Three new medicagenic acid saponins from Polygala micrantha GUILL. & PERR.

Article abstract of DOI:10.1002/hlca.201000331

Three new medicagenic acid saponins, micranthosides A-C (1-3), were isolated from the roots of Polygala micrantha Guill. & Perr., along with six known presenegenin saponins. Their structures were elucidated on the basis of extensive 1D- and 2D-NMR experiments (¹H, ¹³C, DEPT, COSY, TOCSY, NOESY, HSQC, and HMBC) and mass spectrometry as 3-O-β-D- glucopyranosylmedicagenic acid 28-[O-β-D-galactopyranosyl-(1→4)-O- β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2) -β-D-fucopyranosyl] ester (1), 3-O-β-D-glucopyranosylmedicagenic acid 28-[O-6-O-acetyl-β-D-galactopyranosyl-(1→4)-O-β-D-xylopyranosyl- (1→4)-O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl] ester (2), and 3-O-{O-β-D-glucopyranosyl-(1→3)-O-[β-D-glucopyranosyl- (1→6)]-β-D-glucopyranosyl}medicagenic acid 28-{O-β-D- apiofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-O-[β-D- apiofuranosyl-(1→3)]-O-α-L-rhamnopyranosyl-(1→2) -β-D-fucopyranosyl} ester (3). Compounds 1-3 were evaluated against HCT 116 and HT-29 human colon cancer cells, but they did not show any cytotoxicity. Copyright

Full text of DOI:10.1002/hlca.201000331

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Helvetica Chimica Acta – Vol. 94 (2011)
Three New Medicagenic Acid Saponins from Polygala micrantha
Guill. & Perr.
a
b
a
c
by Turibio Kuiate Tabopda ) ), Anne-Claire Mitaine-Offer ), Tomofumi Miyamoto ),
c
b
d
d
Chiaki Tanaka ), Bonaventure Tchaleu Ngadjui ), Jean-FranÅois Mirjolet ), Olivier Duchamp ), and
a
Marie-Aleth Lacaille-Dubois* )
a) Laboratoire de Pharmacognosie, Unit e´ de Mol e´ cules dꢀInt e´ r eˆ t Biologique, UMIB, UPRES-EA 3660,
Facult e´ de Pharmacie, Universit e´ de Bourgogne, 7, Bd. Jeanne dꢀArc, BP 87900, F-21079 Dijon Cedex
(
phone: þ 33-3-80393229; fax: þ 33-3-80393300; e-mail: m-a.lacaille-dubois@u-bourgogne.fr)
b
)
)
D e´ partement de Chimie Organique, Universit e´ de Yaound e´ 1, BP812 Yaound e´ , Cameroun
c
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
d
)
Oncodesign, 20 rue Jean Mazen, BP 27627, F-21076 Dijon Cedex
Three new medicagenic acid saponins, micranthosides A – C (1 – 3), were isolated from the roots of
Polygala micrantha Guill. & Perr., along with six known presenegenin saponins. Their structures were
1
13
elucidated on the basis of extensive 1D- and 2D-NMR experiments ( H, C, DEPT, COSY, TOCSY,
NOESY, HSQC, and HMBC) and mass spectrometry as 3-O-b-d-glucopyranosylmedicagenic acid 28-
[
O-b-d-galactopyranosyl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-fu-
copyranosyl] ester (1), 3-O-b-d-glucopyranosylmedicagenic acid 28-[O-6-O-acetyl-b-d-galactopyrano-
syl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-fucopyranosyl] ester (2),
and 3-O-{O-b-d-glucopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 6)]-b-d-glucopyranosyl}medica-
genic acid 28-{O-b-d-apiofuranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-[b-d-apiofuranosyl-(1 !
3
)]-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-fucopyranosyl} ester (3). Compounds 1 – 3 were evaluated
against HCT 116 and HT-29 human colon cancer cells, but they did not show any cytotoxicity.
Introduction. – Polygala is a genus of ca. 500 species of flowering plants belonging
to the family Polygalaceae, commonly known as milkwort or snakeroot. The genus
includes herbaceous perennial plants, shrubs, and has a subcosmopolitan distribution.
Polygala micrantha Guill. & Perr. is an annual herb up to 30 cm high, recorded from
Senegal to West Cameroon. The plant is said to be used as a purge in Senegal [1].
