Three novel furospirostanol glycosides and a steroidal alkaloid glycoside from the bulbs of Fritillaria camtschatcensis (L.) Ker Gawl., and their cytotoxicity

Article abstract of DOI:10.1080/14786419.2021.1897590

Three novel steroidal glycosides (1–3) and a previously described steroidal alkaloid glycoside (4) have been isolated from the bulbs of Fritillaria camtschatcensis (L.) Ker Gawl. (Liliaceae). The structures of novel compounds 1–3 were characterized based on NMR spectroscopy and chemical transformations. Compounds 1–3 are furospirostanol glycosides bearing a (3S)-3-hydroxy-3-methylglutaryl moiety at C-26 in the aglycone. Compounds 1–4 were evaluated in terms of their cytotoxic activities toward HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human lung small cell carcinoma cells. Only 4 showed moderate cytotoxicity against HL-60, A549, and SBC-3 cells with IC₅₀ values of 22.9, 13.3, and 11.9 μM, respectively. Compound 4 was found to cause necrotic-like cell death in HL-60 cells.

Full text of DOI:10.1080/14786419.2021.1897590

Natural Product Research
Formerly Natural Product Letters
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Three novel furospirostanol glycosides and a
steroidal alkaloid glycoside from the bulbs of
Fritillaria camtschatcensis (L.) Ker Gawl., and their
cytotoxicity
Tomoki Iguchi, Minpei Kuroda, Hiroshi Takayama & Yoshihiro Mimaki
To cite this article: Tomoki Iguchi, Minpei Kuroda, Hiroshi Takayama & Yoshihiro Mimaki (2021):
Three novel furospirostanol glycosides and a steroidal alkaloid glycoside from the bulbs of
Fritillariaꢀcamtschatcensis (L.) Ker Gawl., and their cytotoxicity, Natural Product Research, DOI:
10.1080/14786419.2021.1897590
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1897590
Three novel furospirostanol glycosides and a steroidal
alkaloid glycoside from the bulbs of Fritillaria
camtschatcensis (L.) Ker Gawl., and their cytotoxicity
Tomoki Iguchi, Minpei Kuroda, Hiroshi Takayama and Yoshihiro Mimaki
Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University
of Pharmacy and Life Sciences, Tokyo, Japan
ABSTRACT
ARTICLE HISTORY
Received 20 November 2020
Accepted 26 February 2021
Three novel steroidal glycosides (1–3) and a previously described
steroidal alkaloid glycoside (4) have been isolated from the bulbs of
Fritillaria camtschatcensis (L.) Ker Gawl. (Liliaceae). The structures of
novel compounds 1–3 were characterized based on NMR spectros-
copy and chemical transformations. Compounds 1–3 are furospiros-
tanol glycosides bearing a (3S)-3-hydroxy-3-methylglutaryl moiety at
C-26 in the aglycone. Compounds 1–4 were evaluated in terms
of their cytotoxic activities toward HL-60 human promyelocytic
leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3
human lung small cell carcinoma cells. Only 4 showed moderate
cytotoxicity against HL-60, A549, and SBC-3 cells with IC50 values of
22.9, 13.3, and 11.9 mM, respectively. Compound 4 was found to
cause necrotic-like cell death in HL-60 cells.
KEYWORDS
Fritillaria camtschatcensis;
Liliaceae; bulb;
furospirostanol glycoside;
steroidal alkaloid;
cytotoxicity
1. Introduction
Fritillaria genus plants belonging to the Liliaceae family are distributed in the
temperate regions of the Northern Hemisphere (Tsukamoto 1989). A literature survey
revealed that Fritillaria species, including F. anhuiensis (Shou et al. 2010), F. unibracteata
(Zhang et al. 2011), F. pallidiflora (Shen et al. 2012; Xu et al. 2014), F. meleagris (Matsuo
CONTACT Tomoki Iguchi
iguchit@toyaku.ac.jp
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1897590.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
T. IGUCHI ET AL.
Figure 1. Structures of isolated compounds.
