Two new triterpene saponins from Cyclamen africanum Boiss. & Reuter

Article abstract of DOI:10.1002/hlca.201200041

Two new oleanane-type triterpene saponins, afrocyclamins A and B (1 and 2, resp.), were isolated from a MeOH extract of the roots of Cyclamen africanum Boiss. & Reuter, together with three known triterpenoid saponins, lysikokianoside, deglucocyclamin I, and its dicrotalic acid derivative. The structures were elucidated, on the basis of 1D- and 2D-NMR experiments and mass spectrometry as (3β,20β)-13,28-epoxy-16-oxo-3-{O-β-D- xylopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4) -O-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl}oxy} oleanan-29-al (1) and (3β,16α,20β)-16,28,29-trihydroxy-olean-12- en-3-yl O-4-O-(4-carboxy-3-hydroxy-3-methyl-1-oxobutyl)-β-D-xylopyranosyl- (1→2)-O-β-D-glucopyranosyl-(1→4)-O-[β-D-glucopyranosyl- (1→2)]-α-L-arabinopyranoside (2).

Full text of DOI:10.1002/hlca.201200041

Helvetica Chimica Acta – Vol. 95 (2012)
1573
Two New Triterpene Saponins from Cyclamen africanum Boiss. & Reuter
by Soumeya Bencharif-Betinaa)c), Tomofumi Miyamotob), Chiaki Tanakab), Zahia Kabouchec),
Anne-Claire Mitaine-Offera), and Marie Aleth Lacaille-Dubois*a)
a
´
´
) EA 4267, FDE/UFC, Laboratoire de Pharmacognosie, Faculte de Pharmacie, Universite 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) Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
c
´
´
) Laboratoire dꢀObtention des Substances Therapeutiques, L.O.S.T., Faculte des Sciences,
´
´
Universite de Constantine, Algerie
Two new oleanane-type triterpene saponins, afrocyclamins A and B (1 and 2, resp.), were isolated
from a MeOH extract of the roots of Cyclamen africanum Boiss. & Reuter, together with three known
triterpenoid saponins, lysikokianoside, deglucocyclamin I, and its dicrotalic acid derivative. The
structures were elucidated, on the basis of 1D- and 2D-NMR experiments and mass spectrometry as
(3b,20b)-13,28-epoxy-16-oxo-3-{O-b-d-xylopyranosyl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-
glucopyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}oxy}oleanan-29-al (1) and (3b,16a,20b)-16,28,29-trihy-
droxy-olean-12-en-3-yl O-4-O-(4-carboxy-3-hydroxy-3-methyl-1-oxobutyl)-b-d-xylopyranosyl-(1 ! 2)-
O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabinopyranoside (2).
Introduction. – Cyclamen is a genus of 23 species of flowering plants belonging to
the Primulaceae family [1], many of them being reported to contain pentacyclic
triterpenoids [2], with a wide range of bioactivities, such as somatic embryogenesis [3],
fungicidal, antitumor, and uterocontractile activities [4], and in the treatment against
infertility [5]. As part of our continuing studies on saponins from the genus Cyclamen
[6][7], we chose Cyclamen africanum, an endemic plant of northern Algeria (Bejaia),
which was used in folk medicine against warts. A previous report revealed the presence
of the sapogenin cyclamiretin A [8], but no systematic study on the saponins of this
plant has been reported so far. We describe in this work the isolation and structure
elucidation of two new triterpene saponins named afrocyclamins A and B (1 and 2,
resp.) and of three known saponins from the rhizome MeOH extract of C. africanum.
