Triterpenoid derivatives from Cylicodiscus gabunensis

Article abstract of DOI:10.1248/cpb.58.1100

Three new olean-12-ene derivatives (1-3), together with known urs-12-ene-3β, 28-diol (4) were isolated from the stem root of Cylicodiscus gabunensis. The structures of the new compounds were established by chemical and spectroscopic means as β-amyrin-n-no

Full text of DOI:10.1248/cpb.58.1100

1100
Note
Chem. Pharm. Bull. 58(8) 1100—1102 (2010)
Vol. 58, No. 8
Triterpenoid Derivatives from Cylicodiscus gabunensis
Pierre MKOUNGA,a Alembert Tchinda TIABOU,b and Jacques KOUAM*,a
a Department of Organic Chemistry, Faculty of Sciences, University of Yaounde I; P. O. Box 812, Yaounde, Cameroon: and
b Institute of Medical Research and Medicinal Plants Studies (IMPM); P. O. Box 6163, Yaounde, Cameroon.
Received March 18, 2010; accepted May 6, 2010
Three new olean-12-ene derivatives (1—3), together with known urs-12-ene-3b, 28-diol (4) were isolated
from the stem root of Cylicodiscus gabunensis. The structures of the new compounds were established by chemi-
cal and spectroscopic means as b-amyrin-n-nonyl ether (1), 22a-hydroxyolean-12-en-3b-yl-b-D-galactopyra-
noside (2), and 24-hydroxyolean-12-en-3b-yl-b-D-glucopyranoside (3).
Key words Cylicodiscus gabunensis; Mimosaceae; olean-12-ene; triterpene; b-amyrin derivative; triterpene glycoside
The Cylicodiscus genus belongs to the Mimosaceae family 25.7, 29.7, 29.6, 29.3, 31.9, 22.7 (each CH2), dC 14.4 (CH3)]
comprising 64 genera and 2950 species, mostly tropical.1) suggesting that 1 is a n-nonyl ether derivative of the latter
Cylicodiscus gabunensis HARMS (C. gabunensis), known as compound. Location of the O-n-nonyl group was confirmed
Denya (Ghana), Edum (Gabon), Adoum, Bokoka (Cameroon), by the HMBC spectrum showing correlation between H-1Ј
or Bouemon (Ivory Coast), is a large tree, common in the (dH 3.60) of the O-n-nonyl group and C-3 (dC 78.4) of the b-
rain forests of Sierra Leone to the Cameroon and Gabon.2,3) amyrin moiety. Consequently, the structure of 1 was deter-
In the traditional medicine its stem bark is used for various mined as b-amyrin-n-nonyl ether (1) (see Fig. 1).
therapeutic purposes.4,5) Preliminary bioassays on the crude
Compound 2 was isolated as white powder. Its high resolu-
EtOAc extract from the stem bark of C. gabunensis have tion-FAB-MS (HR-FAB-MS) spectrum exhibited a Mϩ peak
shown antidiarrheal and antimicrobial activities.6) These re- at m/zϭ604.4338 consistent with the molecular formula
sults may be explained by the presence of triterpenoids, a C36H60O7. The positive response to the Lieberman–Burchard
class of phytochemical compounds known for their broad test, as well as inspection of the 13C- and H-NMR spectral
1
spectrum of biological properties.7—10) Recently, we reported data revealed 2 to be a saponin. The H-NMR spectrum of
1
that the stem bark of C. gabunensis contained coumestan and aglycone revealed eight tertiary methyl groups [dH 0.76,
triterpenoid glycosides.6,11) As part of our continuing chemi- 0.86, 0.88, 0.92, 0.95, 0.98, 1.03, 1.08 (each 3H, s)], an
cal investigation of C. gabunensis aimed at the potentially ac- olefinic proton [dH 5.23 (1H, t, Jϭ3.6 Hz)], and two oxy-
tive constituents, phytochemical screening was performed on genated CH groups [dH 3.35 (1H, dd, Jϭ5.2, 11.1 Hz), 3.40
the stem root, resulting in the isolation of three new triter- (1H, dd, Jϭ13.7, 4.3 Hz)]. Moreover, the 13C-NMR and 13C-
penoid derivatives (1—3), and known urs-12-ene-3b, 28-diol DEPT spectra of aglycone indicated characteristic signals of
(4). This paper describes the isolation and structural elucida- two oxygenated C-atoms [dC 81.4, 76.6 (each CH)], and two
tion of these new compounds.
