Novel steroidal glycosides from the whole plants of helleborus foetidus

Article abstract of DOI:10.1248/cpb.c19-01037

Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1-28), including 20 novel spirostanol glycosides (1-20) and a novel furostanol glycoside (21). The structures of the newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage. Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC₅₀ values of 5.9 and 6.6μM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC₅₀ value of 5.5μM.

Full text of DOI:10.1248/cpb.c19-01037

Vol. 68, No. 3
Chem. Pharm. Bull. 68, 273–287 (2020)
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Regular Article
Novel Steroidal Glycosides from the Whole Plants of Helleborus foetidus
Tomoki Iguchi,* Yuka Uchida, Shingo Takano, Akihito Yokosuka, and Yoshihiro Mimaki
Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences;
1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan.
Received November 24, 2019; accepted December 12, 2019
Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1–28),
including 20 novel spirostanol glycosides (1–20) and a novel furostanol glycoside (21). The structures of the
newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage.
Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the
C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated
for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma
cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC50 values of 5.9 and
6.6µM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC50 value of 5.5µM.
Key words Helleborus foetidus; Ranunculaceae; whole plant; steroidal glycoside; cytotoxic activity
(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside
Introduction
Plants from the genus Helleborus are perennials belong- (23),19) (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-yl
ing to the Ranunculaceae family, and are widely distrib- O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylo-
uted throughout Europe.1) Helleborus species are poisonous pyranosyl-(1→3)]-α-L-arabinopyranoside (24),20) (23S,24S)-21-
plants and a series of bufadienolides have been isolated from acetyloxy-3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-yl
H. caucasicus,2,3) H. odorus,4,5) H. thibetanus,6–8) and O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyra-
H. torquatus.9) Recently, various bufadienolides from nosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabino-
H. orientalis and H. foetidus were isolated and identified, pyranoside (25),10) (25R)-26-[(β-D-glucopyranosyl)oxy]-1β,22α-
some of which showed significantly potent cytotoxic activity dihydroxyfurost-5-en-3β-yl β-D-glucopyranoside (26),21) (25R)-
and induced apoptosis via a mitochondria dependent pathway 26-[(β-D-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl
in cultured tumor cells.10,11) Because Helleborus species has O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (27),22) and
been reported to concomitantly contain the glycoside deriva- (25S)-22α,25-epoxy-26-[(β- -glucopyranosyl)oxy]-3β-hydroxy-
D
tives of C27 steroids (spirostanols and furostanols),12–18) further furost-5-en-1β-yl α-L-arabinopyranoside (28),13) respectively
phytochemical examination of the methanolic extract of (Fig. 1).
H. foetidus was performed using whole plants with a specific
Compound 1 was obtained as an amorphous powder and
focus on steroidal glycosides. The isolation of 28 steroidal gly- its molecular formula was determined to be C45H70O19, based
cosides (1–28), including 20 novel spirostanol glycosides (1– on high resolution-electrospray ionization-time of flight (HR-
20) and a novel furostanol glycoside (21) was achieved. The ESI-TOF)-MS and 13C-NMR spectra data. The IR spectrum
structures of the newly identified compounds were determined of 1 showed absorption bands of hydroxy (3392cm−1) and
1
from their two-dimensional NMR spectra and hydrolytic carbonyl groups (1729cm−1). The H- and 13C-NMR spectra
cleavage. The cytotoxic activities of the isolated compounds of 1 indicated the presence of four steroidal methyl groups
were evaluated against HL-60 human promyelocytic leukemia [δH 1.35 (3H, s, Me-19), 1.13 (3H, d, J=7.0Hz, Me-21), 1.02
cells and A549 human lung carcinoma cells.
(3H, s, Me-18), and 0.97 (3H, d, J=6.8Hz, Me-27); δC 16.8
(C-18), 14.9 (C-19), 14.6 (C-21), and 13.0 (C-27)], an olefinic
group [δH 5.60 (1H, brd, J=5.8Hz, H-6); δC 139.3 (C-5) and
Results and Discussion
Whole plants of H. foetidus (fresh weight, 3.3kg) were ex- 124.9 (C-6)], an oxygenated methylene group [δH 3.82 (1H, dd,
tracted using hot MeOH. The concentrated MeOH extract J=11.2, 11.2Hz, H-26ax) and 3.43 (1H, dd, J=11.2, 4.7Hz,
(115g) was fractionated using a Diaion HP-20 porous polymer H-26eq); δC 60.6 (C-26)], five oxygenated methine groups [δH
polystyrene resin column with MeOH/H2O (3:7), MeOH/H2O 4.61 (1H, m, H-16), 4.10 (1H, brs, H-24), 3.90 (1H, m, H-3),
(1:1), MeOH, EtOH, and EtOAc, successively (each solvent 3.83 (1H, dd, J=12.0, 4.1Hz, H-1), and 3.81 (1H, d, J=3.2Hz,
volume 6L). The MeOH eluted fraction was subjected to silica H-23); δC 84.0 (C-1), 83.0 (C-16), 73.0 (C-24), 68.7 (C-23), and
gel column chromatography (CC), octadecylsilanized (ODS) 68.0 (C-3)], and an acetyl group [δH 2.01 (3H, s); δC 170.8 and
silica gel CC, and preparative ODS HPLC to obtain 28 com- 21.0]. Furthermore, signals corresponding to three anomeric
pounds (1–28). Previously characterized compounds (22–28) protons and carbons were observed [δH 6.48 (1H, brs), 4.94
were identified as (23S,24S,25S)-24-[(β-D-fucopyranosyl)- (1H, d, J=7.6Hz), and 4.69 (1H, d, J=7.9Hz); δC 106.7,
1
oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-α-L-rhamnopyranosyl- 100.8, and 100.7]. The H- and 13C-NMR spectral data of 1
(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside were similar to those obtained for 24, except for the signals
(22),19) (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,23-dihy- arising from the F-ring part of the steroidal skeleton. Instead
1
droxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)- of the H- and 13C-NMR signals arising from the C-25(27)
*To whom correspondence should be addressed. e-mail: iguchit@toyaku.ac.jp
© 2020 The Pharmaceutical Society of Japan

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Fig. 1. Structures of Isolated Compounds 1–28
Ara: α-L-arabinopyranosyl; Api: β-D-apiofuranosyl; Fuc: β-D-fucopyranosyl; Gal: β-D-galactopyranosyl; Glc: β-D-glucopyranosyl; Rha: α-L-rhamnopyranosyl; Qui: β-D-
quinovopyranosyl; Ac: acetyl.
