Cytotoxic steroidal saponins from the rhizome of Anemarrhena asphodeloides

Article abstract of DOI:10.1016/j.steroids.2019.108557

Two novel steroidal saponins, timosaponin V and W (1 and 2), together with seven known steroidal saponins (3–9), were isolated from the rhizomes of Anemarrhena asphodeloides Bunge. Their structures were elucidated by extensive 1D NMR and 2D NMR (HSQC, HMBC, ¹H–¹H COSY, and NOESY), and MS analyses. The cytotoxic activities of the isolates were evaluated. Compound 1 showed a significant cytotoxic activity against MCF-7 and HepG2 cell lines with IC₅₀ values of 2.16 ± 0.19 μM and 2.01 ± 0.19 μM, respectively.

Full text of DOI:10.1016/j.steroids.2019.108557

Steroids 155 (2020) 108557
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Cytotoxic steroidal saponins from the rhizome of Anemarrhena asphodeloides
⁎
Yun-Fang Zhao, Jing Zhou, Min-Jie Zhang, Min Zhang, Xue-Feng Huang
Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two novel steroidal saponins, timosaponin V and W (1 and 2), together with seven known steroidal saponins
(3–9), were isolated from the rhizomes of Anemarrhena asphodeloides Bunge. Their structures were elucidated by
extensive 1D NMR and 2D NMR (HSQC, HMBC, H– H COSY, and NOESY), and MS analyses. The cytotoxic
activities of the isolates were evaluated. Compound 1 showed a significant cytotoxic activity against MCF-7 and
HepG2 cell lines with IC50 values of 2.16 ± 0.19 μM and 2.01 ± 0.19 μM, respectively.
Anemarrhena asphodeloides
Steroidal saponin
Timosaponin V
Timosaponin W
Cytotoxic activity
1
1
1. Introduction
2. Experimental
The rhizome of Anemarrhena asphodeloides Bunge, a traditional
2.1. General experimental procedures
Chinese medicine called “Zhi-Mu”, is widely cultivated in China such as
Anhui and Hebei province. A. asphodeloides has the effects of clearing
away heat, purging fire, nourishing yin and moistening dryness [1].
Previous phytochemical investigation showed that it contains a series of
compounds including steroidal saponins [2–6], polysaccharides [7],
flavonoids [8], lignans [9], and xanthones [10]. Pharmacological stu-
dies have found that steroidal saponins from this plant have a variety of
biological activities, such as anti-tumor [11,12], anti-inflammatory
Optical rotations were determined with a JASCO P-1020 digital
polarimeter (Tokyo, Japan). NMR spectra were performed on a Bruker
AV-300 instrument (300 MHz for 1H NMR and 75 MHz for C NMR,
13
5
Zurich, Switzerland) in pyridine‑d with TMS as the internal standard.
ESI-MS spectra were recorded on an Agilent 1100 Series LC/MSD Trap
SL mass spectrometer (Santa Clara, USA). HR-ESI-MS spectra were re-
corded on an Agilent 6520B Q-TOF spectrometer (Santa Clara, USA).
Column chromatography (CC) was performed on macroporous resin
D101 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), silica gel
(200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China),
ODS-AQG12S50 (YMC, Tokyo, Japan) and HW-40 (Tosoh, Tokyo,
Japan). TLC was carried out on silica gel GF254 plates (Qingdao
Haiyang Chemical Co., Ltd., Qingdao, China) and silica gel 60 RP-18
[
13] and anti-fungal [14] activities.
In our previously study, we have reported the isolation and struc-
tural elucidation of several new bioactive steroids from the A. aspho-
deloides which collected in Anhui province. In this paper, in order to
find novel biologically active steroidal saponins in A. asphodeloides from
different habitats, we carried out detailed phytochemical investigation
on the A. asphodeloides which collected in Hebei province. Herein, we
reported the isolation and structure elucidation of two novel steroidal
saponins: timosaponin V (1), timosaponin W (2) and seven known
steroidal saponins: timosaponin BIII (3) [15], timosaponin AII (4) [16],
F254S plates (Merck, Darmstadt, Germany). TLCs spots were visualized
by heating after spraying with 10% H SO -EtOH aqueous. Sugar ana-
2
4
lysis was performed on an Agilent 1200 liquid chromatography (Santa
Clara, USA) with a UV detector and a YMC-Pack ODS-A column
(250 × 4.6 mml, S-5 μm, 12 nm, YMC, Tokyo, Japan).
