A phenanthroindolizidine glycoside with HIF-1 inhibitory activity from Tylophora atrofolliculata

Article abstract of DOI:10.1016/j.phytol.2019.03.003

A phenanthroindolizidine glycoside 6-O-β-D-glucopyranosyl-tylophorinidine (1), the first glycoside of phenanthroindolizidine alkaloid in nature, was isolated from whole plants of Tylophora atrofolliculata (Asclepiadaceae). Its structure was elucidated by means of spectral and chemical evidences. Compound 1 showed potent inhibitory effect (IC₅₀: 69 ± 13 nM) on hypoxia induced factor-1 (HIF-1) activation. It was discovered that glucosylation at C-6 of 1 could lead to reduction in cytotoxicity against normal cells and enhance its selectivity in tumor cells inhibition.

Full text of DOI:10.1016/j.phytol.2019.03.003

Phytochemistry Letters 31 (2019) 39–42
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
A phenanthroindolizidine glycoside with HIF-1 inhibitory activity from
Tylophora atrofolliculata
a
a
b
b
a,⁎
Cheng-Yu Chen , Guo-Yuan Zhu , Tang-Gui Xie , Ping-Chuan Jiang , Jing-Rong Wang ,
a,⁎
Zhi-Hong Jiang
a
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and
Technology, Taipa, Macau, China
b
Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Chinese Medicine and Pharmaceutical Science, Nanning, 530022, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A phenanthroindolizidine glycoside 6-O-β-D-glucopyranosyl-tylophorinidine (1), the first glycoside of phenan-
throindolizidine alkaloid in nature, was isolated from whole plants of Tylophora atrofolliculata (Asclepiadaceae).
Its structure was elucidated by means of spectral and chemical evidences. Compound 1 showed potent inhibitory
effect (IC50: 69 ± 13 nM) on hypoxia induced factor-1 (HIF-1) activation. It was discovered that glucosylation at
C-6 of 1 could lead to reduction in cytotoxicity against normal cells and enhance its selectivity in tumor cells
inhibition.
Phenanthroindolizidine glycoside
6
-O-β-D-glucopyranosyl-tylophorinidine
Hypoxia induced factor-1 (HIF-1)
Cytotoxicity
1
. Introduction
molecular formula was determined as C28
H
33NO
9
by molecular ion at
+
1
13
m/z 528.2203 ([M+H] , calcd 528.2228). The H NMR and C data
(Table 1) collectively indicated signals corresponding to one 1,2,5-tri-
substituted benzene ring, one 1,2,4,5-tetra-substituted benzene ring,
five alcoholic hydroxyl groups, two methoxyl groups, five methylene
groups including two nitrogenated and one oxygenated ones, seven
methine groups including six oxygenated and one nitrogenated. The
Hypoxia induced factor 1 (HIF-1) is a transcription factor greatly
affecting the survival of tumor cells under hypoxia microenvironment
(
Brown, 1999), which could bring about radiotherapy treatment failure
and anticancer drugs resistance (Nagle and Zhou, 2006). HIF-1 inhibi-
tion has thus been recognized as an effective way in cancer therapy,
raising numerous efforts that identify HIF-1 inhibitors from natural
resource. Various natural products have been discovered to exhibit
profound HIF-1 inhibitory effects (Nagle and Zhou, 2012; Du et al.,
anomeric proton [δ
coholic hydroxyl groups [δ
5.13 (d, J =4.8 Hz, 1 H) and 4.74 (t, J =5.4 Hz, 1 H)], together with
H
5.29 (d, J =7.8 Hz, 1 H)] and protons of four al-
H
5.37 (d, J =4.8 Hz, 1 H), 5.19 (br, 1 H),
2
013).
the carbon signals (δ 61.4, 70.5, 73.7, 77.4, 77.7 and 100.8) (Table 1)
C
Tylophora atrofolliculata is a centuries-used folk medicine for the
treatment of rheumatism in China (Jiangsu New Medical College,
were indicative of a β-glucopyranosyl moiety (Feng et al., 2016), whose
absolute configuration was determined to be D- form based on the
LC–MS analysis of derivatives of compound 1’s glucose and standards
(D-glucose, L-glucose) (Feng et al., 2016; Wang and Gao, 2013; Zhu
et al., 2011). The remaining signals corresponding to the aglycone part
suggested the presence of a phenanthroindolizidine moiety (Tanaka
1
977). One group of its bioactive components is phenan-
throindolizidine alkaloids, which have been demonstrated as lead
compounds of anti-tumor agents in our previous studies (Chen et al.,
2
016a, b). Our further phytochemical exploration led to the isolation of
a phenanthroindolizidine glycoside, 6-O-β-D-glucopyranosyl-tylophor-
inidine (1). Herein, we report the structure elucidation of compound 1
together with its capabilities in inhibition of HIF-1 activation and
cancer cells growth.
et al., 2007). Among them, the proton signals at δ 8.21 (d, J =9.6 Hz,
H
1 H), 7.21 (dd, J = 2.4, 9.6 Hz, 1 H), 8.00 (d, J =2.4 Hz, 1 H), 8.30 (s,
1 H) and 7.28 (s, 1 H) collectively suggested the presence of one 1,2,5-
tri-substituted benzene and one 1,2,4,5-tetra-substituted benzene rings
respectively. The two methoxyl groups were decided to be placed at C-3
2
. Results and discussion
and C-7 via the HMBC correlations between OCH -3 and C-3 and be-
3
tween OCH
3
-7 and C-7. The alcoholic hydroxyl group on the aglycone
Compound 1 was obtained as a white amorphous powder. Its
was placed at C-14 through the HMBC correlation between OH-14 and
⁎
Corresponding authors.
E-mail addresses: jrwang@must.edu.mo (J.-R. Wang), zhjiang@must.edu.mo (Z.-H. Jiang).
https://doi.org/10.1016/j.phytol.2019.03.003
Received 24 January 2019; Received in revised form 17 February 2019; Accepted 7 March 2019
1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

