Arboreasides A-E, triterpene saponins from the bark of Cussonia arborea

Article abstract of DOI:10.1021/np8008094

Five new triterpene saponins, arboreasides A-E (1-5), and two known saponins, ciwujianoside C₃ and 23-hydroxyursolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6) -β-D-glucopyranosyl ester, were isolated from the bark of Cussonia arborea. The structures were established using extensive 1D and 2D NMR spectroscopic analyses and mass spectrometry.

Full text of DOI:10.1021/np8008094

J. Nat. Prod. 2009, 72, 1081–1086
1081
Arboreasides A-E, Triterpene Saponins from the Bark of Cussonia arborea
†
,‡
§
⊥
⊥
‡
Guy B. Kougan, Tomofumi Miyamoto, Jean-Fran c¸ ois Mirjolet, Olivier Duchamp, Beibam L. Sondengam, and
,
†
Marie-Aleth Lacaille-Dubois*
Laboratoire de Pharmacognosie, Unit e´ de Mol e´ cules d’Int e´ r eˆ t Biologique, UMIB UPRES-EA 3660, Facult e´ de Pharmacie, UniVersit e´ de
Bourgogne, 7, BouleVard Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France, Laboratoire de Substances Naturelles, D e´ partement de Chimie
Organique, Facult e´ des Sciences, UniVersit e´ de Yaound e´ I, BP 812 Yaound e´ -Cameroun, Graduate School of Pharmaceutical Sciences, Kyushu
UniVersity, Fukuoka 812-8582, Japan, and Oncodesign, 20, Rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France
ReceiVed January 9, 2009
3
Five new triterpene saponins, arboreasides A-E (1-5), and two known saponins, ciwujianoside C and 23-hydroxyursolic
acid 28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranosyl ester, were isolated from the bark
of Cussonia arborea. The structures were established using extensive 1D and 2D NMR spectroscopic analyses and
mass spectrometry.
Cussonia arborea Hochst (Araliaceae) is a tree widely distributed
in the savanna from western into the central and eastern areas of
Africa. Different parts of this plant are used for the treatment of
eye injuries, paralysis, epilepsy, convulsions, spasms, veneral
diseases, diarrhea, dizziness, and women’s infertility, and for social
proton for Glc. The broad singlet of the anomeric proton
1
3
indicated an R-orientation for Rha. In the C NMR spectrum
of 1-5, the downfield chemical shift at δ 82.1-88.9 (Agly-3)
and the upfield chemical shift at δ 176.1-176.5 (Agly-28)
C
C
(Table 1) indicated that 1-5 are bidesmosidic saponins.
Arboreaside A (1) had the molecular formula C H O , as
1
,2
purposes. Previous phytochemical investigations on the genus
5
4
88 23
3
-5
Cussonia revealed the presence of triterpene saponins,
diterpene
determined by HRESIMS (positive-ion mode), showing a pseudo-
molecular ion peak at m/z 1127.5620 [M + Na]+ (calcd for
1127.5614). Its FABMS (negative-ion mode) displayed a quasi-
6
7,8
glycosides, and flavonoids. A literature survey showed that no
significant chemical and biological work has been done on C.
arborea.
-
molecular ion peak at m/z 1103 [M - H] , indicating a molecular
In continuation of our study on the glycosides from Araliaceae
weight of 1104. Further fragment ion peaks were observed at m/z
9,10
-
-
plants,
we describe herein the isolation and structure elucidation
957 [(M - H) - 146] , 633 [(M - H) - 146 - 162 - 162)] ,
-
of five new triterpene glycosides, arboreasides A-E (1-5), as well
as two known saponins from C. arborea bark.
and 471 [(M - H) - 146 - 162 - 162 - 162] , corresponding to
the successive losses of one deoxyhexosyl and three hexosyl
moieties, respectively. The fragment ion peak at m/z 471 is related
to the pseudomolecular ion of the aglycone.
Results and Discussion
The methanol extract of the dried bark of C. arborea was
subjected to multiple chromatographic steps (VLC and MPLC; see
Experimental Section), affording five new bidesmosidic triterpene
saponins, arboreasides A-E (1-5), as well as two known saponins,
The aglycone of 1 was identified as hederagenin by comparison
1
13
of H and C NMR spectroscopic data obtained from the 2D NMR
1
4
spectra of 1 with reported data. The presence of four sugar
residues was confirmed from the observation of four anomeric
1
1
ciwujianoside C
3
and the 28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-
protons at δ
(br s), and 6.00 (d, J ) 8.0 Hz), giving HSQC correlations with
four anomeric carbons at δ 104.0, 105.1, 102.0, and 95.0,
H
4.84 (d, J ) 7.8 Hz), 5.04 (d, J ) 8.3 Hz), 5.60
D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranoside of 23-hydroxyursolic
1
2
acid. Their structures were elucidated using chemical and
spectroscopic methods including 1D and 2D NMR experiments,
HRESIMS, and FABMS.
C
respectively. Complete assignments of the resonances of each sugar
unit were achieved by extensive 1D and 2D NMR analyses.
Evaluation of chemical shifts and spin-spin couplings allowed the
identification of two ꢀ-glucopyranosyl (Glc I and Glc II), one
ꢀ-galactopyranosyl (Gal), and one R-rhamnopyranosyl (Rha) unit
in 1. The absolute configuration of Glc and Gal was determined as
D and that of Rha as L (see above).
