Steroidal glycosides from the bulbs of Lilium speciosum

Article abstract of DOI:10.1016/j.phytol.2020.01.008

Steroidal glycosides (1-21), including eight new spirostanol glycosides (1-8) and a new cholestane glycoside (9), were isolated from the bulbs of Lilium speciosum. The structures of the new compounds were determined based on spectroscopic data analysis and hydrolytic cleavage reactions. The isolated compounds and their aglycones (1a, 8a, and 9a) were evaluated for their cytotoxicity against HL-60 human acute promyelocytic leukemia cells and A549 human lung carcinoma cells. Compounds 12 and 21 exhibited moderate cytotoxic activity toward HL-60 and A549 cells, with IC₅₀ values of 6.9 μM and 5.1 μM against HL-60 cells, and 7.6 μM and 4.4 μM against A549 cells, respectively. Compound 20 only showed moderate cytotoxicity toward A549 cells, with an IC₅₀ value of 6.7 μM.

Full text of DOI:10.1016/j.phytol.2020.01.008

PhytochemistryLetters37(2020)21–28
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Steroidal glycosides from the bulbs of Lilium speciosum
Tomoki Iguchi*, Akihito Yokosuka, Minpei Kuroda, Mayuko Takeya, Mizuki Hagiya,
Yoshihiro Mimaki
Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Steroidal glycosides (1-21), including eight new spirostanol glycosides (1-8) and a new cholestane glycoside (9),
were isolated from the bulbs of Lilium speciosum. The structures of the new compounds were determined based
on spectroscopic data analysis and hydrolytic cleavage reactions. The isolated compounds and their aglycones
(1a, 8a, and 9a) were evaluated for their cytotoxicity against HL-60 human acute promyelocytic leukemia cells
and A549 human lung carcinoma cells. Compounds 12 and 21 exhibited moderate cytotoxic activity toward HL-
60 and A549 cells, with IC50 values of 6.9 μM and 5.1 μM against HL-60 cells, and 7.6 μM and 4.4 μM against
A549 cells, respectively. Compound 20 only showed moderate cytotoxicity toward A549 cells, with an IC50 value
of 6.7 μM.
Lilium speciosum
Liliaceae
Bulb
Spirostanol glycoside
Cholestane glycoside
Cytotoxic activity
1. Introduction
subjected to a Diaion HP-20 porous-polymer polystyrene resin column,
and successively eluted with 30 % MeOH, 50 % MeOH, MeOH, EtOH,
Plants belonging to the genus Lilium are rich sources of phenolic and
steroidal glycosides (Mimaki et al., 1989; Mimaki and Sashida, 1990a,
b, c; Mimaki et al., 1993, 1994; Satou et al., 1996; Mimaki et al., 1999;
Munafo et al., 2010; Hong et al., 2012; Zhou et al., 2012; Matsuo et al.,
2015; Thi et al., 2019). Previously, we have performed chemical ex-
aminations of the bulbs of Lilium speciosum var. rubrum and L. speciosum
froma vestale, and isolated a series of phenylpropanoid sucrose esters, a
glycerol glucoside, and steroidal glycosides (Shimomura et al., 1986;
Mimaki and Sashida, 1991). However, no phytochemical study of the
bulbs of Lilium speciosum species was conducted. L. speciosum is a per-
ennial plant that is mainly distributed in west Japan and is also called
the “Japanese lily” (Tsukamoto, 1989). This phytochemical investiga-
tion of L. speciosum bulbs focused on its steroidal constituents, and re-
sulted in the isolation of 21 steroidal glycosides (1-21), including eight
new spirostanol glycosides (1-8) and a new cholestane glycoside (9).
The structures of the new compounds were determined by spectro-
scopic data analysis and hydrolytic cleavage reactions. The isolated
compounds (1-21) and their aglycones (1a, 8a, and 9a) were evaluated
for their cytotoxic activity against HL-60 human acute promyelocytic
leukemia cells and A549 human lung carcinoma cells.
and EtOAc. The MeOH eluted portion (45 g) was repeatedly fractio-
nated by column chromatography (CC) on silica gel and octadecylsi-
lanized (ODS) silica gel, and by preparative ODS HPLC to obtain 21
steroidal glycosides (1-21). The structures of the known compounds
were identified as (25R,26R)-17α-hydroxy-26-methoxyspirost-5-en-3β-
yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (10) (Mimaki
et al., 1998), (25S)-17α,27-dihydroxyspirost-5-en-3β-yl O-α-L-rhamno-
pyranosyl-(1→2)-β-D-glucopyranoside (11) (Ono et al., 2007), (25R)-
spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside
(12) (Han et al., 1999), (25R,26R)-26-methoxyspirost-5-en-3β-yl O-α-L-
rhamnopyranosyl-(1→2)-β-D-glucopyranoside (13) (Mimaki and
Sashida, 1991), (25S)-27-hydroxyspirost-5-en-3β-yl O-α-L-rhamnopyr-
anosyl-(1→2)-β-D-glucopyranoside (14) (Mimaki and Sashida, 1990a),
(25S)-27-hydroxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-O-
[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside (15) (Mimaki and
Sashida, 1990b), (25S)-27-hydroxyspirost-5-en-3β-yl O-β-D-glucopyr-
anosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-
(1→4)]-β-D-glucopyranoside (16) (Mimaki et al., 1998), (25R)-27-
[((3S)-3-hydroxy-3-methylglutaroyl)oxy]-spirost-5-en-3β-yl
O-α-L-
rhamnopyranosyl-(1→2)-β-D-glucopyranoside (17) (Mimaki et al.,
1993), (25R)-27-[((3S)-3-hydroxy-3-methylglutaroyl)oxy]-spirost-5-en-
3β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-
D-glucopyranoside (18) (Mimaki et al., 1993), (25R)-3-[(O-α-L-rham-
nopyranosyl-(1→2)-β-D-glucopyranosyl)oxy]-5α-spirostan-6-one (19)
(Matsuo et al., 2013), (25R)-26-[(β-D-glucopyranosyl)oxy]-furosta-
2. Results and discussion
L. speciosum bulbs (fresh weight, 8.7 kg) were extracted with hot
MeOH. After removing the solvent, the MeOH extract (340 g) was
⁎ Corresponding author.
E-mail address: iguchit@toyaku.ac.jp (T. Iguchi).
https://doi.org/10.1016/j.phytol.2020.01.008
Received 26 September 2019; Received in revised form 19 December 2019; Accepted 15 January 2020
1874-3900/©2020PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
Fig. 1. Structures of 1, 1a, 2-8, 8a, 9, 9a, and 10-21.
5,20(22)-dien-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyrano-
side (20) (Dong et al., 2001), and (25R)-26-[(β-D-glucopyranosyl)oxy]-
furosta-5,20(22)-dien-3β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glu-
copyranosyl-(1→4)]-β-D-glucopyranoside (21) (Ali et al., 2013), re-
spectively (Fig. 1).
