Antitumor quinazoline alkaloids from the seeds of Peganum harmala

Article abstract of DOI:10.1080/10286020.2015.1042373

A phytochemical study on the methanol extracts from the seeds of Peganum harmala L. led to a new quizonaline alkaloid (S)-vasicinone-1-O-b-D-glucopyranoside (1) and four known ones, (R)-vasicinone-1-O-b-D-glucopyranoside (2), (S)-vasicinone (3), vasicine (4), and deoxyvasicinone (5). Their structures were elucidated by spectroscopic analysis including IR, HR-ESI-MS, 1D and 2D NMR, and specific rotation as well as by comparison of the data with those in the literature. All of the alkaloids were screened for antiproliferative activity against human gastric cancer cells MCG-803 with MTT method. Compounds 1 and 3 exhibited moderate inhibitory activity.

Full text of DOI:10.1080/10286020.2015.1042373

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Antitumor quinazoline alkaloids from
the seeds of Peganum harmala
a
b
c
c
Chun-Hua Wang , Hong Zeng , Yi-Hai Wang , Chuan Li , Jun
c
c
ac
Cheng , Zhi-Jun Ye & Xiang-Jiu He
a
School of Pharmaceutical Sciences, Wuhan University, Wuhan
30071, China
4
b
Xinjiang Production & Construction Corps Key Laboratory of
Protection and Utilization of Biological Resources in Tarim Basin,
Tarim 843300, China
c
School of Pharmacy, Guangdong Pharmaceutical University,
Click for updates
Guangzhou 510006, China
Published online: 13 Jul 2015.
To cite this article: Chun-Hua Wang, Hong Zeng, Yi-Hai Wang, Chuan Li, Jun Cheng, Zhi-Jun Ye &
Xiang-Jiu He (2015) Antitumor quinazoline alkaloids from the seeds of Peganum harmala, Journal of
Asian Natural Products Research, 17:5, 595-600, DOI: 10.1080/10286020.2015.1042373
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042373
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Journal of Asian Natural Products Research, 2015
Vol. 17, No. 5, 595–600, http://dx.doi.org/10.1080/10286020.2015.1042373
Antitumor quinazoline alkaloids from the seeds of Peganum harmala
a
Chun-Hua Wang , Hong Zeng , Yi-Hai Wang , Chuan Li , Jun Cheng ,
b
c
c
c
c
a,c
Zhi-Jun Ye and Xiang-Jiu He *
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; Xinjiang
Production & Construction Corps Key Laboratory of Protection and Utilization of Biological
a
b
c
Resources in Tarim Basin, Tarim 843300, China; School of Pharmacy, Guangdong Pharmaceutical
University, Guangzhou 510006, China
(
Received 31 January 2015; final version received 14 April 2015)
A phytochemical study on the methanol extracts from the seeds of Peganum harmala
L. led to a new quizonaline alkaloid (S)-vasicinone-1-O-b-D-glucopyranoside (1) and
four known ones, (R)-vasicinone-1-O-b-D-glucopyranoside (2), (S)-vasicinone (3),
vasicine (4), and deoxyvasicinone (5). Their structures were elucidated by
spectroscopic analysis including IR, HR-ESI-MS, 1D and 2D NMR, and specific
rotation as well as by comparison of the data with those in the literature. All of the
alkaloids were screened for antiproliferative activity against human gastric cancer cells
MCG-803 with MTT method. Compounds 1 and 3 exhibited moderate inhibitory
activity.
Keywords: Zygophyllaceae; Peganum harmala; quizonaline alkaloids; (S)-vasici-
none-1-O-b-d-glucopyranoside; antiproliferative activity
1
.
Introduction
research on the chemical composition of
P. harmala, considering its traditional
usage and the remarkable pharmacological
activities, especially antitumor activity.
In our present investigation, a new
quinazoline alkaloid, along with four
known ones (Figure 1), was isolated from
the extracts of P. harmala. In addition, a
preliminary screening for the antiproli-
ferative activity against human gastric
cancer cells of all the alkaloids was
conducted.
Peganum harmala L. belongs to the family
of Zygophyllaceae, which is widely
distributed in Central Asia, North Africa,
and Middle East. In China, it mainly
distributed in Northwestern, such as
Xinjiang, Inner Mongolia [1,2]. The
whole plant of P. harmala is a traditional
medicinal herb which has been widely
used for a long time to treat apoplexia,
asthma [3], jaundice, and lumbago [4].
Recent years of researches carried out on
the chemical composition have revealed
that b-carboline and quinazoline alkaloids
are important ingredients accounting for
many pharmacological and therapeutic
effects such as analgesic [5], antibacterial
2.
Results and discussion
Compound 1 was obtained as a white
amorphous powder. Its molecular formula
C H N O was established on the basis
[
6], antiparasitic activity [7], strong
1
7 20 2 7
reversible inhibition activity of mono-
amine oxidase [8], cytotoxicity and anti-
tumor activities [9–11], etc. Therefore, its
of great significance to carry out further
of the HR-ESI-MS at m/z 365.1635
þ
13
[M þ H] and C NMR data. The IR
spectrum showed absorption bands at
2
1
1673 cm
suggesting the presence of
*Corresponding author. Email: hexiangjiu@163.com
q 2015 Taylor & Francis