Compounds 1-5 were isolated as white, amorphous powders.
The sugars obtained by aqueous acid hydrolysis of each
compound were identified by comparison on TLC with authentic
samples as glucose, galactose, and rhamnose (in the case of 1
and 2), glucose, xylose, arabinose, and rhamnose (in the case
of 3), glucose, galactose, arabinose, and rhamnose (in the case
of 4), and glucose, galactose, and rhamnose (in the case of 5).
The absolute configurations of the sugars were determined to
be D for galactose, glucose, and xylose and L for arabinose and
rhamnose by GC analysis of chiral derivatives of the sugars in
the hydrolysate of each compound (see Experimental Section).
The sequence of the oligosaccharidic chains of 1 was determined
by HMBC and NOESY experiments. The HMBC correlations
between the anomeric proton at δ 5.60 (br s, Rha H-1) and the
H
carbon at δ 78.1 (Glc I C-4) and between the proton at δ 5.04
C
H
(d, J ) 8.3 Hz, Glc I H-1) and the carbon at δ 82.1 (Agly C-3)
C
proved the sequence of the sugar chain at C-3 to be R-L-
1
In the H NMR spectra of the compounds, the relatively large
rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl. This sequence was
3
J
H-1,H-2 values of the Gal, Glc, Xyl, and Ara (between 7.0 and
confirmed by the observation of NOESY cross-peaks between the
8
.3 Hz) moieties indicated a ꢀ anomeric proton for Gal, Glc,
protons at δ 5.60 (Rha H-1) and 4.14 (Glc I H-4) and between
H
1
3
and Xyl and an R anomeric proton for Ara, whereas the small
the protons at δH 5.04 (Glc I H-1) and 4.11 (Agly H-3). Other
3
J
H-1,H-2 value (J ) 4.2 Hz) of the Glc indicated an R anomeric
HMBC correlations between the proton at δ 4.84 (d, J ) 7.8 Hz,
H
Gal H-1) and the carbon at δ
proton at δ
76.5 (Agly C-28) and a NOESY cross-peak between the proton
at δ 4.84 (Gal H-1) and the proton at δ 4.54 (Glc II H-6a) allowed
C
68.6 (Glc II C-6) and between the
*
To whom correspondence should be addressed. Tel: +33-3-80393229.
6.00 (d, J ) 6.0 Hz, Glc II H-1) and the carbon at δ
H
C
Fax: +33-3-80393300. E- mail: marie-aleth.lacaille-dubois@u-bourgogne.fr.
1
†
Universit e´ de Bourgogne.
Universit e´ de Yaound e´ I.
Kyushu University.
Oncodesign.
‡
H
H
§
the sequencing of sugars at C-28 as ꢀ-D-galactopyranosyl-(1f6)-
ꢀ-D-glucopyranosyl. Thus, the structure of arboreaside A (1) was
⊥
1
0.1021/np8008094 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/20/2009

1
082 Journal of Natural Products, 2009, Vol. 72, No. 6
Kougan et al.
Chart 1
concludedtobe3-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-
hederagenin-28-O-ꢀ-D-galactopyranosyl-(1f6)-ꢀ-D-glucopyrano-
syl ester.
The molecular formula of arboreaside B (2) was determined as
54 88
C H O23 by HR-ESIMS, showing a pseudomolecular ion peak at
+
m/z 1127.5618 [M + Na] (calcd for 1127.5614). Its FABMS

Triterpene Saponins from Cussonia arborea
Journal of Natural Products, 2009, Vol. 72, No. 6 1083
Table 1. 13C and H NMR Data for the Aglycone Moieties of 1-5
1
a
1
2
3
4
5
position
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
1
2
3
4
5
6
7
8
9
38.3
25.6
82.1
41.2
46.9
17.7
32.1
39.9
47.6
36.4
23.3
122.6
143.8
42.1
32.4
24.1
48.0
41.7
45.8
30.2
36.2
32.6
63.5
13.2
15.7
17.1
25.6
176.5
32.6
23.2
1
0.84, 1.38
1.82, 2.22
4.13 m
38.4
25.6
82.1
42.2
47.7
17.9
32.1
39.5
47.7
36.5
23.4
125.7
138.1
42.2
32.4
24.2
48.1
52.9
39.0
38.7
36.4
33.6
64.0
13.2
15.8
17.2
23.3
176.5
17.0
20.9
0.84, 1.38
1.82, 2.22
4.13 m
38.4
25.9
88.9
39.1
55.4
18.2
32.8
39.5
47.6
36.6
23.3
123.3
143.8
41.8
27.8
23.0
46.7
41.3
45.9
30.3
33.6
32.1
27.8
16.4
15.2
17.1
25.7
176.5
32.8
23.3
0.76, 1.38
1.74, 1.89
3.11
38.4
26.5
88.8
39.5
55.5
18.2
32.8
39.5
47.7
36.9
23.5
123.1
143.8
42.0
27.9
23.0
46.7
41.8
45.9
30.4
33.8
32.1
27.9
16.4
15.2
17.1
25.7
176.5
32.8
23.3
0.82, 1.42
1.78, 1.98
3.14 m
38.6
26.9
88.8
39.5
55.5
18.2
33.6
39.5
48.1
36.4
23.4
122.5
143.5
42.1
27.9