Compound 2 (C51H82O24) was obtained as an amorphous powder,
and its 1H- and 13C-NMR spectra were indicative of the aglycone moiety
of 2 being the same as that of 1. However, the molecular formula of 2
was larger than that of 1 by C6H10O5, which corresponded to one
hexosyl unit. Analysis of the 1H-1H COSY, total correlation spectroscopy
(TOCSY), HSQC, and HSQC-TOCSY spectra of the sugar moiety of 2
revealed that it was the configuration of a 2,4-disubstituted β-D-gluco-
pyranosyl unit [Glc (I)], 3-monosubstituted α-L-rhamnopyranosyl unit
(Rha), and two terminal β-D-glucopyranosyl units [Glc (II) and Glc
(III)]. The HMBC spectrum of 2 exhibited long-range correlations be-
tween H-1ʹʹʹʹ of Glc (III) (δH 5.52) and C-3ʹʹ of Rha (δC 84.0), H-1ʹʹ of
Rha (δH 6.20) and C-2ʹ of Glc (I) (δC 77.3), H-1ʹʹʹ of Glc (II) (δH 5.13)
and C-4ʹ of Glc (I) (δC 82.0), and between H-1ʹ of Glc (I) (δH 4.90) and
C-3 of the aglycone (δC 78.3). Therefore, 2 was identified as (25S)-
Compound 1 was isolated as an amorphous powder. Its molecular
formula was assigned as C45H72O19 based on the high-resolution elec-
trospray ionization time-of-flight mass spectroscopy (HRESITOFMS)
and 13C-NMR spectral data. The 1H-NMR spectral features of the agly-
cone moiety of 1 were closely related to those of 11, showing signals for
two angular methyl groups at δH 1.05 (3H, s, Me-19) and 0.95 (3H, s,
Me-18), a secondary methyl group at δH 1.23 (3H, d, J = 7.1 Hz, Me-
21), two oxymethylene groups at δH 4.06 (1H, dd, J = 11.3, 4.7 Hz, H-
26a) and 3.88 (1H, dd, J = 11.3, 11.3 Hz, H-26b), and δH 3.72 (1H, dd,
J = 10.6, 5.2 Hz, H-27a) and 3.63 (1H, dd, J = 10.6, 7.4 Hz, H-27b),
two oxymethine protons at δH 4.47 (1H, dd, J = 7.1, 6.7 Hz, H-16) and
3.82 (1H, m, W1/2 = 20.7 Hz, H-3), and an olefinic proton at δH 5.27
(1H, br d, J = 4.1 Hz, H-6). Enzymatic hydrolysis of 1 with naringinase
afforded (25S)-spirost-5-ene-3β,17α,27-triol (1a) (Yokosuka and
Mimaki, 2008) as the aglycone, and D-glucose and L-rhamnose as the
carbohydrate moieties. The 1H-1H correlation spectroscopy (COSY) and
heteronuclear single quantum coherence (HSQC) spectra of 1 allowed
the sequential assignments of the 1H- and 13C-NMR signals for each
sugar unit, indicating that the sugar moiety of 1 comprised a 2,4-dis-
ubstituted inner β-D-glucopyranosyl unit [Glc (I): δH 4.91 (1H, d, J =
7.6 Hz, H-1ʹ); δC 99.9, 77.3, 77.6, 81.9, 76.1, and 61.8 (C-1ʹ-6ʹ)], a
terminal α-L-rhamnopyranosyl unit [Rha: δH 6.20 (1H, br s, H-1ʹʹ); δC
101.7, 72.3, 72.7, 74.0, 69.4, and 18.6 (C-1ʹʹ-6ʹʹ)], and a terminal β-D-
glucopyranosyl unit [Glc (II): δH 5.12 (1H, d, J = 7.8 Hz, H-1ʹʹʹ); δC
105.0, 74.9, 78.1, 71.2, 78.4, and 62.0 (C-1ʹʹʹ-6ʹʹʹ)]. In the hetero-
nuclear multiple bond correlation (HMBC) spectrum of 1, long-range
correlations were observed between H-1ʹʹ of Rha (δH 6.20) and C-2ʹ of
Glc (I) (δC 77.3), H-1ʹʹʹ of Glc (II) (δH 5.12) and C-4ʹ of Glc (I) (δC 81.9),
and between H-1ʹ of Glc (I) (δH 4.91) and C-3 of the aglycone (δC 78.2)
in 1. Thus, 1 was deduced as (25S)-17α,27-dihydroxyspirost-5-en-3β-yl
O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-D-glu-
copyranoside.
17α,27-dihydroxyspirost-5-en-3β-yl
O-β-D-glucopyranosyl-(1→3)-O-
α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-D-gluco-
pyranoside.
The 1H- and 13C-NMR spectral data of 3 (C45H70O18) implied that 3
was analogous to 11, including the diglycoside moiety attached to C-3
of the aglycone. However, the 13C-NMR spectrum suggested that a
substituent with six carbon atoms corresponding to the 3-hydroxy-3-
methylglutaroyl (HMG) group was present in 3; the signals arising from
the HMG group included a methyl carbon at δC 28.3 (C-6ʹʹʹ), two me-
thylene carbons at δC 46.4 (C-2ʹʹʹ) and 46.5 (C-4ʹʹʹ), a quaternary carbon
with a hydroxy group at δC 70.0 (C-3ʹʹʹ), the carbonyl carbon of a
carboxy group at δC 174.6 (C-5ʹʹʹ), and an ester carbonyl carbon at δC
171.6 (C-1ʹʹʹ). The HMG group was ascertained to be linked to C-27 of
3
the aglycone based on a JC,H correlation between H2-27 of the agly-
cone at δH 4.07 and 4.00 and C-1ʹʹʹ of the HMG group at δC 171.6. Thus,
3 was identified as (25R)-17α-hydroxy-27-[(3-hydroxy-3-methylglu-
taroyl)oxy]-spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glu-
copyranoside.
Compound 4 (C51H80O22) was shown to be a steroidal glycoside
with an HMG group at the C-27 hydroxy group of the aglycone based on
its 1H- and 13C-NMR spectra, as in the case of 3. Its molecular formula
was the same as that of 18, and the spectroscopic features of 4 were
very similar to those of 18, except for the signals attributable to the
22

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
Fig. 2. Key NOE correlations of the A-, B-, C-, D-, and E-ring parts of 8a.
triglycoside moiety attached to C-3 of the aglycone. The 1H-1H COSY
and HSQC spectra indicated that 4 contained a 2,6-disubstituted inner
β-D-glucopyranosyl unit [Glc (I): δH 4.97 (1H, d, J = 7.3 Hz, H-1ʹ); δC
100.7, 77.5, 79.6, 71.6, 76.9, and 70.0 (C-1ʹ-6ʹ)], a terminal α-L-
rhamnopyranosyl unit [Rha: δH 6.34 (1H, br s, H-1ʹʹ); δC 102.0, 72.5,
72.8, 74.2, 69.5, and 18.7 (C-1ʹʹ-6ʹʹ)], and a terminal β-D-glucopyr-
anosyl unit [Glc (II): δH 5.10 (1H, d, J = 7.8 Hz, H-1ʹʹʹ); δC 105.4, 75.2,
78.4, 71.7, 78.5, and 62.7 (C-1ʹʹʹ-6ʹʹʹ)]. In the HMBC spectrum of 4,
long-range correlations were observed between H-1ʹʹʹ of Glc (II) (δH
5.10) and C-6ʹ of Glc (I) (δC 70.0), H-1ʹʹ of Rha (δH 6.34) and C-2ʹ of Glc
(I) (δC 77.5), and between H-1ʹ of Glc (I) (δH 4.97) and C-3 of the
aglycone (δC 78.4). Therefore, 4 was elucidated as (25R)-27-[(3-hy-
as (25S,26R)-26-methoxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-
(1→2)-β-D-glucopyranoside.