exomethylene group [δH 5.07 and 4.97 (each 1H, brs, H2-27); be (23S,24S,25S)-3β,23,24-trihydroxyspirost-5-en-1β-yl O-(4-
δC 146.1 (C-25) and 112.3 (C-27)] observed in 24, signals from O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
a secondary methyl group [δH 0.97 (3H, d, J=6.8Hz, Me-27); (1→3)]-α-L-arabinopyranoside.
1
δC 13.0 (C-27)] and methine group [δH 1.89 (1H, m, H-25); δC
The H- and 13C-NMR spectra of 2 (C51H80O23) indicated
36.1 (C-25)] were observed in the H- and 13C-NMR spectra of that 2 was analogous to 1, including the triglycoside moiety at-
1. These data suggest that 1 is a saturated derivative of 24 in tached to C-1 of the aglycone. However, the molecular formu-
terms of the C-25(27) exomethylene group. Nuclear Overhaus- la of 2 was larger than that of 1 by a C6H10O4 unit, indicating
er effect spectroscopy (NOESY) of 1 was performed and NOE the presence of an additional hexosyl unit. Acid hydrolysis of
correlations were observed between H-23 (δH 3.81) and H-20 2 yielded L-arabinose, L-rhamnose, D-quinovose, and D-xylose,
1
(δH 2.97)/H-24 (δH 4.10)/H-25 (δH 1.89) as well as between while H-1H COSY and HSQC analysis indicated that 2 con-
1
H-26ax (δH 3.82) and H-16 (δH 4.61). Additionally, the proton tained a terminal β-D-quinovopyranosyl unit [Qui: δH 5.24 (1H,
3
3
spin-coupling constants of JH-23,H-24 and JH-25,H-26ax were 3.2 d, J=7.6Hz, H-1⁗); δC 105.3, 76.2, 78.1, 76.9, 73.0, and 18.6
and 11.2Hz, respectively. Thus, the configurations of C-23, (C-1⁗–6⁗)]. Comparing the 13C-NMR spectrum of 2 with that
C-24, and C-25 were determined as 23S, 24S, and 25S. Acid of 1, the signal attributed to C-24 of the aglycone was shifted
hydrolysis of 1 with 0.2M HCl (dioxane/H2O; 1:1) yielded downfield by 8.4ppm. Additionally, the HMBC spectrum of
L-arabinose, L-rhamnose, and D-xylose carbohydrate moieties. 2 showed a long-range correlation between H-1⁗ of Qui (δH
1
The H-1H correlation spectroscopy (COSY) and heteronuclear 5.24) and C-24 of the aglycone (δC 81.4). Therefore, 2 was
single quantum coherence (HSQC) spectra indicated that the determined to be (23S,24S,25S)-24-[(β-D-quinovopyranosyl)-
sugar moiety of 1 comprised a 2,3-disubstituted inner α-L- oxy]-3β,23-dihydroxyspirost-5-en-1β-yl
O-(4-O-acetyl-α-L-
arabinopyranosyl unit [Ara: δH 4.69 (1H, d, J=7.9Hz, H-1′); rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-
δC 100.8, 72.8, 85.2, 69.9, and 67.3 (C-1′–5′)], a terminal arabinopyranoside.
α-L-rhamnopyranosyl unit [Rha: δH 6.48 (1H, brs, H-1″); δC
Comparison of the 1H- and 13C-NMR spectra of 3
100.7, 72.2, 69.9, 76.5, 66.5, and 18.5 (C-1″–6″)], and a termi- (C50H78O23) with those of 1 suggested that 3 contained the same
nal β-D-xylopyranosyl unit [Xyl: δH 4.94 (1H, d, J=7.6Hz, aglycone moiety as 1 but differed in terms of the sugar moiety
H-1‴); δC 106.7, 74.5, 78.5, 70.9, and 67.1 (C-1‴–5‴)]. In the attached to C-1 of the aglycone. Acid hydrolysis of 3 afforded
1
heteronuclear multiple bond correlation (HMBC) spectrum L-arabinose, D-apiose, L-rhamnose, and D-xylose, while H-1H
of 1, long-range correlations were observed between H-1″ of COSY and HSQC analysis of the sugar moiety in 3 showed
Rha (δH 6.48) and C-2′ of Ara (δC 72.8), H-1‴ of Xyl (δH 4.94) that it consisted of a 2,3-disubstituted α-L-arabinopyranosyl
and C-3′ of Ara (δC 85.2), between H-1′ of Ara (δH 4.69) and (Ara), 3,4-disubstituted α-L-rhamnopyranosyl (Rha), terminal
3
C-1 of the aglycone (δC 84.0). Furthermore, a JC,H correlation β-D-xylopyranosyl (Xyl), and terminal β-D-apiofuranosyl units
was observed between H-4″ of Rha (δH 5.78) and the acetyl [Api: δH 5.95 (1H, brs, H-1⁗); δC 112.1, 77.8, 80.0, 74.8, and
carbonyl carbon (δC 170.8). Accordingly, 1 was determined to 65.2 (C-1⁗–5⁗)], as well as an acetyl group. The α-anomeric

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1
configuration of Rha was determined by the large JC-1,H-1 as
(23S,24S,25S)-3β,21,23,24-tetrahydroxyspirost-5-en-1β-yl
value of 169.7Hz. In the HMBC spectrum of 3, long-range O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D -
correlations were observed between H-1⁗ of Api (δH 5.95) and xylopyranosyl-(1→3)]-α-L-arabinopyranoside,(23S,24S,25S)-24-[(β-
C-3″ of Rha (δC 77.7), H-1″ of Rha (δH 6.49) and C-2′ of Ara D-fucopyranosyl)oxy]-3β,21,23-trihydroxyspirost-5-en-1β-yl
(δC 72.4), H-1‴ of Xyl (δH 4.92) and C-3′ of Ara (δC 85.2), H-4″ O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D -
of Rha (δH 5.90) and the acetyl carbonyl carbon (δC 170.7), xylopyranosyl-(1→3)]-α-L-arabinopyranoside,and(23S,24S,25S)-
and between H-1′ of Ara (δH 4.63) and C-1 of the aglycone (δC 3β,21,23,24-tetrahydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-
84.2). Thus, 3 was determined to be (23S,24S,25S)-3β,23,24- (1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-
trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4- xylopyranosyl-(1→3)]-α-L-arabinopyranoside, respectively.
