Spirostanol and Furostanol Glycosides from the Fresh Tubers of Polianthes tuberosa

Article abstract of DOI:10.1021/np034028a

Six new steroid glycosides-two spirostanols, polianthosides B and C (1, 2), and four furostanols, polianthosides D-G (3-6)-were isolated from the fresh tubers of Polianthes tuberosa, together with seven known spirostanols (7-13) and a known furostanol (14) saponins. Their structures were elucidated on the basis of spectroscopic analysis and the results of acidic and enzymatic hydrolysis. The cytotoxic activities of 1-14 against HeLa cells are reported.

Full text of DOI:10.1021/np034028a

J . Nat. Prod. 2004, 67, 5-9
5
Full Papers
Sp ir osta n ol a n d F u r osta n ol Glycosid es fr om th e F r esh Tu ber s of P olia n th es
tu ber osa
J ian-Ming J in, Ying-J un Zhang,* and Chong-Ren Yang*
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, People’s Republic of China
Received September 24, 2003
Six new steroid glycosidesstwo spirostanols, polianthosides B and C (1, 2), and four furostanols,
polianthosides D-G (3-6)swere isolated from the fresh tubers of Polianthes tuberosa, together with
seven known spirostanols (7-13) and a known furostanol (14) saponins. Their structures were elucidated
on the basis of spectroscopic analysis and the results of acidic and enzymatic hydrolysis. The cytotoxic
activities of 1-14 against HeLa cells are reported.
The genus Polianthes, comprising about 12 species, is
native to Mexico. Polianthes tuberosa L. (Agavaceae), a
well-known ornamental plant, is widely cultivated in the
south of the People’s Republic of China. Its flowers are used
as high-class flavor and the tubers as a Chinese folk
medicine used for the treatment of acute infectious diseases
and pyrogenic inflammations.1 Several steroid sapogenins,
such as hecogenin, 9-dehydroxyhecogenin, and tigogenin,2
as well as glycosides, 29-hydroxystigmast-5-en-3â-yl â-D-
glucoside,3 (22S)-2â,3â,22-trihydroxycholest-5-en-16â-yl â-D-
glucoside,4 and diribofuranosylethyleneglycol,5 and spirostanol
pentaglycosides6 were identified from the underground
parts of P. tuberosa. Our search for bioactive saponins from
P. tuberosa has led to the isolation of six new steroid
glycosidesstwo spirostanols, polianthosides B and C (1, 2),
and four furostanols, polianthosides D-G (3-6)stogether
with eight known saponins (7-14) from the fresh tubers.
We describe herein the structure determination of 1-6 on
the basis of spectroscopic and chemical methods. The
cytotoxic activities of 1-14 against HeLa cells are also
reported.
Resu lts a n d Discu ssion
The fresh tubers of P. tuberosa (24 kg) were extracted
with 80% EtOH under reflux. The EtOH extract was
partitioned between n-butanol and H2O. The n-butanol
layer was concentrated and then chromatographed repeat-
edly over silica gel and RP-8 columns to give compounds
1-6 and eight known steroid saponins, identified as
hecogenin 3-O-â-D-glucopyranosyl-(1f2)-â-D-glucopyrano-
syl-(1f4)-â-D-galactopyranoside (7),7 hecogenin 3-O-â-D-
glucopyranosyl-(1f2)-[â-D-xyloyranosyl-(1f3)]-â-D-glucopy-
ranosyl-(1f4)-â-D-galactopyranoside (8),8 hecogenin 3-O-
â-D-xyloyranosyl-(1f3)-â-D-glucopyranosyl-(1f2)-[â-D-
xyloyr a n osyl-(1f3)]-â-D-glu copyr a n osyl-(1f4)-â-D-
[â-D-xyloyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-â-D-
galactopyranoside (12),9 chlorogenin 3-O-â-D-xyloyranosyl-
(1f3)-â-D-glucopyranosyl-(1f2)-[â-D-xyloyranosyl-(1f3)]-
â-D-glucopyranosyl-(1f4)-â-D-galactopyranoside (13),9 and
26-O-â-D-glucopyranosyl-(25R)-5R-furost-3â,22R,26-triol 3-O-
â-D-glucopyranosyl-(1f2)-[â-D-xylopyranosyl-(1f3)]-â-D-
glucopyranosyl-(1f4)-â-D-galactopyranoside (uttroside B)
(14),12 respectively.
galactopyranoside (9),9 agamenoside F (10),10 tigogenin
3-O-â-D-glucopyranosyl-(1f2)-[â-D-xyloyranosyl-(1f3)]-â-
D-glucopyranosyl-(1f4)-â-D-galactopyranoside (11),11 tigo-
genin 3-O-â-D-xyloyranosyl-(1f3)-â-D-glucopyranosyl-(1f2)-
* To whom correspondence should be addressed. Tel: +86-871-522-3235.
Fax: +86-871-515-0124. E-mail: zhangyj@mail.kib.ac.cn and cryang@
public.km.yn.cn.
10.1021/np034028a CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/12/2003

6
J ournal of Natural Products, 2004, Vol. 67, No. 1
J in et al.
Compound 1 was obtained as a white amorphous powder.
