Two new steroidal saponins from the fresh leaves of Agave sisalana

Article abstract of DOI:10.1002/hlca.201000442

A new furostanol saponin, sisalasaponin C (1), and a new spirostanol saponin, sisalasaponin D (2), were isolated from the fresh leaves of Agave sisalana, along with three other known steroidal saponins and two stilbenes. Their structures were identified as (3β,5α,6α,22α,25R)-3, 26-bis[(β-D-glucopyrano- syl)oxy]-22-hydroxyfurostan-6-yl β-D-glucopyranoside (1), (3β,5α,25R)-12-oxospirostan-3-yl 6-deoxy-α-L-mannopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3) -[β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)] -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (2), (3β,5α,6α,22α,25R)-22-methoxyfurostane-3,6,26-triyl tris-β-D-glucopyranoside, cantalasaponin-1, polianthoside D, (E)- and (Z)-2,3,4′,5-tetrahydroxystilbene 2-O-β-D-glucopyranosides. The last three known compounds were isolated from the fresh leaves of Agavaceae for the first time. The structures of the new compounds were elucidated by detailed spectroscopic analysis, including 1D- and 2D-NMR experiments, and chemical techniques. Copyright

Full text of DOI:10.1002/hlca.201000442

Helvetica Chimica Acta – Vol. 94 (2011)
1351
Two New Steroidal Saponins from the Fresh Leaves of Agave sisalana
a
b
a
b
a
b
a
a
by He-Shui Yu ) ), Peng Zou ), Xin-Bo Song* ), Li-Ping Kang ) ), Yi-Xun Liu ), Xu Pang ),
a
a
a
a
a
b
Jie Zhang ), Jing Fu ), Yang Zhao ), Cheng-Qi Xiong ), Da-Wei Tan ), Li-Juan Zhang ), and
a
Bai-Ping Ma* )
a) Beijing Institute of Radiation Medicine, No. 27 Tai-ping Road, Haidian District, Beijing 100850,
P. R. China (phone: þ 86-10-68210077 ext. 930265; fax: þ 86-10-68214653;
e-mail: mabaiping@sina.com)
b) Tianjin University of Traditional Chinese Medicine, No. 88 Yuquan Road, Nankai District,
Tianjin 300193, P. R. China (phone: þ 86-22-27494976; fax: þ 86-22-27460231;
e-mail: songxinbo@tjutcm.edu.cn)
A new furostanol saponin, sisalasaponin C (1), and a new spirostanol saponin, sisalasaponin D (2),
were isolated from the fresh leaves of Agave sisalana, along with three other known steroidal saponins
and two stilbenes. Their structures were identified as (3b,5a,6a,22a,25R)-3,26-bis[(b-d-glucopyrano-
syl)oxy]-22-hydroxyfurostan-6-yl b-d-glucopyranoside (1), (3b,5a,25R)-12-oxospirostan-3-yl 6-deoxy-a-
l-mannopyranosyl-(1 ! 4)-b-d-glucopyranosyl-(1 ! 3)-[b-d-xylopyranosyl-(1 ! 3)-b-d-glucopyranosyl-
(
1 ! 2)]-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyranoside (2), (3b,5a,6a,22a,25R)-22-methoxyfuros-
tane-3,6,26-triyl tris-b-d-glucopyranoside, cantalasaponin-1, polianthoside D, (E)- and (Z)-2,3,4’,5-
tetrahydroxystilbene 2-O-b-d-glucopyranosides. The last three known compounds were isolated from the
fresh leaves of Agavaceae for the first time. The structures of the new compounds were elucidated by
detailed spectroscopic analysis, including 1D- and 2D-NMR experiments, and chemical techniques.
Introduction. – Plants belonging to the family of Agavaceae with more than 300
species occur natively in the arid and tropical regions of the Western Hemisphere,
particularly Mexico and Central America. In China, several species of the genus Agave,
such as A. americana and A. sisalana, are widely cultivated in the southern parts for the
fiber industry and as important horticultural plants. Agavaceae is known to be a rich
source for steroidal saponins. It is also a source of fiber and steroidal sapogenins.
