New cytotoxic bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch

Article abstract of DOI:10.1080/14786419.2018.1543687

Three new bufadienolides 14β, 16β-dihydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (1), 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→4)-(β-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-β-D-glucosyl-(1→4)-β-D-glucoside (3), together with eight known bufadienolides (4–11) were isolated from the roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by extensive spectroscopic methods and acid hydrolysis. Compounds 1–7 were evaluated for their cytotoxic activity against HCT116, A549 and HepG2 tumor cell lines. Compound 1 exhibited moderate cytotoxicity against HepG2 cells with IC₅₀ value of 15.1 ± 1.72 μM. Compounds 5 and 6 exhibited moderate cytotoxicity against HCT116 cells with IC₅₀ values of 15.12 ± 0.58 μM and 13.17 ± 2.34 μM, respectively.

Full text of DOI:10.1080/14786419.2018.1543687

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New cytotoxic bufadienolides from the roots and
rhizomes of Helleborus thibetanus Franch
Yuze Li, Huawei Zhang, Xiaofei Liang, Bei Song, Xudong Zheng, Rui Wang, Li
Liu, Xiaomei Song & Jianli Liu
To cite this article: Yuze Li, Huawei Zhang, Xiaofei Liang, Bei Song, Xudong Zheng, Rui Wang, Li
Liu, Xiaomei Song & Jianli Liu (2018): New cytotoxic bufadienolides from the roots and rhizomes of
Helleborusꢀthibetanus Franch, Natural Product Research, DOI: 10.1080/14786419.2018.1543687
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1543687
New cytotoxic bufadienolides from the roots and
rhizomes of Helleborus thibetanus Franch
a#
b#
b
a
a
Yuze Li , Huawei Zhang , Xiaofei Liang , Bei Song , Xudong Zheng , Rui
a
a
b
a
Wang , Li Liu , Xiaomei Song and Jianli Liu
a
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education,
b
College of Life Sciences, Northwest University, Xi’an, China; College of Pharmacy, Shaanxi University
of Chinese Medicine, Xianyang, China
ABSTRACT
ARTICLE HISTORY
Three new bufadienolides 14b, 16b-dihydroxy-3b-[b-D-glucopyra-
nosyl-(1!6)-(b-D-glucopyranosyl)oxy]-5a-bufa-20, 22-dienolide (1),
Received 15 August 2018
Accepted 26 October 2018
1
4b-hydroxy-3b-[b-D-glucopyranosyl-(1!4)-(b-D-glucopyranosy-
KEYWORDS
l)oxy]-5a-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-b-D-glu-
cosyl-(1!4)-b-D-glucoside (3), together with eight known
bufadienolides (4–11) were isolated from the roots and rhizomes
of Helleborus thibetanus. Their structures were elucidated by
extensive spectroscopic methods and acid hydrolysis. Compounds
bufadienolide glycosides;
cytotoxic activity; Helleborus
thibetanus Franch; structure
identification
1
–7 were evaluated for their cytotoxic activity against HCT116,
A549 and HepG2 tumor cell lines. Compound 1 exhibited moder-
ate cytotoxicity against HepG2 cells with IC50 value of
1
5.1 ± 1.72 lM. Compounds 5 and 6 exhibited moderate cytotox-
icity against HCT116 cells with IC50 values of 15.12 ± 0.58 lM and
3.17 ± 2.34 lM, respectively.
1
1. Introduction
Helleborus thibetanus Franch., a species in the genus Helleborus of the family
Ranunculaceae, is used as an endemic herbal medicine, known as ‘Tigencao’ or ‘Xiao-
CONTACT Xiaomei Song
Yuze Li and Huawei Zhang contributed equally to this work.
Songxiaom@126.com; Jianli Liu
jlliu@nwu.edu.cn
#
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1543687.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y. LI ET AL.
Figure 1. Structures of compounds 1–11.
tao-er-qi’, in the Qinba Mountains of Shaanxi Province in China (Song and Liu 2011).
