Cytotoxic constituents from the skin of the toad Bufo bufo gargarizans

Article abstract of DOI:10.1080/10286020.2010.545350

To study the chemical composition of the skin of Bufo bufo gargarizans, various chromatographic methods were used in the isolation procedures and the structures of isolated compounds were determined based on NMR and MS analysis. As a result, two new compounds were isolated from its ethanolic extract and characterized as N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea (1) and 19-oxocinobufotalin 3-adipoylarginine ester (2), together with 11 known compounds. Isolated bufadienolides showed significant inhibition effect against human hepatocellular carcinoma cell line SMMC-7721 in vitro.

Full text of DOI:10.1080/10286020.2010.545350

This article was downloaded by: [Dalhousie University]
On: 31 December 2014, At: 08:02
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Cytotoxic constituents from the skin of
the toad Bufo bufo gargarizans
Fu-Kai Wu b c , Ying-Kun Qiu a , Hu-Yi Zhao a , Zhen Wu a , Fa-Mei
Li b , Yong-Tao Jiang d & Ji-Yong Chen d
a Medical College, Xiamen University , Xiamen, 361005, China
b Shenyang Pharmaceutical University , Shenyang, 110016, China
c Xiamen Institute for Drug Control , Xiamen, 361012, China
d Luye Pharmaceutical Co. Ltd. , Yantai, 264003, China
Published online: 28 Jan 2011.
To cite this article: Fu-Kai Wu , Ying-Kun Qiu , Hu-Yi Zhao , Zhen Wu , Fa-Mei Li , Yong-Tao Jiang
& Ji-Yong Chen (2011) Cytotoxic constituents from the skin of the toad Bufo bufo gargarizans ,
Journal of Asian Natural Products Research, 13:02, 111-116, DOI: 10.1080/10286020.2010.545350
To link to this article: http://dx.doi.org/10.1080/10286020.2010.545350
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research
Vol. 13, No. 2, February 2011, 111–116
Cytotoxic constituents from the skin of the toad Bufo bufo gargarizans
Fu-Kai Wubc, Ying-Kun Qiua*, Hu-Yi Zhaoa, Zhen Wua, Fa-Mei Lib*,
Yong-Tao Jiangd and Ji-Yong Chend
b
aMedical College, Xiamen University, Xiamen 361005, China; Shenyang Pharmaceutical
University, Shenyang 110016, China; Xiamen Institute for Drug Control, Xiamen 361012, China;
c
dLuye Pharmaceutical Co. Ltd., Yantai 264003, China
(Received 10 October 2010; final version received 2 December 2010)
To study the chemical composition of the skin of Bufo bufo gargarizans, various
chromatographic methods were used in the isolation procedures and the structures
of isolated compounds were determined based on NMR and MS analysis. As a
result, two new compounds were isolated from its ethanolic extract and characterized
as N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea (1) and 19-oxocinobufota-
lin 3-adipoylarginine ester (2), together with 11 known compounds. Isolated
bufadienolides showed significant inhibition effect against human hepatocellular
carcinoma cell line SMMC-7721 in vitro.
Keywords: Bufo bufo gargarizans; toad skin; N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-
N-methylurea; 19-oxocinobufotalin 3-adipoylarginine ester; cytotoxicity
1. Introduction
bufadienolides, such as the effect of bufalin
on inducing apoptosis in human leukemia
U937 cells by modulating the mitogen-
activated protein kinase pathways, the
inhibitory effect of several bufadienolides
on tumor cell growth, and the inhibitory
effect on the IL-6 activities by 20S,21-
epoxy resibufogenin, have been reported
[3]. In order to find new and bioactive
bufadienolides from Chan’Pi, we investi-
gated the skin of B. b. gargarizans. In our
previous studies, two new bufadienolides
with cytotoxic activity were isolated [4].
