New glycosides of the campesterol derivative from the rhizomes of Tacca chantrieri

Article abstract of DOI:10.1016/j.steroids.2004.11.006

Seven new glycosides of the campesterol derivative (24R,25S)-ergost-5-ene- 3β,26-diol (1-7) were isolated from the rhizomes of Tacca chantrieri (Taccaceae). Their structures were determined by extensive spectroscopic analysis, including 2D NMR data, and a few chemical transformations.

Full text of DOI:10.1016/j.steroids.2004.11.006

Steroids 70 (2005) 257–265
New glycosides of the campesterol derivative from
the rhizomes of Tacca chantrieri
∗
Akihito Yokosuka, Yoshihiro Mimaki ,
Chiseko Sakuma, Yutaka Sashida
Tokyo University of Pharmacy and Life Science, School of Pharmacy,
1432-1, Horinouchi, Hachiouji, Tokyo 192-0392, Japan
Received 20 July 2004; received in revised form 19 October 2004; accepted 4 November 2004
Abstract
Seven new glycosides of the campesterol derivative (24R,25S)-ergost-5-ene-3␤,26-diol (1–7) were isolated from the rhizomes of Tacca
chantrieri (Taccaceae). Their structures were determined by extensive spectroscopic analysis, including 2D NMR data, and a few chemical
transformations.
©
2005 Elsevier Inc. All rights reserved.
Keywords: Taccaceae; Tacca chantrieri; Steroid glycosides; Campesterol glycosides
1
. Introduction
sides of the campesterol derivative (24R,25S)-ergost-5-ene-
␤,26-diol. This paper deals with the structural determination
3
The genus Tacca (Taccaceae) with about 10 species is
of the new compounds by extensive spectroscopic studies,
including 2D NMR data, and a few chemical transforma-
tions.
mainly distributed in the tropical region of Asia, the Pa-
cific Islands, and Australia [1]. Tacca chantrieri Andr e´ is
a perennial plant that grows in southeastern China. Its rhi-
zomes have been used in Chinese folk medicine for the
treatment of gastric ulcer, enteritis, and hepatitis [2]. Pre-
viously, we reported the isolation and structural characteri-
zation of diarylheptanoids, diarylheptanoid glucosides, and
steroidal glycosides, such as spirostan, furostan, pseudo-
furostan, pregnane glycosides, and withanolide glucosides
from the rhizomes of T. chantrieri, as well as their cyto-
toxic activities against cultured tumor and normal cells [3–7].
Further phytochemical analysis of the MeOH extract of T.
chantrieri rhizomes led to the isolation of seven new glyco-
2. Experimental
2.1. General methods
Optical rotation was measured using a JASCO DIP-360
automatic digital polarimeter. IR spectra were recorded on a
JASCO FT-IR 620 spectrophotometer. NMR spectra were
recorded on a Bruker DRX-500 spectrometer (500 MHz
1
for H NMR) or on a Varian Mercury 300 spectrome-
1
ter (300 MHz for H NMR). Chemical shifts are given
∗
as δ values with reference to tetramethylsilane (TMS) as
an internal standard. ESITOFMS were recorded on a Mi-
cromass LCT mass spectrometer. EI-MS was recorded on
Corresponding author. Tel.: +81 426 76 4577;
fax: +81 426 76 4579.
E-mail address: mimakiy@ps.toyaku.ac.jp (Y. Mimaki).
0
039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.11.006

2
58
A. Yokosuka et al. / Steroids 70 (2005) 257–265
a Finnigan MAT TSQ-700 mass spectrometer. Elemental
analysis was carried out using an Elementar Vario EL el-
emental analyzer. Silica gel (200–400 mesh, Fuji-Silysia
Chemical, Aichi, Japan), octadecylsilanized (ODS) silica gel
(75 ␮m, Nacalai Tesque, Kyoto, Japan), and Diaion HP-
2
0 (Mitsubishi-Chemical, Tokyo, Japan) were used for col-
umn chromatography. TLC was carried out on precoated
Kieselgel 60 F254 (0.25 mm thick, Merck, Darmstadt, Ger-
many) and RP-18 F254S (0.25 mm thick, Merck) plates,
and spots were visualized by spraying the plates with
1
0% H2SO4 solution, followed by heating. HPLC was per-
formed using a system composed of a CCPM pump (Tosoh,
Tokyo, Japan), a CCP PX-8010 controller (Tosoh), a RI-
8
010 (Tosoh) or a Shodex OR-2 (Showa-Denko, Tokyo,
Japan) detector, and a Rheodyne injection port. A Cap-
cell Pak C18 UG80 column (10 mm i.d. × 250 mm, 5 ␮m,
Shiseido, Tokyo, Japan) was employed for preparative
HPLC.
2
.2. Plant material
The rhizomes of T. chantrieri were collected from
the Yunnan Province, People’s Republic of China,
in October 1996 and identified by one of the au-
thors (Y.S.). A voucher specimen has been deposited
in our laboratory (Voucher no. TC-96-003, Laboratory
of Medicinal Pharmacognosy; formerly Medicinal Plant
Science).
2
.3. Extraction and isolation
The plant material (dry weight, 7.3 kg) was extracted with
MeOH under reflux (3 L, 2×). The MeOH extract (630 g)
was passed through a Diaion HP-20 (2.2 kg) column, sequen-
tially eluted with 30% MeOH, 50% MeOH, MeOH, EtOH,
and EtOAc (4 L of each). The MeOH eluate portion (115 g)
was chromatographed on a silica gel column, eluted with a
stepwise gradient mixture of CHCl3:MeOH (9:1, 4:1, 3:1,
2
:1, and 1:1; 4 L of each), and finally, with MeOH (4 L).
The CHCl3:MeOH (4:1) eluate portion (40 g) was subjected
to column chromatography on ODS silica gel, eluted with
MeOH:H2O (1:2), and silica gel with CHCl3:MeOH:H2O
(
(
20:10:1), to give 2 (200 mg), 3 (7.6 mg), 4 (32 mg), 5
8.0 mg), 6 (15 mg), and 7 (9.0 mg) in pure form, and 1 and
2
with a few impurities.

A. Yokosuka et al. / Steroids 70 (2005) 257–265
259
Purification of 1 and 2 was established by preparative
HPLC using MeCN:H2O (2:5) to yield 1 (39 mg) and 2
16 mg).