Several species of genus Agave are used in the treatment of scabies, tumors, syphilis,
and dysentery and also as insecticides [1]. The chemical constituents of leaves of several
species have been studied, and several steroidal sapogenins and saponins were found by
several groups [2 – 8]. Previously, we reported two new furostanol saponins from the
fresh leaves of A. sisalana [5]. In our continuing search for new furostanol saponins, we
isolated three furostanol saponins and two spirostanol saponins, including a new
furostanol saponin, 1, and a new spirostanol saponin, 2. Further, polianthoside D, and
(
E)- and (Z)-2,3,4’,5-tetrahydroxystilbene 2-O-b-d-glucopyranosides are reported
from Agavaceae for the first time. Here, we describe the isolation and structure
elucidation of the new steroidal saponins on the basis of extensive spectral analysis,
1
13
1
1
including H- and C-NMR, H, H-COSY, HSQC, HMBC, and HSQC-TOCSY
experiments, as well as chemical evidences.
ꢀ
2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

1
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Helvetica Chimica Acta – Vol. 94 (2011)
Results and Discussion. – The crude extract of the fresh leaves of A. sisalana Perr.
was chromatographed on macroporous resin, SiO , Sephadex LH-20, and octadecyl-
2
silanized (ODS) SiO , and further purified by semi-preparative HPLC to afford the
2
new steroidal saponins 1 and 2, together with (3b,5a,6a,22a,25R)-22-methoxyfuros-
tane-3,6,26-triyl tris-b-d-glucopyranoside, cantalansaporin-1, polianthoside, and (E)-
and (Z)-2,3,4’,5-tetrahydroxystilbene 2-O-b-d-glucopyranosides. The structures were
elucidated by 1D- and 2D-NMR in combination with MS studies.
Sisalasaponin C (1) was obtained as a white amorphous powder, which showed
positive LibermannꢀBurchard and Ehrlich reactions, indicating a furostanol saponin.
The molecular formula was assigned as C H O on the basis of the high-resolution
45
76 20
electrospray ionization mass spectrometry (HR-ESI-MS; m/z 959.4861 ([C H O þ
4
5
76 20
þ
1
13
Na] ); calc. 959.4828), and the H- and C-NMR data. The ESI-QTOF-MS also
ꢀ
showed a quasi-molecular-ion peak at m/z 935.4857 ([M ꢀ H] ), and some character-
ꢀ
istic fragment-ion peaks at m/z 773.4351 ([M ꢀ H ꢀ 162] ), 611.3807 ([M ꢀ H ꢀ 162 ꢀ
ꢀ
ꢀ
1
62] ), and 449.3316 ([M ꢀ H ꢀ 162 ꢀ 162 ꢀ 162] ), attributed to the sequential loss of
three hexose residues. Compound 1 was hydrolyzed with acid to afford d-glucose as the
only sugar component. This was identified by comparison with authentic samples by
1
13
TLC and GC analysis. Based on H- and C-NMR (Table) and 2D-NMR analyses, the
structure of 1 was elucidated as (3b,5a,6a,22a,25R)-3,26-bis(b-d-glucopyranosyloxy)-
2
2-hydroxyfurostan-6-yl b-d-glucopyranoside.
1
The H-NMR spectrum showed signals for four Me groups at d(H) 1.30 (d, J ¼ 6.9,
Me(21)), 0.99 (d, J ¼ 6.6, Me(27)), 0.81 (s, Me(18)), and 0.70 (s, Me(19)). Moreover,
signals for three anomeric H-atom signals at d(H) 4.81 (d, J ¼ 7.8), 4.89 (d, J ¼ 7.8 ) ,
and 5.13 (d, J ¼ 7.8) could be readily assigned. The J values (> 7 Hz) of three anomeric
H-atoms indicated the b-orientation at the anomeric centers for these sugars. The
13
C-NMR spectrum of 1 showed three anomeric C-atom signals at d(C) 101.7, 105.0,
1
13
and 106.2. Analysis of the H- and C-NMR data of 1 in comparison with those in the
literature [9] implied that the aglycone of 1 was the same as that of (3b,5a,6a,25R)-5-
1
1
spirostane-3,6-diyl bis-b-d-glucopyranoside. Furthermore, the H, H-COSY, HSQC,
and HMBC spectra allowed the assignment of the glucose residue at C(3), at C(6), and
at C(26). The chemical shifts of glycosyloxy CH group (H ꢀC(26) and H ꢀC(26)) (D
2
a
b
ab
(d ꢀ d ) < 0.48 for (25R); D > 0.57 for (25S)) were proposed to ascertain the
a
b
ab

Helvetica Chimica Acta – Vol. 94 (2011)
1353
Table. 1H- and 13C-NMR Data ((D )Pyridine) of Compounds 1 and 2. d in ppm, J in Hz.
