Furostanol saponins from Chinese onion induce G2/M cell-cycle arrest and apoptosis through mitochondria-mediate pathway in HepG2 cells

Article abstract of DOI:10.1016/j.steroids.2019.04.003

Phytochemical investigations on the bulbs of Chinese onion led to the isolation of three new furostanol saponins (1, 2, 5) together with seven known furostanol saponins (3, 4, 6–10). Their chemical structures were elucidated on the basis of spectroscopic and chemical methods, including IR, MS, NMR, and GC analyses. The anti-proliferative and anti-inflammatory activities of the isolates were evaluated. Compounds 7–10 showed potential anti-proliferative activities against human cancer cell lines (HepG2, A549, SPC-A-1, MGC80-3, MDA-MB-231, SW620 and CNE-1) with IC₅₀ values below 30 μM. Compounds 4 and 7 could induce G2/M cell-cycle arrest and apoptosis through mitochondria-mediate pathway in HepG2 cells. Compounds 7 and 10 showed strong inhibitory effects against LPS induced NO production in RAW264.7 cells with IC₅₀ values of 2.01 ± 1.40 μM and 2.49 ± 1.54 μM, respectively.

Full text of DOI:10.1016/j.steroids.2019.04.003

Accepted Manuscript
Furostanol saponins from Chinese onion induce G2/M cell-cycle arrest and
apoptosis through mitochondria-mediate pathway in HepG2 cells
Yihai Wang, Xiaomin Yi, Limin Xiang, Yuying Huang, Zhe Wang, Xiangjiu
He
PII:
S0039-128X(19)30068-6
https://doi.org/10.1016/j.steroids.2019.04.003
STE 8398
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
16 November 2018
9 April 2019
12 April 2019
Please cite this article as: Wang, Y., Yi, X., Xiang, L., Huang, Y., Wang, Z., He, X., Furostanol saponins from
Chinese onion induce G2/M cell-cycle arrest and apoptosis through mitochondria-mediate pathway in HepG2 cells,
Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.04.003
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Furostanol saponins from Chinese onion induce G2/M cell-cycle arrest and
apoptosis through mitochondria-mediate pathway in HepG2 cells
a
a
a
Yihai Wang a, b, Xiaomin Yi , Limin Xiang , Yuying Huang a, Zhe Wang , Xiangjiu
He a, b,*
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
China
b
Guangdong Engineering Research Center for Lead Compounds & Drug Discovery,
Guangzhou 510006, China
* Corresponding author: Dr. Xiangjiu He (Tel/Fax: 86-20-3935-2132, E-mail:
hexiangjiu@163.com)
1

ABSTRACT
Phytochemical investigations on the bulbs of Chinese onion led to the isolation of
three new furostanol saponins (1, 2, 5) together with seven known furostanol
saponins (3, 4, 6-10). Their chemical structures were elucidated on the basis of
spectroscopic and chemical methods, including IR, MS, NMR, and GC analyses.
The anti-proliferative and anti-inflammatory activities of the isolates were evaluated.
Compounds 7–10 showed potential anti-proliferative activities against human cancer
cell lines (HepG2, A549, SPC-A-1, MGC80-3, MDA-MB-231, SW620 and CNE-1)
with IC50 values below 30 μM. Compounds 4 and 7 could induce G2/M cell-cycle
arrest and apoptosis through mitochondria-mediate pathway in HepG2 cells.
Compounds 7 and 10 showed strong inhibitory effects against LPS induced NO
production in RAW264.7 cells with IC50 values of 2.01 ± 1.40 μM and 2.49 ± 1.54
μM, respectively.
Keywords: furostanol saponins; Chinese onion; Allium chinense; anti-proliferative
activity; anti-inflammatory activity
2

1. Introduction
The genus Allium, belongs to family Amaryllidaceae (subfamily Allioideae),
comprises more than six hundred different species found throughout North America,
Europe, Asia and North Africa [1]. Members of A. family have been cultivated in the
Middle and Far East for at least five thousand years. Many Allium species possess
characteristic pungent flavor and are prized as foodstuffs or vegetables in widespread
areas of the world [2, 3].
Chinese onion (A. chinense G. Don), also known as oriental onion, is an ancient
vegetable native to China. It is widely cultivated in Asian countries such as China
(common known as “Jiaotou”), Japan (known as “Rakkyo”), Korea, Vietnam, and
Indonesia, for its edible bulbs and leaves, which may be eaten raw or cooked. The
flowers are also edible and may be used as a garnish on salads [4]. In China, the
bulbs of Chinese onion are constantly served as sweet or sour pickles after steeped in
sugar or/and brine. It also can be pickled as a pleasing seasoning or cooked with
other ingredients to afford various featured Chinese cuisine [5]. In Japan, they are
consumed mainly in pickles as side dishes. Chinese onion is also used as a pickled
meal during Vietnamese New Year–Tet celebrations. Additionally, Chinese onion is
also introduced into some similar latitude of Northern America [6].
In China, Chinese onion is also well known as a plant for both food and medicine
suggested by the government. In traditional Chinese medicine, the bulbs of this plant
served as one of the botanical origins of the crude drug “Xiebai”, which is included
in some classic prescription for treating chest pain, stenocardia, as well as heart
3

