Two new bufadienolides from the rhizomes of Helleborus thibetanus Franch

Article abstract of DOI:10.1016/j.fitote.2010.03.009

Two new bufadienolides, named tigencaoside A(1) and tigencaoside B(2), were isolated from the rhizomes of Helleborus thibetanus Franch., along with two known bufadienolides, hellebrigenin (3) and 5β,14β-dihydroxy-19-oxo- 3β-[(α-L-rhamnopyranosyl)oxy]bufa-20,22-dienolide (4). Their structures were elucidated on the basis of extensive spectroscopic analysis. Two new compounds were evaluated for their cytotoxic activities against four strains of cultured tumor cells.

Full text of DOI:10.1016/j.fitote.2010.03.009

Fitoterapia 81 (2010) 636–639
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Two new bufadienolides from the rhizomes of Helleborus thibetanus Franch
a
b
b
a,
c
Jian Yang , Yan-hong Zhang , Fang Miao , Le Zhou ⁎, Wei Sun
a
College of Science, Northwest A&F University, Yangling 712100, PR China
College of Life, Northwest A&F University, Yangling 712100, PR China
Department of Chemistry, Northwest University, Xi'an 710068, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new bufadienolides, named tigencaoside A(1) and tigencaoside B(2), were isolated from
the rhizomes of Helleborus thibetanus Franch., along with two known bufadienolides,
hellebrigenin (3) and 5β,14β-dihydroxy-19-oxo-3β-[(α-L-rhamnopyranosyl)oxy]bufa-20,22-
dienolide (4). Their structures were elucidated on the basis of extensive spectroscopic analysis.
Two new compounds were evaluated for their cytotoxic activities against four strains of
cultured tumor cells.
Received 12 August 2009
Accepted in revised form 9 March 2010
Available online 15 March 2010
Keywords:
Helleborus thibetanus Franch
Bufadienolides
©
2010 Elsevier B.V. All rights reserved.
Tigencaosides A and B
1
. Introduction
chemical constituents of the rhizomes of H. thibetanus and
led to the isolation of two new bufadienolides named
tigencaoside A (1) and tigencaoside B (2), along with two
known bufadienolides (3-4), hellebrigenin (3) and 5β,14β-
dihydroxy-19-oxo-3β-[(α-L-rhamnopyranosyl)oxy]bufa-
Helleborus thibetanus Franch. (the family Ranunculaceae)
commonly known as Tigencao or Xiaotaoqi is a perennial
herb and Chinese special local plant species which is only
distributed in Qinling Mountain in China. Its dried rhizomes
have been used as Chinese folk medicine for the treatment of
cystitis, urethritis and traumatic injury since bufadienoli-
des19th century. Previous phytochemical investigations on
the Helleborus genus have led to the isolation of several
bufadienolides, ecdysteroids, spirostanol saponins, furostanol
saponins and flavonoids [1–15]. Bufadienolides have been
reported to show potent cardiotonic, blood pressure-stimu-
lating and local anaesthetic activities, especially cytotoxic
activity against cultured tumor cells [16]. However, a
literature survey on H. thibetanus indicates that
its phytochemistry has not previously been investigated.
In order to find potentially bioactive secondary metabolites
from H. thibetanus and appraise the ethnomedical proper-
ties, the present study was undertaken to investigate the
2
0,22- dienolide(4) (Fig. 1). Here, we report the isolation and
structure elucidation of two new bufadienolides (1-2).
2
. Experimental
2.1. General
Optical rotations were obtained on a Horiba SEPA-300
digital polarimeter. IR Spectra were taken on a Nexus FT-IR
00 spectrometer with KBr pellets. UV Spectra were per-
4
formed on a SP-2100 UV/VIS spectrometer. NMR spectra were
recorded with a Bruker DRX-500 instrument. ESI-MS and HR-
ESI-MS were measured on Finnigan-MAT 90 and API QSTAR
Pulsar i mass spectrometers respectively. Silica gel (200–300
mesh, Qingdao Marine Chemical Inc., PR China), silica gel 60
(
0.015–0.04 µm, Merck, US), Sephadex LH-20 (Amersham
Biosciences, Sweden) and LSA-30 macroporous resin (Xian
Lanshen Chemical Inc., PR China) were used for column
chromatography. Compounds were visualized on UV light.
Fractions were monitored by TLC and spots were visualized by
⁎
Corresponding author. College of Science, Northwest A&F University,
No.3 Taicheng Road, Yangling 712100, Shaanxi Province, PR China. Tel.:+86
9 87092048; fax: +86 29 87092226.
2
E-mail addresses: vivian54321@gmail.com (J. Yang),
zhoulechem@yahoo.com.cn (L. Zhou).
2 4
heating silica gel plates sprayed with 10% H SO in ethanol.
0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.03.009