Results and Discussion. – The dried and powdered rhizome parts of C. africanum
Boiss. & Reuter were extracted with 70% MeOH. An aliquot of the MeOH extract
was subjected to successive vacuum liquid chromatography (VLC) separations on silica
gel and RP-18 silica gel. Afrocyclamin A (1) and B (2), and the known saponins
lysikokianoside 1, deglucocyclamin I, and its dicrotalic acyl derivative were obtained by
separation of the saponin-containing fraction by medium-pressure liquid chromatog-
raphy (MPLC) on normal and reversed-phase silica gel. The structures of 1 and 2 were
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

1574
Helvetica Chimica Acta – Vol. 95 (2012)
established mainly by 1D- and 2D-NMR spectroscopic methods (1H- and 13C-NMR,
DEPT, COSY, TOCSY, NOESY, HSQC, and HMBC; Tables 1 and 2), in combination
with mass spectrometry (HR-ESI- and FAB-MS).
The high-resolution electrospray-ionization mass spectrum (HR-ESI-MS; positive-
ion mode) of compound 1 exhibited a quasi-molecular ion at m/z 1081.5201 ([M þ
Na]þ) establishing the molecular formula of 1 as C52H84O22. The FAB-MS (negative-
ion mode) of 1 showed a quasi-molecular ion at m/z 1057 ([M ꢀ H]ꢀ). Other fragment
ions at m/z 925 ([M ꢀ H ꢀ 132]ꢀ), 763 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ), 601 ([M ꢀ H ꢀ 132 ꢀ
162 ꢀ 162]ꢀ), and 469 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 162 ꢀ 132]ꢀ) indicated the loss of one
pentosyl, two hexosyl, and one pentosyl units, respectively. The 13C-NMR spectrum
displayed 52 C-atom signals of which 30 were attributed to the aglycone and the
remaining 22 to the sugar moieties. The HSQC spectrum of the aglycone of 1 exhibited
six signals of tertiary Me groups at d(H)/d(C) 1.18(s)/18.0 (C(26)), 1.12(s)/27.3 (C(23)),
1.04(s)/21.9 (C(27)), 1.00(s)/16.0 (C(24)), 0.86(s)/23.3 (C(30)), and 0.70(s)/15.5
(C(25)), which are characteristic of a pentacyclic triterpenoid. In addition, one pair
of geminal H-atoms at d(H) 3.30 (d, J ¼ 8.0 Hz) and 3.82 (d, J ¼ 8.0 Hz) giving
correlations with the downfield-shifted signal at d(C) 74.6 corresponded to the
oxymethylene function located at C(28). Another significant signal at d(H)/d(C) 9.56/
207.1 corresponded to an aldehyde function located at C(29), whereas the carbonyl
signal at d(C) 212.4 corresponded to C(16). Based on the above findings and an

Helvetica Chimica Acta – Vol. 95 (2012)
1575
Table 1. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; C5D5N) of the Aglycones of 1 and 2 from 1D-
and 2D-NMR Experimentsa). d in ppm, J in Hz.
Position
1
2
d(H)
d(C)
d(H)
d(C)
CH2(1)
CH2(2)
0.74, 1.48
1.76, 1.92
3.08 – 3.12 (m)
38.8
26.2
89.0
39.1
55.0
17.0
33.4
43.0
49.9
36.3
18.3
31.2
86.0
49.5
45.4
212.4
54.9
55.6
33.5
47.8
29.1
33.2
27.3
16.0
15.5
18.0
21.9
74.6
207.1
23.3
0.76, 1.53
1.76, 1.92
3.08 – 3.15 (m)
38.7
26.2
89.0
39.2
55.5
18.0
32.9
40.0
46.7
36.0
23.6
124.0
144.2
41.8
33.9
73.0
39.5
42.0
42.7
35.7
31.4
27.8
27.6
16.0
15.7
16.5
27.0
69.0
66.5
27.7
HꢀC(3)
C(4)
HꢀC(5)
CH2(6)
CH2(7)
0.57 (d, J ¼ 9.5)
0.62 – 0.64 (m)
1.25 (d, J ¼ 9.2)b)
1.34b)
b
b
b),
)