et al. 2013), F. shuchengensis (Huang et al. 2013), and F. thunbergii (Suh et al. 2018) are
rich sources of steroidal alkaloids. F. camtschatcensis (L.) Ker Gawl. grows in Northern
Japan and a number of steroidal alkaloids have been isolated from this plant (Kaneko
et al. 1978; Kaneko, Nakaoka, et al. 1981; Kaneko, Tanaka, et al. 1981). In a previous study,
we accomplished the isolation of two phenolic compounds, regaloside A and 3,60-di-O-fer-
uloylsucrose, which are widely found in plants of the Lilium genus, and two steroidal alka-
loids, solanidine glycoside and kuroyurinidine. Kuroyurinidine is a C-nor-D-homo steroidal
alkaloid with three axial hydroxy groups at the C-2, C-3, and C-6 positions in the steroidal
skeleton (Sashida et al. 1989; Mimaki and Sashida 1990). As a continuation of our previous
study, a phytochemical investigation was conducted on the bulbs of F. camtschatcensis
with a focus on steroidal glycosides, which resulted in the isolation of three novel furospir-
ostanol glycosides (1–3) and a previously described steroidal alkaloid glycoside (4). The
structures of the novel compounds were characterized based on one- and two-dimen-
sional NMR spectroscopy and chemical transformations. Compounds 1–4 were evaluated
for their cytotoxic activities toward HL-60 human promyelocytic leukemia cells, A549
human lung adenocarcinoma cells, and SBC-3 human lung small cell carcinoma cells.
2. Results and discussion
The bulbs of F. camtschatcensis (fresh weight, 2.3 kg) were extracted using MeOH
(20 L) at 60 ꢀC, and the solvent was removed under reduced pressure. The MeOH
extract (130 g) was loaded onto a Diaion HP-20 porous polymer polystyrene resin col-
umn and eluted using MeOH/H2O (3:7, v/v), EtOH, and EtOAc. The EtOH eluted portion
(25 g) was repeatedly fractionated using silica gel column chromatography (CC), octa-
decylsilanized (ODS) silica gel CC, and preparative ODS HPLC to collect 4 compounds
(1–4). Compound 4 was identified as solanidine 3-O-a-L-rhamnopyranosyl-(1!2)-b-D-
glucopyranoside (Kitajima et al. 1982) (Figure 1).
Compound 1 was obtained as an amorphous powder. Its molecular formula was
determined to be C51H80O22 based on high resolution-electrospray ionization-time of
flight mass spectroscopy (HR-ESI-TOF-MS) and 13C-NMR spectroscopy. The IR spectrum
of 1 indicated the presence of hydroxy (3539 cmꢁ1) and carbonyl (1739 and
1
1709 cmꢁ1) groups. The H- and 13C-NMR spectra of 1 showed signals for four ster-
oidal methyl groups [dH 1.30 (s, Me-27), 1.06 (d, J ¼ 6.9 Hz, Me-21), 1.02 (s, Me-19), and

NATURAL PRODUCT RESEARCH
3
0.79 (s, Me-18); dC 24.2 (C-27), 19.3 (C-19), 16.1 (C-18), and 15.1 (C-21)], an olefinic
group [dH 5.26 (br d, J ¼ 4.7 Hz, H-6); dC 140.7 (C-5) and 121.8 (C-6)], two oxygenated
methine groups [dH 4.64 (q-like, J ¼ 7.6 Hz, H-16) and 3.86 (m, H-3); dC 81.1 (C-16) and
78.1 (C-3)], an oxygenated methylene group [dH 4.40 (d, J ¼ 10.7 Hz, H-26a) and 4.36
(d, J ¼ 10.7 Hz, H-26b); dC 70.9 (C-26)], an acetal carbon [dC 120.3 (C-22)], an oxygen-
ated quaternary carbon [dC 82.4 (C-25)], and three anomeric protons and carbons [dH
6.24 (br s), 5.13 (d, J ¼ 7.9 Hz), and 4.94 (d, J ¼ 7.4 Hz); dC 105.2, 101.7, and 100.0]. In
addition, the 13C-NMR spectrum showed the presence of a six carbon atom substitu-
ent, whose signals were observed at dC 171.5 (ester carbonyl, C-10000), 46.7 and 46.4
(methylene carbons, C-20000 and C-40000), 70.0 (quaternary carbon with a hydroxy group,
C-30000), 174.6 (carbonyl carbon of a carboxy group, C-50000), and 28.3 (methyl carbon, C-
1
6
0000). The signals in the H-NMR spectrum that could be assigned to this substituent
involved two isolated spin systems consisting of two methylene groups [dH 3.17 and
3.15 (each d, J ¼ 14.7 Hz, H2-20000); dH 3.23 and 3.20 (each d, J ¼ 15.0 Hz, H2-40000)] and a
deshielded methyl group [dH 1.80 (s)]. These data suggested that the substituent was
3-hydroxy-3-methylglutaryl (HMG). Subsequent acid hydrolysis of 1 using 1 M HCl
(dioxane/H2O, 1:1, v/v) yielded (25S)-spirost-5-ene-3b,25-diol (1a) (Saijo et al. 1983) as
the aglycone, and D-glucose and L-rhamnose as carbohydrate moieties. Identification
of the monosaccharides was conducted by direct HPLC analysis of the hydrolysate,
using a combination of refractive index and optical rotation detector. (25S)-26-
Hydroxyfurospirostanols are known to be converted into their corresponding (25S)-
hydroxyspirostanols under acidic conditions (Tschesche and Richert 1964). Thus, the
genuine aglycone of 1 was elucidated to be (25S)-22,25-epoxy-furost-5-ene-3b,26-diol.