Polygalaceae species have already been investigated, resulting in the isolation of
presenegenin [2][3] and medicagenic acid glycosides [4][5], but no previous
phytochemical study has been undertaken on P. micrantha. In continuation of our
studies of saponin constituents of medicinal plants of the Polygalaceae family [6 – 8], we
describe in this article the isolation and structure elucidation of three new medicagenic
acid saponins named micranthosides A – C (1 – 3; Fig. 1) and six known presenegenin
glycosides from the 70% MeOH extract of the roots of P. micrantha. The cytotoxicity of
compounds 1 – 3 was evaluated against two human colon cancer cell lines, HT-29 and
HCT 116.
Results and Discussion. – A 70% aqueous MeOH extract of the roots of P.
micrantha was submitted to vacuum liquid chromatography (VLC) and further
ꢁ
2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

Helvetica Chimica Acta – Vol. 94 (2011)
915
Fig. 1. Compounds 1 – 3, isolated from Polygala micrantha
fractionated by repeated medium-pressure liquid chromatography (MPLC) yielding
micranthosides A – C (1 – 3), and the known polygalasaponin XXVIII [9], polygala-
saponin XXXV [10], desacylsenegasaponin B [11], polygalasaponins XLV and XLVI
[
12], and arilloside A [13]. Their structures were established mainly by 1D- and 2D-
1
13
NMR experiments ( H, C, DEPT, COSY, TOCSY, NOESY, HSQC, and HMBC, see
1
13
Tables 1 and 2) and mass spectrometry. H- and C-NMR signals of the aglycone
assigned from 2D-NMR experiments of 1 – 3 were in good agreement with those of
medicagenic acid (¼(2b,3b,4a)-2,3-dihydroxyolean-12-ene-23,28-dioic acid; Table 1)
[
4][5]. The monosaccharides obtained by acid hydrolysis of each compound were
identified by TLC comparison with standard sugars as glucose, fucose, rhamnose,
xylose, and galactose in the case of 1 and 2, and as glucose, fucose, rhamnose, xylose,
and apiose in the case of 3. The d-configuration for apiose, galactose, glucose, fucose,
and xylose, and the l-configuration for rhamnose were determined by GC analysis (see
Exper. Part). The b-orientation of the anomeric center of the glucosyl, xylosyl, and
fucosyl moieties in their pyranose form was supported by the relatively large J values of
1
their anomeric H-atoms (J ¼ 6.9 – 8.1 Hz). For the rhamnosyl moiety, the large J(C,H)
value (165 – 168 Hz) and three-bond-coupled strong HMBCs from the anomeric H-
atom to C(3) and C(5) (dihedral angle between HꢀC(1) and C(3), and between
HꢀC(1) and C(5) ca. 1808) indicated that the anomeric H-atom was equatorial thus
possessing an a-pyranoid anomeric form [14].
Micranthoside A (1) was obtained as an amorphous powder. The high-resolution
electrospray-ionization mass spectrum (HR-ESI-MS; positive-ion mode) exhibited a
þ
quasimolecular-ion peak at m/z 1273.5834 ([M þ Na] ) consistent with the molecular
formula C H NaO . The negative-ion-mode FAB-MS showed a quasimolecular-ion
59
94
28
ꢀ
peak at m/z 1249 ([M ꢀ H] ), and two other important fragment-ion peaks at m/z 1087
[M ꢀ H ꢀ 162] ) and 925 ([M ꢀ H ꢀ 162 – 162] ), corresponding to the loss of one and
ꢀ
ꢀ
(

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 1. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; (D )pyridine) of the Aglycones of 1 – 3 from
5
a
b
1
D- and 2D-NMR Experiments ) ). d in ppm, J in Hz.