3 and 4 were similar to each other and to those of
terrestrosin I (26-O-â-D-glucopyranosyl-(25R,S)-5R-furost-
3â,22R,26-triol-12-one 3-O-â-D-galactopyranosyl-(1f2)-â-
D-glucopyranosyl-(1f4)-â-D-galactopyranoside),15 which was
isolated from Tribulus terrestris. In the 1H NMR spectrum,
3 showed the presence of five sugar units and 4 showed
six anomeric proton signals. These observations suggested
that 3 was a furostanol pentaglycoside, and 4, a hexa-
glycoside with the same aglycone as 3. The J values (>5
Hz) of the anomeric protons indicated the â-orientation for
each anomeric center of the sugar units.
Comparing the 13C NMR data of the sugar moieties of 3
with those of 14, it was indicated that 3 had the same sugar
moieties as 14; that is, a â-D-glucopyranosyl unit was
attached at C-26 of the aglycone and the same sugar chain
as 14 was linked to C-3 of the aglycone. This was further
confirmed by the three-bond 1H-13C long-range correla-
tions. In the HMBC spectrum of 3, correlations of δH 4.84
(H-Gal-1) with δC 77.8 (C-3 of aglycone), δH 5.14 (H-Glc-1)
with δC 80.0 (C-Gal-4), δH 5.51 (H-Glc′-1) with δC 81.4 (C-
Glc-2), and δH 5.18 (H-Xyl-1) with δC 87.0 (C-Glc-3), as well
as δH 4.77 (H-Glc26-1) with δC 75.3 (C-26 of aglycone), were
observed. Moreover, enzymatic hydrolysis of 3 with â-glu-
cosidase yielded hecogenin 3-O-â-D-glucopyranosyl-(1f2)-
[â-D-xyloyranosyl (1f3)]-â-D-glucopyranosyl-(1f4)-â-D-
galactopyranoside (8). On the basis of the above evidence,
3 was determined to be 26-O-â-D-glucopyranosyl-(25R)-5R-
furost-3â,22R,26-triol-12-one 3-O-â-D-glucopyranosyl-(1f2)-
[â-D-xylopyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-â-D-
galactopyranoside, named polianthoside D.
Its molecular formula was assigned as C56H92O27 on the
basis of the 13C NMR data and negative ion HRFABMS
([M - H]-, m/z 1195.5709). The negative ion FABMS also
showed fragment ion peaks at m/z 1033 [M - H -
162(hexosyl)]-, 901 [M -H -132(pentosyl)-162(hexosyl)]-,
739 [M -H -132(pentosyl)-162(hexosyl)-162(hexosyl)]-,
and 577 [M - H - 132(pentosyl) - 162(hexosyl) - 162-
(hexosyl) - 162(hexosyl)]-. The 1H NMR spectrum of 1
showed two three-proton singlet signals at δ 0.81 and 0.63
and two three-proton doublet signals at δ 1.13 (J ) 6.8 Hz)
and 0.69 (J ) 5.2 Hz), characteristic of the spirostanol
skeleton, as well as signals for five anomeric protons at δ
4.86, 5.10, 5.19, 5.55, and 5.08. Acid hydrolysis of 1 with 1
M HCl produced tigogenin, which was identified by normal-
and reversed-phase TLC comparison with an authentic
sample, and D-xylose, D-glucose, and D-galactose as sugar
residues determined by GC analysis. The 13C NMR data
of the sugar moiety were closely related to those of 10,
suggesting that both compounds had the same sugar
linkages. In the HMBC spectrum of 1, correlations of δH
4.86 (H-Gal-1) with δC 77.9 (C-3), δH 5.10 (H-Glc-1) with
δC 80.0 (C-Gal-4), δH 5.19 (H-Glc′′-1) with δC 88.3 (C-Glc-
3), δH 5.55 (H-Glc′-1) with δC 81.0 (C-Glc-2), and δH 5.08
(H-Xyl-1) with δC 87.1 (C-Glc′-3) were observed. In addition,
the IR spectrum showed absorptions at 982, 921, 898, and
865 cm-1, among which the band at 898 cm-1 was stronger
than the band at 921 cm-1, indicating the R configuration
at C-25.13 On the basis of the above evidence, 1 was
determined to be tigogenin 3-O-â-D-xylopyranosyl-(1f3)-
â-D-glucopyranosyl-(1f2)-[â-D-glucopyranosy-(1f3)]-â-D-
glucopyranosyl-(1f4)-â-D-galactopyranoside, named poli-
anthoside B.
The sequence of the sugars and binding sites at the
aglycone of 4 were determined by 2D NMR experiments.
The 13C chemical shifts due to each sugar unit were
assigned by the HMQC-TOCSY spectrum. In the HMBC
spectrum of 4, the following correlations were observed: δH
4.83 (H-Gal-1) with δC 77.8 (C-3 of aglycone), δH 5.14 (H-
Glc-1) with δC 79.8 (C-Gal-4), δH 5.53 (H-Glc′-1) with δC
80.8 (C-Glc-2), δH 5.11 (H-Xyl-1) with δC 86.8 (C-Glc-3), δH
5.05 (H-Xyl′-1) with δC 86.8 (C-Glc′-3), and δH 4.77 (H-Glc26-
1) with δC 75.2 (C-26 of aglycone). In addition, enzymatic
hydrolysis of 4 with â-glucosidase yielded hecogenin 3-O-
â-D-xyloyranosyl-(1f3)-â-D-glucopyranosyl-(1f2)-[â-D-
xyloyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-â-D-galacto-
pyranoside (9). Therefore, polianthoside E (4) was eluci-
dated as 26-O-â-D-glucopyranosyl-(25R)-5R-furost-3â,22R,-
26-triol-12-one3-O-â-D-xylopyranosyl-(1f3)-â-D-glucopyranosyl-
(1f2)-[â-D-xylopyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-
â-D-galactopyranoside.