J. Yang et al. / Fitoterapia 81 (2010) 636–639
637
Fig. 1. The structures of 1–4.
Table 1
1
H NMR and 13C NMR Spectral Data of 1 and 2 (500 and 125 MHz, DMSO, J in
2
.2. Plant material
Hz and δ in ppm).
The rhizomes of H. thibetanus Franch. were collected from
Position
1
2
Qinling Mountain, Shaanxi province, PR China in August 2006.
The plants were identified by Dr. Fang Miao, a co-author of
this paper. The voucher specimen (No.200602638) was
deposited in botanic specimen center of Northwest A&F
University, Yangling, PR China.
δ (C) δ (H)
δ (C) δ (H)
1
2
36.7 0.88–0.92 (1H, m)
36.7 0.88–0.93 (1H, m)
1.65–1.68 (1H, m)
29.0 1.37–1.40 (1H, m)
1.72–1.74 (1H, m)
1
.62–1.66 (1H, m)
29.0 1.38–1.40 (1H, m)
.72–1.74 (1H, m)
1
3
4
76.8 3.53–3.56 (1H, m)
34.0 1.16–1.20 (1H, m)
76.8 3.53–3.57 (1H, m)
34.0 1.14–1.18 (1H, m)
1.64–1.66 (1H, m)
2
.3. Extraction and isolation
1.59–1.63 (1H, m)
5
6
43.7 0.97–1.02 (1H, m, H
28.5 1.11–1.14 (1H, m)
β
)
)
43.6 0.98–1.02 (1H, m, H
28.5 1.13–1.16 (1H, m)
1.29–1.32 (1H, m)
β
)
)
The dried and powdered rhizomes of H. thibetanus (2.4 kg)
were exhaustively extracted with 95% ethanol (2×15 L) at
0 °C assisted by ultrasonicfor 30 min for each time, and the
combined extracts were concentrated in vacuo. This residue
465.2 g) was suspended in H O and then successively
1.27–1.30 (1H, m)
4
7
27.1 0.93–0.99 (1H, m)
2.01 (1H, d, J 10.1)
27.1 0.92–0.95 (1H, m)
2.01 (1H, d, J 10.7)
41.2 1.37–1.41 (1H, m, H
49.0 0.82–0.85 (1H, m)
35.4
8
9
41.2 1.36–1.40 (1H, m, H
49.0 0.78–0.83 (1H, m)
35.4
β
β
(
2
partitioned with petroleum ether (PE), EtOAc, and BuOH.
The BuOH extract (248.8 g) was dissolved in 2 000 ml
distilled water, and passed through a column packed with
LSA-30 macroporous resin with a rate of 0.5 ml/s. The column
was firstly eluted with water to remove saccharide (detected
by α-naphthol test), and then subsequently eluted with 30%
aqueous ethanol, 50% aqueous ethanol and 95% aqueous
ethanol to afford fractions Fr.1-4 respectively. Fr.2 (50%
ethanol portion, 29.8 g) was subjected to column chroma-
1
1
0
1
20.8 1.02–1.08 (1H, m)
1.36–1.40 (1H, m)
39.7 1.18–1.23 (1H, m)
20.8 1.07–1.09 (1H, m)
1.38–1.41 (1H, m)
39.7 1.20–1.23 (1H, m)
1.38–1.42 (1H, m)
12
1.38–1.42 (1H, m)
1
1
3
4
48.7
83.1 4.17 (1H, s, OH)
42.7 1.57 (1H, d, J 14.2, Hβ)
2.48–2.49 (1H,
48.7
83.1 4.17 (1H, s, OH)
42.7 1.57 (1H, d, J 14.2, Hβ)
2.48–2.49 (1H,
15
t–like, H
α
)
t–like, H
70.4 4.38–4.42 (1H, m, H
4.51 (1H, d, J 3.75, OH)
α
)
1
6
7
70.5 4.38–4.42 (1H, m, H
α
)
α
)
tography over silica gel and eluted with CHCl
:1 to 0:1, v/v) to give subfractions Fr.2.1–2.4. Fr.2.1 was
subjected to be repeated CC over silica gel 60 (stepwise,
CHCl -MeOH from 15:1 to 12:1) to afford 1 (39 mg) and 2
24 mg). Fr.3 (70% ethanol portion, 36.6 g) was further
separated into subfractions Fr.3.1–3.3 by CC (SiO , stepwise,
CHCl -MeOH-H O from 9:1:0.1 to 2:1:0.1). Fr.3.1 was
subjected to CC (stepwise, CHCl –MeOH from 9:1 to 4:1)
3
–MeOH (from
4.51 (1H, d, J 3.90, OH)
9
1
57.5 2.62 (1H, d, J 8.1, H
16.8 0.63 (3H, s)
12.0 0.71 (3H, s)
118.7
α
)
57.5 2.62 (1H, d, J 8.0, H
16.8 0.63 (3H, s)
12.0 0.70 (3H, s)
118.7
α
)
18
19
20
21
22
3
(
150.3 7.46 (1H, d, J 1.5)
151.5 8.08 (1H, dd, J 2.4, 9.7) 151.5 8.08 (1H, dd, J 2.1, 9.7)
111.0 6.11 (1H, d, J 9.7)
161.7
100.6 4.20 (1H, d, J 7.8)
73.5 2.83–2.88 (1H, m)
76.8 3.09–3.12 (1H, m)
70.1 2.98–3.02 (1H, m)
76.3 3.02–3.05 (1H, m)
61.1 3.38–3.41 (1H, m)
150.3 7.46 (1H, d, J 1.5)
2
3
2
23
24
1
2
3
4
111.0 6.11 (1H, d, J 9.8)
161.7
3
′
′
′
′
100.4 4.28 (1H, d, J 7.8)
73.2 2.94–3.00 (1H, m)
74.7 3.23–3.28 (1H, m)
80.8 3.24–3.28 (1H, m)
75.0 3.23–3.28 (1H, m)
61.0 3.34–3.38 (1H, m)
3.67–3.72 (1H, m)
103.2 4.23 (1H, d, J 7.8)
73.3 2.96–3.01 (1H, m)
76.6 3.13–3.18 (1H, m)
70.0 2.99–3.04 (1H, m)
76.5 3.10–3.14 (1H, m)
60.5 3.55–3.58 (1H, m)
3
and Sephadex LH-20 chromatography (CHCl –MeOH, 1:1) to
yield 3 (30 mg) and 4 (121 mg).
2
.4. Acid hydrolysis
5′
6
′
3.61–3.65 (1H, m)
The compounds 1 or 2 (each 10 mg) was refluxed with
mol/L HCl for 2 h respectively. The reaction mixture was
1
2
3″
4″
5
6
″
″
4
diluted with H
layer was neutralized with Na
ca. 2 mL. D-glucose in the aqueous layer was identified by co-
TLC with authentic sample using CHCl –MeOH–H O (16:9:1)
2
O and extracted with EtOAc. The aqueous
2
CO and then concentrated to
3
″
″
3
2

