Two new biologically active triterpenoidal saponins acylated with salicylic acid from Albizia adianthifolia

Article abstract of DOI:10.1021/np020391q

Two new oleanane-type triterpene saponins, adianthifoliosides A (1) and B (2), were isolated from a 95% ethanolic extract of roots of Albizia adianthifolia. Their structures were elucidated mainly by using a combination of 600 MHz 1D and 2D NMR techniques (COSY, NOESY, TOCSY, HSQC, and HMBC) and by FABMS and HRESIMS. Compounds 1 and 2 were characterized as glycosides of acacic acid acylated by an o-hydroxybenzoyl unit. The crude saponin mixture (CSM), compounds 1 and 2 together with 3 and 4 (prosapogenins obtained from the mild alkaline hydrolysate of the CSM), were evaluated for immunomodulatory activity on the Jurkat T cell line and for hemolytic property against sheep erythrocytes. Compound 2 and, to a lesser extent, 1 and 3 were found to exhibit a dose-dependent immunomodulatory effect in the concentration range 10^-2-10 μM, whereas 4 showed a lymphoproliferative activity in the same concentration range. Among the compounds tested, only 1 and 2 were found to be hemolytic.

Full text of DOI:10.1021/np020391q

3
72
J . Nat. Prod. 2003, 66, 372-377
Tw o New Biologica lly Active Tr iter p en oid a l Sa p on in s Acyla ted w ith Sa licylic
Acid fr om Albizia a d ia n th ifolia
†
‡
§
,†
Mohamed Haddad, Tomofumi Miyamoto, V e´ ronique Laurens, and Marie-Aleth Lacaille-Dubois*
Laboratoire de Pharmacognosie, Unit e´ MIB J E 2244, Facult e´ de Pharmacie, Universit e´ de Bourgogne, 7, Bd. J eanne d’Arc,
BP 87900, 21079 Dijon Cedex, France, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, J apan, and
Groupe Immunologie Compar e´ e, UMR CNRS 5548, Facult e´ des Sciences, Universit e´ de Bourgogne, 6, Bd. Gabriel,
2
1079 Dijon Cedex, France
Received August 30, 2002
Two new oleanane-type triterpene saponins, adianthifoliosides A (1) and B (2), were isolated from a 95%
ethanolic extract of roots of Albizia adianthifolia. Their structures were elucidated mainly by using a
combination of 600 MHz 1D and 2D NMR techniques (COSY, NOESY, TOCSY, HSQC, and HMBC) and
by FABMS and HRESIMS. Compounds 1 and 2 were characterized as glycosides of acacic acid acylated
by an o-hydroxybenzoyl unit. The crude saponin mixture (CSM), compounds 1 and 2 together with 3 and
4
(prosapogenins obtained from the mild alkaline hydrolysate of the CSM), were evaluated for
immunomodulatory activity on the J urkat T cell line and for hemolytic property against sheep erythrocytes.
Compound 2 and, to a lesser extent, 1 and 3 were found to exhibit a dose-dependent immunomodulatory
-
2
effect in the concentration range 10 -10 µM, whereas 4 showed a lymphoproliferative activity in the
same concentration range. Among the compounds tested, only 1 and 2 were found to be hemolytic.
The genus Albizia comprises about 150 species widely
Compound 1 was obtained as an amorphous powder. The
distributed in the tropics, with the greatest diversity in
Africa and Central and South America. Albizia species
HRESI mass spectrum (positive-ion mode) exhibited a
quasimolecular ion peak at m/z 1714.7490 [M + Na] (calcd
121
1714.7465), consistent with a molecular formula of C79H -
1
+
have been reported to contain alkaloids, steroids, triter-
2
penoid saponins, and flavonols. Some saponins such as
NO38. Upon acid hydrolysis with 2 N TFA at 120 °C, 1
afforded the aglycon 6, which was identified as acacic acid
lactone (the 21,28-lactone derivative of acacic acid obtained
under the experimental conditions used), by comparison
3
,4
1 2 9
julibrosides J , J , and J from Albizia julibrissin possess
various biological effects, such as inhibitory activity against
the KB cancer cell line in vitro.