O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
(1→3)]-α-L-arabinopyranoside.
The 1H- and 13C-NMR spectra of 9 (C55H86O28) were
similar to those obtained for 8, except for the presence of
Compound 4 (C56H88O27) exhibited 1H- and 13C-NMR the signals attributed to one more monosaccharide unit. The
spectral features similar to those of 3, except for the signals molecular formula of 9 was larger than that of 8 by C5H8O4,
arising from the F-ring part of the steroidal skeleton. The corresponding to a single pentosyl unit. Acid hydrolysis of
molecular formula of 4 featured an additional C6H10O4 com- 9 yielded L-arabinose, D-apiose, L-rhamnose, and D-xylose.
pared to that of 3. From acid hydrolysis of 4, L-arabinose, Analysis of the corresponding 1H-1H COSY and HSQC
D-apiose, D-fucose, L-rhamnose, and D-xylose were obtained. spectra of the sugar moieties of 9 indicated the presence of
A terminal β-D-fucopyranosyl unit [Fuc: δH 5.13 (1H, d, an additional terminal α-L-arabinopyranosyl unit [Ara′: δH
J=7.9Hz, H-1″‴); δC 105.8, 73.1, 75.1, 72.6, 71.3, and 17.1 5.11 (1H, d, J=7.7Hz, H-1″‴); δC 106.4, 73.6, 74.8, 69.9, and
1
(C-1″‴–6″‴)] was detected by H-1H COSY and HSQC spec- 67.2 (C-1″‴–5″‴)]. A long-range correlation was observed
tral analysis of 4. Comparing the 13C-NMR spectrum of 4 between H-1″‴ of Ara′ (δH 5.11) and C-24 of the aglycone
with that of 3, the signal assigned to C-24 of the aglycone (δC 81.7) in the HMBC spectrum. Thus, 9 was identified
3
was shifted downfield by 8.4ppm and a JC,H correlation as
(23S,24S,25S)-24-[(β-D-arabinopyranosyl)oxy]-3β,21,23-
was observed between H-1″‴ of Fuc (δH 5.13) and C-24 of trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-
the aglycone (δC 81.4) in the HMBC spectrum. Therefore, O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
4 was elucidated as (23S,24S,25S)-24-[(β-D-fucopyranosyl)- (1→3)]-α-L-arabinopyranoside.
1
oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-
Analysis of the H- and 13C-NMR spectra of 10 (C56H88O28)
(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D- and subsequent comparison with those of 9 revealed that
xylopyranosyl-(1→3)]-α-L-arabinopyranoside.
the aglycone and tetraglycoside attached to C-1 of the agly-
The H- and 13C-NMR spectra of 5 (C49H78O23) were es- cone were identical to those of 9 but differed in terms of
sentially analogous to those of 22, but remarkable differences the monosaccharide linked to C-24 of the aglycone. Instead
were observed for the signals assigned to the monosaccha- of the signals for an arabinopyranosyl moiety, signals at-
ride linked to C-24 of the aglycone between the two glyco- tributed to a β-D-fucopyranosyl (Fuc) residue were observed.
sides. Acid hydrolysis of 5 yielded L-arabinose, D-glucose, Acid hydrolysis of 10 yielded L-arabinose, D-apiose, D-fucose,
1
L-rhamnose, and D-xylose. Analysis of the associated H-1H L-rhamnose, and D-xylose. The linkage of the Fuc moiety to
1
COSY and HSQC spectra indicated the presence of a termi- C-24 of the aglycone was confirmed by an HMBC correla-
nal β-D-glucopyranosyl unit [Glc: δH 5.33 (1H, d, J=7.7Hz, tion between H-1″‴ of Fuc (δH 5.13) and C-24 of the aglycone
H-1⁗); δC 105.6, 76.1, 78.3, 69.5, 78.3, and 63.0 (C-1⁗–6⁗)]. (δC 81.7). Thus, 10 was assigned as (23S,24S,25S)-24-[(β-D-
The HMBC spectrum of 5 showed a long-range correlation fucopyranosyl)oxy]-3β,21,23-trihydroxyspirost-5-en-1β-yl O-β-
between H-1⁗ of Glc (δH 5.33) and C-24 of the aglycone (δC D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-
81.6). Accordingly, 5 was determined to be (23S,24S,25S)-24- (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.
