Polyhydroxylated steroidal constituents from the fresh rhizomes of Tupistra yunnanensis

Article abstract of DOI:10.1016/j.steroids.2005.04.003

Six new polyhydroxylated steroidal saponins, tupistrosides A-F (1-6), together with nine known steroids, were isolated from the fresh rhizomes of Tupistra yunnanensis. Their structures were elucidated to be (25S)-1β,4β,5β-trihydroxy-spirostane-3β-yl O-α-l-arabinopyranoside (1), 1β,24β-dihydroxy-spirost-5,25(27)- dien-3α-yl O-β-d-glucopyranoside (2), (22S,25S)-1α,2β, 3α,5α-tetrahydroxy-furo-spirostane-26-yl O-β-d-glucopyranoside (3), 1β,3α,22 ξ-trihydroxy-furost-5,25(27)-dien-26-yl O-β-d-glucopyranoside (4), 26-O-β-d-glucopyranosyl-1β,22- dihydroxy-furost-5-en-3α-yl O-β-d-glucopyranoside (5) and 22-methoxy-1β,2β,3β,4β,5β,7α-hexahydroxy-furost- 25(27)-en-6-one-26-yl O-β-d-glucopyranoside (6), respectively, by means of spectroscopic analysis and the results of acid hydrolysis.

Full text of DOI:10.1016/j.steroids.2005.04.003

Steroids 70 (2005) 732–737
Polyhydroxylated steroidal constituents from the fresh
rhizomes of Tupistra yunnanensis
Qing-Xiong Yang, Ying-Jun Zhang∗, Hai-Zhou Li, Chong-Ren Yang∗∗
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, PR China
Received 5 December 2003; received in revised form 1 April 2005; accepted 4 April 2005
Available online 23 May 2005
Abstract
Six new polyhydroxylated steroidal saponins, tupistrosides A–F (1–6), together with nine known steroids, were isolated from the fresh rhi-
zomes of Tupistra yunnanensis. Their structures were elucidated to be (25S)-1␤,4␤,5␤-trihydroxy-spirostane-3␤-yl O-␣-l-arabinopyranoside
(1), 1␤,24␤-dihydroxy-spirost-5,25(27)-dien-3␣-yl O-␤-d-glucopyranoside (2), (22S,25S)-1␣,2␤,3␣,5␣-tetrahydroxy-furo-spirostane-26-yl
O-␤-d-glucopyranoside (3), 1␤,3␣,22 ξ-trihydroxy-furost-5,25(27)-dien-26-yl O-␤-d-glucopyranoside (4), 26-O-␤-d-glucopyranosyl-1␤,22-
dihydroxy-furost-5-en-3␣-yl O-␤-d-glucopyranoside (5) and 22-methoxy-1␤,2␤,3␤,4␤,5␤,7␣-hexahydroxy-furost-25(27)-en-6-one-26-yl O-
␤-d-glucopyranoside (6), respectively, by means of spectroscopic analysis and the results of acid hydrolysis.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Tupistra yunnanensis; Liliaceae; Steroidal saponins; Tupistrosides A–F; NMR spectroscopy; Acid hydrolysis
1. Introduction
and chemical characterization of six new polyhydroxylated
steroidal saponins, named tupistrosides A–F (1–6), together
with nine known steroids from this plant are presented.
The genus Tupistra (Liliaceae), comprising about 26
species is mainly distributed in Asia. Among which, about 17
species are growing in the People’s Republic of China, par-
ticularly in the southwestern part [1]. Because of their similar
morphologic characteristics, some species were substituted
for each other as a traditional Chinese medicine, called
“Xin-Bu-Gan” [2]. Tupistra yunnanensis Wang et Tang is
endemic to Yunnan province of China. Its rhizomes are used
as a remedy for stomach upset, injuries from falls and frac-
ture. To the best of our knowledge, no chemical investigation
appears to have been performed on this plant, although some
species of genus Tupistra were studied for their chemical
components and bioactivities [3–16]. As a part of a contin-
uing study for the discovery of novel secondary metabolites
from liliaceous plants [7–10,17–20], the fresh rhizomes of
Tupistra yunnanensis were investigated. Here, the isolation
2. Experimental
2.1. General methods
Optical rotations were measured on a SEPA-3000 auto-
matic digital polarimeter. NMR spectra were measured in
pyridine-d5 unless otherwise noted and recorded on a Bruker
AM-400 (for 1H NMR and 13C NMR) and DRX-500 (for 2D
NMR) instrument with TMS as internal standard; IR spectra
were measured on a Bio-Rad FTS-135 spectrometer in KBr
pellets. FABMS (negative ion mode) spectra were recorded
on a VG Auto Spec-300 spectrometer.
