N. Andriamisaina et al.
Phytochemistry 160 (2019) 78–84
+
Na] at m/z 927, which indicated a molecular weight of 904.
polarimeter. NMR spectra of compounds 2, 4, and 5 were performed
1
13
H and C NMR signals of the aglycone of 5 were almost super-
using a Varian INOVA 600 (Agilent Technologies) at the operating
1
imposable with those of 4, excepted two methylenic signals at δ
C
/δ
H
frequency of 600 MHz. The operating conditions were as follows: H:
3
5.1/1.92 (CH
2
-1) and 19.0/1.06, 2.04 (CH
2
-11) in 5 instead of two
frequency, 600 MHz; sweep width, 8 kHz; sampling point, 66 k; spectral
1
3
secondary alcoholic functions at C-1 and C-11 positions in 4. These data
allowed the identification of the aglycone as (22S,23S)-5α-cholest-24-
ene-3β,16β,22,23-tetraol, which is also an unusual cholestane-type
derivative. Two Glcs and one Rha were identified by 2D NMR and GC
width, 7804 Hz, accumulation, 32 pulses; temperature, 304 K. C:
frequency, 150 MHz; sweep width, 32 kHz; sampling point, 160 k;
spectral width, 30,000 Hz, accumulation, 8000 pulses; temperature,
304 K. Samples 2 and 5 were dissolved in C
5
D N (200 μL) using 5 mm
5
analysis. The HMBC correlations at δ
H
/δ
C
4.81 (d, J = 7.9 Hz) (GlcI H-
micro-sample tube (SHIGEMI Co., Ltd., Japan). Chemical shifts were
referenced to C N signals (δ 7.22, δ 123.8). Sample 4 was dissolved
in CD OD and chemical shifts were referenced to CD OD signals (δ
3.30, δ 49.0). Conventional pulse sequences were used for gMQF-
1
)/78.0 (C-3), 4.96 (d, J = 7.2 Hz) (GlcII H-1)/84.7 (C-16), and the
5
D
5
H
C
NOESY cross-peak at δ
H
/δ
H
4.81 (GlcI H-1)/3.96 (H-3) proved the O-
3
3
H
heterosidic linkage between GlcI and C-3, and between GlcII and C-16.
C
The (1 → 4) linkage between Rha and GlcII was determined by the
COSY, TOCSY, NOESY, gHSQC, and gHMBC. The mixing time in the
NOESY experiment was set to 500 ms. TOCSY spectra were acquired
using the standard MLEV17 spin-locking sequence and 60 ms mixing
time. TOCSY, NOESY and HSQC spectra were recorded using phase-
sensitive mode. The size of the acquisition data matrix was 2048 × 256
words in f2 and f1, respectively, and zero filling up to 2k in f1 was made
prior to Fourier transformation. Sine-bell or Shifted sine-bell window
functions, with the corresponding shift optimized for every spectrum,
were used for resolution enhancement, and baseline correction was
applied in both dimensions. The NMR spectra of 1 and 3 were recorded
with a Varian VNMR-S 600 MHz spectrometer equipped with 3 mm
triple resonance inverse and 3 mm dual broadband probes. Spectra are
NOESY correlation at δ
H
/δ 6.29 (br s) (Rha H-1)/4.18 (t, J = 8.8 Hz)
H
(
GlcII H-4).
The structure of compound 5 was thus established as (22S,23S)-3β-
[
(β-D-glucopyranosyl)oxy]-22,23-dihydroxy-5α-cholest-24-en-16β-yl
O-α-L-rhamnopyranosyl (1 → 4)-β-D-glucopyranoside (Fig. 1).
The Ornithogalum species are rich in spirostane and cholestane-type
glycosides, such as O. thyrsoides (Kuroda et al., 2002, 2004; 2006; Cao
et al., 2012), and O saundersiae (Kubo et al., 1992; Kuroda et al., 1995,
1
999; 2001; Mimaki et al., 1996, 1997; Iguchi et al., 2017). From a
chemotaxonomic point of view, it was not surprising to find these types
of aglycones in 1–5 even if they were unusual in compounds 4 and 5.