The roots and rhizomes of H. thibetanus are commonly used as folk medicine to treat
cystitis, urethritis, sores and traumatic injury (An et al. 2013; Yang et al. 2010). Previous
phytochemical investigation on Helleborus illustrated that steroids including bufadieno-
lides, phytoecdystones and steroidal saponins (Bassarello et al. 2008; Braca et al. 2004;
Meng et al. 2001; Muzashvili et al. 2011; Tsiftsoglou et al. 2018) were the main compo-
nents. Pharmacological studies suggested that this plant possesses antitumour, anti-
bacterial, immune-regulation and cytotoxic properties (Littmann et al. 2008; Rosselli
et al. 2009). Previous studies disclosed the presence of bufadienolides, spirostanol gly-
cosides, spirostanol sulfonate, pregane and phytoecdystones in the rhizomes of H. thi-
betanus (Yang et al. 2010; Zhang et al. 2016a; Zhang et al. 2016b; Zhang et al. 2017).
The bufadienolides of H. thibetanus had both A/B cis and A/B trans ring fusion modes,
and some of them were found to exhibited potent antitumour activity. (Cheng et al.
2
014; Ma et al. 2018). As part of our research project to explore more diverse bioactive
leading compounds from the medicinal herbs of Qinba mountains of China (Chai et al.
014; Li et al. 2015; Song et al. 2015), the chemical constituents and pharmacological
2
studies of H. thibetanus were studied, and three new bufadienolides 14b, 16b-dihy-
droxy-3b-[b-D-glucopyranosyl-(1!6)-(b-D-glucopyranosyl)oxy]-5a-bufa-20,22-dienolide
(
1),
14b-hydroxy-3b-[b-D-glucopyranosyl-(1!4)-(b-D-glucopyranosyl)oxy]-5a-bufa-20,
2
2-dienolide (2) and hellebrigenin-3-O-b-D-glucosyl-(1!4)-b-D-glucoside (3), along
with eight known bufadienolides 16b-hydroxyhellebrigenin-3-O-a-L-rhamnoside (4)
Watanabe et al. 2003), hellebrigenin-3-O-b-D-glucoside (5) (Watanabe et al. 2003),
(
deglucohellebrin (6) (Watanabe et al. 2003), hellebrin (7) (Yang et al. 2010), 14b-
hydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa-20, 22-dienolide (8) (Cheng et al. 2014),
1
4b-hydroxy-3b-[b-D-glucopyranosyl-(1!6)-(b-D-glucopyranosyl)oxy]-5a-bufa-20,
22-
dienolide (9) (Zhang et al. 2014), 14b, 16b-dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-
bufa-20, 22-dienolide (10) (Cheng et al. 2014), 14b, 16b-dihydroxy-3b-[b-D-glucopyrano-
syl-(1!4)-(b-D-glucopyranosyloxy)]-5b-bufa-20, 22-dienolide (11) (Yang et al. 2010)
(Figure 1) were isolated from the rhizomes of H. thibetanus. Their structures were

NATURAL PRODUCT RESEARCH
3
elucidated on the basis of extensive spectroscopic analysis. Compounds 1–7 were eval-
uated for their cytotoxic activity against HCT116, A549 and HepG2 tumor cell lines.