2.2. Plant material
∗
∗∗
Corresponding author. Tel.: +86 871 5223235; fax: +86 871 5150124.
Co corresponding author.
The rhizomes of Tupistra yunnanensis Wang et Tang was
E-mail addresses: zhangyj@mail.kib.ac.cn (Y.-J. Zhang),
cryang@hotmail.com (C.-R. Yang).
collected at Luquan, in the northeast of Yunnan province,
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.04.003

Q.-X. Yang et al. / Steroids 70 (2005) 732–737
733
Table 1
China, and identified by Prof. Li Heng (Kunming Institute of
13C NMR spectral data of compounds 1–6 (pyridine-d5)
Botany, Chinese Academy of Sciences). A voucher specimen
is deposited in the herbarium of Kunming Institute of Botany,
Chinese Academy of Sciences.
Position
1
2a
3
4
5a
75.0
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
22-OCH3
73.5
32.9
78.0
67.5
78.4
29.4
28.5
34.9
44.9
42.5
22.0
40.8
40.1
56.4
32.2
81.3
62.9
16.6
14.9
42.3
16.3
109.8
26.4
26.2
27.6
65.2
17.6
76.2
77.9
68.2
71.9
39.3
74.7
36.0
28.9
34.9
45.5
45.7
21.8
40.1
40.7
56.1
32.2
81.0
62.7
16.5
13.9
38.9
15.2
75.5
38.1
66.6
40.8
76.5
67.9
75.5
71.2
86.3
36.7
75.4
39.1
139.9
125.5
33.0
32.9
51.6
44.9
24.9
39.1
41.3
58.0
32.9
82.8
63.8
16.9
13.3
42.9
14.8
112.6
41.3
67.8
148.6
65.2
106.7
36.7
74.9
39.0
139.8
125.5
32.3
32.9
51.5
44.8
24.8
41.4
41.2
57.9
32.9
82.4
65.2
16.9
13.3
41.2
16.1
113.6
31.4
28.7
34.9
75.9
17.3
2.3. Extraction and isolation
140.1
124.7
32.5
32.9
51.4
44.7
24.4
40.6
41.1
57.1
32.7
81.2
64.1
16.8
13.3
40.8
16.4
110.8
30.2
28.5
147.3
72.2
110.4
The fresh rhizomes of Tupistra yunnanensis (10.0 kg)
were extracted with MeOH under reflux (3 L × 30 L) for
three times (4, 3 and 3 h, respectively). After removal of the
solvent, the concentrated residue was partitioned between
n-BuOH (3 mL × 800 mL) and H2O (3 L). The n-BuOH
phase (96 g) was fractionated on a silica gel column with
CHCl3–MeOH–H2O (7:3:1, lower layer) and gave four frac-
tions (1–4). Fr. 1 (12.5 g) was chromatographed on silica gel
(375 g) with a gradient of CHCl3–MeOH (20:1–10:1) to give
211.1
75.5
37.9
41.1
50.2
21.9
39.3
40.9
49.7
31.6
81.4
64.1
16.3
11.6
40.8
16.1
112.5
31.9
28.2
146.9
72.1
111.3
47.4
∆
25(27) pentologenin (50 mg), convallagenin B (60 mg), epir-
uscogenin (50 mg) and tupisgenin (80 mg). Fr. 2 (20.0 g) and
Fr. 3 (10.5 g) were supplied separately to silica gel column
(400 and 200 g, respectively) chromatography (CC) eluting
with CHCl3–MeOH–H2O (8:2:1, lower layer and 7:2:1) and
repeated Rp-8 CC with 70–90% MeOH to yield 1 (200 mg),
2 (50 mg), 1␤,2␤,3␤,4␤,5␤,7␣-hexahydroxy-spirost-25(27)-
en-6-one (100 mg) and 25(R)-1␤-hydroxy-spirost-5-en-3␣-
yl O-␤-d-glucopyranoside (1.0 g) from Fr. 2 and 3
(50 mg), 25(S)-22-methoxy-1␤,3␤,4␤,5␤,26-pentahydroxy-
furost-26-yl O-␤-d-glucopyranoside (150 mg), and a mix-
ture from Fr. 3, respectively. The mixture was refluxed
in 30% aqueous acetone to give 4 (40 mg). Fr. 4 (24.0 g)
was subjected to silica gel CC (360 g) eluting with
CHCl3–MeOH–H2O (7:2:1) and repeated Rp-8 CC eluting
with 50–70% MeOH to furnish tupstroside G (30 mg), wat-
toside D (50 mg), 6 (60 mg), and a mixture, which yielded 5
(3.0 g) by refluxing in 30% aqueous acetone.