timosaponin
P (5) [17], timosaponin AIII (6) [18], anemar-
rhenasaponin I (7) [19], anemarrhenasaponin Ia (8) [19], and timo-
pregnane A (9) [18] (Fig. 1). In addition, the cytotoxic activities of the
isolates were evaluated, and compounds 1 and 6 showed significant
cytotoxic activities.
2.2. Plant material
The rhizomes of Anemarrhena asphodeloides Bunge were collected in
August 2016 from Chengde of Hebei province. The plant material was
identified by Dr. Minjian Qin, Department of Resources Science of
Traditional Chinese Medicines, China Pharmaceutical University,
China. A voucher specimen (No.2016080001) was deposited at the
⁎
Corresponding author.
E-mail address: hxf@cpu.edu.cn (X.-F. Huang).
https://doi.org/10.1016/j.steroids.2019.108557
Received 18 September 2019; Received in revised form 4 December 2019; Accepted 12 December 2019
Available online 20 December 2019
0039-128X/ © 2019 Elsevier Inc. All rights reserved.

Y.-F. Zhao, et al.
Steroids 155 (2020) 108557
Fig. 1. Structures of compounds 1–9.
Department of Natural Medicinal Chemistry, China Pharmaceutical
University.
(100:20:2–100:50:8, v/v/v) to yield compound 2 (4.0 mg) and com-
pound 9 (9.0 mg). ZA-4 was subjected to a silica gel CC using CH
MeOH (20:1–1:0, v/v) as eluent to afford five fractions (ZA-4-1~ZA-
–5). ZA-4–3 (CH Cl -MeOH, 5:1, v/v) precipitated a large amount of
2 2
Cl -
4
2
2
2.3. Extraction and isolation
white powder, then compound 6 (5.0 g) was purified by filtrating and
washing with MeOH. ZA-4–2 was fractionated by an ODS CC and eluted
The dried rhizomes of A. asphodeloides (10 kg) were extracted three
times with 75% EtOH at reflux (50 L × 3 h). The combined extract was
filtered and concentrated under vacuum to give a residue (3.09 kg, not
with MeOH-H
and 8 (19.1 mg). ZA-4–4 was separated by an ODS CC with MeOH-H
30:70–100:0, v/v) to afford compound 1 (8.0 mg), then the residue
2
O (30:70–100:0, v/v) to give compounds 7 (78.7 mg)
2
O
(
thoroughly dry). Then, the crude extract was suspended in H
extracted with petroleum ether, EtOAc and 1-butanol, respectively. The
-butanol extract (600 g) was subjected to a D101 macroporous resin
column and eluted with EtOH-H O (1:9, 3:7, 1:1, 7:3, 19:1, v/v) to
obtain five fractions (ZA-1~ZA-5). ZA-3 was subjected to an ODS
column using MeOH-H O (30:70–100:0, v/v) as eluent to afford six
fractions (ZA-3-1~ZA-3-6). ZA-3–5 (MeOH-H O, 7:3, v/v) precipitated
2
O and
was purified on a HW-40 column with CH
compound 4 (26.5 mg).
2 2
Cl -MeOH (1:1, v/v) to yield
1
2
2.3.1. Timosaponin V (1)
2
5
−3.39 (c = 0.10, pyridine); 1H NMR (pyr-
White powder; [α]
D
2
1
3
idine‑d , 300 MHz) and C NMR (pyridine‑d , 75 MHz) spectra data
5
5
−
2
a large amount of white powder, then compound 3 (1.0 g) was obtained
by filtrating and washing with MeOH. ZA-3-2 was purified on an ODS
73 17
see Table 1; HR-ESI-MS m/z 885.4873 [M−H] (calcd for C45H O ,
885.4853).