Table 1
1
H and 13C NMR spectral data for the sugar moieties of 1 in C5D5N at 303 K
1H
J (Hz)
13C
(
Position
ꢀ
ꢀ
ꢀ
ꢀ
1
2
3
4
5.06 d
7.9
102.6
75.4
78.6
71.7
78.4
62.9
2
.4. Compound 1
4.06 dd
4.31 dd
4.28 dd
4.00 m
4.57 br d
4.42 dd
8.5, 7.9
8.5, 8.1
8.1, 8.0
Amorphous solid: [␣]28 −20.0 (MeOH; c 0.10). ESI-
◦
D
5ꢀ
+
TOFMS m/z: 1573 [M + Na] . (Found: C, 50.41; H, 7.92;
ꢀ
6
a
b
12.0
12.0, 3.9
calc. for C70H118O37·6H2O: C, 50.65; H, 7.89). IR νmax
−1
1
(
film) cm : 3425 (OH), 2932 (CH), 1070, 1029. H NMR
ꢀꢀ
ꢀꢀ
1
2
3
4
5
6
4.83 d
7.8
102.9
84.6
77.9
71.3
76.8
70.0
(
C D N): δ 5.36 (1H, br d, J = 5.5 Hz, H-6), 3.98 (1H, m, H-
5
5
4.05 dd
4.27 dd
4.12 dd
4.04 m
4.80 br d
4.25 br d
9.1, 7.8
9.1, 9.1
9.1, 8.6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
), 0.98 (3H, d, J = 6.7 Hz, Me-27), 0.96 (3H, d, J = 6.5 Hz,
Me-21), 0.95 (3H, s, Me-19), 0.81 (3H, d, J = 6.7 Hz, Me-28),
1
0
.66 (3H, s, Me-18). H NMR signals for the sugar moieties:
a
b
11.4
11.4
13
Table 1. C NMR: Tables 2 and 3.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
1
2
3
4
5
6
5.20 d
8.1
106.3
76.4
76.2
80.9
76.7
62.0
2
.5. Acid hydrolysis of 1
4.06 dd
4.22 dd
4.36 dd
3.89 m
4.61 br d
4.43 br d
9.1, 8.1
9.2, 9.1
9.2, 8.3
A solution of 1 (38 mg) in 1 M HCl (dioxane:H2O, 1:1;
◦
2
mL)washeatedat95 Cfor1 hunderanAratmosphere. Af-
ꢀꢀ
a
b
11.6
11.6
ter cooling, the reaction mixture was neutralized by passage
through an Amberlite IRA-93ZU (Organo, Tokyo, Japan)
column and chromatographed on Diaion HP-20, eluted with
H2O:MeOH (3:2) followed by Me2CO:EtOH (1:1), to yield
a sugar fraction (14 mg) and an aglycone fraction. The agly-
cone fraction was chromatographed on silica gel, eluted
with CHCl3:MeOH (19:1), to give (24R,25S)-ergost-5-ene-
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
1
2
3
4
5
6
5.16 d
8.2
104.5
74.2
76.5
80.8
76.4
62.0
4.09 dd
4.21 dd
4.26 dd
3.95 m
4.48 br d
4.30 dd
8.4, 8.2
9.1, 8.4
9.1, 8.2
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀ
ꢀꢀꢀ
a
b
12.7
12.7, 3.9
3
␤,26-diol (1a, 6.0 mg). The sugar fraction was dissolved
in H2O (1 mL) and passed through a Sep-Pak C18 cartridge
Waters, Milford, MA, USA), which was then analyzed by
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
1
2
3
4
5
6
5.13 d
8.2
104.9
74.7
78.2
71.5
78.4
62.4
(
4.06 dd
4.19 dd
4.17 dd
3.99 m
4.52 br d
4.28 br d
8.7, 8.2
9.1, 8.7
9.1, 8.0
HPLC under the following conditions: column, Capcell Pak
NH2 SG80 (4.6 mm i.d. × 250 mm, 5 ␮m, Shiseido); solvent,
MeCN:H2O (17:3); flow rate, 0.8 mL/min; detection, RI and
OR. Identification of d-glucose present in the sugar fraction
was carried out by comparison of its retention time and opti-
cal rotation with those of an authentic sample; tR (min) 12.00
ꢀꢀꢀꢀꢀ
a
b
11.1
11.1
ꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
2
3
4
5
6
5.01 d
8.0
105.1
74.7
76.6
81.0
76.7
61.7
4.03 dd
4.23 dd
4.33 dd
3.85 m
4.53 br d
4.43 br d
8.8, 8.0
8.9, 8.8
8.9, 8.5
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
(d-glucose, positive optical rotation).
2
.6. Compound 1a
a
b
11.2
11.2
Amorphous solid: [␣]2 −32.0 (CHCl3; c 0.10). EIMS
8
D
◦
ꢀꢀꢀꢀꢀꢀꢀ
ꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀ
ꢀ
1
2
3
4
5
6
5.17 d
8.1
104.9
74.7
78.2
71.5
78.4
62.4
+
+
m/z: 416 [M] . HRESITOFMS m/z: 399.3608 [M + H–H2O]
ꢀꢀꢀꢀꢀꢀ
3.92 dd
4.21 dd
4.19 dd
4.00 m
4.52 br d
4.30 dd
8.4, 8.1
8.6, 8.4
8.6, 8.6
−
1
(C28H47O requires 399.3627). IR νmax (film) cm : 3253
1
(OH), 2927 (CH), 1454, 1378, 1029. H NMR (C D N):
5
5
ꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀ
δ 5.44 (1H, br d, J = 4.8 Hz, H-6), 3.85 (1H, m, H-3), 1.16
3H, d, J = 6.8 Hz, Me-27), 1.07 (3H, s, Me-19), 0.99 (3H,
d, J = 6.5 Hz, Me-21), 0.92 (3H, d, J = 6.8 Hz, Me-28), 0.68
3H, s, Me-18). 13C NMR: Table 2.
a
b
10.3
10.3, 4.4
(
(
solution of 1a (1.6 mg) in CH2Cl2 (1.0 mL), and the mixture
was stirred at room temperature for 12 h. The reaction mix-
ture was poured into ice–water and the whole mixture was ex-
tracted with AcOEt (10 mL, 3×). Evaporation of the solution
followed by silica gel column chromatography, eluted with
hexane:AcOEt (4:1), gave 1a-R (1.0 mg). Using 1a (1.5 mg)
and (S)-MTPA (7.5 mg), the same procedures yielded 1a-S
2
.7. Preparation of (R)-MTPA ester (1a-R) and
(S)-MTPA ester (1a-S) of 1a
(R)-2-Methoxy-2-trifluoromethylphenylacetic acid [(R)-
MTPA, 8.1 mg], 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (EDC·HCl, 6.5 mg), and 4-
(1.0 mg).