C.-Y. Chen, et al.
Phytochemistry Letters 31 (2019) 39–42
1
1
Table 1
C-14, as well as H- H COSY correlation between OH-14 and H-14. The
NMR Spectroscopic Data (DMSO-d
6
) for Compound 1.
(J in Hz)
structure of 1 resembles that of tylophorinidine (2) (Tanaka et al.,
2
007; Huang et al., 2004) except for an additional glucose which was
Position
δ
H
δ
C
connected to C-6 by the HMBC correlation between H-1′ and C-6
Fig. 1). The small coupling constant (J =1.8 Hz) and strong NOE
1
2
3
4
5
6
7
8
9
8.21, d (9.6)
126.9
116.4
157.8
103.9
108.7
146.9
149.7
104.6
54.0
(
7.21, dd (9.6, 2.4)
correlation between H-13a and H-14 led to the cis configuration of both
hydrogens (Zhen et al., 2002; Stærk et al., 2000). The negative Cotton
effect at 271 nm in the CD spectrum indicated the S configuration at C-
13a (Zhen et al., 2002; Stærk et al., 2000; Damu et al., 2009), resulting
in the S configuration at C-14 accordingly. Therefore, compound 1 was
elucidated as 6-O-β-D-glucopyranosyl-tylophorinidine (Fig. 2). To the
best of our knowledge, this is the first glucoside of phenan-
throindolizidine type alkaloid isolated from nature.
8.00, d (2.4)
8.30, s
7.28, s
4.60, d (15.6); 3.51, d (15.6)
3.34, m; 2.39, m
1.85, m
1
1
1
1
1
4
4
8
8
1
1
1
55.3
2
22.1
3
2.20, m; 1.86, m
2.43, m
24.4
Compound 1 showed potent inhibitory effect on HIF-1 activation
induced by hypoxia in low nanomolar scale (IC50: 69 ± 13 nM). The
3a
4
65.4
4.96, dd (1.8, 9.6)
64.0
potency was even higher than the positive control digoxin (IC50
:
a
130.9
123.9
125.6
126.6
130.4
125.3
56.0
b
122 ± 3 nM) (Mi et al., 1992). As the aglycone of 1, compound 2(7)
showed a lower IC50 value (5 ± 1 nM) than that of 1, suggested that
glucosylation at C-6 of 1 could decrease HIF-1 inhibitory effects.
Compounds 1 and 2 were further evaluated for their cytotoxic ac-
tivities against a panel of cancer [human ovary (A2780) and liver
(HepG2) carcinoma] and normal [human ovary (Hospeic) and liver
(LO2)] cells. As shown in Table 2, compound 1 exhibited weaker cy-
totoxic effects against both cancer and normal cells than those of 2. The
IC50 values of 1 determined on A2780, HepG2, Hospeic and LO2 are
a
b
4a
4b
OCH
3
3
-3
-7
3.98
OCH
3.97
55.7
1
1
2
3
4
5
6
2
3
4
6
4-OH
4.70, d (9.6)
5.29, d (7.8)
'
100.8
73.7
77.4
70.5
77.7
61.4
a
a
'
3.38 , m
'
3.39 , m
0
.41, 4.58, 14.30 and 21.40 μM respectively, which is higher than those
'
3.19, m
'
3.62, ddd (1.8, 6.6, 9.6)
3.78, ddd (1.6, 5.4, 12.0); 3.49, m
5.37, d (4.8)
of 2 calculated on the four corresponding cell lines (A2780: 0.04 μM;
HepG2: 1.49 μM; Hospeic: 0.46 μM; LO2: 2.06 μM). These data in-
dicated that glucosylation at C-6, resulting in higher hydrophilicity,
reduce the cytotoxicity against both normal and tumor cells.
'
'-OH
'-OH
'-OH
'-OH
5.19, br
5.13, d (4.8)
4.74, t (5.4)
The selectivity index (SI) was measured by the proportion of the
IC50 values of normal cells relative to that of their tumor counterparts.
As shown in Table 2, the SI values of compound 1 are significant higher
than those of 2 for both A2780 (1: 34.9; 2: 11.5) and HepG2 (1: 4.7; 2:
a
Overlapped signals.
1
.4) cells, suggesting that 1 is more selective in inhibiting both cancer
cell lines. Glucosylation strategy has been adopted in several cytotoxic
drugs (including ifosfamide and taxol), resulting in lower toxicity and
higher cancer cell inhibitory selectivity in vitro (Zhang et al., 2008;
Calvaresi and Hergenrother, 2013; Pohl et al., 1995; He et al., 2016).
Consistent with these previous results, our data suggested glucosylation
at C-6 could significantly enhance tumor cell inhibitory selectivity.
3
. Conclusion
Fig. 1. Structure of 6-O-β-D-glucopyranosyl-tylophorinidine (1) and its agly-
cone tylophorinidine (2).
This investigation led to the isolation of a new phenan-
throindolizidine glycoside, named 6-O-β-D-glucopyranosyl-tylophor-
inidine (1), with glycosylation at C-6 of the aglycone part. Biological
activity evaluation suggested that 1 demonstrated more potent HIF-1
inhibitory effect than digoxin. SAR analyses indicated that glycosyla-
tion at C-6 of 1 reduced its cytotoxicity against normal cells, but en-
hance cancer cell inhibitory selectivity in vitro. Above all, this is the first
glucoside of phenanthroindolizidine type alkaloid from natural re-
source. Moreover, our results suggested that 6-O-β-D-glucopyranosyl-
tylophorinidine (1) could be a promising lead compound in the devel-
opment of anti-cancer drugs.
4
. Material and methods
4.1. General experimental procedures
Optical rotations were determined using a Rudolph Research
Analytical Autopol I Autometic polarimeter. CD spectra were measured
on a Chirascan Circular Dichroism spectrometer. UV data were re-
corded using
a Shimadzu UV-2700 UV/vis spectrophotometer.
1
1
Fig. 2. Key correlations observed in H– H COSY and HMBC (H→C) spectra of
HRESIMS were recorded on an Agilent 6230 ESI-TOF mass spectro-
meter. NMR spectral data were obtained from Bruker Ascend™ 600
1
.
40