5
1
2
d(H)
d(C) d(H)
d(C)
CH (1)
0.79 – 0.83 (m), 1.46 – 1.53 (m)
1.64 – 1.68 (m), 2.02 – 2.08 (m)
3.91 – 3.98 (m)
37.6 0.69 – 0.74 (m), 1.26 – 1.30 (m)
29.9 1.55 ), 1.98 – 2.01 (m)
77.0 3.85 – 3.90 (m)
28.6 1.30 – 1.35 (m), 1.75 – 1.80 (m)
51.0 0.80 – 0.85 (m)
36.6
29.7
77.1
34.6
44.5
28.6
31.5
34.3
55.5
36.3
38.0
212.8
55.4
55.9
31.7
79.7
54.3
16.1
11.7
42.6
13.9
109.3
31.8
29.2
30.6
67.0
17.3
2
a
CH (2)
2
HꢀC(3)
CH (4)
1.43 – 1.50 (m), 3.40 (d, J ¼ 5.4)
2
HꢀC(5)
1.21 – 1.27 (m)
HꢀC(6) or CH (6) 3.60 – 3.64 (m)
79.7 1.04 – 1.10 (m), 1.04 – 1.10 (m)
2
a
CH (7)
1.21 – 1.27 (m), 2.53 – 2.57(m)
1.46 – 1.53 (m)
0.50 – 0.54 (m)
41.1 1.55 ), 2.06 – 2.10 (m)
2
CH (8)
34.0 1.72 – 1.76 (m)
53.9 0.85 – 0.90 (m)
36.7
21.2 2.20 – 2.25 (m), 2.33 – 2.37 (m)
40.1
41.5
2
HꢀC(9)
C(10)
CH (11)
1.21 – 1.27 (m), 1.29 – 1.38 (m)
1.02 – 1.09 (m), 1.65 – 1.69 (m)
2
CH (12) or C(12)
2
C(13)
HꢀC(14)
1.02 – 1.09 (m)
1.29 – 1.35 (m), 1.90 – 1.92 (m)
4.75 – 4.80 (m)
1.85 – 1.89 (m)
0.81 (s)
56.3 1.35 – 1.39 (m)
a
a
CH (15)
32.3 1.54 ), 1.60 )
2
a
HꢀC(16)
HꢀC(17)
Me(18)
Me(19)
HꢀC(20)
Me(21)
C(22)
81.0 4.45 )
63.9 2.75 (dd, J ¼ 7.4, 7.2)
16.8 1.07 (s)
0.70 (s)
13.4 0.66 (s)
2.04 – 2.08 (m)
1.30 (d, J ¼ 6.6)
40.1 1.89 – 1.95 (m)
16.4 1.34 (d, J ¼ 7.2)
110.6
a
a
CH (23)
1.98 ), 2.01 )
37.2 1.91 (t, J ¼ 6.6)
2
a
a
a
a
CH (24)
1.67 ), 2.02 )
28.4 1.54 ), 1.55 )
2
a
HꢀC(25)
1.90 – 1.92 (m)
3.60 – 3.64 (m), 3.91 – 3.98 (m)
0.99 (d, J ¼ 6.6)
Glc
34.3 1.57 )
CH (26)
75.3 3.46 – 3.49 (m), 3.54 – 3.58 (m)
17.5 0.68 (d, J ¼ 6.0)
Gal
2
Me(27)
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
5.13 (d, J ¼ 7.8)
101.7 4.84 (d, J ¼ 7.8)
75.5 4.38 – 4.41 (m)
78.1 4.08 – 4.13 (m)
71.8 4.52 – 4.62 (m)
78.7 3.97 – 4.01 (m)
102.5
73.1
75.4
79.9
75.6
60.7
4.00 – 4.06 (m)
a
4.25 )
4.20 – 4.22 (m)
3.91 – 3.98 (m)
4.32 – 4.38 (m), 4.42 – 4.55 (m)
Glc
a
a
CH (6)
62.7 4.21 ), 4.62 )
Glc’
2
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