638
J. Yang et al. / Fitoterapia 81 (2010) 636–639
and n-BuOH–HOAc–H
2
O (BAW, 4:1:5, upper layer) as devel-
from Vietnamese toad venom [17]. The NMR data of 1 were in
good agreement with the literature values except for the
signals of an additional glucose moiety, leading to the
aglycone of 1 as desacetylbufotalin. Based on the correlation
between H-1′ (δ 4.20, d, J=7.8 Hz) and C-3 (δ 76.8) in HMBC
and the downfield shift of C-3 (δ 67.9) of desacetylbufotalin
[18], the glucose moiety was assigned to be attached to 3-OH
group.
opers and spraying with aniline o-phthalic acid solution.
Tigencaoside A (1), white amorphous powder; [α]2
1.0
:
D
−
11.0° (c 0.052, MeOH); UVmax (MeOH): 293 (logε 3.62),
2
1
17 (3.53) nm; IR bands (KBr): 3425, 2923, 2854, 1693, 1118,
−1
1
13
081, 1027 m
; H and C NMR data: see Table 1; ESI-MS
−
−
(
(
negative) m/z 563 [M–H] , 401[M–H–162] ; HR-ESI-MS
positive) m/z 564.2931 [M] (Calcd. for C30
+
H
44
O
10,
5
64.2934).
The linkage positions of two hydroxyl groups in the
aglycone were substantiated by HMBC spectrum. In the
HMBC spectrum, the correlations between H-17 (δ 2.62) and
C-16 (δ 70.5) , and between H-16 (δ 4.38–4.42) and C-17 (δ
57.5) showed that one hydroxyl group was attached to C-16,
and the correlations between C-14 (δ 83.1)/14-OH (δ 4.17),
H-18 (δ 0.63), H-8 (δ 1.36–1.40 ) and H-15β (δ 1.57) showed
that the other hydroxyl group was located at C-14. Based on
the long range correlations between H-18 (δ 0.63)/C-12 (δ
21.0
Tigencaoside B (2), white amorphous powder; [α]
11.0° (c 0.026, MeOH); UVmax (MeOH): 290 (logε 3.56),
06 (3.43) nm; IR bands (KBr): 3423, 2926, 2853, 1715, 1158,
079, 1043; H and C NMR data: see Table 1; ESI-MS
D
:
−
2
1
1
13
+
(
positive) m/z 726 [M] ; HR-ESI-MS (negative) m/z 725.3426
−
[M–H] (Calcd. for C36
H
53
O
15, 725.3384).
3
. Results and discussion
3
9.7), C-13 (δ 48.7), C-14 (δ 83.1) and C-17 (δ 57.5), as well as
Compound 1, obtained as a white amorphous powder, has
a molecular formula of C30 10 based on HR–ESI–MS,
showing a molecular ion peak at m/z 564.2931 (C30
calc. 564.2934). Its UV spectrum exhibited absorption bands at
max (log ε): 293 (3.62) and 217 (3.53)nm due to the presence
of a conjugated system. The IR analysis of 1 showed the
between H-19 (δ 0.71) and C-1 (δ 36.7), C-10 (δ 35.4), C-5 (δ
43.7) and C-9 (δ 49.0), two methyl groups at δ 0.63 and 0.71
were assigned to be at C-13 and C-10, respectively. The
assignments (Table 1) of all C-atoms and H-atoms of 1 were
carried out by H, H-COSY and HSQC as well as in conjunction
with comparison with literature values [17,18].
The stereochemistry of 1 was confirmed by the ROESY
analysis. The results were showed in Fig. 2. The cross
relationships between 14-OH (δ 4.17)/H-18 (δ 0.63) and
H-8 (δ 1.36-1.40) established the β-configuration of 14-OH.