C.-Y. Chen, et al.
Phytochemistry Letters 31 (2019) 39–42
Table 2
Cytotoxicities of 1 and 2 against both cancer and normal cells.
Compounds
Cytotoxicity (μM)
SIc
Cytotoxicity (μM)
LO2a
SIc
Hospeica
A2780b
HepG2b
1
2
14.30 ± 1.01
0.46 ± 0.11
0.41 ± 0.09
0.04 ± 0.01
34.9
11.5
21.40 ± 1.14
2.06 ± 0.80
4.58 ± 0.47
1.49 ± 0.31
4.7
1.4
Values are means ± SD, where SD = standard deviation; all experiments were independently performed at least three times.
a
b
c
Normal cells, including ovary (Hospeic) and liver (LO2) cells.
Cancer cells, including ovary (A2780) and liver (HepG2) carcinoma cells.
Selectivity index (SI), referring to the IC50 value measured for the cytotoxicity of the alkaloids on normal cells divided by that measured for cancer cells.
spectrometer equipped with a cyro platform. Silica gel (Davisil®,
glucose derivative from acid hydrolysates of compound 1 is 20.1 min.
The retention time of the derivatives of D-glucose and L-glucose is 20.2
and 19.2 min, respectively.
4
0˜63 μm, Germany) using solvent signals (DMSO-d
6
: δ
H
3.41/δ 39.9)
C
as references, ODS (Waters, Preparative C18 125 Å, 55˜105 μm,
American) were used for column chromatography. Silica gel plates
(
Merck, DC Kieselgel 60 F254, Germany) were used for TLC analyses.
4.5. Biological assay
High-performance liquid chromatography (HPLC) was carried out on an
Waters 1525–2489 apparatus with a semi-preparative column (Waters,
XBridge® Prep C18, 5 μm, 250 × 10 mm, American). Solvents for HPLC
separation are HPLC grade. Tylophorinidine (2) was isolated in our
previous study (Chen et al., 2016b).
HIF-1-mediated reporter gene assays in T47D cells were performed
according to our previous protocol (Chen et al., 2016b). The cytotoxi-
city assays were carried out in accordance with previous literature
protocol (Lin et al., 2008). Detail of each protocol could be seen in
Supporting Information.
4.2. Plant material
Acknowledgement
Whole plants of T. atrofolliculata were collected at Wuping Village,
Jingxi, Guangxi Province, China in 2012 and identified by Dr. Zhifeng
Zhang (Faculty of Chinese Medicine, Macau University of Science and
Technology). A voucher specimen (No. MUST-TA201302) was de-
posited at State Key Laboratory of Quality Research in Chinese
Medicine, Macau University of Science and Technology.
The authors would like to thank the Macao Science and Technology
Development Fund for the financial support (015/2017/AFJ to Z.H.J.
and 023/2016/AFJ to J.R.W.).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2019.03.003.
4
.3. Extraction and isolation
Whole plants of T. atrofolliculata (5.5 kg) were refluxed with MeOH
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