Results and Discussion
The crude methanolic extract of the stem root of C.
gabunensis was successively extracted with hexane, and
EtOAc to give 15.8 g and 122 g of extract, respectively. A
portion of the EtOAc extract was subjected to column chro-
matography (CC) as described in the Experimental to afford
compounds 1, 2, 3, and 4.
Compound 1 was isolated as white powder and gave a pos-
itive Lieberman–Burchard test for triterpenoids. Combined
with broad-band-decoupled 13C-NMR and distortionless en-
hancement by polarization transfer (DEPT) analysis, its mo-
lecular formula was determined as C39H68O by high resolu-
tion-electron ionization-MS (HR-EI-MS) spectrum, showing
a molecular ion peak (Mϩ) at m/zϭ552.5271.
The IR spectrum exhibited absorptions at 1650 cmϪ1 (dou-
ble bond), and 1357 cmϪ1 (ether function). Interpretation of
1
1H–1H shift correlation spectroscopy (COSY), H-detected-
heteronuclear multiple-quantum coherence (HMQC), and
1H-detected heteronuclear multiple-bond connectivity
1
(HMBC) spectra led assignments of H- and 13C-NMR data
as shown in Table 1. The 13C-NMR spectrum of 1 was almost
superimposable with that of b-amyrin12) except that the for-
mer disclosed additional O-n-nonyl signals [dC 63.1, 26.3,
Fig. 1. Structures of Isolated Triterpenoid Derivatives 1—3
∗ To whom correspondence should be addressed. e-mail: kouamjac@yahoo.fr
© 2010 Pharmaceutical Society of Japan

August 2010
1101
Table 1. The 1H-NMR (dH in ppm, 500 MHz) and 13C-NMR (dC in ppm, 125 MHz) Data of Compounds 1—3
1
2
3
No.
d
H (mult, J)a)
dCa) (DEPT)
38.6 (CH2)
27.4 (CH2)
d
H (mult, J)b)
dCb) (DEPT)
36.6 (CH2)
27.3 (CH2)
d
H (mult, J)b)
dCb) (DEPT)
1
2
1.10 (1H, t-like, 10.9)
1.60 (1H , m)
1.12 (1H, m)
1.59 (1H, m)
2.22 (1H, m)
1.88 (1H, m)
1.11 (1H, dd, 13.2, 12.2)
1.65 (1H, m)
38.4 (CH2)
27.7 (CH2)
2.01 (1H, m)
2.23 (1H, m)
1.59 (1H, m)
1.92 (1H, m)
3
4
5
6
3.21 (1H, dd, 4.4, 10.6)
78.4 (CH)
38.7 (C)
3.35 (1H, dd, 5.2, 11.1)
81.4 (CH)
37.55 (C)
55.3 (CH)
18.5 (CH2)
3.63 (1H, dd, 5.1, 10.2)
81.9 (CH)
42.8 (C)
0.69 (1H, d, 11.6)
1. 40 (1H, m)
55.2 (CH)
18.5 (CH2)
0.75 (1H, d, 11.2)
1.50 (1H, m)
0.75 (1H, d, 11.3)
1.49 (1H, m)
55.8 (CH)
19.7 (CH2)
1.29 (1H, m)
1.33 (1H, m)
1.34 (1H, m)
7
1.26 (1H, m)
32.7 (CH2)
1.27 (1H, m)
32.9 (CH2)
1.27 (1H, m)
32.9 (CH2)
1.01 (1H, d, 12.3)
1.04 (1H, d, 12.6)