b),
)
C(8)
HꢀC(9)
1.02
1.56 – 1.60 (m)
C(10)
b
CH2(11)
CH2(12) or HꢀC(12)
C(13)
b),
)
0.96, 1.72
5.36
1.52, 1.94
C(14)
b
CH2(15)
C(16) or HꢀC(16)
C(17)
HꢀC(18)
CH2(19)
C(20)
CH2(21)
CH2(22)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
1.91b)
b),
)
4.58
1.78
2.40 – 2.44 (m)
2.50 – 2,54 (m), 1.72
b
b),
)
1.80, 1.92 – 1.96 (m)
2.12 – 2.16 (m), 1.74
1.52, 2.28
1.12 (s)
1.00 (s)
0.74 (s)
0.86 (s)
1.70 (s)
3.56, 3.65 (2d, each J ¼ 11.0)
3.82 (d, J ¼ 10.4), 3.92
1.19 (s)
b
b),
)
1.12 (s)
1.00 (s)
0.70 (s)
1.18 (s)
1.04 (s)
CH2(28)
CHO(29) or CH2(29)
Me(30)
3.30, 3.82 (2d, each J ¼ 8.0)
9.56 (s)
0.86 (s)
a) Overlapped signals are reported without designated multiplicity. b) Not determined.
extensive 1D- and 2D-NMR analysis, the aglycone of 1 was identified as cyclamigenin
B (¼(3b,20b)-13,28-epoxy-3-hydroxy-16-oxooleanan-29-al), previously reported from
1
C. europaeum [9], Ardisia gigantifolia [10] and A. mamillata [11]. The H-NMR
spectrum of 1 showed four anomeric H-atom signals at d(H) 5.38 (d, J ¼ 8.0 Hz), 4.86
(d, J ¼ 7.6 Hz), 4.82 (d, J ¼ 8.6 Hz), and 4.72 (d, J ¼ 5.9 Hz), correlated in the HSQC
spectrum with four anomeric C-signals at d(C) 103.8, 103.4, 106.8, and 104.2,
respectively, indicating the presence of four sugar units. Evaluation of spinꢀspin
couplings and chemical shifts from the 2D-NMR spectra allowed the identification of
two b-glucopyranosyl (Glc I and Glc II), one b-xylopyranosyl (Xyl), and one a-
arabinopyranosyl (Ara) unit. The b-orientation at the anomeric center of Glc and Xyl
and the a-orientation at the anomeric center of Ara, in their pyranose form, was

1576
Helvetica Chimica Acta – Vol. 95 (2012)
Table 2. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; C5D5N) of the Sugar Moieties of 1 and 2 from
1D- and 2D-NMR Experimentsa). d in ppm, J in Hz.
Position
1
2
d(H)
d(C)
d(H)
d(C)
3-O-Sugars:
Ara
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
CH2(5)
Glc I
4.72 (d, J ¼ 5.9)
4.43 (t-like, J ¼ 9.0)
4.22
104.2
78.8
72.9
78.2
64.0
4.72 (d, J ¼ 5.9)
4.42 (t-like, J ¼ 9.0)
4.20
104.2
78.8
72.8
78.8
64.0
4.18
4.18
4.60 (br. d, J ¼ 10.4), 3.72
4.60, 3.70 (br. d, J ¼ 11.2)
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
Glc II
5.38 (d, J ¼ 8.0)
3.97 (t, J ¼ 9.6)
4.16
4.04
3.96
103.8
75.5
76.2
71.0
77.2
62.2
5.38 (d, J ¼ 7.0)
3.97
4.16
4.04
3.96
103.8
75.2
76.2
71.0
77.2
62.2
4.46, 4.24
4.46, 4.24
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
Xyl
4.86 (d, J ¼ 7.6)
103.4
84.2
76.5
70.0
77.3
61.9
4.84 (d, J ¼ 7.6)
104.1
84.8
76.5
70.0
77.2
61.7
3.77 (d, J ¼ 8.5)
3.78 (t, J ¼ 8.5)
4.10
4.06
3.75
4.10
4.06
3.75
4.34 (d, J ¼ 10.2), 4.17
4.34 (br. d, J ¼ 11.6), 4.17
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
CH2(5)
Acid at Xyl C(4)
C(1)
4.82 (d, J ¼ 8.6)
3.90
4.00
4.06
4.47, 3.68
106.8
75.5
76.8
70.3
66.9
4.82 (d, J ¼ 8.6)
3.90
4.00
5.18
4.46, 3.68
106.5
75.5
76.7
72.0
66.9
170.0
46.8
70.0
47.0
172.3
27.5
HꢀC(2)
3.02 (d, J ¼ 12.3), 2.82 (d, J ¼ 12.1)
C(3)
b
HꢀC(4)
C(5)
Me(6)
b),
)
1.46
a) Overlapped signals are reported without designated multiplicity. b) Not determined.