6
In a previous contribution, we reported the isolation and
structure determination of two prosapogenins (3 and 4)
isolated from the butanol extract of the mild alkaline
hydrolysate of the crude saponin fraction of the roots of
Albizia adianthifolia (Schumach.) W. F. Wight (Mimo-
of its NMR data with literature values. The native aglycon
was characterized as acacic acid from the 2D NMR spectra
from 1. The sugars obtained from the saponin hydrolysate
were identified as glucose, fucose, rhamnose, arabinose,
and xylose by comparison with authentic samples. How-
ever, the presence of an N-acetamido group [IR 1639 and
5
saceae). The further investigation of the saponins of this
-
1
1
plant, obtained as a complex mixture, afforded two new
acylated triterpene saponins, adianthifoliosides A (1) and
B (2), from the 95% ethanolic extract of the roots of A.
adianthifolia. This paper deals with the isolation and
structure elucidation of these new acylated triterpene
glycosides (1 and 2) and the evaluation of the immuno-
modulatory activity of 1-4 (Chart 1) and the crude saponin
mixture (CSM) on J urkat T cell proliferation (human T cell
leukemia) and the hemolytic activity of 1-4 on sheep
erythrocytes.
1570 cm ; H NMR δ
H
2.13 (3H, s, MeCO) and δ
H
8.91
22.9 and 171.9],
together with the H and C NMR data of the C-2 of the
Glc (δ 4.40 and δ 56.8), suggested the presence of one
2-(acetylamino)-2-deoxyglucose (Glc NAc) unit. In addition,
1
3
(1H, d, J ) 8.8 Hz, NH); C NMR δ
C
1
13
1
H
C
1
alkaline hydrolysis of 1 afforded salicylic acid (SA, o-
1
hydroxybenzoic acid, 7), identified by comparison of its H
and 13C NMR data with those reported, and a prosapoge-
7
nin whose spectroscopic NMR data were in good agreement
5
with those of 4. The above data suggested that 1 is a 21-
acyl-3,28-bisdesmoside. This was confirmed by the observa-
tion of glycosylation- and acylation-induced shifts in the
Resu lts a n d Discu ssion
1
3
C NMR spectrum at δ
6.5 (downfield shift of C-21), and δ
C
88.7 (downfield shift of C-3), δ
174.4 (upfield shift
C
The 95% ethanolic extract of the roots of A. adianthifolia
was purified by precipitation with diethyl ether, yielding
a crude saponin mixture, which was then dialyzed for 2
days. The powder obtained was submitted to column
chromatography over Sephadex LH-20 and was separated
by repeated medium-pressure liquid chromatography
7
C
of C-28).
1
The H NMR spectrum of 1 displayed signals for seven
anomeric protons at δ 6.06 (br s), 5.83 (d, J ) 7.5 Hz),
H
5.77 (br s), 5.10 (d, J ) 7.5 Hz), 4.91 (d, J ) 7.5 Hz), 4.90
(d, J ) 7.8 Hz), and 4.79 (d, J ) 8.0 Hz), which correlated
(MPLC) over normal Si gel, yielding compounds 1 and 2.
Their structures were elucidated mainly by 1D and 2D
NMR spectroscopy (COSY, TOCSY, NOESY, HSQC, and
HMBC) and by FABMS and HRESIMS.
C
with the carbon signals at δ 109.8, 94.9, 101.2, 104.7,
103.8, 105.6, and 102.5, respectively, in the HSQC spec-
trum. Starting from the anomeric proton of each sugar unit,
all the protons within each spin system were delineated
using COSY with the aid of TOCSY and NOESY spectra.
*
To whom correspondence should be addressed. Tel: 0033-3-80393229.
Fax: 0033-3-80393300. E-mail: malacd@u-bourgogne.fr.
After assignments of the protons, the C NMR resonances
13
†
Universit e´ de Bourgogne, J E 2244.
Kyushu University.
Universit e´ de Bourgogne, UMR CNRS 5548.
‡
of each sugar unit were identified by HSQC and further
confirmed by HMBC. The COSY and TOCSY spectra
§
1
0.1021/np020391q CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/21/2003

Triterpenoidal Saponins from Albizia
J ournal of Natural Products, 2003, Vol. 66, No. 3 373
Ch a r t 1
The H and 13C NMR signals of 1 assigned from the 2D
NMR experiments were almost superimposable with those
of 4, previously characterized as acacic acid 3-O-â-D-
xylopyranosyl-(1f2)-â-D-fucopyranosyl-(1f6)-2-(acetylami-
no)-2-deoxy-â-D-glucopyranoside, except for the presence of
the additional salicylic acid unit and an oligosaccharidic
chain at C-28.