1.05 (1H, d, 12.2)
8
9
38.2 (C)
39.9 (C)
40.6 (C)
1.59 (1H, dd, 12.2, 4.2)
47.7 (CH)
36.2 (C)
1.63 (1H, dd, 12.4, 5.3)
47.6 (CH)
36.9 (C)
1.63 (1H, dd, 12.3, 5.1)
47.0 (CH)
36.7 (C)
10
11
1.76 (1H, m)
2.01 (1H, m)
5.16 (1H, t, 3.5)
23.5 (CH2)
1.79 (1H, m)
2.03 (1H, m)
5.23 (1H, t, 3.6)
23.5 (CH2)
1.77 (1H, m)
2.05 (1H, m)
5.19 (1H, t, 3.9)
23.3 (CH2)
12
13
14
15
121.7 (CH)
145.2 (C)
41.7 (C)
122.5 (CH)
143.9 (C)
42.1 (C)
121.7 (CH)
145.2 (C)
41.7 (C)
1.03 (1H, dd, 11.3, 5.4)
1.48 (1H, m)
26.2 (CH2)
1.03 (1H, dd, 10.8, 4.6)
1.52 (1H, m)
25.9 (CH2)
1.02 (1H, dd, 10.5, 5.5)
1.50 (1H, m)
26.9 (CH2)
16
1.44 (1H, m)
27.2 (CH2)
1.48 (1H, m)
29.8 (CH2)
1.47 (1H, m)
26.1 (CH2)
1.05 (1H, dd, 11.3, 5.7)
1.07 (1H, 10.8, 4.9)
1.06 (1H, dd, 10.5, 4.8)
17
18
19
32.6 (C)
37.4 (C)
32.4 (C)
2.15 (1H, dd, 9.7, 3.6)
1.89 (1H, d, 3.6)
47.2 (CH)
46.7 (CH2)
2.18 (1H, dd, 10.2, 4.1)
1.92 (1H, d, 4.1)
44.7 (CH)
46.2 (CH2)
2.15 (1H, dd, 9.9, 3.8)
1.88 (1H , d, 3.8)
1.63 (1H, d, 9.9)
47.3 (CH)
46.8 (CH2)
1.64 (1H, d, 9.7)
1.66 (1H, d, 10.2)
20
21
31.1 (C)
30.5 (C)
31.1 (C)
1.69 (1H, dd, 11.6, 5.2)
1.14 (1H, m)
34.8 (CH2)
1.96 (1H, d, 4.3)
41.5 (CH2)
1.70 (1H, dd, 11.8, 5.1)
1.16 (1H, m)
34.7 (CH2)
1.58 (1H, d, 13.7)
3.40 (1H, dd, 13.7, 4.3)
22
1.68 (1H, 11.6, 5.8)
1.12 (1H, m)
37.1 (CH2)
76.6 (CH)
1.71 (1H, dd, 11.8, 5.3)
1.18 (1H, m)
37.1 (CH2)
23
24
1.14 (3H, s)
28.2 (CH3)
15.5 (CH3)
0.88 (3H, s)
0.76 (3H, s)
28.1 (CH3)
15.6 (CH3)
0.86 (3H, s)
22.3 (CH3)
64.4 (CH2)
0.94 (3H, s)
3.35 (1H, d, 11.2, 24a-H)
4.24 (1H, d, 11.2, 24b-H)
1.10 (3H, s)
25
26
27
28
29
30
1Ј
0.99 (3H, s)
16.3 (CH3)
16.7 (CH3)
0.98 (3H, s)
15.6 (CH3)
16.9 (CH3)
20.1 (CH3)
28.2 (CH3)
32.2 (CH3)
25.4 (CH3)
Gal. 100.7 (CH)
73.5 (CH)
16.1 (CH3)
16.8 (CH3)
26.0 (CH3)
28.5 (CH3)
33.3 (CH3)
23.7 (CH3)
Glc. 101.5 (CH)
74.5 (CH)
0.87 (3H, s)
1.03 (3H, s)
0.88 (3H, s)
1.25 (3H, s)
26.1 (CH3)
1.08 (3H, s)
1.32 (3H, s)
0.83 (3H, s)
28.4 (CH3)
0.86 (3H, s)
1.13 (3H, s)
0.83 (3H, s)
33.2 (CH3)
0.95 (3H, s)
0.88 (3H, s)
0.97 (3H, s)
23.7 (CH3)
0.92 (3H, s)
0.94 (3H, s)
n-nonyl. 3.60 (2H, t, 4.4)
1.45 (2H, m)
1.31 (2H, m)
1.29 (2H, m)
1.30 (2H, m)
1.26 (2H, m)
n-nonyl. 63.1 (CH2)
26.3 (CH2)
Gal. 4.80 (1H, d, 7.8)
4.33 (1H, dd, 8.9, 8.1)
4.04 (1H, dd, 9.2, 9.1)
4.54 (1H, dd, 9.2, 9.3)
3.83 (1H, m)
Glc. 5.32 (1H, d, 7.9)
3.50 (1H, dd, 8.8, 7.8)