23.0
46.7
41.3
45.9
30.3
32.4
32.1
27.9
16.7
15.7
17.2
25.7
176.1
32.7
23.3
0.78, 1.45
, 1.82
3.31
b
1.50
1.20, 1.55
1.66, 1.76
1.54
1.22, 1.52
1.66, 1.76
0.63 m
1.15, 1.33
1.24, 1.36
0.64 d (11.9)
1.16, 1.34
1.26, 1.34
0.68
1.26, 1.34
b
1.20,
1.62
1.62
1.49 m
1.52 m
1.62 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0.80, 1.82
5.35 br s
0.80, 1.82
5.34 br s
1.09, 1.80
5.34 br s
1.10, 1.82
5.35 br s
1.82, 1.84
5.35 br s
1.22, 1.58
1.84, 1.94
1.22, 1.58
1.84, 1.94
1.15, 2.19
1.94, 2.00
1.14, 2.19
1b .20, 2.19
b
, 1.82
3.05 br d (13.0)
1.12, 1.62
2.38 d (10.7)
1.29 m
0.79 m
b1.66, 1.80
3.09
1.15, 1.66
3.09
1.16, 1.64
3.08 br d (13.1)
1.14, 1.62
b
1.63, 1.80
1.12, 1.56
3.56 d (11.2), 4.16
0.84 s
0.83 s
0.99 s
1.01, 1.24
1.63, 1.80
1.09 s
0.92 s
0.76 s
1.24,
1.26, 1.50
1.66, 1.79
1.22 s
0.92 s
0.88 s
1.66, 1.79
1.14 s
0.96 s
0.78 s
1.00 s
3.58 d (11.0), 4.18
0.88 s
0.84 s
1.01 s
1.04 s
0.98 s
1.15 s
1.02 s
1.16 s
1.12 s
1.16 s
0.82 s
0.79 s
13
0.84
0.81 d (5.7)
0.82 s
0.81 s
0.81 s
0.82 s
0.82 s
0.82 s
a
6
5
00 MHz ( H), 125 MHz ( C), pyridine-d , δ (ppm), coupling constants (J) in Hz are given in parentheses, overlapped signals are reported without
b
designated multiplicity. Not determined.
(
1
negative-ion mode) displayed a quasimolecular ion peak at m/z
The aglycone of 3 was identified as oleanolic acid by comparison
-
1
13
103 [M - H] , indicating a molecular weight of 1104. Further
of H and C NMR spectroscopic data obtained from the 2D NMR
-
16
fragment ion peaks were observed at m/z 957 [(M - H) - 146] ,
6
spectra of 3 with reported data.
-
33 [(M - H) - 146 - 162 - 162)] , and 471 [(M - H) - 146
The presence of six sugar residues was confirmed from the
-
-
162 - 162 - 162] , corresponding to the successive loss of
observation of six anomeric protons at δ 4.88 (d, J ) 6.5 Hz),
H
one desoxyhexosyl and three hexosyl moieties, respectively.
4.96 (d, J ) 4.2 Hz), 5.12 (d, J ) 7.1 Hz), 5.13 (d, J ) 7.1 Hz),
5.65 (br s), and 6.09 (d, J ) 8.0 Hz), giving correlations in the
1
13
Comparison of the H and C NMR data of 2 with 1 (Tables 1
and 2) showed that they differ only in the aglycone part. Thus, a
C
HSQC spectrum with six anomeric carbons at δ 104.0, 103.8,
set of olefinic carbon signals at δ
and a tertiary carbon signal at δ
instead of δ
Furthermore, two tertiary carbons at δ
0), showing HSQC correlations with protons at δ
C
125.6 (C-12) and 138.0 (C-13)
52.8 (C-18) was observed in 2
103.0, 106.2, 102.2, and 95.2, respectively. Complete assignments
of each sugar by extensive 1D and 2D NMR spectroscopic analysis
allowed the identification of one R-glucopyranosyl (Glc I), two
ꢀ-glucopyranosyl (Glc II and Glc III), one ꢀ-xylopyranosyl (Xyl),
one R-rhamnopyranosyl (Rha), and one R-arabinopyranosyl (Ara)
moiety. The absolute configuration of Xyl and Glc was determined
as D and that of Rha and Ara as L (see above).
C
C
122.6 (C-12), 143.8 (C-13), and 41.7 (C-18) in 1.
39.0 (C-19) and 38.7 (C-
1.29 (m, H-19)
C
2
H
and 0.79 (m, H-20), respectively, and a COSY correlation between
these two resonances, are in good agreement with an ursane-type
skeleton. Aditionally, the proton at δ
showing a long-range correlation in the HMBC spectrum with the
carbons at δ 125.6 (C-12), 138.0 (C-13), 48.0 (C-17), and 39.0
C-19) confirmed the proposed skeleton. Thus, the structure of the
aglycone of 2 was elucidated as 23-hydroxyurs-12-en-28-oic acid,
H
2.38 (d, J ) 10.7 Hz, H-18)
The sequence of the oligosaccharidic chains of 3 was determined
by HMBC and NOESY experiments. HMBC correlations between
C
the proton at δ
at δ 84.0 (Xyl C-3), between the proton at δ
Xyl H-1) and the carbon at δ 79.2 (Glc I C-2), and between the
proton at δ 4.96 (d, J ) 4.2, Glc I H-1) and the carbon at δ 88.9
H
5.12 (d, J ) 7.1 Hz, Glc II H-1) and the carbon
(
C
H
5.13 (d, J ) 7.1,
C
4
,15
which was confirmed by extensive 2D NMR experiments.