5
96
C.-H. Wang et al.
Figure 1. Structures of compounds 1–5 isolated from P. harmala L.
1
amide functionality. The H NMR spec-
trum of 1 in DMSO-d6 indicated the
present of four aromatic protons at dH
8
7
120.9; seven oxygenated carbon signals at
dC 101.7, 77.3, 76.9, 76.7, 73.6, 70.1, and
61.2; and two methylene carbon signals at
3
1
.15–8.17 (1H, m), 7.82–7.85 (1H, m),
.71–7.72 (1H, m), and 7.55–7.57 (1H,
dC 43.9 and 28.3, respectively. The
NMR signal at d 101.7 was assignable to
C
C
1
the anomeric carbon of a sugar. The H
m), which revealed a typical pattern for
0
0
13
and C NMR spectra of compound 1 were
AA BB splitting system of an ortho-
disubstituted benzene ring. An oxygenated
methine proton signal at dH 5.18–5.20
(1H, m) and two methylene proton signals
at d 4.07–4.13 (1H, m), 3.98–4.03 (1H,
similar to those of (S)-vasicinone (com-
pound 3), except for the signals of a sugar
(d 101.7, 77.3, 76.7, 73.6, 70.1, 61.2) and
C
the downfield-shifted C-1 signal at d 76.9
C
H
m), 2.49–2.57 (1H, m), and 2.18–2.24
(1H, m) were observed. The anomeric
compared with dC 71.3 of compound 3.
Therefore, compound 1 was assumed to
be a vasicinone glycoside. The saccharidic
chain of compound 1 was determined to be
D-glucosyl group by acid hydrolysis and
proton at dH 4.74 (1H, d, J ¼ 7.8 Hz)
suggested the presence of a sugar unit in
compound 1.
1
3
13
The C NMR spectrum showed 17
carbon signals, including a benzene ring at
dC 148.6, 134.4, 127.4, 126.9, 125.9, and
TLC analysis, as well as its C NMR data,
which were all in line with that of
saccharidic chain hydrolysed from

Journal of Asian Natural Products Research
597
the vasicinone and glucopyranosyl was
established by analysis of the HMBC
0
correlations (Figure 2) between H-1 at dH
4.74 and C-1 at d 76.9, H-1 at d 5.20 and
C-1 at d 101.7. The stereo structure of
C H
0
C
the chiral carbon (C-1) was determined as
S-form by the specific rotation value of
2
8.43 (c ¼ 0.35, CHCl ) after enzymatic
3
hydrolysis by emulsin, which is in
accordance with those in the literature
[
12]. Therefore, the chemical structure of
compound 1 was determined to be
S)-vasicinone-1-O-b-D-glucopyranoside,
(
which was a new alkaloid as far as we
knew. The proton and carbon signals were
1
13
assigned by H, C NMR, HSQC, and
HMBC experiments (Table 1).
Four known compounds were ident-
ified as (R)-vasicinone-1-O-b-D-glucopyr-
anoside (2) [13], (S)-vasicinone (3) [14],
vasicine (4) [15], and deoxyvasicinone (5)
[16] by comparing their spectroscopic data
with the corresponding literature.
Figure 2. Key HMBC correlations of
compound 1.
compound 2 and D-glucose standards. The
stereo structure of the anomeric carbon
was confirmed to be b-form based on its
coupling constant of the anomeric proton
All the five compounds were evaluated
for their antiproliferative activity against
human gastric cancer cells MCG-803
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
signal at d 4.74 (1H, d, J ¼ 7.8 Hz) in the
H
1
H NMR spectrum. The linkage between
1 13
Table 1. H and C NMR spectral data for compounds 1 and 2 in DMSO-d6.
1
2
Position
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
1
2
3
4
5.18–5.20 (m)
2.18–2.24 (m), 2.49–2.57 (m)
3.98–4.03 (m), 4.07–4.13 (m)
76.9
28.3
43.9
5.27–5.29 (m)
2.16–2.22 (m), 2.46–2.53 (m)
3.94–3.97 (m), 4.09–4.13 (m)
76.5
27.1
43.6
–
–
159.9
120.9
125.9
126.9
134.4
127.4
148.6
158.2
101.7
73.6
–
–
159.9
120.7
125.9
126.9
134.5
127.2
148.7
158.1
100.7
73.0
4
5
6
7
8
8
9
1
2
3
4
5
6
a
a
8.15–8.17 (m)
7.53–7.57 (m)
7.82–7.85 (m)
7.71–7.72 (m)
–
8.12–8.14 (m)
7.51–7.54 (m)
7.80–7.84 (m)
7.67–7.68 (m)
–
a
–
–
0
4.74 (d, 7.8)
2.98–3.01 (m)
3.04–3.17 (m)
3.05–3.08 (m)
3.17–3.21 (m)
3.45–3.49 (m), 3.71–3.74 (m)
4.43 (d, 7.8)
3.02–3.06 (m)
3.16–3.20 (m)
3.06–3.10 (m)
3.14–3.16 (m)
3.42–3.46 (m), 3.58–3.62 (m)
0
0
0
0
0
76.7
70.1
77.3
61.2
76.2
70.0
77.1
61.0