3.44 (1H, dd, 9.1, 8.8)
3.42 (1H, dd, 9.7, 9.1)
3.43 (1H, m)
2Ј
3Ј
25.7 (CH2)
76.4 (CH)
77.4 (CH)
4Ј
29.7 (CH2)
70.5 (CH)
71.2 (CH)
5Ј
29.6 (CH2)
76.8 (CH)
77.8 (CH)
6Ј
29.3 (CH2)
4.62 (1H, m)
61.5 (CH2)
3.79 (1H, m)
62.1 (CH2)
4.15 (1H, m)
4.10 (1H, d, 10.5)
7Ј
8Ј
9Ј
1.27 (2H, m)
1.35 (2H, m)
0.79 (3H, t, 3.5)
31.9 (CH2)
22.7 (CH2)
14.4 (CH3)
a) Measured in CDCl3; b) Measured in DMSO-d6.
olefinic C-atoms [dC 122.5 (CH), 143.9 (C)]. Comparison of tation with those of an authentic sample.
1
the H- and 13C-NMR assignments of 2, which were estab-
Meanwhile, the configuration of the D-galactopyranosyl
lished by analysis of the H–1H COSY, HMQC, and HMBC unit was deduced to be b from the coupling constant value
1
spectra, with reported data suggested that the aglycone moi- observed for the H-1Ј of Gal (Jϭ7.8 Hz). HMBC correla-
ety was olean-12-ene-3b, 22-diol (a-sophoradiol).13)
tions from H-1Ј (dH 4.80) of galactose unit to C-3 (dC 81.4)
Compound 2 was subjected to acid hydrolysis with 15% of the aglycone confirmed the attachment of the D-galactopy-
HCl/MeOH, to yield an aglycone, identified as a-sophora- ranosyl group to C-3. On the basis of the above data, the
diol by comparison of its physical and spectral data with structure of compound 2 was elucidated to be 22a-hydroxy-
those published,13) along with a sugar component, identified olean-12-en-3b-yl-b-D-galactopyranoside (2) (see Fig. 1).
by direct HPLC analysis. The sugar was confirmed as D-
Compound 3 was obtained as colourless needles. Its mo-
galactose by comparison of its retention time and optical ro- lecular formula was deduced as C36H60O7 by HR-FAB-MS

1102
Vol. 58, No. 8
Compound 1: White powder, mp 99—101 °C; [a]D22 ϩ57° (cϭ0.25,
[m/zϭ604.4338 (Mϩ)] and confirmed by broad band decou-
pled 13C-NMR and 13C-DEPT analysis. The olean-12-ene
glycoside nature of 3 was evident from the positive response
to the Lieberman–Burchard test combined with the 13C- and
1
CHCl3); IR (KBr) cmϪ1: 1650, 1380, 1357, 1034; H-NMR (see Table 1);
13C-NMR (see Table 1); HR-EI-MS m/zϭ552.5271 (Mϩ) (Calcd for
C39H68O: m/zϭ552.5270).
Compound 2: White powder; mp 285—286 °C; [a]D22 ϩ62° (cϭ0.02,
MeOH); IR (KBr) cmϪ1 3545—3250, 1450, 1385, 1010; 1H-NMR (see
Table 1); 13C-NMR (see Table 1); HR-FAB-MS m/zϭ604.4338 (Mϩ) Calcd
for C36H60O7: m/zϭ604.4339).