4.81 (d, J ¼ 7.8)
105.0 5.12 (d, J ¼ 7.8)
75.2 4.30 – 4.34 (m)
78.7 4.08 – 4.13 (m)
71.8 3.70 – 3.73 (m)
104.8
80.8
88.1
70.6
77.5
63.0
4.00 – 4.06 (m)
a
4.27 )
a
4.23 )
a
3.91 – 3.98 (m)
78.1 3.79 )
a
a
a
a
CH (6)
4.23 ), 4.45 )
Glc
62.9 3.97 ), 4.41 )
Glc’’
2
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
4.89 (d, J ¼ 7.8)
106.2 5.17 (d, J ¼ 7.8)
104.2
75.6
76.5
78.0
77.3
61.1
4.00 – 4.06 (m)
78.0 3.98 – 4.02 (m)
a
a
4.23 )
78.1 4.08 )
4.20 – 4.22 (m)
3.85 – 3.90 (m)
71.8 4.30 – 4.34 (m)
a
78.7 3.79 )
62.7 4.04 ), 4.22 )
a
a
a
a
CH (6)
4.33 ), 4.43 )
2

1
354
Helvetica Chimica Acta – Vol. 94 (2011)
Table (cont.)
1
2
d(H)
d(C)
d(H)
d(C)
Rha
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
5.78 (br. s)
102.7
72.6
72.7
74.0
70.4
18.5
a
4.63 )
a
4.51 )
4.30 – 4.34 (m)
4.91 – 4.95 (m)
1.68 (d, J ¼ 6.0)
Glc’’’
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
5.57 (d, J ¼ 7.2)
104.0
75.1
87.0
69.1
78.2
62.1
a
4.06 )
a
4.05 )
a
4.06 )
a
3.80 )
4.29 ), 4.47 )
a
a
CH (6)
2
Xyl
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
5.09 (d, J ¼ 7.2)
3.91 – 3.95 (m)
4.06 )
106.2
75.4
77.7
70.8
67.1
a
4.08 – 4.13 (m)
a
a
CH (5)
3.52 ), 4.19 )
2
a) Overlapped with other signals.
orientation of the Me(27) group of furostane-type steroidal saponin [10][11]. In the
1
1
H, H-COSY and HSQC spectrum of 1, the signals of the H-atoms at C(26) were
observed at d(H) 3.63 (H ꢀC(26)) and 3.96 (H ꢀC(26)), with a chemical-shift
a
b
difference (D ) of 0.33 (D (d ꢀ d ) < 0.48), suggesting (25R)-configuration of 1. The
ab
ab
a
b
complete assignment of the glycosidic NMR signals was performed by analyses of
1
1
H, H-COSY, HSQC, and HMBC data. In the HMBC spectrum of 1 (Fig.), the HMBC
cross-peaks d(H) 4.81 (Glc HꢀC(1))/d(C) 75.3 (C(26)), d(H) 5.13 (Glc HꢀC(1))/d(C)
7
5.3 (C(3)), and d(H) 4.89 (Glc HꢀC(1))/d(C) 79.7 (Glc HꢀC(6)) were observed,
which confirmed the sugar sequence and the glycosylation position.