asthma [7]. Pharmacological studies have shown that preparations from “Xiebai”
show a number of biological activities, including antiatherogenic, hypolipidemic,
anti-platelet aggregation, antihypertension, antioxidant, and analgesic effects [5].
In the past decades, Chinese onion has attracted increasing attention in the field of
food, nutraceuticals and phytomedicine because of its wide health benefits. It has
been reported to possess a wide range of biological activities such as
anti-hyperlipidemic activity [8], anti-tumor activity [9, 10], and anti-Alzheimer’s
disease [11, 12]. Phytochemical investigates on Chinese onion have led to the
isolation of sulfur-containing compounds which responsible for its onion-like flavor
[13], nitrogen-containing constituents [14], and steroidal saponins [15-19]. As an
important class of natural products discovered in many advanced plants, steroidal
saponins are found to possess a variety of biological activities, such as
antiproliferation, hemolytic, anti-platelet aggregation, anti-inflammatory, and
anti-bacterial activities [20].
In our previously study, spirostanol saponins isolated from Chinese onion exert
pronounced anti-inflammatory and anti-proliferative activities [19]. In our
continuing efforts to screen the bioactive constituent of Chinese onion, further
phytochemical study was carried out. As a result, ten furostanol saponins including
three new were isolated and identified from the bulbs of Chinese onion. Some
saponins exhibit potential anti-proliferative, and anti-inflammatory activities and
could induce G2/M cell-cycle arrest and apoptosis through the mitochondria-mediate
pathway in HepG2 cells.
4

2. Experimental
2.1 General Experimental Procedures.
Optical rotations were determined with a JASCO P-1020 polarimeter (Tokyo,
Japan). IR spectra (4000–450 cm–1) of the saponins (as pellets in KBr) were recorded
using a 100 FT-IR spectrometer (Perkin Elmer Inc., Waltham, MA). All NMR
spectra were acquired on a Bruker Avance III 500 MHz digital NMR spectrometer
(Bruker BioSpin GmbH, Switzerland). HR-ESI-MS data were acquired on a
UPLC-Q-TOF Micro mass spectrometer equipped with an ESI source (Waters Corp.,
Milford, MA). Preparative HPLC was carried out on a Waters HPLC system (Waters
Corp., Milford, MA), including a 2535 pump and a 2489 UV/Visible detector. The
separation was performed on a 5C18-MS-II (10ID×250 mm) semi-preparative HPLC
column or 5C18-AR-II (20ID×250 mm) preparative HPLC column (Cosmosil,
Nacalai Tesque, Kyoto, Japan). Microplate reader (KHB-ST-360) was product of
Kehua Technologies, Inc. (Shanghai, China).
2.2 Chemicals and Reagents.
Macroporous resin (D101) was purchased from Xian Lanxiao Resin Corporation
Ltd. (Xian, China). Silica gel (200-300 mesh, Anhui Liangchen Silicon Material Co.
Ltd., Luan, China) and ODS (40-60 μm, Merck KGaA, Darmstadt, Germany) were
used for column chromatography. HPLC-grade methanol was the product of
Oceanpak Alexative Chemical Co., Ltd. (Gothenburg, Sweden). The standard sugar
reagents for GC analysis (D-glucose, D-galactose, L-arabinose, and D-xylose),
5

lipopolysaccharides (LPS) and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were obtained from Sigma-Aldrich Chemical Corp. (St. Louis, MO).
Fetal bovine serum (FBS) and cell culture medium were provided by Gibco Life
Technologies (Grand Island, NY). Antibodies for western blot assay (PARP,
c-Caspase-3, c-Caspase-9, Bcl-2, Bax and β-actin) were purchased from Cell
Signaling Technology (Danvers, MA, USA).
2.3 Plant Material.
Chinese Onion (122.9 Kg) were collected from Shuan County of Jiangxia District
(Wuhan, China) and were identified as A. chinense G. Don by Prof. X.J. He of
Guangdong
Pharmaceutical
University.
A
voucher
specimen
(No.
GDPU-NPR-2012-ACG) was deposited in the School of Pharmacy, Guangdong
Pharmaceutical University (Guangzhou, China).
2.4 Extraction and Isolation.
Air dried bulbs of Chinese onion were pulverized and then extracted with 3
volumes of 60% (v/v) methanol at reflux (2 h×3). The combined filtrate was
concentrated under vacuum at 55 ºC to remove the methanol and then suspended in
distilled water and applied to a macroporous resin column (D101, 100×1200 mm),
which was eluted with methanol/water (0:100, 30:70, 100:0, v/v) successively. The
methanol/water (100:0, v/v) fraction (152.1 g) was then subjected to a silica gel
chromatography (200-300 mesh, 100 × 1100 mm) and eluted with a CHCl3/MeOH
6