The correlation between 14-OH (δ 4.17)/16-OH (δ 4.51)
proved the β-configuration of 16-OH and the α-configuration
of H-16. Based on the cross relationships between H-18
(δ 0.63)/H-22 (δ 8.08) and H -16α (δ 4.38–4.42)/H-17 (δ 2.62),
H-17 was determined as α-configuration. Moreover, the
correlations of the protons of β-orientated Me-19 with H-5
(δ 0.97–1.02) and H-8 (δ 1.36–1.40) established H-5 and H-
8 as β-configuration. Based on the evidences above, 1 was
identified as 14β,16β-dihydroxy-3β-[(β-D-glucopyranosyl)
oxy]bufa-20,22-dienolide, named tigencaoside A.
44
H O
44 10
H O ,
1
1
λ
−1
−1
presence of OH (3425 cm ), C=C (1629 cm ) and C=O
−1
(
(
1693 cm ) functional groups. The 1H NMR spectrum
Table 1) showed the presence of three olefinic protons at δ
6
9
.11 (d, J=9.7 Hz), 7.46 (d, J=1.5 Hz), 8.08 (dd, J=2.4,
.7 Hz) and two methyl resonances at δ 0.63, 0.71, supporting
13
the fact that 1 had a bufadienolide skeleton. The C NMR
spectrum (Table 1) revealed a total of 30 carbon atoms and
their multiplicity assignments using DEPT established
3 2
the presence of two methyl (CH ), nine methylene (CH ),
fourteen methenyl (CH) groups, and five quaternary C-atoms,
of which five signals from δ 77 to 61, and a methine signal at δ
1
05.0 (C-1′) as well as in conjunction with the fragment peak
−
at m/z 401 [M–H–162] in ESI-MS (negative) suggested the
presence of one hexose moiety in the molecule of 1. The sugar
moiety was determined as D-glucose by co-TLC of acid
hydrolyzate with an authentic sample. Large coupling con-
stant of the anomeric proton at δ 4.20 (d, J=7.8 Hz) pointed
out to the β-configuration of D-glucose unit.
Compound 2, obtained as a white amorphous powder,
possessed a molecular formula C36
54
H O15, as deduced by HR-
ESI-MS (negative) showing a quasi-molecular ion peak ([M-
–
53
H] ) at m/z 725.3426 (C36H O15, calc.725.3384). Comparison
1
of the 1H and C NMR spectra for 2 with those of 1 and the
13
The aglycone of 1 was determined by comparison of its H
and 13C NMR data with that of desacetylbufotalin isolated
co-TLC of the acid hydrolyzate of 2 with authentic sample of
Fig. 2. Key HMBC (left) and ROESY (right) correlations of 1 and 2.

J. Yang et al. / Fitoterapia 81 (2010) 636–639
639
Table 2
823 were determined using the MTT assay method [9,20].
Their IC50 values are given in Table 2.
Cytotoxic activities of two new bufadienolides against 3LL, MCF-7, QGY-7701
and BGC-823.
IC50(μg/ml)
Acknowledgement
3
LL
MCF-7
QGY-7701
BGC-823
This work was supported by the National Natural Science
Foundation of China (NNSF; No. 30571402, 30771454).
Tigencaoside A
Tigencaoside B
153.45
68.56
105.23
86.45
128.76
56.54
253.12
78.75
References
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The cytotoxic activities of the compounds 1 and 2 against
four human cancer cell lines: 3LL, MCF-7, QGY-7701 and BGC-