We obtained another two new compounds
from its ethanolic extract, named N-[2-
hydroxy-2-(4-hydroxyphenyl)ethyl]-N-
methylurea (1) and 19-oxocinobufotalin 3-
adipoylarginine ester (2), together with 11
known compounds. Moreover, the isolated
bufadienolides showed significant inhi-
bition effect against human hepatocellular
carcinoma cell line SMMC-7721 in vitro.
Toads, particularly members of the genus
Bufo, are identified as a convenient and
useful source of granular gland secretions
that commonly contain biogenic amines,
bufodienolides, alkaloids, steroids, pep-
tides, and proteins. Including genus Bufo,
the different amphibian skin possessed a
wide range of biologically active sub-
stances with a therapeutic potential. In the
traditional Chinese medicine, amphibian
skin had been used for the alleviation of
human sufferings. Chan’Pi, the skin of a
Chinese toad (Bufo bufo gargarizans),
whose extract preparation known as ‘Hua
Chan Su’ was used in the treatment of
various diseases, including cancer, arrhyth-
mia, and heart diseases [1]. Many of these
effects are attributed to bufadienolides [2],
one of the active compounds in this drug.
Recently, new biological activities of
*Corresponding authors. Email: qyk@xmu.edu.cn; fameili@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2010.545350
http://www.informaworld.com

112
F.-K. Wu et al.
the signals attributed to a para-substituted
2. Results and discussion
benzene ring at d 156.3, 134.1, 127.0, and
114.7, was found in the low field of 13C
NMR spectrum. In the 1H–1H COSY,
HSQC, and DEPT experiments, the carbon
and proton signals could be attributed one
by one. Long-range correlations between
H-1 (d 3.27 and 3.20) and NZC H3 (d
35.8), CvO (d 159.4), C-10 (d 134.1),
which constructed the fragments above
into a whole structure as shown in Figure 1,
could be observed in the HMBC spectrum.
The relative configuration of the structure
was further confirmed by the NOESY
correlations that are shown in Figure 1.
Thus, the structure of 1 was elucidated as N-
[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-
methylurea.
Compound 1 was isolated as a white
powder. ESI-MS gave its quasi-molecular
ion at m/z 211 [M þ H]þ, corresponding to
the molecular formula C10H14N2O3 in the
1
HR-ESI-MS measurement. The H NMR
spectrum (DMSO-d6, 400 MHz) of 1
showed the presence of para-substituted
benzene ring signals at d 7.13 (2H, d,
J ¼ 8.4 Hz) and 6.73 (2H, d, J ¼ 8.4 Hz)
as well as a signal at d 2.70 (3H, s), which
was attributed to NZCH3 in high field.
Three signals of labile protons at d 9.30
(1H, br s), 5.78 (2H, br s), and 5.40 (1H, d,
J ¼ 3.6 Hz) could be observed, which
disappeared after adding D2O. Besides,
typical ABX conjugation system proton
signals belonging to the fragment of
ZCH(OH)ZCH2ZN could be observed
at d 4.63 (1H, m), 3.27 (1H, dd, J ¼ 14.2,
7.8 Hz), and 3.20 (1H, dd, J ¼ 14.2,
4.6 Hz). The 13C NMR and DEPT spectra
furnished 10 carbon signals, including a
methyl, a methylene, five methenyls and
three quaternary carbons. The signal at d
159.4 due to an urea carbonyl, as well as
For compound 2, ESI-MS gave its
quasi-molecular ion peak at m/z 757
[M þ H]þ. The extract molecular weight
[757.3658, (M þ H)þ, calcd 757.3655] and
the molecular formula (C38H52N4O12)
were given by high-resolution ESI-MS
measurement. The UV spectrum of 2
showed absorption maximum due to an
1H–1H COSY
HMBC
O
O
HO
HO
NH2
N
N
NH2
2
NOESY
1'
2'
3'
6'
5'
4'
OH
O
OH
O
1
NH
5''
O
H2N
NH
OHC
H
4''
O
3''
2'' 1''
O
O
H
N
5'
6' 4'
3'
H
1'
O
2'
OH
HOOC
O
2
1
Figure 1. Structures and key H–1H COSY, HMBC, NOE correlations of compounds 1 and 2.