2. Results and discussion
Compound 1 was isolated as a white amorphous powder. Its molecular formula was
þ
determined as C H O from the HR-ESI-MS at m/z 749.3364 [M þ Na] (calcd
36 54 15
C H O Na 749.3360). Its IR spectrum revealed absorption bands for hydroxyl
3383 cm ) and carbonyl groups (1711 cm ). The H and C NMR spectra of 1 indi-
3
6
54 15
ꢀ
1
ꢀ1
1
13
(
cated the presence of a 2H-pyran-2-one unit [d 6.34 (1H, d, J ¼ 9.7 Hz, H-23), 7.54 (1H,
H
d, J ¼ 1.6 Hz, H-21), 8.55 (1H, dd, J ¼ 2.5, 9.8 Hz, H-22); d 113.1, 119.9, 151.1, 151.8,
C
1
62.8], two angular methyls [d 0.68 (3H, s, H-19), 1.02 (3H, s, H-18); d 12.7, 17.8], two
H
C
oxygenated methines [dH 4.08 (1H, H-3), 4.82 (1H, t, J ¼ 5.5 Hz, H-16); d 78.0, 73.0],
C
one oxygenated quaternary carbon (d 85.0) and two anomeric signals [d 5.01 (1H, d,
C
H
J ¼ 7.8 Hz, H-1’), 5.18 (1H, d, J ¼ 7.8 Hz, H-1’’); d 102.9, 105.9]. These evidences indi-
C
1
13
cated that compound 1 possessed a bufadienolide skeleton. The H and C NMR
spectroscopic data of 1 were similar to those of the known compound 3b, 14b, 16b-
trihydroxy-5a-bufa-20, 22-dienolide (Zhang et al. 2014), except for the obvious down-
field shift of C-3 (dC-3 ¼ þ 7.1), which resulted from glycosylation shifts. The b-config-
uration (Zhang et al. 2012) of the glucose was deduced from the relative large
coupling constants of the anomeric protons (J ¼ 7.8 Hz). The two glucosyl moieties
were identified as D-glucose by acid hydrolysis of 1, followed by TLC comparison with
a reference compound and optical rotation determination (Hudson and Dale 2002). In
addition, the HMBC correlation (Table S1 and Figure S1) between H-1’ (d 5.01) and C-
H
3
(d 78.0), as well as between H-1" (d 5.18) and C-6’ (d 70.7) indicated the terminal
C H C
Glc was linked at C-6 of the inner Glc, which was linked at C-3 of the aglycone.
Meanwhile, in the NOESY spectrum, the NOE correlations of H-3/H-1a, H-2a, H-4a and
H-5; H-19/H-1b, H-2b, H-4b and H-8 were observed and the lack of correlation
between H-5 and H-19, confirming the a-configuration of H-3 and H-5 and the sugar
residue to be located at the 3b-position. A key NOE correlation of H-16/H-12a showed
the b-configuration of 16-OH. Thus, the structure of 1 was assigned as 14b, 16b-dihy-
droxy-3b-[b-D-glucopyranosyl-(1!6)-(b-D-glucopyranosyl)oxy]-5a-bufa-20, 22-dienolide.
Compound 2 was isolated as a white amorphous powder. Its molecular formula
þ
was determined as C H O from the HR-ESI-MS at m/z 711.3588 [M þ H] (calcd
3
6 54 14
1
13
C H O 711.3592). The H and C NMR spectra of 2 indicated the presence of a
3
6 55 14
2
H-pyran-2-one unit [d 6.38 (1H, d, J ¼ 9.7 Hz, H-23), 7.49 (1H, brs, H-21), 8.26 (1H, dd,
H
J ¼ 1.3, 9.7 Hz, H-22); d 115.5, 123.7, 147.9, 149.6, 162.4], two angular methyls [d 0.66
C
H
(3H, s, H-19), 0.89 (3H, s, H-18); d 12.4, 17.4], one oxygenated methines [d 4.01 (1H,
C
H
H-3), d 77.6], one oxygenated quaternary carbon (d 84.6) and two anomeric signals
C
C
[
dH 5.03 (1H, d, J ¼ 7.8 Hz, H-1’), 5.23 (1H, d, J ¼ 7.8 Hz, H-1’’); d 102.1, 105.2]. These
C
evidences indicated that compound 2 possessed a bufadienolide skeleton. Comparison
of the NMR, HR-ESI-MS data of 2 and 1, compound 2 exihibited spectroscopic features
similar to those of 1, except sugar chain difference and an absence of 16-OH. The pro-
ton and carbon NMR signals of [d 2.49 (1H, ca., H-15a), 2.16 (1H, ca., H-15b); d 43.6],
H
C
[
d 4.82 (1H, t, J ¼ 5.5 Hz, H-16); d 73.0] and [d 2.82 (1H, d, J ¼ 7.7 Hz, H-17); d 59.5]
H
C
H
C