The assignments of the 1H and 13C NMR signals of 1
from the TOCSY, HSQC, and HMBC spectra showed that
1
indicated the presence of seven monosaccharide units, and
one of them was an R-rhamnopyranosyl (Rha) from its
8
,9
typical pattern in the COSY-LR spectrum. After inter-
pretation of the H and 13C NMR signals, the other sugars
were identified as two â-glucopyranosyl (Glc) units, one
â-fucopyranosyl (Fuc) unit, one â-xylopyranosyl (Xyl) unit,
one 2-(acetylamino)-2-deoxy-â-glucopyranosyl (GlcNAc) unit,
and one arabinofuranosyl unit, respectively. The R-config-
uration of this latter sugar was clarified by observation of
NOESY correlations between H-1, H-3, and H-5. The
absolute configurations of these sugar residues were de-
termined to be D-glucose, D-xylose, D-fucose, L-rhamnose,
L-arabinose, and 2-(acetylamino)-2-deoxy-â-D-glucopyra-
nose by GC analysis of chiral derivatives of sugars in the
acid hydrolysate (see Experimental Section).10
1
7
the four remaining sugars were a terminal Glc (Glc′ ), a
2
disubstituted Glc (Glc-1,2)(Glc′ ), a terminal Araf, and a
1
trisubstituted Rha (Rha-1,3,4). A correlation in the HSQC
spectrum at δ /δH 94.9/5.83 (d, J ) 7.5 Hz) showed that
C
1
the Glc′ residue is linked to the carboxylic group of the
aglycon by an ester linkage. This conclusion was supported

3
74 J ournal of Natural Products, 2003, Vol. 66, No. 3
Haddad et al.
Ta ble 1. 13C NMR and 1H NMR Data of the Aglycons of 1 and
by the upfield shift of C-28 at δ
the free carboxylic acid observed in 4 at δ
long-range correlations observed in the HMBC spectrum
C
174.4, in comparison with
a,b
2
(C5D5N)
C
180.0 ppm. The
1
2
1
between the H NMR signals at δ
H
(Rha-1) 5.77 and the
-2) 76.9, between δ (Glc′ -1)
(Rha-3) 81.2, and between δ (Araf-1) 6.06 and
C
(Rha-4) 77.6 showed that the tetrasaccharide residue
position
DEPT
δC
δH
δC
δH
1
3
C NMR signals at δ
.10 and δ
C
(Glc′
1
H
2
1
2
3
4
5
6
7
8
9
1
11
12
1
1
1
16
17
1
1
2
2
2
23
24
2
2
2
2
CH2
CH2
CH
C
CH
CH2
CH2
C
CH
C
CH2
CH
C
38.0
26.2
88.7
39.0
55.8
18.2
33.3
40.1
46.9
36.8
23.3
122.7
143.0
41.2
35.1
73.1
51.4
40.3
47.3
35.2
76.5
35.7
27.5
16.5
15.2
16.8
26.7
174.4
28.6
18.7
0.94, 1.52
nd , nd
3.29
38.0
0.94, 1.51
5
δ
C
H
c
88.8
39.6
55.1
18.3
32.8
39.5
46.1
37.0
22.8
122.7
142.7
41.5
34.8
73
3.30
O-a-L-arabinofuranosyl-(1f4)-[â-D-glucopyranosyl-(1f3)]-
a-L-rhamnopyranosyl-(1f2)-â-D-glucopyranosyl was linked
to the acacic acid unit at C-28.
The comparison of the 13C NMR chemical shifts of 4 and
0.84
1.33, nd
1.66, nd
0.77
nd, nd
1.57, nd
1
permitted the position of the linkage of the salicylic acid
1.78
1.73
on the aglycon to be located. In 1, when compared to 4,
the signals for C-20, C-21, and C-22 were displayed upfield
by -2.1 ppm, downfield by + 2.6 ppm, and upfield by -6.5
ppm, respectively, as a consequence of the acylation at
C-21.
0
1.95, nd
5.57
1.94, nd
5.57
3
4
5
C
CH2
CH
C
CH
CH2
C
CH
CH2
CH3
CH3
CH3
CH3
CH3
COOH
CH3
CH3
1.91, 2.09
5.07
1.90, 2.11
5.08
From these data, compound 1 was assigned as 16R-
hydroxy-21â-[(2-hydroxybenzoyl)oxy]-3â-[(O-â-D-xylopyra-
nosyl-(1f2)-O-â-D-fucopyranosyl-(1f6)-2-(acetylamino)-2-
deoxy-â-D-glucopyranosyl)oxy]olean-12-en-28-oic acid 28-
O-R-L-arabinofuranosyl-(1f4)-O-[â-D-glucopyranosyl-(1f3)]-
51.2
41.3
47.3
34.8
76.1
8
9
0
1
2
3.27
1.33, 2.82
3.38
1.30
6.09
2.13, 2.68
1.08
0.88
0.82
6.10
O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranosyl ester.
