Journal of Ethnopharmacology 162 (2015) 7986

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

In vivo evaluation of the antitussive, expectorant and bronchodilating

effects of extract and fractions from aerial parts of Peganum
harmala linn
Wei Liu a, Xuemei Cheng a,b, Yongli Wang a,b, Shuping Li a, Tianhui Zheng a, Yingying Gao a,
Guofeng Wang a, Shenglan Qi a, Jingxin Wang a, Jiayi Ni a, Zhengtao Wang a,b,
Changhong Wang a,b,n
a
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, The MOE Key Laboratory for Standardization of Chinese
Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, 1200 Cailun Rood, Shanghai 201203, China
b
Shanghai R&D Centre for Standardization of Chinese Medicines, 199 Guoshoujing Road, Shanghai 201210, China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Aerial parts of Peganum harmala Linn (APP) is used as traditional
Received 15 June 2014 medical herb in Uighur medicine in China, and it is traditionally used for treatment of cough and asthma.
Received in revised form The aim of the present study is to evaluate the antitussive, expectorant and bronchodilating effects of
2 December 2014
extract and fractions (alkaloids and avonoids) from APP, and to support its folk use with scientic
Accepted 23 December 2014
evidence, and lay a foundation for its further researches.
Available online 31 December 2014
Materials and methods: APP was extracted with 50% ethanol by reux, extracts were concentrated in
Keywords: vacuum to afford total extract of APP (EXT). EXT was separated to provide alkaloid fraction (ALK) and
Peganum harmala L. avonoid fraction (FLA) by macroporous resin. Antitussive evaluations were carried out with cough
Antitussive
models in mice and guinea pigs induced by ammonia liquor, capsaicin, and citric acid. Phenol red
Expectorant
secretion experiments in mice were performed to evaluate the expectorant activity. Bronchodilating
Bronchodilating
Alkaloids activities were evaluated with a bronchoconstrictive challenge induced by acetylcholine chloride and
Flavonoids histamine in guinea pigs.
Results: In all the three antitussive tests, the EXT and ALK could signicantly inhibit the frequency of cough,
and prolong the cough latent period in animals. High dose of EXT (1650 mg/kg) and ALK (90 mg/kg) in mice
and in guinea pigs created therapeutic activities as good as that of codeine phosphate (30 mg/kg). EXT could
signicantly increase phenol red secretion in mice for 0.64, 1.08 and 1.29 fold averagely at dosages of 183,
550, and 1650 mg/kg, ALK for 0.63, 0.96, 1.06 fold averagely at dosages of 10, 30, and 90 mg/kg, and
ammonium chloride (1500 mg/kg, standard expectorant drug) for 0.97 fold, comparing with control group.
Aminophylline could dramatically prolong the preconvulsive time for 162.28% in guinea pigs, EXT for 67.34%,
101.96% and 138.00% at dosages of 183, 550, and 1650 mg/kg, ALK for 55.47%, 97.74% and 126.77% at dosages
of 10, 30, and 90 mg/kg, and FLA for 84.69%, 95.94% and 154.52% at dosages of 10, 30, and 90 mg/kg,
comparing with pretreatment.
Conclusions: APP is an effective traditional folk medicine for the treatment of cough with potent antitussive,
expectorant and bronchodilating activities. The alkaloid fraction is proved to be the most effective
components of APP. The alkaloids from APP may be valuable lead compounds for drug development of
respiratory diseases.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Coughing is one of common symptoms associated with many
respiratory diseases such as asthma, chronic bronchitis, pneumonia
n
Corresponding author at: The Institute of Traditional Chinese Medicine,
Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Tel.: 86 21 51322511; fax: 86 21 51322519.
E-mail address: wchcxm@hotmail.com (C. Wang).
and so on (Irwing and Madison, 2000; Ge et al., 2009). At present, the
antitussives, expectorants, mucolytics, bronchodilators, and glucocor-
ticoids can usually be used to treat cough (Prez et al., 2008).
However, available therapies to treat cough are limited for lack of
effective and safe medications and coughing remains among the
most common complaints for which patients seek medical attention
(Zhang et al., 2009; Wang et al., 2012). In traditional Chinese
medicines, many medicine herbs are used for hundreds of years to
treat respiratory complaints such as cough, asthma, expectoration,

http://dx.doi.org/10.1016/j.jep.2014.12.046
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
80 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986
bronchial inammatory, and pneumonia, and have shown less or no
side effects as being compared to synthetic drugs (Jiangsu New
Medical College, 1977; Shang et al., 2010). However, they still cannot
be accepted by most advanced countries as therapeutic agents,
although many of todays new drugs come directly or indirectly
from traditional medicines. A major reason is lack of chemical and
pharmacological investigation on them (Newman and Cragg, 2007;
Shang et al., 2010). So it is worthy to search for effective medicines
from traditional medicines for treatment of cough (Akah et al., 2003;
Chu et al., 2007; Yang et al., 2008).