The chemical formula of 7 (C39H62O13) was the same as that of 14,
the spectral features of 7 were closely related to those of 14, suggesting
that 7 was a stereoisomer of 14 with regard to the configuration at C-
25. The nuclear Overhauser enhancement spectroscopy (NOESY)
spectrum of 7 showed NOE correlations between H-20 (δH 1.92) and
Me-18 (δH 0.81)/H-23ax (δH 1.51), and between H-23ax and H2-27 (δH
4.16 and 3.97). Accordingly, 7 was elucidated as (25R)-27-hydro-
xyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyrano-
side.
Compound 8 (C45H72O19) was isolated as an amorphous powder.
The 1H-NMR spectrum of 8 exhibited signals for two angular methyl
groups [δH 0.88 (3H, s, Me-18) and 0.78 (3H, s, Me-19)], two secondary
methyl groups [δH 1.21 (3H, d, J = 7.2 Hz, Me-21) and 0.67 (3H, d, J
= 5.4 Hz, Me-27)], and three anomeric protons [δH 6.18 (1H, br s),
5.10 (1H, d, J = 7.9 Hz), and 4.93 (1H, d, J = 7.3 Hz)]. The IR
spectrum (1710 cm−1) and 13C-NMR spectrum (δC 209.4) indicated the
presence of a carbonyl group in 8. Enzymatic hydrolysis of 8 with
naringinase yielded the aglycone (8a), and D-glucose and L-rhamnose as
the carbohydrate moieties. The 1H- and 13C-NMR spectra of 8a re-
sembled those of (25R)-3β-hydroxy-5α-spirostan-6-one (laxogenin)
(Baba et al., 2000), except for the signals arising from the D- and E-ring
parts of the steroid skeleton. The molecular formula of 8a was one
oxygen atom more than that of laxogenin, and an oxygenated qua-
ternary carbon (δC 89.8) was observed in the 13C-NMR spectrum of 8a.
In the HMBC spectrum of 8a, long-range correlations were observed
from H-14 (δH 2.29), H-15α (δH 2.12), H-16 (δH 4.44), Me-18 (δH 0.93),
H-20 (δH 2.27), Me-21 (δH 1.24) to δC 89.8, which was assigned to C-17.
When the 13C-NMR spectrum of 8a was compared to that of laxogenin,
C-13, C-16, and C-20 were shifted downfield, whereas C-14 and C-21
were shifted upfield. The above data indicated the presence of the C-
17α hydroxy group. The C-3β and C-17α configurations, and A/B-trans
ring junction of 8a were ascertained by NOE correlations, as shown in
Fig. 2. Thus, 8a was established as (25R)-3β,17α-dihydroxy-5α-spiro-
stan-6-one. As for the sugar moiety of 8, the 1H- and 13C-NMR, and
HMBC spectra indicated that the triglycoside linked to C-3 of the
aglycone was the same as that of 1. Compound 8 was determined as
(25R)-17α-hydroxy-3-[(O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-gluco-
pyranosyl-(1→4)]-β-D-glucopyranosyl)oxy]-5α-spirostan-6-one.
The 1H-NMR spectrum of 9 (C45H74O18) exhibited signals for four
methyl groups [δH 1.10 (3H, d, J = 6.9 Hz, Me-21), 1.09 (3H, d, J =
6.7 Hz, Me-27), 0.77 (3H, s, Me-19) and 0.56 (3H, s, Me-18)] and three
anomeric protons [δH 6.20 (1H, d, J = 1.2 Hz), 5.12 (1H, d, J = 7.9
Hz), and 4.96 (1H, d, J = 7.2 Hz)]. The IR spectrum (1708 cm−1) and
13C-NMR spectrum (δC 213.9 and 209.5) indicated the presence of two
carbonyl groups in 9. Enzymatic hydrolysis of 9 with naringinase gave
droxy-3-methylglutaroyl)oxy]-spirost-5-en-3β-yl
O-α-L-rhamnopyr-
anosyl-(1→2)-O-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside.
The 1H- and 13C-NMR spectral data of 5 (C57H90O27) showed close
similarity to those of 18, including the signals attributed to the trigly-
coside moiety and HMG group, which were attached to C-3 and C-27 of
the aglycone, respectively. However, the molecular formula of 5 was
larger than that of 18 by C6H10O5, and the presence of one more
terminal β-D-glucopyranosyl unit [Glc (III): δH 5.28 (1H, d, J = 7.9 Hz,
H-1ʹʹʹʹʹ); δC 98.5, 75.3, 78.4, 71.6, 78.3, and 63.0 (C-1ʹʹʹʹʹ-6ʹʹʹʹʹ)] in 18
was verified based on 1H- and 13C-NMR spectral analysis. On compar-
ison of the 13C-NMR spectrum of 5 with that of 18, it was found that the
signal assigned to C-3ʹʹʹʹ of the HMG moiety was shifted downfield by
6.8 ppm, whereas those assigned to C-2ʹʹʹʹ and C-4ʹʹʹʹ had moved upfield
3
by 2.4 and 2.1 ppm, respectively. Furthermore, a JC,H correlation was
observed between H-1ʹʹʹʹʹ of Glc (III) (δH 5.28) and C-3ʹʹʹʹ of the HMG
moiety (δC 76.7). These data implied that C-3ʹʹʹʹ of the HMG moiety was
the position where the additional β-D-glucopyranosyl unit was linked.
Thus, 5 was established as (25R)-[[3-(β-D-glucopyranosyl)oxy-3-me-
thylglutaroyl]oxy]-spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-
O-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside. The absolute con-
figuration of the HMG moiety in 3-5 have not yet been determined
owing to their low yields. In the previous reports (Hong et al., 2012;
Mimaki et al., 1993), the absolute configuration of the HMG moiety of
related steroidal glycosides isolated from L. brownii var. viridulum, and
L. regale and L. henryi was determined to be S. Therefore, the config-
uration of the HMG moiety in 3-5 was also speculated to be S.