Compound 1 is a new compound1
1,17-25
and was given the
27.5
16.7
15.3
16.9
26.7
174.5
28.7
18.7
1.13
1.06
0.87
1.02
1.73
trivial name adianthifolioside A.
5
6
7
8
Compound 2 was obtained as an amorphous powder.
1.02
1.73
HRESIMS (positive-ion mode) exhibited a quasimolecular
+
ion peak at m/z 1835.7700 [M + Na] (calcd 1835.7727),
consistent with a molecular formula of C83
H
128
O
43. Upon
29
30
0.88
1.02
0.85
1.03
acid hydrolysis with 2 N TFA at 120 °C, 2 afforded the
aglycon 6, again identified as acacic acid lactone by
a
Multiplicities were assigned from DEPT spectra. b Assign-
1
13
comparison of its H and C NMR data with those
ments were based on the HMBC, HSQC, and DEPT experiments
6
13
1
c
reported. The sugars obtained from the saponin hydroly-
(150 MHz for C and 600 MHz for H NMR). nd: not determined.
sate were identified as glucose, fucose, rhamnose, arabi-
nose, and xylose, by comparison with authentic samples
on TLC. Alkaline hydrolysis of 2 afforded salicylic acid (7),
(1f2)-â-D-fucopyranosyl-(1f6)-[â-D-glucopyranosyl-(1f2)]-
â-D-glucopyranose moiety of 3 and 5. Thus, compound 2
was concluded to be 16R-hydroxy-21â-[(2-hydroxybenzoyl)-
oxy]-3â-[(O-â-D-glucopyranosyl-(1f2)-O-[O-â-D-xylopyranos-
yl-(1f2)-O-â-D-fucopyranosyl-(1f6)]-â-D-glucopyranosyl)oxy]-
olean-12-en-28-oic acid 28-O-R-L-arabinofuranosyl-(1f4)-
1
13
identified by comparison of its H and C NMR data with
7
those reported, and a prosapogenin (5), whose NMR data
were almost similar to those of the prosapogenin previously
isolated from the alkaline hydrolysate of the Albizia cortex
saponin mixture.1 The above data suggested that 2 should
be a 21-acyl-3,28-O-bisdesmoside. This was confirmed by
the observation of glycosylation- and acylation-induced
2
O-[â-D-glucopyranosyl-(1f3)]-O-R-L-rhamnopyranosyl-(1f2)-
â-D-glucopyranosyl ester. As a new compound,1
1,17-25
2 was
given the trivial name adianthifolioside B. It is interesting
to note that 1 and 2 contain a salicilyl substituent, not
reported before among plant triterpene glycosides.
shifts in the 13C NMR spectrum at δ
of C-3), δ 76.1 (downfield shift of C-21), and δ
upfield shift of C-28).
88.8 (downfield shift
174.5
C
C
C
(
Since triterpene saponins from plants have been reported
1
11,13
The H NMR spectrum of 2 displayed signals for eight
anomeric protons at δ 6.09 (br s), 5.85 (d, J ) 7.5 Hz),
.72 (br s), 5.30 (d, J ) 7.5 Hz), 5.10 (d, J ) 7.5 Hz), 4.90
d, J ) 7.8 Hz), 4.80 (d, J ) 8.0 Hz), and 4.72 (d, J ) 7.5
to exert immunostimulatory activities,
the CSM and
H
compounds 1-4 were tested in an in vitro lymphocyte
1
4
5
proliferation assay. The cellular proliferation was mea-
3
(
sured by H-thymidine incorporation in J urkat tumor cell
1
3
Hz), which correlated with the C NMR signals at δ
C
109.8,
lines (human T cell leukemia). Compound 2 showed a
9
4.8, 101.3, 103.8, 104.7, 105.6, 102.5, and 104.2, respec-
concentration-dependent immunomodulatory effect (Figure
-
2
tively, in the HSQC spectrum. Evaluation of spin-spin
couplings and chemical shifts and interpretation of exten-
sive 2D NMR allowed the identification of four â-glucopy-
ranosyl units, one â-fucopyranosyl unit, one â-xylopyranos-
yl unit, one R-rhamnopyranosyl unit, and one R-arabino-
furanosyl unit, respectively. The absolute configuration of
sugar residues was determined to be D-glucose, D-xylose,
D-fucose, L-rhamnose, and L-arabinose by GC analysis of
chiral derivatives of sugars in the acid hydrolysate (see
Experimental Section).10
1). At a concentration of 10 µM, compound 2 exerted a
weak but significant lymphoproliferative activity with a
stimulation index (SI) of 1.21, and at 1 µM it showed an
inhibition of this effect. Furthermore, 1 and the CSM
displayed a cytotoxic effect to lymphocytes in culture (from
1 µM for 1). Compound 3 showed a weak cytotoxic effect
from 5 µM, whereas 4 exhibited a lymphoproliferative effect
in the concentration range 1-10 µM and no cytotoxic effect
at all tested concentrations.