gradient elution (100:0 to 0:100, v/v) to give 14 fractions (Frs. 1-14). Fr. 10 (4.19 g)
was purified on an ODS column (30 × 300 mm) eluted gradient with MeOH/H2O
(20:80 to 100:0, v/v), to give seven subfractions (Subfrs. 10.1–10.7). Subfr. 10.5 was
subjected to a preparative HPLC column (20 × 250 mm) which eluted with 65% (v/v)
methanol in water at the flow rate of 8.0 mL/min. Compounds 1 (12.0 mg) and 5
(32.0 mg) were obtained at the retention time of 17.3 min and 35.9 min respectively.
Fr.12 (9.18 g) was separated into ten subfrs. (Subfrs. 12.1–12.10) with an ODS
column (30 × 300 mm) eluted gradient with MeOH/H2O (from 10:90 to 90:10, v/v).
Subfrs. 12.7 (3.63 g) was chromatographed on a preparative HPLC column (20 ×
250 mm) eluted with 60% (v/v) methanol in water (flow rate: 8.0 mL/min), to afford
compounds 2 (160 mg) and 3 (130 mg). Fr. 13 (8.50 g) was chromatographed on an
ODS column (30×300 mm) eluted gradient with MeOH/H2O (10:90 to 90:10, v/v)
and afford ten subfrs. (Subfr. 13.113.10). The Subfr. 13.6 (1.56 g) was finally
purified by a semi-preparative HPLC column (10×250 mm) eluted with 55% (v/v)
methanol in water at the flow rate of 4.0 mL/min to afford compounds 7 (67.7 mg)
and 8 (37.4 mg) at the retention times of 41.6 and 48.4 min respectively. Fr.14 (98.66
g) was separated into nine subfrs. (Subfrs. 14.1–14.9) with a flash ODS column
(60.8×190 mm) eluted gradient with MeOH/H2O (from 10:90 to 90:10, v/v).
Compound 4 (10.85g) was obtained from the Subfrs. 14.2. Subfr. 14.4 (1.56 g) was
further separated by ODS column (30 × 300 mm) eluted gradient with MeOH/H2O
(50:50 to 90:10, v/v) and yield seven subfrs. (Subfr. 14.4.114.4.7). Subfr. 14.4.5
(744 mg) was finally purified by a preparative HPLC column (20 × 250 mm) eluted
7

with 50% (v/v) methanol in water (Flow rate: 8.0 mL/min), to afford compound 10
(270.6 mg) at the retention time of 64.2 min. Subfr. 14.4.6 (620 mg) was also
purified by preparative HPLC column (20×250 mm) eluted with 55% methanol (v/v)
in water (Flow rate: 8.0 mL/min), compound 6 (15.7 mg) was obtained at the
retention time of 35.5 min. Subfr. 14.5 (1.00 g) was subjected to a semi-preparative
HPLC column (10×250 mm) and eluted with 55% (v/v) methanol in water (Flow
rate: 4.0 mL/min), compound 9 (101.0 mg) was obtained at the retention time of 17.7
min.
26-O-β-D-glucopyranosyl 3β,22α,26-trihydroxy-25(R)-5α-furostan-6-one (1):
White amorphous powder; [α]D14 –41.9 (c 0.23, MeOH); IR νmax (KBr) cm : 3426
1
(OH), 2937(CH), 1706 (C=O); 1H NMR (500 MHz, pyridine-d5) and 13C NMR (125
1
MHz, pyridine-d5) data for the aglycone moiety see Table 1; H NMR (500 MHz,
pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data of sugar moieties, see Table
2; HR-ESI-Q-TOF-MS (positive) m/z 633.3634 [M + Na]+, calcd. for C33H54O10Na.
633.3615.
26-O-β-D-glucopyranosyl
3β,22α,26-trihydroxy-25(R)-5α-furostan-6-one
3-O-β-D-glucopyranoside (2): White amorphous powder; [α]1D4 –38.9 (c 0.35, MeOH);
1
IR νmax (KBr) cm : 3343 (OH), 2941 (CH), 1708 (C=O), 1076; 1H NMR (500 MHz,
13
pyridine-d5) and C NMR (125 MHz, pyridine-d5) data for the aglycone moiety see
1
13
Table 1; H NMR (500 MHz, pyridine-d5) and C NMR (125 MHz, pyridine-d5)
data of sugar moieties, see Table 2; HR-ESI-Q-TOF-MS m/z 795.4170 [M + Na]+,
calcd. for C39H64O15Na 795.4143.
8