1
[(β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl
The H- and 13C-NMR spectra of 11 (C56H88O29) suggested
O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]- that it was analogous to 8, including the tetraglycoside moi-
α-L-arabinopyranoside.
ety attached to C-1 of the aglycone. The molecular formula
The 1H- and 13C-NMR spectra of 6 (C45H70O20), 7 of 11 was larger than that of 8 by C6H10O5, corresponding
(C51H80O24), and 8 (C50H78O24) were closely related to those to a single hexosyl unit. Acid hydrolysis of 11 yielded L-
of 1, 23, and 3, respectively. The molecular formula of 6 was arabinose, D-apiose, D-galactose, L-rhamnose, and D-xylose.
larger than that of 1 (C45H70O19) by a single oxygen atom, The 1H-1H COSY and HSQC spectral analysis of 11 indi-
and only differences observed in the H- and 13C-NMR sig- cated the presence of a terminal β-D-galactopyranosyl unit
1
nals were those assigned to Me-21/C-21. The methyl signals [Gal: δH 4.90 (1H, d, J=7.9Hz, H-1″‴); δC 105.4, 72.5, 75.6,
[δH 1.13 (3H, d, J=7.0Hz); δC 14.6] in 1 were displaced by 70.2, 77.0, and 62.4 (C-1″‴–6″‴)], and an HMBC correla-
hydroxymethyl signals [δH 4.22 (1H, brd, J=10.1Hz) and tion was observed between H-1″‴ of Gal (δH 4.90) and C-21
4.01 (1H, dd, J=10.1, 6.7Hz); δC 62.3] in 6. Similarly, the of the aglycone (δC 70.0). Therefore, 11 was determined
molecular formulas of 7 and 8 were larger than those of to be (23S,24S,25S)-21-[(β-D-galactopyranosyl)oxy]-3β,23,24-
23 (C51H80O23) and 3 (C50H78O23) by a single oxygen atom, trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-
and the C-21 methyl groups were hydroxylated, as indi- O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
1
cated by the H- and 13C-NMR spectra [7: δH 4.20 (1H, brd, (1→3)]-α-L-arabinopyranoside.
J=10.4Hz) and 4.01 (1H, dd, J=10.4, 7.1Hz); δC 62.5; 8:
The 1H- and 13C-NMR spectra of 12 (C61H96O33) were
δH 4.19 (1H, m), 3.98 (1H, m); δC 62.3)]. Therefore, 6, 7, similar to those obtained for 11, but the molecular formula
and 8 were identified as the corresponding C-21 hydroxy of 12 was larger than that of 11 by C5H8O4. Acid hydro-
derivatives of 1, 23, and 3, respectively, establishing them lysis of 12 afforded L-arabinose, D-apiose, D-galactose,

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Table 4. 13C-NMR Data for the Sugar and Acetyl Moieties of 1–21
All spectra were measured in C5D5N (1, 8, 18, and 19: 125MHz; 2–7, 9–17, 20, and 21: 150MHz).
L-rhamnose, and D-xylose. The 1H-1H COSY and HSQC (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.
spectral analysis of 12 indicated the presence of signals cor-
Compound 13 (C61H98O33) was obtained as an amorphous
1
responding to an additional terminal α-L-arabinopyranosyl powder and its H- and 13C-NMR spectra showed the same
(Ara′) unit. The HMBC spectrum of 12 contained aglycone unit as 7, also indicating that the sugar moieties
a
long-range correlation from H-1‴‴ of Ara′ (δH 5.10) to attached to C-1 and C-24 of the aglycone of 13 were coin-
C-24 of the aglycone (δC 81.4). Thus, 12 was assigned cident with those of 22. Additionally, a 2-substituted β-D-
as
(23S,24S,25S)-24-[(β-D-arabinopyranosyl)oxy]-21-[(β-D- galactopyranosyl unit [Gal: δH 4.98 (1H, d, J=7.4Hz, H-1⁗);
galactopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-β- δC 103.4, 81.6, 75.2, 69.7, 77.0, and 62.2 (C-1⁗–6⁗)] and a ter-
D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)- minal β-D-glucopyranosyl unit [Glc: δH 5.40 (1H, d, J=7.6Hz,

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H-1″‴); δC 106.1, 76.5, 78.1, 71.4, 78.6, and 62.5 (C-1″‴–6″‴)] with those of 18 showed considerable structural similarity.
1
were detected in the H-1H COSY and HSQC spectral analysis Furthermore, the presence of an additional acetyl group to
of 13. Acid hydrolysis of 13 afforded L-arabinose, D-fucose, D- that attached at C-21 of the aglycone was indicated by the
galactose, D-glucose, L-rhamnose, and D-xylose. In the HMBC 1H- and 13C-NMR spectra [δH 1.92 (3H, s); δC 170.8 and 20.9].
spectrum of 13, long-range correlations were observed be- The ester linkage at aglycone C-21 was formed from acetic
tween H-1″‴ of Glc (δH 5.40) and C-2⁗ of Gal (δC 81.6) as well acid, as indicated by the long-range correlations between
as between H-1⁗ of Gal (δH 4.98) and C-21 of the aglycone the H2-21 methylene protons [δH 4.37 (m) and 4.34 (m)] and
(δC 69.9). Thus, 13 was identified as (23S,24S,25S)-24-[(β- acetyl carbonyl carbon (δC 170.8). Thus, 19 was identified as
D-fucopyranosyl)oxy]-21-[O-β-D-glucopyranosyl-(1→2)-(β-D- (23S,24S)-21-acetyloxy-24-[(α-L-arabinopyranosyl)oxy]-3β,23-
galactopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-α- dihydroxyspirosta-5,25(27)-dien-1β-yl
O-(4-O-acetyl-α-L-
L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L- rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-
arabinopyranoside.
arabinopyranoside.
