Structural characterization of cholestane rhamnosides from ornithogalum saundersiae bulbs and their cytotoxic activity against cultured tumor cells

Article abstract of DOI:10.3390/molecules22081243

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1-12) and seven known compounds (13-19). The structures of the new compounds (1-12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1-19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10-12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC₅₀ value of 0.16 μM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Full text of DOI:10.3390/molecules22081243

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molecules
Article
Structural Characterization of Cholestane
Rhamnosides from Ornithogalum saundersiae
Bulbs and Their Cytotoxic Activity against Cultured
Tumor Cells
Tomoki Iguchi, Minpei Kuroda *, Rei Naito, Tomoyuki Watanabe, Yukiko Matsuo,
Akihito Yokosuka and Yoshihiro Mimaki
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji,
Tokyo 192-0392, Japan; y091015@toyaku.ac.jp (T.I.); 7811imsn@jcom.zaq.ne.jp (R.N.);
tmyk.w14@gmail.com (T.W.); matsuoy@toyaku.ac.jp (Y.M.);
yokosuka@toyaku.ac.jp (A.Y.); mimakiy@toyaku.ac.jp (Y.M.)
* Correspondence: kurodam@toyaku.ac.jp; Tel.: +81-42-676-4575
Received: 30 June 2017; Accepted: 17 July 2017; Published: 25 July 2017
Abstract: Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental
perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides,
some of which were structurally unique and showed potent cytotoxic activity against cultured
tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic
extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides
(1–12) and seven known compounds (13–19). The structures of the new compounds (1–12) were
identified via NMR-based structural characterization methods, and through a sequence of chemical
transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of
the isolated compounds (1–19) and the derivatives (1a and 6a) against HL-60 human promyelocytic
leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10
16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent
cytotoxicity with an IC50 value of 0.16 M against HL-60 cells and induced apoptotic cell death via a
mitochondrion-independent pathway.
–12,
µ
Keywords: Ornithogalum saundersiae; Asparagaceae; Liliaceae; cholestane rhamnoside; cytotoxicity;
HL-60 cells; A549 cells; apoptosis
1. Introduction
Ornithogalum saundersiae (Baker), of the Asparagaceae (formerly Liliaceae) family, is an ornamental
perennial plant native to South Africa. Our systematic phytochemical examinations of a methanolic
extract of Ornithogalum saundersiae bulbs resulted in the isolation of 29 new cholestane glycosides
including 16,23-epoxy-5
β-cholestane glycoside [
1], 22-homo-23-norcholestane glycosides [2–6], and
polyoxycholestane glycosides [
7–12]. Some cholestane glycosides, especially those with an aromatic
acyl group, showed potent cytotoxic activity against cultured tumor cell lines, including HL-60 human
promyelocytic leukemia cells.
We first isolated 3
xylopyranosyl)-(1 3)-(2-O-acetyl-
called OSW-1, from O. saundersiae bulbs in 1992 [
β
,16
β
,17
α
-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-
-L-arabinopyranoside), an acylated cholestenol glycoside commonly
]. The cytotoxic activity of OSW-1 against tumor
β-D-
→
α
9
cells was approximately 10–100 times more potent than that of clinically used anticancer agents,
with low toxicity against healthy cells [10]. The structural diversity and potent cytotoxic activity
of the cholestane glycosides obtained from O. saundersiae bulbs prompted us to carry out a further
Molecules 2017, 22, 1243; doi:10.3390/molecules22081243
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Molecules 2017, 22, 1243
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phytochemical examination of the plant. As a result, 12 new (
1
–
12) and seven known cholestane
glycosides (13
structural characterization methods, and through a sequence of chemical transformations followed
by spectroscopic and chromatographic analysis. The cytotoxic activity of 19 against HL-60 cells
–19) (Figure 1) were isolated. The new compounds (1–12) were identified via NMR-based
1–
and A549 human lung adenocarcinoma cells, and the apoptosis-inducing properties of 11, were
also investigated.
Figure 1. Structures of 1–19, 1a, and 6a.
2. Results and Discussion
2.1. Isolation and Structure Determination of 1–19
Fresh bulbs obtained from O. saundersiae were extracted with hot MeOH. The MeOH extract was
passed through a porous polymer polystyrene resin (Diaion HP-20) column. The EtOH-eluted fraction
was then subjected to column chromatography (CC) using silica gel and octadecylsilanized (ODS) silica
gel, and preparative high-performance liquid chromatography (HPLC), yielding compounds
1
–
19. The
-yl
structures of the known compounds were identified as (22S)-3 ,11 ,22-trihydroxycholest-5-en-16β
β
α
α
-L-rhamnopyranoside
2-O-acetyl- -L-rhamnopyranoside
2,3-di-O-acetyl- -L-rhamnopyranoside
2-O-acetyl-3-O-p-methoxybenzoyl-
5-en-16 -yl 2-O-acetyl-3-O-3,4,5-trimethoxybenzoyl-
(22S)-3 ,11 ,22-trihydroxycholesta-5,24-dien-16 -yl -L-rhamnopyranoside
(22S)-3β,11α,22-trihydroxycholesta-5,24-dien-16β-yl 2,3-di-O-acetyl-α-L-rhamnopyranoside (19) [7].