The negative ion HRFABMS assigned the molecular
formulas of 5 and 6 as C61H102O32 and C62H104O33 (5: m/z
1345.6194 [M - H]-, 6: m/z 1375.6420 [M - H]-),
respectively. Acid hydrolysis of 5 and 6 gave D-glucose,
D-galactose, and D-xylose as sugar residues determined by
GC analysis, and tigogenin, which was confirmed by 13C
NMR data.14 The 13C chemical shifts of the aglycone of 5
and 6 were identical to those of 14 and terrestrosin H (26-
O-â-D-glucopyranosyl-(25R,S)-5R-furost-3â,22R,26-triol 3-O-
â-D-galactopyranosyl-(1f2)-â-D-glucopyranosyl-(1f4)-ga-
lactopyranoside) isolated from Tribulus terrestris.15 Com-
paring the 13C chemical shifts of the sugar moieties of 5
with those of 4 indicated that 5 had the same sequence of
sugar linkage as 4. The sugar sequence and linkage
position to the aglycone of 6 were determined by the HMBC
spectrum, which showed correlations of δH 4.86 (H-Gal-1)
with δC 77.8 (C-3 of aglycone), δH 5.11 (H-Glc-1) with δC
80.1 (C-Gal-4), δH 5.54 (H-Glc′-1) with δC 81.0 (C-Glc-2),
δH 5.08 (H-Glc′′-1) with δC 88.5 (C-Glc-3), δH 5.07 (H-Xyl-
The molecular formula of 2 was established as C57H94O28
on the basis of its negative ion HRFABMS ([M]- m/z
1226.5870). The negative ion FABMS also showed fragment
ion peaks at m/z 1063 [M - H - 162(hexosyl)]- and 901
[M - H - 162(hexosyl) - 162(hexosyl)]- and suggested a
hexosyl unit as the terminal sugar moiety. The 1H NMR
spectrum of 2 exhibited four characteristic methyl signals
for the spirostanol skeleton at δ 0.80, 0.62, 1.12, and 0.68,
as well as five anomeric proton signals. Acid hydrolysis of
2 yielded tigogenin as the aglycone by TLC comparison and
D-glucose and D-galactose as sugar residues by GC analysis.
The HMQC-TOCSY spectrum assigned the13C NMR
chemical shifts for each sugar unit. The HMBC, 1H-1H
COSY, and ROESY spectra determined the sugar linkage
sequence. In the HMBC spectrum, correlations of δH 4.86
(H-Gal-1) with δC 77.5 (C-3 of aglycone), δH 5.11 (H-Glc-1)
with δC 80.0 (C-Gal-4), δH 5.51 (H-Glc′-1) with δC 81.0 (C-
Glc-2), δH 5.19 (H-Glc′′-1) with δC 88.5 (C-Glc-3), and δH
5.13 (H-Glc′′′-1) with δC 87.5 (C-Glc′-3) were observed.
Therefore, polianthoside C (2) was characterized as tigo-
genin 3-O-â-D-glucopyranosyl-(1f3)-â-D-glucopyranosyl-
(1f2)-[â-D-glucopyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-
â-D-galactopyranoside.
Compounds 3-6 were obtained as white amorphous
powders. The DEPT spectrum showed a characteristic
quaternary carbon signal around δ 110. These observations
suggested that 3-6 were furostanol glycosides.
The molecular formulas of 3 and 4 were determined to
be C56H92O29 and C61H100O33, respectively, by HRFABMS.
Acid hydrolysis of 3 and 4 yielded hecogenin, which was
confirmed by direct comparison of the 13C chemical shifts
with those of the reference data,14 and D-glucose, D-
galactose, and D-xylose as sugar residues determined by
GC analysis. The 13C NMR data of the aglycone moiety of

Spirostanol Glycosides from Polianthes tuberosa
J ournal of Natural Products, 2004, Vol. 67, No. 1
7
Ta ble 1. 13C NMR Data of Compounds 1 and 2
was done by spraying the plates with 5% anisaldehyde-sulfric
acid, followed by heating.
aglycone
1
2
sugar
1
2
P la n t Ma ter ia l. The fresh tubers of P. tuberosa L. cv
Double were obtained from Kunming Qianhui Seed and
Seedling Limited Company during J une 2001.