Sisalasaponin D (2) was obtained as a white amorphous powder, which gave
positive LiebermannꢀBurchard and negative Ehrlich reagent tests, suggesting a
spirostanol saponin. The HR-ESI-MS showed a peak at m/z 1379.6148, which indicated
þ
a formula C H O ([M þ Na] ; calc. 1379.6090). The ESI-QTOF-MS showed a peak
62
100 32
ꢀ
at m/z 1355.6251 ([M ꢀ H] ), and significant fragment-ion peaks at m/z 1223.5770
ꢀ
ꢀ
(
[M ꢀ H ꢀ 132] ), 1061.5205 ([M ꢀ H ꢀ 132 ꢀ 162] ), 915.4227 ([M ꢀ H ꢀ 132 ꢀ
ꢀ ꢀ
1
1
1
62 ꢀ 146] ), 753.3995 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146 ꢀ 162] ), 591.3532 ([M ꢀ H ꢀ
ꢀ
32 ꢀ 162 ꢀ 146 ꢀ 162 ꢀ 162] ), and 429.2926 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146 ꢀ 162 ꢀ
ꢀ
62 ꢀ 162] ), which were attributed to loss of xylose, glucose, rhamnose, and galactose
present in the molecule, in agreement with the results of the acid hydrolysis of 2, which
afforded d-xylose, d-galactose, d-glucose, and l-rhamnose as the sugar components.
1
13
On the basis of extensive H- and C-NMR (Table), and 2D-NMR analyses, the

Helvetica Chimica Acta – Vol. 94 (2011)
1355
Figure. Key HMBCs for compounds 1 and 2
structure of 2 was elucidated as (3b,5a,25R)-12-oxospirostan-3-yl 6-deoxy-a-l-
mannopyranosyl-(1 ! 4)-b-d-glucopyranosyl-(1 ! 3)-[b-d-xylopyranosyl-(1 ! 3)-b-d-
glucopyranosyl-(1 ! 2)]-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyranoside.
1
The H-NMR spectrum of 2 exhibited two singlet Me signals at d(H) 0.66 (s) and
1
.07 (s), and three doublet Me signals at d(H) 0.68 (d, J ¼ 6.0), 1.34 (d, J ¼ 7.2), and
1.68 (d, J ¼ 6.0), corresponding to the typical spirostanol Me(19), Me(18), Me(27),
and Me(21) Me H-atoms, and rhamnose Me H-atoms. Moreover, signals for six
anomeric H-atoms at d(H) 4.84 (d, J ¼ 7.2), 5.12 (d, J ¼ 7.8), 5.57 (d, J ¼ 7.2), 5.17 (d,
13
J ¼ 7.8), 5.78 (s), and 5.09 (d, J ¼ 7.2) could be readily assigned. The C-NMR
spectrum of 2 showed six anomeric C-atom signals at d(C) 102.5, 104.8, 104.0, 104.2,
1
02.7, and 106.2. Other characteristic features were the hemiketalic function at C(22)
1
(d(C) 109.3) and the C(12)¼O group (d(C) 213.0). The above H-NMR data and a

1
356
Helvetica Chimica Acta – Vol. 94 (2011)
13
comparison of the C-NMR signals of the aglycone moiety of 2 with those described in
the literature [12] indicated the structure of the aglycone to be hecogenin.