Compound 6 (C40H64O13) was obtained as an amorphous powder
and had the same molecular formula as 13. The 1H- and 13C-NMR
spectral properties of 6 were very similar to those of 13, except for the
signals arising from the F-ring, suggesting that 6 was a stereoisomer of
13 at C-25. In the rotatory-frame Overhauser enhancement spectro-
scopy (ROESY) spectrum of 6, ROE correlations were observed between
H-23ax (δH 1.85) and H-20 (δH 1.96)/H-24eq (δH 1.46)/Me-27 (δH
1.09), Me-27 and H-24eq, and between H-24ax (δH 2.12) and H-23eq
(δH 1.40)/H-25 (δH 1.93)/H-26 (δH 4.88). Therefore, 6 was formulated
23

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
Fig. 3. Key HMBC correlations of 1-8, 8a, and 9.
(25R)-3β,26-dihydroxy-5α-cholesta-6,22-dione (Shimomura et al.,
1988) (9a), D-glucose and L-rhamnose. The sugar sequence attached to
C-3 of the aglycone of 9 was determined to be identical to 1 by analysis
of the 1H- and 13C-NMR, and HMBC spectra of 9 (Fig. 3). Thus, 9 was
formulated as (25R)-26-hydroxy-3-[(O-α-L-rhamnopyranosyl-(1→2)-O-
[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranosyl)oxy]-5α-cholesta-
6,22-dione.
data were obtained on a Waters-Micromass LCT mass spectrometer
(Waters, Manchester, UK). CC was performed by Diaion HP-20 (50
mesh, Mitsubishi-Chemical, Tokyo, Japan), silica gel Chromatorex BW-
300 (300 mesh, Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel
COSMOSIL 75C18-OPN (75 μM particle size, Nacalai Tesque, Kyoto,
Japan). TLC were conducted on precoated silica gel 60 F254 or RP18
F254S plates (0.25 mm thick, Merck, Darmstadt, Germany), and the
spots were visualized by spraying the plates with 10 % H2SO4 aqueous
solution, followed by heating. HPLC was performed with a system
consisting of a CCPM pump (Shimadzu, Kyoto, Japan), an RI-8021
(Tosoh, Tokyo, Japan) or a Shodex OR-2 (Showa-Denko, Tokyo, Japan)
detector, and a Rheodyne injection port (Rohnert Park, CA, USA). A
TSK gel ODS-100Z column (10 mm i.d. × 250 mm, 5 μm; Tosoh) was
used for preparative HPLC. The following materials and biochemical-
grade reagents were used for the cell culture assays: Spectra Classic
microplate reader (Tecan, Salzburg, Austria); 96-well microplate (Iwaki
Glass, Chiba, Japan); HL-60 cells (JCRB 0085) and A549 cells (JCRB
0076) (Human Science Research Resources Bank, Osaka, Japan); fetal
bovine serum (FBS), 0.25 % trypsin ethylenediaminetetraacetic acid
(EDTA) solution, RPMI-1640 medium, minimum essential medium
(MEM), cisplatin, and MTT (Sigma, St. Louis, MO, USA); penicillin G
sodium salt and streptomycin sulfate (Gibco, Gland Island, NY, USA);
paraformaldehyde and phosphate-buffered saline (PBS) (Wako Pure
Chemical Industries, Osaka, Japan).
The isolated compounds (1-21) and the aglycones (1a, 8a, and 9a)
were evaluated for their cytotoxic activity against HL-60 and A549
cells, using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay. It was found that 12 and 21 ex-
hibited moderate cytotoxic activity toward HL-60 and A549 cells, with
IC50 values of 6.9 0.12 μM and 5.1 0.031 μM against HL-60 cells,
and 7.6 0.14 μM and 4.4 0.063 μM against A549 cells, respec-
tively. Compound 20 only showed moderate cytotoxicity toward A549
cells, with an IC50 value of 6.7 0.26 μM. Cisplatin was used as a
positive control and gave IC50 values of 1.1 μM and 2.0 μM against HL-
60 and A549 cells, respectively. The other compounds did not show any
significant cytotoxic activity against HL-60 and A549 cells (IC50 > 10
μM).
3. Experimental
3.1. General
Optical rotations were obtained using a JASCO P-1030 (JASCO,
Tokyo, Japan) automatic digital polarimeter. The IR spectra were re-
corded on a JASCO FT-IR 620 (JASCO). NMR spectra were recorded on
a DRX-500 (500 MHz for 1H-NMR) or a DPX-600 (600 MHz for 1H-
NMR) spectrometer (Bruker, Karlsruhe, Germany), using standard
Bruker pulse programs at 300 K. Chemical shifts are reported in δ with
reference to tetramethylsilane as an internal standard. HRESITOFMS
3.2. Plant material
L. speciosum bulbs were purchased from a garden center of Fuji-
engei (Okayama, Japan) in October 2014. A voucher specimen has been
deposited at the herbarium of the Tokyo University of Pharmacy and
Life Sciences (KS-2014-010).
24

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
3.3. Extraction and isolation
(3H, s, Me-19), 0.95 (3H, s, Me-18). For 1H-NMR spectral data of the
sugar and HMG moieties, see Table 1. For 13C-NMR spectral data, see
L. speciosum bulbs (fresh weight, 8.7 kg) were extracted with MeOH
(9 L × 3) and the extracts were concentrated using an evaporator. The
MeOH extract (340 g) was fractionated using the Diaion HP-20 column
with MeOH/H2O (3:7) (18 L), MeOH/H2O (1:1) (12 L), MeOH (12 L),
EtOH (6 L), and EtOAc (6 L), successively, as the eluent. The MeOH
eluate fraction (45 g) was separated by ODS silica gel CC with MeCN/
H2O (2:3) to obtain four fractions; Fraction (Fr.) 1 (1.2 g), Fr. 2 (31.5 g),
Fr. 3 (0.9 g), and Fr. 4 (10.9 g). Fr. 2 was subjected to silica gel CC
eluted with CHCl3/MeOH/H2O (90:10:1, 40:10:1, 20:10:1) to give four
subfractions (Frs. 2-1–4). Fraction 2-2 was purified by silica gel CC
eluted with CHCl3/MeOH/H2O (40:10:1, 20:10:1), ODS silica gel CC
eluted with MeCN/H2O (3:7, 1:2, 2:3) and MeOH/H2O (7:3), and pre-
parative HPLC using MeCN/H2O (3:7) or MeOH/H2O (3:2, 7:3) to ac-
quire 3 (3.3 mg), 7 (4.0 mg), 11 (12 mg), 14 (8.0 mg), and 17 (7.6 mg).