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
CH (1)
1.30, 2.18
4.75
4.56
43.9 1.30, 2.18
69.5 4.75
86.4 4.56
53.0
43.9 1.31, 2.27 (d, J ¼ 12.6)
69.5 4.65
86.4 4.62
53.0
52.1 2.03
20.5 1.87, 1.98
33.0 1.65, 1.71
40.0
48.6 1.74
36.2
44.0
69.8
87.0
53.1
52.0
20.6
31.9
40.0
48.1
36.6
23.8
122.3
144.1
42.1
28.4
22.4
46.8
41.6
45.9
30.2
33.4
32.0
185.4
14.4
16.7
17.2
25.8
176.0
32.8
23.2
2
HꢀC(2)
HꢀC(3)
C(4)
HꢀC(5)
2.02
52.1 2.02
c
c
CH (6)
1.77 )
20.5 1.77 )
2
c
c
CH (7)
)
33.0
40.0
)
2
C(8)
HꢀC(9)
2.20
48.6 2.20
36.2
23.8 1.86, 2.05
122.0 5.37 (br. s)
143.8
C(10)
CH (11)
1.86, 2.05
23.8 1.88, 2.01
122.0 5.32 (br. s)
143.8
2
HꢀC(12) 5.37 (br. s)
C(13)
C(14)
42.0
42.0
CH (15) 1.44c)
28.2 1.44c)
22.4 1.96, 2.03
46.6
28.2 1.58, 1.88
22.4 1.96, 2.03
46.6
41.4 3.01 (br. d, J ¼ 10.5)
45.2 1.14, 1.63
30.4
33.4 1.01, 1.22
31.8 1.58, 1.65
186.0
14.5 1.85 (s)
16.5 1.42 (s)
17.0 1.03 (s)
25.6 1.21 (s)
176.8
2
CH (16) 1.96, 2.03
2
C(17)
HꢀC(18) 3.03 (br. d, J ¼ 12.6)
41.4 3.12 (br. d, J ¼ 12.0)
45.2 1.14, 1.64
30.4
CH (19) 1.14, 1.64
2
C(20)
CH (21)
c)
33.4 c)
31.8 1.68, 1.84
186.0
14.5 1.82 (s)
16.5 1.40 (s)
17.0 1.00 (s)
25.6 1.20 (s)
176.8
2
CH (22) 1.68, 1.84
2
C(23)
Me(24)
Me(25)
Me(26)
Me(27)
C(28)
1.82 (s)
1.40 (s)
1.00 (s)
1.20 (s)
Me(29)
Me(30)
0.69 (s)
0.75 (s)
32.6 0.69 (s)
23.4 0.75 (s)
32.6 0.77 (s)
23.4 0.72 (s)
a) Multiplicities were assigned from DEPT spectra. ) Overlapped H-NMR signals are reported without
designated multiplicity. ) Not determined.
b
1
c
1
two hexosyl units, respectively. The H-NMR spectrum of 1 (Table 2) showed five
anomeric-H-atom signals at d(H) 4.81 (d, J ¼ 7.4 Hz), 4.87 (d, J ¼ 8.1 Hz), 5.01 (d, J ¼
7
.9 Hz), 5.86 (d, J ¼ 8.1 Hz), and 6.32 (br. s) which were correlated in the HSQC
spectrum with five anomeric-C signals at d(C) 106.3, 103.4, 104.1, 94.0, and 100.9,
respectively, indicating the presence of five sugar units. The ring H- and C-atoms of the
sugar moieties were assigned starting from the readily identifiable anomeric H-atoms
by means of COSY, TOCSY, NOESY, HSQC, and HMBC experiments. Evaluation of
spinꢀspin couplings and chemical shifts allowed the identification of one b-glucopyr-
anosyl (Glc I), one b-fucopyranosyl (Fuc), one a-rhamnopyranosyl (Rha), one b-
galactopyranosyl (Gal), and one b-xylopyranosyl (Xyl) unit. Analysis of HMBC data
concerning the connectivities between the sugar residues and the medicagenic acid

Helvetica Chimica Acta – Vol. 94 (2011)
917
Table 2. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; (D )pyridine) of the Sugar Moieties of 1 – 3
5
a
b
from 1D- and 2D-NMR Experiments ) ). d in ppm, J in Hz.