Kuroda et al. (2001), have described the cytotoxic activity of cholestane
glycosides from O. saundersiae against leukemia HL-60 cells in the nM
range. We thus investigated the potential cytotoxic effect of the cho-
lestane-type glycosides 3–5, and the two spirostane-type derivatives 1
and 2. They were tested on human lung carcinoma A-549 and human
promyelocytic leukemia HL-60 cell lines at different concentrations
recorded in C
5
D N. Solvent signals were used as internal standard
5
(C N: δ = 7.21, δ = 123.5 ppm), and all spectra were recorded at
5
D
5
H
C
T = 35 °C. Pulse sequences were taken from Varian pulse sequence li-
brary (gCOSY; gHSQCAD and gHMBCAD with adiabatic pulses CRISIS-
HSQC and CRISIS-HMBC). TOCSY spectra are acquired using DIPSI
spin-lock and 150 ms mixing time. Mixing time in ROESY experiments:
(
0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, 200 μM) by the XTT
300 ms. The carbon type (CH
3
, CH , CH) was determined by DEPT
2
assay (Zhang et al., 2018), with cisplatin as positive control at a con-
centration of 10 μM.
experiments, and coupling constants (J) were measured in Hz.
HR-ESIMS and ESIMS (positive-ion mode) of 2, 4 and 5 was carried
out on a Bruker micrOTOF II mass spectrometer and ESIMS (positive-
ion mode) for 1 and 3 on a Finnigan LCQ Deca. GC analysis was carried
out on a thermoquest gas chromatograph using a DB-1701 cap. column
(30 m × 0.25 mm, i.d.) (J and W Scientific); detection by FID; detector
temperature, 250 °C, injection temperature, 230 °C, initial temperature
was maintained at 80 °C for 5 min and then raised to 270 °C at 15 °C/
min; carrier gas, He (Pertuit et al., 2017). For the extractions, a R.E.U.S
ultrasonic apparatus (US frequency 24 KHz, Power 200 W), and a MARS
6 microwave apparatus (CEM) were used. Isolation of compounds was
carried out using column chromatography (CC) on Sephadex LH-20
(550 mm × 20 mm, GE Healthcare Bio-Sciences AB), and vacuum li-
quid chromatography (VLC) on silica gel 60 (Merck, 60–200 μm) and
reversed-phase RP-18 silica gel (75–200 μm, Silicycle). Medium pres-
sure liquid chromatography (MPLC) was performed on silica gel 60
(Merck, 15–40 μm) and reversed-phase RP-18 silica gel (75–200 μm,
Silicycle), with a Gilson M 305 pump (25 SC head pump, M 805
manometric module), a Büchi glass column (230 mm × 15 mm and
460 mm × 15 mm), and a Büchi precolumn (110 mm × 15 mm). TLC
(Silicycle) and HPTLC (Merck) were carried out on precoated silica gel
plates 60F254. The spray reagent for saponins was vanillin reagent (1%
In this preliminary assay, compounds 1–5 were inactive against A
5
49 cells at all tested concentrations. They were inactive on HL-60 cells
except compound 1 which exhibited a moderate cytotoxicity of 30% at
1
0 μM. Furthermore, we administrated all compounds (1–5) in combi-
nation with 10 μM cisplatin in A-549 cells and observed a slight po-
tentiation of cisplatin cytotoxicity only by compound 1 from 63%
survival (cisplatin alone) to 47% (cisplatin in combination with 1 at
1
0 μM) (Gaidi et al., 2002). These preliminary results showed the low
sensitivity of these two cell lines to the steroid saponins isolated from O.
dubium.
The results on the two spirostane derivatives 1 and 2, glycosylated
at the C-1 position, are in accordance with those reported in the review
of Podolak et al. (2010) in which the C-1 glycosylated spirostane de-
rivatives showed no activity. The results about the inactive cholestane-
type glycosides 3–5 were also in accordance with those reported by
Tang et al. (2013). These authors described the evaluation of the cy-
totoxicity of deacylated derivatives from O. saundersiae. The native
compounds possessed a diglycoside moiety at the C-16 position of the
aglycon with an aromatic acyl group and an acetyl function. The dea-
cylated molecules were significantly less potent than the native ones.
This indicated that the acyl groups attached at the diglycosyl moiety
was a structural requirement for the potent activity. In our case, 3–5
possess a structure closely related to the compounds of O. saundersiae,
without the acylated glycosyl group at the C-16 position of the aglycon.
These preliminary results together with those of the literature, showed
that the cytotoxic activity of such cholestane-type saponins might be
connected with a certain degree of lipophilicity.
vanillin in EtOH–H
2
SO , 50:1).
4
3.2. Plant material
Ornithogalum dubium Houtt. (Asparagaceae), was purchased in 2015
from Jardiland (Dijon) and identified by Pr. Marie-Aleth Lacaille-
Dubois. A voucher specimen (No. 20151009) was deposited in the
herbarium of the Laboratory of Pharmacognosy, Université de
Bourgogne Franche-Comté, Dijon, France.
3
. Experimental
3.1. General experimental procedures
3.3. Extraction and isolation
Optical rotations values were recorded on an AA-10R automatic
Fresh bulbs of Ornithogalum dubium (224 g) were submitted to
81