2
CC using MeOH-H O (30:70–100:0, v/v) as eluent to give compound 5
(
(
8.4 mg). ZA-3-3 was separated by repeated ODS CC with MeOH-H
30:70–100:0, v/v) and silica gel CC with CH Cl -MeOH-H
2
O
O
2.3.2. Timosaponin W (2)
25
−4.67 (c = 0.10, pyridine); 1H NMR
2
2
2
White powder; [α]
D
2

Y.-F. Zhao, et al.
Steroids 155 (2020) 108557
2.4. Acid hydrolysis of compounds 1–2 and sugar analysis
Table 1
NMR data of compounds 1 and 2 in pyridine‑d (δ in ppm, J in Hz).
5
Compounds 1 and 2 (each 2.0 mg) were individually dissolved in
NO.
1
2
2
M HCl-1, 4-dioxane (1:1, 1.0 mL) and held at 80 °C for 6 h. The
δ
C
δ
H
δ
C
δ
H
3
liberated aglycone was extracted with CHCl (0.5 mL × 3). The aqu-
eous residue was evaporated to dryness under nitrogen protection to
yield the monosaccharides. The residue was dissolved in 1, 4-dioxane
1
2
31.21
27.59
1.49 m
.91 m
1.59 m
.83 m
4.32 m
1.83 m
31.34
27.20
1.47 m
1.80 m
1.09 m
2.00 m
4.34 m
1.80 m
1.82 m
2.16 m
1.23 m
1.84 m
0.95 m
1.86 m
1.49 m
1.28 m
–
0.92 m
1.20 m
1.06 d (6.2)
1.66 d (12.3)
–
1.04 m
1.33 m
1.97 m
4.55 m
1.80 m
0.77 s
1
(80 μL), then 0.5 M PMP-CH
added to the mixture. After reacting at 70 °C for 0.5 h, 0.3 M HCl was
added to neutralize and then the mixture was extracted with CHCl
3
OH (80 μL) and 0.3 M NaOH (80 μL) were
1
3
4
75.25
30.88
75.89
31.34
3
(
3 × 0.5 mL). The aqueous residue was dried and then dissolved with
1
.96 m
2
MeOH-H O for analysis of the sugar moieties by HPLC. The following
was the analysis condition: mobile phase A: acetonitrile-20 mM am-
monium acetate solution (15:85, v/v), mobile phase B: acetonitrile-
5
6
36.44
27.41
2.45 d (12.5)
1.79 m
37.34
27.39
1
.85 m
1.93 m
.17 m
7
26.86
27.15
20 mM ammonium acetate solution (40:60, v/v), gradient elution: 0% B
2
to 60% B for 30 min with the rate of 1 mL/min, injection volume: 10 μL,
detection wavelength: 254 nm. As a result, sugars obtained from
compounds were confirmed to be D-galactose, D-glucose, L-rhamnose by
comparison of the retention times of standard monosaccharides: D-ga-
8
9
36.05
40.72
35.71
21.60
1.47 m
1.33 m
–
35.90
40.61
35.64
21.53
1
1
0
1
1.18 m
1
.33 m
R R
lactose (t = 24.375 min), D-glucose (t = 23.380 min), L-rhamnose
1
2
40.72
1.10 m
40.66
(t
R
= 19.653 min).
1
.71 m
1
1
1
3
4
5
41.32
56.93
32.62
–
1.10
1.45 m
41.20
56.83
32.39
2.5. Cytotoxic activity
2
.00 m
MCF-7 (human breast cancer cells) and HepG2 (human hepatocel-
1
1
1
1
2
2
2
2
6
7
8
9
0
1
2
3
81.80
63.44
16.98
24.38
42.91
15.30
110.10
27.30
4.63 dd (15.0, 7.3)
1.86 m
0.84 s
1.01 s
1.93 m
1.17 d (6.8)
–
1.17 m
82.08
62.84
16.95
24.40
42.86
15.13
111.60
34.52
lular carcinoma cells) were obtained from Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences. Cancer cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) (Gibco, Invitrogen Corporation, Carlsbad, CA) sup-
plemented with 10% fetal bovine serum (Royal, Lanzhou, China),
100U/ml benzyl penicillin, and 100U/ml streptomycin in a humidified
0.98 s
1.96 m
1.12 d (6.8)
–
1.95 m
2.12 m
4.84 dt (10.4, 5.0)
1
.24 m
1.36 m
.41 m
2
environment with 5% CO at 37 °C.