(dimethylamino)pyridine (4-DMAP, 2.0 mg) were added to a

2
60
A. Yokosuka et al. / Steroids 70 (2005) 257–265
Table 2
Table 3
1
3C NMR spectral data for the aglycone moiety of compounds 1, 1a, and
–7 in C5D5N
13
C NMR spectral data for the sugar moieties for compounds 2–7 in C5D5N
2
Position
2
3
4
5
6
7
Position
1
1a
37.9
32.6
71.3
43.5
2
3
4
5
6
7
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
2
3
4
5
6
102.5
75.3
78.4
71.7
78.6
62.8
–
–
–
–
–
–
102.5
75.3
78.5
71.6
78.6
62.8
102.6
75.3
78.5
71.7
78.6
62.8
102.6
75.3
78.6
71.7
78.6
62.8
102.6
75.3
78.6
71.7
78.5
62.9
1
2
3
4
5
6
7
8
9
37.5
30.3
78.1
39.4
37.5
30.2
78.2
39.3
37.8
29.9
71.3
43.5
37.5
30.2
78.2
39.3
37.5
30.2
78.2
39.3
37.4
30.2
78.2
39.3
37.5
30.3
78.3
39.3
140.9 142.0 140.9 141.9 140.9 140.9 140.9 140.9
122.0 121.2 121.9 121.3 121.9 122.0 121.9 121.9
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
2
3
4
5
6
102.9
84.5
77.8
71.2
76.8
70.0
102.9
84.5
77.9
71.2
76.8
70.0
102.9
84.5
77.9
71.2
76.9
69.9
103.1
84.0
77.8
71.3
76.8
70.0
104.3
73.9
88.5
69.7
76.7
69.7
103.1
84.8
78.1
71.5
78.1
62.6
32.1
32.1
50.4
36.9
21.3
40.0
42.5
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.4
34.5
37.9
73.9
12.0
14.8
32.2
32.2
50.5
36.9
21.4
40.0
42.5
56.9
24.5
28.5
56.3
12.0
19.6
36.2
19.0
34.1
31.8
34.1
40.8
66.0
12.2
14.7
32.1
32.0
50.3
36.9
21.3
39.9
42.4
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.4
34.5
37.8
73.9
12.0
14.7
32.2
32.2
50.5
36.9
21.4
40.0
42.5
56.9
24.5
28.5
56.3
12.0
19.6
36.1
19.0
34.1
31.4
34.5
37.9
74.0
12.0
14.7
32.2
32.0
50.3
36.9
21.3
39.9
42.4
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.4
34.5
37.8
73.9
12.0
14.7
32.2
32.0
50.3
36.9
21.3
39.9
42.4
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.5
34.4
37.8
74.0
11.9
14.6
32.2
32.0
50.3
36.9
21.3
39.9
42.4
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.6
34.1
37.7
73.9
11.7
14.4
32.2
32.1
50.4
36.9
21.3
39.9
42.5
56.8
24.5
28.5
56.2
12.0
19.4
36.1
19.0
34.1
31.4
34.6
38.0
74.0
12.2
14.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
1
2
3
4
5
6
106.2
76.3
76.2
81.3
76.7
62.1
106.2
76.3
76.3
81.3
76.7
62.1
106.2
76.3
76.3
81.5
76.7
62.1
106.5
76.9
78.0
71.4
78.4
62.6
105.9
75.5
78.1
71.4
78.4
62.5
106.3
76.4
76.3
81.3
76.7
62.1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
1
2
3
4
5
6
105.0
74.7
78.1
71.5
78.4
62.4
105.1
74.7
78.2
71.5
78.4
62.4
105.0
74.7
78.4
71.5
78.4
62.4
105.1
74.7
76.6
81.0
76.5
62.0
105.1
74.7
76.5
81.0
76.6
61.9
105.0
74.7
78.2
71.4
78.5
62.6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
1
2
3
4
5
6
105.0
74.7
76.6
81.0
76.5
61.9
105.1
74.7
76.6
81.0
76.5
61.9
105.4
75.2
78.1
71.7
78.4
62.7
104.9
74.7
78.1
71.4
78.6
62.4
104.9
74.7
78.1
71.6
78.4
62.4
2
.8. Compound 1a-R
ꢀꢀꢀꢀꢀꢀ
1
2
3
4
5
6
104.9
74.7
78.1
71.5
78.4
62.4
104.9
74.7
78.2
71.5
78.4
62.4
ꢀ
ꢀꢀꢀꢀꢀ
Amorphous solid: 1H NMR (300 MHz, CDCl3): δ
.39–7.53 (5H, m, Ph-H), 5.35 (1H, br d, J = 5.1 Hz, H-6),
.20 (2H, d-like, J = 6.3 Hz, H2-26), 3.55 (3H, s, OMe), 3.52
ꢀꢀꢀꢀꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
7
4
(
0
1H, m, H-3), 1.01 (3H, s, Me-19), 0.89 (3H, d, J = 6.3 Hz),
.83 (3H, d, J = 6.9 Hz), 0.77 (3H, d, J = 6.9 Hz), 0.66 (3H, s,
Me-18).
The Et2O extract was chromatographed on silica gel, eluted
with hexane:AcOEt (4:1), to give 1a-T (1.5 mg).