5
98
C.-H. Wang et al.
Table 2. Antiproliferative activity against
MCG-803 of the alkaloids (mean ^ SEM).
3.2. Plant material
The dry seeds of P. harmala were
collected in Kalpin County, Xinjiang,
China, in 2012, and identified by Prof.
Xiangjiu He, the School of Pharmacy,
Guangdong Pharmaceutical University.
The specimen (No. GDPU-NPR-
2013001) was deposited in the Department
of Medicinal Chemistry, School of
Pharmacy, Guangdong Pharmaceutical
University.
Compound
IC50 (mmol/l)
1
2
84.11 ^ 3.69
.100
3
4
94.5 ^ 10.47
.100
.100
5
Cisplatin
10.22 ^ 1.43
diphenyltetrazolium bromide (MTT)
method. The antiproliferative activity
was shown in Table 2. Compounds 1 and
3 exhibited a moderate inhibitory activity
with IC50 values of 84.11 ^ 3.69 and
3
.3. Extraction and isolation
The dried seeds of P. harmala (11 kg) were
powdered and then extracted three times
with 70% aqueous methanol under reflux.
After filtration and evaporation of the
solvent by rotary evaporator under reduced
pressure, the residues (2.3 kg) were sus-
pended in water and acidified to pH 1.0
with HCl (10%). Lipophilic impurities
9
4.5 ^ 10.47 mmol/l, respectively.
3
.
Experimental
3
.1. General experimental procedures
Optical rotation was recorded using P-
020 polarimeter (Jasco, Tokyo, Japan).
The IR spectra were obtained on Spectrum
00 FT-IR Spectrometer (PerkinElmer,
1
were removed with CHCl extraction and
3
the aqueous fraction was alkalized to pH
7.0 with NH ·H O. The total alkaloids
were extracted four times with CHCl and
1
3
2
Fremont, CA, USA). The NMR spectra
were recorded on a Bruker AV-500
spectrometer, using TMS as an internal
standard (Bruker, Bremen, Germany). The
ESI-MS were measured with ESI-Q-TOF-
MS Spectrometer (Waters, Milford, CT,
USA). Semi-preparative HPLC was car-
ried out on a Dynamax model HPXL
Solvent Delivery System with a UV
detector (Rainin, Oakland, CA, USA),
using a Cosmosil 5C -MS-II, 20ID
3
n-BuOH sequentially. The n-BuOH frac-
tion (220 g) was separated into 19 fractions
(B1–B19) by silica gel CC (Column
chromatograph) (200–300 mesh, 100
£ 1700 mm) eluted with CHCl /MeOH/
3
TEA (Triethylamine) (100:0:0.1 to
0:100:0.1, v/v/v), in increasing order of
polarity. Fraction B14 (7.3 g) was sub-
jected to RP-18 chromatography and
eluted with MeOH/H O (10:90 to 100:0)
2
1
8
£ 250 mm packed column (Nacalai, Naka-
gyo-ku, Japan). Sephadex LH-20 was
product of the Pharmacia Biothech AB
successively to yield 13 fractions (B14-1
to B14-13). Subfraction B14-8 (617.0 mg)
was fractionated over Sephadex LH-20
(
Uppsala, Sweden). Silica gel column
(20 £ 1100 mm) eluted by MeOH/H O
2
chromatography was performed with silica
gel (Liangchen Silicon Material Co. Ltd.,
Anhui, l u´ an, China). TLC was performed
with silica gel GF254 (Liangchen Silicon
Material Co. Ltd., Anhui, l u´ an, China).
MCG-803 cells were purchased from cell
library of Sun Yat-sen University Labora-
tory Animal Center.
(1:1) to yield a mixture (98.0 mg) that
was further separated by HPLC using the
Cosmosil 5C -MS-II with MeOH/H O
1
8
2
(20:80) to isolate compounds 1 (Rt
23.7 min, 37.0 mg) and 2 (Rt 21.2 min,
18.0 mg), which is conducted with the
flowrate of 4.0 ml/min and detected under
210 nm.