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
3
-O-Sugars:
Glc I
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
5.01 (d, J ¼ 7.9)
3.86
4.16 – 4.20 (m)
3.96
3.84
4.06, 4.35
104.1 5.01 (d, J ¼ 7.9)
74.6 3.86
76.9 4.16 – 4.20 (m)
70.7 3.96
77.4 3.84
62.0 4.06, 4.35
104.2 4.94 (d, J ¼ 6.9)
74.7 3.84
77.3 4.13
70.5 4.10
77.3 3.92
103.5
74.5
86.6
70.8
77.7
69.3
CH (6)
61.9 3.96, 4.51
2
Glc II
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
4.77 (d, J ¼ 7.9)
3.88
4.14
3.92
3.76 – 3.82 (m)
4.15, 4.40
104.3
74.8
77.3
71.0
77.7
62.0
CH (6)
2
Glc III
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
5.14 (d, J ¼ 7.6)
3.91
4.12
4.02
3.88
104.6
75.5
77.3
71.0
77.7
62.0
CH (6)
4.06, 4.15
2
2
8-O-Sugars:
Fuc
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
5.86 (d, J ¼ 8.1)
94.0 5.86 (d, J ¼ 8.1)
73.6 4.58
76.2 4.05
72.5 3.93
71.8 3.82
94.0 5.79 (d, J ¼ 8.1)
73.6 4.63
76.2 4.10
72.5 3.93
71.8 3.87
94.5
73.2
76.4
73.2
72.1
16.5
4.58
4.05
3.93
3.82
1.38 (d, J ¼ 5.3)
16.5 1.38 (d, J ¼ 5.1)
16.5 1.40 (d, J ¼ 6.0)
Rha
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
6.32 (br. s)
4.73
4.45 – 4.52 (m)
4.10 – 4.16 (m)
4.40
100.9 6.32 (br. s)
70.8 4.73
71.6 4.45 – 4.52 (m)
84.8 4.10 – 4.16 (m)
67.7 4.40
100.9 6.45 (br. s)
70.8 4.82
71.6 4.41
84.8 4.30 – 4.34 (m)
67.7 4.37
17.8 1.60 (d, J ¼ 5.7)
101.1
71.5
81.2
78.9
67.5
18.2
1.60 (d, J ¼ 5.7)
17.8 1.60 (d, J ¼ 5.7)
Xyl
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
4.81 (d, J ¼ 7.4)
106.3 4.80 (d, J ¼ 7.4)
75.2 3.96
76.6 3.98
106.3 5.05 (d, J ¼ 7.0)
75.2 3.84
76.6 3.84
104.6
74.5
87.0
69.0
3.96
3.98
4.27
77.0 4.27
77.0 3.94
CH (5)
3.35 (t, J ¼ 9.8), 4.25 64.2 3.35 (t, J ¼ 9.8), 4.25 64.2 3.32 (t, J ¼ 10.0), 4.06 65.8
2
Gal
HꢀC(1)
HꢀC(2)
HꢀC(3)
4.87 (d, J ¼ 8.1)
103.4 4.82 (d, J ¼ 8.1)
70.8 4.34
74.0 4.00
103.6
71.0
74.1
4.36
4.04

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 2 (cont.)
1
2
3
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
HꢀC(4)
HꢀC(5)
4.26
4.06
4.08, 4.31
69.8
76.2
62.0
4.23
4.06
69.6
76.5
63.6
20.5, 170.8
CH (6)
4.62, 4.86 – 4.88 (m)
1.92 (s)
2
AcOꢀC(6)
Api I
HꢀC(1)
HꢀC(2)
HꢀC(3)
5.90 (d, J ¼ 2.6)
4.68
–
4.26, 4.46
4.00, 4.03
111.1
78.0
79.3
74.4
64.0
CH (4)
2
CH (5)
2
Api II
HꢀC(1)
HꢀC(2)
HꢀC(3)
5.95 (d, J ¼ 2.6)
4.78
–
4.12, 4.37
4.03, 4.05
111.1
77.0
79.8
74.1
64.7
CH (4)
2
CH (5)
2
a) Multiplicities were assigned from DEPT spectra. ) Overlapped H-NMR signals are reported without
b
1
designated multiplicity.
aglycone indicated that 1 was a bisdesmoside. Correlations were observed between
d(H) 4.56 (Agly HꢀC(3)) and d(C) 104.1 (Glc I C(1)) and between d(H) 5.01 (d, J ¼
7
.9 Hz, Glc I HꢀC(1)) and d(C) 86.4 (Agly C(3)). Thus a Glc unit was O-linked at
C(3). The remaining sugar units constituted a tetrasaccharide linked at C(28) of the
aglycone through an ester bond. Interglycosidic linkages were determined by the
HMBCs between d(H) 5.86 (d, J ¼ 8.1 Hz, Fuc HꢀC(1)) and d(C) 176.8 (Agly C(28)),
between d(H) 4.58 (Fuc HꢀC(2)) and d(C) 100.9 (Rha C(1)), between d(H) 4.81 (d,
J ¼ 7.4 Hz, Xyl HꢀC(1)) and d(C) 84.8 (Rha C(4)), and between d(H) 4.87 (Gal
HꢀC(1)) and d(C) 77.0 (Xyl C(4)) as well as reverse HMBCs between d(H) 6.32 (Rha
HꢀC(1)) and d(C) 73.6 (Fuc C(2)), between d(H) 4.10 – 4.16 (Rha HꢀC(4)) and d(C)
1
06.3 (Xyl C(1)), and between d(H) 4.27 (Xyl HꢀC(4)) and d(C) 103.4 (Gal C(1)).