1
2
3
4
5
6
7
8
37.4 (t)
30.6 (t)
77.9 (d)
35.0 (t)
44.8 (d)
29.1 (t)
32.6 (t)
35.4 (d)
54.6 (d)
36.0 (s)
21.5 (t)
40.3 (t)
41.0 (s)
56.6 (d)
32.3 (t)
81.3 (d)
63.2 (d)
16.8 (q)
12.5 (q)
42.5 (d)
15.2 (q)
37.2 (t) Gal 1
102.6(d)
73.2 (d)
75.5 (d)
80.0 (d)
75.6 (d)
60.9 (t)
102.5(d)
73.2 (d)
75.4 (d)
80.0 (d)
75.6 (d)
60.7 (t)
30.0 (t)
77.5 (d)
34.9 (t)
44.7 (d)
29.0 (t)
2
3
4
5
6
Extr a ct a n d Isola tion . The fresh tubers of P. tuberosa (24
kg) were extracted with hot 80% EtOH three times for 4 h,
and the combined extract was concentrated under reduced
pressure. Then concentrated extract was partitioned between
n-butanol and H2O. Half the n-butanol layer (450 g) was
chromatographed on silica gel with CHCl3-MeOH-H2O
(7:3:0.5) and gave four fractions (I-IV). Fraction 3 (60 g) was
subjected to silica gel (CHCl3-MeOH-H2O) and RP-8 (MeOH-
H2O) column chromatography to afford 7 (570 mg), 8 (4.9 g),
9 (420 mg), 10 (210 mg), and 11 (2 g). Fraction 4 (150 g) was
repeatedly chromatographed on silica gel with CHCl3-MeOH-
H2O and RP-8 with MeOH-H2O to yield 1 (70 mg), 2 (34 mg),
3 (740 mg), 4 (2.5 g), 5 (3.6 g), 6 (90 mg), 12 (482 mg), 13 (300
mg), and 14 (520 mg). Since the furostanol glycoside generally
exists as an equilibrium mixture of 22-methoxy and 22-
hydroxy forms with the presence of MeOH, furostanol glyco-
sides 3-6 and 14 afforded their 22-hydroxy forms after being
32.5 (t) Glc 1
104.8 (d) 104.8 (d)
35.3 (d)
54.5 (d)
35.9 (s)
21.3 (t)
40.2 (t)
2
3
4
5
6
81.0 (d)
88.3 (d)
70.9 (d)
77.6 (d)
63.2 (t)
81.0 (d)
88.5 (d)
70.8 (d)
77.5 (d)
63.1 (t)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
40.8 (s) Glc′ 1
104.1 (d) 104.1 (d)
56.5 (d)
32.2 (t)
80.2 (d)
63.1 (d)
16.7 (q)
2
3
4
5
6
75.2 (d)
87.1 (d)
69.2 (d)
78.2 (d)
62.2 (t)
106.1 (d)
75.5 (d)
77.7 (d)
70.9 (d)
67.2 (t)
74.8 (d)
87.5 (d)
69.3 (d)
78.0 (d)
62.1 (t)
12.4 (q) Xyl 1
42.1 (d)
15.1 (q)
2
3
4
5
refluxed with 70% aqueous acetone for 10 h.16
Com p ou n d 1: white amorphous powder; [R]D
109.5 (s) 109.3 (s)
18.3
-52.04°
32.0 (t)
29.4 (t)
30.8 (d)
67.1 (t)
17.5 (q)
31.9 (t)
(c 0.0221, pyridine); IR (KBr) νmax 3412, 2929, 1455, 1373, 1158,
1073, 982, 921, 898, 865 cm-1 (absorption: 898 > 921); 1H
NMR (pyridine-d5) δ 3.58 (1H, H-3), 4.46 (1H, q-like, J ) 8.3
Hz, H-16), 0.81 (3H, s, H-18), 0.63 (3H, s, H-19), 1.13 (3H, d,
J ) 6.8 Hz, H-21), 3.53, 3.50 (1H each, H-26), 0.69 (3H, d, J )
5.2 Hz, H-27), 4.86 (d, J ) 7.4 Hz, H-Gal-1), 5.10 (1H, d, J )
10.3 Hz, H-Glc-1), 5.55 (1H, br, H-Glc′-1), 5.19 (1H, d, J ) 7.8
Hz, H-Glc′′-1), 5.08 (1H, d, J ) 7.6 Hz, H-Xyl-1); 13C NMR
(pyridine-d5), see Table 1; FABMS (negative mode) m/z 1195
[M - H]-, 1033 [M - H - 162(hexosyl)]-, 901 [M - H - 132-
(pentosyl) - 162(hexosyl)]-, 739 [M - H - 132(pentosyl) -
162(hexosyl) - 162(hexosyl)]-, 577 [M - H - 132(pentosyl) -
162(hexosyl) - 162(hexosyl) - 162(hexosyl)]-; HRFABMS m/z
1195.5709 [M - H]- (calcd for C56H91O27, 1195.5748).