H
C
According to the above data, arboreoside B (2) was determined to
be 3-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl 23-hy-
droxyurs-12-en-oic acid 28-O-ꢀ-D-galactopyranosyl-(1f6)-ꢀ-D-
glucopyranosyl ester.
(Agly C-3) proved the sugar chain sequence at C-3 to be
ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-xylopyranosyl-(1f2)-R-D-glucopy-
ranosyl. This was confirmed by NOESY cross-peaks between the
proton at δ
between the proton at δ
5.13 (Xyl H-1), and between the proton at δ
the proton at δ 3.11 (Agly H-3). Other HMBC correlations between
the proton at δ 5.65 (Rha H-1) and the carbon at δ 78.1 (Glc III
C-4), between the proton at δ 4.88 (Ara H-1) and the carbon at
68.5 (Glc III C-6), and between the proton at δ 6.09 (Glc III
H-1) and the carbon at δ 176.5 (Agly C-28) suggested the sequence
H
5.12 (Glc II H-1) and the proton at δ
4.46 (Glc I, H-2) and the proton at δ
4.96 (Glc I H-1) and
H
3.99 (Xyl H-3),
Arboreaside C (3) had the molecular formula C64
H
104
O
30 by
H
H
HRESIMS ([M + Na]+ at m/z 1375.6519, calcd for 1375.6510).
H
The FABMS (negative-ion mode) displayed a quasimolecular ion
H
-
peak at m/z 1351 [M - H] , indicating a molecular weight of 1352.
H
C
Further fragment ion peaks were observed at m/z 779 [(M - H) -
H
1
1
62 - 132 - 132 - 146)]- and 455 [(M - H) - 162 - 132 -
δ
C
H
-
32 - 146 - 162 - 162] , corresponding to the loss of three
C
hexosyl, one deoxyhexosyl, and two pentosyl moieties. The
fragment ion peak at m/z 455 is related to the pseudomolecular ion
of aglycone.
at C-28 to be [R-L-arabinopyranosyl-(1f6)]-[R-L-rhamnopyranosyl-
(1f4)]-ꢀ-D-glucopyranosyl. This was confirmed by the HMBC
reverse correlation between the proton at δ 4.23 (Glc III H-6b)
H

1
084 Journal of Natural Products, 2009, Vol. 72, No. 6
Kougan et al.
Table 2. 13C and H NMR Data for Sugar Moieties of 1-5
1
a
1
2
3
4
5
position
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
3
1
2
3
4
5
-O
Glc I
105.1
75.7
77.6
78.1
77.4
Glc I
105.1
75.7
77.4
78.1
77.4
Glc I
103.8
79.2
77.1
70.0
77.8
Ara
Glc I
106.1
75.6
77.6
70.2
77.5
5.04 d (8.3)
3.97
4.20
4.16
3.85
5.02 d (8.3)
3.97
4.20
4.16
3.85
4.96 d (4.2)
4.46
4.13 m
4.24
104.4
79.7
73.0
78.1
64.6
4.86 d (6.4)
4.52 t (6.6)
4.29
4.21
3.75 l d (9.7)
4.82 d (8.0)
3.96 m
4.21
4.21
3.99
3.85
4
.22
6
60.7
3.94
60.7
3.94
4.07
62.0
4.27
4.42
60.8
3.95
4.06
4
.07
Xyl
Glc I
105.0
75.6
77.5
71.0
77.7
1
2
3
4
5
106.2
75.9
84.0
71.2
66.8
5.13 d (7.1)
4.00
3.99
4.09
3.65 t (10.9)
5.10 d (7.6)
3.98 m
4.11
4.12
3.73
4
.36
6
60.9
3.98, 4.10
Glc II
103.0
75.3
76.2
70.7
77.4
60.7
1
2
3
4
5
6
5.12 d (7.1)
3.97 m
4.00
4.04
3.68 m
3.97, 4.07
Rha
Rha
Rha
1
2
3
4
5
6
2
1
2
3
4
5
6
102.0
71.7
71.8
73.1
69.8
17.9
Glc II
95.0
73.1
77.6
70.9
77.4
68.6
Gal
5.60 br s
4.54 m
4.42 m
4.21
4.72
1.56 d (5.9)
102.0
71.7
71.8
73.1
69.4
17.9
Glc II
95.0
73.1
77.6
70.9
77.4
68.4
Gal
5.60 br s
4.54
4.42 m
4.21
4.72
1.56 d (5.9)
102.2
71.9
72.0
73.8
70.0
17.9
Glc II
95.2
73.3
77.5
70.5
77.5
68.6
Gal
5.66 br s
4.56 m
4.44 m
4.24
4.76 m
1.60 d (6.1)
8-O
Glc III
95.2
73.2
77.8
78.1
77.4
68.5
Ara
104.0
74.6
75.9
70.2
64.0
Gal
6.00 d (8.0)
4.03
4.16
4.07
3.98
6.05 d (8.0)
4.03
4.16
4.07
3.98
6.09 d (8.0)
4.05
4.23