These linkages were also confirmed by the following NOESY correlations: Fuc
HꢀC(2)/Rha HꢀC(1), Rha HꢀC(4)/Xyl HꢀC(1), and Xyl HꢀC(4)/Gal HꢀC(1). All of
these data were consistent with the structure 3-O-b-d-glucopyranosylmedicagenic acid
2
8-[O-b-d-galactopyranosyl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-a-l-rhamnopyra-
nosyl-(1 ! 2)-b-d-fucopyranosyl] ester for 1, which was called micranthoside A.
Micranthoside B (2) was obtained as an amorphous powder. The HR-ESI-MS
(
positive-ion mode) exhibited a quasimolecular-ion peak at m/z 1315.5940 ([M þ
þ
Na] ) consistent with the molecular formula C H NaO , with 42 mass units more
61
96
29
than 1. The negative-ion-mode FAB-MS showed a quasimolecular-ion peak at m/z 1291
ꢀ
ꢀ
(
[M ꢀ H] ), and two other important fragment-ion peaks at m/z 1249 ([M ꢀ H ꢀ 42] )
ꢀ
and 1087 ([M ꢀ H ꢀ 42 ꢀ 162] ), indicating the elimination of an Ac and a hexosyl
1
13
moiety. The full assignment of all the H- and C-NMR signals obtained from 2D-
NMR analysis showed that 2 differed from 1 only by the presence of one Ac group at

Helvetica Chimica Acta – Vol. 94 (2011)
919
C(6) of Gal (Table 2). This was supported by the observation in the HSQC spectrum of
deshielded signals of Gal CH (6) at d(H) 4.62 and 4.86 – 4.88 and d(C) 63.6 in
2
comparison with those of the OH-unsubstituted Gal CH (6) of 1. Thus, the structure of
2
2
was established as 3-O-b-d-glucopyranosylmedicagenic acid 28-[O-6-O-acetyl-b-d-
galactopyranosyl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-a-l-rhamnopyranosyl-(1 !
2
)-b-d-fucopyranosyl] ester.
Micranthoside C (3) was obtained as an amorphous powder. The HR-ESI-MS
(
positive-ion mode) exhibited a quasimolecular-ion peak at m/z 1699.7197 ([M þ
þ
Na] ) consistent with the molecular formula C H NaO . The negative-ion-mode
75
120
41
ꢀ
FAB-MS showed a quasimolecular-ion peak at m/z 1675 ([M ꢀ H] ), and important
ꢀ
ꢀ
fragment-ion peaks at m/z 1513 ([M ꢀ H ꢀ 162] ), 1381 ([M ꢀ H ꢀ 162 ꢀ 132] ), and
ꢀ
1
057 ([M ꢀ H ꢀ 162 ꢀ 132 ꢀ 162 ꢀ 162] ) indicating the elimination of one hexosyl, one
1
pentosyl, and two hexosyl moieties, respectively. The H-NMR spectrum of 3 showed
eight anomeric-H-atom NMR signals at d(H) 4.77 (d, J ¼ 7.9 Hz), 4.94 (d, J ¼ 6.9 Hz),
5
.05 (d, J ¼ 7.0 Hz), 5.14 (d, J ¼ 7.6 Hz), 5.79 (d, J ¼ 8.1 Hz), 5.90 (d, J ¼ 2.6 Hz), 5.95
(
d, J ¼ 2.6 Hz), and 6.45 (br. s, 1 H) which gave correlations, in the HSQC spectrum
with eight anomeric-C signals at d(C) 104.3, 103.5, 104.6, 104.6, 94.5, 111.1, 111.1, and
01.1, respectively, indicating the presence of eight sugar units. From the extensive 1D-
and 2D-NMR experiments, these sugar units were identified as three b-glucopyranosyl
Glc I, Glc II, and Glc III), one b-fucopyranosyl (Fuc), one a-rhamnopyranosyl (Rha),
1
(
one b-xylopyranosyl (Xyl), and two b-apiofuranosyl moieties (Api I and Api II).
