Journal of Asian Natural Products Research

ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20

A new indole alkaloid from the traditional Chinese

medicine Chansu

Ying-Hui Dai, An-Dong Wang, Yu-Lin Chen, Ming-Yu Xia, Xin-Yi Shao, Dong-
Chun Liu & Dong Wang

To cite this article: Ying-Hui Dai, An-Dong Wang, Yu-Lin Chen, Ming-Yu Xia, Xin-Yi Shao, Dong-
Chun Liu & Dong Wang (2017): A new indole alkaloid from the traditional Chinese medicine
Chansu, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1339697

To link to this article: http://dx.doi.org/10.1080/10286020.2017.1339697

Published online: 19 Jun 2017.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ganp20

Download by: [The UC San Diego Library] Date: 20 June 2017, At: 02:29
Journal of Asian Natural Products Research, 2017
https://doi.org/10.1080/10286020.2017.1339697

A new indole alkaloid from the traditional Chinese medicine

Chansu
Ying-Hui Daia#, An-Dong Wanga#, Yu-Lin Chena, Ming-Yu Xiab, Xin-Yi Shaoa,
Dong-Chun Liua and Dong Wanga
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;
b
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China

ABSTRACT ARTICLE HISTORY

A new indole alkaloid N′-formylserotonin (1), along with five known Received 16 May 2017
indole alkaloids N′-methylserotonin (2), 5-hydroxy-1H-indole-3- Accepted 5 June 2017
carbaldehyde (3), N-acetylserotonin (4), 6-hydroxy-1-oxo-3,4-dihydro- KEYWORDS
β-carboline (5), and bufoserotoin C (6), were isolated from the water Toad venom; Chansu;
extract of traditional Chinese medicine Chansu. Their structures were N′-formylserotonin;
elucidated on the basis of spectral analyses. The cytotoxicities of 1–6 bufoserotonin C; cytotoxic
against human lung adenocarcinoma epithelial cells A549 were tested activity
using the MTT method. Compound 6 exhibited stronger cytotoxic
effect than 5-FU, and 1–5 showed no cytotoxic effects. Bufoserotonin
C is one of the cytotoxic components in water-soluble extract of
Chansu.

1. Introduction
Chansu is a product of the venom or skin gland secretion of giant toads, including Bufo
bufo gargarizans Cantor and B. melanostictus Schneider [1]. It is an important traditional
Chinese medicine used in China and other Asian countries for a long time in clinic to treat a
number of diseases, such as sore throat, pains, heart failure, cancers [2]. The major chemical
components of Chansu, include bufotoxins, bufogenins, and bufotenines. Bufotoxins, which
exist only in fresh toad secretions, are almost hydrolyzed completely and corresponding
bufogenins are obtained during the drying process [3]. As a kind of lipid-soluble compo-
nents, bufogenins have been generally considered to be the main active substance because
of their significant biological activities such as cardiotonic, hypertensive, and anti-tumor

CONTACT  Dong Wang  [email protected]

#
These authors contributed equally to this work.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
2   Y.-H. DAI ET AL.

effects [4,5]. However, the anti-tumor preparations of Chansu or toad skin, such as Chansu
injection and cinobufacini injection, are all water-soluble agents which only contain trace
amounts of bufogenins [6,7]. Therefore, we hypothesize that the water-soluble components
of Chansu also have anti-tumor activity. In order to further investigate the anti-tumor
components, the water-soluble extract of Chansu was isolated to obtain six indole alkaloids.
Among them, 1 was a new compound, and 3 and 4 were isolated for the first time from
Chansu. In addition, the cytotoxic effects against A549 cancer cells of these compounds
were evaluated. Herein, we report the isolation, structural elucidation and bioactivity of
these compounds.