1
1
The H- and 13C-NMR spectra of 14 (C62H98O34) and 15
Analysis of the H- and 13C-NMR spectra of 20 (C52H78O25)
(C68H108O38) indicated their relation to 8 and 10, respectively, and comparison with those of 19 showed that the aglycone
containing a diglycoside composed of Glc and Gal at C-21 with an acetyloxy group at C-21 and monosaccharide at C-24
1
of the aglycone, as in 13. The molecular formula of 14 and were identical to those observed in 19. However, the H- and
15 were both larger than those of 8 and 10, respectively, by 13C-NMR signals for the apiofuranosyl group linked to C-3
C12H20O10 and acid hydrolysis of 14 yielded L-arabinose, D-ga- of the rhamnopyranosyl unit in the sugar moiety attached to
1
lactose, D-glucose, L-rhamnose, and D-xylose. In contrast, acid the aglycone C-1 were not observed. The H- and 13C-NMR
hydrolysis of 15 afforded L-arabinose, D-fucose, D-galactose, spectra of the triglycoside moiety agreed with those observed
D-glucose, L-rhamnose, and D-xylose. In the HMBC spectra in the concomitantly isolated spirostanol triglycosides 1, 2, 6,
of 14 and 15, long-range correlations were observed between and 7. Thus, 20 was identified as (23S,24S)-21-acetyloxy-24-
H-1‴‴ of Glc [δH 5.38 (14); 5.38 (15)] and C-2″‴ of Gal [δC 81.6 [(α-L-arabinopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-
(14); 81.8 (15)] as well as between H-1″‴ of Gal [δH 4.98 (14); dien-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-
4.97 (15)] and C-21 of the aglycone [δC 69.8 (14); 69.8 (15)]. D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.
Accordingly, 14 and 15 were identified as (23S,24S,25S)-21-
Compound 21 was obtained as an amorphous powder with
[O-(β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl)- a molecular formula was of C38H62O14 as determined by HR-
1
oxy]-3β,23,24-trihydroxyspirost-5-en-1β-ylO-β-D-apiofuranosyl- ESI-TOF-MS and 13C-NMR data. In the H- and 13C-NMR
(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D- spectra of 21, signals corresponding to four steroidal methyl
xylopyranosyl-(1→3)]-α-L-arabinopyranoside and (23S,24S,25S)- groups [δH 1.27 (3H, d, J=7.1Hz, Me-21), 1.21 (3H, s, Me-19),
24-[(β-D-fucopyranosyl)oxy]-21-[O-(β-D-glucopyranosyl- 0.95 (3H, d, J=6.5Hz, Me-27), and 0.90 (3H, s, M-18); δC
(1→2)-β-D-galactopyranosyl)oxy]-3β,23-dihydroxyspirost- 17.4 (C-27), 16.7 (C-18), 16.3 (C-21), and 14.7 (C-19)], an
5-en-1β-yl
O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L- olefinic group [δH 5.55 (1H, brd, J=4.6Hz, H-6); δC 139.5
rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L- (C-5) and 124.7 (C-6)], two anomeric protons and associ-
arabinopyranoside, respectively. ated carbons [δH 4.79 (1H, d, J=7.7Hz) and 4.75 (1H, d,
The NMR spectral features of 16 (C56H86O27), 17 J=7.1Hz); δC 104.8 and 102.2], and a hemiacetal carbon [δC
(C50H76O24), and 18 (C55H84O28) were related to those of 110.6 (C-22)] were observed. These data suggested that 21 was
4, 8, and 9, respectively. However, instead of containing a 22-hydroxyfurostanol glycoside and its enzymatic hydrolysis
three-proton doublet signals for Me-27, as observed in the with naringinase yielded (25R)-3β-hydroxyspirost-5-en-1β-yl
1H- and 13C-NMR spectra of 4, 8, and 9, signals arising α-L-arabinopyranoside (21a)22) and D-glucose. In the as-
from an exomethylene group [δH 5.23 (brs) and 5.08 (brs) sociated HMBC spectrum, a long-range correlation was
(16); 5.05 (brs) and 4.95 (brs) (17); 5.20 (brs) and 5.06 (brs) observed between H-1″ of the β-D-glucopyranosyl unit (δH
(18); δC 113.7 (16); 112.3 (17); 113.9 (18)] and quaternary 4.79) and C-26 of the aglycone (δC 75.2). Accordingly, 21
carbon [δC 143.8 (16); 146.2 (17); 143.7 (18)] were observed. was identified as (25R)-26-[(β-D-glucopyranosyl)oxy]-3β,22α-
Therefore, 16, 17, and 18 correspond to the C-25/27 dehy- dihydroxyfurost-5-en-1β-yl α-L-arabinopyranoside.
droxy derivatives of 4, 8, and 9, respectively, as addition-
Compounds 12, 13, and 15 are spirostanol trisdesmosides,
ally confirmed by long-range correlations from H2-27 to which are types of steroidal glycosides rarely observed in the
C-24, C-25, and C-26 in the associated HMBC spectra. plant kingdom.