2
4
26.64
73.24
Cell toxicity was measured by a colorimetric assay using MTT as
described previously [20]. Each experiment was carried out in tripli-
cate. Control group was cultured with culture media containing 0.1%
dimethylsulfoxide. Absorbance (A) was measured at 570 nm. Cell via-
bility (I) was calculated according to the following equation:
1
2
2
5
6
27.97
65.53
1.59 m
3.38 d (10.8)
32.14
64.44
2.28 m
3.53 brd (10.6)
3.95 brd (9.8)
1.34 d (7.1)
4.93 d (7.6)
4.69 m
4.29 m
4.59 m
4.05 t (6.0)
4.43 m
4.46 m
4
.08 m
2
7
16.70
102.00
77.02
76.80
71.09
77.45
62.95
1.09 d (6.2)
4.95 d (7.1)
4.81 m
4.50 m
4.40 m
10.32
102.99
82.32
75.63
70.25
77.02
62.60
Gal-1′
Gal-2′
Gal-3′
Gal-4′
Gal-5′
Gal-6′
Cell viability(I) = (1−Aexperimental group /A control group) × 100%
Paclitaxel was used as a positive control.
4.01 t (5.8)
4.37 m
3. Results and discussion
4
.40 m
Glc-1″
Glc-2″
Glc-3″
Glc-4″
Glc-5″
Glc-6″
102.40
79.79
79.71
73.61
77.67
63.92
5.77 d (7.0)
4.20 m
4.21 m
4.08 m
3.68 m
106.59
77.38
78.44
71.92
78.83
62.90
5.31 d (7.7)
4.11 m
4.22 t (9.0)
4.33 m
3.87 m
4.49 m
4.52 m
5.06 d (7.7)
4.08 m
4.28 m
4.33 m
Compound 1 was obtained as a white powder. Its HR-ESI–MS
showed quasi-molecular ion peak at m/z 885.4873 ([M−H] ), (calcd
−
for C45
unsaturation. The H NMR spectrum of compound 1 (Table 1) displayed
three anomeric proton signals at δ 4.95 (1H, d, J = 7.1 Hz, H-1′), 5.77
(1H, d, J = 7.0 Hz, H-1″) and 6.35 (1H, br s, H-1‴), two angular methyl
signals at δ 0.84 (3H, s, H-18) and 1.01 (3H, s, H-19), and three
doublet signals at δ 1.17 (3H, d, J = 6.8 Hz, H-21), 1.09 (3H, d,
J = 7.1 Hz, H-27) and 1.86 (3H, d, J = 6.2 Hz, Rha-H-6‴). The
NMR spectrum of compound 1 (Table 1) showed three anomeric carbon
signals at δ 102.00 (C-1′), 102.40 (C-1″) and 102.79 (C-1‴), and five
methyl carbon signals at δ 16.98 (C-18), 24.38 (C-19), 15.30 (C-21),
6.70 (C-27) and 19.51 (Rha-C-6‴). The observations above suggested
73
H O17 is 885.4853, −2.26 ppm) indicating nine degrees of
1
4.27 m
4
.40 m
H
Glc-1‴
Glc-2‴
Glc-3‴
Glc-4‴
Glc-5‴
Glc-6‴
101.58
75.77
79.10
72.12
78.87
63.19
H
H
13
C
3.95 m
4.43 m
4
.49 m
C
Rha-1‴
Rha-2‴
Rha-3‴
Rha-4‴
Rha-5‴
Rha-6‴
102.79
72.91
73.19
74.85
69.98
19.51
6.35 s
4.83 m
4.78 m
4.40 m
5.06 dd (9.4, 6.2)
1.86 d (6.2)
C
1
that compound 1 was a steroidal glycoside with three sugar moieties.