Journal of Asian Natural Products Research
113
a-pyrone group at 293.2 nm. The IR
absorption bands at 3428, 2939, 1736,
1639, and 1541 cm21 reveal the existence
of hydroxyl, methyl, and a-pyrone groups.
Other known compounds were ident-
1
ified by comparing their H and 13C NMR
spectral data with those reported in
literature. They were characterized as
cinobufagin (3) [6], bufalin (4) [7],
cinobufotalin (5) [8,9], gamabufotalin (6)
[10], marinobufagin (7) [11], deacetylcino-
bufagin (8) [9], de-O-acetylcinobufotalin
(9) [9], bufotalinin (10) [7], bufotoxin (11)
[12], cinobufotalitoxin (12) [9], and cino-
bufagin 3-adipoylarginine ester (13) [13].
Most of the compounds, isolated from
the skin of B. b. gargarizans, showed
significant cytotoxicities against the
human hepatocellular carcinoma cell line,
SMMC-7721. The results are shown in
Table 3.
1
The H (dH 8.18, 7.53, and 6.42) and 13C
NMR (d 161.4, 152.6, 148.8, 116.5, and
113.9) spectral data in low field of 2 also
indicated the presence of a mono-substi-
tuted a-pyrone residue. All the data
suggested that compound 2 was a bufadie-
nolide. Signals of two methyls (d 20.2 and
17.1) and five carbons bearing oxygen (d
75.2, 72.6, 72.2, 69.6, and 59.8) appeared in
the 13C NMR spectrum of 2. The carbonyl
signal (d 208.3) in the lowest field of the 13
C
NMR spectrum was attributed to an
aldehyde, which showed correlation with
the proton singlet (d 10.49) in HSQC
spectrum. The bufadienolide core of 2 was
identified as 19-oxocinobufotalin, whose
1H and 13C NMR spectral data were
assigned by comparing with those of
cinobufotalin (5) and further confirmed by
the experiment of DEPT and 2D NMR,
except for the acylation shifts around C-3
and C-16 of 2. The arginine structure of 2
was established by comparison of its NMR
chemical shifts and proton–proton corre-
lations with those of 12. Remaining signals
(d 173.1, 29.3, 24.0, 29.2, 34.8, and 173.0)
in 13C NMR spectrum belonged to an
adipic acid fragment by comparing with
corresponding signals in 13. After acid
hydrolysis, the three fragments 19-oxoci-
nobufotalin, arginine, and adipic acid
mentioned above were obtained. On the
basis of 1H–1H COSY, HSQC, and DEPT
experiments, the carbon and proton signals
can be attributed one by one. Furthermore,
long-range correlations between H-100
(d 4.86) and C-60 (d 173.0), as well as H-3
(d 5.30) and C-10 (d 173.1) in the HMBC
spectrum, revealed the linkage of the three
fragments. Thus, compound 2 was eluci-
dated as 19-oxocinobufotalin 3-adipoylar-
ginine ester, which had been applied for
patent protection for its significant inhibi-
tory activity against the SMMC-7721
cancer cell line [5].
3. Experimental
3.1 General experimental procedures
Melting points were determined on an XT-
4 micromelting point apparatus and are
uncorrected. Optical rotations were
measured on a Perkin-Elmer digital
polarimeter. UV spectra were recorded
on a Shimadzu UV-260 spectrometer. IR
spectra were determined on a Perkin-
Elmer 683 infrared spectrometer in KBr
pellets. NMR spectra were taken with
TMS as an internal standard on a Bruker
Avance 400 FT-NMR spectrometer.
