26), 1.15 (3H, s, CH3-1); HR-MS [M+Na]+ m/z 601.4073 (calcd. for
C34H58O7Na, 601.4080).
Fig. 6. Effect of the tested compounds on the viability of PBMCs. (a) Sapelenin G (1);
(b) Sapelenin H (2); (c) Sapelenin I (3); (d) Sapelenin J (4). (Concentrations used:
0.41, 1.23, 3.70, 11.11, 33.33, and 100 lM).
3.6.4. Acetylation of ekeberin D2 (5)
The same reaction was performed with 5 (10 mg, 0.020 mmol)
to afford the expected ekeberin D2 diacetate (5b) (5.7 mg, 48.7%)
of ekeberin D2 (5) as a major colorless oil; ekeberin D2 diacetate
isocratic for 5 min, the system returned to its initial condition (90%
A) within 0.5 min, and was equilibrated for 4.5 min.
(5b), ½a 2D0
ꢃ
¼ þ10ꢄ (c 0.005, MeOH); 1H NMR (500 MHz, CDCl3):
5.06 (m, 3H), 4.85 (dd, J = 2.2, 10.2), 4.72 (dd, J = 2.6, 10.2), 3.63
(t, J = 8.0), 2.03 (s, 3H), 1.99 (s, 3H), 1.52 (m, 9H), 1.13 (s, 3H),
1.12 (s, 3H), 1.11 (s, 6H), 1.03 (s, 3H); HR-MS [M+Na]+ m/z
601.4073 (calcd. for C34H58O7Na, 601.4080).
3.5. Physico-chemical constants of 1–4
Sapelenin G (1): oil, ½a D20
ꢃ
þ 260 (c 0.01, MeOH); IR (KBr); mmax
1H
3.6.5. Hydrolysis of sapelenin H (2)
3386 (OH), 1660 (C@C), 1447, 1373, 1160, 1069, 1008 cmꢀ1
;
To a solution of sapelenin H (2, 10 mg, 0.021 mmol), 1 N H2SO4
(5 ml) was added in one portion. The mixture was stirred at room
temperature until the HPLC analysis revealed the disappearance of
the starting epoxide (10 h). The reaction medium was then ex-
NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) are shown
in Table S1 (Supplementary data); HR-MS [M+H]+m/z 513.4152
(calcd. for C30H57O6, 513.4155).

86
S.F. Kouam et al. / Phytochemistry 83 (2012) 79–86
tracted with dichloromethane (5 ꢁ 3 ml), and the organic solution
was decanted. The solvent was evaporated at reduced pressure and
the extract subjected to the preparative HPLC to afford the natural
product ekeberin D2 (5, 10.1 mg, 97.3%).
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Part of this work was supported by the Dortmunder Gambrinus
Fellowship (to S.F.K.) and the International Foundation of Science
(Grant No. F/4893-1). This work was also supported by the German
Academic Exchange Service (DAAD) initiative ‘‘Welcome to Africa’’.
The authors (S.K., M.L., and M.S.) are grateful to the Ministry of
Innovation, Science, Research and Technology of the State of North
Rhine-Westphalia, Germany and the German Research Foundation
(DFG) for funding a high-resolution mass spectrometer. We thank
Sebastian Malchow and Tobias Goldmann for excellent technical
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
06.004.