Acid hydrolysis and HPLC analysis of compound 1 afforded D-galactose,
D-glucose and L-rhamnose and the relative ratio was 1:1:1. The β-con-
figuration of the galactose and glucose were determined according to
3
the coupling constants ( J1,2 > 7 Hz) of the anomeric protons. The α-
5 5
pyridine‑d , 75 MHz) spectra
, 300 MHz) and 13C NMR (pyridine‑d
(
C
configuration of the rhamnose was inferred by δ 73.19 (C-3‴) and
−
data see Table 1; HR-ESI-MS m/z 917.4757 [M−H] (calcd for
19, 917.4752).
6
9.98 (C-5‴) [21]. After acid hydrolysis of 1, the NMR and MS spec-
45 73
C H O
trum of the aglycone were similar to sarsasapogen [22]. The config-
uration of H-5 was confirmed as β based on the NOESY correlations of
H-19 (δ
H
H
1.01) with H-5 (δ 2.45) (Fig. 2). In the HSQC spectrum,
3

Y.-F. Zhao, et al.
Steroids 155 (2020) 108557
Fig. 2. Key HMBC and NOESY correlations of compounds 1 and 2.
according to the two proton signals of δ
H
4.08 (m, 1H) and 3.38 (d, 1H,
J = 10.8 Hz) of C-26, the 25S configuration was deduced [23]. In
5.31)/C-2′ (δ
C
82.32). Thus, the structure of compound 2 was de-
termined as 24-O-β-D-glucopyranosyl-(24S,25R)-3β, 24β-dihydroxy-5β-
addition, the 25S configuration could also be confirmed by δ
C
16.70 (C-
spirostane-3-O-β-D-glucopyranosyl-(1
named tiomosaponin W.
→
2)-β-D-galactopyranoside,
27) [24]. Therefore, the aglycone of compound 1 was determined as
(
25S)-5β-spirostane-3β-ol.
It has been reported that the sugars linked to the steroidal saponins
in A. asphodeloides are mainly glucose, galactose, mannose, and xylose.
In this study, the compound 1 was isolated for the first time from A.
asphodeloides. Moreover, the particulary glycosylation at C-24 on the
ring F observed for compound 2 is noteworthy.
The compounds (1–9) were evaluated for in vitro cytotoxic activ-
ities against MCF-7 and HepG2 cell lines (Table2), and paclitaxel was
used as positive control. The results showed that compounds 1 and 6
exhibited significant cytotoxic activities against MCF-7 and HepG2 cell
The HMBC correlations H-1′ (δ
H
4.95)/C-3 (δ
C
75.25); H-1″ (δ
H
5
.77)/C-2′(δ
C
77.02) and H-1‴ (δ
H
6.35)/C-2″ (δ 79.79) confirmed the
C
sequence of the sugar chain at C-3. So, the attachment sequence of the
sugar chain at C-3 was elucidated to be α-L-rhamnopyranosyl-(1 → 2)-β-
D-glucopyranosyl-(1 → 2)-β-D-galactopyranoside. Thus, the structure of
compound 1 was determined as (25S)-5β-spirostane-3β-ol-3-O- α-L-
rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-galactopyr-
anoside, named tiomosaponin V.
Compound 2 was obtained as a white powder. Its HR-ESI–MS
showed a quasi-molecular ion peak at m/z 917.4757 [M−H] , (calcd
lines, with IC50 values ranging from 2.01
±
0.19 μM to
−
2.22 0.25 μM. Compound 4 showed a good cytotoxic activity
±
for C45
unsaturation. The H NMR spectrum of compound 2 (Table 1) displayed
4.93 (1H, d, J = 7.6 Hz, H-1′), 5.06
1H, d, J = 7.7 Hz, H-1‴) and 5.31 (1H, d, J = 7.7 Hz, H-1″); two
H
73
O
19 is 917.4752, −0.54 ppm) indicating nine degrees of
against HepG2 cells with an IC50 values of 13.98 ± 0.43 μM. Com-
pounds 3, 5, 7, 8, and 9 showed moderate cytotoxic activities against
HepG2 cells with IC50 values below 65 μM. Compound 2 was inactive
toward the two cell lines with IC50 > 100 μM. Comparing the cyto-
toxicity of compounds 1, 2, and 4, it indicated that the aglycon struc-
ture, the type of monosaccharide and the position of sugar moiety have
an effect on the activity. Compounds 3, 5, 7, and 8 showed lower cy-
totoxicities than compounds 1, 4, and 6, which suggested that the cy-
totoxicity of spirostanol saponins is stronger than that of saponins
without F-ring.