HR-ESI-MS were measured on a Bruker
FTMS Apex III spectrometer and ESI-MS
on a Finnigan LCQ Advantage spec-
trometer. Column chromatography was
performed on silica gel (Marine Chemical
Factory, Qingdao, China), Sephadex LH-
20 (Amersham Pharmacia Biotech AB,
Uppsala, Sweden), Cosmosil 75 C18-OPN
(Nakalai Tesque Co. Ltd., Toyko, Japan).
TLC was conducted on silica gel GF254
(Marine Chemical Factory, Qingdao,
China) and RP-18 F254 (Merck, Darm-
stadt, Germany) plates. Detection was
done by spraying 1% Ce(SO4)2 on 10%
aqueous H2SO4, followed by heating.
HPLC was performed using an ODS

114
F.-K. Wu et al.
column (Phenomenex LUNA C18
20 £ 250 mm, Los Angeles, America).
water and alcohol, and they were H2O
eluent, 30% EtOH eluent, and 95% EtOH
eluent, respectively. N-[2-Hydroxy-2-(4-
hydroxyphenyl)ethyl]-N-methylurea (1,
27 mg) was isolated from the H2O residue.
The residue of 95% EtOH eluent was
further separated using repeated ODS
(eluted successively with H2O, 30, 50,
70% MeOH and MeOH) and preparative
HPLC (Phenomenex LUNA C18
20 £ 250 mm, CH3CN–H2O 35:65) to
give four bufatoxins, characterized as 19-
oxocinobufotalin 3-adipoylarginine ester
(2, 29 mg), bufotoxin (11, 46 mg), cinobu-
fotalitoxin (12, 350 mg), and cinobufagin
3-adipoylarginine ester (13, 15 mg).
3.2 Animal material
The skin of B. b. gargarizans was
purchased in Shandong Province, China,
in April 2005, and identified by Dr Yanyan
Zhao, School of Pharmacy, Yantai Univer-
sity. A voucher specimen (20050201-CA)
has been deposited at the Medical College,
Xiamen University, Xiamen, China.
3.3 Extraction and isolation
The dried skin of B. b. gargarizans (20 kg)
was finely minced and extracted with 95%
EtOH under reflux. Evaporation of the
solvent under reduced pressure gave the
EtOH extract (660 g). The EtOH extract
was partitioned with acetic ether and
H2O. Removal of the solvent under
reduced pressure from the acetic ether
and H2O-soluble fractions yielded 450 and
200 g of residues, respectively. The acetic
ether soluble fraction (120 g) was sub-
jected to normal-phase silica gel column
[1.2 kg, CHCl3–MeOH (100:0 ! 0:100,
v/v)] to give 24 fractions. Each fraction
was subjected to further separation
using repeated ODS (eluted successively
with H2O, 30, 50, 70% MeOH and
MeOH), silica gel [eluted successively
with CHCl3 –acetone (20:1 ! 15:1 !
10:1 ! 5:1 ! acetone, v/v)], Sephadex
LH-20 (eluted with MeOH) column
chromatographies, and preparative HPLC
(Phenomenex LUNA C18 20 £ 250 mm,
CH3CN–H2O 45:55) to give eight com-
pounds, identified as cinobufagin (3,
878 mg), bufalin (4, 124 mg), cinobufota-
lin (5, 42 mg), gamabufotalin (6, 56 mg),
marinobufagin (7, 24 mg), deacetylcino-
bufagin (8, 23 mg), de-O-acetylcinobufo-
talin (9, 19 mg), and bufotalinin (10,
20 mg). Macroporous resin column
(HPD-100) was applied in the separation
of water-soluble extract. Three fractions
were afforded after eluted gradually with
3.3.1 N-[2-Hydroxy-2-(4-hydroxyphenyl)
ethyl]-N-methylurea (1)
25
White powder; mp 168–1708C; ½aꢀD
MeOH
max
þ50.0 (c ¼ 1.0, MeOH); UV l
nm
(log 1) (MeOH): 224.6 (3.81); IR nmax
(KBr): 3432, 2925, 2855, 1642,
1542 cm21
;
1H NMR (DMSO-d6,
400 MHz); and 13C NMR (DMSO-d6,
100 MHz) spectral data are shown in
Table 1; positive ion ESI-MS: m/z 211
(M þ H)þ; HR-ESI-MS: m/z 211.1074
[M þ H]þ (calcd for C10H15N2O3,
211.1077).