2
.9. Compound 1a-S
2.11. Compound 1a-T
Amorphous solid: 1H NMR (300 MHz, CDCl3): δ
.39–7.53 (5H, m, Ph-H), 5.35 (1H, br d, J = 5.4 Hz, H-
), 4.30 (1H, dd, J = 10.8, 6.6 Hz, H-26a), 4.09 (1H, dd,
J = 10.8, 7.2 Hz, H-26b), 3.55 (3H, s, OMe), 3.53 (1H, m,
H-3), 1.01 (3H, s, Me-19), 0.89 (3H, d, J = 6.3 Hz), 0.82
1
Amorphous solid: H NMR (500 MHz, CDCl3): δ 7.79
5H, m, Ph-H), 5.35 (1H, br d, J = 5.1 Hz, H-6), 4.20 (2H,
7
6
(
dd, J = 6.3, 2.4 Hz, H2-26), 3.55 (3H, s, OMe), 3.52 (1H, m,
H-3), 1.01 (3H, s, Me-19), 0.89 (3H, d, J = 6.3 Hz), 0.83 (3H,
d, J = 6.9 Hz), 0.77 (3H, d, J = 6.9 Hz), 0.66 (3H, s, Me-18).
(
3H, d, J = 6.9 Hz), 0.77 (3H, d, J = 6.9 Hz), 0.66 (3H, s,
Me-18).
2.12. Conversion of 1a-T to campesterol
2
.10. Preparation of 26-O-tosylate (1a-T) of 1a
A solution of 1a-T (1.5 mg) was treated with LiAlH4
10 mg) in THF (0.3 mL) at 0 C for 5 h. The reaction mix-
◦
(
Compound 1a (2.4 mg) was dissolved in pyridine (0.3 mL)
ture was poured into 10% H2SO4 solution (1.0 mL) and then
extracted with Et2O (10 mL, 3×). The Et2O extract was chro-
matographed on silica gel, eluted with hexane:AcOEt (4:1),
to give campesterol (1.0 mg).
containing p-toluenesulfonyl chloride (p-TsCl, 4.7 mg) and
4
6
-DMAP (2.0 mg), and it was stirred at room temperature for
h. The reaction mixture was poured into water (3.0 mL),
and the whole mixture was extracted with Et2O (10 mL, 3×).

A. Yokosuka et al. / Steroids 70 (2005) 257–265
261
2
.13. Compound 2
2.18. Acid hydrolysis of 4
Amorphous solid: [␣]25 −34.0 (MeOH; c 0.10). ESI-
◦
D
A solution of 4 (1.9 mg) was subjected to acid hydroly-
sis as described for 1 to give 1a (0.4 mg) and a sugar frac-
tion (0.9 mg). HPLC analysis of the sugar fraction under the
same conditions as described for 1 showed the presence of
+
TOFMS m/z: 1411 [M + Na] . (Found: C, 53.46; H, 8.23.
Calc. for C H108O32·3H2O: C, 53.25; H, 7.96.) IR νmax
64
−1
1
(
(
film) cm : 3224 (OH), 2931 (CH), 1076, 1028. H NMR
C D N): δ 5.34 (1H, br d, J = 4.4 Hz, H-6), 5.23 (1H, d,
5
5
d-glucose: t (min) 11.83 (d-glucose, positive optical rota-
R
ꢀ
ꢀꢀ
ꢀꢀꢀꢀ
J = 7.8 Hz, H-1 ), 5.19 (1H, d, J = 8.1 Hz, H-1 ), 5.18
tion).
ꢀ
ꢀꢀꢀꢀꢀ ꢀ
), 5.06 (1H, d, J = 7.7 Hz, H-1 ),
(
1H, d, J = 8.1 Hz, H-1
ꢀꢀꢀꢀꢀ
.01 (1H, d, J = 7.8 Hz, H-1 ), 4.83 (1H, d, J = 7.8 Hz, H-
5
1
0
2.19. Compound 5
ꢀ
ꢀ
), 3.96 (1H, m, H-3), 0.96 (3H, d, J = 6.5 Hz, Me-27),
.95 (3H, d, J = 5.6 Hz, Me-21), 0.92 (3H, s, Me-19), 0.79
Amorphous solid: [␣]25 −34.0 (MeOH; c 0.10). HR-
◦
D
+
(
3H, d, J = 6.7 Hz, Me-28), 0.64 (3H, s, Me-18). 13C NMR:
ESITOFMS m/z: 1227.6464 [M + H] (C H99O27 requires
227.6374). IR νmax (film) cm : 3174 (OH), 2935 (CH),
073, 1029. H NMR (C D N): δ 5.34 (1H, br d, J = 4.4 Hz,
H-6), 5.30 (1H, d, J = 7.7 Hz, H-1 ), 5.18 (1H, d, J = 7.9 Hz,
H-1 ), 5.07 (1H, d, J = 7.7 Hz, H-1 ), 5.02 (1H, d,
J = 7.8 Hz, H-1 ), 4.86 (1H, d, J = 7.6 Hz, H-1 ), 3.95
1H, m, H-3), 0.99 (3H, d, J = 6.8 Hz, Me-21), 0.96 (3H,
58
−
1
Tables 2 and 3.
1
1
1
5
5
2
.14. Acid hydrolysis of 2
ꢀꢀꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀ
A solution of 2 (67 mg) was subjected to acid hydrolysis
as described for 1 to give 1a (9.5 mg) and a sugar fraction
44 mg). HPLC analysis of the sugar fraction under the same
conditions as in the case of 1 showed the presence of d-
glucose: tR (min) 12.00 (d-glucose, positive optical rotation).
ꢀꢀꢀꢀ
ꢀꢀ
(
(
d, J = 6.4 Hz, Me-27), 0.93 (3H, s, Me-19), 0.82 (3H,
13
d, J = 6.8 Hz, Me-28), 0.64 (3H, s, Me-18). C NMR:
Tables 2 and 3.
2
.15. Compound 3
2.20. Acid hydrolysis of 5
Amorphous solid: [␣]25 −48.0 (MeOH; c 0.10). HR-
◦
D
+
A solution of 5 (1.8 mg) was subjected to acid hydrolysis
ESITOFMS m/z: 1227.6351 [M + H] (C H99O27 requires
227.6374). IR νmax (film) cm : 3422 (OH), 2926 (CH),
029. H NMR (C D N): δ 5.41 (1H, br d, J = 4.4 Hz, H-6),
58
−
1
as described for 1 to give 1a (0.6 mg) and a sugar fraction
(1.0 mg). HPLC analysis of the sugar fraction under the same
conditions as in the case of 1 showed the presence of d-
glucose: tR (min) 11.83 (d-glucose, positive optical rotation).