29.3 (t) Glc′′ 1
104.5 (d) 104.5 (d)
30.7 (d)
67.0 (t)
17.4 (q)
2
3
4
75.5 (d)
78.7 (d)
71.7 (d)
78.5 (d)
62.5 (t)
75.5 (d)
78.6 (d)
71.6 (d)
78.5 (d)
62.4 (t)
105.4 (d)
75.6 (d)
78.6 (d)
71.6 (d)
78.5 (d)
62.6 (t)
5
6
Glc′′′ 1
2
3
4
5
6
1) with δC 86.9 (C-Glc′-3), and δH 4.77 (H-Glc26-1) with δC
75.2 (C-26 of aglycone). Furthermore, enzymatic hydrolysis
of 5 and 6 with â-glucosidase yielded 12 and tigogenin 3-O-
â-D-xylopyranosyl-(1f3)-â-D-glucopyranosyl-(1f2)-[â-D-
glucopyranosyl-(1f3)]-â-D-glucopyranosyl-(1f4)-â-D-galac-
topyranoside, identified by its 1H and 13C NMR data,
respectively. On the basis of the above evidence, polian-
thosides F (5) and G (6) were deduced to be 26-O-â-D-
glucopyranosyl-(25R)-5R-furost-3â,22R,26-triol 3-O-â-D-
xylopyr a n osyl-(1f3)-â-D-glu copyr a n osyl-(1f2)-[â-D-
xylopyr a n osyl-(1f3)]-â-D-glu copyr a n osyl-(1f4)-â-D-
galactopyranoside, and 26-O-â-D-glucopyranosyl-(25R)-5R-
furost-3â,22R,26-triol 3-O-â-D-xylopyranosyl-(1f3)-â-D-
glucopyranosyl-(1f2)-[â-D-glucopyranosyl-(1f3)]-â-D-
glucopyranosyl-(1f4)-â-D-galactopyranoside, respectively.
Compounds 1-14 were tested for in vitro cytotoxicity
against HeLa cells. The IC50 values are listed in Table 3.
It is noticed that most of the saponins (3, 4, and 7-10)
with a carbonyl group at C-12 of the aglycone showed
stronger cytotoxicities (IC50 4.02-8.61 µg/mL) against HeLa
cells than saponins 1, 2, 5, and 14, with no carbonyl group
attached at the aglycone (IC50 > 18.83 µg/mL).
Acid Hyd r olysis of 1. Compound of 1 (2 mg) was refluxed
with 1 mol/L HCl-dioxane (1:1, v/v, 4 mL) on a water bath
for 6 h. The reaction mixture was evaporated to dryness. The
dry reaction mixture was partitioned between CHCl3 and H2O
four times. The CHCl3 extract was concentrated and identified
as tigogenin by normal- and reversed-phase TLC comparison
with authentic sample. The sugar residues were diluted in 5
mL of pyridine without water and treated with 0.5 mL of
trimethyl-chlorsilan (TMCS, Fluka) at room temperature for
30 min. The reaction mixture was evaporated to dryness under
reduced pressure. The mixture of trimethylsilylated deriva-
tives of the monosaccharides was diluted in 0.5 mL of ether
without water and then analyzed by GC. GC condition: AC-5
capillary column (30 m × 0.25 mm i.d.); detector FID (270 °C);
column temperature 180-260 °C, rate 5° C/min. tR (second):
692 (D-glucose), 653 (D-galactose), and 510 (D-xylose).
19.8
Com p ou n d 2: white amorphous powder; [R]D
-32.79°
(c 0.0183, pyridine); IR (KBr) νmax 3402, 2923, 1606, 1451, 1383,
1158, 1074, 983, 922, 899, 867 cm-1 (absorption: 899 > 922);
1H NMR (pyridine-d5) δ 3.58 (1H, H-3), 4.46 (1H, q-like, J )
10.2 Hz, H-16), 0.80 (3H, s, H-18), 0.62 (3H, s, H-19), 1.12 (3H,
d, J ) 6.9 Hz, H-21), 3.56, 3.48 (1H each, H-26), 0.68 (3H, d,
J ) 4.3 Hz, H-27), 4.86 (1H, d, J ) 7.3 Hz, H-Gal-1), 5.11 (1H,
d, J ) 7.7 Hz, H-Glc-1), 5.51 (1H, br, H-Glc′-1), 5.19 (1H, d, J
) 7.7 Hz, H-Glc′′-1), 5.13 (1H, d, J ) 9.8 Hz, H-Glc′′′-1); 13C
NMR (pyridine-d5), see Table 1; FABMS (negative mode) m/z
1225 [M - H]-, 1063 [M - H - 162(hexosyl)]-, 901 [M - H -
162(hexosyl) - 162(hexosyl)]-; HRFABMS m/z 1226.5870 [M
- H]- (calcd for C57H94O27, 1226.5932).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with a HORIBA SEPA-300 high-sensitive
polarimeter. IR (KBr) spectra were measured on a Bio-Rad
FTS-135 spectrophotometer. NMR spectra were recorded on
a Bruker DRX-500 instrument (500 MHz for 1H NMR, and
125 MHz for 13C NMR) at 25 °C, using TMS as an internal
standard. The negative ion and high-resolution FAB mass
spectra were recorded on a VG AutoSpec-3000 mass spectrom-
eter using glycerol as matrix. Precoated silica gel plates
(Qingdao Haiyang Chemical Co.) were used for TLC. Detection
Acid Hyd r olysis of 2. Compound of 2 (2 mg) was subjected
to acid hydrolysis as described for 1 to give tigogenin, by TLC
comparison with authentic sample, and D-glucose and D-
galactose as sugar moieties by GC analysis.