Peganum harmala Linn (Zygophyllaceae) grows spontaneously
in the arid and semiarid areas north-west China, and also
distributed in North Africa and the Middle East (Farouk et al.,
2008; Cheng et al., 2010). The seeds and aerial parts of Peganum
harmala have been commonly used as traditional folk medicine to
treat various ailments, including cough, asthma, rheumatism,
hypertension, diabetes and jaundice in the Xinjiang Uygur and
Mongolian Autonomous Regions of China for a long time (Chinese
Pharmacopoeia Committee, 1998; Zheng et al., 2009). It is also a
well-known and effective herbal medicine in Turkey, Iran, Algeria
and Morocco (Kartal et al., 2003; Hemmateenejad et al., 2006;
Farouk et al., 2008; Bensalem et al., 2014). Peganum harmala is
used as an effective herb to treat cough and asthma in the folk
medicine, there are only a few of preliminary studies on the seeds
of Peganum harmala in the past years (Hider et al., 1981; Nie et al.,
2004). But, rare investigation has been conducted on the aerial
parts of Peganum harmala (APP). Therefore, a series of experiments
are designed to evaluate the antitussive, expectorant and bronch-
odilating effects of the extract and two mainly fractions (alkaloids
and avonoids) from APP. The aim is to conrm its traditional
function of APP and to provide scientic evidence for the discovery
of new antitussive, expectorant; and bronchodilating drugs
from APP.
2. Materials and methods
2.1. Reagents
Codeine phosphate, phenol red, ammonium chloride and ammo-
nia liquor were purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). Capsaicin, aminophylline, acetylcholine (ACh)
chloride, histamine phosphate were purchased from Sigma Aldrich
Co. (St. Louis, MO, USA). The standard compounds of vasicine,
harmaline, harmine, deoxypeganetin, peganetin were isolated pre-
viously from the APP in our laboratory and characterized by NMR
and mass spectral data and comparison with literature values. The
purities of these compounds were determined to be more than 98%
by HPLC analysis.
2.2. Collection and preparation of plant material
APP was collected in Urumqi, Xinjiang Uygur Autonomous Region,
China in August 2011 and authenticated by Professor Changhong
Wang, the Institute of Chinese Materia Medica, Shanghai University
of Traditional Chinese Medicine. Fresh herbs were dried in shade for
a week. The voucher specimens (Voucher number: PH-XJ1104) were
deposited at the Herbarium of Shanghai R&D Center for Standardiza-
tion of Traditional Chinese, Shanghai, China.
Dried APP (2500 g) was sheared into segments, extracted with
50L of 50% ethanol (v/v) thrice in reux conditions by 100L-
extraction tank, each for 2 h. Extracts was combined, ltered, and
concentrated under reduced pressure at 45 1C to afford 10L
concentrated extract of APP. A portion of the concentrated extract
(3L) was dessicated in vacuum to afford extract of APP (EXT,
187.5 g). And the other concentrated extract (7 l) was separated
and prepared alkaloid fraction (ALK) and avonoid fraction (FLA)
by macroporous resin column chromatography (15  150 cm; 4 l),
being eluted with a gradient system of waterethanol (100:0,
80:20, 20:80). Finally, three different fractions (water, 20% ethanol,
80% ethanol) of the eluted solutions were concentrated under
reduced pressure at 45 1C and dessicated in vacuum. The water
fraction (388.1 g) mainly contains polysaccharides, hydrosoluble
pigment, tannin and so on (the fraction may not be the active part
of APP, and no follow studies), the 20% ethanol fraction (35.6 g)
mainly contains alkaloids, and the 80% ethanol fraction (34.5 g)
mainly contains avonoids. The contents of alkaloids (vasicine,
harmaline and harmine) and avonoids (deacetylpeganetin and
peganetin) of prepared EXT, ALK and FLA were determined by
previous method (Wen et al., 2014).
2.3. Animals and drug administration
All animals, including ICR mice of either sex (body weight 2025 g)
for ammonia or capsaicin induced mice cough studies, ICR mice of
either sex (body weight 2025 g) for phenol red secretion study, and
guinea pigs of either sex (body weight 200300 g) for citric acid
induced guinea pig cough experiment and bronchodilating tests were
purchased from Experimental Animal Center, Shanghai University of
Traditional Chinese Medicine, China.
All animals were housed with free access to food and water.
The animals were maintained on a 12 h lightdark cycles (light on
from 7:00 to 19:00) at environmental temperature (2224 1C) and
6065% relative humidity for 7 days. Before the experiments, all
animals were fasted for 12 h and fed with water. All animal
experimental protocols were in accordance with the regulations
of experimental animal administration issued by the State Com-
mittee of Science and Technology of Peoples Republic of China on
November 14th, 1988.
