Potent AChE and BChE inhibitors isolated from seeds of Peganum harmala Linn by a bioassay-guided fractionation

Article abstract of DOI:10.1016/j.jep.2015.03.070

Ethnopharmacological relevance Seeds of Peganum harmala Linn are traditionally used as folk medical herb in Uighur medicine in China to treat disorders of hemiplegia and amnesia. Previously studies have proved that dominating alkaloids in P. harmala show significant inhibitory activities on the cholinesterase. Aim of the study The aim of the present study is to isolate trace ingredients from seeds of P. harmala and evaluate its inhibitory activities on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Materials and methods For sake of screening effective cholinesterase inhibitors, trace compounds were isolated from seeds of P. harmala through a bioassay-guided fractionation and their structures were determined via detailed spectral analysis. The inhibitory activities on AChE and BChE were assessed using an improved Ellman method by UPLC-ESI-MS/MS to determine the common final product choline. Results The activity-guided fractionation led to the isolation of two new alkaloids 2-aldehyde-tetrahydroharmine (10), 2-carboxyl-3,4-dihydroquinazoline (19), one syringin structure analog 1-O-β-D-xylopyranose sinapyl alcohol (22), and along with 19 known compounds. Compounds acetylnorharmine (6), harmic acid methy ester (7), harmine N-oxide (13), 6-methoxyindoline (14), syringin (21) were first found from genus Peganum and compounds 3-hydroxylated harmine (4), 1-hydroxy-7-methoxy-β-carboline (5) were new natural products. The results showed that the 2-aldehyde-tetrahydroharmine (10) has a potential inbibitive effect on both AChE and BChE with IC₅₀ values of 12.35±0.24 and 5.51±0.33 μM, respectively. Deoxyvasicine (15) and vasicine (16) showed the strongest BChE inhibitory activity with IC₅₀ values of 0.04±0.01 and 0.1±0.01 μM. The analysis of the structure-activity relationship indicated that the saturation of pyridine ring and the presence of substitution at indole ring, C-1, C-3, C-7 and N-2, for β-carbolines, were essential for effective inhibition of both AChE and BChE and the five-membered ring between C-2 and N-3 as well as the substituent groups sited at C-4 and C-9, for quinazolines, were important to both the AChE/BChE-inhibitory activity. Conclusions Bioassay-guided fractionation has led to the isolation of AChE and BChE inhibitors from the seeds of P. harmala. These results are in agreement with the traditional uses of the seeds of P. harmala.

Full text of DOI:10.1016/j.jep.2015.03.070

Journal of Ethnopharmacology 168 (2015) 279–286
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
Potent AChE and BChE inhibitors isolated from seeds of Peganum
harmala Linn by a bioassay-guided fractionation
Yadi Yang a,b,c, Xuemei Cheng , Wei Liu a,b,c, Guixin Chou a,b,c,d, Zhengtao Wang a,b,c,d
Changhong Wang
a
d,
n
,
a,b,c,d,nn
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Rood, Shanghai 201203, China
The MOE Key Laboratory for Standardization of Chinese Medicines, 1200 Cailun Rood, Shanghai 201203, China
The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicine, 1200 Cailun Rood, Shanghai 201203, China
Shanghai R&D Centre for Standardization of Chinese Medicines, 199 Guoshoujing Road, Shanghai 201203, China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 October 2014
Received in revised form
Ethnopharmacological relevance: Seeds of Peganum harmala Linn are traditionally used as folk medical herb in
Uighur medicine in China to treat disorders of hemiplegia and amnesia. Previously studies have proved that
dominating alkaloids in P. harmala show significant inhibitory activities on the cholinesterase.
Aim of the study: The aim of the present study is to isolate trace ingredients from seeds of P. harmala and
evaluate its inhibitory activities on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).
Materials and methods: For sake of screening effective cholinesterase inhibitors, trace compounds were
isolated from seeds of P. harmala through a bioassay-guided fractionation and their structures were
determined via detailed spectral analysis. The inhibitory activities on AChE and BChE were assessed using
an improved Ellman method by UPLC-ESI-MS/MS to determine the common final product choline.
Results: The activity-guided fractionation led to the isolation of two new alkaloids 2-aldehyde-
10 March 2015
Accepted 13 March 2015
Available online 8 April 2015
Keywords:
Alkaloids
Peganum harmala
Cholinesterase inhibitors
Acetylcholinesterase
Butyrylcholinesterase
tetrahydroharmine (10), 2-carboxyl-3,4-dihydroquinazoline (19), one syringin structure analog 1-O-β-D-
xylopyranose sinapyl alcohol (22), and along with 19 known compounds. Compounds acetylnorharmine (6),
harmic acid methy ester (7), harmine N-oxide (13), 6-methoxyindoline (14), syringin (21) were first found
from genus Peganum and compounds 3-hydroxylated harmine (4), 1-hydroxy-7-methoxy-β-carboline (5)
were new natural products. The results showed that the 2-aldehyde-tetrahydroharmine (10) has a potential
inbibitive effect on both AChE and BChE with IC50 values of 12.3570.24 and 5.5170.33 mM, respectively.
Deoxyvasicine (15) and vasicine (16) showed the strongest BChE inhibitory activity with IC50 values of
0.0470.01 and 0.170.01 mM. The analysis of the structure-activity relationship indicated that the saturation
of pyridine ring and the presence of substitution at indole ring, C-1, C-3, C-7 and N-2, for -carbolines, were
β
essential for effective inhibition of both AChE and BChE and the five-membered ring between C-2 and N-3 as
well as the substituent groups sited at C-4 and C-9, for quinazolines, were important to both the AChE/BChE-
inhibitory activity.
Conclusions: Bioassay-guided fractionation has led to the isolation of AChE and BChE inhibitors from the
seeds of P. harmala. These results are in agreement with the traditional uses of the seeds of P. harmala.