1H-1H correlation spectroscopy (COSY) and heteronuclear single quantum coherence
(HSQC) spectra of the sugar moieties in 1 revealed that it contained a 2,4-disubstituted
b-D-glucopyranosyl unit [Glc (I): dH 4.94 (d, J ¼ 7.4 Hz, H-10); dC 100.0, 77.3, 77.7, 82.0,
76.2, and 61.9 (C-10–C-60)], a terminal a-L-rhamnopyranosyl unit [Rha: dH 6.24 (br s, H-
100); dC 101.7, 72.4, 72.7, 74.1, 69.4, and 18.6 (C-100–C-600)], and a terminal b-D-glucopyr-
anosyl unit [Glc (II): dH 5.13 (d, J ¼ 7.9 Hz, H-1000); dC 105.2, 75.0, 78.2, 71.2, 78.5, and
62.1 (C-1000–C-6000)]. The heteronuclear multiple bond correlation (HMBC) spectrum of 1
3
exhibited JC,H correlations between H-100 of Rha (dH 6.24) and C-20 of Glc (I) (dC 77.3),
H-1000 of Glc (II) (dH 5.13) and C-40 of Glc (I) (dC 82.0), and between H-10 of Glc (I) (dH
4.94) and C-3 of the aglycone (dC 78.1). The ester linkage formed between the HMG
group and C-26 atom in the aglycone was ascertained based on the HMBC correla-
tions observed between the H2-26 oxymethylene protons of the aglycone (dH 4.40 and
4.36) and the C-10000 carbonyl group of the HMG moiety (dC 171.5). The absolute con-
figuration at C-30000 in the HMG moiety was determined using the following proce-
dures. Compound 1 was treated with (S)-(–)-1-phenylethylamine (PEA) using 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMT-MM) to afford the (S)-(–)-PEA
conjugate derivative (1b) of 1. Compound 1 b was then subjected to alkaline metha-
nolysis with 1% NaOMe to obtain the methyl HMG amide derivative (1c). The physico-
chemical and spectroscopic data of 1c were in agreement with those of the methyl
HMG amide derivative (10a) obtained from (25R)-6b-hydroxy-2-[[(3S)-3-hydroxy-3-meth-
ylglutaryl]oxy]-5a-spirostan-3b-yl
O-b-D-glucopyranosyl-(1!2)-O-[b-D-xylopyranosyl-
(1!3)]-O-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside (10), which was previously

4
T. IGUCHI ET AL.
Figure 2. Chemical transformations of 1 and 10.
isolated from Allium albopilosum bulbs in our laboratory (Mimaki et al. 1993) (Figure
2). Therefore, the absolute configuration of the asymmetric center in the HMG moiety
was determined to be S, and 1 was elucidated to be (25S)-26-O-[(3S)-3-hydroxy-3-
methylglutaryl]-22,25-epoxy-furost-5-en-3b-yl
glucopyranosyl-(1!4)]-b-D-glucopyranoside.