2.  Results and discussion

2.1.  Structure elucidation
Compound 1 was obtained as yellow amorphous powder. The positive and negative ESI-MS
of 1 showed the quasi-molecular ions at m/z 205.2 [M + H]+, 227.1 [M + Na]+, and 202.9
[M–H]−, and the molecular formula C11H12N2O2 was established by HR-ESI-MS at m/z
205.0981 [M + H]+. In the 1H NMR (600 MHz, DMSO-d6) spectrum, four aromatic pro-
ton signals at δH 7.11 (1H, d, J = 8.6 Hz, H-7), 7.04 (1H, d, J = 2.3 Hz, H-2), 6.81 (1H, d,
J = 2.3 Hz, H-4), and 6.58 (1H, dd, J = 8.6, 2.3 Hz, H-6) indicated a typical 3, 5-disubstituted
indole moiety (Table 1). Combined with two methylene signals at δH 3.32 (2H, t, J = 7.5 Hz,
H-11), 2.73 (2H, t, J = 7.5 Hz, H-10), it was suggested that 1 is a derivative of serotonin.
The 13C NMR (150 MHz, DMSO-d6) spectrum showed 11 carbon signals, containing eight
olefinic carbon signals at δC 102.6 (C-4), 111.0 (C-3), 111.7 (C-6), 112.1 (C-7), 123.6 (C-2),
128.3 (C-9), 131.2 (C-8), 150.6 (C-5) and two methylene carbon signals at δC 25.7 (C-10),
38.4 (C-11), which also indicated the existence of 5-hydroxyserotonin skeleton. In addition,
one carbonyl signal at δC 161.4 (C-13) was shown in 13C NMR spectrum, and the reactive
hydrogen signal of 1-NH at δH 10.48 (1H, s) was also given by 1H NMR spectrum of 1.
These data indicated that an aldehyde group should be linked to N-12. It is also confirmed
by HMBC correlations between H-11 and C-13, and between the proton (8.01, 1H, s) of
aldehyde group and C-11 (Figure 1). Therefore, the chemical structure of 1 was charac-
terized as N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)formamide, named N′-formylserotonin.

Table 1. 1H and 13C NMR spectral data of 1 in DMSO-d6.

Position δH (J in Hz) δC HMBC*
1 10.48 (1H, s) C-3, C-9
2 7.04 (1H, d, 2.3) 123.6 C-3, C-8, C-9, C-10
3 111.0
4 6.81 (1H, d, 2.3) 102.6 C-5, C-6
5 150.6
6 6.58 (1H, dd, 8.6, 2.3) 111.7 C-5, C-4
7 7.11 (1H, d, 8.6) 112.1 C-5, C-9
8 131.2
9 128.3
10 2.73 (2H, t, 7.5) 25.7 C-2, C-3, C-9, C-11
11 3.32 (2H, t, 7.5) 38.4 C-3, C-10, C-13
12 8.05 (1H, brs)
13 8.01 (1H, s) 161.4 C-11
*
HMBC correlations are from proton(s) stated to the indicated carbon.
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH   3

Figure 1. Key HMBC correlations of 1.

Five known compounds were identified as, N′-methylserotonin (2) [8], 5-hydroxy-1H-in-
dole-3-carbaldehyde (3) [9], N-acetylserotonin (4) [10], 6-hydroxy-1-oxo-3, 4-dihy-
dro-β-carboline (5) [11], and bufoserotonin C (6) [12], by comparing their NMR spectral
data with those reported in the literature.

2.2.  Cytotoxic activities

The cytotoxic effects of 1–6 were evaluated against human lung adenocarcinoma epithelial
cells A549 using the MTT method. The results showed compound 6 exhibited cytotoxic
effects with IC50 34.3 μM, stronger than that of positive control 5-FU (IC50, 48.65 μM).
Unfortunately, the others showed no cytotoxic activity against A549 with IC50 values more
than 100 μΜ.
Up to now, only three bufotenines, including bufobutanoic acid, bufopyramide, and
bufothionine, showed cytotoxic activities against the murine leukemia cell line P388, human
hepatocellular carcinoma cell lines SMMC-7721 or BEL-7402 [13,14]. The structure of
bufopyramide was reassigned as bufoserotonin C by Emma K. Davison and Jonathan Sperry
when they tried to synthesize putative bufopyramide. They found the NMR data for the
natural product bufopyramide match that was obtained from a synthetic sample of bufo-
serotonin C, confirming that the two natural products are the same compound [15]. This
means bufoserotonin C should possess cytotoxic effect and this deduction is proven to be
true by our research. Bufoserotonin C is one of the cytotoxic components in water-soluble
extract of Chansu, and the conjugated heterocyclic moiety in N-12 is the key structure for
cytotoxic effect.