supported by the relatively large J values of their anomeric H-atoms (J ¼ 5.9 – 8.6 Hz).
The absolute configuration was determined to be d for Glc and Xyl and l for Ara by
GC analysis (see Exper. Part). HMBC Cross-peaks at d(H)/d(C) 4.72 (Ara HꢀC(1))/
89.0 (Agly C(3)), 5.38 (Glc I HꢀC(1))/78.8 (Ara C(2)), 4.86 (Glc II HꢀC(1))/78.2 (Ara
C(4)), and 4.82 (Xyl HꢀC(1))/84.2 (Glc II C(2)) allowed the oligosaccharide sequence
Xyl-(1 ! 2)-Glc II-(1 ! 4)-[Glc I-(1 ! 2)]-Ara to be linked at Agly C(3). These
linkages were also confirmed by the following NOESY cross-peaks: 4.72 (Ara

Helvetica Chimica Acta – Vol. 95 (2012)
1577
HꢀC(1))/3.08 – 3.12 (Agly HꢀC(3)), 5.38 (Glc I HꢀC(1))/4.43 (Ara HꢀC(2)), 4.86 (Glc
II HꢀC(1))/4.18 (Ara HꢀC(4)), and 4.82 (Xyl HꢀC(1))/3.77 (Glc II HꢀC(2)). From the
above evidence, the structure of 1 was elucidated as (3b,20b)-13,28-epoxy-16-oxo-3-
{{O-b-d-xylopyranosyl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-
(1 ! 2)]-a-l-arabinopyranosyl}oxy}oleanan-29-al, a new compound named afrocycla-
min A.
On the basis of HR-ESI-MS (positive-ion mode: m/z 1229.6780 ([M þ Na]þ)) and
13C-NMR data, the molecular formula of compound 2 was established as C58H94O26.
The negative-ion-mode FAB-MS exhibited a quasi-molecular ion at m/z 1205 ([M ꢀ
H]ꢀ). Other fragment ions at m/z 1061 ([M ꢀ H ꢀ 144]ꢀ), 929 ([M ꢀ H ꢀ 144 ꢀ
132]ꢀ), 767 ([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162]ꢀ), 605 ([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162 ꢀ 162]ꢀ),
and 473 ([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162 ꢀ 162 ꢀ 132]ꢀ) corresponded to the successive loss
of one unit of 144 (C6H12O5) suggesting the presence of an acid, one pentosyl, two
hexosyl, and one pentosyl moiety. The 13C-NMR spectrum of 2 displayed 58 C-signals,
of which 30 could be readily assigned to a triterpene skeleton and the 28 remaining to
the saccharide and acid portion. The HSQC spectrum corresponding to the aglycone
part of 2 revealed the presence of six tertiary Me groups as s at d(H)/d(C)1.70(s)/27.0
(Me(27)), 1.19(s)/27.7 (Me(30)), 1.12(s)/ 27.6 (Me(23)), 1.00(s)/16.0 (Me(24)), 0.86(s)/
16.5 (Me(26)), and 0.74(s)/15.7 (Me(25)), together with the signals for an olefinic C-
atom at d(H)/d(C) 5.36(s)/124.0 (C(12)) and a quaternary C-atom at d(C) 144.2
(C(13)). These data suggested an olean-12-ene skeleton for the aglycone of 2.