In addition, the hemolytic activities of the CSM and
Almost all NMR signals of 2 (Tables 1 and 2) were
superimposable on those of 1 except for those of the
oligosaccharidic moiety linked at C-3. The signals of this
chain assigned from 2D NMR experiments were almost
superimposable with those of the 3-O-â-D-xylopyranosyl-
compounds 1-4 were evaluated on sheep erythrocytes
1
5
according to a procedure adapted from the litterature.
Strong hemolytic activity is considered to be a typical
characteristic of saponins, although some saponins show
only weak or no hemolytic effect at all. This activity is often

Triterpenoidal Saponins from Albizia
J ournal of Natural Products, 2003, Vol. 66, No. 3 375
Ta ble 2. 1H and 13C NMR Data of Sugar and Acid Moieties of Compounds 1 and 2 (C5D5N)a,b
1
2
1
2
position
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
3
Glc
2
3
4
-O-sugar
28-O-sugar
-1 4.72 (d, J ) 7.5 Hz) 104.2 Glc′ -1
1
NAc-1 4.91 (d, J ) 7.5 Hz) 103.8 Glc
1
1
5.83 (d, J )7.5 Hz)
94.9 5.85 (d, J ) 7.5 Hz) 94.8
4.40
4.30
3.93
3.90
4.19
56.8
74.5
73.1
75.0
69.0
2
3
4
5
6
4.17
3.88
3.91
4.04
4.20
4.49
80.3
77.9
71.0
77.1
68.7
2
3
4
5
6
3.90
3.83
3.91
3.84
4.00
4.23
76.9 3.89
78.1 3.87
70.5 3.92
78.9 3.84
61.2 4.00
4.21
75.5
77.0
70.6
78.2
61.6
5
6
4
.50
8.91 (d, J ) 8.8 Hz) 171.9
2.13 22.9
4.79 (d, J ) 8.0 Hz) 102.5 Glc
NHCOCH
NHCOCH
Fuc-1
3
3
2
-1 5.30 (d, J ) 7.5 Hz) 103.8 Rha-1
5.77 (brs)
4.98
4.65
4.33
4.32
101.2 5.72 (brs)
69.8 4.99
81.2 4.69
77.6 4.33
68.6 4.34
101.3
69.9
81.2
77.7
68.7
18.4
2
3
4
5
6
4.25
3.90
3.92
3.65
1.37
80.8
74.5
71.4
70.7
16.4
2
3
4
5
6
3.92
3.83
4.06
4.07
4.08
76.3
78.1
70.1
77.1
61.2
2
3
4
5
6
1.65
18.2 1.66
4
.30
Xyl-1
4.90 (d, J ) 7.8 Hz) 105.6 Fuc-1 4.80 (d, J ) 8.0 Hz) 102.5 Ara
f
-1
-1
6.06 (brs)
4.79
4.58
4.56
4.10
109.8 6.09 (brs)
83.1 4.80
77.4 4.55
84.4 4.56
61.4 4.10
4.35
109.8
83.2
77.6
84.4
61.7
2
3
4
5
4.01
4.03
4.08
3.48
74.1
77.1
70.7
66.2
2
3
4
5
6
4.26
4.08
3.98
3.66
1.37
80.9
74.1
73.0
71.0
16.4
2
3
4
5
4
.33
4.32
Xyl-1 4.90 (d, J ) 7.8 Hz) 105.6 Glc′
2
5.10 (d, J ) 7.5 Hz) 104.7 5.10 (d, J ) 7.5 Hz) 104.7
2
3
4
5
3.89
4.03
4.05
3.50
74.4
77.3
69.8
66.1
2
3
4
5
6
3.89
4.32
4.34
3.81
4.05
74.5 3.90
79.9 4.30
70.3 4.10
77.4 4.05
61.3 4.07
4.33
74.4
79.3
70.6
77.1
61.5
4
.16
4
.30
2
1
2
3
4
5
6
7
1-O- acyl
110.1
151.7
110.2
151.8
115.4
134.1
116.8
131.0
167.9
6.62
7.24
7.03
7.83
115.4 6.62
134.2 7.25
117.0 7.04
130.9 7.86
167.9
a
Assignments were based on COSY, TOCSY, NOESY, HSQC, and HMBC experiments (150 MHz for 13C and 600 MHz for 1H NMR).