4.17 m
3.98
4.23, 4.56
6.10 d (8.0)
4.06 t (8.0)
4.19
4.24
4.00
95.1
72.9
75.9
70.0
76.6
68.5
Glc II
104.2
78.1
77.6
72.0
76.8
6.09 d (8.0)
4.06 m
4.00
4.74 m
3.42
4.21, 4.54
4.21, 4.54
4.24, 4.56
4.22, 4.55
1
2
3
4
5
104.0
74.4
75.7
70.0
76.5
4.84 d (7.8)
3.83
4.00
4.18
3.50 m
104.1
74.4
75.7
69.8
76.5
4.84 d (7.8)
3.83
4.00
4.18 m
3.50 m
4.88 d (7.6)
3.83
4.02
4.20
3.82 l d (8.3)
104.0
74.6
75.9
67.8
76.8
4.90 d (7.8)
3.85 t (8.5)
4.02
4.32
3.53
4.88 d (7.8)
4.18
4.20
4.42
3.52
4
.23
6
62.0
4.21, 4.35
l d (11.5)
62.0
4.21, 4.35
l d (11.5)
62.0
4.25, 4.34
62.5
4.25 m, 4.46 m
Rha
1
2
3
4
5
6
102.2
71.8
72.0
73.2
69.9
17.9
5.65 br s
4.56
4.42
4.23
4.76 m
1.58 d (6.1)
Rha
1
2
3
4
5
6
a
102.1
72.0
72.9
73.2
69.6
18.0
5.64 br s
4.55 m
4.06 m
4.04 m
4.74 m
1.58 d (5.9)
1
13
6
00 MHz ( H), 125 MHz ( C), pyridine-d
5
, δ (ppm), coupling constants (J) in Hz are given in parentheses, overlapped signals are reported without
designated multiplicity.
and the carbon at δ
between the proton at δ
(
C
104.0 (Ara C-1) and by NOESY cross-peaks
4.88 (Ara H-1) and the proton at δ 4.56
Glc III H-6a) and between the proton at δ 5.65 (Rha H-1) and
4.17 (Glc III, H-4). Thus, the structure of
The molecular formula of arboreaside D (4) was determined as
+
H
H
59 96
C H O26 by HRESIMS, showing the [M + Na] ion peak at m/z
H
1243.613 (calcd for 1243.6088). Its FABMS (negative-ion mode)
-
the proton at δ
H
displayed a quasimolecular ion peak at m/z 1219 [M - H] ,
arboreaside C (3) was elucidated as 3-O-ꢀ-D-glucopyranosyl-(1f3)-
-D-xylopyranosyl-(1f2)-R-D-glucopyranosyloleanolic acid 28-O-
R-L-arabinopyranosyl-(1f6)]-[R-L-rhamnopyranosyl-(1f4)]-ꢀ-D-
glucopyranosyl ester.
indicating a molecular weight of 1220. Further fragment ion peaks
-
ꢀ
[
were observed at m/z 749 [(M - H) - 162 - 162 - 146] and
-
455 [(M - H) - 162 - 162 - 146 - 132 - 162] , corresponding
to the successive loss of two hexosyl, one deoxyhexosyl, one

Triterpene Saponins from Cussonia arborea
Journal of Natural Products, 2009, Vol. 72, No. 6 1085
pentosyl, and one hexosyl moiety, respectively. The fragment ion
peak at m/z 455 is related to the pseudomolecular ion of aglycone.
the sugar chain at C-28 as R-L-rhamnopyranosyl-(1f2)-ꢀ-D-
glucopyranosyl-(1f6)-ꢀ-D-galactopyranosyl. The structure of ar-
boreaside E (5) was thus elucidated as 3-O-ꢀ-D-glucopyranosy-
loleanolic acid 28-O-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-glucopyrano-
syl-(1f6)-ꢀ-D-galactopyranosyl ester.
Extensive studies of 1D and 2D NMR spectra data led to the
1
6
identification of the aglycone of 4 as oleanolic acid.
1
The H NMR spectrum of 4, showing five anomeric protons at
4.86 (d, J ) 6.4 Hz), 4.90 (d, J ) 7.8 Hz), 5.10 (d, J ) 7.6
δ
H
Hz), 5.66 (br s), and 6.10 (d, J ) 8.0 Hz), giving correlations in
the HSQC spectrum with five anomeric carbons at δ 104.4, 104.0,
05.0, 102.2, and 95.2 respectively, indicated the presence of five
sugar moieties. Units of one R-rhamnopyranosyl (Rha), one
R-arabinopyranosyl (Ara), two ꢀ-glucopyranosyl (Glc I and Glc
II), and one ꢀ-galactopyranosyl (Gal) were identified. The absolute
configuration of the sugar residues was D for Glc and Gal and L
for Rha and Ara (see above).