1
three anomeric proton signals at δ
H
(
H
angular methyl signals at δ 0.77 (3H, s, H-18) and 0.98 (3H, s, H-19);
H
two doublet signals at δ 1.12 (3H, d, J = 6.8 Hz, H-21) and 1.34 (3H,
1
3
d, J = 7.1 Hz, H-27). The C NMR spectrum of compound 2 (Table 1)
101.58 (C-1‴), 102.99 (C-
′) and 106.59 (C-1″); four methyl carbon signals at δ 16.95 (C-18),
4.40 (C-19), 15.13 (C-21) and 10.32 (C-27). The observations above
showed three anomeric carbon signals at δ
C
1
2
C
suggested that compound 2 was a steroidal glycoside with three sugar
moieties. Acid hydrolysis and HPLC analysis of compound 2 gave D-
glucose and D-galactose and the relative ratio was 2:1. The β-config-
uration of the galactose and glucose were determined according to the
4
. Conclusion
In conclusion, two novel steroidal saponins 1 and 2 together with
3
coupling constants ( J1,2 > 7 Hz) of the anomeric protons. Comparing
seven known steroidal saponins were isolated from the rhizomes of
the NMR data of compound 2 with timosaponin A III [18], it displayed
similarity except for variation at the ring F. These indicated that the
hydroxyl group attached to C-24. In addition, the conclusion was fur-
Table 2
a
The cytotoxic activities of compounds 1–9. (IC50/μM.)
ther confirmed by the HMBC correlations from H-26a (δ
73.24) and H-27 (δ 1.34) to C-24 (δ 73.24). The NOESY corre-
lations of H-24 (δ 4.84)/H-26b (δ 3.95) suggested that H-24 was a-
H
3.53) to C-24
Compounds
Cell lines
(
δ
C
H
C
H
H
MCF-7
HepG2
oriented (Fig. 2). Therefore, the hydroxyl group at C-24 was β-oriented,
and the stereochemistry of C-24 was determined as S. Moreover, the
data of ring F was consistent with literature [25], which can be proved
that absolute configuration of C-25 is R-configuration. Therefore, the
aglycone of compound 2 was elucidated as (24S, 25R)-5β-spirostane-3β,
1
2
3
4
5
6
7
8
9
2.16 ± 0.19
> 100
> 100
> 100
> 100
2.05 ± 0.12
> 100
> 100
> 100
6.92 ± 0.82
2.01 ± 0.19
> 100
61.91 ± 2.60
13.98 ± 0.43
56.28 ± 3.91
2.22 ± 0.25
35.75 ± 3.98
42.22 ± 3.42
45.71 ± 4.22
4.71 ± 0.56
2
4β-diol.
The connectivity of the sugar chain was deduced based on the
HMBC correlations. Firstly, the HMBC cross-peaks between H-1‴ (δ
.06) and C-24 (δ 73.24) indicated a glucosyl residue attached to C-24.
Moreover, the linkages of the sugar units at C-3 were ascertained from
the HMBC correlations H-1′ (δ 4.93)/C-3 (δ 75.89) and H-1″ (δ
H
b
5
C
Paclitaxel (nM)
a
IC50 valve based on triplicate five points, presented as the mean ± S.D.
H
C
H
b
Positive control.
4

Y.-F. Zhao, et al.
Steroids 155 (2020) 108557
Anemarrhena asphodeloides Bunge. The cytotoxic activities of the iso-
lates were evaluated. Compounds 1 and 6 exhibited significant cyto-
toxic activities against MCF-7 and HepG2 cell lines. The aglycon
structure and the variations of the sugar moiety of furostanol saponins
could affect their cytotoxicity. Compounds 1 and 6 have similar struc-
tures and pharmacological activities. In-depth studies and comparisons
of their structure-activity relationships have a good effect on the anti-
tumor mechanism of steroidal saponins.
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Acknowledgment
This work was supported by the National Natural Science
Foundation of China (grant number 81573560). We are grateful to the
members of the analytical group of China Pharmaceutical University for
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