1
1
5
1
1
5
5
ꢀ
ꢀꢀ
.22 (1H, d, J = 7.8 Hz, H-1 ), 5.19 (1H, d, J = 7.8 Hz, H-
ꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀ
), 5.17(1H, d, J = 7.8 Hz, H-1
), 5.02(1H, d, J = 8.0 Hz,
ꢀ
ꢀꢀꢀꢀ
ꢀꢀ
H-1 ), 4.83 (1H, d, J = 7.7 Hz, H-1 ), 3.96 (1H, m, H-3),
.96 (3H, d, J = 6.5 Hz, Me-27), 0.96 (3H, d, J = 6.5 Hz, Me-
2
.21. Compound 6
0
2
0
1), 1.06 (3H, s, Me-19), 0.80 (3H, d, J = 6.7 Hz, Me-28),
Amorphous solid: [␣]25 −36.0 (MeOH; c 0.10). HR-
◦
.67 (3H, s, Me-18). 13C NMR: Tables 2 and 3.
D
+
ESITOFMS m/z: 1227.6473 [M + H] (C H99O27 requires
227.6374). IR νmax (film) cm : 3243 (OH), 2934 (CH),
029. H NMR (C D N): δ 5.35 (1H, br d, J = 4.5 Hz, H-6),
.29 (1H, d, J = 7.8 Hz, H-1 ), 5.17 (1H, d, J = 7.8 Hz, H-
), 5.06 (1H, d, J = 7.7 Hz, H-1 ), 5.03 (1H, d, J = 7.8 Hz,
H-1 ), 4.77 (1H, d, J = 7.7 Hz, H-1 ), 3.94 (1H, m, H-3),
58
−
1
1
1
5
1
2
.16. Acid hydrolysis of 3
1
5
5
ꢀ
ꢀꢀ
A solution of 3 (2.0 mg) was subjected to acid hydroly-
ꢀ
ꢀꢀꢀꢀ
ꢀ
ꢀ
sis as described for 1 to give 1a (0.3 mg) and a sugar frac-
tion (1.6 mg). HPLC analysis of the sugar fraction under the
same conditions as in the case of 1 showed the presence of
d-glucose: tR (min) 11.40 (d-glucose, positive optical rota-
tion).
ꢀꢀꢀ
ꢀꢀ
0
(
0
.94 (3H, d, J = 6.6 Hz, Me-21), 0.94 (3H, s, Me-19), 0.89
3H, d, J = 6.8 Hz, Me-27), 0.83 (3H, d, J = 6.8 Hz, Me-28),
.64 (3H, s, Me-18). 13C NMR: Tables 2 and 3.
2.22. Acid hydrolysis of 6
2
.17. Compound 4
Amorphous solid: [␣]25 −16.0 (MeOH; c 0.10). HR-
◦
A solution of 6 (1.9 mg) was subjected to acid hydrolysis
D
+
ESITOFMS m/z: 1249.6266 [M + Na] (C H98O27Na re-
quires 1249.6193). IR νmax (film) cm : 3356 (OH), 2936
CH), 1071. H NMR (C D N): δ 5.34 (1H, br d, J = 4.4 Hz,
H-6), 5.23 (1H, d, J = 7.8 Hz, H-1 ), 5.20 (1H, d, J = 7.8 Hz,
as described for 1 to give 1a (0.5 mg) and a sugar fraction
(1.0 mg). HPLC analysis of the sugar fraction under the same
conditions as in the case of 1 showed the presence of d-
glucose: tR (min) 11.51 (d-glucose, positive optical rotation).
58
−
1
1
(
5 5
ꢀ
ꢀꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀꢀ
H-1 ), 5.08 (1H, d, J = 7.6 Hz, H-1 ), 5.07 (1H, d,
ꢀ
ꢀꢀ
J = 7.5 Hz, H-1 ), 4.84 (1H, d, J = 7.8 Hz, H-1 ), 3.96 (1H, m,
H-3), 0.96 (3H, d, J = 6.8 Hz, Me-27), 0.95 (3H, d, J = 6.2 Hz,
Me-21), 0.92 (3H, s, Me-19), 0.80 (3H, d, J = 6.7 Hz, Me-28),
2.23. Compound 7
Amorphous solid: [␣]25 −44.0 (MeOH; c 0.10). HR-
◦
D
+
0
.64 (3H, s, Me-18). 13C NMR: Tables 2 and 3.
ESITOFMS m/z: 1087.5688 [M + Na] (C H88O22Na re-
52

2
62
A. Yokosuka et al. / Steroids 70 (2005) 257–265
1
quires 1087.5665). IR νmax (film) cm 1: 3186 (OH), 2932
−
group next to the C-25 chiral center, and the H NMR cou-
pling pattern differences of the H2-26 protons were inspected.
The H2-26 protons of 1a-R were observed as a d-like signal
at δ 4.20 (J = 6.3 Hz), whereas those of 1a-S were observed
as a dd pattern at δ 4.30 (J = 10.8, 6.6 Hz) and 4.09 (J = 10.8,
1
(
CH), 1073, 1028. H NMR (C D N): δ 5.35 (1H, br d,
J = 4.7 Hz, H-6), 5.27 (1H, d, J = 7.8 Hz, H-1 ), 5.20 (1H, d,
J = 7.9 Hz, H-1 ), 5.06 (1H, d, J = 7.7 Hz, H-1 ), 4.90 (1H, d,
J = 7.7 Hz, H-1 ), 3.96 (1H, m, H-3), 1.00 (3H, d, J = 6.8 Hz,
Me-27), 0.95 (3H, d, J = 6.5 Hz, Me-21), 0.94 (3H, s, Me-
5
5
ꢀ
ꢀꢀ
ꢀ
ꢀꢀꢀ
ꢀ
ꢀ
ꢀ
7
.2 Hz). Application of these spectral data to the empirical
rule reported by Yasuhara et al. [9] allowed us to confirm that
the C-25 configuration was exclusively S. The configurations
at the C-24 position and other points of the steroidal skeleton
were established by the following chemical transformations.