4
Y. LI ET AL.
in 1, were replaced by [d 1.87 (1H, ca., H-15a), 1.99 (1H, ca., H-15b); d 33.1], [d 2.15
H
C
H
(
1
1H, ca., H-16a), 1.86 (1H, ca., H-16b); d 29.7] and [d 2.47 (1H, dd, J ¼ 6.1, 9.3 Hz, H-
C
H
1
1
7); d 51.7] in 2, which was supported by H - H COSY, HMQC, HMBC and NOESY
C
spectra. In addition, the HMBC correlation between H-1’ (d 5.03) and C-3 (d 77.6), as
H
C
well as between H-1" (d 5.23) and C-4’ (d 81.8) indicated the terminal Glc was linked
H
C
at C-4 of the inner Glc, which was linked at C-3 of the aglycone. Similarly as compound
, the results of the acid hydrolysis procedure and analysis of detail NOESY spectra
data showed (Figure S2) the structure of compound 2 was deduced as 14b-hydroxy-
1
3
b-[b-D-glucopyranosyl-(1!4)-(b-D-glucopyranosyl)oxy]-5a-bufa-20, 22-dienolide.
Compound 3 was isolated as a white amorphous powder. Its molecular formula
þ
was determined as C H O from the HR-ESI-MS at m/z 763.3155 [M þ Na] (calcd
3
6 52 16
1
13
C H O Na 763.3153). The H and C NMR spectra of 3 indicated the presence of a
3
6 52 16
2
H-pyran-2-one unit [d 6.36 (1H, d, J ¼ 9.6 Hz, H-23), 7.48 (1H, d, J ¼ 1.6 Hz, H-21), 8.25
H
(1H, dd, J ¼ 2.5, 9.7 Hz, H-22); d 115.8, 123.5, 147.9, 149.9, 162.6], one aldehyde hydro-
C
gen [d 10.45 (1H, s, H-19); d 209.1], one angular methyl [d 0.89 (3H, s, H-18); d
C
H
C
H
1
7.5], one oxygenated methine [d 4.08 (1H, H-3); d 73.6], two oxygenated quaternary
H C
carbons (d 73.9, 84.7) and two anomeric signals [d 5.04 (1H, d, J ¼ 7.9 Hz, H-1’), 5.22
C
H
(
1H, d, J ¼ 7.9 Hz, H-1’’); d 101.4, 105.6]. These evidences indicated that compound 3
C
1 13
possessed a bufadienolide skeleton. The H and C NMR spectroscopic data of 3 were
similar to those of 5, except for the sugar chain difference. And the HMBC correlation
between H-1’ (d 5.04) and C-3 (d 73.6), as well as between H-1" (d 5.22) and C-4’
H
C
H
(dC 82.1) indicated the terminal Glc was linked at C-4 of the inner Glc, which was
linked at C-3 of the aglycone. The b-configuration (Zhang et al. 2012) of the glucose
was deduced from the relative large coupling constants of the anomeric protons
(J ¼ 7.9 Hz). The two glucosyl moieties were identified as D-glucose by acid hydrolysis
of 3, followed by TLC comparison with a reference compound and optical rotation
determination (Hudson and Dale 2002). Meanwhile, in the NOESY spectrum (Figure
S3), the NOE correlations of H-3/H-4, H-4a/H-7a and H-9, and H-19/H-8, indicated
a-axial configurations of H-3 and b-orientation of H-19, 3-OH and 5-OH, which sup-
ported the A/B cis ring junction pattern. Thus, the structure of 3 was formulated as
hellebrigenin-3-O-b-D-glucosyl-(1!4)-b-D-glucoside.
Compounds 1–7 were evaluated for their cytotoxic activity against HCT116, A549
and HepG2 tumor cell lines, 5-fluorouracil was used as positive control. As shown in
Table S2, compound 1 exhibited moderate cytotoxicity against HepG2 cells with IC50
value of 15.1 ± 1.72 lM, compounds 5 and 6 exhibited moderate cytotoxicity against
HCT116 cells with IC50 values of 15.12 ± 0.58 lM and 13.17 ± 2.34 lM, respectively.
Compounds 3, 5–7 shared the same aglycone, but exhibited different activities. This
suggested that the structural differences such as the category and the number of the
oligosaccharide at C-3 played a role in terms of antitumor effect.