&
2015 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
Morris, 2001). Since the introduction of the first cholinesterase
inhibitor (ChEI) in 1997, most clinicians and probably most
patients would consider the cholinergic drugs, donepezil, galanta-
mine and rivastigmine, to be the first line pharmacotherapy for
moderate AD (Birks, 2006). Recent studies indicated that the main
cause of the loss of cognitive functions in AD patients is a
continuous decline of the cholinergic neurotransmission in cortical
and other regions of the human brain (Schuster et al., 2010). The
drugs above can block the acetylcholinesterase (AChE) and inhibit
the breakdown of acetylcholine (ACh) accordingly. The selective
inhibitors on butyrylcholinesterase (BChE) could elevate extracel-
lular levels of ACh in the brain and improve cognitive performance
Alzheimer's disease (AD) is the most common age-related
neurodegenerative disease and has become an urgent public
health problem in most areas of the world (Grutzendler and
n
Corresponding author.
nn
Corresponding author at: The Institute of Chinese Materia Medica, Shanghai
University of Traditional Chinese Medicine, Shanghai 201203, China. Tel.: þ86 21
5
1322511; fax: þ86 21 51322519.
E-mail addresses: chengxuemei1963@163.com (X. Cheng),
wchcxm@hotmail.com (C. Wang).
http://dx.doi.org/10.1016/j.jep.2015.03.070
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

280
Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
in rodents without the classic adverse side effects induced by the
selective or nonselective inhibitors on AChE, such as nausea and
vomiting (Hartmann et al., 2007).
Alto, CA, USA) coupled with an analytical column (Diamonsil C18,
5 mm, i.d.4.6 ꢀ 250 mm).
AChE from Electrophorus electricus, BChE from equine serum,
acetylcholine (Ach) chloride, butyrocholine (BCh) chloride, choline
(Ch) chloride, chlormequat (internal standard, IS), galantamine
and harmane were purchased from Sigma-Aldrich (MO, USA).
HPLC-grade methanol was obtained from Fisher Co. (NJ, USA).
Formic acid was obtained from Tedia Inc. (OH, USA). Water was
produced with a Milli-Q Academic System (Millipore, Billerica,
MA). Other reagents were of analytical grade.
Ever since two AChE inhibitors derived from natural products
(
galantamine and rivastigmine) currently being licensed to alle-
viate cognitive symptoms in dementia, extensive research has
been directed towards the identification of other AChE inhibitors,
with the majority of these arising from the plant kingdom
(Williams et al., 2011). While structurally diverse, these strongest
AChE inhibitors are primarily alkaloids (Houghton et al., 2006;
Williams et al., 2011). Peganum harmala is a perennial herb
growing in Africa, the Middle East, India, South America, Mexico,
southern America and China (Kartal et al., 2003; Cheng et al.,
2.2. Plant material
2
010). In China, P. harmala has been used as a folk medicine since
The seeds of P. harmala were collected in wild in Urumuqi,
Xinjiang Uyghur Autonomous Region, China, in September 2009.
The plant material was authenticated by professor Chang-hong
Wang and the voucher specimen (No. PH09-1) was deposited at
the Herbarium of the Shanghai R&D Center for Standardization of
Traditional Chinese Medicine, Shanghai, China.
antiquity among the Uighur, Kazakh, and Mongolia for the treat-
ment of cold, asthma, malaria, rheumatism, lumbago, hemiplegia,
forgetfulness, and some skin diseases (Liu et al., 2013; Cheng et al.,
2
010). Previous reports have indicated that P. harmala show a
potential therapeutic effect on AD, due to the cholinesterase
inhibitory activities of harmine, harmaline, harmalol, harmol,
and vasicine presented in plant (Rook et al., 2010; Zheng et al.,
2.3. Extraction and isolation
2
009; Zhao et al., 2013; Frost et al., 2011; Liu et al., 2014). It was
determined that the total alkaloids content (mainly harmine and
harmaline) in dry seeds of P. harmala could accumulated up to 4.3%
and 5.6% (w/w) (Herraiza et al., 2010; Wang et al., 2002).
Many trace ingredients isolated from plants such as paclitaxel
may have strong biological activities (Rowinsky and Donehower,
The dried seeds of P. harmala (400 kg) were extracted three
times with 85% EtOH (3200 L) at 85 1C after immersion overnight.
The combined extracts were concentrated under vacuum to afford
a viscous residue (114 kg, total ethanol extract, TEE). The concen-
trated extracts (TEE) were first defatted by using petroleum ether
to remove liposoluble constituents and fatty oil (17 kg, petroleum
ether extract, PEE). The residual extracts (97 kg, total alkaloid
extract, TAE) were dissolved in 4% HCl (80 L) and centrifuged to
remove non-alkaloid extracts (54.6 kg, NAE). Then the filtrate was
adjusted to pH 11 by using NaOH solution and stand overnight to
precipitate the crude alkaloids (21.4 kg, abundant alkaloid extract,
AAE), which mainly contained compounds 3 and 8. The rest alkali
1
995). A few studies have reported that some abundant alkaloids
from P. harmala have potent inhibitory activities on AChE and BChE
Zhao et al., 2013; Zheng et al., 2009; Liu et al., 2014). More
(
interesting result is that some selective BChE inhibitors, such as
vasicine, deoxyvasicine, deoxyvasicinone, and vasicinone, have been
found from P. harmala (Zhao et al., 2013; Liu et al., 2014), which
prompted us to find other trace alkaloids as potent AChE/BChE
inhibitors from P. harmala. The present study performed a bioactive
guided isolation of the trace ingredients from P. harmala and
determined their inhibitory activities against AChE and BChE, using
a UPLC-ESI-MS/MS method established by our team earlier (Liu et al.,
solutions (70 L) were extracted with CH
2
Cl
2
(3 ꢀ 70 L) and led to
800 g concentrated extracts (trace alkaloid extract, TrAE). Then the
extract (TrAE) was subjected to silica gel (5 kg) column and
gradient eluted with mixture of AcOEt–MeOH (100:0, 50:1, 20:1,
10:1, 0:100, v/v). The next series of effluents were concentrated
under vacuum to obtain Fr. A to Fr.G. Compounds 18 (5 g) and 16
2014). It would provide some guidance for the design and synthesis
or semi-synthesis of potential inhibitors on AChE or BChE.