Indicated doses of extract (EXT) and fractions (ALK and FLA)
were suspended and diluted with 0.5% carboxylmethylcellulose
(CMC-Na) solution and administrated orally. The animal dose was
extrapolated from human daily dose by a simple conversion based
on body weight. And these doses did not show any toxicity
properties by a preliminary experiment (Data not show). CMC-
Na solution (0.5%) was taken as control.
2.4. Antitussive activity against ammonia induced cough in mice
The test of ammonia liquor induced cough in mice was carried
out as described before (Wang et al., 2012) with slight modication.
Mice (110) were numbered and individually placed into a 500 ml
special glass chamber which was placed upside down, and exposed
to 0.1 ml 25% NH4OH (loaded in a glass plate, diameter 28 mm,
height 10 mm) for 45 s. The latent period (the period from the start
to the onset of cough) and the frequency of cough within 2 min
were recorded by a trained observer. During observation, only
typical cough reection, characterized by obvious contraction of
the abdominal cavity and successively distinctive opening of mouth
was counted. Then, the latent period (the period from the start to
the onset of cough) and the frequency of cough within 2 min were
recorded before treatment. After 24 h recovery, these mice were
divided into 11 groups of 10 each randomly. Animals in Group
1 were administrated with 0.5% CMC-Na, animals in group 2
(positive control) were received 30 mg/kg of codeine phosphate,
animals in groups 35 were treated with 183.3, 550, 1650 mg/kg of
EXT, animals in groups 68 were treated with 10, 30, 90 mg/kg of
ALK, and animals in groups 911 were treated with 10, 30, 90 mg/kg
of FLA. One hour after administration, mice were exposed to
ammonia again as described above. The latent period and the
frequency of cough before and after treatment were compared.
 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986
2.5. Antitussive activity against capsaicin induced cough in mice
The test of capsaicin induced cough in mice was performed as
described before (Zhang et al., 2009) with slight modication.
Briey, mice (110) were placed individually into a 500 ml special
glass chamber and sprayed with the nebulized capsaicin solution
(100 mol/l) for 10 s, the latent period and the frequency of cough
within 2 min were recorded. After 24 h recovery, these mice were
divided into 11 groups of 10 each randomly. Animals in group
1 were administrated with 0.5% CMC-Na, animals in group 2
(positive control) were received 30 mg/kg of codeine phosphate,
animals in groups 35 were treated with 183.3, 550, 1650 mg/kg of
EXT, animals in groups 68 were treated with 10, 30, 90 mg/kg of
ALK, and animals in groups 911 were treated with 10, 30, 90 mg/kg
of FLA. One hour after administration, mice were exposed to
capsaicin again as described above. The latent period and the
frequency of cough before and after treatment were compared
2.6. Antitussive activity against citric acid induced cough in guinea
pigs
The test of citric acid induced cough in guinea pigs was carried
out as previously reported (Ge et al., 2009) with slight modica-
tion. Guinea pigs (110) were placed individually in a 3L transparent
chamber, and then sprayed with 33% citric acid solution for 1 min,
the period of the rst cough since spraying (latent period) and the
frequency of cough within 5 min were recorded. Only animals
with a cough frequency between 8 and 30 were kept for the next
round of evaluation. After 24 h recuperation, these guinea pigs
were divided into 11 groups of 10 each randomly. Animals in group
1 were administrated with 0.5% CMC-Na, animals in group 2
(positive control) were received 30 mg/kg of codeine phosphate,
animals in groups 35 animals were treated with 183.3, 550,
1650 mg/kg of EXT, animals in groups 68 were treated with 10,
30, 90 mg/kg of ALK, and animals in groups 911 were treated
with 10, 30, 90 mg/kg of FLA. The second round of antitussive
challenge was performed just 1 h after administration, and all the
protocols were strictly the same with the rst round. The latent
period and the frequency of cough before and after treatment
were compared.
2.7. Expectorant activity
Phenol red secretion experiments were carried out as described
(Han et al., 2010) to evaluate the expectorant activity of extract and
fractions of APP. Mice were divided into 11 groups of 10 each
randomly. Animals in group 1 were administrated with 0.5% CMC-
Na, animals in group 2 (positive control) were received 1500 mg/kg
of ammonium chloride, animals in groups 35 were treated with
183.3, 550, 1650 mg/kg of EXT, animals in groups 68 were treated
with 10, 30, 90 mg/kg of ALK, and animals in groups 911 were
treated with 10, 30, 90 mg/kg of FLA. After administration 30 min
later, mice were injected intraperitoneally with 5% phenol red
physiologic saline solution (500 mg/kg). Another 30 min later, mice
were sacriced without damaging the trachea. The trachea between
the thyroid cartilage and the main stem bronchi was removed and
put into 1.5 ml of physiologic saline solution. The solution was
vibrated for 30 min to resolve the phenol red. Then the solution was
removed 100 l in a 96-well plate, and put into 100 l of 0.1 M
NaOH. Optical density of the mixture was measured immediately at
546 nm on a microplate reader (Power wave XS, Bio-Tek Instru-
ments, Winooski, VT, USA). The amount of phenol red was deter-
mined by the regression curve.