1
1
The H, H-COSY, HSQC, HMBC, and HSQC-TOCSY spectra allowed the
assignment of the saccharide moiety at C(3). HSQC-TOCSY was used to solve the
severe overlapping of the H-atom signals for the six sugar sequences of 2. On the
1
13
HꢀC(1) track through the anomeric H, C correlations at d(H/C) 5.17/104.2, four
cross-peaks were observed; with d(C) 78.0 (CH), 77.3 (CH), 76.5 (CH), and 75.6 (CH),
13
these C chemical shifts showed correlations in HSQC with the signals at d(H) 4.34
m), 3.79 (m), 4.08 (m), and 3.99 (m), respectively. The signal at d(H) 3.99 (m)
(
exhibited correlation with the signal at d(C) 61.1 in HSQC-TOCSY, and the signal at
d(C) 61.1, which showed correlations with signals at d(H) 3.52 (m) and 4.19 (m) in the
1
13
HSQC spectrum, was assigned to C(6) of a glucose. The anomeric H/ C correlations at
13
d(H/C) 5.09/106.2 led to four cross-peaks, with C chemical shifts of d(C) 75.4 (CH),
1
7
7.7 (CH), 70.8 (CH), and 67.1 (CH ). The signal at d(C) 67.1, which showed J(C,H)
2
correlations with d(H) 3.52 (m) and 4.19 (m) in the HSQC spectrum, was assigned to
C(5) of a xylose. The anomeric H-atom signal at d(H) 5.78 showed correlation peaks
13
with the C signals at d(C) 102.7 and 72.7; Me H-atom signal at d(H) 1.68 exhibited
13
correlation peaks with the C signals at d(C) 74.0, 72.6, 70.4, and 18.5; and the signal at
1
1
d(H) 4.63 (m) showed correlation peaks with the signal at d(H) 4.51 (m) in H, H-
COSY, indicating the presence of one terminal rhamnose. The anomeric H-atom signal
13
at d(H) 4.84 displayed correlation peaks with the C signals at d(C) 102.5, 73.1, 75.6;
1
1
other signals of the galactose were assigned by H, H-COSY and HSQC. The cross-
peaks of other anomeric H-atoms at d(H) 5.12 and 5.57 were also observed in HSQC
and TOCSY, respectively. In the HMBC spectrum (Fig.), the anomeric H-atom signal
of galactose (d(H) 4.84, HꢀC(1) of Gal) showed correlations with C(3) of the aglycone
(d(C) 77.1), whereas other anomeric H-atom signals, i.e., at d(H) 5.12 (Glc’), 5.17
(Glc’’), 5.57 (Glc’’’), 5.78 (Rha), and 5.09 (Xyl), showed correlations with C(4) of Gal
(d(C) 79.9), C(3) of (Glc’) (d(C) 88.1), C(2) of (Glc’) (d(C) 80.8), C(4) of (Glc’’) (d(C)
7
8.0), and C(3) of (Glc’’’) (d(C) 87.0), respectively. The full assignments of these sugar
1
1
signals were confirmed by H, H-COSY, HSQC, HMBC, and HSQC-TOCSY experi-
ments.
Comparison of the physicochemical properties and NMR data with those reported
in the literature allowed us to identify (3b,5a,6a,22a,25R)-methoxy-5-furostane-3,6,26-
triyl tris-b-d-glucopyranoside [6], cantalasaponin-1 [13], polianthoside D [14], and
(
E)- and (Z)-2,3,4’,5-tetrahydroxystilbene 2-O-b-d-glucopyranosides [15]. The last
three compounds are reported from the Agavaceae for the first time.
This work was supported by the Major Program of Municipal Natural Science Foundation of Beijing
(
7090001). The authors are grateful to Mrs. Yan Xue and Mr. He-Bing Chen, National Center of
Biomedical Analysis, for recording the NMR and mass spectra.
Experimental Part
General. Column chromatography (CC): macroporous resin SP825 (Mitsubishi Chemical, Japan),
silica gel (SiO ; Qingdao Haiyang Chemical Co., Ltd., P. R. China), and ODS SiO (120 ꢂ, 50 mm, YMC,
2
2
Japan). HPLC: Waters2695 Alliance system (Waters, USA) equipped with a YMC-Pack ODS-A C18
column (YMC, 4.6 mm i.d. ꢁ250 mm, ODS, 5 mm, Japan). GC: Agilent 6890 gas chromatograph

Helvetica Chimica Acta – Vol. 94 (2011)
1357
equipped with an H flame ionization detector; HP-5 cap. column (30 m ꢁ 0.25 mm ꢁ 0.25 mm; Agilent,
2
USA). Optical rotations: Perkin-Elmer 343 polarimeter. HR-ESI-MS: 9.4 T Q-FT-MS Apex Qe (Bruker
UNITY
1
13
Co.). NMR spectra: Varian
INOVA 600 (599.8 MHz for H and 150.8 MHz for C); the chemical
shifts in d [ppm] scale with Me Si as an internal standard. ESI-QTOF-MS: Synapt MS system (Waters,
4
USA), mass accuracy was maintained using a lock spray with leucine enkephalin for negative-ion mode
ꢀ
(
[M ꢀ H] 554.2615) at a concentration of 200 pg/ml and a flow rate of 50 ml/min as reference. The full
scan data acquisition range was 100 to 1500 Da.