Com p ou n d 3: white amorphous powder; [R]D18.1 -23.21 (c
0.0474, pyridine); IR (KBr) νmax 3407, 2927,1704, 1373, 1160,
1071, 1040, 894 cm-1; 1H NMR (pyridine-d5) δ 3.77 (1H, H-3),

8
J ournal of Natural Products, 2004, Vol. 67, No. 1
J in et al.
Ta ble 2. 13C NMR Data of Compounds 3-6
aglycone
3
4
5
6
sugar
3
4
5
6
1
2
3
4
5
6
7
8
36.8 (t)
29.8 (t)
77.8 (d)
34.8 (t)
44.6 (d)
28.7 (t)
31.9 (t)
34.5 (d)
55.8 (d)
36.4 (s)
38.2 (t)
213.3 (s)
55.9 (s)
56.0 (d)
31.9 (t)
79.8 (d)
55.2 (d)
16.4 (q)
11.9 (q)
41.3 (d)
15.4 (q)
111.0 (s)
37.2 (t)
28.5 (t)
34.4 (d)
75.3 (t)
17.6 (q)
36.7 (t)
29.7 (t)
77.8 (d)
34.7 (t)
44.6 (d)
28.7 (t)
31.8 (t)
34.3 (d)
55.7 (d)
36.4 (s)
38.1 (t)
213.2 (s)
55.8 (s)
56.0 (d)
31.8 (t)
79.8 (d)
55.1 (d)
16.4 (q)
11.8 (q)
41.3 (d)
15.4 (q)
110.8 (s)
37.2 (t)
28.5 (t)
34.3 (d)
75.2 (t)
17.5 (q)
37.3 (t)
30.0 (t)
77.8 (d)
34.9 (t)
44.8 (d)
29.1 (t)
32.6 (t)
35.4 (d)
54.6 (d)
35.9 (s)
21.4 (t)
40.8 (t)
40.3 (s)
56.5 (d)
32.6 (t)
81.3 (d)
64.0 (d)
16.9 (q)
12.4 (q)
41.2 (d)
16.6 (q)
110.8 (s)
37.3 (t)
28.5 (t)
34.4 (d)
75.3 (t)
17.6 (q)
37.3 (t)
30.0 (t)
77.8 (d)
34.9 (t)
44.8 (d)
29.1 (t)
32.5 (t)
35.4 (d)
54.6 (d)
35.9 (s)
21.4 (t)
40.8 (t)
40.3 (s)
56.5 (d)
32.5 (t)
81.2 (d)
64.0 (d)
16.9 (q)
12.4 (q)
41.2 (d)
16.6 (q)
110.8 (s)
37.3 (t)
28.5 (t)
34.3 (d)
75.2 (t)
17.6 (q)
Gal 1
2
3
4
5
6
Glc 1
2
3
4
5
6
Glc′ 1
2
3
4
5
6
Xyl 1
2
3
4
102.5 (d)
73.3 (d)
75.6 (d)
80.0 (d)
75.4 (d)
60.8 (t)
105.2 (d)
81.4 (d)
87.0 (d)
70.6 (d)
77.6 (d)
63.1 (t)
104.9 (d)
76.2 (d)
77.8 (d)
71.1 (d)
78.6 (d)
62.5 (t)
105.0 (d)
75.2 (d)
78.6 (d)
70.8 (d)
67.4 (t)
102.5 (d)
73.2 (d)
75.2 (d)
79.8 (d)
75.2 (d)
60.8 (t)
105.0 (d)
80.8 (d)
86.8 (d)
70.5 (d)
77.6 (d)
63.0 (t)
104.0 (d)
75.2 (d)
86.8 (d)
69.2 (d)
78.5 (d)
62.2 (t)
105.0 (d)
75.2 (d)
78.6 (d)
70.8 (d)
67.2 (t)
106.2 (d)
75.4 (d)
77.3 (d)
70.8 (d)
67.2 (t)
102.6 (d)
73.2 (d)
75.2 (d)
79.8 (d)
75.3 (d)
60.8 (t)
104.9 (d)
80.8 (d)
86.9 (d)
70.5 (d)
77.6 (d)
63.0 (t)
104.0 (d)
75.2 (d)
86.9 (d)
69.2 (d)
78.5 (d)
62.2 (t)
104.9 (d)
75.3 (d)
78.6 (d)
70.8 (d)
67.3 (t)
106.2 (d)
75.5 (d)
77.6 (d)
70.8 (d)
67.2 (t)
102.5 (d)
73.2 (d)
75.2 (d)
80.1 (d)
75.5 (d)
60.8 (t)
104.9 (d)
81.0 (d)
88.5 (d)
70.8 (d)
77.6 (d)
62.9 (t)
104.0 (d)
75.2 (d)
86.9 (d)
69.2 (d)
78.5 (d)
62.2 (t)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
5
Xyl′ 1
2
3
4
106.1 (d)
75.2 (d)
77.6 (d)
70.8 (d)
67.2 (t)
104.6 (d)
75.5 (d)
78.6 (d)
71.8 (d)
78.5 (d)
62.5 (t)
104.9 (d)
75.5 (d)
78.6 (d)
71.7 (d)
78.5 (d)
62.9 (t)
5
Glc′′1
2
3
4
5
6
Glc26
1
105.0 (d)
75.5 (d)
78.5 (d)
71.8 (d)
78.6 (d)
62.9 (t)
105.0 (d)
75.4 (d)
78.6 (d)
71.8 (d)
78.6 (d)
62.9 (t)
104.9 (d)
75.5 (d)
78.6 (d)
71.8 (d)
78.5 (d)
62.9 (t)
2
3
4
5
6
Com p ou n d 4: white amorphous powder; [R]D18.1 -23.53 (c