Fraction 2-3 was separated by silica gel CC eluted with CHCl3/MeOH/
H2O (40:10:1, 20:10:1), ODS silica gel CC eluted with MeCN/H2O (1:2,
3:7) and MeOH/H2O (7:3, 2:1, 3:2), and preparative HPLC using
MeCN/H2O (3:7) to collect 1 (61 mg), 8 (78 mg), 9 (14 mg), 15 (4.8
mg), 18 (168 mg), and 20 (2.1 mg). Fraction 2-4 was subjected to silica
gel CC eluted with CHCl3/MeOH/H2O (30:10:1), ODS silica gel eluted
with MeCN/H2O (1:2, 3:7) and MeOH/H2O (2:1, 3:2,), and preparative
HPLC using MeCN/H2O (3:7, 7:13) and MeOH/H2O (2:1) to obtain 2
(6.7 mg), 4 (4.5 mg), 5 (6.9 mg), 16 (5.9 mg), and 21 (6.2 mg). Fr. 4
was chromatographed on silica gel eluted with CHCl3/MeOH/H2O
(60:10:1, 30:10:1), ODS silica gel CC eluted with MeOH/H2O (3:1, 4:1),
and preparative HPLC using MeOH/H2O (4:1, 3:1) to furnish 6 (3.9
mg), 10 (7.6 mg), 12 (7.3 mg), 13 (37 mg), and 19 (6.9 mg).
Table 2. HRESITOFMS m/z: 921.4467 [M
+
Na]+ (calcd for
C45H70O18Na: 921.4460).
3.3.4. (25R)-27-[(3-hydroxy-3-methylglutaroyl)oxy]-spirost-5-en-3β-yl O-
α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→6)]-β-D-
glucopyranoside (4)
25
Amorphous powder; [α]D -29.0 (c 0.05, MeOH); IR (film) νmax
:
3355 (OH), 2926 (CH), 1730 and 1714 (C=O) cm−1
;
1H-NMR (500
MHz, C5D5N): δH 5.31 (1H, br d, J = 5.1 Hz, H-6), 4.48 (1H, m, H-16),
4.06 (1H, dd, J = 10.9, 3.1 Hz, H-27a), 4.02 (1H, dd, J = 10.9, 10.9
Hz, H-27b), 3.96 (1H, m, W1/2 = 23.0 Hz, H-3), 3.90 (1H, dd, J = 11.2,
3.7 Hz, H-26a), 3.72 (1H, dd, J = 11.2, 11.2 Hz, H-26b), 1.12 (3H, d, J
= 7.0 Hz, Me-21), 1.05 (3H, s, Me-19), 0.80 (3H, s, Me-18). For 1H-
NMR spectral data of the sugar and HMG moieties, see Table 1. For 13C-
NMR spectral data, see Table 2. HRESITOFMS m/z: 1067.5039 [M +
Na]+ (calcd for C51H80O22Na: 1067.5039).
3.3.5. (25R)-[[3-(β-D-glucopyranosyl)oxy-3-methylglutaroyl]oxy]-spirost-
5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-
β-D-glucopyranoside (5)
25
Amorphous powder; [α]D -17.2 (c 0.05, MeOH); IR (film) νmax
:
3388 (OH), 2926 (CH), 1727 and 1716 (C=O) cm−1
;
1H-NMR (600
MHz, C5D5N): δH 5.28 (1H, br d, J = 4.7 Hz, H-6), 4.48 (1H, m, H-16),
4.03 (1H, m, H-27a), 3.95 (1H, br d, J = 11.3 Hz, H-27b), 3.87 (1H, m,
W1/2 = 22.1 Hz, H-3), 3.86 (1H, dd, J = 11.1, 3.7 Hz, H-26a), 3.68
(1H, dd, J = 11.1, 11.1 Hz, H-26b), 1.11 (3H, d, J = 6.9 Hz, Me-21),
1.05 (3H, s, Me-19), 0.81 (3H, s, Me-18). For 1H-NMR spectral data of
the sugar and HMG moieties, see Table 1. For 13C-NMR spectral data,
see Table 2. HRESITOFMS m/z: 1229.5587 [M + Na]+ (calcd for
3.3.1. (25S)-17α,27-dihydroxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-
(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside (1)
C57H90O27Na: 1229.5567).
25
Amorphous powder; [α]D -70.5 (c 0.10, MeOH); IR (film) νmax
:
3390 (OH), 2934 (CH) cm−1; 1H-NMR (600 MHz, C5D5N): δH 5.27 (1H,
br d, J = 4.1 Hz, H-6), 4.47 (1H, dd, J = 7.1, 6.7 Hz, H-16), 4.06 (1H,
dd, J = 11.3, 4.7 Hz, H-26a), 3.88 (1H, dd, J = 11.3, 11.3 Hz, H-26b),
3.82 (1H, m, W1/2 = 20.7 Hz, H-3), 3.72 (1H, dd, J = 10.6, 5.2 Hz, H-
27a), 3.63 (1H, dd, J = 10.6, 7.4 Hz, H-27b), 2.29 (1H, q, J = 7.1 Hz,
H-20), 1.23 (3H, d, J = 7.1 Hz, Me-21), 1.05 (3H, s, Me-19), 0.95 (3H,
s, Me-18). For 1H-NMR spectral data of the sugar moiety, see Table 1.
For 13C-NMR spectral data, see Table 2. HRESITOFMS m/z: 939.4557
[M + Na]+ (calcd for C45H72O19Na: 939.4566).
3.3.6. (25S,26R)-26-methoxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-
(1→2)-β-D-glucopyranoside (6)
25
Amorphous powder; [α]D -45.5 (c 0.05, MeOH); IR (film) νmax
:
3397 (OH), 2926 (CH) cm−1; 1H-NMR (600 MHz, C5D5N): δH 5.34 (1H,
br d, J = 4.7 Hz, H-6), 4.88 (1H, d, J = 2.3 Hz, H-26), 4.68 (1H, m, H-
16), 3.97 (1H, m, W1/2 = 20.5 Hz, H-3), 3.50 (3H, s, OMe), 1.15 (3H, d,
J = 7.0 Hz, Me-21), 1.09 (3H, d, J = 6.8 Hz, Me-27), 1.07 (3H, s, Me-
19), 0.83 (3H, s, Me-18). For 1H-NMR spectral data of the sugar moiety,
see Table 1. For 13C-NMR spectral data, see Table 2. HRESITOFMS m/z:
775.4252 [M + Na]+ (calcd for C40H64O13Na: 775.4245).
3.3.2. (25S)-17α,27-dihydroxyspirost-5-en-3β-yl
O-β-D-glucopyranosyl-
(1→3)-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-
3.3.7. (25R)-27-hydroxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→
D-glucopyranoside (2)
2)-β-D-glucopyranoside (7)
25
25
Amorphous powder; [α]D -52.2 (c 0.05, MeOH); IR (film) νmax
:
Amorphous powder; [α]D -58.3 (c 0.10, MeOH); IR (film) νmax
:
3389 (OH), 2928 (CH) cm−1; 1H-NMR (600 MHz, C5D5N): δH 5.43 (1H,
br d, J = 4.9 Hz, H-6), 4.48 (1H, dd, J = 7.0, 6.7 Hz, H-16), 4.06 (1H,
m, W1/2 = 19.9 Hz, H-3), 4.06 (1H, dd, J = 10.9, 4.2 Hz, H-26a), 3.90
(1H, dd, J = 10.9, 10.9 Hz, H-26b), 3.74 (1H, dd, J = 10.7, 5.1 Hz, H-
27a), 3.66 (1H, dd, J = 10.7, 7.4 Hz, H-27b), 2.30 (1H, q, J = 7.1 Hz,
H-20), 1.25 (3H, d, J = 7.1 Hz, Me-21), 1.10 (3H, s, Me-19), 0.94 (3H,
s, Me-18). For 1H-NMR spectral data of the sugar moiety, see Table 1.