26-O-β-D-glucopyranosyl 3β,26-dihydroxy-25(R)-5α-furostan-20(22)-en-6-one
(5): White amorphous powder; [α]1D4 –20.1 (c 0.50, MeOH); IR νmax (KBr) cm : 3279
1
1
(OH), 2948 (CH), 2911, 2868, 1708 (C=O), 1449, 1070; H NMR (500 MHz,
13
pyridine-d5) and C NMR (125 MHz, pyridine-d5) data for the aglycone moiety see
1
13
Table 1; H NMR (500 MHz, pyridine-d5) and C NMR (125 MHz, pyridine-d5)
data of sugar moieties, see Table 2; HR-ESI-Q-TOF-MS m/z 615.3491 [M + Na]+,
calcd. for C33H52O9Na 615.3509.
2.5 Acid Hydrolysis and GC Analysis.
The hydrolysis and GC analysis of he isolated saponins were performed as a
reported method [21]. Briefly, each saponin (1-2 mg) was dissolved in 2 M HCl (5
mL) and held at 90 °C for 28 h. The liberated aglycone was extracted with EtOAc
(5 mL × 3) and then the aqueous residue was evaporated at 60 °C under reduced
pressure. Then 600 μL C5D5N and 5 mg NH2OH·HCl were added to the residue and
the mixture was heated at 90 °C for 30 min. After cooling, Ac2O (300 μL) was added
to the mixtures and homogenized, followed by holding at 90 °C for 1 h. After
cooling, the reaction mixture was analyzed using GC with standard aldononitrile
peracetates as reference samples under the following conditions: Agilent 6820
(G1176A) Gas Chromatograph equipped with a FID detector (detection temperature
300 ºC), column: Agilent DB-5MS(30 m×0.250 mm×0.25 μm), column temperature:
150ºC (hold 2min) - 300℃ (hold 10 min) with the rate of 15℃/min, carrier gas: N2,
injection temperature: 250℃, injection volume:0.5-2 μL. As a result, the acetate
9

derivatives of D-glucose, D-galactose, L-arabinose, and D-xylose were detected at
the following retention times: tR (min) = 10.13 for L-arabinose, 10.23 for D-xylose,
12.10 for D-glucose and 12.33 for D-galactose.
2.6 Anti-proliferative Assay.
Human cancer cell lines, including HepG2, MDA-MB 231, A549, SPC-A-1,
CNE-1, MGC80-3, and a human non-neoplastic cell line MRC-5 were obtained from
the Chinese Academy of Sciences Cell Bank (Shanghai, China). Cell culture and cell
viability assay were carried out according to the reported protocol [22]. Briefly, cells
were cultured in RPMI 1640 or DMEM supplemented with 10% FBS, 10 mmol/L
Hepes, 50 units/mL penicillin, 50 μg/mL streptomycin, and 100 μg /mL gentamicin
and were maintained at 37 °C in 5% CO2. A total of 2.5 × 104 cells were placed in
each well of the 96-well flat-bottom plate. After 12 h of incubation, the growth
medium was replaced by media containing various concentrations of the test samples.
Cell viability was determined at 48 h from the MTT absorbance at 490 nm. At least
three replications for each sample were tested.
2.7 Western Blot Analysis.
HepG2 cells (2 × 105) were treated for 24 h with the indicated concentrations of
compound 4 or 7, then harvested and washed with ice-cold PBS. The cells were lysed
in ice-cold RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride according to
the manufacturer's instructions. The lysates were collected and centrifuged at 12,000
× g at 4 °C for 10 min to remove insoluble debris. Protein concentration in the
10

supernatants was determined with the BCA protein assay (Beyotime biotechnology).
Aliquots of the supernatants (20 μg of protein per lane) were run on 12% SDS
polyacrylamide gel. Proteins were transferred onto Immobilon-P polyvinylidene
fluoride (PVDF) membranes (0.45 µm). Membranes were blocked at room
temperature for 1 h with 5% skimmed milk in TBS containing 0.1% Tween-20
(TBST). After four 5 min washing in TBST, membranes were incubated overnight
with primary antibodies (1:2000 in TBST) at 4 °C. Subsequent to washing,
membranes were incubated for 1 h at room temperature with horseradish
peroxidase-conjugated secondary antibody at 1:10000 in TBST, and then finally the
bands were detected using ECL and visualized using the FluorChem E detection
instrument from ProteinSimple (Santa Clara, CA). The values shown were quantified,
normalized to the internal control β-actin, and then the densitometry was performed
using the Alpha View image acquisition and analysis software from ProteinSimple.
Phosphorylated proteins were shown as the ratio of detected phospho-protein/total
protein.
2.8 Bioassay for Inhibitory Effect on LPS-induced NO Production.
Macrophage RAW264.7 cells were purchased from the Chinese Academy of
Sciences Cell Bank (Shanghai, China) and cultured as previously described [19]. NO
−
produced in the culture medium was measured by assaying the content of NO2 via
the Griess reaction [23].
2.9 Statistical Analysis.
11

Data were presented as mean ± SD. Statistical analysis was performed with
one-way ANOVA followed by Dunnett’s test using GraphPad Prism software. The
difference with p value < 0.05 was perceived to be statistically significant.
3. Results and discussion
3.1 Structural elucidation of the isolated steroidal saponins.
In this research, bulbs of Chinese onion were collected, dried and extracted with
60% methanol. The obtained extract was then successively chromatographed on
D101 macroporous resin, silica gel, ODS C.C. and finally purified by RP-HPLC to
obtain 3 new steroidal saponins (1, 2, 5,), as well as 7 known congeners (Figure 1).
Compound 1 was obtained as a white amorphous powder and showed a purple
colouration with Ehrlich agent. This suggested 1 is a furostanol saponin. Its
molecular formula was determined as C33H54O10 by HR-ESI-MS with the ion of m/z
633.3634 [M + Na]+ (calcd. 633.3615). The IR spectrum displayed absorption bands
1
1
for hydroxyl (3426cm ) and carbonyl (1706 cm ) functionalities. Acid hydrolysis
1
of 1 only afforded D-glucose. The H NMR spectrum displayed two angular methyl
signals at δH 0.75 and 0.84, two secondary methyl signals at δH 1.34 (d, J = 6.9 Hz,
3H) and 0.96 (d, J = 6.7 Hz, 3H), indicative of the methyl groups of the furostanol
skeleton (Table 1). An anomeric proton at δH 4.82 (d, J = 7.8 Hz, 1H) and some
overlapping signals from δH 3.58 to 4.93 consistent with the acid hydrolysis results
revealed the presence of a D-glucose in the molecule. In the 13C NMR spectrum, the
quaternary carbon signal at δC 110.9 was assigned to C-22 of the furostanol skeleton.
The carbonyl carbon signal at δC 210.5 was due to be located at the C-6 position.
12