O-a-L-rhamnopyranosyl-(1!2)-O-[b-D-
Compound 2 was obtained as an amorphous powder and its molecular formula
(C51H80O22; HR-ESI-TOF-MS m/z: 1045.5238 [M þ H]þ, calculated for 1045.5219) was
1
determined to be the same as 1. The H- and 13C-NMR spectra obtained for 2 were
very similar to those of 1, except for the signals assigned to the H2-26 methylene and
Me-27 methyl protons, and the C-23, C-26, and C-27 carbon atoms in the aglycone
moiety. All the other signals, including those corresponding to the triglycoside and
HMG moieties, appeared at almost the same position in 1 and 2. Thus, 2 was pre-
sumed to be a stereoisomer of 1 in regard to the C-25 configuration of the aglycone.
Acid hydrolysis of 2 with 1 M HCl (dioxane/H2O, 1:1, v/v) afforded (25R)-spirost-5-ene-
3b,25-diol (2a) (Faini et al. 1984), D-glucose, and L-rhamnose. Accordingly, 2 was eluci-
dated to be (25R)-26-O-[(3S)-3-hydroxy-3-methylglutaryl]-22,25-epoxy-furost-5-en-3b-yl
O-a-L-rhamnopyranosyl-(1!2)-O-[b-D-glucopyranosyl-(1!4)]-b-D-glucopyranoside.
1
The H- and 13C-NMR spectra of 3 were closely related to those of 1, except for the sig-
nals corresponding to the sugar moiety attached to the C-3 atom of the aglycone. The
molecular formula of 3 was lower than that of 1 by C6H10O5, which corresponded to a
hexosyl unit. Thus, 3 was proposed to share the same aglycone with 1, including the
HMG moiety attached at C-26, but differed in terms of the structure of the sugar moiety.
1
Based on the H- and 13C-NMR spectral data, the sugar moiety of 3 was consisted of a 2-
monosubstituted b-D-glucopyranosyl unit [Glc: dH 5.03 (d, J¼ 7.4 Hz); dC 100.4, 77.8, 79.6,
71.8, 78.2, and 62.6 (C-10–C-60)] and a terminal a-L-rhamnopyranosyl unit [Rha: dH 6.38 (br
s, H-100); dC 102.0, 72.5, 72.8, 74.1, 69.4, and 18.7 (C-100–C-600)]. In the HMBC spectrum of 3,
long-range correlations were observed between H-100 of Rha (dH 6.38) and C-20 of Glc (dC
77.8), and between H-10 of Glc (dH 5.03) and C-3 of the aglycone (dC 78.0). Thus, 3 was

NATURAL PRODUCT RESEARCH
5
identified to be (25S)-26-O-[(3S)-3-hydroxy-3-methylglutaryl]-22,25-epoxy-furost-5-en-3b-yl
O-a-L-rhamnopyranosyl-(1!2)-b-D-glucopyranoside.
The cytotoxic activities of 1–4 against HL-60, A549, and SBC-3 cells were evaluated
using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
The dose-response curves shown in Figure S16 (Supplementary material) indicate that
4 exhibited moderate cytotoxic activity against HL-60, A549, and SBC-3 cells with IC50
values of 22.9, 13.3, and 11.9 mM, respectively. Compounds 1–3 did not exhibit any
cytotoxicity at sample concentrations up to 40 mM. The morphology of HL-60 cells
treated with 4 (50 mM) or cisplatin (3 mM) for 3, 6, 12, 24, 48, and 72 h were observed
using a fluorescence microscopy after treatment with 40,6-diamidino-2-phenylindole
dihydrochloride (DAPI) (Figure S17, Supplementary material). The characteristics of the
apoptotic cells including nuclear chromatin condensation and nuclear disassembly
were observed in HL-60 cells treated with cisplatin. On the other hand, cell membrane
disruption was observed in HL-60 cells treated with 4. Furthermore, HL-60 cells treated
with 4 (50 mM) for 3, 6, 12, 24, 48, and 72 h were treated with oxazole yellow dimer
(YOYO-1), which stains the impermeable cell membrane (Matsuo et al. 2017). The
green fluorescence images suggest that the cell membrane of HL-60 cells was dis-
rupted by 4 in a short period of time (Figure S18, Supplementary material). These
observations suggested that 4 causes necrotic-like cell death in HL-60 cells.