Experimental Section
C
General Experimental Procedures. Optical rotations were recorded
on an AA-OR automatic polarimeter. IR spectra (KBr disk) were
recorded on a Perkin-Elmer 281 spectrometer. 1D and 2D NMR spectra
were recorded in pyridine-d using a Varian INOVA-600 (600 MHz)
NMR spectrometer. Solvent signals were used as internal standard
1
5
(pyridine-d
are in Hz. HRESIMS (positive-ion mode) was carried out on a Q-TOF
-micromass spectrometer and FABMS (negative-ion mode, glycerol
5 H C
: δ 7.21, δ 123.5 ppm), and the coupling constants (J)
1
HMBC correlations between the proton at δ
H-1) and the carbon at δ 78.1 (Ara C-4), between the proton at
5.10 (d, J ) 7.6 Hz, Glc I H-1) and the carbon at δ 79.7 (Ara
C-2), and between the proton at δ 4.86 (d, J ) 6.4 Hz, Ara H-1)
and the carbon at δ 88.8 (Agly C-3) suggested the sequence at
H
5.66 (br s, Rha
matrix) on a Jeol-SX-102 mass spectrometer. GC analysis was carried
out on a Thermoquest gas chromatograph. TLC and HPTLC were
carried out on precoated silica gel plates 60F254 (Merck)
C
δ
H
C
H
(
CHCl
3
-MeOH-H O-HOAc, 65:32:6.5:0.5 and 70:30:5:0.5). Sa-
2
C
ponins were detected with the Komarowsky reagent. Isolations were
carried out using column chromatography (CC) on silica gel 60 (Merck,
70-200 µm), CC on Sephadex LH-20, vacuum-liquid chromatography
(VLC) on reversed-phase RP-18 (25-40 µm), medium-pressure liquid
chromatography (MPLC) on silica gel 60 (Merck, 15-40 µm) (Gilson
C-3 as [R-L-rhamnopyranosyl-(1f4)]-[ꢀ-D-glucopyranosyl-(1f2)]-
R-L-arabinopyranosyl. This was confirmed by NOESY correlations
between the proton at δ
(
H
5.66 (Rha H-1) and the proton at δ
Ara H-4), between the proton at δ 5.10 (Glc I H-1) and the proton
4.52 (Ara H-2), and between the proton at δ 4.86 (Ara H-1)
3.14 (Agly H-3). Other HMBC correlations
between the proton at δ 4.90 (Gal H-1) and the carbon at δ 68.6
Glc II C-6) and between the proton at δ 6.10 (Glc II H-1) and
176.5 (Agly C-28) and NOESY cross-peaks
4.90 (d, J ) 7.8 Hz, Gal H-1) and the
4.24 (Glc II H-6a) allowed the sugar sequence at C-28
H
4.21
H
13
apparatus), and flash chromatography (Combiflash, Serlabo; silica gel
Redisep flash column, 15-40 µm, 3.5 × 14 cm, 40 g).
at δ
H
H
and the proton at δ
H
Plant Material. The bark of C. arborea was collected in the village
Bangoua near Bangangt e´ located in the Nd e´ Division of the Western
Highlands of Cameroon in April 2007 and identified by Dr. P. Nana,
botanist of the National Herbarium of Cameroon (NHC), Yaound e´ ,
where a voucher specimen (N1545) was deposited.
H
C
(
H
the carbon at δ
C
between the proton at δ
proton at δ
H
H
Extraction and Isolation. Dried and finely powdered bark of C.
arborea (1.0 kg) was macerated with MeOH (3 L) for 48 h. After
evaporation of the solvent in vacuo, a dark residue of 140 g was
obtained. A 13 g aliquot of this MeOH extract was submitted to VLC
as ꢀ-D-galactopyranosyl-(1f6)-ꢀ-D-glucopyranosyl. Thus, arborea-
side D (4) was elucidated as 3-O-[R-L-rhamnopyranosyl-(1f4)]-
[
ꢀ-D-glucopyranosyl-(1f2)]-R-L-arabinopyranosyloleanolic acid 28-
on RP-18 silica gel using H
2
O (3 × 100 mL), MeOH-H O (5:5, 3 ×
2
O-ꢀ-D-galactopyranosyl-(1f6)-ꢀ-D-glucopyranosyl ester.
1
00 mL), and MeOH (200 mL). The MeOH-soluble portion was
The molecular formula of arboreoside E (5) was determined as
C H O22 by HRESIMS, showing the pseudomolecular ion peak
54 88
evaporated to dryness to afford a yellowish powder (7.6 g). An aliquot
of 2.5 g was then fractionated by flash chromatography on silica gel
+
[
(
1
M + Na] at m/z 1111.5671 (calcd for 1111.5665). The FABMS
3 2
using CHCl -MeOH-H O (62:32:6.5) as eluent, affording 10 fractions,
negative-ion mode) displayed a quasimolecular ion peak at m/z
F-1 (66.3 mg), F-2 (114.4 mg), F-3 (22.1 mg), F-4 (223.5 mg), F-5
(24.7 mg), F-6 (110.2 mg), F-7 (120 mg), F-8 (90.7 mg), F-9 (80.4
mg), and F-10 (10 mg).
-
087 [M - H] , indicating a molecular weight of 1088. Further
-
fragment ion peaks were observed at m/z 925 [(M - H) - 162] ,
-
6
1
17 [(M - H) - 162 - 146 - 162] , and 455 [(M - H) - 162 -
F-8 (90.7 mg) was purified by successive MPLC on RP-18 silica
-
46 - 162 - 162] , corresponding to the sucessive loss of one
gel eluted with MeOH-H
6.8 mg), and 4 (3.5 mg). F-9 (80.4 mg) was purified under the same
conditions to yield 3 (10 mg). F-4 (223.5 mg) was purified by repeated
MPLC on silica gel using gradient system of solvent,
CHCl O (70:30:5 f 80:20:2), followed by MPLC on RP-
O gradient system to yield 5 and
2
O (40% f 100%), yielding 1 (29.1 mg), 2
(
hexosyl, one deoxyhexosyl, and two hexosyl moieties, respectively.