Comparing the 13C NMR data of 1 with those of the known compound chinenoside I,
which was also isolated from Chinese onion, it displayed similarity except for
variation at the A ring. These indicated the hydroxyl group at C-3 of 1 was free and
the only one sugar moiety in the molecule linked to C-26 position of the aglycone.
Furthermore, the cross-peaks observed in the HMBC spectrum between the H-1′ (δH
4.82) of the glucose and C-26 (δC 75.5) of the aglycone disclosed the D-glucose
attached to C-26 of the aglycone (Fig. 2). The α-configuration of C-22 hydroxyl
group of the aglycone was deduced from the semi-ketal carbon signal at δC 110.9
instead of δC 115.5 for β-configuration, and further evidenced by the ROESY
correlation between H-20 (δH 2.22) and H-23 (δH 2.02).[24, 25] The 25-R
configuration was inferred by the chemical shift difference (0.34 ppm) between
equatorial proton signal (δH 3.94) and axial proton signal (δH 3.60) of H-26, a few
smaller than 0.48 ppm.[25, 26] Therefore, the structure of 1 was identified to be
26-O-β-D-glucopyranosyl 3β,22α,26-trihydroxy-25(R)-5α-furostan-6-one.
Compound 2 was obtained as a white amorphous powder with the molecular
formula of C39H64O15 on the basis of HR-ESI-MS with the ion of m/z 795.4170 [M +
1
13
Na]+ (calcd. 795.4143). The H and C NMR signals of the aglycone moiety of 2
were almost identical to those of 1, suggesting the aglycone of 2 to be laxogenin
(Table 1). The inferred molecular formula has an additional 162 mass unit than that
of 1, corresponding to a hexose unit. Acid hydrolysis of 2 only gave D-glucose, but
two anomeric protons at δH 5.05 (d, J = 7.6 Hz, 1H) and 4.82 (d, J = 7.8 Hz, 1H),
and two anomeric carbons at δC 105.1 and 102.5 indicated the presence of two
13

glucosyl units. The downfield chemical shift of C-3 (δC 77.1) by +6.9 ppm indicated
the glycosylation of C-3 position. The linkages of the sugar units were further
verified in the HMBC experiment by the correlations between H-1 (δH 5.05) of one
glucose and C-3 (δC 77.1), and the H-1 of the other glucose with C-26 (δC 75.5)
(Figure 2). Therefore, the structure of 2 was elucidated as 26-O-β-D-glucopyranosyl
3β,22α,26-trihydroxy-25(R)-5α-furostan-6-one 3-O-β-D-glucopyranoside.
Compound 5 was isolated as a white amorphous powder, which showed a purple
colouration with Ehrlich agent. Its molecular formula was inferred as C33H54O10 by
HR-ESI-MS with the ion of m/z 615.3491 [M+Na]+ (calcd. 615.3509). The IR
1
spectrum displayed absorption bands for hydroxyl (3479 cm ) and carbonyl (1708
1
1
cm ) functionalities. Acid hydrolysis of 5 only afforded D-glucose. The H NMR
spectrum of 5 displayed characteristic signals for four methyls at δH 1.63 (s, 3H),
1.01 (d, J = 6.6 Hz, 3H), 0.74 1 (s, 3H), and 0.65 (s, 3H). An anomeric proton signal
was observed at δH 4.84 (d, J = 7.7 Hz, 1H) suggesting the presence of a D-glucose
and the β anomeric orientation of the glucose was inferred. In the 13C NMR spectrum,
four steroidal methyl groups were observed at δC 17.6 (Me-27), 14.5 (Me-18), 13.4
(Me-19), and 12.0 (Me-21) respectively, while the anomeric carbon of D-glucose
was determined at δC 105.1, which giving correlations with the signals of δH 4.84 in
the HSQC spectrum. Two olefinic carbons at δC 152.7and 103.7 indicated a Δ20(22)
unsaturated aglycone for 5, which were consistent with the literature value for a
Δ20(22) furostanol saponin [16]. These were further supported by the HMBC
correlations between H-21 (δH 1.63) and C-20 (δC 103.7), C-22 (δC 152.7), C-17 (δC
14