1
1H-NMR spectral data analysis. Comparison of the H- and
13C-NMR assignments of 3, which were established under
the same conditions as in the case of 1 and 2, with reported
data suggested that aglycone moiety and carbohydrate unit
were olean-12-ene-3b, 24-diol14) and D-glucose,15,16) respec-
tively. Acid hydrolysis followed by spectroscopic analysis of
the aglycone and direct HPLC analysis of the sugar compo-
nent, confirmed this suggestion. Furthermore, the configura-
tion of the D-glucopyranosyl moiety was regarded to be b by
the J value of its anomeric proton signal at d 5.32 (d,
Jϭ7.9 Hz). The site of glycosylation was suggested by a
downfield shift observed for C-3 (dC 81.9 in 3, this signal ap-
peared at 80.7 ppm in olean-12-ene-3b, 24-diol), and con-
firmed by HMBC experiments showing correlations between
Compound 3: Colourless needles; mp 258—260 °C; [a]D22 ϩ78° (cϭ0.02,
MeOH); IR (KBr) cmϪ1: 3550—3250, 1450, 1390, 1010; 1H-NMR (see
Table 1); 13C-NMR (see Table 1); HR-FAB-MS m/zϭ604.4338 (Mϩ) Calcd
for C36H60O7: m/zϭ604.4339).
Acid Hydrolysis and Identification of Sugars Compounds 2 and 3
(6 mg each) were separately refluxed with 15% HCl/MeOH (6 ml) at 80 °C
for 4 h. After cooling, each reaction mixture was concentrated and the
residue partitioned with CH2Cl2/H2O. The organic layer was concentrated to
dryness to yield 2.9 mg of white material from 2 (M2) and 2.4 mg of white
material from 3 (M3). After purification by preparative-TLC, M2 and M3
yielded respectively white powder identified as a-sophoradiol and colourless
needles identified as olean-12-ene-3b, 24-diol, by comparison of its physical
and spectroscopic data with those published.13,14) The aqueous layer was
evaporated and the residue was analysed by HPLC under the following con-
H-1 (dH 5.32) of glucose unit and C-3 (dC 81.9) of the agly- ditions: column, Aminex HPX-87H (7.8 mm i.d.ϫ300 mm); solvent, 5 mM
H2SO4; flow rate, 0.6 ml/min; detection, refractive index and optical rotation.
The sugar was confirmed as D-galactose (D-glucose) by comparison of its re-
tention time and optical rotation with those of an authentic sample: retention
times (min), 9.62 (D-galactose, positive optical rotation), 8.98 (D-glucose,
cone. Consequently, the structure of 3 was determined as
24-hydroxyolean-12-en-3b-yl-b-D-glucopyranoside (3) (see
Fig. 1). The known compound urs-12-ene-3b, 28-diol (4)
was isolated as white powder and identified by comparison
positive optical rotation).
with the reported data.12)
Acknowledgment We are indebted to Dr. Kirk Marat and Dr. Philip
Gregory Hultin of the Department of Chemistry, Winnipeg, MB R3T 2N2,
University of Manitoba-Canada for technical assistance.
Experimental
General Procedures Melting points were determined on X-4 digital
micro-melting point apparatus and were uncorrected. Optical rotations were
measured with a Perkin-Elmer 341 digital polarimeter. IR spectra were
recorded with KBr pellets on a Perkin-Elmer 577 spectrometer. HPLC was
performed by using a system comprised of a CCPM pump, a CCP PX-8010
controller, an RI-8010 detector and a Shodex OR-2 detector, and a Rheo-
dyne injection port with a 20 ml sample loop. The EI-MS was recorded on a
JEOLMSRoute massspectrometer. The FAB-MS was obtained with a Kratos
MS 25 instrument with a DS-55 data system, and collision gas Xe (ion gun
conditions 6 kV and 10 mA). The NMR spectra were recorded with a Bruker
AMX-500 (500 MHz for 1H-NMR and 125 MHz for 13C-NMR). Samples
were run in DMSO-d6 or CDCl3. Chemical shifts were given in (ppm) with
tetramethylsilane as an internal standard, and coupling constants (J) were re-
ported in Hertz (Hz). CC was performed using a silica-gel (Kieselgel 60,
70—230 mesh, 230—400 mesh, Merck, Germany), Sephadex LH-20 (Phar-
macia), and TLC using a pre-coated silica-gel 60 F254 (0.25 mm, Merck,
Germany).
References
1) Coates P. K. (revised and updated by Meg C. P.) “Trees of Southern
Africa,” 3rd ed., Struik, South Africa, 2002.