The fragment ion peak at m/z 455 is related to the pseudomolecular
ion of aglycone.
a
3
-MeOH-H
2
The 1D and 2D NMR spectroscopic data led to the conclusion
1
8 silica gel with a MeOH-H
2
that 5 is a bidesmosidic saponin having an oleanolic acid as
11
3
ciwujianoside C .
1
6
1
aglycone (Tables 1 and 2). The H NMR spectrum of 5 showed
four anomeric protons at δ 4.82 (d, J ) 8.0 Hz), 4.88 (d, J ) 7.8
Hz), 5.64 (br s), and 6.09 (d, J ) 8.0 Hz), which gave correlations
in the HSQC spectrum with four anomeric carbons at δ 106.1,
04.2, 98.4, and 95.1, respectively. 2D NMR data allowed the
F-3 (22.1 mg) was obtained as a pure compound identified as 28-
O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glu-
copyranoside of 23-hydroxyursolic acid, identified by comparison of
the reported spectroscopic data.
Acid Hydrolysis and GC Analysis. Each compound (3 mg) was
H
1
2
C
1
hydrolyzed with 2 N aqueous TFA (5 mL) for 3 h at 95 °C. After extraction
identification of one R-rhamnopyranosyl (Rha), two ꢀ-glucopyra-
nosyl (Glc I and Glc II), and one ꢀ-galactopyranosyl (Gal) unit.
The absolute configuration was D for Glc and Gal and L for Rha
with CH
2
Cl
2
(3 × 5 mL), the aqueous layer was repeatedly evaporated to
dryness with MeOH until neutral and then analyzed by TLC over silica
gel (CHCl -MeOH-H O, 8:5:1) by comparison with authentic samples.
The trimethylsilyl thiazolidine derivatives of the sugar residue of each
3
2
(
see above).
17
The substitution of Agly C-3 by the terminal Glc I compared to
compound were prepared and analyzed by GC. The absolute configura-
tions were determined by comparing the retention times with thiazolidine
derivatives prepared in a similar way from standard sugars (Sigma-Aldrich).
The following sugars were detected: D-glucose, D-galactose, L-rhamnose
for 1 and 2; D-glucose, D-xylose, L-arabinose, and L-rhamnose for 3;
D-glucose, D-galactose, L-arabinose, and L-rhamnose for 4, and D-glucose,
oleanolic acid was suggested by HMBC correlations between the
proton at δ 4.82 (d, J ) 8.0 Hz, Glc I H-1) and the carbon at δ
8.8 (Agly C-3). This was confirmed by a NOESY cross-peak
H
C
8
between the proton at δ
.31 (Agly H-3).
Additional HMBC correlations between the proton at δ
br s, Rha H-1) and the carbon at δ
H
4.82 (Glc I H-1) and the proton at δ
H
3
D-galactose, and L-rhamnose for 5.
H
5.64
21
Arboreaside A (1): white, amorphous powder; [R]
D
-33.5 (c 0.2,
-1 1
(
C
78.1 (Glc II C-2), between
4.88 (d, J ) 7.8 Hz, Glc II H-1) and the carbon
6.09 (d, J )
176.1 (Agly C-28) established
MeOH); IR (KBr) νmax 3440, 1726, 1655, 1260, 1074 cm ; H NMR
the proton at δ
H
13
5
and C NMR (pyridine-d ), see Tables 1 and 2; FABMS (negative-
-
-
at δ
C
68.5 (Gal C-6), and between the proton at δ
H
ion mode) m/z 1103 [M - H] , 957 [(M - H) - 146] , 633 [(M - H)
-
8
.0 Hz, Gal H-1) and the carbon at δ
C
- 146 - 162 - 162)] , 471 [(M - H) - 146 - 162 - 162 - 162];

1
086 Journal of Natural Products, 2009, Vol. 72, No. 6
Kougan et al.
+
HRESIMS (positive-ion mode) m/z 1127.5620 [M + Na] (calcd for
(2) Adjanohoun, E.; Ahiji, M. R. A.; Ak e´ Assi, L.; Dramane, K.; Elewude,
J. A.; Fadoju, S. O.; Gbile, Z. O.; Goudot e´ , E.; Johnson, C. L. A.;
Keita, A.; et al. Traditional Medicine and Pharmacopoeia, Contribu-
tion to Ethnobotanical and Floristic Studies in Western Nigeria;
Commission of Scientific and Technical Research/Organisation of
African Unity: Lagos, 1991.
C
54
H
88
O
23Na, 1127.5614).
Arboreaside B (2): white, amorphous powder; [R]
MeOH); IR (KBr) νmax 3450, 1736, 1649, 1250, 1072 cm ; H NMR
21
D
+52.2 (c 0.05,
-1 1
and 1 C NMR (pyridine-d
3
5
), see Tables 1 and 2; FABMS (negative-
-
-
ion mode) m/z 1103 [M - H] , 957 [(M - H) - 146] , 633 [(M - H)
(
(
3) Dubois, M. A.; Ilyas, M.; Wagner, H. Planta Med. 1986, 52, 80–83.
4) Tapondjou, L. A.; Lontsi, D.; Sondengam, B. L.; Shaheen, F.;
Choudhary, M. I.; Atta-ur-Rahman; Van Heerden, F. R.; Park, H. J.;
Lee, K. T. J. Nat. Prod. 2003, 66, 1266–1269.