Compound 1a was treated with p-TsCl to give the 26-O-tosyl
ester (1a-T), which was then reduced with LiAlH4, afford-
ing (24R)-ergost-5-en-3␤-ol, that is, campesterol. The struc-
ture of 1a was determined as (24R,25S)-ergost-5-ene-3␤,26-
diol.
1
9), 0.77 (3H, d, J = 6.8 Hz, Me-28), 0.65 (3H, s, Me-18).
C NMR: Tables 2 and 3.
1
3
2
.24. Acid hydrolysis of 7
A solution of 7 (2.0 mg) was subjected to acid hydroly-
sis as described for 1 to give 1a (0.4 mg) and a sugar frac-
tion (1.2 mg). HPLC analysis of the sugar fraction under
the same conditions as in the case of 1 showed the pres-
ence of d-glucose: tR (min) 11.47 (d-glucose, positive optical
rotation).
The severe overlapping of the proton signals for the sugar
moieties in 1 excluded the possibility of a complete assign-
ment in a straightforward way using the conventional 2D
1
1
NMR methods, such as the H- H COSY, 2D TOCSY, and
HSQC spectra. Analysis of the 1D TOCSY spectra followed
by interpretation of the 1H- H COSY, HSQC-TOCSY,
and HMBC spectra allowed us to solve the exact sugar
sequences of the sugar moieties and their linkage positions
to the aglycone. The 1H NMR subspectra of individual
monosaccharide units were obtained by using selective
irradiation of easily identifiable anomeric proton signals
as well as by irradiation of other nonoverlapping proton
signals in a series of 1D TOCSY experiments [10–12].
Subsequent analysis of the H- H COSY spectrum resulted
in the sequential assignments of all the proton resonances
due to the seven glucosyl units, including identification of
their multiplet patterns and coupling constants as shown in
Table 1. The HSQC and HSQC–TOCSY spectra correlated
the proton resonances with those of the corresponding one-
bond coupled carbons, leading to unambiguous assignments
of the carbon shifts (Table 1). Comparison of the carbon
chemical shifts thus assigned with those of reference methyl
␣-d- and ␤-d-glucosides [13], taking into account the known
effects of O-glycosylation shift, indicated that 1 contained
1
3
. Results and discussion
The dried rhizomes of T. chantrieri were extracted with
MeOH, and the MeOH extract was passed through a Di-
aion HP-20 column, eluted with MeOH–H2O mixtures fol-
lowed by MeOH. The MeOH eluted portion was subjected
to multiple chromatographic steps over silica gel and ODS
silica gel as well as to preparative HPLC to give compounds
1
1
1
–7.
Compound 1 was obtained as an amorphous solid, and its
molecular formula was determined as C70H118O37 by com-
+
13
bined ESITOFMS ([M + Na] ion at m/z 1573), C NMR
with DEPT spectra, and elemental analysis. The H NMR
spectrum showed two three-proton singlet signals at δ 0.95
and 0.66, and three three-proton doublet signals at δ 0.98
1
(J = 6.7 Hz), 0.96 (J = 6.5 Hz), and 0.81 (J = 6.7 Hz), which
were recognized as typical steroid methyls [4,6,8]. Signals
for seven anomeric protons at δ 5.20 (d, J = 8.1 Hz), 5.17
(
(
d, J = 8.1 Hz), 5.16 (d, J = 8.2 Hz), 5.13 (d, J = 8.2 Hz), 5.06
d, J = 7.9 Hz), 5.01 (d, J = 8.0 Hz), and 4.83 (d, J = 7.8 Hz)
ꢀ
three terminal ␤-d-glucopyranosyl moieties (Glc : δC 102.6,
ꢀ
ꢀꢀꢀꢀ
were also observed. Acid hydrolysis of 1 with 1 M HCl in
dioxane:H2O (1:1) gave a C28-sterol as the aglycone (1a,
C28H48O2) and d-glucose. d-Glucose was identified by di-
rect HPLC analysis of the hydrolysate, which was performed
on an aminopropyl-bonded silica gel column. Detection was
carried out using a combination of refractive index (RI) and
optical rotation (OR) detectors. The above data, along with
the seven anomeric carbon signals at δ 106.3, 105.1, 104.9,
75.4, 78.6, 71.7, 78.4, 62.9; Glc : δC 104.9, 74.7, 78.2,
71.5, 78.4, 62.4; Glc : δC 104.9, 74.7, 78.2, 71.5, 78.4,
62.4), three C-4 substituted ␤-d-glucopyranosyl moieties
ꢀ
ꢀꢀꢀꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀꢀꢀꢀ
(Glc : δC 106.3, 76.4, 76.2, 80.9, 76.7, 62.0; Glc : δC
ꢀ
ꢀꢀꢀꢀꢀ
: δC 105.1, 74.7,
104.5, 74.2, 76.5, 80.8, 76.4, 62.0; Glc
76.6, 81.0, 76.7, 61.7), and a C-2 and C-6 disubstituted
ꢀ
ꢀ
␤-d-glucopyranosyl moiety (Glc : δC 102.9, 84.6, 77.9,
71.3, 76.8, 70.0). The ␤-orientations of the anomeric centers
of all the glucosyl moieties were supported by the relatively
large J values of their anomeric protons (7.8–8.2 Hz). In
the HMBC spectrum, the anomeric proton of the termi-
1
04.9, 104.5, 102.9, and 102.6, suggested that 1 was a C28-
sterol heptaglucoside.
The structure of 1a, except for the absolute configurations
at C-24 and C-25, was identified as ergost-5-ene-3␤,26-diol
ꢀ
nal glucosyl unit (Glc ) at δ 5.06 exhibited a long-range
1
13
[8] by analysis of its H, C, and 2D NMR spectra. In order
correlation with C-3 of the aglycone at δ 78.1, indicating
that one glucosyl unit was attached at the C-3 hydroxyl
group of the aglycone. Consequently, an oligoglucoside
composed of six glucosyl units was presumed to be linked
to determine the absolute configuration at C-25, 1a was con-
vertedtothediastereomericpairof(R)-MTPA(1a-R)and(S)-
MTPA (1a-S) esters with regard to the C-26 primary hydroxyl

A. Yokosuka et al. / Steroids 70 (2005) 257–265
263
Fig. 1. HMBC correlations of the sugar moieties of 1.
to the C-26 hydroxyl group of the aglycone. Further HMBC
signals due to C-3 and C-26 of the aglycone residue were
observed at δ 78.2 and 73.9, respectively, indicating that
ꢀ
ꢀꢀ
ꢀꢀ
correlations from δ 5.20 (H-1 of Glc ) to C-2 of Glc at δ
ꢀ
ꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀ
8
4.6, δ 5.17 (H-1 of Glc
) to C-4 of Glc
at δ 81.0,
the sugar linkages were at both C-3 and C-26, as in 1.