0.0340, pyridine); IR (KBr) νmax 3435, 2928, 1704, 1424, 1375,
1161, 1075, 1040, 894 cm-1; 1H NMR (pyridine-d5) δ 3.77 (1H,
H-3), 4.52 (1H, m, H-16), 0.63 (3H, s, H-18), 1.11 (3H, s, H-19),
1.52 (3H, d, J ) 6.7 Hz, H-21), 4.21, 3.84, (1H each, m, H-26),
0.96 (3H, d, J ) 6.5 Hz, H-27), 4.83 (1H, d, J ) 7.0 Hz, H-Gal-
1), 5.14 (1H, d, J ) 7.0 Hz, H-Glc-1), 5.53 (1H, d, J ) 6.2 Hz,
H-Glc′-1), 5.05 (1H, d, J ) 7.0 Hz, H-Xyl′-1), 5.11 (1H, d, J )
7.7 Hz, H-Xyl-1), 4.77 (1H, d, J ) 7.7 Hz, H-Glc26-1); 13C NMR
(pyridine-d5), see Table 2; FABMS (negative mode) m/z 1360
[M]-, 1228 [M - 132(pentosyl)]-, 1066 [M - 132(pentosyl) -
162(hexosyl)]-, 934 [M - 132(pentosyl) - 162(hexosyl) -
132(pentosyl)]-, 771 [M - H - 132(pentosyl) - 162(hexosyl)
- 132(pentosyl) - 162(hexosyl)]-; HRFABMS m/z 1359.6039
[M - H]- (calcd for C61H99O33, 1359.6069).
Acid Hyd r olysis of 4. Compound 4 (206 mg) was subjected
to acid hydrolysis as described for 3 to afford hecogenin (163
mg), and D-glucose, D-galactose, and D-xylose as sugar moieties
by GC analysis.
En zym a tic Hyd r olysis of 4. Compound 4 (75 mg) was
subjected to enzymatic hydrolysis with â-glucosidase as de-
scribed for 3 to give 9 (18 mg).
Ta ble 3. Cytotoxic Activities of 1-14 against Hela Cells
IC50 IC50
compound (µg/mL) compound (µg/mL) compound (µg/mL)
IC50
1
2
3
4
5
>20
>20
6
7
8
9
5.36
8.61
8.17
4.02
5.10
11
12
13
14
3.54
7.20
7.50
18.83
0.75
7.86
5.21
20.00
10
cisplatin
4.47 (1H, m, H-16), 0.65 (3H, s, H-18), 1.11 (3H, s, H-19), 1.51
(3H, d, J ) 6.0 Hz, H-21), 4.21, 3.82, (1H each, m, H-26), 0.96
(3H, d, J ) 6.3 Hz, H-27), 4.84 (1H, d, J ) 6.6 Hz, H-Gal-1),
5.14 (1H, d, J ) 6.9 Hz, H-Glc-1), 5.51 (1H, d, J ) 6.3 Hz,
H-Glc′-1), 5.18 (1H, d, J ) 7.4 Hz, H-Xyl-1), 4.77 (1H, d, J )
7.7 Hz, H-Glc26-1); 13C NMR (pyridine-d5), see Table 2; FABMS
(negative mode) m/z 1228 [M]-, 1066 [M - 162(hexosyl)]-, 771
[M - H - 132(pentosyl) - 162(hexosyl) - 162(hexosyl)]-;
HRFABMS m/z 1227.5735 [M - H]- (calcd for C56H91O29
1227.5646).
,
Acid Hyd r olysis of 3. Compound 3 (35 mg) was refluxed
with 1 mol/L HCl-dioxane (1:1, v/v, 4 mL) on a water bath
for 6 h. The reaction mixture was evaporated to dryness. The
dry reaction mixture was partitioned between CHCl3 and H2O
four times. The CHCl3 extract was concentrated and chro-
matographed on silica gel to give hecogenin (23 mg), identified
by 13C NMR comparison with the reference data, and D-glucose,
D-galactose, and D-xylose as sugar moieties by GC analysis.
En zym a tic Hyd r olysis of 3. Compound 3 (28 mg) and
â-glucosidase (Sigma) in a NaOAc-HOAc buffer (pH 5.0) were
incubated at 25 °C for 16 h. The resulting precipitate was
treated with n-butanol. The n-butanol extract was purified on
silica gel to give 8 (6 mg).