Therefore, 16, 17, and 18 were identified as (23S,24S)-24-[(β-
The cytotoxic activities of 1–28 were evaluated against
D-fucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-dien- HL-60 and A549 cells using the 3-(4,5-dimethylthiazol-
1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamno- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method.
pyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabino- Compound 7 showed cytotoxic activity against HL-60 and
pyranoside,(23S,24S)-3β,21,23,24-tetrahydroxyspirosta-5,25(27)- A549 cells with IC50 values of 5.9 0.46 and 6.6 0.17 µM,
dien-1β-yl
O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L- respectively. In contrast, 19 was selectively cytotoxic against
rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L- only A549 cells with an IC50 value of 5.5 0.61 µM. Cisplatin
arabinopyranoside, and (23S,24S)-24-[(α-L-arabinopyranosyl)- was used as a positive control, yielding IC50 values of 1.9 and
oxy]-3β,21,23-trihydroxyspirosta-5,25(27)-dien-1β-yl
O-β-D- 3.7 µM for HL-60 and A549 cells, respectively. Compounds 1–
apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)- 6, 8–18, and 20–28 did not show cytotoxicity against HL-60
(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, and A549 cells at concentrations of up to 10µM.
respectively.
1
Comparison of the H- and 13C-NMR spectra 19 (C57H86O29)

Vol. 68, No. 3 (2020)
Chem. Pharm. Bull.
285
eluted with MeOH/H2O (3:2) and MeCN/H2O (1:3; 1:4), and
Experimental
General Optical rotations were measured on a JASCO preparative HPLC using MeCN/H2O (1:2; 3:7) to furnish 4
P-1030 automatic digital polarimeter (Jasco, Tokyo, Japan). (72.1mg), 5 (2.3mg), 7 (30.1mg), 8 (13.0mg), 20 (8.5mg), and
IR spectra were obtained using a JASCO FT-IR 620 spec- 26 (85.4mg). Fraction 14 was fractionated by ODS silica gel
trophotometer (Jasco). NMR spectra were recorded on a CC using MeOH/H2O (3:2; 11:9; 1:1) and MeCN/H2O (1:3;
Bruker DRX-500 or AV-600 spectrometer (Bruker, Karlsruhe, 1:4), and preparative HPLC using MeCN/H2O (3:7; 1:3) to
Germany) using standard Bruker pulse programs. Chemi- acquire 16 (16.2mg), 17 (6.1mg), 19 (28.6mg), 22 (16.6mg),
cal shifts (δ) were given with reference to tetramethylsilane and 27 (4.9mg). Fraction 15 was separated by ODS silica
(TMS) as an internal standard. HR-ESI-TOF-MS data were gel CC eluted with MeOH/H2O (11:9; 1:1) and MeCN/H2O
obtained using a Water-Micromass LCT mass spectrometer (1:3; 1:4), and preparative HPLC using MeCN/H2O (1:3) to
(Waters-Micromass, Manchester, U.K.). CC was conducted by afford 9 (26.9mg) and 10 (6.8mg). Fraction 16 was chromato-
Diaion HP-20 (Mitsubishi-chemical, Tokyo, Japan), silica gel graphed on ODS silica gel using MeOH/H2O (11:9; 9:11) and
Chromatorex BW-300 (Fuji-Silysia Chemical, Aichi, Japan), MeCN/H2O (1:3; 1:4) to collect 11 (3.9mg) and 18 (46.3mg).
and ODS silica gel COSMOSIL 75C18-OPN (Nacalai Tesque, Fraction 17 was subjected to silica gel CC eluted with CHCl3/
Kyoto, Japan). TLC was carried out precoated silica gel 60F254 MeOH/H2O (20:10:1; 10:10:1), ODS silica gel CC eluted
or RP18 F254S plates (0.25mm thick) (Merck, Darmstadt, Ger- with MeOH/H2O (1:1) and MeCN/H2O (1:3; 1:4), and pre-
many), and the spots were detected by spraying the plates parative HPLC using MeCN/H2O (1:3; 1:4) to furnish 12
with 10% H2SO4 aqueous solution and then heating. HPLC (12.9mg), 13 (7.0mg), 14 (19.9mg), and 15 (15.9mg).
was conducted using a system consisting of a CCPM pump
Compound 1 Amorphous powder; [α]D25 −94.4 (c=0.10,
(Shimadzu, Kyoto, Japan), an RI-8021 (Tosoh, Tokyo, Japan) MeOH); IR νmax (film) cm−1: 3392 (OH), 2926 (CH), 1729
or a Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector, and (C=O); 1H- (500MHz, C5D5N) and 13C-NMR (125MHz,
a Rheodyne injection port (Rohnert Park, CA, U.S.A.). A TSK C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
gel ODS-100Z column (10mm i.d. ×250mm, 5µm) (Tosoh) (500MHz, C5D5N) and 13C-NMR (125MHz, C5D5N) data
was used for the preparative HPLC. Purities of the isolated of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
compounds were verified by MS, TLC, and NMR spectra. TOF-MS m/z: 937.4412 [M+Na]+ (Calcd for C45H70NaO19:
The following materials and biochemical grade reagents were 937.4409).
used for the cell cultures and cytotoxicity assays: SH-1300
Compound 2 Amorphous powder; [α]D25 −14.5 (c=0.05,
Lab microplate reader (CORONA ELECTRIC, Ibaraki, Japan); MeOH); IR νmax (film) cm−1: 3370 (OH), 2925 (CH), 1730
96-well flat-bottom plate (Iwaki Glass, Chiba, Japan); fetal bo- (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
vine serum (FBS), 0.25% trypsin-ethylenediaminetetraacetic C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
acid (EDTA) solution, RPMI-1640 medium, minimum essen- (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
tial medium (MEM), cisplatin, and MTT (Sigma, St. Louis, of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
MO, U.S.A.); paraformaldehyde and phosphate-buffered saline TOF-MS m/z: 1083.4980 [M+Na]+ (Calcd for C51H80NaO23:
(PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, 1083.4988).
Japan); penicillin G sodium salt and streptomycin sulfate
Compound 3 Amorphous powder; [α]D25 −23.4 (c=0.10,
(Gibco, Gland Island, NY, U.S.A.); HL-60 cells (JCRB0085) MeOH); IR νmax (film) cm−1: 3375 (OH), 2930 (CH), 1727
and A549 cells (JCRB0076) (Human Science Research Re- (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
sources Bank, Osaka, Japan).