3. Experimental
3.1. General experimental procedures
Optical rotation indices were determined in methanol on a Rudolph Autopol II digital
polarimeter (Rudolph, Hackettstown, NJ, USA). The IR spectra were recorded on a

NATURAL PRODUCT RESEARCH
5
TENSOR-27 instrument (Bruker, Rheinstetten, Germany). ESI-MS was performed on a
Quattro Premier instrument (Waters, Milford, MA, USA). The HR-ESI-MS spectra were
recorded on an Agilent Technologies 6550 Q-TOF (Santa Clara, CA, USA). 1D and 2D
NMR spectra were recorded on Bruker-AVANCE 400 instrument (Bruker, Rheinstetten,
Germany) with TMS as an internal standard. The analytical HPLC was performed on a
Waters 2695 Separations Module coupled with a 2996 Photodiode Array Detector
and a Accurasil C18 column (4.6 mm ꢁ250 mm, 5 mm particles, Ameritech, Chicago,
IL, USA). Semipreparative HPLC was performed on a system comprising an LC-6AD
pump (Shimadzu, Kyoto, Japan) equipped with a SPD-20A UV detector and a
Ultimate XB-C18 (10 mm ꢁ250 mm, 5 mm particles) or YMC-Pack-ODS-A (10 mm
ꢁ
250 mm, 5 mm particles). D101 was from Sunresin New Materials Co. Ltd. (Xi’an,
China). Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation
Qingdao, China).
(
3.2. Plant material
The roots and rhizomes of H. thibetanus were collected from the Taibai region of
Qinba Mountains in Shaanxi Province, China, in June 2016, and identified by senior
experimentalist Jitao Wang. A voucher specimen (herbarium No. 20160915) has been
deposited in the Medicinal Plants Herbarium (MPH), Shaanxi University of Chinese
Medicine, Xianyang, China.
3.3. Extraction and isolation
The air-dried and powdered underground parts of H. thibetanus (15 kg) were
ꢂ
extracted with 60% EtOH (15 L) three times at 80 C. After removing the solvent, the
concentrated residue was successively partitioned with petroleum ether and n-
BuOH. The n-BuOH extract (500 g) was subjected to column chromatography (CC)
on silica gel with gradient elution (CHCl -MeOH-H O, 100:0:0-65:35:10), which
3
2
yielded ten fractions (Fr.1-10). Fr.3 (80 g) was subjected to column chromatography
CC) on silica gel, eluting with gradient solvent system (CHCl -MeOH, 100:0-50:50) to
(
3
yield five fractions (Fr.3-1–Fr.3-5). Fr.3-3 (220 mg) were purified by HPLC with
CH CN-H O (35:65) as mobile phase to afford 4 (50 mg), 5 (62 mg), 6 (14 mg). Fr.4
3
2
(
60 g) was subjected to column chromatography (CC) on silica gel, eluting with gra-
dient solvent system (CHCl -MeOH, 100:0-50:50) to yield six fractions (Fr.4-1–Fr.4-6).
3
Fr.4-2 (220 mg) were purified by HPLC (YMC-Pack-ODS-A, 10 mm ꢁ250 mm, 5 lm
ꢀ
1
particles, flow rate: 1.0 mLꢃmin ) with CH CN-H O (30:70) as mobile phase to afford
3
2
8
(13 mg), 10 (11 mg), 9 (15 mg) and 11 (21 mg). Fr.6 (65 g) were loaded on a silica
gel column and eluted with a gradient solvent system (CHCl -MeOH, 100:0-50:50) to
3
yield four fractions (Fr.6-1–Fr.6-4). Fr.6-2 was purified by HPLC with CH CN-H O
3
2
(
25:75) as the mobile phase to obtain 2 (100 mg) and 7 (12 mg). Fr.6-4 was purified
by HPLC with CH CN-H O (27:73) as the mobile phase to obtain 1 (10 mg) and
3
2
3
(8 mg).

6
Y. LI ET AL.
3
5
.3.1. 14b, 16b-dihydroxy-3b-[b-D-glucopyranosyl-(1!6)-(b-D-glucopyranosyl)oxy]-
a-bufa-20, 22-dienolide (1)
27:2
A white amorphous powder, ½aꢄ -16.9 (c 1.1, MeOH), IR (KBr) mmax: 3382, 2941, 1711,
D
1
13
1055. H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ) spectral data,
5
5
þ
see Table S1; m/z 749.3364 [M þ Na] (calcd. for C H O Na 749.3360).