Multiplicities were assigned from DEPT spectra. Coupling constants in Hz. Overlapped signals are reported without designated
b
1
13
multiplicities.
H and C chemical shifts of substituted residues are italicized.
3
F igu r e 1. Effect of compounds 1-4 on the cellular proliferation of J urkat cells measured by H-thymidine incorporation (see Experimental Section).
SI ) stimulation index. Each column represents the mean ( SE (n ) 4).
related to the amphiphilic character of the saponin and is
influenced by the affinity of the aglycon to cholesterol in
cell membranes, allowing the formation of pores. In this
test, since the CSM was shown to exhibit a good hemolytic
activity (HC50 ) 12 µg/mL), we have tested the pure
compounds. Among them, 1 exhibited the highest hemolytic
activity (HD50 ) 17.5 µg/mL), followed by 2, which was less
active than 1 (HD50 ) 48 µg/mL), whereas 3 and 4 were
not hemolytic at the concentration tested (HD50 > 400 µg/
mL). These data suggested the importance of the N-
acetamido group in 1 for the hemolytic activity compared
with 2. The results of this above test correlated well with

3
76 J ournal of Natural Products, 2003, Vol. 66, No. 3
Haddad et al.
those of the cytotoxicity observed for 1 and 2 against J urkat
cells in the concentration range 1-10 µM. Moreover,
concerning 3, which is not hemolytic up to 400 µg/mL (282
µM) but cytotoxic from 10 µM, it could be thought that
another mechanism than the membrane effect is at the
origin of the cytotoxicity. Since avicins (closely related to
submitted to repeated MPLC column chromatography on Si
gel 60 (15-40 µm) using as eluent CHCl
3
2
-MeOH-H O (8:5:1
and 6:4:1), affording compounds 1 (13 mg) and 2 (14 mg).
Ad ia n th ifoliosid e A (1): white amorphous powder; [R]20
D
-
20° (c 0.1, MeOH); IR (KBr) νmax 3500-3300 (OH), 2928 (CH),
1
735 (CdO ester), 1718 (CdO carboxylic acid), 1570 and 1639
-
1
1
(CO-NH), 1580, 1260, 1090 cm ; H NMR (pyridine-d
5
, 600
, 150 MHz), see Tables 1 and
; long-range correlations in the HMBC spectrum used for
3
) were shown to induce inhibition of the J urkat cell
1
3
MHz) and C NMR (pyridine-d
2
defining the aglycon, as previously reported; negative FABMS
5
1
6
proliferation by induction of apoptosis, we are currently
investigating this possible mechanism involved in the
cytotoxic effect of 3 against J urkat T cells.
5
-
(
1
glycerol matrix) m/z 1690 [M - H] , 1088 [(M - H) - 162 -
-
32 - 146 - 162] ; HRESIMS positive mode m/z 1714.7490
+
[
M + Na] (calcd for C79
H
121NO38Na, 1714.7465); TLC R
f
0.29
Exp er im en ta l Section
(system b), brown spot by spraying with Komarowsky reagent.
Ad ia n th ifoliosid e B (2): white amorphous powder; [R]20
D
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were taken with a Perkin-Elmer 241 polarimeter. IR spectra
-
1
1
20° (c 0.1, MeOH); IR (KBr) νmax 3500-3300 (OH), 2927 (CH),
735 (CdO ester), 1718 (CdO carboxylic acid), 1580, 1260,
(
KBr disks) were recorded on a Perkin-Elmer 281 spectropho-
-
1
1
13
090 cm
;
H NMR (pyridine-d
5
, 600 MHz) and C NMR
1
1
tometer. The 1D and 2D NMR spectra ( H- H COSY, TOCSY,
NOESY, HSQC, and HMBC) were performed using a UNITY-
(pyridine-d
5
, 150 MHz), see Tables 1 and 2; long-range
correlations in the HMBC spectrum used for defining the
6
00 spectrometer at the operating frequency of 600 MHz on a
5
aglycon, as previously reported; negative FABMS (glycerol
Varian INOVA 600 instrument equipped with a SUN 4 L-X
-
matrix) m/z 1811 [M - H] ; HRESIMS positive mode m/z
1
13
computer system (600 MHz for H and 150 MHz for
C
+
1
R
835.7700 [M + Na] (calcd for C83
f
H
128
O
43Na, 1835.7727); TLC
spectra). Conventional pulse sequences were used for COSY,
HSQC, and HMBC. TOCSY spectra were acquired using the
standard MLEV17 spin-locking sequence and 90 ms mixing
time. The mixing time in the NOESY experiment was set to
0.2 (system b), brown spot by spraying with Komarowsky
reagent.