3. Experimental
3.1.  General experimental procedures
UV spectra were recorded on a Shimadzu UV-1700 Spectrophotometer (Shimadzu Co.,
Kyoto, Japan). IR spectra were recorded on a Bruker IFS 55 FTIR spectrometer on KBr
pellets (Bruker Co., Karlsruhe, Germany). NMR spectra were recorded on Bruker ARX-
600 spectrometer (chemical shift values are presented as δ values with TMS as the internal
standard; Bruker Co., Billerica, MA, U.S.A). HR-ESI-MS data were recorded on a Waters
Xevo G2 Q-TOF mass spectrometer (Waters Co., Milford, MA, U.S.A). Semi-preparative
HPLC was performed on a Model LC-10ATVP system consisting of two LC-10AT HPLC
pumps with a SPD-10Avp detector (Shimadzu Co., Kyoto, Japan), and a Phenomenex Luna
C18 column (250 mm × 10.00 mm, 5 μm). Column chromatography (CC) was performed
4   Y.-H. DAI ET AL.

using 101 macroporous resin (0.3–1.25  mm, Cangzhou Bon Adsorber Technology Co.,
Ltd, Cangzhou, China), and Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech
AB, Uppsala, Sweden). Human lung adenocarcinoma epithelial cells A549 were purchased
from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A). 5-Flauouracil
(5-FU) is the product of Sigma (the purity was more than 99% detected by HPLC).

3.2. Material
The crude drug Chansu was collected from Linyi, Shandong Province, China, in March
2010 and was authenticated by the Associate Professor Dong Wang from Shenyang
Pharmaceutical University.

3.3.  Extraction and isolation

The dried and roughly powdered Chansu (250  g) was extracted with dichloromethane
(7 × 2.5 L) under reflux. The solvent was removed in vacuo to afford dichloromethane extract
(50 g). The residue (200 g) was extracted six times by distilled water (6 × 2 L) with an ultra-
sonator (200 W, 59 kHz, 30 min), and concentrated in vacuo to obtain crude water extract
(120 g). The crude water extract was suspended in 1 L distilled water, and partitioned three
times with 1 L n-butanol saturated with water. Collecting water phase and concentrating
under vacuum afforded water extract (100 g). The water extract was dispersed with 0.5 L
distilled water, and ethanol was added to a final concentration of 75% (v/v), then kept for
12 h at 4 °C. The filtrate was evaporated to provide water-soluble low-molecular components
(WSLM) (50 g). The WSLM was subjected to 101 macroporous resin, adsorbed overnight,
and then eluted with water-ethanol (100:0, 70:30, 40:60, 5:95, v/v) successively to yield four
fractions (Frs. 1–4). Fr. 2 (9.4 g) was applied to Sephadex LH-20 (methanol-water, 60:40,
v/v) and was further separated by semi-preparative HPLC using MeOH-H2O (10:90) as
an eluent (5 ml/min) to afford compounds 1 (6.2 mg, Rt 30 min), 2 (3.2 mg, Rt 62 min),
3 (2.1 mg, Rt 33 min), 4 (4.7 mg, Rt 37 min), and 5 (4.2 mg, Rt 50 min). Fr. 3 (4.6 g) was
applied to Sephadex LH-20 (methanol-water, 40:60, v/v), followed by semi-preparative
HPLC (MeOH-H2O, 20:80; 5 ml/min), to afford compound 6 (3.2 mg, Rt 62 min).

3.3.1. N′-Formylserotonin (1)
Yellow amorphous powder from MeOH-H2O (1:9); UV (CH3CN) λmax (log ε): 199 (4.38),
222 (4.36), and 279 (3.74) nm. IR (KBr) vmax (cm−1): 3397, 1630, 1548,1232, and 1191; 1H
NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) spectral data are shown
in Table 1. Positive ESI-MS: m/z 205.2 [M + H]+, 227.1 [M + Na]+, and negative ESI-MS:
m/z 202.9 [M–H]−; HR-ESI-MS: m/z 205.0981 [M + H]+ (calcd for C11H13N2O2, 205.0977).