64.5) (Figure 2). The 1H and 13C NMR spectrum of 5 (Table 1, Table 2) were similar
to those of chinenoside III, except for the absence of the signals for the
arabinopyranosyl-(1→6)-glucopyranosyl residue linked to C-3 position of the
aglycon and the significant upfield chemical shift by 6.6 ppm of the signal due to
C-3 of the aglycone. These suggested the hydroxyl group at C-3 of 5 was free and
the sugar moiety in the molecule linked to C-26 position of the aglycone. The
1
difference observed in H NMR chemical shifts of the germinal protons H-26a and
H-26b (δHa - δHb = 0.33) supported R configuration of C-25 since this difference is
usually larger than 0.57 ppm in 25S furostane-type steroidal saponins and smaller
than 0.48 ppm in 25R compounds. Thus, the structure of 5 was determined as
26-O-β-D-glucopyranosyl 3β,26-dihydroxy-25(R)-5α-furostan-20(22)-en-6-one.
The known compounds were identified as 26-O-β-D-glucopyranosyl 3β,22,26-
tridyroxy
25(R)-5α-furostan-6-one
3-O-α-L-arabinopyranosyl(1→6)-β-D-
glucopyranoside (3) [18], 26-O-β-D-glucopyranosyl 3β,22,26-tridyroxy-25(R)-5α-
furostan-6-one 3-O-β-D-xylopyranosyl(1→4)-[α-L-arabinopyranosyl (1→6)]-β-D-
glucopyranoside (4) [18], 26-O-β-D-glucopyranosyl 3β,26-didyroxy-25(R)-5α-
furostan-20(22)-en-6-one
3-O-β-D-xylopyranosyl(1→4)-[α-L-arabinopyranosyl
(1→6)]-β-D-glucopyranoside (6) [16], (25R)-26-O-β-D-glucopyranosyl-5α-furost-
3-β,26-didyroxy- 3-O-{O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} (7)
[27], tomatoside A (8) [27], macorstemonoside C (9) [28], (25R)-26-O-β-D-
glucopyranosyl-22-hydroxy-furostane-2α,3β,26-triol-3-O-β-D-glucopyranosyl (1→2)
15

-[β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D- galactopyranoside
(10), by comparing the NMR data with those reported in the literature.
3.2 Anti-proliferative activity.
All the isolated compounds were evaluated for their anti-proliferation against
human hepatocellular carcinoma (HepG2), human lung cancer (A549 and SPC-A-1),
human gastric adenocarcinoma (MGC80-3), human breast cancer (MDA-MB-231),
human colon cancer (SW620), and human nasopharyngeal carcinoma (CNE-1) cells
and human non-neoplastic cells (MRC-5). The anti-proliferative results were
summarized in Table 3. Among the tested compounds, compounds 7–10 showed
potent anti-proliferative activities against all the seven tumor cell lines, with IC50
values below 30 μM, except compound 7 was inactive against SW620 cells and
compound 9 was inactive to MGC80-3 and MDA-MB-231 cells. Compound 7
exhibited a significant selective inhibition against cancer cells except for SW620
cells with IC50 values ranging from 12.14 to 26.63 μM, in comparison with the
normal cells MRC-5 (IC50 > 100 μM). Compounds 1–6 bearing 6-one in the A-ring
were inactive (IC50 > 100 μM) except compound 4 displayed mild inhibition against
HepG2 and CNE-1 cells with IC50 values of 64.01 ± 4.54 μM and 40.99 ± 2.82 μM
respectively. These results revealed that functionalization of the aglycone
significantly influenced the cytotoxicity of furostanol saponins. This is similar to the
results observed in the spirostanol saponins as previously reported [29]. On the other
hand, the variations in the sugar moiety of furostanol saponins also could
16

considerably affect their cytotoxicity. This could be illustrated by, when an
additional D-glucose was incorporated to the C3-OH of the inner glucose in
compound 8, the inhibition significantly increased compared with compound 7,
especially when tested to CNE-1, MDA-MB-231, and SW620 cells. Although
further SAR study focused on functionalization as well as the variation of the sugar
moiety should be carried out, the current results allowed us to conclude that
cytotoxicity of furostanol saponins was influenced both by the aglycone and the
sugar moiety.
3.3 Effects on apoptotic and cell cycle arrest in HepG2 cells.
Apoptosis, known as a physiological cell suicide process, is a main type of cell
death way that is considered an ideal way. In this study, we investigated if compound
4 and 7 could induce apoptosis on HepG2 cells. As shown in Figure 3, compound 4
(75 and 150 μM) and compound 7 (20 and 40 μM) were selected to determine
whether the cytotoxicity of the cancer cells was a result of apoptosis via an annexin
V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) binding assay. Flow
cytometric analyses showed enhanced apoptosis of HepG2 cells treated with high
concentration of compound 7, based on the formation of a significant accumulation
of early apoptosis and late apoptosis in HepG2 cells.
There is evidence that apoptosis is related to cell cycle arrest. Compounds that can
induce cell cycle arrest and apoptotic cell death are generally considered to be potent
anticancer drugs. In order to investigate the effect of compounds 4 and 7 on the cell
17