Comparison of the C-atom chemical shifts of the sugar units thus assigned with those of
the literature [4 – 7] taking into account the known effects of O-glycosylation, indicated
that 3 contained two terminal Glc (Glc II and Glc III), two terminal Api (Api I and Api
II), a C(3)-substituted Xyl, a C(3)- and C(4)-disubstituted Rha, a C(3)- and C(6)-
disubstituted Glc (Glc I), and a C(2)-substituted Fuc unit. The downfield shift of Rha
C(3) from d(C) 71.6 in 1 to 81.2 in 3 and the upfield shift of Rha C(4) from d(C) 84.8 in
1
to 78.9 in 3, suggested Rha to be C(3)- and C(4)-disubstituted [7]. This was also
confirmed by the HMBC spectrum (Fig. 2) showing long-range correlations between
d(H) 6.45 (br. s, Rha HꢀC(1)) and d(C) 81.2 (Rha C(3)), 73.2 (Fuc C(2)), and 67.5
(
Rha C(5)), and between d(H) 4.30 – 4.34 (Rha HꢀC(4)) and d(C) 104.6 (Xyl C(1)),
8
1.2 (Rha C(3)), and 67.5 (Rha C(5)). Further analysis of NOESY cross-peaks
between d(H) 5.90 (Api I HꢀC(1)) and d(H) 4.41 (Rha C(3)) and between d(H) 5.05
Xyl HꢀC(1)) and d(H) 4.30 – 4.34 (Rha HꢀC(4)) established that Api I and Xyl were
(
linked to Rha by a (1 ! 3) and (1 ! 4) linkage, respectively. Moreover, the NOESY
correlations between d(H) 5.95 (Api II HꢀC(1)) and d(H) 3.84 (Xyl HꢀC(3)) and
between d(H) 3.84 (Xyl HꢀC(3)) and d(H) 5.05 (Xyl HꢀC(1)) established that Xyl
C(3) was substituted by Api II. Thus, it was revealed that one Fuc, one Rha, one Xyl,
and two Api (Api I and Api II) were attached to Agly C(28) and that Agly C(3) was
linked to three Glc residues. The connection of the Glc moieties at Agly C(3) was
deduced by the NOESY and HMBC data. The terminal Glc II and Glc III moieties
were ascertained to be linked to C(3) and C(6), respectively, of the inner Glc I by
NOESY correlations between d(H) 4.77 (Glc II HꢀC(1)) and d(H) 4.13 (Glc I
HꢀC(3)), and between d(H) 5.14 (Glc III HꢀC(1)) and d(H) 3.96 and 4.51 (Glc I
CH (6)). Furthermore, NOESY correlations between d(H) 4.94 (Glc I HꢀC(1)), 4.13
2
(Glc I HꢀC(3)), and 4.62 (Agly HꢀC(3)) established that the trisaccharide structure

9
20
Helvetica Chimica Acta – Vol. 94 (2011)
was attached at C(3) of the aglycone. In the HMBC spectrum, the correlation between
d(H) 4.77 (Glc II HꢀC(1)) and d(C) 86.6 (Glc I C(3)) confirmed the (1 ! 3) linkage
between Glc II and Glc I. Accordingly, the structure of 3 was formulated as 3-O-{O-b-
d-glucopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 6)]-b-d-glucopyranosyl}medi-
cagenic acid 28-{O-b-d-apiofuranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-[b-d-
apiofuranosyl-(1 ! 3)]-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-fucopyranosyl} ester. To
the best of our knowledge, this is the first saponin with a trisaccharide moiety attached
at C(3) of the aglycone isolated from the Polygalaceae family. On the contrary,
saponins with two (1 ! 2)-linked glucose units at C(3) of the aglycone have been
isolated from many Polygalaceae species [6].
Fig. 2. Selective HMBCs of 3
Compounds 1 – 3 were evaluated against two human colon cancer cells, HCT 116
and HT-29, by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) method [15] with paclitaxel as positive control, and all of them were
inactive at the concentration of 100 mm.