For 13C-NMR spectral data, see Table 2. HRESITOFMS m/z: 1101.5099
[M + Na]+ (calcd for C51H82O24Na: 1101.5094).
3433 (OH), 2935 (CH) cm−1; 1H-NMR (600 MHz, C5D5N): δH 5.31 (1H,
br d, J = 4.8 Hz, H-6), 4.53 (1H, m, H-16), 4.16 (1H, br d, J = 10.5 Hz,
H-27a), 4.14 (1H, dd, J = 11.2, 2.7 Hz, H-26a), 4.01 (1H, br d, J =
11.2 Hz, H-26b), 3.97 (1H, dd, J = 10.5, 6.8 Hz, H-27b), 3.94 (1H, m,
W1/2 = 20.2 Hz, H-3), 1.11 (3H, d, J = 7.0 Hz, Me-21), 1.05 (3H, s,
Me-19), 0.81 (3H, s, Me-18). For 1H-NMR spectral data of the sugar
moiety, see Table 1. For 13C-NMR spectral data, see Table 2. HRESIT-
OFMS m/z: 761.4093 [M + Na]+ (calcd for C39H62O13Na: 761.4088).
3.3.8. (25R)-17α-hydroxy-3-[(O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-
3.3.3. (25R)-17α-hydroxy-27-[(3-hydroxy-3-methylglutaroyl)oxy]-
glucopyranosyl-(1→4)]-β-D-glucopyranosyl)oxy]-5α-spirostan-6-one (8)
25
spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (3)
Amorphous powder; [α]D -57.5 (c 0.05, MeOH); IR (film) νmax
:
25
Amorphous powder; [α]D -42.9 (c 0.05, MeOH); IR (film) νmax
:
3388 (OH), 2925 (CH), 1710 (C=O) cm−1
;
1H-NMR (600 MHz,
3405 (OH), 2931 (CH), 1725 and 1715 (C=O) cm−1
;
1H-NMR (600
C5D5N): δH 4.41 (1H, dd, J = 7.3, 7.3 Hz, H-16), 3.90 (1H, m, W1/2 =
MHz, C5D5N): δH 5.30 (1H, br d, J = 4.9 Hz, H-6), 4.44 (1H, dd, J =
7.4, 6.4 Hz, H-16), 4.07 (1H, dd, J = 11.5, 5.3 Hz, H-27a), 4.00 (1H,
dd, J = 11.5, 7.9 Hz, H-27b), 3.87 (1H, m, W1/2 = 19.6 Hz, H-3), 3.83
(1H, dd, J = 11.0, 4.6 Hz, H-26a), 3.72 (1H, J = 11.0, 11.0 Hz, H-26b),
2.27 (1H, q, J = 7.2 Hz, H-20), 1.22 (3H, d, J = 7.2 Hz, Me-21), 1.09
21.6 Hz, H-3), 3.49 (1H, dd, J = 10.4, 3.8 Hz, H-26a), 3.46 (1H, dd, J
= 10.4, 10.4 Hz, H-26b), 2.23 (1H, q, J = 7.2 Hz, H-20), 1.21 (3H, d, J
= 7.2 Hz, Me-21), 0.88 (3H, s, Me-18), 0.78 (3H, s, Me-19), 0.67 (3H,
d, J = 5.4 Hz, Me-27). For 1H-NMR spectral data of the sugar moiety,
see Table 1. For 13C-NMR spectral data, see Table 2. HRESITOFMS m/z:
25

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
Table 1
1H-NMR spectral data for the sugar and HMG moieties of 1-9 in C5D5N.
1
2
3
4
5
Positions
δH
J (Hz)
Positions
Glc (I) 1′
3′
δH
J (Hz)
Positions δH
J (Hz)
Positions
Glc (I) 1′
δH
J (Hz)
Positions
δH
J (Hz)
Glc (I) 1′ 4.91 d
2′ 4.17 dd
3′ 4.20 dd
4′ 4.21 dd
5′ 3.83 m
7.6
4.90 d
2′ 4.17 m
4.18 m
7.3
Glc 1′
5.04 d
7.2
4.97 d
2′ 4.21 dd
3′ 4.24 dd
4′ 4.22 dd
5′ 4.00 m
7.3
Glc (I) 1′ 4.96 d
2′ 4.18 dd
3′ 4.19 dd
4′ 4.21 dd
5′ 3.86 m
7.7
8.3, 7.6
8.3, 8.3
8.3, 8.3
2′ 4.28 dd
3′ 4.31 dd
4′ 4.19 dd
5′ 3.90 m
6′a 4.51 dd
9.1, 7.2
9.1, 9.1
9.1, 9.1
8.8, 7.3
8.8, 8.8
8.8, 8.8
9.0, 7.7
9.0, 9.0
9.0, 9.0
4′ 4.21 dd
5′ 3.77 m
8.8, 8.8
6′a 4.51 dd
12.1, 3.6
6′a 4.52 br d 11.8
12.1, 2.1
12.1, 5.7
6′a 4.75 br d 10.9
6′a 4.53 br d 12.8
4.47 br d 12.8
b
4.42 br d 12.1
b
4.45 br d 11.8
b
4.37 dd
b
4.33 br d 10.9
b
Rha 1′′ 6.20 br s
Rha 1′′
2′′
6.20 br s
Rha 1′′
2′′
6.39 br s
4.82 br s
4.64 dd
4.37 dd
5.00 m
Rha 1′′
2′′
6.34 br s
4.79 br s
Rha 1′′ 6.26 br s
2′′ 4.75 br s
3′′ 4.60 dd
4′′ 4.35 dd
5′′ 4.95 m
2′′ 4.73 br d 3.0
4.96 br d 3.3
3′′ 4.57 dd
4′′ 4.33 dd
5′′ 4.91 m
6′′ 1.74 d
9.2, 3.0
9.2, 9.2
3′′
4.74 dd
4.54 dd
4.96 m
1.70 d
9.5, 3.3 3′′
9.3, 3.1
9.3, 9.3
3′′
4.62 dd
4.35 dd
4.99 m
1.78 d
9.3, 3.4
9.2, 3.5
9.2, 9.2
4′′
9.5, 9.5 4′′
4′′
9.3, 9.3
6.3
5′′
5′′
6′′
5′′
6.1