120.3
33.1
33.9
83.9
77.5
24.4
3-O-Glc
(Ara)
102.6
73.2
75.2
71.2
67.5
1ꢀ
102.9
74.9
78.2
71.7
77.9
62.9
103.2
75.0
78.1
71.6
77.8
62.8
104.5
75.4
78.1
71.6
77.8
62.8
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
26-O-Glc-1ꢀ
105.4
75.3
78.4
71.6
78.5
62.6
103.9
75.2
78.6
71.8
78.6
62.8
103.9
75.2
78.5
71.8
78.7
62.9
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
a
2.3.1. Tupistroside A (1)
White amorphous powder, [α]D = −56.4◦ (c = 0.4,
MeOH). FABMS (negative ion mode): m/z 595 [M − H]−,
463 [M − Ara − H]−. HRFABMS (negative ion mode):
m/z 595.3430 [M (C32H52O10)− H]− (Calc. 595.3482).
IR (KBr): νmax 3410 (br, OH), 2951 (C–H), 920, 878,
Measured in CD3OD.
silica gel CC (4.8 g) eluting with CHCl3–MeOH (20:1–10:1)
to give a pure compound, convallagenin B (1a, 10 mg), iden-
tified by the comparison with authentic samples through
spectral and TLC properties. The aqueous layer was con-
centrated to dryness and then chromatographed over a sil-
ica gel column with CHCl3–MeOH–H2O (7:3:0.5) to yield
l-arabinose [(3.2 mg), [α]D = +23.5◦ (c = 0.17, H2O), Rf 0.2
[CHCl3–MeOH–H2O, 7:3:0.5].
850 (intensity 920 > 878, 25(S) spirostanol) cm−1 [21]. H
1
NMR (pyridine-d5): δ 0.81 (s, CH3-18), 1.02 (d, J = 7.0 Hz,
CH3-27), 1.14 (d, J = 6.8 Hz, CH3-21), 1.20 (s, CH3-19),
2.50 (dd, J = 13.1, 13.2 Hz, H-2), 3.35 (d, J = 10.9 Hz,
H-26), 4.06 (d, J = 10.7 Hz, H-26), 3.78 (brd, J = 11.3 Hz,
H-5ꢀ), 4.08 (dd, J = 11.0, 2.0 Hz, H-26), 4.16 (dd, J = 8.6,
7.6 Hz, H-2ꢀ), 4.42 (dd, J = 11.3, 5.1 Hz, H-5ꢀ), 4.54 (m,
H-16), 4.95 (brs, H-1), 5.05 (d, J = 7.6 Hz, H-1ꢀ). 13C NMR
(pyridine-d5); see Table 1.
2.3.3. Tupistroside B (2)
White amorphous powder, [α]D = −60.5◦ (c = 0.4,
MeOH). FABMS (negative ion mode): m/z 605 [M − H]−.