3.4.  Cytotoxic assays

Human lung adenocarcinoma epithelial cells A549 were cultured in a RPMI-1640 medium
(Gibco, Grand Island, NY, U.S.A) and supplemented with 10% fetal bovine serum (FBS)
(Dalian Biological Reagent Factory, Dalian, China) and 0.03% L-glutamine (Gibco) at 37 °C
in 5% CO2. Compounds 1–6 were dissolved in DMSO separately to make stock solutions,
then diluted in cell culture medium at different concentrations, and used immediately. In
all assays, the final concentrations of DMSO in the culture medium were less than 0.01%.
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH   5

The in vitro cytotoxic activities were assessed by a standard MTT-based colorimetric

assay. Briefly, tumor cells in exponential growth were seeded at 5 × 104 cells/well in 96-well
plates (Nunc, Roskilde, Denmark) for 12 h, then treated with compounds at final concen-
trations of 0.1, 1, 10, and 100 μM. After incubation for 0, 12, 24, and 48 h, cell growth was
measured at different time points by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-
zolium bromide (MTT) assay with a plate reader (Tecan, Grödig, Austria) [16]. DMSO
was used as blank sample, while 5-fluorouracil (5-FU) was used as positive control. The
inhibition ratio (%) was calculated using the following formula:
( )
mean survival of treated group
Inhibiton ratio (%) = 1 − × 100% (1)
mean survival of control

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by the National Natural Science Foundation of China [grant
number 81303196]; Natural Science Foundation of Liaoning Province of China [grant number
2015020741]; Scientific Research Foundation for Returned Scholars, Shenyang Pharmaceutical
University [grant number GGJJ2015101].

References
  [1] Committee for the Pharmacopoeia of P.R. China, Pharmacopoeia of P.R. China, Vol. I. (China
Medical Science Press, Beijing, 2015), p. 383.
  [2] Y. Liang, A.H. Liu, S. Qin, J.H. Sun, M. Yang, P. Li, and D.A. Guo, J. Pharm. Biomed. Anal. 46,
442 (2008).
  [3] J. He, Y. Li, N. Si, H.Y. Zhao, B.L. Bian, and H.J. Wang, Acta Pharm. Sin. 49, 1446 (2014).
  [4] Y.M. Wang, Z.Y. Li, J.J. Wang, X.Y. Wu, H.M. Gao, and Z.M. Wang, J. Asian Nat. Prod. Res. 17,
364 (2015).
  [5] Y. Zhang, Y.K. Qiu, K. Liu, Y.T. Jiang, J.Y. Chen, and D.Q. Dou, Chin. Tradit. Herb. Drugs. 37,
1905 (2006).
  [6] L.X. Yang, H.Y. Zhao, S.F. Yuan, Y.J. Li, B.L. Bian, and H.J. Wang, Chin. J. Exp. Tradit. Med.
Formulae. 19, 87 (2013).
  [7] 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).
  [8] P. Zhang, Z. Cui, Y.S. Liu, and Y. Sheng, J. Shenyang Pharm. Univ. 23, 216 (2006).
  [9] P. Sauleau, M.T. Martin, M.E.T.H. Dau, D.T.A. Youssef, and M.-L. Bourguet-Kondracki, J. Nat.
Prod. 69, 1676 (2006).
[10] S.W. Yang and G.A. Cordell, J. Nat. Prod. 60, 230 (1997).
[11] R.H. Liu, H. Luo, Y.L. Li, M. Yang, X.K. Xu, H.L. Li, Y.H. Shen, C. Zhang, J. Su, and W.D. Zhang,
Chem. Nat. Compd. 45, 599 (2009).
[12] R.H. Liu, H. Luo, Y.L. Li, M. Yang, H.L. Li, Y.H. Shen, C. Zhang, J. Su, and W.D. Zhang, Helv.
Chim. Acta. 90, 2427 (2007).
[13] Y. Kamano, H. Morita, R. Takano, A. Kotake, T. Nogawa, H. Hashima, K. Takeya, H. Itokawa,
and G.R. Pettit, Heterocycles 50, 499 (1999).
[14] R.F. Xie, Z.C. Li, B. Gao, Z.N. Shi, and X. Zhou, J. Ethnopharmacol. 141, 692 (2012).
[15] E.K. Davison and J. Sperry, Org. Biomol. Chem. 13, 7911 (2015).
[16] M.Y. Xia, M.W. Wang, Z. Cui, S.I. Tashiro, S. Onodera, M. Minami, and T. Ikejima, J. Asian
Nat. Prod. Res. 8, 335 (2006).