cycle phase distribution in HepG2 cells, propidium iodide (PI)/RNase Staining
Buffer detection kit was used in the flow cytometric analysis (Figure 4). The results
showed that treatment with compound 4 (75 or 150 μM) or compound 7 (40 μM) for
48 h increased subG1 phase (a sign of fragmentation related to apoptosis) in HepG2
cells. Compound 7 promoted cell-cycle arrest in G2/M phase in HepG2 cells. There
was no obvious G1, S or G2 arrest after treatment by compound 7. These results
demonstrated that compounds 4 and 7 may be responsible for inducing apoptosis of
HepG2 cells.
3.4 Effects on apoptosis signaling.
To explore the mechanism by which induces apoptosis of HepG2 cell line we
determined the correlative proteins of mitochondria-mediate intrinsic apoptosis
including PARP, caspase-9, caspase-3, Bcl-2, and Bax. As shown in Figure 5, the
activation of caspase-9 (the initiator caspases) in turn an increase in the levels of
cleaved-caspase-3 (the effector caspase) and its substrates (PARP) was observed
after treatment with compound 4 (10, 50, 100 μM) or compound 7 (10, 20, 50 μM)
for 24 h, respectively. Treatments with compound 7 or compound 4 resulted in
down-regulation of the level of Bcl-2 and up-regulation of the level of Bax.
Therefore, the ratio of Bcl-2/Bax was significantly decreased in
a
concentration-dependent manner compared with control cells. Thus, both
compounds 4 and 7 activate the caspase cascade, and increase the ratio of expression
levels of Bax to Bcl-2 finally triggering the execution of apoptosis.
18

3.5 Anti-inflammatory activity.
Inflammation is a normal biological response to injury and infection, and involves
the recruitment of the immune system to neutralize invading pathogens, repair
injured tissues, and promote wound healing. Nitric oxide (NO) is a well-known
endogenous cellular signaling molecule and served as an important mediator in many
physiological processes. However, pharmacological studies have demonstrated that
inflammation is related to overproduction of NO. During chronic or excessive
activation of the immune system, NO and pro-inflammatory cytokines such as
interleukin 6 and interleukin 1β are released, which are implicated as a promoter of
the severity of a series of diseases such as cancer, diabetes, and stroke.
In this study, the isolated saponins were also evaluated for their inhibitory effects
on LPS induced NO production in murine macrophage RAW264.7 cells. Cell
viability was determined by the MTT method to define whether inhibitory activities
were due to their cytotoxicity and no obvious cytotoxicity against RAW 264.7 cells
was observed at the tested concentrations. As shown in Table 4, compounds 7 and 10
showed strong inhibitory effects against NO production with IC50 values of 2.01 ±
1.40 μM and 2.49 ± 1.54 μM respectively, followed by compound 6 (IC50 = 25.74 ±
0.79 μM). Compounds 1, 3 and 4 displayed moderate inhibition with IC50 values
ranging from 70.77 to 82.10 μM. It is interesting to note that, compared with
compound 7, an additional β-D-glucosyl at C-3 of the glucose in compound 8
induced significant reduction of the NO inhibitory activity. When another D-glucosyl
19

was introduced to the C-4 position of the inner glucose of compound 9 with the
presence of a methoxy at the C-22 position, it is inactive. However, when a
D-glucosyl was incorporated to the C-4 position of the inner glucose of compound
10 with the presence of a hydroxyl group at the C-2 position, it displayed potential
inhibitory activity with an IC50 value of 2.49 ± 1.54 μM. These results indicated that
the NO inhibitory activity of furstanol saponins influenced by the sugar chain as well
as the aglycone.
In summary, Chinese onion is an ancient medicinal vegetable and widely
cultivated and consumed in Asian countries. It has been reported to possess serious
health benefits such as anti-hyperlipidemic activity, anti-tumor activity, and
anti-Alzheimer’s disease. In this study, ten furostanol saponins, including three new
compounds, were isolated and identified. Some saponins exhibit potential
anti-proliferative and anti-inflammatory activities. Further study suggested that some
saponins could induce G2/M cell-cycle arrest and apoptosis through
mitochondria-mediate pathway in HepG2 cells. Results of this study provided a basis
for further development and utilization of this ancient vegetable as functional foods
or natural source of chemopreventive agents for cancer.
Conflicts of interest
The authors declare that there was no competing interest.
Acknowledgements
This research was supported by Guangdong Province Universities and Colleges
20

Pearl River Scholar Funded Scheme (2017), the Doctoral Program of Higher
Education, Ministry of Education of the People’s Republic of China (No.
20130141110050), and the project (2015A020211027) of Guangdong Provincial of
Science and Technology Department.
Supplementary data
The 1D and 2D NMR, HR-ESI-MS and IR spectra of the new compounds 1, 2,
and 5. This material can be found as supplementary material.
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25