Compound was obtained as an amorphous solid, with a specific rotation of 7.1 (MeOH),
(
13
)
[
)
(
7
],
(22S)-3
(22S)-3
], (22S)-3
β
,11
α
α
α
,22-trihydroxycholest-5-en-16
,22-trihydroxycholest-5-en-16
,22-trihydroxycholest-5-en-16
β
β
β
-yl
-yl
-yl
α
(
14
[
11],
β
,11
,11
α
15
)
[7
β
α
-L-rhamnopyranoside (16) [
7], (22S)-3
β,11
α
,22-trihydroxycholest-
17 ],
and
β
α
-L-rhamnopyranoside
(
)
[7
β
α
β
α
(
18
)
[7],
1
−
and the molecular formula C41H68O14 based on the high-resolution electrospray ionization mass
spectrometry (HRESIMS, m/z 807.4518 [M + Na]+, calcd. for 807.4507) and 13C-NMR spectra. The
1H-NMR spectrum of
1 showed signals for five steroidal methyl groups at δH 1.31 (s, Me-19), 1.23
(d, J = 6.8 Hz, Me-21), 1.05 (d, J = 6.3 Hz, Me-26), 1.03 (d, J = 6.3 Hz, Me-27), 0.99 (s, Me-18); an
olefinic proton at δH 5.44 (br d, J = 4.9 Hz, H-6); and two anomeric protons at δH 5.09 (d, J = 7.7 Hz,
H-1 of
β
-D-glucopyranosyl (Glc)) and 5.05 (br s, H-1 of
α
-L-rhamnopyranosyl (Rha)). Furthermore,
1
the presence of an acetyl group in
1
was revealed via IR (1729 cm−1), H-NMR (δH 2.03, s, 3H),
and 13C-NMR (δC 170.4 C=O and 21.0 Me) spectroscopy (Table 1 and Table 3). Treatment of
3% NaOMe in MeOH afforded a deacyl derivative (1a), and subsequent acid hydrolysis of 1a with
1 M HCl in dioxane/H2O (1:1) yielded (22S)-cholest-5-ene-3 ,11 ,16 ,22-tetrol [ ] as the aglycone,
1 with
β
α
β
7

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and D-glucose and L-rhamnose as the carbohydrate moieties. The identification of D-glucose and
L-rhamnose, including their absolute configurations, was carried out by direct HPLC analysis of the
hydrolysate. These data implied that
1 was a (22S)-cholest-5-ene-3β,11α,16β,22-tetrol diglycoside
bearing an acetyl group. In the heteronuclear multiple bond correlation (HMBC) spectrum of
1,
long-range correlations were observed between the anomeric proton (H-1) of Glc at δH 5.09 and C-3 of
the aglycone at δC 78.2, between H-1 of Rha at δH 5.05 and C-16 of the aglycone at δC 82.7, and between
H-2 of Rha at δH 5.66 and the carbonyl carbon of the acetyl group at δC 170.4. All these data indicate that
compound
2-O-acetyl-α-L-rhamnopyranoside.
Compound (C43H70O15) was shown to be closely related to
spectra, differing from
1.98 (each 3H, s); δC 170.2 (C=O) and 20.5 (Me), and δC 170.0 (C=O) and 20.6 (Me))
(Table 1 and Table 3). Alkaline degradation of with 3% NaOMe in MeOH furnished
1a In the HMBC spectrum of , correlation peaks were observed between H-2 of Rha at
1
structure is (22S)-3
β
-[(
β
-D-glucopyranosyl)oxy]-11
α
,22-dihydroxycholest-5-en-16
β-yl
1
2
1
by the H- and 13C-NMR
1
only by the presence of two acetyl groups moieties (δH 2.07 and
2
.
2
δH 5.62 and the carbonyl carbon of one acetyl group at δC 170.0, and between H-3 of Rha
at δH 5.76 and the carbonyl carbon of another acetyl group at δC 170.2. The structure
of
2 was assigned as (22S)-3β-[(β-D-glucopyranosyl)oxy]-11α,22-dihydroxycholest-5-en-16β-yl
2,3-di-O-acetyl-α-L-rhamnopyranoside.