Furthermore, two CH2OH and one CHOH moieties were observed in the HSQC
spectrum at d(H)/d(C) 3.82 and 3.92/66.5 (C(29)), 3.56 and 3.65/69.0 (C(28)), and 4.58/
73.0 (C(16)). The HMBC cross-peaks of the aglycone part of 2 between d(H) 1.19
(Me(30)) and d(C) 42.7 (C(19)), 35.7 (C(20)), 31.4 (C(21)), and 66.5 (C(29)) indicated
that one primary-alcohol function was located at C(29). A close comparison between
the signals of the aglycones of 1 and 2 showed that the characteristic signals of the
13,28-epoxy bridge at d(C) 86.0 (C(13)), 74.6 (C(28)), and 54.9 (C(17)), and of the keto
group at d(C) 212.4 (C(16)) in 1 were replaced by the signals of a C(12)¼C(13) bond at
d(C) 124.0 (C(12)) and 144.2 (C(13)), of a primary-alcohol function at d(C) 69.0
(C(28)), and of a secondary-alcohol function at d(C) 73.0 (C(16)) in 2. This data
suggested the aglycone of 2 to be dihydrocyclamiretin D or pridentigenin E
(¼(3b,16a,20b)-olean-12-ene-3,16,28,29-tetrol) isolated from Primula denticulata
[12]. The relative configuration of the aglycone of 2 was established by the NOESY
data. Namely the cross-peaks d(H)/d(H) 1.19 (Me(30))/2.50 – 2.54 (m, HaxꢀC(19)),
2.50 – 2.54 (m, HaxꢀC(19))/1.70 (Me(27)), 1.19 (Me(30))/1.74 (HaxꢀC(21)), and 2.50 –
2.54 (m, HaxꢀC(19))/1.74 (HaxꢀC(21)) confirmed the a-equatorial orientation of
Me(30) and, consequently, the b axial orientation of the CH2OH group at C(20). The
NOESY cross-peaks d(H)/d(H) 4.58 (HꢀC(16))/3.65 (CH2(28)) and 4.58 (HꢀC(16))/
2.14 (HeqꢀC(21)) confirmed the b-equatorial orientation of HꢀC(16) and, conse-
quently, the a-axial orientation of OHꢀC(16). These data confirmed the identification
of the aglycone of 2 as (3b,16a,20b)-olean-12-ene-3,16,28,29-tetrol (also named olean-
12-ene-3b,16a,28,30-tetraol), which was recently reported as the aglycone of heder-
ifolioside E isolated from C. hederifolium [13]. The HSQC spectrum of 2 showed four
anomeric H-atom signals at d(H) 5.38 (d, J ¼ 7.0 Hz), 4.84 (d, J ¼ 7.6 Hz), 4.82 (d, J ¼
8.61 Hz), and 4.72 (d, J ¼ 5.9 Hz) which correlated with four anomeric C-signals at

1578
Helvetica Chimica Acta – Vol. 95 (2012)
d(C) 103.8, 104.1, 106.5, and 104.2, indicating the presence of four sugar units. All the
1H- and 13C-NMR signals corresponding to the oligosaccharide part of 2 were almost
superimposable to those of 1 (Xyl-(1 ! 2)-Glc II-(1 ! 4)-[Glc I-(1 ! 2)]-Ara-), except
for the deshielded signals of Xyl HꢀC(4) at d(H) 5.18/d(C) 72.0, indicating an acylation
at this position. The presence of a dicrotalic acid (¼ 3-hydroxy-3-methylpentanedioic
acid) acyl group was ascertained by the observation of a set of additional signals in the
1D- and 2D-NMR spectra corresponding to a 4-carboxy-3-hydroxy-3-methyl-1-
oxobutyl moiety, which were in good agreement with literature data [6]. This was
confirmed by the observation of a fragment ion at m/z 1061 ([M ꢀ H ꢀ 144]ꢀ) in the
FAB-MS (negative-ion mode). From the above evidence, 2 was elucidated as
(3b,16a,20b)-16,28,29-trihydroxyolean-12-en-3-yl
O-4-O-(4-carboxy-3-hydroxy-3-
methyl-1-oxobutyl)-b-d-xylopyranosyl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-
glucopyranosyl-(1 ! 2)]-a-l-arabinopyranoside, which we called afrocyclamin B.