(80 g) were found as precipitate and filtered out from Fr. D and Fr.
F, respectively.
Fr. D (21 g) was dissolved in 30% MeOH and the precipitate was
filtrated and discarded. The filtrate (300 mL) was fractionated by
2
. Materials and methods
MCI gel with the elution of MeOH–H
10:0, v/v) to obtain 9 fractions. Fr.D.4 (0.8 g) gave compound 22
10 mg) after purifying by ODS column chromatography under
isocratic elution with acetonitrile–H O (8: 92). Fr.D.5 (1.2 g) gave
compound 20 (66.8 mg) after purifying by preparative HPLC under
isocratic elution with MeOH–H O (4.5:5.5). Fr.D.8 (0.8 g) and Fr.D.9
2
O (3:7, 4:6, 6:4, 7:3, 9:1, and
2
.1. General experimental procedures
(
UV spectra were determined using a TU-1901 spectrophot-
2
ometer (Purkinje General Instrument Co., Ltd., Beijing, China).
Scanning IR spectroscopy was performed on a Thermo Nicolet
2
3
80 FT-IR spectrometer (Thermo Electron Corp., San Jose, Calif.,
(0.6 g) were subjected to Sephadex LH-20 (MeOH) to afford
compound 5 (16.6 mg) and compound 12 (10 mg).
USA) using KBr pellets. Optical rotations were measured on
Autopol VI (Rudolph Research Analytical). 1 and 2-D NMR spectra
Fr.E (40 g) was subjected to silica gel (800 g) column chroma-
tography with the elution of AcOEt–MeOH (100:0, 40:1, 15:1,
10:1.5, 0:100, v/v) and 12 fractions were collected. Fr.E.4 (4 g)
was further separated by Sephadex LH-20 (250 g), eluted with
MeOH to give three fractions. Fr.E.4.1 was then purified by
1
were recorded at Bruker AV-400 instrument at 400 ( H) and
1
00 MHz (13C) in MeOD or DMSO soln., with TMSi as an internal
standard. ESI-MS were taken at Bruker Daltonics, Inc. APEXIII 7.0 T
FTMS and HR-EI-MS were taken at Finnigan LC QDECA mass
spectrometers. Analytical and preparative TLC was performed on
silica gel plates (HSGF254, Yantai Jiangyou Guijiao Kaifa Co., Ltd.,
PR China). The spots were visualized by exposure to UV radiation
under 254 nm and 365 nm. Column chromatography (CC) was
preparative HPLC with the eluent of MeOH–H
compound 7 (12 mg). Compound 10 (7.5 mg) was obtained from
Fr.E.11 (7 g) by MCI gel eluted with MeOH–H O (3:7, 4:6, 6:4, 7:3,
1:9, v/v). In addition, compound 21 (23 mg) was separated from Fr.
E.9 by MCI gel eluted with MeOH–H O (4:6).
Fr.G (60 g) was fractionated by silica gel (1.5 kg) column chromato-
graphy by applying mixture of CH CL –MeOH (20:1, 15:1, 10:1, 1:1,
2
O (1:1) to give
2
performed on silica gel (SiO
2
; 200–300 mesh; Qingdao Haiyang,
2
Co., Ltd., PR China), Sephadex LH-20 (Amersham Biosciences, GE
Health Care) and MCI gel CHP-20P (Mitsubishi Chemical Corpora-
tion). High performance liquid chromatography (HPLC) was per-
formed using an Agilent 1200 series (Agilent Technologies, Palo
2
2
0:100, v/v) as eluent and 7 fractions were collected. Fr.G.1 (2 g) was
fractionated by silica gel (60 g) column chromatography with the

Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
281
eluent of petroleum ether: EtOAc (15:1, 1:1, 0:1) to afford six fractions.
2.3.3. Compound 22
MeOH
max
Fr.G.1.2 was then subjected to Sephadex LH-20 (MeOH) to obtain
compound 6 (15 mg). Compound 14 (49.1 mg) was obtained from Fr.
G.2 (3.4 g) by silica gel column chromatography with the elution of
Amorphous white powder: UV
λ
nm (log
ε
): 221 (4.43);
KBr
ꢁ1
IR
ν
cm : 3419, 2917, 2613, 1589, 1508, 1467, 1421, 1338, 1245,
max
þ
1130, 1085, 1025, 960, 632. HR-ESI-MS m/z [Mþ þNa] 365.1221,
1
13
AcOEt. Fr.G.5 was subjected to MCI gel (500 g) with the MeOH–H
3:7, 4:6, 7:3, 9:1, v/v) as eluent to give 18 fractions. Compound 19
40 mg, white crystal) was crystallized out from Fr.G.5.4 after being
2
O
22 8
calcd. 365.1207, C16H O ; H and C NMR are shown in Table 1.