Table 1 1H and 13C NMR Data (500 and 125 MHz, in Pyr-d5) for aglycone moieties
of compounds 1, 2 and 5
1
2
5
No. δC
δH (J in Hz)
1.63o
δC
δH (J in Hz)
1.58o
δC
δH (J in Hz)
1.63o
1
37.4
37.4
37.2
1.15o
1.05o
1.16o
2
32.0
2.05o
29.7
2.09o
31.6
1.91o
1.91o
1.56o
1.44 m
3.84 m
2.31 m
1.81o
3
4
70.2
31.4
3.85 m
3.31 m
1.90o
77.1
27.3
4.05o
70.2
31.4
2.37 m
1.72o
5
6
7
57.2
210.6
47.1
2.28 m
56.7
2.09o
57.1
2.28o
209.9
210.5
2.31 dd (12.7, 4.2) 47.1
1.97o
2.35 dd (13.1, 4.3) 47.2
1.95o
2.36 dd (13.0, 4.6)
2.02 d (12.5)
1.83o
8
37.7
54.0
41.2
21.8
1.90o
1.14o
37.7
54.0
41.1
21.8
1.99o
1.10o
37.4
53.9
41.2
22.0
1.14o
9
10
11
1.52 m
1.28o
1.50 m
1.18o
1.54 m
1.27 m
1.73 m
1.19o
12
40.0
1.75 m
1.15o
40.0
1.73 m
1.11o
39.6
13
14
15
41.7
56.7
32.3
41.7
56.7
32.3
44.2
55.0
34.3
1.24o
1.22 m
1.05 m
1.98o
1.67o
1.92o
1.36 m
1.33o
1.37o
16
17
18
19
20
21
22
23
81.1
64.0
16.9
13.5
40.8
16.7
110.9
37.2
4.93 m
81.1
64.0
16.8
13.3
40.8
16.7
110.9
37.0
4.92 q (7.2)
1.95o
84.4
64.5
14.5
13.4
103.7
12.0
152.7
23.8
4.75 m
2.46 d (10.3)
0.65 s
1.95o
0.84 s
0.82 s
0.75 s
0.62 s
0.74 s
2.22 dd (8.7, 4.5)
1.34 d (7.1)
2.22 q (6.7)
1.33 d (6.9)
1.63 s
2.02o
2.02o
2.02o
1.65o
2.03o
2.03o
2.05o
1.67o
2.20 m
2.20 m
2.04o
24
28.6
28.6
31.9
1.64o
26

25
26
34.5
75.5
1.90o
34.5
75.5
1.91o
33.6
75.2
1.93o
3.94 dd (9.3, 6.9)
3.60 dd (9.7, 5.8)
0.96 d (6.6)
3.94 dd (9.3, 7.1)
3.60 dd (9.4, 6.0)
0.97 d (6.6)
3.94 dd (9.2, 7.1)
3.61 dd (9.6, 5.8)
1.01 d (6.6)
27
17.7
17.7
17.6
o Signals are overlapped.
27

Table 2 1H and 13C NMR Data (500 and 125 MHz, in Pyr-d5) for sugar moieties of compounds 1, 2
and 5
1
2
5
No.
C-3:
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
102.5 5.05 d (7.3)
75.6
78.8
72.1
78.7
63.2
4.00o
4.24o
4.19o
3.91o
4.62 dd (11.6, 1.8)
4.27 m
C-26:
1
2
3
4
5
6
105.2 4.82 d (7.8)
105.1 4.82 d (7.8)
105.1 4.84 d (7.7)
75.4
78.8
71.9
78.7
63.0
4.04 t (8.2)
4.27o
75.4
78.8
72.0
78.7
63.1
4.00o
75.4
78.8
71.9
78.7
63.0
4.05 t (8.1)
4.26o
4.24o
4.23o
4.19o
4.24o
3.95o
3.91o
3.97 m
4.58 dd (11.9, 2.1)
4.39 dd (11.7, 5.4)
4.55 dd (11.7, 2.2)
4.30 m
4.58 dd (11.9, 1.8)
4.39 dd (11.7, 5.4)
o Signals are overlapped.
28

Table 3 Anti-proliferative activity of compounds 1-10 a, b
IC50 (μM)
Compd. HepG2
SPC-A-1
A549
> 100
CNE-1
MGC80-3
MDA-MB-231 SW620
MRC-5
> 100
64.01 ± 4.54 > 100
40.99 ± 2.82 > 100
> 100
> 100
> 100
4
12.14 ± 1.61 19.67 ± 1.25 16.91 ± 1.04 12.30 ± 1.95 26.63 ± 1.12 25.42 ± 3.49
5.44 ± 0.45 22.59 ± 3.50 11.38 ± 0.86 2.45 ± 0.19 26.93 ± 2.30 4.16 ± 1.25
> 100
7
12.27 ± 2.52 4.74 ± 0.52
1.76 ± 0.54 9.28 ± 1.44
10.68 ± 1.45 11.71 ± 2.21
10.68 ± 1.45
8
6.91 ± 0.35 25.07 ± 0.99 13.70 ± 1.81 4.09 ± 0.48 > 100
> 100
9
3.77 ± 0.53 22.83 ± 1.38 12.06 ± 1.00 4.15 ± 1.09 19.72 ± 1.63 13.61 ± 1.69
10
Cisplatin c3.48 ± 0.31 7.22 ± 0.71 4.07 ± 0.40 7.14 ± 0.77 10.22 ± 1.43 13.61 ± 1.69
a Values are the mean ± SD; n=3.
b Compounds 1–3, 5 and 6 are inactive (IC50 > 100 μM).
c Cisplatin was used as positive control.
29