-
-
-
146 - 162 - 162] , 471 [(M - H) - 146 - 162 - 162 - 162] ;
+
HRESIMS (positive-ion mode) m/z 1127.5620 [M + Na] (calcd for
23Na, 1127.5614).
Arboreaside C (3): white, amorphous powder; [R]
MeOH); IR (KBr) νmax 3445, 1730, 1640, 1250, 1076 cm ; H NMR
54 88
C H O
21
D
+20.9 (c 0.2,
(5) Harinantenaina, L.; Kasai, R.; Yamasaki, K. Chem. Pharm. Bull. 2002,
50, 1290–1293.
(6) Harinantenaina, L.; Kasai, R.; Yamasaki, K. Chem. Pharm. Bull. 2002,
50, 1122–1123.
-
1 1
and 1 C NMR (pyridine-d
3
5
), see Tables 1 and 2; FABMS (negative-
-
ion mode) m/z 1351 [M - H] , 779 [(M - H) - 162 - 132 - 132 -
-
-
(7) Haruna, A. K.; Ilyas, M.; Mustapha, A. Ghana J. Chem. 1990, 1, 160–
1
46] , 455 [(M - H) - 162 - 132 - 132 - 146 - 162 - 162] ;
+
1
65.
HRESIMS (positive-ion mode) m/z 1375.6519 [M + Na] (calcd for
30Na, 1375.6510).
Arboreaside D (4): white, amorphous powder, [R]
MeOH); IR (KBr) νmax 3435, 1728, 1655, 1255, 1080 cm ; H NMR
and 13C NMR (pyridine-d
), see Tables 1 and 2; FABMS (negative-
(
(
8) Trotin, F.; Bezanger-Beauquesne, L.; Pinkas, M. C. R. S e´ ances Acad.
Sci., Ser. D: Sci. Nat. 1969, 268, 855–858.
9) Mitaine-Offer, A. C.; Tapondjou, L. A.; Lontsi, D.; Sondengam, B. L.;
Choudhary, M. I.; Atta-ur-Rahman; Lacaille-Dubois, M. A. Biochem.
Syst. Ecol. 2004, 32, 607–610.
64 104
C H O
2
1
D
+25 (c 0.16,
-1 1
5
-
-
ion mode) m/z 1219 [M - H] , 749 [(M - H) - 162 - 162 - 162] ;
(10) Tapondjou, L. A.; Mitaine-Offer, A. C.; Miyamoto, T.; Lerche, H.;
Mirjolet, J. F.; Guilbaud, N.; Lacaille-Dubois, M. A. Biochem. Syst.
Ecol. 2006, 34, 887–889.
+
HRESIMS (positive-ion mode) m/z 1243.613 [M + Na] (calcd for
59 96
C H O26Na, 1243.6088).
Arboreaside E (5): white, amorphous powder; [R]
2
1
(11) Jiang, W.; Li, W.; Han, L.; Liu, L.; Zhang, Q.; Zhang, S.; Nikaido,
D
+30 (c 0.22,
-1
T.; Koike, K. J. Nat. Prod. 2003, 66, 1266–1269.
(12) Dovgii, I. I.; Grishkovets, V. I.; Kachala, V. V.; Shaskhov, A. S. Chem.
Nat. Compd. 2005, 41, 200–204.
MeOH); MeOH); IR (KBr) νmax 3435, 1735, 1660, 1260, 1078 cm
;
1
13
5
H NMR and C NMR (pyridine-d ), see Tables 1 and 2; FABMS
-
-
(
[
-
negative-ion mode) m/z 108 [M - H] , 925 [(M - H) - 162] , 617
(
13) Avunduk, S.; Mitaine-Offer, A. C.; Alankus-Caliskan, O.; Miyamoto,
-
(M - H) - 162 - 146 - 162] , 455 [(M - H) - 162 - 146 - 162
T.; Senol, S. G.; Lacaille-Dubois, M. A. J. Nat. Prod. 2008, 71, 141–
-
+
162] ; HRESIMS (positive-ion mode) m/z 1111.5671 [M + Na]
1
45.
(
calcd for C54
H
88
O
22Na, 1111.5665).
(
14) Jhoo, J.-W.; Sang, S.; He, K.; Cheng, X.; Zhu, N.; Stark, R. E.; Zheng,
Q. Y.; Rosen, R. T.; Ho, C.-T. J. Agric. Food. Chem. 2001, 49, 5969–
5974.
Acknowledgment. We are grateful to Dr. Tamze Victorine and Mr.
Simo David of the Laboratory of Chemistry, IMPM Yaound e´ , for
providing facilities for plant extraction.
(15) Mahato, B. S.; Kundu, P. A. Phytochemistry 1994, 37, 1517–1575.
(
16) Seebacher, W.; Simic, N.; Weis, R.; Saf, R.; Kunert, O. Magn. Reson.
Chem. 2003, 41, 636–638.
References and Notes
(17) Haddad, M.; Miyamoto, T.; Laurens, V.; Lacaille-Dubois, M. A. J.
Nat. Prod. 2003, 66, 372–377.
(
1) Burkill, H. M. The Useful Plants of West Tropical Africa; Royal
Botanic Gardens: Kew, 1985; Vol 1.
NP8008094