36 54 15
3
.3.2.
14b-hydroxy-3b-[b-D-glucopyranosyl-(1!4)-(b-D-glucopyranosyl)oxy]-5a-
bufa-20, 22-dienolide (2)
26:8
A white amorphous powder, ½aꢄ -36.0 (c 1.3, MeOH), IR (KBr) mmax: 3382, 2943, 1709,
D
1
13
1056. H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ) spectral data,
5
5
þ
see Table S1; m/z 711.3588 [M þ H] (calcd. for C H O 711.3592).
36 55 14
3
.3.3. Hellebrigenin-3-O-b-D-glucosyl-(1!4)-b-D-glucoside (3)
2
D
7:0
A white amorphous powder, ½aꢄ -19.5 (c 0.7, MeOH), IR (KBr) mmax: 3407, 2944,
1
13
1715, 1630, 1056. H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-
5
þ
d ) spectral data, see Table S1; m/z 763.3155 [M þ Na]
(calcd. for
5
C H O Na 763.3153).
3
6 52 16
3.4. Acid hydrolysis of compounds 1-3 and absolute sugar configuration
determination
The solutions of compounds 1 (4 mg), 2 (3 mg) and 3 (5 mg) were hydrolyzed with 2N
ꢂ
HCl (5 mL) for 5 h at 80 C, respectively. The reaction mixtures were concentrated and
dried by N , and then water (5 mL) was added and the mixtures were extracted with
2
EtOAc (3 ꢁ 5 mL). The aqueous layers of 1–3 were subjected to CC over silica gel
eluted with MeCN-H O (8:1) to yield D-glucose, which was determined by TLC compari-
2
son (MeCN-H O, 6:1) with the authentic sugar and the optical rotation determination
2
2
D
0:0
½
aꢄ
þ49.2 (c 0.16, H O).
2
3.5. Cytotoxicity experiments
The cytotoxic activity assays towards the HCT116, A549 and HepG2 tumor cell lines
were measured by the MTT method in vitro, using 5-fluorouracil as positive control.
4
ꢀ1
Briefly, 1 ꢁ 10 mL cells were seeded into 96-well plates and allowed to adhere
for 24 h. Compounds 1–7 were dissolved in DMSO and diluted with complete
ꢀ
1
ꢀ1
medium to six concentration levels (from 0.001 mmolꢃL to 0.3 mmolꢃL ) for inhib-
ꢂ
ition rate determination. After incubation at 37 C for 4 h, the supernatant was
removed before adding DMSO (100 lL) to each well. 5-Fluorouracil (5-Fu) was used
as positive control. The inhibition rate (IR) and IC50 were calculated. Values are
mean ± SD, n ¼ 3,
ꢅꢅ p < 0.01 vs. DMEM control. Compounds 1–7 showed cytotox-
icity against human HCT116, A549 and HepG2 cell lines; the IC50 values are shown
in Table S2.

NATURAL PRODUCT RESEARCH
7
4. Conclusion
Three new bufadienolides 14b, 16b-dihydroxy-3b-[b-D-glucopyranosyl-(1!6)-(b-D-glu-
copyranosyl)oxy]-5a-bufa-20, 22-dienolide (1), 14b-hydroxy-3b-[b-D-glucopyranosyl-
(
1!4)-(b-D-glucopyranosyl)oxy]-5a-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-b-D-
glucosyl-(1!4)-b-D-glucoside (3), together with eight known bufadienolides (4–11)
were isolated from the roots and rhizomes of H. thibetanus. Compounds 1, 5 and 6
showed moderate cytotoxicity.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This project was financially supported by the National Natural Science Foundations of China
(grant number 81503195); the Natural Science Foundation of Shaanxi Province (grant number
2
016JQ8030); Key R&D Program of Shaanxi Province (grant number 2017SF-360); and the project
was supported by the Open Research Fund of Key Laboratory of Basic and New Herbal
Medicament Research, Shaanxi university of Chinese medicine (grant number
2
017KF02, 17JS030).
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