(
(
2.4. Acidic hydrolysis of compounds 22
stood overnight at room temperature. Fr.G.5.12 was further purified by
preparative TLC with the developing solvent system of AcOEt–MeOH–
ammonia (10:1:0.2) and yielded compound 13 (32.9 mg). Fr.G.6 (8 g)
was subjected to silica gel (200 g), employed with AcOEt–MeOH–
ammonia (10:1:0, 10:1:0.5) as developing solvent system to afford
nine sub-fractions. Fr.G.6.8 yielded compound 4 (44.8 mg) after
concentration and being extracted with 30% MeOH. Fr.G.6.9 was
loaded on MCI gel to afford compounds 15 (2 g) and 17 (13.7 mg)
A soln. of 22 (1–2 mg) in 3 M aq. CF
mL aq. borane-4-methylmorpholine (50
0 1C. After cooling, 50 of aq. borane-4-methylmorpholine
80 mg/mL) was added to the mixture and heated for 60 min at
20 1C in oil bath. The mixture was cooled and added with 100 L aq.
borane-4-methylmorpholine (80 mg/mL) and then concentrated
until anhydrous. Acetic anhydride/anh. (100 L) and CF COOH
100 L) were added to the hydrolyzate and the mixture was allowed
to react for 20 min at 50 1C. After addition of 1 mL of CHCl , the
mixture was washed three times with saturated Na CO solution and
O, successively. The organic phase was dried (Na SO ) and
subjected to GC/MS (Thermo TR-5 MS, 60 mꢀ 0.25 mm ꢀ 2.5 mm);
3
COOH (200
μ
L) and 80 mg/
μL) was heated for 5 min at
8
(
1
μL
μ
μ
3
eluted with MeOH–H
Fr.G.7 (10 g) was applied to MCI gel using MeOH–H
:6, 5:5, 6:4, 7:3, 9:1) to afford eleven fractions. Fr.G.7.7 was then
subjected to Sephadex LH-20 and eluted with MeOH to yield
fractions. Fr.G.7.7.2 was then loaded on a reverse-phase C18-ODS
column with the eluent of MeOH–H O (15: 85) to yield compound
1 (36.4 mg).
2
O (3:7, 8:2, respectively).
(
μ
2
O (2:8, 3:7,
4
3
2
3
H
2
2
4
2
2
1
1
carrier gas He, flow rate 1 mL/min; oven temp.: 180–220 C (4 C/min),
1
1
1
1
1
2
20 C for 2 min, 220–270 C (1 C/min), and 270 C for 1 min. By
comparison with the retention times of authentic samples, the
configuration (D) of the sugar moieties was determined.
2
.3.1. Compound 10
2
L
0
Amorphous white powder:
α
ν
:ꢁ41.31(C 0.17 MeOH),
MeOH
max
KBr
ꢁ1
UV
2
λ
nm (log
ε
): 226 (4.49); IR
cm : 3277, 2967, 2874,
max
2.5. Biological activities
828, 1655, 1644, 1630, 1428, 1216, 1249, 1195, 1154, 1036, 805; HR-
þ
1
EI-MS m/z [MþH] 245.1318, calcd. 245.1290, C14
H
16 2 2
N O ; H and
The AChE and BChE inhibitory activities of fractions and
isolated compounds were assessed using a UPLC-ESI-MS/MS
method previously established by our lab. (Liu et al., 2014),
employing galantamine as reference compounds.
13
C NMR shown in Table 1.
2
.3.2. Compound 19
All the tested extracts were dissolved in DMSO to obtain 10 mg/mL
solutions, and the test compounds were dissolved in 0.2% DMSO to
obtain 10 mM or 5 mM solutions. All the stock solutions were diluted
to a series of concentrations with 20 mM sodium phosphate buffer
solution (pH 7.6) before the experiment.
MeOH
max
Colorless acicular crystal: UV
λ
nm (log
ε
): 220 (4.12);
KBr
ꢁ1
IR
ν
cm : 3535, 3159, 1650, 1579, 1501, 1623, 1395, 1369,
max
þ
1
294, 1223, 757; HR-EI-MS m/z [MþH]
177.0677, calcd.
1
13
177.0664, C
H
9 8
N
2
O
2
; H and C NMR are shown in Table 1.
Table 1
1
3C and 1H NMR data of compounds 10, 19, and 22.
Position
Compound 10
Compound 19
Compound 22
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
1
45.8
5.42 (1H, q, 6.60)
135.2
1
2
3
4
4
5
6
7
8
9
a
a
132.1
154.0
154.4
104.9
134.9
42.4
23.3
3.51 (1H, m) 3.92(1H, m)
2.75 (2H, overlapped)
6.76 (1H, s)
6.76 (1H, s)
42.1
4.71 (2H, s)
118.1
126.8
127.0
128.8
117.8
154.8
119.4
109.7
157.6
95.8
7.26 (1H, d, 8.6)
6.66 (1H, dd, 8.6, 2.16)
7.16 (1H, overlapped)
7.16 (1H, overlapped)
7.24 (1H, t, 7.13)
104.9
154.4
131.3
129.9
63.5
6.57 (2H, d, 15.84)
6.34 (1H, dt, 15.84, 5.56)
4.24 (2H, d, 5.52)
3.86 (3H, s)
6.84 (1H, d, 1.76)
7.36 (1H, d, 7.88)
1
0
133.6
107.3
122.4
138.8
19.5
56.7
56.7
1
1
3.86 (3H, s)
1
2
1
3
1
4
1.49 (3H, d, 6.68)
8.17 (1H, s)
3.89 (3H, s)
1
5
163.9
56.0
16
1
2
3
4
5
'
'
'
'
'
104.9
74.3
76.0
71.0
66.3
5.01 (1H, d, 6.24)
3.58 (1H, dd, 7.68, 6.44)
3.48 (1H, overlapped)
3.61 (1H, overlapped)
3.99 (1H, dd, 11.72, 4.6)
3.21 (1H, dd, 11.72, 8.28)

282
Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
Fig. 1. The AChE/BChE inhibitory activities (the IC50 values are expressed as mg/mL) of the extract fractions from seeds of Peganum harmala (TEE: total ethanol extracts; PEE:
petroleum ether extract; NAE: non-alkaloid extract; TAE: total alkaloids extract, TrAE: trace alkaloid extract. Fr.A–Fr.G: the fractions separated from TrAE).