Plant Material. The material was collected from Sanya County of Hainan Province, P. R. China, in
August 2005, and was identified as leaves of Agave sisalana Perr. by Prof. L.-J. Z., Tianjing University of
Traditional Chinese Medicine. Avoucher specimen (No. 040123) has been deposited with the Herbarium
of the Beijing Institute of Radiation Medicine, Beijing.
Extraction and Isolation. The fresh leaves of Agave sisalana Perr. (74.0 kg) were extracted three
times with 50% aq. EtOH at 1208 for 1 h each time (3 ꢁ 200 l). The combined extract was concentrated
under reduced pressure. CC of the extract was performed on macroporous resin (AB-8) and eluted with a
gradient mixture of Me CO/H O (1:9, 1:1, and 8 :2) to give three fractions, Frs. A – C. Fr. B (1120 g) was
2
2
chromatographed on macroporous resin (SP825) and eluted with a gradient mixture of Me CO/H O
2
2
(
(
3 :20, 5 :20, 7:20, and 20 :20) to give four fractions, Frs. B (182 g), B (208 g), B (202 g), and B
1 2 3 4
330 g). A part of Fr. B (208 g) was chromatographed on ODS SiO (50 mm) and eluted with MeOH/
2
2
H O (38 :62, 45 :55, and 70 :30), the part eluted with MeOH/H O (70 :30) was chromatographed on SiO
2
2
2
and eluted with CHCl /MeOH/H O (60 :35 :10; lower phase); combination of Frs. 8 – 11 gave
3
2
compounds 1 (147 mg) and (3b,5a,6a,22a,25R)-22-methoxyfurostane-3,6,26-triyl tris-b-d-glucopyrano-
side (23 mg). A part of Fr. B (310 g) was chromatographed on SiO and eluted with CHCl /MeOH/H O
4
2
3
2
(
70 :25 :10, lower phase); recrystallization yielded cantalasaponin-1 (235 mg). A part of Fr. B (200 g)
2
was chromatographed on ODS SiO (50 mm) and eluted with MeOH/H O (38 :62, 45 :55, and 70 :30).
2
2
The part eluted with MeOH/H O 45 :55 was chromatographed on ODS SiO and eluted with MeOH/
2
2
H O (35 :65, 38 :62, 40 :60, 44 :56, and 70 :30), and polianthoside (46 mg) was obtained from the part
2
that eluted with MeOH/H O 44 :56 by semi-prep. HPLC with MeCN/H O 18 :82. A part of Fr. B (180 g)
2
2
3
was chromatographed on ODS SiO (50 mm) and eluted with Me CO/H O (20 :80, 24 :76, and 50 :50);
2
2
2
the part eluted with Me CO/H O 20 :80 was chromatographed on Sephadex LH-20 and eluted with
2
2
Me CO/H O 50 :50 to afford (E)- and (Z)-2,3,4’,5-tetrahydrostilbene 2-O-b-d-glucopyranosides (46.7
2
2
and 16.3 mg, resp.) were obtained by semi-prep. HPLC with Me CO/H O 23 :77. Fr. C (20 g) was
2
2
chromatographed on SiO2 with a gradient mixture of CHCl /MeOH/H O (100 :20 :5, 70 :30 :10,
3
2
6
5 :35 :10, lower phase) to give five fractions, Frs. C – C . A part of Fr. C (2 g) was chromatographed on
1 5 1
ODS SiO (50 mm) and eluted with MeOH/H O (50 :50, 80 :20, and 100 :0); the second fraction was
2
2
recrystallized to yield compound 2 (13 mg).
Sisalasaponin C ( ¼ (3b,5a,6a,22a,25R)-3,26-Bis[(b-d-glucopyranosyl)oxy]-22-hydroxyfurostan-6-
2
0
1
yl b-d-Glucopyranoside; 1). White amorphous power. [a] ¼ ꢀ40.3 (c ¼ 0.064, pyridine). H- and
D
13
ꢀ
ꢀ
C-NMR: see the Table. ESI-QTOF-MS (neg.): 935.4857 ([M ꢀ H] ), 773.4351 ([M ꢀ H ꢀ 162] ),
ꢀ
ꢀ
6
11.3807 ([M ꢀ H ꢀ 162 ꢀ 162] ), 449.3316 ([M ꢀ H ꢀ 162 ꢀ 162 ꢀ 162] ). HR-ESI-MS (pos.): 959.4861
þ þ
(
[M þ Na] , C H NaO20 ; calc. 959.4828).