3.3.2 19-Oxocinobufotalin 3-
adipoylarginine ester (2)
25
White powder; mp 184–1858C; ½aꢀD
MeOH
max
þ70.0 (c ¼ 1.0, MeOH); UV l
nm
(log 1) (MeOH): 295.2 (3.68); IR nmax
(KBr): 3428, 2939,1736, 1639,
1541 cm21; H NMR (C5D5N, 400 MHz);
and 13C NMR spectral data (C5D5N,
100 MHz) are shown in Table 2; positive
ion ESI-MS: m/z 757 (M þ H)þ, HR-ESI-
MS: m/z 757.3658 [M þ H]þ (calcd for
C38H53N4O12, 757.3655).
1
3.3.3 Acid hydrolysis of compound 2
Compound 2 (2.0 mg) in 10% HCl–
dioxane (1:1, 1 ml) was heated at 808C

Journal of Asian Natural Products Research
Table 1. 1H and 13C NMR spectral data of compound 1.
115
No.
1H NMR
13C NMR
1
2
10
20, 60
30, 50
40
vCO
NZCH3
phZOH
ZNH2
2-OH
3.27 (1H, dd, J ¼ 14.2, 7.8 Hz) and 3.20 (1H, dd, J ¼ 14.2, 4.6 Hz)
56.8
71.3
4.63 (1H, m)
134.1
127.0
114.7
156.3
159.4
35.8
7.13 (2H, d, J ¼ 8.4 Hz)
6.73 (2H, d, J ¼ 8.4 Hz)
2.70 (3H, s)
9.30 (1H, br s)
5.78 (2H, br s)
5.40 (1H, d, J ¼ 3.6 Hz)
for 4 h in a water bath. The reaction
mixtures were neutralized with Ag2CO3,
filtered, and then extracted with CHCl3
(1 ml £ 3). After concentration, 19-oxoci-
nobufotalin was found in the CHCl3 layer
and the H2O layer was examined by TLC
with n-BuOH–AcOH–H2O (4:1:5, upper
layer) as development solvent and com-
pared with arginine and suberic acid.
3.4 MTT assay
Tumor cells (1 £ 104 cells/ml) were
seeded to RPMI 1640 medium, sup-
plemented with 10% heat-inactivated
FBS and kanamin (0.1 mg/ml) at 378C in
a humidified atmosphere containing 5%
CO2 for 24 h. Test compounds, as well as
the positive control, taxol, were added to
this culture and incubated at 378C for
Table 2. 1H and 13C NMR spectral data of compound 2.
No.
1H NMR
13C NMR
No.