For the AChE and BChE assay, 10 mL of the solutions of test extracts
or compounds, 40 mL enzyme preparation (with final concentrations:
3.1.1. Activity-guided isolation of compounds
The total ethanol extracts (TEE) of the dried seeds of P. harmala
were first treated with petroleum ether in order to remove the oil
(PEE). In order to remove the non-alkaloids ingredients (NAE), the
skimmed residue was first dissolved in acid to dissolve alkaloid
components and filtrated. Then the most abundant alkaloids (AAE)
such as harmaline and harmine were precipitated from the acid
solution by alkalization. Some trace alkaloid ingredients would be
retained in the alkaline solution section (Liu et al., 2013). The trace
alkaloid extract (TrAE) was obtained from the alkalization solution by
dichloromethane extraction. The extracts obtained above were assayed
on AChE/BChE using a UPLC-ESI-MS/MS method and calculated the
IC50 values to evaluate their inhibitory activity (Fig. 1). A high
inhibitory activity (AChE IC50¼0.481 mg, BChE IC50¼0.205 mg) of the
total ethanol extracts (TEE) was observed. The total alkaloid extract
(TAE) and the trace alkaloid extract (TrAE) which all were yielded from
the total ethanol extracts (TAE) also exhibited a strong inhibitory
activities against AChE with IC50 values of 0.218 mg and 3.194 mg, BChE
with IC50 values of 0.935 mg and 1.424 mg, respectively. While the
petroleum ether extract (PEE) and the non-alkaloid extract (NAE)
showed much weaker inhibitory activity on both AchE and BChE.
The total alkaloids extract (TAE), which mainly containing
harmine and harmaline, have been proved potential inhibitory
activity on AChE and BChE (Zhao et al., 2013). The trace alkaloid
extract (TrAE), which has been removed off most harmine and
harmaline and enriched a mass of other trace alkaloids according
to principle of dissolution, became as the target for further
separation of active compounds with potential inhibitory activity
on AChE and BchE. The trace alkaloid extract (TrAE) were sub-
jected to silica gel column and eluted with AcOEt–MeOH to give
7 fractions. The concentrated extracts of every fractions were
tested on AChE/BChE inhibitory activity. From the results shown
in Fig. 1, the fractions A–C did not show a desired inhibitory
activity on AChE and BChE, while the fractions D–G showed
potential inhibitory activities (AChE IC50¼0.151, 0.236, 0.103,
0.264 mg, BChE IC50¼0.508, 0.052, 0.056, 0.159 mg, respectively).
After multiple separation processes by normal and reverse column
chromatogram and preparative HPLC of fractions D–G, total 22
compounds containing 20 alkaloids were obtained (Fig. 2), and
their structures were elucidated on the basis of spectra data using
0.0035 unit/mL for AChE, or 0.008 unit/mL for BChE,) were mixed
and pre-incubated in ice bath for 15 min. To the mixture, 50 mL
substrate solutions (final concentrations 1 mg/mL for acetylcholine
chloride, or 1.5 mg/mL for butyrocholine chloride) was added and
incubated for 30 min at room temperature, the reaction was termi-
nated by the addition of 300 mL acetonitrile (0°C and added 300 ng/
mL chlormequat as internal standard) immediately, and the super-
natant was used for analysis.
For controls, the test solutions were replaced by the corre-
sponding volume of phosphate buffer solutions (pH 7.6) or
galantamine solution in concentrations corresponding to their
IC50 values (50% of inhibition). For blanks, the test solutions and
the enzyme preparation were all replaced by the corresponding
volume of phosphate buffer solutions (pH 7.6).
The separation was performed on a Waters-ACQUITYTMUPLC
system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC
HSS T3 column (100 mm ꢀ 2.1 mm, 1.8 mm) maintained at 40 1C.
The mobile phase consisted of A (0.1% formic acid) and B
(
methanol) at a flow rate of 0.3 mL/min, using an isocratic elution
with 98% A and 2% B. The injection volume was 5 mL using a partial
loop with needle overfill mode (Liu et al., 2014).
The assays were conducted in triplicate, and all tabulated
results were expressed as means7 7SD, and the IC50 values were
calculated from concentration-response curves by a nonlinear
regression analysis using Prism software (GraphPad Software,
San Diego, CA).
3
. Results and discussion
3.1. Isolation and characterization of compounds
Two new alkaloids (10, 19) and one new syringin structure
analog (22) were isolated from P. harmala, along with 18 known
alkaloids (1–9, 11–18, 20) and syringin (21). Besides, compounds 6,
7
, 13, 14, 21 were first found from P. harmala and compound 4, 5
were new natural products. Their structures were elucidated on
1
13
the basis of spectroscopy data using H and C NMR, 2D NMR
1
1
1
13
1
1
(
HMQC, HMBC, H– H COZY, ROESY), as well as HR-ESI-MS.
H and C NMR, 2D (HMQC, HMBC, Hꢁ H COSY, ROESY) NMR.

Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
283
Fig. 2. Structures of compounds separated and identified from seeds of P. harmala.
3.1.2. Characterization of the three new compounds
1,2-disubstituted benzene ring [
δ
H
7.16 (1H, m, overlapped, H-5),
2
-Aldehyde-tetrahydroharmine (10) was obtained as a white
7.16 (1H, m, overlapped, H-6), 7.24 (1H, t, J¼ ¼7.13 Hz, H-7) and
amorphous powder, with the molecular formula as C14
H N O
16 2 2
7.36 (1H, d, J¼7.88 Hz, H-8)], together with the characteristic
þ
determined by HR-ESI-MS (m/z [MþH] 245.1318, calcd. 245.1290).