HRFABMS (negative ion mode): m/z 605.3326 [M
(C33H50O10)− H]− (Calc. 605.3325). IR (KBr): νmax 3410,
2.3.2. Acid hydrolysis of 1
A solution of 1 (60 mg) in 2N HCl–MeOH (1:1, 20 mL)
was heated at 95 ◦C for 5 h. The reaction mixture was neu-
tralized with NaHCO3 (1N) and extracted with CHCl3 three
times. The CHCl3 phase (40 mg) was chromatographed on
2939, 1650, 1100, 980, 835 cm−1 1H NMR (CD3OD):
.
δ 0.84 (s, CH3-18), 0.98 (d, J = 6.8 Hz, CH3-21), 1.03 (s,

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Q.-X. Yang et al. / Steroids 70 (2005) 732–737
CH3-19), 3.15 (t, J = 7.8 Hz, H-2ꢀ), 3.64 (dd, J = 5.4, 11.7 Hz,
H-6ꢀ), 3.76 (dd, J = 4.3, 11.7 Hz, H-1), 3.85 (dd, J = 2.4,
11.7 Hz, H-6ꢀ), 3.92 (d, J = 12.2 Hz, H-26), 4.04 (dd, J = 3.0,
3.9 Hz, H-3), 4.20 (d, J = 12.2 Hz, H-26), 4.32 (d, J = 7.8 Hz,
H-1ꢀ), 4.40 (dd, J = 3.0, 7.4 Hz, H-24), 4.87 (brs, H-27), 5.08
(brs, H-27), 5.47 (d, J = 4.7 Hz, H-5). 13C NMR (CD3OD);
see Table 1.
2.3.9. Tupistroside E (5)
White amorphous powder, [α]D = −48.5◦ (c = 0.3, pyri-
dine). FABMS (negative ion mode): m/z 772 [M]−,
609 [M–Glc − H]−. HRFABMS (negative ion mode): m/z
771.4150 [M (C39H64O15)− H]− (Calc. 771.4163). IR
(KBr): νmax 3418, 2902, 1650, 1455, 1078, 1024 cm−1. H
1
NMR (CD3OD): δ 0.83 (d, J = 5.5 Hz, CH3-27), 0.86 (s, CH3-
18), 1.00 (d, J = 6.7 Hz, CH3-21), 1.10 (s, CH3-19), 4.20
(d, J = 7.8 Hz, 3-O-Glc H-1ꢀ), 4.24 (d, J = 7.5 Hz, 26-O-Glc
H-1ꢀ), 5.47 (d, J = 5.1 Hz, H-6). 13C NMR (CD3OD); see
Table 1.
2.3.4. Acid hydrolysis of 2
A solution of 2 (2 mg) in 2N HCl–MeOH (1:1, 1 mL)
was heated at 95 ◦C for 5 h. The reaction mixture was neu-
tralized with NaHCO3 (1N) and evaporated. TLC analysis
on a silica gel HPTLC silica gel 50000 F254 plate using n-
BuOH-i-PrOH–H2O (10:5:4, homogenous) as development
and anisaldehyde-H2SO4 as detection, compared with the
authentic samples, indicated the presence of d-glucose (Rf
0.29).
2.3.10. Acid hydrolysis of 5
Compound 5 (80 mg) was subjected to acid hydroly-
sis as described for 1 to give epiruscogenin (5a, 14 mg),
by comparison with an authentic samples through spectral
and TLC properties and d-glucose [(4.4 mg), [α]D = +26.1◦
(c = 0.2, H2O), Rf 0.29 (n-BuOH-i-PrOH–H2O (10:5:4, ho-
mogenous))].