6′′
6.3
8.1
1.78 d
5.9
6′′
6′′ 1.77 d
6.2
Glc (II)1′′′ 5.12 d
2′′′ 4.04 dd
3′′′ 4.25 dd
4′′′ 4.26 dd
5′′′ 3.96 m
7.8
Glc (II) 1′′′
5.13 d
HMG 1′′′
－
Glc (II) 1′′′ 5.10 d
7.8
Glc (II) 1′′′
5.14 d
7.7
8.3, 7.8
8.3, 8.3
8.3, 8.3
2′′′ 4.06 dd
3′′′ 4.22 dd
4′′′ 4.29 dd
5′′′ 3.95 m
8.8, 8.1 2′′′a
3.15 d
3.12 d
－
3.20 d
3.18 d
－
14.3
14.3
2′′′
3′′′
4′′′
5′′′
6′′′a
b
4.04 dd
4.23 dd
4.14 dd
3.93 m
8.4, 7.8 2′′′
8.4, 8.4 3′′′
8.4, 8.4 4′′′
5′′′
4.07 dd
4.29 dd
4.23 dd
3.98 m
8.6, 7.7
8.6, 8.6
8.6, 8.6
8.8, 8.8
8.8, 8.8 3′′′
4′′′a
b
14.2
14.2
6′′′a 4.44 dd
13.4, 3.1
13.4, 4.9
6′′′a 4.46 br d 12.7
a
4.52 br d 11.8
4.33 br d 11.8
6′′′a
b
4.48 br d 11.6
4.35 br d 11.6
b
4.31 dd
b
4.35 br d 12.7
5′′′
6′′′
1.78 s
Glc (III) 1′′′′ 5.52 d
7.9
HMG 1′′′′
2′′′′a
b
－
HMG 1′′′′
2′′′′a
b
－
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′a
b
4.11 dd
4.21 dd
4.33 dd
3.90 m
8.9, 7.9
8.9, 8.9
8.9, 8.9
3.16 d
3.12 d
－
3.21 d
3.17 d
－
14.3
14.3
3.30 d
3.20 d
－
15.2
15.2
3′′′′
3′′′′
4′′′′a
b
15.3
15.3
4′′′′a
b
3.47 d
3.37 d
－
1.89 s
15.4
15.4
4.42 br d 11.8
4.39 br d 11.8
5′′′′
5′′′′
6′′′′
1.78 s
6′′′′
Glc (III) 1′′′′′ 5.28 d
7.9
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′a
b
4.02 dd
4.19 dd
4.28 dd
3.97 m
4.52 dd
4.32 dd
8.5, 7.9
8.5, 8.5
8.5, 8.5
11.9, 2.0
11.9, 4.4
6
7
8
9
Positions
δH
J (Hz)
Positions
δH
J (Hz)
Positions
δH
J (Hz)
Positions
δH
J (Hz)
Glc 1′
2′
5.04 d
7.5
Glc 1′
2′
5.06 d
7.2
Glc 1′
2′
4.93 d
7.3
Glc 1′
2′
4.96 d
7.2
4.27 dd
4.31 dd
4.19 dd
3.90 m
4.51 dd
4.37 dd
9.0, 7.5
9.0, 9.0
9.0, 9.0
4.29 dd
4.31 dd
4.19 dd
3.91 m
4.53 dd
4.37 dd
9.1, 7.2
9.1, 9.1
9.1, 9.1
4.18 m
4.17 m
4.16 dd
3.83 m
4.51 br d
4.48 br d
4.20 dd
4.22 dd
4.27 dd
3.86 m
8.3, 7.2
8.9, 8.3
8.9, 8.9
3′
3′
3′
3′
4′
4′
4′
8.8, 8.8
4′
5′
5′
5′
5′
6′a
b
11.7, 2.2
11.7, 4.4
6′a
b
11.8, 2.1
11.8, 4.5
6′a
b
12.4
12.4
6′a
b
4.52 br d
4.33 br d
11.9
11.9
Rha 1′′
2′′
6.38 br s
4.82 dd
4.64 dd
4.36 dd
5.00 m
1.78 d
Rha 1′′
2′′
6.40 br s
4.82 br d
4.65 dd
4.37 dd
5.01 m
Rha 1′′
2′′
6.18 br s
4.69 dd
4.58 dd
4.30 dd
4.88 m
1.74 d
Rha 1′′
2′′
6.20 d
1.2
3.3, 1.4
9.2, 3.3
9.2, 9.2
3.1
3.4, 1.4
9.3, 3.4
9.3, 9.3
4.72 dd
4.60 dd
4.32 dd
4.92 m
1.77 d
3.4, 1.2
9.4, 3.4
9.4, 9.4
3′′
3′′
9.3, 3.1
9.3, 9.3
3′′
3′′
4′′
4′′
4′′
4′′
5′′
5′′
5′′
5′′
6′′
6.2
6′′
1.79 d
6.2
6′′
6.2
6′′
6.2
Glc 1′′′
2′′′
5.10 d
7.9
Glc 1′′′
2′′′
5.12 d
7.9
4.03 dd
4.21 dd
4.25 dd
3.96 m
4.44 dd
4.31 dd
8.5, 7.9
9.0, 8.5
9.0, 9.0
4.06 dd
4.23 dd
4.26 dd
3.97 m
4.46 dd
4.31 dd
8.6, 7.9
8.6, 8.6
8.6, 8.6
3′′′
3′′′
4′′′
4′′′
5′′′
5′′′
6′′′a
b
11.9, 2.3
11.9, 4.9
6′′′a
b
11.9, 2.2
11.9, 5.5
500 MHz (4 and 9), 600 MHz (1-3 and 5-8).
939.4564 [M + Na]+ (calcd for C45H72O19Na: 939.4565).
C5D5N): δH 4.44 (1H, dd, J = 7.4, 6.2 Hz, H-16), 3.83 (1H, m, W1/2 =
20.0 Hz, H-3), 3.51 (1H, dd, J = 10.6, 2.8 Hz, H-26a), 3.48 (1H, dd, J
= 10.6, 10.6 Hz, H-26b), 2.44 (1H, dd, J = 12.2, 3.5 Hz, H-7β), 2.31
(1H, m, H-4α), 2.29 (1H, m, H-14), 2.27 (1H, q, J = 7.2 Hz, H-20), 2.25
(1H, br d, J = 11.8 Hz, H-5), 2.22 (1H, ddd, J = 12.9, 12.9, 3.7 Hz, H-
3.3.9. (25R)-3β,17α-dihydroxy-5α-spirostan-6-one (8a)
25
Amorphous powder; [α]D -45.1 (c 0.05, MeOH); IR (film) νmax
:
3291 (OH), 2928 (CH), 1702 (C=O) cm−1
;
1H-NMR (600 MHz,
26

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
Table 2
13C-NMR spectral data of 1-8, 8a, and 9.