45
76
Sisalasaponin D ( ¼ (3b,5a,25R)-12-Oxospirostan-3-yl 6-Deoxy-a-l-mannopyranosyl-(1 ! 4)-b-d-
glucopyranosyl-(1 ! 3)-[b-d-xylopyranosyl-(1 ! 3)-b-d-glucopyranosyl-(1 ! 2)]-b-d-glucopyranosyl-
2
0
1
(
1 ! 4)-b-d-galactopyranoside; 2). White amorphous power. [a] ¼ ꢀ60.4 (c ¼ 0.086, pyridine). H- and
D
1
3
ꢀ
ꢀ
C-NMR: see the Table. ESI-QTOF-MS (neg.): 1355.6251 ([M ꢀ H] ), 1223.5770 ([M ꢀ H ꢀ 132] ),
ꢀ
ꢀ
1
1
1
061.5205 ([M ꢀ H ꢀ 132 ꢀ 162] ), 915.4227 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146] ), 753.3995 ([M ꢀ H ꢀ 132 ꢀ
ꢀ
ꢀ
62 ꢀ 146 ꢀ 162] ), 591.3532 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146 ꢀ 162 ꢀ 162] ), 429.2926 ([M ꢀ H ꢀ 132 ꢀ
ꢀ
þ
þ
32
62 ꢀ 146 ꢀ 162 ꢀ 162 ꢀ 162] ). HR-ESI-MS (pos.): 1379.6148 ([M þ Na] , C H NaO ; calc.
6
2
100
1
379.6090).
Acid Hydrolysis. Compound 1 (2.0 mg) was treated with 1m HCl (dioxane/H O, 1:1, 2 ml) at 1008 for
2
1
.5 h. The mixture was neutralized with Ag CO , and the solvent was thoroughly driven out under N
2
3
2
overnight. The residue was extracted with CHCl and H O. Then, in the monosaccharide mixture, glucose
3
2
from 1 was detected by TLC analysis on a cellulose plate with BuOH/AcOEt/C H N/H O 6 :1:5 :4 and
5
5
2
aniline-o-phthalic acid for detection, comparing with the authentic sample (glucose: Rf 0.46).

1
358
Helvetica Chimica Acta – Vol. 94 (2011)
Furthermore, the sugar residue in pyridine (1 ml) was added to l-cysteine methyl ester hydrochloride
3.0 mg), and the mixture was kept at 608 for 1 h. Then, HMDS/TMCS (hexamethyldisilazane/
trimethylchlorosilane; 0.6 ml) was added to the mixture and kept at 608 for 0.5 h. The supernatant
1.0 ml) was analyzed by GC (Agilent Technologies 6890 GC was the equipment carrying H2 flame
(
(
ionization detector and HP-5 cap. column (30 m ꢁ 0.25 mm ꢁ 0.25 mm; column temp., 1808/2508;
programmed increase, 38/min, carrier gas, N (1 ml/min); injection and detector temp., 2508; injection
2
volume, 4.0 ml; split ratio, 1:50). The retention times (tR [min]) of the derivatives of the authentic
standards were: tR 17.95 (d-glucose derivative), tR 18.39 (l-glucose derivative), tR 19.57 (d-galactose
derivative), and tR 19.18 (l-galactose derivative), tR 14.53 (l-rhamnose derivative), tR 13.00 (d-xylose
derivative), and tR 13.66 (l-xylose derivative). The tR value of the derivative of d-glucose (d-glucose
derivative) for compound 1 was 17.93. The same procedures carried out for 2 (ca. 2.0 mg) provided the tR
values of 17.94, 14.53, 19.57, and 13.00 for d-glucose, l-rhamnose, d-galactose, and d-xylose, resp.
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Received December 2, 2010