2.3.5. Tupistroside C (3)
White amorphous powder, [α]D = −67.2◦ (c = 0.4,
MeOH). FABMS (positive ion mode): m/z 643 [M + H]+,
625 [M–H2O + H]+, 607 [M–2H2O + H]+, 481 [M–Glc + H]+,
463 [M–Glc–H2O + H]+. HRFABMS (negative ion mode):
m/z 641.3537 [M (C33H54O12)− H]− (Calc. 641.3537). IR
2.3.11. Tupistroside F (6)
White amorphous powder, [α]D = −51.7◦ (c = 0.2, pyri-
dine). FABMS (negative ion mode): m/z 703 [M − H]−,
541 [M–Glc − H]−. HR FABMS (negative ion mode):
m/z 701.3386 [M (C34H54O15)− H]− (Calc. 701.3384). IR
(KBr): νmax 3399, 2945, 2913, 1712 (C O), 1647 (C C)
1
(KBr): νmax 3449, 3263, 2936, 1665, 1407, 1043 cm−1. H
1
cm−1. H NMR (pyridine-d5): δ 0.81 (s, CH3-18), 1.15 (d,
NMR (pyridine-d5): δ 0.80 (s, CH3-18), 1.06 (d, J = 6.5 Hz,
CH3-21), 1.39 (s, CH3-27), 1.55 (s, CH3-19), 2.20 (dd,
J = 2.9, 14.9 Hz, Ha-4), 2.50 (dd, J = 3.8, 14.9 Hz, Hb-4),
4.11 (dd, J = 2.9, 3.8 Hz, H-2), 4.31 (dd, J = 1.8, 3.8 Hz, H-1),
4.38 (dd, J = 5.1, 11.9 Hz, H-6ꢀ), 4.52 (dd, J = 2.0, 11.9 Hz,
H-6ꢀ), 4.67 (td, J = 2.6, 3.8 Hz, H-3), 4.75 (m, H-16), 4.94
(d, J = 7.48 Hz, H-1ꢀ). 13C NMR (pyridine-d5); see Table 1.
J = 6.8 Hz, CH3-21), 1.43 (s, CH3-19), 3.22 (s, 22-OCH3),
4.92 (d, J = 8.0 Hz, H-1ꢀ), 5.06 (brs, H-27), 5.33 (brs, H-27).
13C NMR (pyridine-d5); see Table 1.
2.3.12. Acid hydrolysis of 6
Compound 6 (80 mg) was subjected to acid hydrolysis
as described for 1 to give 1␤,2␤,3␤,4␤,5␤,7␣-hexahydroxy-
spirost-25(27)-en-6-one (6a, 12 mg), by comparison with an
authentic samples through spectral and TLC properties and
d-glucose [(2.4 mg), [α]D = +22.1◦(c = 0.1, H2O), Rf 0.29 (n-
BuOH-i-PrOH–H2O (10:5:4, homogenous))].
2.3.6. Acid hydrolysis of 3
Compound 3 (2 mg) was subjected to acid hydrolysis as
described for 2. TLC analysis indicated the presence of d-
glucose.
2.3.7. Tupistroside D (4)
3. Results and discussion
White amorphous powder, [α]D = −50.6◦ (c = 0.3, pyri-
dine). FABMS (negative ion mode): m/z 607 [M − H]−,
445 [M–Glc − H]−. HRFABMS (negative ion mode): m/z
607.3436 [M (C33H52O10)− H]− (Calc. 607.3482). IR
(KBr): νmax 3430, 2942, 1651, 1450, 1080 cm−1. 1H NMR
(pyridine-d5): 0.96 (s, CH3-18), 1.26 (d, J = 6.9 Hz, CH3-21),
1.34 (s, CH3-19), 4.04 (dd, J = 7.4, 7.8 Hz, H-2ꢀ), 4.20 (over-
lapped, H-26), 4.59 (d, J = 13.0 Hz, H-26), 4.87 (d, J = 7.4 Hz,
H-1ꢀ), 5.03 (brs, H-27), 5.32 (brs, H-27), 5.63 (d, J = 5.3 Hz,
H-6). 13C NMR (pyridine-d5); see Table 1.