Positions
1
2
3
4
5
6
7
8
8a
37.0
9
1
2
37.5
30.1
78.2
38.8
37.5
30.2
78.3
39.1
37.6
37.5
30.0
78.4
39.1
37.5
30.1
78.1
38.9
37.5
37.5
36.7
29.2
76.4
26.5
56.3
36.7
29.3
76.4
26.4
56.3
30.2
78.2
39.0
141.0
121.7
32.3
31.8
50.2
37.2
20.9
32.1
45.2
53.0
32.4
90.1
90.1
17.1
19.4
44.8
9.6
30.2
77.9
38.9
140.8
121.7
32.3
31.6
50.2
37.1
21.0
39.8
40.4
56.6
32.1
81.2
62.7
16.3
19.4
42.4
14.9
109.8
27.2
21.6
36.2
60.6
61.4
30.2
78.0
39.0
141.0
121.7
32.4
31.8
50.4
37.2
21.1
39.8
40.5
56.7
32.2
81.5
62.7
16.3
19.4
42.2
14.9
111.9
25.6
26.4
31.7
99.5
10.5
55.8
31.8
70.0
31.2
56.9
210.1
46.9
38.1
53.6
40.9
21.4
32.0
45.8
52.9
31.3
89.6
89.8
17.2
13.2
44.7
9.7
3
4
5
140.7
121.7
32.4
32.3
50.2
37.1
20.9
32.0
45.1
52.9
31.7
90.0
90.1
17.1
19.4
44.8
9.7
140.8
122.0
32.4
32.3
50.2
37.1
21.0
32.1
45.1
53.0
31.8
90.1
90.2
17.2
19.5
44.9
9.7
140.9
121.6
32.1
31.1
50.1
37.1
21.0
39.8
40.4
56.5
32.2
81.2
62.8
16.3
19.4
41.9
14.9
109.4
30.3
21.4
35.5
63.0
66.1
140.8
121.8
32.3
31.6
50.3
37.1
21.1
39.8
40.4
56.6
32.1
81.2
62.8
16.3
19.4
41.9
14.9
109.4
31.1
23.6
35.4
63.0
66.2
6
209.4
46.8
37.9
53.5
40.9
21.3
31.8
45.7
52.9
31.2
89.6
89.8
17.2
13.1
44.7
9.7
209.5
46.6
37.6
53.7
40.9
21.5
39.5
43.1
56.0
24.3
27.6
52.3
12.1
13.0
49.4
16.6
213.9
39.8
27.7
36.1
67.3
17.2
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
110.2
31.7
23.5
38.9
63.9
64.3
110.2
31.8
23.6
39.0
63.9
64.4
109.9
31.4
23.2
35.3
62.9
66.0
109.8
32.0
28.7
30.3
66.6
17.1
109.8
31.9
28.7
30.4
66.6
17.2
Glc (I)
99.9
77.3
77.6
81.9
76.1
61.8
Glc (I)
100.0
77.3
Glc
100.4
77.9
79.6
71.9
78.0
62.7
Glc (I)
100.7
77.5
Glc (I)
100.0
77.3
Glc
100.3
77.8
79.6
71.8
78.2
62.6
Glc
100.4
77.9
79.7
71.9
78.3
62.7
Glc (I)
99.0
77.6
77.5
82.0
76.2
61.9
Glc (I)
99.2
77.5
77.6
82.0
76.3
62.0
1′
2′
3′
4′
5′
6′
77.5
79.6
77.7
82.0
71.6
82.1
76.2
76.9
76.2
62.1
70.0
61.9
Rha
101.7
72.3
72.7
74.0
69.4
18.6
Rha
101.7
71.9
84.0
73.2
69.3
18.6
Rha
102.0
72.6
72.9
74.2
69.5
18.7
Rha
102.0
72.5
72.8
74.2
69.5
18.7
Rha
101.8
72.4
72.8
74.1
69.5
18.6
Rha
102.0
72.5
72.8
74.1
69.5
18.7
Rha
102.1
72.6
72.9
74.2
69.5
18.7
Rha
101.9
72.3
72.7
74.0
69.4
18.6
Rha
101.9
72.3
72.7
74.1
69.5
18.7
1′′
2′′
3′′
4′′
5′′
6′′
Glc (II)
105.0
74.9
Glc (II)
107.0
75.0
HMG
171.6
46.4
Glc (II)
105.4
75.2
Glc (II)
105.2
75.0
Glc (II)
105.1
74.9
Glc (II)
105.2
74.9
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
78.1
78.4
70.0
78.4
78.5
78.2
78.3
71.2
71.2
46.5
71.7
71.2
71.2
71.2
78.4
78.4
174.6
28.3
78.5
78.4
78.4
78.5
62.0
61.9
62.7
62.1
62.0
62.1
Glc (III)
105.2
76.0
HMG
171.6
46.4
HMG
171.4
43.9
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
78.5
70.0
76.7
71.3
46.5
44.3
78.4
174.6
28.3
173.8
25.2
62.3
Glc (III)
98.5
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′
75.3
78.4
71.6
78.3
63.0
125 MHz (4 and 9), 150 MHz (1-3, 5-8, and 8a).
12α), 2,12 (1H, m, H-15α), 2.05 (1H, br dd, J = 14.2, 12.4 Hz, H-2α),
2.03 (1H, m, H-7α), 2.01 (1H, m, H-8), 1.91 (1H, ddd, J = 12.5, 11.8,
11.8 Hz, H-4β), 1.73 (1H, ddd, J = 12.2, 12.2, 5.0 Hz, H-23a), 1.67
(1H, m, H-2β), 1.66 (1H, m, H-11α), 1.65 (1H, m, H-1β), 1.64 (1H, m,
H-12β), 1.59 (1H, m, H-24a), 1.56 (1H, m, H-25), 1.52 (1H, m, H-24b),
1.51 (1H, m, H-23b), 1.45 (1H, ddd, J = 13.3, 13.3, 6.2 Hz, H-15β),
27

T. Iguchi, et al.
PhytochemistryLetters37(2020)21–28
1.35 (1H, dddd, J = 12.9, 12.9, 12.9, 3.7 Hz, H-11β), 1.24 (3H, d, J =
7.2 Hz, Me-21), 1.21 (1H, br dd, J = 12.9, 12.9 Hz, H-9), 1.13 (1H, ddd,
J = 14.2, 14.2, 4.1 Hz, H-1α), 0.93 (3H, s, Me-18), 0.79 (3H, s, Me-19),
0.67 (3H, d, J = 5.3 Hz, Me-27). For 13C-NMR spectral data, see
Declaration of Competing Interest
The authors declare no conflict of interest associated with this
manuscript.
Table 2. HRESITOFMS m/z: 469.2931 [M
+
Na]+ (calcd for
C27H42O5Na: 469.2930).
Appendix A. Supplementary data
3.3.10. (25R)-26-hydroxy-3-[(O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2020.01.008.
glucopyranosyl-(1→4)]-β-D-glucopyranosyl)oxy]-5α-cholesta-6,22-dione
(9)
25
Amorphous powder; [α]D -49.5 (c 0.10, MeOH); IR (film) νmax
:
References
3327 (OH), 2926 (CH), 1708 (C=O) cm−1
;
1H-NMR (500 MHz,
C5D5N): δH 3.92 (1H, m, W1/2 = 20.2 Hz, H-3), 3.77 (1H, dd, J = 10.5,
6.0 Hz, H-26a), 3.73 (1H, dd, J = 10.5, 6.0 Hz, H-26b), 1.10 (3H, d, J
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28