2) Le Thomas A., “Mimosaceae in Flore du Gabon,” ed. by Aubreville,
Orstom, Paris, 1969.
3) Chudnoff M., “Ag, Handbook,” USDA Foresr Service, Washington
D.C., 1984, p. 607.
4) Tane P., Bergquist K.-E., Téné M., Ngadjui B. T., Ayafor J. F., Olov S.,
Tetrahedron, 51, 11595—11600 (1995).
5) Adjanohoun J. E., Aboubakar N., Dramane K., Ebot M. E., Ekpere J.
A., Enow-Orock E. G., “Contribution to Ethnobotanical and Floristic
Studies in Cameroon,” Traditional Medicine and Pharmacopoeia,
Technical and Research Commission of Organisation of African Unity
(OAU/STRC), Lagos, Nigeria 1996, p. 241.
6) Kouam J., Tane P., Meli L. A., Noundou S. X., Muhammad I. C.,
Fomum Z. T., Nat. Prod. Commun., 2, 835—840 (2007).
7) Morinaga O., Fukuda N., Tanaka H., Shoyama Y., Glycobiology, 15,
1061—1066 (2005).
8) Katerere D. R., Gray A. I., Nash R. J., Waigh R. D., Phytochemistry,
63, 81—83 (2003).
9) Gaidi G., Correia M., Chauffert B. , Beltramo J.-L., Wagner H., La-
caille D. M.-A., Planta Med., 68, 70—72 (2002).
10) Pawar R. S., Bhutani K. K., Phytomedecine, 12, 391—393 (2005).
11) Kazie N., Kouam J., Tane P., Kuete V., Watchueng J., Fomum Z. T.,
Nat. Prod. Commun., 4, 931—934 (2009).
12) Shashi B. M., Asish P. K., Phytochemistry, 37, 1517—1575 (1994).
13) Ndom J.-C., Kouam, Vardamides J. C., Wansi J. D., Kamdem A. W.,
Mbafor J. T., Fomum Z. T., Bull. Chem. Soc. Ethiop., 15, 151—156
(2001).
14) Tanaka R., Tabuse M., Matsunaga S., Phytochemistry, 27, 3563—3567
(1988).
15) Nguyen X. N., Nguyen H. T., Phan V. K., Chau V. M., Yan D., Jae-Hee
H., Hee-Kyoung K., Young H. K., Chem. Pharm. Bull., 57, 986—989
(2009).
Plant Material The stem root of Cylicodiscus gabunensis HARMS was
collected in May 2002 on Mount Eloudem, Yaounde-Cameroon. The plant
was identified at the National Herbarium, Yaounde, where a voucher speci-
men is deposited (No. 21574/SRF/CAM).
Extraction and Isolation The air-dried, powdered stem root of C.
gabunensis (4.8 kg) was immersed in MeOH (25 l) and kept for 72 h. The
MeOH extract was filtered and concentrated to dryness under reduced pres-
sure. The crude extract (225 g) was successively extracted with n-hexane,
(5ϫ500 ml) and EtOAc (5ϫ500 ml) to give 15.8 g and 122 g of extract, re-
spectively and 67.5 g of residue. A 90 g portion of the EtOAc extract was
subjected to CC on silica gel (400 g) using a gradient solvent system of
hexane, hexane–EtOAc, EtOAc, EtOAc–MeOH and MeOH in increasing po-
larity. A total of 200 fractions of 250 ml each were collected and combined
on the basis of TLC analysis leading to six main series I—VI. Series III
(9.5 g) [fractions 20—89] was rechromatographed on silica gel, using
hexane–EtOAc (70 : 30) to give fifty fractions (F1—F50). Fractions F3 and
F7 afforded compound 1 (200 mg) and 4 (150 mg) respectively. Series V
(3 g) [fractions 150—190] was separated on Sephadex LH-20 eluted with
MeOH to give twenty fractions [F1—F20]. Fraction F18 was further purified
by preparative TLC, using MeOH/CH2Cl2/cyclohexane (1.5 : 5 : 3.5) to give
compounds 2 (32 mg) and 3 (28 mg).
16) Hao-Bin H., Xu-Dong Z., Hong C., Xiao-Qiang G., Huai-Sheng H.,
Chem. Pharm. Bull., 57, 1000—1003 (2009).