UV absorption at 226 and 268 nm suggested the presence of an
indole ring, and the IR spectrum indicated the presence of NH
C
carbon signals at δ 154.0 (C-2), 42.1 (C-4), 118.1 (C-4a) and
132.1 (C-1a) implied a 2-substituted-3,4-dihydroquinazoline ring
1
1
structure. Furthermore, Hꢁ H COZY correlations (Fig. 3) from
7.16 (H-5) to 7.36 (H-8) and HMBCs (Fig. 3) of H-4 ( 4.71) with
C-2 ( 154.0), C-5 ( 126.8), C-4a ( 118.1), and C-1a ( 132.1)
δ
H
ꢁ
1
ꢁ1
ꢁ1
(
3277 cm ), carbonyl (1655 cm ), and benzene ring (1630 cm
)
δ
H
δ
H
1
groups. The H NMR signals (Table 1) of an ABX coupling benzene
ring [
6.84 (1H, d, J¼1.9 Hz, H-8), 6.66 (1H, dd, J¼8.6, 1.9 Hz, H-6)
and 7.26 (1H, d, J¼8.6 Hz, H-5)], together with five quaternary
carbons at 157.6 (C-7), 133.6 (C-10), 107.3 (C-11), 122.4 (C-12)
and 138.8 (C-13) confirmed a 2,3,6-trisubstituted indole ring struc-
ture. HMBC correlations of H-15 (1H, 8.17, s) with C-1 ( 45.8)
and C-3 ( 42.4) suggested their connection through a nitrogen
atom. Hꢁ H COZY correlations (Fig. 3) from H-4 (
.51 and 3.92) in combination with HMBCs of H-1(
133.6), and H-4 ( 2.75) with C-11 ( 107.3) demonstrated that
compound 10 had a 1,2,3,4-tetrahydro- -carboline skeleton. In addi-
tion, the HMBCs of H-14 ( 1.49) with C-1 ( 45.8) and C-10 (
33.6), and H-OMe ( 3.89) with C-7 ( 157.6) indicated the methyl
δ
C
δ
C
δ
C
δ
C
δ
H
confirmed the above skeleton. Subtracting the structure of 3,4-
dihydroquinazoline ring from molecular formula calculated by HR-
δ
C
ESI-MS led to a carboxyl, and the diamagnetic shift (C-9,
C
δ 154.8)
suggested its attachment to C-2 ( 153.9). Based on the above
δ
C
δ
H
δ
C
evidences, the structure of 19 was as shown in Fig. 3, and named
as 2-carboxyl-3,4-dihydroquinazoline.
δ
C
1
1
δ
H
2.75) to H-3 (
δ
H
1-O-
β-D-xylopyranose sinapyl alcohol (compound 22) was
3
H
δ 5.42) with C-10
obtained as a white amorphous powder, with the molecular
(δ
C
δ
H
δ
C
formula C16
H
22
O
8
determined by the HR-ESI-MS (m/z 365.1221,
þ
1
β
[MþNa] , calcd. 365.1207). The H NMR signals (Table 1) of
δ
H
δ
H
δ
C
δ
C
H
6.76 (2H, s, H-3,5) and two methoxy group signals (6H, δ 3.86, s,
1
δ
H
δ
C
H-10,11) of magnetic equivalence demonstrated the presence of a
and methoxyl was connected with C-1 and C-7, respectively. The
absolute configuration of compound 10 was established as 1S by
X-ray crystallographic analysis (Fig. 4). Accordingly, the structure of
compound 10 was determined as shown in Fig. 3, and named as (1S)
symmetrical quadri-substituted benzene ring stucture. In addition,
a trans-disubstituted alkenyl [
(1H, dt, J¼15.84, 5.56 Hz)] was also observed. Hꢁ H COZY data
from H-9 ( 4.24) to H-8 ( 6.34) and HMBC of H-7 ( 6.57)
with C-4 ( 134.98) indicated the presence of a sinapyl alcohol
structure. The NMR characteristics were very similar to those of
δ 6.57 (2H, d, J¼15.84 Hz), 6.34
H
1 1
δ
H
δ
H
δ
H
2-aldehyde-tetrahydroharmine.
δ
C
2
-Carboxyl-3,4-dihydroquinazoline (19) was isolated as a color-
1
13
less acicular crystal with the molecular formula established as
syringin (21). However, H and C NMR data revealed a β-
þ
C
9
H
8
N
2
O
2
by HR-ESI-MS (m/z 177.0677, [M þH] , calcd. 177.0664).
xylopyranose moiety with anomeric proton at
δ
H
5.01 (1H, d,
5.01) to C-1
ꢁ
1
0
The IR spectrum indicated the presence of OH (3160 cm ),
carbonyl (1651 cm ) groups. The H NMR signals (Table 1) of a
J¼6.24, H-1 ). HMBC data (Fig. 2) from H-1' (
δ
H
ꢁ
1
1
C
(δ 135.25) demonstrated that the β-xylopyranose moiety was

284
Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
Fig. 3. The 1H– H COSY and selected HMBC of compounds 10, 19, 22.
1
3.2. Cholinesterase inhibition evaluations
The IC50 values of inhibitive activity against AChE and BChE of
the 22 compounds were evaluated and shown in Table 2.
It was observed that compounds of harmane (1), harmol
(
2), harmalol (9), deoxyvasicine (15) and vasicine (16) were able
to strongly inhibit AChE and BChE activity with IC50 values of 3.64,
.90, 3.45, 2.37, 3.38 mM for AChE and IC50 values of 1.04, 0.35, 0.66,
.04, 0.10 mM for BChE, respectively. The BChE IC50 values dis-
1
0
played by deoxyvasicine (15) and vasicine (16) were lower than
that of galantamine at the same test conditions. Moreover,
harmine (3) and harmaline (8) were selective AChE inhibitors
with the IC50 values 1.21 mM and 1.95 mM, respectively. Besides,
Fig. 4. The X-ray crystallographic structure of compound 10.
2-aldehyde-tetrahydroharmine (10), 3-hydroxylated harmine (4)
and tetrahydroharmine (11) also showed weak inbibitive effect on
both AChE and BChE.