Compound
3 (C49H74O16) and 4 (C51H78O18) were suggested to be essentially analogous to 2
based on the 1H- and 13C-NMR spectra; however, the signals for one acetyl group attached to the
rhamnosyl group were not present. Instead, the signals assignable to a p-methoxybenzoyl group and a
3,4,5-trimethoxybenzoyl group were detectable in the 1H- and 13C-NMR spectra of
3
and
with 3% NaOMe in MeOH, methyl p-methoxybenzoate and 1a
, while methyl 3,4,5-trimethoxybenzoate and 1a were obtained from . HMBC
revealed long-range correlations between H-2 of Rha at δH 5.78 (dd, J = 3.0, 1.9 Hz) and
4, respectively.
Upon alkaline methanolysis of
were obtained from
analysis of
3 and 4
3
4
3
the carbonyl carbon of the acetyl group at δC 169.9, and between H-3 of Rha at δH 6.05 (dd, J = 9.4,
3.0 Hz) and the carbonyl carbon of the p-methoxybenzoyl group at δC 165.8. Similarly, correlation
peaks were observed between H-2 of Rha at δH 5.81 (dd, J = 3.3, 1.7 Hz) and the carbonyl carbon
of the acetyl group at δC 170.0, and between H-3 of Rha at δH 6.08 (dd, J = 9.6, 3.3 Hz) and the
carbonyl carbon of the 3,4,5-trimethoxybenzoyl group at δC 165.9 in that of
and were elucidated as (22S)-3 -[( -D-glucopyranosyl)oxy]-11 ,22-dihydroxycholest-5-en-16
2-O-acetyl-3-O-p-methoxybenzoyl- -L-rhamnopyranoside and (22S)-3 -[(
11 ,22-dihydroxycholest-5-en-16 -yl 2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-
respectively.
Compound
displayed signals arising from two tertiary methyl groups at δH 1.41 and 1.00 (each s, Me-19,
4. Thus, the structures of
3
4
β
β
α
β-yl
α
β
β-D-glucopyranosyl)oxy]-
α
β
α
-L-rhamnopyranoside,
5
(C35H56O9) was obtained as an amorphous powder. The 1H-NMR spectrum of
5
Me-18); a secondary methyl group at δH 1.26 (d, J = 6.8 Hz, Me-21); two methyl groups on a
double bond at δH 1.78 (Me-27) and 1.73 (Me-26); two olefinic protons at δH 5.82 (br t, J = 7.2 Hz,
H-24) and 5.65 (br d, J = 4.7 Hz, H-6); an anomeric proton at δH 5.03 (br s, H-1 of Rha); and the
methyl group of an acetyl moiety at δH 2.03 (s). These spectral features of
those of 18, and alkaline degradation of with 3% NaOMe in MeOH yielded 18. A long-range
correlation was observed between the H-2 of Rha at δH 5.65 (br d, J = 2.2 Hz) and the carbonyl
carbon of the acetyl group at δC 170.6 in the HMBC spectrum of , providing evidence that the acetyl
group was located at C-2 of the -L-rhamnopyranosyl moiety. The structure of was defined as
(22S)-3β,11α,22-trihydroxycholesta-5,24-dien-16β-yl 2-O-acetyl-α-L-rhamnopyranoside.
Compound
(C43H68O15), for which 1H-NMR analysis showed two anomeric proton signals
at δH 5.07 (d, J = 7.7 Hz, H-1 of Glc) and 5.03 (br s, H-1 of Rha), was assumed to be a bisdesmosidic
cholestane glycoside closely related to . The two compounds differed only in the signals of the
5 showed similarity to
5
5
α
5
6
2
methyl groups assignable to Me-26 and Me-27. The two three-proton doublets for Me-26 and
1
Me-27 observed in the H-NMR spectrum of
2 were displaced by two three-proton singlets at δH

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1.77 and 1.73 in that of
6, suggesting that
6
was the C-24/C-25 dehydro derivative of
2. This was
confirmed by alkaline methanolysis of
6, followed by enzymatic hydrolysis using β-D-glucosidase,
to yield 18 and D-glucose. The linkage position of the glucosyl moiety of
an HMBC correlation between H-1 of Glc and C-3 of the aglycone at δC 78.1. The structure of
was elucidated as (22S)-3 -[( -D-glucopyranosyl)oxy]-11 ,22-dihydroxycholesta-5,24-dien-16 -yl
2,3-di-O-acetyl-α-L-rhamnopyranoside.