The authors are grateful to the Conseil R e´ gional de Bourgogne for financial support.
Experimental Part
General. Column chromatography (CC)/medium-pressure liquid chromatography (MPLC): silica
gel 60 (SiO ; 15 – 40 mm; Merck), RP-18 (Silicycle; 75 – 200 mm), Gilson pump M 305, Bꢀchi column
2
(
F
460 ꢁ 25 mm and 460 ꢁ 15 mm), Bꢀchi precolumn (110 ꢁ 15 mm). TLC and HP-TLC: SiO plates 60
2
(Merck); solvent systems: CHCl /MeOH/H O 80 :20 :2, 70 :30 :5, and 60 :32 :7; spray reagent:
3 2
254
vanillin reagent (2% mixture of conc. H SO soln. and 1% vanillin in EtOH). Optical rotation: Perkin-
2
4
Elmer-241 polarimeter. GC Analysis: Thermoquest gas chromatograph; DB-1701 cap. column (30 m ꢁ
.25 mm (i.d.); J & W Scientific); detection by FID; detector temp. 2508, injection temp. 2308, initial
temp. maintained at 808 for 5 min and then raised to 2708 at 158/min; carrier gas He. 1D- and 2D-NMR
0

Helvetica Chimica Acta – Vol. 94 (2011)
921
1
1
Spectra ( H, H-COSY, TOCSY, NOESY, HSQC, and HMBC): Varian-Unity-Inova-600 spectrometer,
1
13
equipped with a Sun-4L-X computer system; at 600 ( H) and 150 MHz ( C); conventional pulse
sequences for COSY, HSQC, and HMBC, TOCSY by using the standard MLEV17 spin-locking sequence
13
and 90-ms mixing time; mixing time in NOESY experiment, 500 ms; C multiplicities by DEPT
experiments; chemical shifts d in ppm, J in Hz; (D )pyridine solns. (d(C) 150.3, 135.9, and 123.9). FAB-
5
MS: Jeol-SX-102 spectrometer; neg.-ion mode; glycerol matrix; in m/z. HR-ESI-MS: micromass
Quattro-LS instrument; neg.-ion mode; in m/z.
Plant Material. The roots of P. micrantha were collected in the village Bangoua near Bangangt e´ City
located in the Nd e´ division of the Western highlands of Cameroon in July 2009, and identified by P. Nana,
botanist at the National Herbarium of Cameroon (NHC), Yaound e´ , where a voucher specimen
(
57326 HNC) was deposited.
Extraction and Isolation. Dried and finely powdered roots of P. micrantha (180 g) were extracted
three times under reflux in MeOH/H O 7:3 (1500 ml) for 3 h. After evaporation, a dark residue of 25 g
2
was obtained. A fraction (10 g) of this extract was submitted to VLC (RP-18, MeOH/H O gradient (each
2
eluent 250 ml): Frs. 1 – 6. Fr. 6 (MeOH/H O 50 :50; 1.6 g) was fractionated by MPLC (SiO , CHCl /
2
2
3
MeOH/H O 80 :20 :2, 70 :30 :5, and 60 :32 :7) and MPLC (RP-18, MeOH/H O 40 :60 ! 100 :0)): 1
2
2
(
8 mg), 2 (7.7 mg), 3 (35 mg), arilloside A (11 mg), polygalasaponin XXXV (5.7 mg), desacylsenega-
saponin B (4.3 mg), polygalasaponin XXVIII (4.5 mg), and polygalasaponins XLV and XLVI (9.3 mg).
2b,3b,4a)-3-(b-d-Glucopyranosyloxy)-2-hydroxyolean-12-ene-23,28-dioic Acid 28-[O-b-d-Galacto-
(
pyranosyl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-6-deoxy-b-d-
2
5
1
galactopyranosyl] Ester (1): White amorphous powder. [a] ¼ þ1.76 (c ¼ 0.43, MeOH). H- and
D
13
þ
þ
28
C-NMR ((D )pyridine): Tables 1 and 2. HR-ESI-MS (pos.): 1273.5834 ([M þ Na] , C H NaO ; calc.
5
59 94
ꢀ
ꢀ
ꢀ
1
273.5829). FAB-MS (neg.): 1249 ([M ꢀ H] ), 1087 ([M ꢀ H ꢀ 162] ), 925 ([M ꢀ H ꢀ 162 ꢀ 162] ).