The MeOH extract of the fresh rhizomes of Tupistra
yunnanensis was suspended in H2O and extracted with
n-BuOH. The n-BuOH fraction was chromatographed
on silica gel and Rp-8 to afford compounds 1–6 (Fig. 1)
and nine known compounds. On the basis of the spec-
troscopic evidence and by comparison with the literature
values, the known compounds were identified as ∆25(27)
pentologenin [22], epiruscogenin [22], 1␤,2␤,3␤,4␤, 5␤,7␣-
hexahydroxy-spirost-25(27)-en-6-one [22], tupisgenin
[22], 25(S)-22-methoxy-1␤, 3␤,4␤,5␤,26-pentahydroxy-
furost-26-yl O-␤-d-glucopyranoside [23], convallagenin
B (1a) [24], 25(R)-1␤-hydroxy-spirost-5-en-3␣-yl O-␤-d-
glucopyranoside [25], wattoside D [8], tupstroside G [19],
respectively.
2.3.8. Acid hydrolysis of 4
Compound 4 (2 mg) was subjected to acid hydrolysis as
described for 2. TLC analysis indicated the presence of d-
glucose.

Q.-X. Yang et al. / Steroids 70 (2005) 732–737
735
Fig. 1. New polyhydroxylated steroidal saponins isolated from Tupistra yunnanensis.
Compound 1 was obtained as a white amorphous pow-
der. Its molecular formula was assigned as C32H52O10 on
the basis of the 13C NMR data (Table 1) and negative ion
HRFABMS ([M − H]−, m/z 595.3430). The 1H NMR spec-
trum of 1 showed four steroidal methyl groups at δ 1.14
(J = 6.8 Hz), 1.20 (s), 1.02 (J = 7.0 Hz) and 0.81 (s), and
one doublet anomeric proton signal at δ 5.05 (J = 7.6 Hz)
[26]. Acid hydrolysis of 1 with 2N HCl afforded conva-
llagenin B (1a), which was confirmed by direct compari-
son of the 13C chemical shifts with literature data [24] and
l-arabinose ([α]D = +23.5◦). In the 13C NMR spectrum of
1, the downfield shift of C-3 (δ 78.0) and the upfield shift
of C-2 and C-4 (δ 32.9, 67.5) [convallagenin B: δ 71.4,
33.6 and 68.3, respectively] indicated that the arabinopy-
ranosyl unit was linked at C-3 of the aglycone. This was
confirmed by the long-range correlation between δ 5.05
(H-1ꢀ of arabinose) and δ 78.0 (C-3 of aglycone) in the
HMBC spectrum. Therefore, the structure of 1 was deter-
mined to be (25S)-1␤,4␤, 5␤-trihydroxy-spirostane-3␤-yl O-
␣-l-arabinopyranoside (1), named as tupistroside A.
Compound 2 was shown to have the molecular for-
mula C33H50O10 on the basis of HRFABMS ([M − H]− m/z
605.3326) and 13C DEPT spectrum. Acid hydrolysis of 2
afforded d-glucose. The typical methyl signals at δ 0.84 (s),
1.03 (s) and 0.95 (d, J = 6.8 Hz) in the 1H NMR spectrum and
olefinic carbon signals at δ 139.9 and 125.5 and a quaternary

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Q.-X. Yang et al. / Steroids 70 (2005) 732–737
carbon signal at δ 112.6 (C-22) in the 13C NMR spectrum
indicated that 2 was a ꢀ5-spirostanol. Moreover, instead of
a doublet methyl proton signal, the presence of the olefinic
carbon signals at δ 148.6 (q) and 106.7 (d) suggested that an
additional olefinic bond was present between C-25 and C-27
[27]. In addition, an anomeric proton signal was also noted
at δ 4.32 (d, J = 7.80 Hz) in the 1H NMR spectrum.
In the 13C NMR spectrum of 2, 33 carbon signals were ob-
served, six of which were due to the glucose. This implied a
C27H40O5 molecular formula for the aglycone portion, sug-
gesting that the aglycone was a spirostanol with three hy-
droxyl groups. The locations of the hydroxyl groups, as well
as the sugar moiety on the aglycone, were determined by
detailed analysis of the HMQC and HMBC spectra. In the
HMBC spectrum of 2, the long-range correlations of δ 1.03
(H-19) with δ 76.2 (C-1), δ 2.23 (H-4) with δ 75.4 (C-3), δ
4.42 (H-24) with δ 148.6 (C-25) and δ 4.21 (H-26) with δ 67.8
(C-24) revealed that the three hydroxyl groups were linked at
C-1, C-3 and C-24 of the aglycone. HMBC correlation of the
anomeric proton signal at δ 4.32 with δ 75.4 (C-3) proved the
location of the glucopyranosyl moiety at C-3 of aglycone.