3. Experimental
3.1. Compound 1
Amorphous powder; [a]D25 ꢁ101.2 (c 0.10, MeOH); IR mmax (film) cmꢁ1: 3539 (OH), 1739,
1709 (C ¼ O); 13C-NMR spectral data of the aglycone moiety (150 MHz, C5D5N): dC 37.4,
30.1, 78.1, 38.9, 140.7, 121.8, 32.1, 31.6, 50.2, 37.1, 21.0, 39.8, 40.5, 56.4, 32.2, 81.1, 62.4,
16.1, 19.3, 38.5, 15.1, 120.3, 33.0, 33.5, 82.4, 70.9, 24.2 (C-1–C-27); 13C-NMR spectral data of
the sugar and HMG moieties (150 MHz, C5D5N): 100.0, 77.3, 77.7, 82.0, 76.2, 61.9 [C-10–C-60
of Glc (I)], 101.7, 72.4, 72.7, 74.1, 69.4, 18.6 (C-100–C-600 of Rha), 105.2, 75.0, 78.2, 71.2, 78.5,
62.1 [C-1000–C-6000 of Glc (II)], 171.5, 46.7, 70.0, 46.4, 174.6, 28.3 (C-10000–C-60000 of HMG).
3.2. Compound 2
Amorphous powder; [a]D25 ꢁ60.4 (c 0.10, MeOH); IR mmax (film) cmꢁ1: 3450 (OH), 1728,
1715 (C ¼ O); 13C-NMR spectral data of the aglycone moiety (150 MHz, C5D5N): dC 37.5,
30.1, 78.1, 38.9, 140.8, 121.8, 32.2, 31.6, 50.2, 37.1, 21.0, 39.8, 40.5, 56.5, 32.2, 80.9, 62.6,
16.1, 19.4, 38.6, 15.1, 120.3, 34.0, 33.7, 82.3, 69.7, 26.3 (C-1–C-27); 13C-NMR spectral data of
the sugar and HMG moieties (150 MHz, C5D5N): 100.0, 77.4, 77.7, 82.0, 76.2, 61.9 [C-10–C-60
of Glc (I)], 101.8, 72.4, 72.8, 74.1, 69.4, 18.6 (C-100–C-600 of Rha), 105.2, 75.0, 78.3, 71.2, 78.5,
62.1 [C-1000–C-6000 of Glc (II)], 171.6, 46.5, 70.0, 46.4, 174.7, 28.3 (C-10000–C-60000 of HMG).
3.3. Compound 3
Amorphous powder; [a]D25 ꢁ69.2 (c 0.10, MeOH); IR mmax (film) cmꢁ1: 3405 (OH), 1730
(C ¼ O); 13C-NMR spectral data of the aglycone moiety (150 MHz, C5D5N): dC 37.5, 30.2,

6
T. IGUCHI ET AL.
78.0, 39.0, 140.8, 121.7, 32.2, 31.6, 50.2, 37.1, 21.0, 39.8, 40.5, 56.4, 32.2, 81.1, 62.4, 16.1,
19.4, 38.5, 15.1, 120.3, 33.0, 33.5, 82.5, 70.9, 24.2 (C-1–C-27); 13C-NMR spectral data of
the sugar and HMG moieties (150 MHz, C5D5N): 100.4, 77.8, 79.6, 71.8, 78.2, 62.6 (C-
10–C-60 of Glc), 102.0, 72.5, 72.8, 74.1, 69.4, 18.7 (C-100–C-600 of Rha), 171.5, 46.7, 70.0,
46.4, 174.6, 28.3 (C-1000–C-6000 of HMG).
See supplementary material for others.
4. Conclusions
Three novel steroidal glycosides (1–3) and a known steroidal alkaloid glycoside (4)
were isolated from the bulbs of F. camtschatcensis. The structures of 1–3 including the
absolute configuration of the HMG moiety were determined using one- and two-
dimensional NMR spectroscopy and chemical transformations. Compound 4 exhibited
moderate cytotoxicity against HL-60, A549, and SBC-3 cells, and caused necrotic-like
cell death in HL-60 cells.
Disclosure statement
The authors declare no conflict interest.
Funding
This work was supported by JSPS KAKENHI Grant Number JP19K23809.
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