The three known compounds isolated besides 1 and 2 were identified by
comparison of their 1D- and 2D-NMR and MS data with those reported in the
literature as lysikokianoside 1 (¼(3b,16a)-13,28-epoxy-16-hydroxyoleanan-3-yl O-b-d-
xylopyranosyl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-(1 !
2)]-a-l-arabinopyranoside), isolated previously from C. persicum [7] and Lysimachia
sikokiana [14], deglucocyclamin I (¼(3b,16a,20b)-13,28-epoxy-3-{{O-b-d-xylopyrano-
syl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabi-
nopyranosyl}oxy}-16-hydroxyoleanan-29-al) [6][8], and its dicrotalic acyl derivative
(3b,16a,20b)-3-{{O-4-O-(4-carboxy-3-hydroxy-3-methyl-1-oxobutyl)-b-d-xylopyrano-
syl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabi-
nopyranosyl}oxy}-13,28-epoxy-16-hydroxyoleanan-29-al, isolated previously from C.
trocopteranthum [6]. Deglucocyclamin I, isolated for the first time from C. africanum,
has been obtained from several Cyclamen species (C. mirabile [4], C. trocopteranthum
[6], C. persicum [7], C. greacum [8], C. hederifolium [13], C. repandum [15], C.
neapolitanum [16], C. europaeum [17], and C. coum var coum [18]). This compound
may represent a chemotaxonomic marker for the genus Cyclamen.
Experimental Part
General. Vacuum liquid chromatography (VLC)/medium-pressure liquid-chromatography
(MPLC): SiO2 60 (Merck, 15 – 40 mm), RP-18 (Silicycle; 75 – 200 mm). MPLC: Gilson M 303 pump,
Bꢀchi glass column (460 ꢁ 15 mm and 230 ꢁ 15 mm), Bꢀchi precolumn (110 ꢁ 15 mm). Anal. TLC: SiO2
plates 60F254 (Silicycle); HP-TLC on SiO2 60F254 (Merck); detection of saponins with vanillin reagent
(1% vanillin in EtOH/H2SO4 50 :1). GC: Termoquest gas chromatograph, DB-1701 cap. column (30 m ꢁ
0.25 mm, i.d.; J&W Scientific) [7]; FI detection; detector temp. 2508, injection temp. 2308, initial temp.
808 for 5 min and then increase to 2708 at the rate of 158/min; carrier gas He. Optical rotations: AA-OR
automatic polarimeter. For 1D and 2D-NMR spectra (1H,1H-COSY, TOCSY, NOESY, HSQC, and
HMBC), see [6]. MS: Q-TOF 1-micromass spectrometer (pos.-ion mode HR-ESI) and Jeol-SX-102 mass
spectrometer (neg.-ion mode FAB, glycerol matrix) in m/z.
Plant Material. The roots of Cyclamen africanum Boiss. & Reuter, were collected near Constantine
(Algeria) in 2008 and identified by Prof. Debellaire, Faculty of Chemistry, University of Annaba. A
voucher specimen (LOST ca05.08) has been deposited with the Phytochemistry Department, Mentouri
University, Constantine, Algeria.