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
13
δ 5.16 (H-1 of Glc ) to C-4 of Glc at δ 80.9, δ 5.13
H-1 of Glc ) to C-4 of Glc at δ 80.8, δ 5.01 (H-1 of
Using the same procedures as described for 1, all the
C
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
(
NMR signals for the sugar moieties of 2 were assigned
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
ꢀꢀꢀꢀ
Glc
) to C-6 of Glc at δ 70.0, and δ 4.83 (H-1 of Glc ) to
to three terminal ␤-d-glucopyranosyl units (Glc , Glc ,
ꢀ
ꢀꢀꢀꢀꢀ
C-26 of the aglycone at δ 73.9 confirmed the hexaglucoside
sequence as Glc-(1 → 4)-Glc-(1 → 4)-Glc-(1 → 2)-[Glc-
and Glc
), two C-4 substituted ␤-d-glucopyranosyl
units (Glc and Glc ), and a C-2 and C-6 disubstituted
ꢀ
ꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀ
ꢀ
(
1 → 4)-Glc-(1 → 6)]-Glc, which was attached at C-26 of
␤-d-glucopyranosyl unit (Glc ). In the HMBC spectrum of
2, long-range correlations were observed from δ 5.23 (H-1 of
the aglycone. Accordingly, the structure of 1 was elucidated
as (24R,25S)-26-[(O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-
glucopyranosyl-(1 → 4)-O-␤-d-glucopyranosyl-(1 → 2)-O-
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀꢀꢀ
Glc ) to C-2 of Glc at δ 84.5, δ 5.19 (H-1 of Glc ) to C-4
ꢀ
ꢀꢀ
ꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
of Glc at δ 81.3, δ 5.18 (H-1 of Glc
) to C-4 of Glc at
δ 81.0, δ 5.06 (H-1 of Glc ) to C-3 of the aglycone at δ 78.2,
δ 5.01 (H-1 of Glc ) to C-6 of Glc at δ 70.0, and δ 4.83
(H-1 of Glc ) to C-26 of the aglycone at δ 73.9. Thus, the
structure of 2 was characterized as (24R,25S)-26-[(O-␤-d-
glucopyranosyl-(1 → 4)-O-␤-d-glucopyranosyl-(1 → 2)-O-
[O-␤-d-glucopyranosyl-(1 → 4)-␤-d-glucopyranosyl-(1 →
6)]-␤-d-glucopyranosyl)oxy]ergost-5-en-3␤-yl ␤-d-gluco-
ꢀ
[
O-␤-d-glucopyranosyl-(1 → 4)-␤-d-glucopyranosyl-(1 →
ꢀ
ꢀꢀꢀꢀ
ꢀꢀ
6
)]-␤-d-glucopyranosyl)oxy]ergost-5-en-3␤-yl ␤-d-gluco-
pyranoside (Fig. 1).
Compound 2 was analyzed as C H108O32 on the basis
ꢀ
ꢀ
64
+
13
of the ESITOFMS (m/z 1411 [M + Na] ), C-NMR with
DEPT spectral, and elemental analysis data. The deduced
molecular formula was less than that of 1 by a C H10O ,
6
5
1
corresponding to the lack of one hexose unit. The H NMR
spectrum of 2 showed signals for six anomeric protons
at δ 5.23 (d, J = 7.8 Hz), 5.19 (d, J = 8.1 Hz), 5.18 (d,
J = 8.1 Hz), 5.06 (d, J = 7.7 Hz), 5.01 (d, J = 7.8 Hz), and
pyranoside.
1
The H NMR spectrum of compound 3 (C H98O27)
58
showed signals for five anomeric protons at δ 5.22 (d,
J = 7.8 Hz), 5.19 (d, J = 7.8 Hz), 5.17 (d, J = 7.8 Hz), 5.02 (d,
J = 8.0 Hz), and 4.83 (d, J = 7.7 Hz), together with signals
for five steroid methyl groups at δ 0.96 (d, J = 6.5 Hz),
0.96 (d, J = 6.5 Hz), 1.06 (s), 0.80 (d, J = 6.7 Hz), and 0.67
(s). Acid hydrolysis of 3 with 1 M HCl in dioxane-H2O
4
.83 (d, J = 7.8 Hz), as well as signals for five steroid methyl
groups at δ 0.96 (d, J = 6.5 Hz), 0.95 (d, J = 5.6 Hz), 0.92 (s),
0
1
.79 (d, J = 6.7 Hz), and 0.64 (s). Acid hydrolysis of 2 with
M HCl in dioxane-H2O (1:1) resulted in the production
13
1
of 1a and d-glucose. In the C NMR spectrum of 2, the
(1:1) yielded 1a and d-glucose. When all of the H and

2
64
A. Yokosuka et al. / Steroids 70 (2005) 257–265
13C NMR signals of 3 were compared with those of 2,
the two compounds completely agreed as to the signals
arising from the pentaglucosyl group attached at C-26 of
the aglycone. However, the signals assignable to the ␤-d-
glucopyranosyl residue bound to C-3 of the aglycone could
Compound 6 (C H98O27) gave 1a and d-glucose
58
1
13
following acid hydrolysis. The H and C NMR spectra
of 6 was almost superimposable on those of 5 except
for the signals due to the glucosyl unit attached at C-26
ꢀ
ꢀ
13
of the aglycone (Glc ). In the C NMR spectrum of 6,
1
13
glycosylation shifts were recognized in the signals due
not be observed in the H and C NMR spectra of 3, and
the C-3 carbon signal of 3 was shifted upfield by 6.9 ppm in
comparison with that of 2. The above chemical and spectral
data implied that 3 was the C-3 deglucosyl derivative
of 2 and allowed the structural determination of 3 to be
made as (24R,25S)-3␤-hydroxyergost-5-en-26-yl O-␤-d-
glucopyranosyl-(1 → 4)-O-␤-d-glucopyranosyl-(1 → 2)-O-
ꢀ
ꢀ
to C-3 and C-6 of Glc , and HMBC correlations were
ꢀ
ꢀꢀ
observed between H-1 of the terminal glucosyl unit (glc )
at δ 5.29 and C-3 of Glc at δ 88.5, and between H-1
of the C-4 glucosylated glucosyl unit (Glc ) at δ 5.03
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀ
ꢀ
and C-6 of Glc at δ 69.7. The structure of 6 was shown
to be (24R,25S)-26-[(O-␤-d-glucopyranosyl-(1 → 3)-O-
[
6
O-␤-d-glucopyranosyl-(1 → 4)-␤-d-glucopyranosyl-(1 →
[
6
O-␤-d-glucopyranosyl-(1 → 4)-␤-d-glucopyranosyl-(1 →
)]-␤-d-glucopyranosyl)oxy]ergost-5-en-3␤-yl ␤-d-gluco-
)]-␤-d-glucopyranoside.