1H NMR
13C NMR
1
2
3
4
5
6
7
8
2.66 (1H, m) and 1.89 (1H, m)
1.69 (2H, m)
5.30 (1H, br s)
18.8
26.1
69.6
36.5
72.6
37.0
24.1
33.5
41.9
54.6
22.1
39.5
45.0
72.2
59.8
75.2
50.2
17.1
208.3
116.5
152.6
148.8
113.9
161.4
10
173.1
29.3
24.0
29.2
34.8
173.0
54.8
30.6
25.1
41.5
20
1.26 (2H, m)
1.97 (2H, m)
1.26 (2H, m)
2.32 (1H, m)
30
2.37 (1H, m) and 1.93 (1H, m)
40
500
2.37 (1H, m) and 1.83 (1H, m)
1.70 (1H, m) and 1.19 (1H, m)
2.68 (1H, m)
600
100
4.86 (1H, br s)
1.60 (2H, m)
2
9
1.85 (1H, m)
300
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
400
1.64 (1H, m) and 1.46 (1H, m)
1.70 (1H, m) and 1.47 (1H, m)
500
158.9
20.2
169.9
178.1
COCH3
COCH3
ZCOOH
3.95 (1H, br s)
5.64 (1H, d, J ¼ 9.3 Hz)
3.04 (1H, d, J ¼ 9.3 Hz)
0.82 (3H, s)
10.49 (1H, s)
7.53 (1H, br s)
8.18 (1H, br d, J ¼ 9.9 Hz)
6.42 (1H, d, J ¼ 9.9 Hz)

116
F.-K. Wu et al.
Table 3. Cytotoxicities of isolated bufadienolides (IC50, mg ml21).
Compound
2
3
4
5
6
7
11
12
Taxol
0.02
SMMC-7721
2.7
0.38
0.12
0.26
0.0001
4.72
0.27
1.95
further 120 h without medium change. Cell
viability was then evaluated by the MTT-
reducing test and compared with that of
the control culture, where the cells were
treated without adding test compounds.
References
[1] Y. Zhang, Y.K. Qiu, and K. Liu, Chin.
Tradit. Herb. Drugs 37, 1905 (2006).
[2] Y. Zhang, Y.K. Qiu, and J.Y. Chen,
J. Shenyang Pharm. Univ. 24, 484 (2007).
[3] B. Giri, A. Gomes, and A. Debnath,
Toxicon 48, 388 (2006).
[4] H.Y. Zhao, F.K. Wu, Y.K. Qiu, Z. Wu,
Y.T. Jiang, and J.Y. Chen, J. Asian Nat.
Prod. Res. 12, 793 (2010).
[5] Y.K. Qiu, Y.T. Jiang, J.Y. Chen, J. He,
and Z. Li, CN Patent No. 101177445A (14
May 2008).
[6] M. Ye, G.Q. Qiu, H.Z. Guo, and D.A.
Guo, Appl. Environ. Microbiol. 70, 3521
(2004).
3.5 Statistics
Values are shown as mean ^ SE. Statisti-
cal analysis was done by Student’s t-test
and one-way ANOVA, and p , 0.05 was
considered significant.
[7] R. Verpoorte, Q.K. Phan, and S.A.
Baerheim, J. Nat. Prod. 43, 347 (1980).
[8] X.W. Yang, Z.T. Xing, J.R. Cui, and Y.L.
Fan, J. Beijing Med. Univ. 33, 199 (2001).
[9] M. Ye, L.L. Ning, J.X. Zhan, H.Z. Guo,
and D.A. Guo, J. Mol. Catal. B-Enzym.
22, 89 (2003).
3.6 Biological activity
Most of the compounds showed significant
cytotoxicities against the SMMC-7721
cancer cell lines. The results are shown
in Table 3.
[10] Y.J. Chen, J. Xiang, W. Gu, and M.J. Xu,
China J Chin. Mater. Med. 23, 620
(1988).
[11] M. Ye, H. Guo, H.Z. Guo, J. Han, and
D.A. Guo, J. Chromatogr. B 838, 86
(2006).
[12] R.P. George, K. Yoshiaki, D. Pavel, I.
Masuo, and C.K. John, J. Org. Chem. 52,
3573 (1987).
[13] M. Ye, J. Han, H.Z. Guo, and D.A. Guo,
Magn. Reson. Chem. 40, 786 (2002).
Acknowledgements
The project was supported by the Natural
Science Foundation of Fujian Province of
China (No. 2010J05087). The authors grate-
fully acknowledge Li Shen, Center of Analysis
and Test, School of Pharmacy, Yantai Univer-
sity, for her kind help in determination of their
samples.