Extraction and Isolation. The air-dried powdered roots of Cyclamen africanum (5 kg) were extracted
with 70% MeOH (3 ꢁ 1000 ml) for 3 h under reflux to give 345 g of a crude extract after evaporation. A

Helvetica Chimica Acta – Vol. 95 (2012)
1579
fraction (10 g) of this extract was submitted to VLC (SiO2 60 (15 – 40 mm), CHCl3/MeOH/H2O 70 :23 :4,
60 :32 :7, and 0 :100 :0 (each eluent 400 ml)): Fractions 1 – 10. Fr. 1 – 3 (2.07 g; eluted with CHCl3/MeOH/
H2O 70 :23 :4) were separated by VLC (RP-18, MeOH/H2O 0 :100 ! 100 :0): Frs. 1 – 3.1, 1 – 3.2, and 1 –
3.3. Fr. 1 – 3.2 (860 mg; eluted with MeOH/H2O 1:1) was submitted to successive MPLC (RP-18, MeOH/
H2O gradient) and MPLC (SiO2 60 (15 – 40 mm), CHCl3/MeOH/H2O 70 :23 :4, 60 :32 :7, and 0 :100 :0): 1
(10 mg). Fr. 1 – 3.3 (640 mg eluted with MeOH) was fractionated by successive MPLC (RP-18, MeOH/
H2O gradient): 2 (6 mg), lysikokianoside 1 (23 mg), deglucocyclamin I (75 mg), and deglucocyclamin I
dicrotalic acyl derivative (8 mg).
Afrocyclamin A (¼{(3b,20b)-13,28-Epoxy-16-oxo-3-{{O-b-d-xylopyranosyl-(1 ! 2)-O-b-d-gluco-
pyranosyl-(1 ! 4)-O-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}oxy}oleanan-29-al; 1): White
1
amorphous powder. [a]2D5 ¼ ꢀ2.5 (c ¼ 0.50, MeOH). H- and 13C-NMR (C5D5N): Tables 1 and 2. FAB-
MS (neg.): 1057 ([M ꢀ H]ꢀ), 925 ([M ꢀ H ꢀ 132]ꢀ), 763 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ), 601 ([M ꢀ H ꢀ 132 ꢀ
162 ꢀ 162]ꢀ), 469 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 162 ꢀ 132]ꢀ). HR-ESI-MS (pos.): 1081.5201 ([M þ Na]þ,
C52H84NaOþ22 ; calc. 1081.5195).
Afrocyclamin B (¼(3b,16a,20b)-16,28,29-Trihydroxyolean-12-en-3-yl O-4-O-(4-Carboxy-3-hy-
droxy-3-methyl-1-oxobutyl)-b-d-xylopyranosyl-(1 ! 2)-O-b-d-glucopyranosyl-(1 ! 4)-O-[b-d-glucopyr-
anosyl-(1 ! 2)]-a-l-arabinopyranoside; 2): White amorphous powder. [a]2D5 ¼ ꢀ17.0 (c ¼ 0.43, MeOH).
1H- and 13C-NMR: Tables 1 and 2. FAB-MS (neg.): 1205 ([M ꢀ H]ꢀ), 1061 ([M ꢀ H ꢀ 144]ꢀ), 929 ([M ꢀ
H ꢀ 144 ꢀ 132]ꢀ), 767 ([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162]ꢀ), 605 ([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162 ꢀ 162]ꢀ), 473
([M ꢀ H ꢀ 144 ꢀ 132 ꢀ 162 ꢀ 162 ꢀ 132]ꢀ). HR-ESI-MS (pos.): 1229.6780 ([M þ Na]þ, C58H94NaOþ26
;
calc. 1229,6786).