The J coupling patterns (H-1: ␦ 3.76, dd, J = 4.3, 11.7 Hz;
H-3: δ 4.04, dd, J = 3.0, 3.9 Hz; H-24: δ 4.42, dd, J = 3.0,
7.4Hz) measured in the JRES spectrum, determined the con-
figuration of the hydroxyl groups as 1␤, 3␣ and 24␤. On the
basis of the above evidence, tupistroside B (2) was deter-
mined to be 1␤,24␤-dihydroxy-spirost-5,25(27)-dien-3␣-yl
O-␤-d-glucopyranoside.
Compound 3 had a molecular formula C33H54O12 on the
basis of HRFABMS ([M − H]− m/z 641.3537). The 1H NMR
spectrum showed four typical steroidal methyl signals at δ
1.55 (s), 1.39 (s), 1.06 (d, J = 6.5 Hz) and 0.80 (s) [26]. The
13CNMRspectrumexhibitedtheC-22carbonoftheaglycone
at δ 120.3, characteristic of the furospirostanol skeleton [28].
In addition, the observation of an anomeric proton signal at
δ 4.94 (d, J = 7.5 Hz), together with acidic hydrolysis of 3
affording d-glucose, suggested that 3 were a furospirostanol
glucoside.
In the FAB mass spectrum of 3, the presence of
the fragment ion peaks at m/z 643 [M + H]+, 625
[M–H2O + H]+, 607 [M–2H2O + H]+, 481 [M-162 + H]+, 463
[M-162–H2O + H]+, 445 [M-162–2H2O + H]+ and 427 [M-
3H2O + H]+ indicated the existence of several hydroxyl
groups in the aglycone. The locations of the hydroxyl groups
were determined to be at C-1, C-2, C-3, C-5 and C-26, respec-
tively, on the basis of the 1H–1H COSY correlations from δ
4.31 (H-1) to δ 4.11 (H-2), H-2 to δ 4.67 (H-3) and H-3 to δ
2.50 and 2.20 (H-4), and the long-range correlations observed
in the HMBC spectrum (Fig. 2). In addition, the HMBC cor-
relation of the anomeric proton at δ 4.94 with δ 77.5 (C-26)
revealed that the glucopyranosyl unit was linked to the C-26
OH of the aglycone.
Fig. 2. The important 1H–13C long-range correlations in the HMBC spec-
trum of 3.
NOE between H-1 and H-19 (δ 1.55, s) assigned H-1 as
␤-orientation. Since only the A/B trans fusion and the 3-
␣ OH can satisfy the above properties, H-3 was assigned
to have an ␤-orientation and 5-OH an ␣-orientation. The ␣-
orientation of H-2 was concluded by the J values observed
from the JRES spectrum (H-1: δ 4.31, dd, J = 1.8, 3.8 Hz;
H-2: δ 4.11, dd, J = 2.9, 3.8 Hz; H-3: δ 4.67, td, J = 2.9,
3.8 Hz; H-4: δ 2.20, dd, J = 2.9, 14.9 Hz and δ 2.50, dd,
J = 3.8, 14.9 Hz), which also confirmed the above conclu-
sion. Moreover, the chemical shift of the C-27 (δ 24.4) and
the other carbons in the F-ring (Table 1) suggested that 3 be-
longed to the 25S series [22]. Therefore, 3 was deduced to
be (22S,25S)-1␣,2␤,3␣,5␣-tetrahydroxy-furospirostane-26-
yl O-␤-d-glucopyranoside, and named tupistroside C (3).
Compounds 4–6 were obtained as white amorphous pow-
ders and showed a positive reaction to the Ehrlich reagent.
The DEPT spectrum showed a characteristic quaternary car-
bon signal around δ 111. Acid hydrolysis of 4–6 yielded
d-glucose as the sugar residue. These observations sug-
gested that 4–6 were 26-O-d-glucopyranosyl furostanols.