β
-carboline is a tricyclic structure of indole ring fused to a
pyridine ring with the nitrogen 2-sited. From the AChE and BChE
inhibitory potencies of -carboline alkaloids (compound 1–13) are
attached to C-1 of the sinapyl alcohol aglycone. Acidic hydrolysis of
2 gave D-xylose as the sugar moiety. Based on the above
2
β
evidences, compound 22 was determined to be 1-O-
β
-D-xylopyr-
shown in Table 2, it could be found that the compounds 1, 2 and 3
anose sinapyl alcohol.
were more active for AChE, which demonstrated that the presence of
In addition, 12 known
Kusurkar and Goswami, 2004), harmol (2) (Duan et al., 1998a),
harmine (3) (Duan et al., 1998a,b), 3-hydroxylated harmine (4)
Zhao et al., 2012), 1-hydroxy-7-methoxy- -carboline (5) (Ghazala,
980), acetylnorharmine (6) (Hashimoto and Kawanishi, 1976),
β
-caboline alkaloids of harmane (1)
methoxyl or hydroxide at C-7 of the β-carbolines led to an increase in
(
potency for AChE. Furthermore, compounds 3 and 8 have a more
effective inhibitory activity against AChE than compounds 2 and 9,
respectively. It suggested that methoxyl (as an electron-withdrawing
(
1
β
group sited on C-7) would make the β-carboline skeleton more
harmic acid methy ester (7) (Hashimoto and Kawanishi, 1975),
harmaline (8) (Duan et al., 1998a), harmalol (9), tetrahydrohar-
mine (11), harmalanine (12) (Salimuzzaman et al., 1988), harmine
N-oxide (13) (Hashimoto and Kawanishi, 1975), one known indole
alkaloid 6-methoxyindoline (14), five known quinazoline alkaloids
of deoxyvasicine (15) (Duan et al., 1998a), vasicine (16) (Duan
et al., 1998a,b), dexyvasicinone (17) (Duan et al., 1998b), vasicinone
inhibitory potency on AChE than a hydroxy.
A further observation was that the compound 2 and 9 gave the
AChE selectivity index (S.I., defined as IC50 BChE/IC50 AChE affinity
ratio) of 0.18 and 0.19, respectively. Both of them displayed more
potent inhibition on BChE than the compounds which has a
methoxy sited on C-7, such as compounds 3, 5,8, etc. It implied
that the hydroxy at C-7 would make the
a higher inhibition on BChE.
β-carboline skeleton have
(18) (Duan et al., 1998a,b), pegamine (20) (Ma et al., 1999), and one
other type compound of syringin (21) (Gao et al., 2012) were
isolated. Their structures were identified by comparison of their
spectroscopic data with those reported in the literatures or the
authentic compounds.
Simple indoles with substitution of methoxy, carboxy or
hydroxy at the benzene ring showed a low percent of inhibitory
activity on AChE. Adding a side chain at the pyrrole ring, such as
serotonin, β-carbolines and quinolines (the bioisostere of indole),

Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
285
Table 2
The half Inhibition concentration (IC50) and selectivity index (S.I.) of the compounds investigated against AChE and BChE.
Compounds
No.
IC50 (mM7SD)
S.I.a
Name
AChE
BChE
1
2
3
4
5
6
7
8
9
Galantamine
Harmane
Harmol
Harmine
3-hydroxylated harmine
1-hydroxy-7-methoxy-β-carboline
Acetylnorharmine
Harmic acid methy ester
Harmaline
0.0570.00
3.6470.19
1.9070.02
1.2170.04
23.6171.30
7.1970.47
4.0470.07
14.170.74
1.9570.08
3.4570.08
12.3570.24
22.9570.56
16.9570.56
41000
67.8578.60
2.3770.40
3.3870.03
35.1671.34
76.6078.46
344.80740.82
155.1077.04
31.4772.25
41000
0.2570.00
1.0470.05
0.3570.03
2.7970.27
3.2570.06
5.1570.23
26.572.82
21.4870.87
5.3870.64
0.6670.09
5.5170.33
10.4170.63
3.2470.29
4 1000
21.3771.41
0.0470.01
0.1070.00
17.8270.93
10.2072.08
41000
5.28
0.29
0.18
2.30
0.14
0.72
6.56
1.52
2.76
0.19
0.45
0.45
0.19
/
1
0
Harmalol
1
1
(1S)2-aldehyde-tetrahydroharmine
Tetrahydroharmine
Harmalanine
Harmine N-oxide
6-methoxyindoline
Deoxyvasicine
Vasicine
Dexyvasicinone
Vasicinone
2-carboxyl-3,4-dihydroquinazoline
Pegamine
1
2
1
3
1
4
1
5
/
1
6
0.02
0.03
0.51
0.13
/
0.68
0.09
/
1
7
1
8
1
9
20
2
1
105.70713.50
2.7770.12
41000
2
2
2
3
Syringin
1-O-β-D-xylopyranose sinapyl alcohol
IC50 values were determined by regression analyses and expressed as the means7SD of three replicate determinations.
/
: no S.I. values.
a
S.I. is the AChE selectivity index defined as IC50 BChE/IC50 AChE affinity ratio.
improved the inhibitory activity on AChE significantly (Khorana
et al., 2012). While, the presence of pyridine ring substituted at
indole ring seemed to be important to inhibitory activities on
AChE/BChE, concluding from the lower activity of compound 14
compound 3 implied that the methyl on C1 would make the
β
-carbolines showing selective AChE inhibition.
When the S.I. values of compounds 3 and 4 were compared, it
was observed that the hydroxyl sited on C-3 would cut down
obviously the inhibitory activity on AChE but slightly on the BChE.
In consideration of other natural and synthetic compounds in
the literature (Torres et al., 2012), quaternalization of the pyridine
(
AChE IC50¼67.850 mM, BChE IC50¼21.37 mM) and comparing to
that of -carbolines (compounds 1–13).