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
Plant Material The whole plants of H. foetidus were pur- (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
chased from Fuji-engei (Okayama, Japan). A voucher speci- of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
men was deposited at the herbarium of the Tokyo University TOF-MS m/z: 1069.4832 [M+Na]+ (Calcd for C50H78NaO23:
of Pharmacy and Life Sciences (KS-2014-011).23)
1069.4832).
Extraction and Isolation The whole plants of H. foetidus
Compound 4 Amorphous powder; [α]D25 −31.7 (c=0.11,
(fresh weight, 3.3kg) were extracted with hot MeOH (10L). MeOH); IR νmax (film) cm−1: 3375 (OH), 2927 (CH), 1727
After solvent was removed in vacuo, the concentrated MeOH (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
extract (115g) was fractionated by Diaion HP-20 column and C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
then successively eluted with MeOH/H2O (3:7), MeOH/H2O (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
(1:1), MeOH, EtOH, and EtOAc (each solvent volume 6L). The of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
MeOH eluate fraction (10g) was chromatographed on silica gel TOF-MS m/z: 1215.5405 [M+Na]+ (Calcd for C56H88NaO27:
eluted with stepwise gradient of CHCl3/MeOH/H2O (90:10:1, 1215.5411).
40:10:1, 20:10:1) and finally MeOH alone to get 17 fractions
Compound 5 Amorphous powder; [α]D25 −8.7 (c=0.14,
(Frs. 1–17). Fraction 10 was subjected to silica gel CC eluted MeOH); IR νmax (film) cm−1: 3374 (OH), 2925 (CH); 1H-
with CHCl3/MeOH/H2O (90:10:1; 40:10:1) and EtOAc/ (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data of
1
MeOH/H2O (80:10:1; 50:10:1; 40:10:1), and ODS silica the aglycone moiety, see Tables 1, 2; H- (600MHz, C5D5N)
gel CC eluted with MeOH/H2O (3:2; 1:1) and MeCN/H2O and 13C-NMR (150MHz, C5D5N) data of the sugar moieties,
(7:3; 1:1; 3:7; 1:3) to yield 1 (35.7mg), 21 (85.4mg), 23 see Tables 3, 4; HR-ESI-TOF-MS m/z: 1057.4819 [M+Na]+
(72.1mg), and 24 (2.9mg). Fraction 11 was separated by ODS (Calcd for C49H78NaO23: 1057.4832).
silica gel CC using MeOH/H2O (4:1; 3:2) and MeCN/H2O
Compound 6 Amorphous powder; [α]D25 −30.5 (c=0.05,
(1:3) to obtain 2 (5.8mg), 3 (27.0mg), 6 (3.3mg), 25 (18.1mg), MeOH); IR νmax (film) cm−1: 3364 (OH), 2925 (CH), 1725
and 28 (6.7mg). Fraction 13 was chromatographed on silica (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
gel eluted with CHCl3/MeOH/H2O (30:10:1), ODS silica gel C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-

286
Chem. Pharm. Bull.
Vol. 68, No. 3 (2020)
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 953.4354 [M+Na]+ (Calcd for C45H70NaO20: (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
953.4358).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 7 Amorphous powder; [α]D25 −42.0 (c=0.10, TOF-MS m/z: 1409.5863 [M+Na]+ (Calcd for C62H98NaO34:
MeOH); IR νmax (film) cm−1: 3383 (OH), 2922 (CH), 1728 1409.5837).
(C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
Compound 15 Amorphous powder; [α]D25 −32.0 (c=0.10,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3373 (OH), 2931 (CH), 1731
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1099.4934 [M+Na]+ (Calcd for C51H80NaO24: (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
1099.4937).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 8 Amorphous powder; [α]D25 −13.9 (c=0.10, TOF-MS m/z: 1555.6422 [M+Na]+ (Calcd for C68H108NaO38:
MeOH); IR νmax (film) cm−1: 3389 (OH), 2924 (CH), 1729 1555.6416).
(C=O); 1H- (500MHz, C5D5N) and 13C-NMR (125MHz,
Compound 16 Amorphous powder; [α]D25 −69.1 (c=0.10,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3389 (OH), 2932 (CH), 1732
(500MHz, C5D5N) and 13C-NMR (125MHz, C5D5N) data (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1085.4775 [M+Na]+ (Calcd for C50H78NaO24: (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
1085.4781).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 9 Amorphous powder; [α]D25 −62.5 (c=0.10, TOF-MS m/z: 1213.5251 [M+Na]+ (Calcd for C56H86NaO27:
MeOH); IR νmax (film) cm−1: 3388 (OH), 2925 (CH), 1731 1213.5254).
(C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
Compound 17 Amorphous powder; [α]D25 −94.4 (c=0.05,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3375 (OH), 2925 (CH), 1731
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1217.5203 [M+Na]+ (Calcd for C55H86NaO28: (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
1217.5203).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 10 Amorphous powder; [α]D25 −41.9 (c=0.05, TOF-MS m/z: 1083.4623 [M+Na]+ (Calcd for C50H76NaO24:
MeOH); IR νmax (film) cm−1: 3389 (OH), 2925 (CH), 1731 1083.4624).
(C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
Compound 18 Amorphous powder; [α]D25 −125.4 (c=0.10,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3404 (OH), 2923 (CH), 1728
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (500MHz, C5D5N) and 13C-NMR (125MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1231.5364 [M+Na]+ (Calcd for C56H88NaO28: (500MHz, C5D5N) and 13C-NMR (125MHz, C5D5N) data
1231.5360).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 11 Amorphous powder; [α]D25 −8.8 (c=0.05, TOF-MS m/z: 1215.5081 [M+Na]+ (Calcd for C55H84NaO28:
MeOH); IR νmax (film) cm−1: 3357 (OH), 2926 (CH), 1731 1215.5047).