1
pyranoside.
The H NMR spectrum of compound 4 (C H98O27)
58
Compound 7 (C H88O22) was suggested to correspond
showed five anomeric proton signals at δ 5.23 (d, J = 7.8 Hz),
.20 (d, J = 7.8 Hz), 5.08 (d, J = 7.6 Hz), 5.07 (d, J = 7.5 Hz),
52
to 4 without the terminal glucosyl unit attached at C-6 of
5
the inner glucosyl unit on the basis of its spectral properties
and the results of acid hydrolysis. This was confirmed by
HMBC correlations between H-1 of the terminal glucosyl
and 4.84 (d, J = 7.8 Hz), along with five steroid methyl proton
signals at δ 0.96 (d, J = 6.8 Hz), 0.95 (d, J = 6.2 Hz), 0.92 (s),
0
.80 (d, J = 6.7 Hz), and 0.64 (s). On the basis of the spectral
ꢀ
ꢀꢀꢀ
unit (Glc ) at δ 5.20 and C-4 of the inner glucosyl unit
properties of 4 and the results of acid hydrolysis, giving 1a
and d-glucose, 4 was shown to be a steroid glycoside closely
related to 2; however, the sugar chain attached at C-26
of the aglycone was made up of four ␤-d-glucopyranosyl
units and differed from that of 2 by the absence of one
glucosyl unit. The four glucosyl units were identified as
ꢀ
ꢀꢀ
ꢀꢀꢀ
(
Glc ) at δ 81.3, and between H-1 of Glc at δ 5.27 and
C-2 of the glucosyl unit linked to C-26 of the aglycone
ꢀ
ꢀ
(
Glc ) at δ 84.8. The structure of 7 was formulated as
24R,25S)-26-[(O-␤-d-glucopyranosyl-(1 → 4)-O-␤-d-glu-
(
copyranosyl-(1 → 2)-␤-d-glucopyranosyl)oxy]ergost-5-en-
ꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀꢀ
3␤-yl ␤-d-glucopyranoside.
two terminal ␤-d-glucopyranosyl units (Glc and Glc ),
ꢀ
ꢀꢀ
Compounds 1–7 are new glycosides of the campesterol
a C-4 substituted ␤-d-glucopyranosyl unit (Glc ), and a
C-2 and C-6 disubstituted ␤-d-glucopyranosyl unit (Glc ).
In the HMBC spectrum of 4, long-range correlations were
ꢀ
ꢀ
derivative (24R,25S)-ergost-5-ene-3␤,26-diol. Phytosterols
and their monoglucosides, such as campesterol, stigmasterol
and ␤-sitosterol, and their 3-O-glucoside are widely present
in the plant kingdom. However, to the best of our knowl-
edge, 1–7 isolated from T. chantrieri are the first representa-
tive oligoglucosides of a phytosterol derivative, having sugar
moieties with a total of four to seven glucose units. The bis-
desmosidic nature of these structures, except for 3, is also
notable.
ꢀ
ꢀꢀ
ꢀꢀ
observed from δ 5.23 (H-1 of Glc ) to C-2 of Glc at δ
ꢀꢀꢀꢀ ꢀꢀꢀ
8
4.5, δ 5.20 (H-1 of Glc ) to C-4 of Glc at δ 81.5, δ 5.08
ꢀꢀꢀꢀꢀ ꢀꢀ
H-1 of Glc ) to C-6 of Glc at δ 69.9, and δ 4.84 (H-1
(
ꢀ
ꢀ
of Glc ) to C-26 of the aglycone at δ 73.9. Thus, the struc-
ture of 4 was formulated as (24R,25S)-26-[(O-␤-d-gluco-
pyranosyl-(1 → 4)-O-␤-d-glucopyranosyl-(1 → 2)-O-[␤-d-
glucopyranosyl-(1 → 6)]-␤-d-glucopyranosyl)oxy]ergost-5-
en-3␤-yl ␤-d-glucopyranoside.
The molecular formula of compound 5 (C H98O27)
58
References
was the same as that of 4, and acid hydrolysis of 5 gave
1
13
1
a and d-glucose. Comparison of the H and C NMR
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ꢀ
ꢀꢀ
ꢀꢀꢀꢀꢀ
glucopyranosyl units (Glc and Glc ), a C-4 substituted
ꢀꢀꢀꢀ
␤
-d-glucopyranosyl unit (Glc ), and a C-2 and C-6
disubstituted ␤-d-glucopyranosyl unit (Glc ). In the HMBC
spectrum of 5, long-range correlations were observed from
[
4] Yokosuka A, Mimaki Y, Sashida Y. Spirostanol saponins from the
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ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
δ 5.30 (H-1 of Glc ) to C-2 of Glc at δ 84.0, δ 5.18 (H-1
[5] Yokosuka A, Mimaki Y, Sashida Y. Two new steroidal glycosides
from Tacca chantrieri. Nat Med (Tokyo) 2002;56:208–11.
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ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
of Glc ) to C-4 of Glc at δ 81.0, δ 5.02 (H-1 of Glc )
ꢀ
ꢀ
ꢀꢀ
to C-6 of Glc at δ 70.0, and δ 4.86 (H-1 of Glc ) to C-26
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