P r osa p ogen in 3. The spectral data were in full agreement
5
with previously published data for this compound.
5
00 ms. The carbon type (CH
DEPT experiments. All chemical shifts (δ) are given in ppm,
and the samples were solubilized in pyridine-d (δ 150.3,
55.9, 123.9). The fast-atom bombardment mass spectrum
FABMS) (negative-ion mode, thioglycerol matrix) was ob-
3 2
, CH , CH) was determined by
P r osa p ogen in 5. The spectral data were in full agreement
12
with previously published data for this compound.
5
C
Deter m in a tion of Su ga r Com p on en ts. A solution of 1
1
(
and 2 (7 mg of each) in H
COOH (5 mL) were refluxed on a water bath for 3 h. After
this period, the reaction mixture was diluted with H O (15 mL)
and extracted with CH Cl Cl
2 3
O (2 mL) and 2 N aqueous CF -
tained on a J EOL SX 102 mass spectrometer. HRESIMS was
carried out on a Q-TOF 1 micromass spectrometer. GC analysis
was carried out on a Termoquest gas chromatograph using a
DB-1701 capillary column (30 m × 0.25 mm, i.d.) (J & W
Scientific); detection, FID; detector temperature, 250 °C;
injection temperature, 230 °C; initial temperature was main-
tained at 80 °C for 5 min and then raised to 270 °C at the rate
of 15 °C/min; carrier gas, He. TLC and HPTLC employed
precoated Si gel plates 60 F254 (Merck). The following TLC
2
2
2
(3 × 5 mL). The combined CH
2
2
extracts were washed with H O and then evaporated to
2
dryness in vacuo. Evaporation of the solvent gave acacic acid
lactone 6 (1.5 mg) (co-TLC with an authentic sample, solvent
c). After repeated evaporations to dryness of the aqueous layer
with MeOH until neutral, the sugars were analyzed by Si gel
TLC by comparison with standard sugars (solvent system d);
for sugars of 1 and 2: TLC R
f
(glucose) 0.30, R
f
(rhamnose) 0.50,
R
f
(arabinose) 0.56, R (xylose) 0.45, and R
f
f
(fucose) 0.49. Fur-
solvent systems were used: for saponins (a) CHCl
AcOH-H O, 15:8:3:2, (b) CHCl -MeOH-H O, 65:40:10; for
sapogenins (c) toluene-Me CO, 4:1; for monosaccharides (d)
CHCl -MeOH-H O, 8:5:1. Spray reagents for the saponins
were Komarowsky reagent, a mixture (5:1) of p-hydroxybenz-
aldehyde (2% in MeOH) and H SO 50%; for the sugars,
3
-MeOH-
thermore, the residue of sugars was dissolved in anhydrous
2
3
2
pyridine (100 µL), and L-cysteine methyl ester hydrochloride
2
(0.06 mol/L) was added. The mixture was stirred at 60 °C for
3
2
1
h, then 150 µL of HMDS-TMCS (hexamethyldisilazane-
trimethylchlorosilane, 3:1) was added, and the mixture was
stirred at 60 °C for another 30 min. The precipitate was
centrifuged off, and the supernatant was concentrated under
2
4
diphenylaminephosphoric acid reagent. Isolations were carried
out using a medium-pressure liquid chromatography (MPLC)
system [Gilson pump M 305, head pump 25 SC, manometric
module M 805, Autoinjector 234, B u¨ chi column (460 × 25 mm
and 460 × 15 mm), B u¨ chi precolumn (110 × 15 mm)].
a N
and H
2
stream. The residue was partitioned between n-hexane
O (0.1 mL each), and the hexane layer (1 µL) was
2
analyzed by GC. D-Glucose, D-xylose, D-fucose, L-rhamnose,
L-arabinose, and 2-(acetylamino)-2-deoxy-â-D-glucopyranose for
1 were detected in each case (1 and 2) by co-injection of the
hydrolysate with standard silylated samples, giving single
peaks at 18.64, 13.46, 12.14, 13.13, 11.88, and 21.55 min,
respectively. In the same manner, identification of D-glucose,
D-xylose, D-fucose, L-rhamnose, and L-arabinose was carried
out for 2, giving single peaks at 18.60, 13.46, 12.12, 13.13, and
11.90 min, respectively.