Compound (C49H72O16) and (C51H76O18) were suggested to be the corresponding C-24/C-25
dehydro derivatives of and , respectively, based on their spectral properties (Tables 2 and 3). This
assumption was subsequently verified by chemical and spectroscopic analysis. When and were
treated with 3% NaOMe, yielded methyl p-methoxybenzoate and 6a, while furnished methyl
3,4,5-trimethoxybenzoate and 6a. In the HMBC spectra of , long-range correlations were observed
6 was ascertained by
6
β
β
α
β
7
8
3
4
7
8
7
8
7
between H-2 of Rha at δH 5.78 (dd, J = 2.7, 1.4 Hz) and the carbonyl carbon of the acetyl group
at δC 170.0, and between H-3 of Rha δH 6.08 (dd, J = 9.7, 2.7 Hz) and the carbonyl carbon of the
p-methoxybenzoyl group at δC 165.9. Similarly in 8, correlation peaks were observed between H-2 of
Rha at δH 5.81 (dd, J = 3.1, 1.6 Hz) and the carbonyl carbon of the acetyl group at δC 170.1, and between
H-3 of Rha at δH 6.11 (dd, J = 9.6, 3.1 Hz) and the carbonyl carbon of the 3,4,5-trimethoxybenzoyl group
at δC 166.0. The structures of
dihydroxycholesta-5,24-dien-16
(22S)- 3 -[( -D-glucopyranosyl)oxy]-11
(3,4,5-trimethoxybenzoyl)-α-L-rhamnopyranoside, respectively.
Compound was obtained as an amorphous solid and shown to have the molecular formula
C49H78O19 based on HRESIMS (m/z: 993.5050 [M + Na]+, calcd. 993.5035) and 13C-NMR spectral data.
The deduced molecular formula of was higher than that of
by C6H10O4, and the 1H-NMR spectrum
showed signals for three anomeric protons at δH 6.39 and 5.07 (each, br s), and 5.07 (d, J = 7.3 Hz),
and the methyl groups of two 6-deoxy sugars at δH 1.75 (3H 2, d, J = 6.0 Hz). Acid hydrolysis
of was compared with
yielded L-rhamnose and D-glucose. When the 13C-NMR spectrum of
that of (Table 3), a set of signals corresponding to one more terminal -L-rhamnopyranosyl unit
7
and
-yl 2-O-acetyl-3-O-p-methoxybenzoyl-
,22-dihydroxycholesta-5,24-dien-16
8
were assigned as (22S)-3
β
-[(
β
-D-glucopyranosyl)oxy]-11
-L-rhamnopyranoside and
-yl 2-O-acetyl-3-O-
α,22-
β
α
β
β
α
β
9
9
6
×
9
9
6
α
(Rha’) (δC 102.1 (CH), 72.6 (CH), 72.9 (CH), 74.2 (CH), 69.5 (CH), and 18.7 (Me)) were observed. The
resonances attributable to C-2 of the inner Glc unit and its neighboring carbons varied, while all the
other signals remained almost unaffected. In the HMBC spectrum of
observed between H-1 of Rha at δH 6.39 (br s) and C-2 of Glc at δC 77.9, and between H-1 of Glc at
δH 5.07 (J = 7.3 Hz) and C-3 of the aglycone at δC 78.2. Thus, the structure of was determined as
(22S)-3 -[( -L-rhamnopyranosyl-(1 2)- -D-glucopyranosyl)oxy]-11 ,22-dihydroxycholesta-5,24-dien-
16β-yl 2,3-di-O-acetyl-α-L-rhamnopyranoside.
Compound 10 (C43H70O14) was suggested to be a bisdesmosidic cholestane glycoside closely
related to , both possessing acetyl groups at C-2 and C-3 of the -L-rhamnopyranosyl moiety attached
to the aglycone’s C-16 hydroxy group. However, the molecular formula of 10 contained one oxygen
atom less than , the hydroxymethine
. When the 13C-NMR spectrum of 10 was compared with that of
carbon signal observed at δC 67.8 (C-11) in the spectrum of was displaced by a methylene carbon signal
at δC 21.1 in that of 10, indicating that 10 was the C-11 dehydroxy derivative of . Alkaline methanolysis
of 10 yielded the structure (22S)-3 -[( -D-glucopyranosyl)oxy]-22-hydroxycholest-5-en-16
9, long-range correlations were
9
β
α
→
β
α
2
α
2
2
2
2
β
β
β
-yl
-yl
α
-L-rhamnopyranoside (10a), which we have previously isolated from Galtonia candicans [13]. The
structure of 10 was assigned as (22S)-3β-[(β-D-glucopyranosyl)oxy]-22-hydroxycholest-5-en-16
β
2,3-di-O-acetyl-α-L-rhamnopyranoside.
Compound 11 (C33H54O8) was a cholestane rhamnoside with similar spectral properties to
those of 18. However, the molecular formula of 11 lacked one oxygen atom in comparison to 18
and the 13C signals assignable to C-11 differed between the two compounds. The hydroxymethine
carbone signal observed at δC 68.1 (C-11) in the 13C-NMR spectrum of 18 was displaced by a
methylene carbon signal at δC 21.1 in that of 11, allowing 11’s structure to be determined as
(22S)-3β,22-dihydroxycholesta-5,24-dien-16β-yl α-L-rhamnopyranoside.
,