Their molecular formulae were assigned to be C33H52O10,
C39H64O15 and C34H54O15, respectively, on the basis of the
negative-ion HRFABMS.
1
The H NMR spectrum of 4 showed one anomeric pro-
ton signal at δ 4.87 (d, J = 7.4 Hz), indicating the presence of
one glucopyranosyl unit in the molecule. Comparing the 13
C
NMR spectral data of 4 with those of epiruscogenin [25(R)-
5-en-spirost-1␤, 3␣-diol] suggested that the chemical shifts
due to the A–D rings were in good agreement. Significant dif-
ferences were observed for their F-ring. The olefinic carbons
signals at δ 147.3 (q) and 110.4 (d) in the 13C NMR spectrum
were assigned to C-25 and C-27, which was confirmed by the
long range correlations of δ 4.20 (H-26) with δ 147.3 (C-25),
δ 5.03 and 5.32 (H-27) with δ 72.2 (C-26), 28.5 (C-24) and
C-25 in the HMBC spectrum. Moreover, the HMBC corre-
lations of the anomeric proton signal at δ 4.87 with δ 72.2
(C-26) indicated that the glucopyranosyl unit was attached at
C-26 of the aglycone. Thus, tupistroside D (4) was deduced
to be 1␤,3␣,22 ξ-trihydroxy- furost-5,25(27)-dien-26-yl O-
␤-d-glucopyranoside.
The configurations of C-1, C-2, C-3 and C-5 were iden-
tified through the following steps. The observation of the
small correlation (J = 1.8 Hz) between H-1 and H-3 sug-
gested that H-1 and H-3 were in a W-coupling system. The
Compound 5 exhibited two anomeric proton signals at δ
1
4.20 (d, J = 7.8Hz) and 4.24 (d, J = 7.5Hz) in the H NMR
spectrum, suggesting that two sugar units existed in the

Q.-X. Yang et al. / Steroids 70 (2005) 732–737
737
molecule. Acid hydrolysis of 5 afforded epiruscogenin (5a)
and d-glucose. The sugar linkage position of 5 was deter-
mined by the HMBC spectrum, in which correlations of
one anomeric proton signal at δ 4.20 with δ 74.9 (C-3),
and another anomeric proton at δ 4.24 with δ 76.0 (C-26)
were observed. Thus, tupistroside E (5) was determined to
be 26-O-␤-d-glucopyranosyl-1␤,22-dihydroxy-furost-5-en-
3␣-yl O-␤-d-glucopyranoside.
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from Tupistra wattii. Acta Botan Yunnan 1995;17:341–4.
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Compound 6 showed only one anomeric proton signal at
δ 4.92 (d, J = 8.0 Hz) in the 1H NMR spectrum. The appear-
ance of a methoxyl signal around δ 47.4 in the 13C NMR
spectrum and the downfield shift of C-22 to δ 112 suggested
that the 22-OH was methylated. When treated with 2N HCl,
6 gave 1␤,2␤,3␤,4␤,5␤,7␣-hexahydroxy-spirost-25(27)-en-
6-one (6a) [22] as the aglycone, which were identified on the
basis of their spectroscopic evidence and by comparison with
literature values. Therefore, tupistroside F (6) were deduced
to be 22-methoxy-1␤,2␤,3␤,4␤,5␤,7␣-hexahydroxy-furost-
25(27)-en-6-one-26-yl O-␤-d-glucopyranoside.
In this work, 15 polyhydroxylated steroidal saponins and
sapogenins were obtained from the fresh rhizomes of Tupis-
tra yunnanensis among which 1–6 were newly isolated
molecules. It was known that genus Tupistra was close to
genera Convallaria, Aspidistra, Rohdea and Reineckea, and
all of them are in rich of steroidal saponins with the poly-
hydroxynated skeletons [24,29–31]. The results discussed in
this paper will give some chemical evidences for the chemo-
taxonomy of Liliaceae.
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Acknowledgements
The authors are grateful to members of the Analytical
Group in State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, for
measurements of all spectra.
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