P la n t Ma ter ia l. The roots of Albizia adianthifolia (Schu-
mach.) W. F. Wight (Mimosaceae) were collected in April 1990
at Lamto, Ivory Coast, and identified by Mr. N. Konan,
Tropical Ecology Station, Lamto. A voucher specimen (No. 16-
9
0) is deposited in the Herbarium of the Laboratory of
Pharmacognosy, Faculty of Pharmacy, University of Bur-
gundy, France.
Extr a ction a n d Isola tion . Dried, powdered roots (200 g)
were macerated for 4 h with 3 L of 95% ethanol and further
refluxed for 24 h (3 × 3 L). After cooling, the ethanolic solution
was filtrated and evaporated to dryness, and the 95% ethanolic
extract was obtained (20 g). This extract was suspended in
Alk a lin e Hyd r olysis. Compounds 1 and 2 (5 mg of each)
were refluxed with 5% aqueous KOH (10 mL) for 2 h. The
reaction mixture was adjusted to pH 6 with dilute HCl and
extracted successively with Et
saturated n-BuOH (3 × 10 mL). The combined Et
were washed (H O). Evaporation of the Et O layer yielded
2
O (3 × 10 mL) and H
2
O-
O extracts
2
H
2
O (400 mL) and submitted to a partition against n-BuOH
2
2
1
3
saturated with H
reduced pressure of the solvent, 7 g of a n-BuOH extract was
2
O (3 × 400 mL). After evaporation under
salicylic acid 7 (1 mg) (TLC, C NMR), identified by compari-
7
son with literature data, whereas evaporation of the n-BuOH
obtained. This was solubilized in MeOH (10 mL) and precipi-
extract yields the prosapogenin 4 (3 mg) (TLC, 13C NMR) from
1, which was identified by comparison of its spectral data with
tated in Et
2
O (3 × 250 mL), yielding 2.5 g of a crude saponin
5
fraction. This resulting mixture was suspended in water,
dialyzed for 2 days, and lyophilized, yielding a crude saponin
mixture (CSM). An aliquot (1.8 g) of this mixture was fraction-
ated by column chromatography over Sephadex LH-20 and
those reported in the literature, and a prosapogenin 5 (2.5
mg) from 2, previously isolated from the alkaline hydrolysate
of the crude saponin mixture of Albizia cortex by comparison
1
2
of its spectral data with those reported in the literature.

Triterpenoidal Saponins from Albizia
J ournal of Natural Products, 2003, Vol. 66, No. 3 377
P r olifer a tion Assa y. The effect of 1-4 on the cell prolif-
(12) Ikeda, T.; Fujiwara, S.; Kinjo, J .; Nohara, T.; Ida, Y.; Shoji, J .; Shingu,
T.; Isobe, R.; Kajimoto, T. Bull. Chem. Soc. J pn. 1995, 68, 3483-
3490.
eration in J urkat cells was evaluated according to a method
1
4
previously described in the literature.
(
13) Lacaille-Dubois, M. A. In Immunomodulatory Agents from Plants;
Wagner, H., Ed.; Birkh a¨ user Verlag: Basel, 1999; pp 243-272.
14) Gaidi, G.; Miyamoto, T.; Laurens, V.; Lacaille-Dubois, M. A. J . Nat.
Prod. 2002, 65, 1568-1572.
Hem olysis Assa y. The hemolytic activity of CSM and 1-4
was evaluated on sheep erythrocytes according to a procedure
(
1
5
adapted from the literature.
(
(
15) Woldemichael, G. M.; Wink, M. Phytochemistry 2002, 60, 323-327.
16) Haridas, V.; Higuchi, M.; J ayatilake, G. S.; Bailey, D., Mujoo, K.;
Blake, M. E., Arntzen, C. J .; Gutterman, J . U. Proc. Natl. Acad. Sci.,
U.S.A. 2001, 98, 5821-5826.
Ack n ow led gm en t. We thank Prof. F. Tillequin, Univer-
sity of Paris V, for helpful discussions.
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NP020391Q