(C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
Compound 19 Amorphous powder; [α]D25 −110.3 (c=0.10,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3393 (OH), 2924 (CH), 1733
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (500MHz, C5D5N) and 13C-NMR (125MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1247.5299 [M+Na]+ (Calcd for C56H88NaO29: (500MHz, C5D5N) and 13C-NMR (125MHz, C5D5N) data
1247.5309).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 12 Amorphous powder; [α]D25 −22.7 (c=0.10, TOF-MS m/z: 1257.5111 [M+Na]+ (Calcd for C57H86NaO29:
MeOH); IR νmax (film) cm−1: 3357 (OH), 2930 (CH), 1732 1257.5152).
(C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
Compound 20 Amorphous powder; [α]D25 −43.6 (c=0.025,
C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H- MeOH); IR νmax (film) cm−1: 3406 (OH), 2923 (CH), 1720
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data (C=O); 1H- (600MHz, C5D5N) and 13C-NMR (150MHz,
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI- C5D5N) data of the aglycone moiety, see Tables 1, 2; 1H-
TOF-MS m/z: 1379.5753 [M+Na]+ (Calcd for C61H96NaO33: (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data
1379.5732).
of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-
Compound 13 Amorphous powder; [α]D25 −17.7 (c=0.10, TOF-MS m/z: 1125.4730 [M+Na]+ (Calcd for C52H78NaO25:
MeOH); IR νmax (film) cm−1: 3364 (OH), 2927 (CH); 1H- 1125.4730).
(600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data of
Compound 21 Amorphous powder; [α]D25 −10.7 (c=0.11,
the aglycone moiety, see Tables 1, 2; H- (600MHz, C5D5N) MeOH); IR νmax (film) cm−1: 3364 (OH), 2925 (CH); 1H-
1
and 13C-NMR (150MHz, C5D5N) data of the sugar moieties, (600MHz, C5D5N) and 13C-NMR (150MHz, C5D5N) data of
see Tables 3, 4; HR-ESI-TOF-MS m/z: 1381.5883 [M+Na]+ the aglycone moiety, see Tables 1, 2; H- (600MHz, C5D5N)
1
(Calcd for C61H98NaO33: 1381.5888).
and 13C-NMR (150MHz, C5D5N) data of the sugar moieties,
Compound 14 Amorphous powder; [α]D25 −34.1 (c=0.10, see Tables 3, 4; HR-ESI-TOF-MS m/z: 765.4035 [M+Na]+
MeOH); IR νmax (film) cm−1: 3360 (OH), 2925 (CH), 1730 (Calcd for C38H62NaO14: 765.4037).

Vol. 68, No. 3 (2020)
Chem. Pharm. Bull.
287
Acid Hydrolysis of 1–20 Compound 1 (10.0mg), 2
Supplementary Materials The online version of this ar-
(2.0mg), 3 (3.6mg), 4 (4.0mg), 5 (1.9mg), 6 (1.1mg), 7 ticle contains supplementary materials.
(10.0mg), 8 (5.0mg), 9 (5.5mg), 10 (2.3mg), 11 (2.5mg), 12
(4.7mg), 13 (4.0mg), 14 (5.0mg), 15 (5.0mg), 16 (4.8mg), 17
(2.4mg), 18 (10.0mg), 19 (5.0mg), and 20 (2.8mg) were inde-
pendently dissolved in 0.2M HCl or 0.5M HCl (dioxane/H2O,
1:1) and heated at 95°C for 30min under an Ar atmosphere.
Each reaction mixture was neutralized by passing through
an Amberlite IRA-96 (Organo, Tokyo, Japan) column, was
subjected to Diaion HP-20 CC eluted with MeOH/H2O (2:3)
and finally Me2CO/EtOH (1:1) to obtain sugar fractions
(3.3mg from 1; 0.79mg from 2; 1.4mg from 3; 2.9mg from 4;
0.77mg from 5; 0.38mg from 6; 4.5mg from 7; 2.8mg from 8;
2.3mg from 9; 0.92mg from 10; 0.8mg from 11; 2.3mg from
12; 2.0mg from 13; 2.5mg from 14; 2.6mg from 15; 2.1mg
from 16; 0.94mg from 17; 3.8mg from 18; 1.9mg from 19;
and 1.0mg from 20). The sugar fractions were analyzed by
HPLC under the following conditions: column, Capcell Pak
NH2 UG80 (4.6mm i.d. ×250mm, 5µm, Shiseido, Tokyo,
Japan); solvent, MeCN/H2O (17:3); detection, refractive index
and optical rotation; flow rate, 1.0mL/min. D-Apiose in 3, 4,
8–12, and 14–19, L-arabinose in 1–20, D-fucose in 4, 7, 10, 13,
15, and 16, D-galactose in 11–15, D-glucose in 5 and 13–15,
D-quinovose in 2, L-rhamnose in 1–20, and D-xylose in 1–20
were identified by comparing their retention times (tR) and op-
tical rotations with those of authentic samples: D-apiose (6.66,
positive optical rotation), L-rhamnose (7.64, negative optical
rotation), D-fucose (8.42, positive optical rotation), D-quino-
vose (8.78, positive optical rotation), L-arabinose (9.57, posi-
tive optical rotation), D-xylose (9.92, positive optical rotation),
D-galactose (15.63, positive optical rotation), and D-glucose
(16.14, positive optical rotation).
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Conflict of Interest The authors declare no conflict of
interest.