Journal of Asian Natural Products Research
599
The CHCl3 fraction (200 g) was
separated into 14 fractions (C1–C14) by
silica gel CC (200–300 mesh, 100
pH 5.0) respectively. The aglycone was
extracted with CHCl for three times and
3
then CHCl was removed. The aglycones
3
£
1500 mm) eluted with CHCl /MeOH/
3
were then dissolved in CHCl3 and
prepared to solutions (0.35 g/100 ml).
Optical rotations of solutions were
detected within 30 min. The aglycone of
compound 1 was determined to be S-
Vasicinone on the base of its optical
TEA (100:0:0.1 to 0:100:0.1, v/v/v), in
increasing order of polarity. Fraction C6
(4.3 g) was applied to a Sephadex LH-20
CC (20 £ 1100 mm) eluted by CHCl /
3
MeOH (2:1), followed by recrystallization
to obtain compound 3 (40.0 mg). Fraction
C7 (10 g) was submitted to a silica gel CC
eluted with a gradient solvent of CHCl3/
MeOH (80:1 to 0:1) to yield five fractions
2
0
rotation with ½aꢀ 2 8:43 (c ¼ 0.35,
D
CHCl ), which is in accordance with data
3
in literature. The aglycone of compound 2
was determined to be R-vasicinone with
2
D
0
(
(
(
C7-1 to C7-5). Fraction C7-2 (1 g), C7-3
70 mg) were applied to Sephadex LH-20
½aꢀ þ 6:1
(c ¼ 0.35,
CHCl3)
accordingly.
20 £ 1100 mm) column with CHCl /
3
MeOH (2:1) to yield compounds 4
(32.0 mg) and 5 (15.0 mg), respectively.
3
.5 Antiproliferative assay
Antiproliferative activities of the com-
pounds against human gastric cancer
cells MCG-803 were evaluated using the
3.3.1. (S)-Vasicinone-1-O-b-D-
glucopyranoside (1)
3
-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-
White amorphous powder (MeOH-H O);
2
tetrazolium bromide (MTT) method [17].
Cisplatin was used as the positive control.
2
0
20
D
½
aꢀ 2 23 (c ¼ 0.2, MeOH); ½aꢀ 2 8:43
D
(
c ¼ 0.35, CHCl ) (aglycone); IR(KBr)
3
y
: 3395, 2883, 1673, 1630, 1470,
max
2
1
1
13
; H and C NMR spectral
Acknowledgments
1
data, see Table 1; HR-ESI-MS: m/z
075 cm
The authors are thankful to Prof. Dr. Hao Gao,
Jinan University, for his generous help in the
measurement of optical rotation.
þ
3
C H N O , 365.1444).
65.1365
[M þ H]
(calcd
for
1
7 21 2 7
Disclosure statement
3
.3.2. (R)-Vasicinone-1-O-b-D-
glucopyranoside (2)
White amorphous powder (MeOH-H O);
No potential conflict of interest was reported by
the authors.
2
2
0
20
½
aꢀ 2 29 (c ¼ 0.2, MeOH); ½aꢀ þ 6:1
D
D
Funding
(
c ¼ 0.35, CHCl ) (aglycone); IR(KBr)
3
This work was financially supported by the
National Natural Science Foundation of China
y
: 3419, 2879, 1687, 1669, 1632, 1474,
max
2
1
1 13
; H and C NMR spectral
1084 cm
data, see Table 1; HR-ESI-MS: m/z
[
grant number U1203103]; the key project of
Xinjiang Production & Construction Corps Key
Laboratory of Protection and Utilization of
Biological Resources in Tarim Basin [grant
number BRZD1205].
þ
3
C H N O , 365.1349).
65.1365
[M þ H]
(calcd
for
1
7 21 2 7
3.4. Enzymatic hydrolysis for
determination of the stereo structure
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(
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