(
2b,3b,4a)-3-(b-d-Glucopyranosyloxy)-2-hydroxyolean-12-ene-23,28-dioic Acid 28-[O-6-O-Acetyl-
b-d-galactopyranosyl-(1 ! 4)-O-b-d-xylopyranosyl-(1 ! 4)-O-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-6-
2
5
1
deoxy-b-d-galactopyranosyl] Ester (2): White amorphous powder. [a] ¼ ꢀ1.21 (c ¼ 0.43, MeOH). H-
D
13
þ
þ
and C-NMR ((D )pyridine): Tables 1 and 2. HR-ESI-MS (pos.): 1315.5940 ([M þ Na] , C H NaO29
;
5
61 96
ꢀ
ꢀ
ꢀ
calc. 1315.5935). FAB-MS (neg.): 1291 ([M ꢀ H] ), 1249 ([M ꢀ H ꢀ 42] ), 1087 ([M ꢀ H ꢀ 42 ꢀ 162] ).
2b,3b,4a)-3-{O-b-d-Glucopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 6)]-b-d-glucopyranosyl-
(
oxy}-2-hydroxyolean-12-ene-23,28-dioic Acid 28-{O-b-d-apiofuranosyl-(1 ! 3)-O-b-d-xylopyranosyl-
(
1 ! 4)-O-[b-d-apiofuranosyl-(1 ! 3)]-O-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-6-deoxy-b-d-galacto-
2
5
1
1
3
pyranosyl] Ester (3): White amorphous powder. [a]
D
¼ ꢀ6.52 (c ¼ 0.43, MeOH). H- and C-NMR
þ þ
(
1
1
(D )pyridine): Tables 1 and 2. HR-ESI-MS (pos.): 1699.7197 ([M þ Na] , C H NaO ; calc.
5
75 120
41
ꢀ
ꢀ
ꢀ
699.7203). FAB-MS (neg.): 1675 ([M ꢀ H] ), 1513 ([M ꢀ H ꢀ 162] ), 1381 ([M ꢀ H ꢀ 162 ꢀ 132] ),
ꢀ
057 ([M ꢀ H ꢀ 162 ꢀ 132 ꢀ 162 ꢀ 162] ).
Acid Hydrolysis and GC Analysis. Each compound (3 mg) was hydrolyzed with 2n aq. CF COOH
3
(
5 ml) for 3 h at 958. After extraction with CH Cl (3 ꢁ 5 ml), the aq. layer was repeatedly concentrated
2
2
with MeOH until neutral, and then analyzed by TLC (SiO , CHCl /MeOH/H O 8 :5 :1) by comparison
2
3
2
with authentic samples. Furthermore, the residue of the sugars was dissolved in anh. pyridine (100 ml),
and l-cysteine methyl ester hydrochloride (0.06 mol/l) was added. The mixture was stirred at 608 for 1 h,
then 150 ml of HMDS/Me SiCl (hexamethyldisilazane/trimethylchlorosilane 3 :1) was added, and the
3
mixture was stirred at 608 for another 30 min. The precipitate was centrifuged off, and the supernatant
was concentrated under an N stream. The residue was partitioned between hexane (1 ml) and H O
2
2
(
0.1 ml), and the hexane layer (1 ml) was analyzed by GC [16]. d-Glucose, d-galactose, d-xylose, l-
rhamnose, and d-fucose were detected from 1 and 2 by co-injection of the hydrolysate with standard
silylated samples with t 18.60, 17.20, 13.45, 13.15, and 12.17 min, respectively. Identification of d-glucose,
R
d-xylose, l-rhamnose, d-fucose, and d-apiose was carried out for 3, giving peaks at tR 18.64, 13.47, 13.15,
1
2.12 min, and 11.55, resp.
MTT Cytotoxicity Assay. The bioassay was carried out according to the method described by
Carmichael and co-workers [15] with two human colorectal cancer cell lines (HCT 116 and HT-29)
provided by the Oncodesign, Dijon, France. Paclitaxel was used as pos. control, and exhibited IC50 values
of 2.40 and 4.27 nm against HCT 116 and HT-29, resp.

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Helvetica Chimica Acta – Vol. 94 (2011)
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Received August 31, 2010