β
Compounds 3, 8 and 11 showed a progressively lower AChE/
BChE-inhibitory activity. Moreover, compound 2 showed more
AChE/BChE-inhibitory than compound 9. All of above results
suggested that the nonsaturation of pyridine ring was very
essential for both the cholinesterase inhibition. Further observa-
tion on the results, the compound 8 had a higher S.I. value of 2.76
than the compound 11 (S.I., 0.45). It implied that the saturated
bond of C1–N2 would make the compound to have a BChE-
selectivity inhibitory activity. The compound 2 had a similar S.I.
value with compound 9 and also a similar S.I. value was found
between the compound 3 and 8. It indicated that the double bond
between C3 and C4 would not contribute to the change of
cholinesterase inhibition selectivity.
nitrogen of the β-carboline nucleus leads to an increase in potency
for both cholinesterase inhibitions. However, compound 13, which
is quaternalized in the pyridine nitrogen, shows non-inhibitory
activity in present test with the IC50 values above 1000 mM on both
the AChE and BChE.
For quinazoline alkaloids obtained in P. harmala, the structure-
function relationship seemed simple. Compared to the compounds
15 and 17, the compounds 19 and 20 had no five-membered ring
between N-3 and C-2. Both of them exhibit weak inhibitory
activities on either AChE or BChE. It suggested that the five-
membered ring between C-2 and N-3 was important to both the
AChE/BChE-inhibitory activities.
When comparing with compound 11, the compound 10 with an
aldehyde group substituted at tetrahydropyridine nitrogen and the
compound 12 with an unsaturated ring between C-1 and N-2
displayed the increased IC50 values on both AChE and BChE. It
suggested that the electron withdrawing group sited on N-2 would
It was observed that compounds 15 and 16 have a much more
potential inhibitory activity than compounds 17 and 18 on both
AChE and BChE. Therefore, the non-substitution on C-4 of the
quinazolines is important to the cholinesterase inhibitory activities.
Compared with the compound 16, the compound 15 has a
higher inhibitory activity on both AChE and BChE. Furthermore,
the compound 17 is more active than compound 18 on both AChE
and BChE. Thus, the hydroxy sited on C-9 might decrease the
AChE/BChE inhibitory activity of the quinazolines.
In the process of drug development, toxicity and safety of drug
is one of the important factors that should be considered first. The
acute toxicity of the seeds of the genus Peganum growing in China,
including Peganum harmala, Peganum multisectum, Peganum nigel-
lastrum, and Peganum variety, have been determined previously
(Zhao, 2011). The results indicated that the strength of the toxicity
is in order of P. nigellastrum4 4P. variety4 4P. multisec-
tum4 4P. harmala, and the toxicity potency is proportional to
make the β-carboline more active on cholinesterase inhibition. In
addition, the similar S.I. between compounds 11 and 10 implied
that the aldehyde group substitute on N-2 made the compound 10
show corresponding BChE-selective inhibition as compound 11.
Moreover, the compound 12 had a lower S.I. value than compound
11, which meant that the unsaturated ring between C-1 and N-2
would observably improve the selective inhibition on BChE of
compound 11.
When comparing the IC50 values of the compounds 3, 6, 5 and
7
to each other, it could be found that the methyl cited on C-1 of
the -carboline mother nucleus would improve both the AChE and
BChE inhibitory activity. Besides, the highest S.I. value 2.303 of
β

286
Y. Yang et al. / Journal of Ethnopharmacology 168 (2015) 279–286
the harmaline content in the extract of plants. According to
previous reports, the half lethal dose (LD50 of harmine
417.45 mg/kg) is about 2 times larger than that of harmaline
144.07 mg/kg) (Massoud et al., 2002; Wang, 2002). In order to
evaluate the potential for these new compounds becoming drug
candidates, it is very important to further understand their
toxicity. This will be conducted in a follow-up study.
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4
. Conclusion
For sake of screening effective cholinesterase inhibitors, a
bioactive guided isolation of the trace ingredients from P. harmala
led to two new alkaloids 2-aldehyde-tetrahydroharmine (10) and
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9 known compounds (1–9, 11–18, 20–21, 23) were obtained.
Among them, the new compound 2-aldehyde-tetrahydroharmine
10) has a potential inbibitive effect on both AChE and BChE, and
β
1
(
two known compounds deoxyvasicine (15) and vasicine (16) show
the strongest BChE inhibitory activity. The analysis of the
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Acknowledgments
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Lehmann, J., Winckler, T., 2010. Bivalent beta-carbolines as potential multi-
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The authors gratefully acknowledge the award from the Key
Program of Joint Funds of the National Natural Science Foundation
of China and Xinjiang Uygur Autonomous Region of China (No.
U1130303), the National Nature Science Foundation of China (No.
Rowinsky, E.K., Donehower, R.C., 1995. Paclitaxel (Taxol). N.Engl. J. Med. 332 (15),
1004–1014.
Salimuzzaman, S., Obaid, Y.K., Shaheen, F., Bina, Shaheen, S., 1988. Studies in the
chemical constituents of the seeds of Peganum harmala: isolation and structure
8
1173119), the Key Project of Ministry of Science and Technology of the
elucidation of two β-carboline lactams - harmalanine and harmalacidine.
Heterocycles 27 (6), 1401–1410.
People's Republic of China (2012ZX09103201-051), and the Program of
Shanghai Subject Chief Scientist (13XD1403500) awarded to Professor
Chang-hong Wang for financial support of this study.
Schuster, D., Spetea, M., Music, M., Rief, S., Fink, M., Kirchmair, J., Schütz, J., Wolber,
J., Wolber, G., Langer, T., Stuppner, H., Schmidhammer, H., Rollinger, J.M., 2010.
Morphinans and isoquinolines: acetylcholinesterase inhibition, pharmacophore
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Appendix A. Supporting information
Rumjanek, V.M., 2012. Structural insights into cholinesterases inhibition by
harmane β-carboliniumderivatives: a kinetics – molecular modeling approach.
Phytochemistry 81, 24–30.
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2015.03.070.
Wang, C.H., 2002. The study on harmine hydrochloride capsule [D]. Xinjiang
Medical University, pp. 126–130.
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of harmine and harmaline of Peganum harmala in different parts and different
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