Acid Hydrolysis and GC Analysis. Each compound (2 mg) was hydrolyzed with 2n aq. CF3COOH
(5 ml) for 3 h at 958. After extraction with CH2Cl2 (3 ꢁ 5 ml), the aq. layer was repeatedly concentrated
to dryness with MeOH until neutral, and then analyzed by TLC (SiO2, CHCl3/MeOH/H2O 8 :5 :1) by
comparison with authentic samples. The (trimethylsilyl)thiazolidine derivatives of the sugar residue of
each compound were prepared and analyzed by GC [19]. The absolute configurations were determined
by comparing the retention times with thiazolidine derivatives prepared in a similar way from standard
sugars (SigmaꢀAldrich). The following sugars were detected: d-glucose, d-xylose, and l-arabinose for
both 1 and 2.
REFERENCES
[1] L. Affre, J. D. Thomson, M. Debussche, Bot. J. Linn. Soc. 1995, 118, 309.
[2] S. T. Bennett, J. M. Grimshaw, Plant Syst. Evol. 1991, 176, 135.
[3] A.-K. Pueschel, H.-G. Schwenkel, T. Winkelmann, Plant Cell Tiss. Org. 2003, 72, 43.
˙
[4] I. ꢃalis¸, M. E. S¸atana, A. Yꢂrꢂker, P. Kelican, R. Demirdamar, R. AlaÅam, N. Tanker, H. Rꢂegger,
O. Sticher, J. Nat. Prod. 1997, 60, 315.
[5] Z. Jia, K. Koike, T. Ohmoto, M. Ni, Phytochemistry 1994, 37, 1389.
[6] G. Mihci-Gaidi, S. Ozbey, I. Orhan, B. Sener, T. Miyamoto, J.-F. Mirjolet, O. Duchamp, A.-C.
Mitaine-Offer, M.-A. Lacaille-Dubois, Planta Med. 2010, 76, 218.
[7] G. Mihci-Gaidi, D. Pertuit, T. Miyamoto, J.-F. Mirjolet, O. Duchamp, A.-C. Mitaine-Offer, M.-A.
Lacaille-Dubois, Nat. Prod. Commun. 2010, 5, 1023.
[8] G. Reznicek, J. Jurenitsch, W. Robien, W. Kubelka, Phytochemistry 1989, 28, 825.
[9] R. O. Dorchai, J. B. Thomson, Tetrahedron Lett. 1963, 26, 2223.
[10] Q. Q. Gong, L. H. Mu, P. Liu, S. L. Yang, B. Wang, Y. L. Feng, Chin. Chem. Lett. 2010, 21, 449.
[11] J. Huang, H. Zhang, N. Shimizu, T. Takeda, Chem. Pharm. Bull. 2003, 51, 875.
[12] A. Viqar Uddin, S. Qazi Najmus, K.Usmanghani, Phytochemistry 1980, 9, 1875.
[13] H. Altunkeyik, D. Gꢂlcemal, M. Masullo, O. Alankus-Caliskan, S. Piacente, T. Karayildirim,
Phytochemistry 2012, 73, 127.
[14] H. Kohda, O. Takeda, S. Tanaka, Chem. Pharm. Bull. 1989, 37, 3304.
[15] S. DallꢀAcqua, I. Castagliuolo, P. Brun, F. Ditadi, G. Palu, G. Innocenti, Carbohydr. Res. 2010, 345,
709.

1580
Helvetica Chimica Acta – Vol. 95 (2012)
[16] A. Huseyin, Doga 1977, 1, 148.
[17] R. Tschesche, H. J. Mercker, G. Wuff, Justus Liebigs Ann. Chem. 1969, 721, 194.
[18] I. ꢃalis¸, A. Yꢂrꢂker, N. Tanker, A. D. Wright, O. Sticher, Planta Med. 1997, 63, 166.
[19] S. Hara, H. Okabe, K. Mihashi, Chem. Pharm. Bull. 1987, 35, 501.
Received January 30, 2012