Com p ou n d 5: white amorphous powder; [R]D19.8 -37.18 (c
0.0390, pyridine); IR (KBr) νmax 3413, 2927, 1699, 1373, 1161,
1074, 1040, 894 cm-1; 1H NMR (pyridine-d5) δ 3.77 (1H, H-3),
4.94 (1H, q-like, J ) 7.3 Hz, H-16), 0.86 (3H, s, H-18), 0.61
(3H, s, H-19), 1.32 (3H, d, J ) 6.9 Hz, H-21), 4.38, 3.93 (1H
each, m, H-26), 0.96 (3H, d, J ) 6.8 Hz, H-27), 4.91 (1H, d, J
) 6.9 Hz, H-Gal-1), 5.17 (1H, d, J ) 7.7 Hz, H-Glc-1), 5.56
(1H, d, J ) 7.7 Hz, H-Glc′-1), 5.08 (1H, d, J ) 7.3 Hz, H-Xyl′-
1), 5.14 (1H, d, J ) 7.7 Hz, H-Xyl-1), 4.87 (1H, d, J ) 7.7 Hz,
H-Glc26-1); 13C NMR (pyridine-d5), see Table 2; FABMS (nega-
tive mode) m/z 1346 [M]-, 1214 [M - 132(pentosyl)]-, 1051

Spirostanol Glycosides from Polianthes tuberosa
J ournal of Natural Products, 2004, Vol. 67, No. 1
9
[M - H - 132(pentosyl) - 162(hexosyl)]-, 919 [M - H - 132-
(pentosyl) - 162(hexosyl) - 132(pentosyl)]-, 757 [M - H -
132(pentosyl) - 162(hexosyl) - 132(pentosyl) - 162 (hexosyl)]-;
The cells were further incubated for 72 h, and then cell growth
was evaluated by the MTT method. The OD value was read
on a plate reader at a wavelength of 570 nm. The cytotoxicity
was expressed as IC50 value (µg/mL), which was the mean of
three determinations and reduced the viable cell number by
50%.
HRFABMS m/z 1345.6194 [M - H]- (calcd for C61H101O32
,
1345.6276).
Acid Hyd r olysis of 5. Compound 5 (100 mg) was subjected
to acid hydrolysis as described for 3 to afford tigogenin (72
mg), D-glucose, D-galactose, and D-xylose.
Ack n ow led gm en t. The authors are grateful to members
of the Analytical Group in State Key Laboratory of Phy-
tochemistry and Plant Resources in West China, Kunming
Institute of Botany, for measurements of all spectra. We are
also grateful to Professor Xingcong Li (University of Missis-
sippi) for his help.
En zym a tic Hyd r olysis of 5. Compound 5 (86 mg) was
subjected to enzymatic hydrolysis with â-glucosidase as de-
scribed for 3 to give 12 (20 mg).
Com p ou n d 6: white amorphous powder; [R]D19.7 -35.26 (c
0.0390, pyridine); IR (KBr) νmax 3412, 2927, 1704, 1649, 1453,
1
1425, 1376, 1160, 1075, 1040, 894 cm-1; H NMR (pyridine-
d5) δ 3.78 (1H, H-3), 4.92 (1H, m, H-16), 0.86 (3H, s, H-18),
0.63 (3H, s, H-19), 1.31 (3H, d, J ) 6.3 Hz, H-21), 4.34, 3.81
(1H each, m, H-26), 0.96 (3H, d, J ) 6.6 Hz, H-27), 4.86 (1H,
d, J ) 4.4 Hz, H-Gal-1), 5.11 (1H, d, J ) 5.5 Hz, H-Glc-1),
5.54 (1H, H-Glc′-1), 5.18 (1H, d, J ) 7.7 Hz, H-Glc′′-1), 5.07
(1H, H-Xyl-1), 4.78 (1H, d, J ) 7.7 Hz, H-Glc26-1); 13C NMR
(pyridine-d5), see Table 2; FABMS (negative mode) m/z 1376
[M]-, 1214 [M - 162(hexosyl)]-, 1082 [M - 132(pentosyl) -
162(hexosyl)]-, 757 [M - H - 132(pentosyl) - 162(hexosyl) -
162(hexosyl) - 162(hexosyl)]-; HRFABMS m/z 1375.6420 [M
- H]- (calcd for C62H103O33, 1375.6381).
Acid Hyd r olysis of 6. Compound 6 (32 mg) was subjected
to acid hydrolysis as described for 3 to afford tigogenin (22
mg), and D-glucose, D-galactose, and D-xylose.
En zym a tic Hyd r olysis of 6. Compound 6 (21 mg) was
subjected to enzymatic hydrolysis with â-glucosidase as de-
scribed for 3 to gave tigogenin 3-O-â-D-xylopyranosyl-(1f3)-
â-D-glucopyranosyl-(1f2)-[â-D-glucopyranosyl-(1f3)]-â-D-glu-
copyranosyl-(1f4)-â-D-galactopyranoside (4 mg), identified by
1H and 13C NMR data.
Cytotoxicity a ga in st HeLa Cells. The cytotoxicity against
HeLa cells was determined using the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric
assay on 96-well microplates.17 Briefly, 196 µL of treated HeLa
cell suspension (2 × 104 cells/mL in the RPMI 1640 medium)
was placed in each well of a 96-well flat-bottom plate and
incubated in 5% CO2/air for 12 h at 37 °C. After incubation, 4
µL of DMSO solution containing the sample was added to give
the final concentration of the samples (25, 20, 15, 10, 5, 2.5,
1.25, 0.80 µg/mL); 4 µL of DMSO was added into control wells.
Refer en ces a n d Notes
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(11) Wang, Y.; Kazuhiro, O.; Ryoji, K.; Yamasaki, K. Phytochemistry 1996,
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NP034028A