81
2.8. Bronchodilating activity
To evaluate the bronchodilating activity of extract and fractions
of APP, a bronchoconstrictive challenge induced by acetylcholine
chloride and histamine in guinea pigs was conducted (Xu et al.,
1991). Briey, guinea pigs (200300 g body weight) of either sex
were placed individually into a 3L glass chamber, and sprayed with
mixture of 2% acetylcholine chloride and 0.1% histamine (1:1, v/v)
for 20 s. The time from the spraying of solution to the onset of
tumble was recorded (preconvulsive time). Animals with precon-
vulsive time between 30 and 120 s were considered as eligible.
After 24 h recovery, eligible animals (110) were divided randomly
into 11 groups, Animals in group 1 were administrated with 0.5%
CMC-Na, animals in group 2 (positive control) were received
50 mg/kg of aminophylline, animals in groups 35 were treated
with 183.3, 550, 1650 mg/kg of EXT, animals in groups 68 animals
were treated with 10, 30, 90 mg/kg of ALK, and animals in groups
911 were treated with 10, 30, 90 mg/kg of FLA. One hour after
administration, guinea pigs were subjected to bronchoconstrictive
challenge as preformed previously.
2.9. Statistical analysis
Data were expressed as mean 7standard error (S.E.M). Paired
samples t test were used to evaluate the differences between
results of before and after drug administration in the same groups.
Statistical signicances of differences between different treatment
groups were assessed by one-way analysis of variance (ANOVA).
All calculations were conducted in software of SPSS 18.0. A level of
Po 0.05 was taken as statistically signicant.
3. Results
3.1. Preparation of extract and different fractions of APP
The solution of concentrated extract was separated and pre-
pared ALK and FLA by macroporous resin column chromatography.
The chromatogram of different fractions determined by HPLC was
shown in Fig. 1. The contents of index components, vasicine,
harmaline, harmine, deacetylpeganetin and peganetin, were deter-
mined of 2.58%, 0.02%, 0.07%, 0.90% and 1.25% in EXT, respectively
by HPLC using reported method (Wen et al., 2014). The content of
vasicine is 52.47% in ALK and the contents of deacetylpeganetin
and peganetin are 17.00% and 23.45% in FLA. In addition, the
contents of harmaline and harmine (beta-carboline alkaliods) are
0.33% and 1.20% in FLA, respectively.
3.2. Antitussive effects
In order to evaluate the antitussive effects of APP extract and
different fractions, three different animal models including mice
cough models induced by ammonia and capsaicin as well as
guinea pig cough model induced by citric acid are adopted.
The effects of different extract and fractions from APP on
ammonia induced cough in mice are shown in Fig. 2. The
frequency of cough within 2 min are decreased by 37.09%, 37.49%
and 49.88% by treatments with EXT at dosages of 183.3, 550, and
1650 mg/kg (P o0.05), and by 31.74%, 39.19% and 41.72% (P o0.05)
by treatments with ALK at dosages of 10, 30, and 90 mg/kg,
respectively, compared with that of before drug administration.
However, FLA have not been observed obviously decrease in the
frequency of cough within 2 min (P 40.05) at dosages of 10, 30,
and 90 mg/kg. Given the fact that the frequency of cough is
decreased for 38.11% by treatment with codeine phosphate
(30 mg/kg) in same condition, it could be concluded that ALK
82 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986

Fig. 1. The typical HPLC chromatograms of extract of aerial parts of Peganum harmala (A), the water fraction of aerial parts of Peganum harmala (B), 20% ethanol fraction
(alkaloids fraction) of aerial parts of Peganum harmala (C), 80% ethanol fraction (avonoids fraction) of aerial parts of Peganum harmala (D) and reference compounds (E,
vasicine, harmaline, harmine, deacetylpeganetin and peganetin).
 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986 83

and EXT are more effective than that of codeine phosphate at
middle and high dosages. In addition, compared with the cough
frequency of control mice, all of EXT and ALK administrations
groups show signicantly decrease in cough frequency (Fig. 2A,
P o0.05). The latent periods are signicantly prolonged for 36.93%,
54.14% and 77.29% by treatments with EXT at dosages of 183.3,
550, and 1650 mg/kg (P o0.05), and for 37.09%, 72.11% and 72.58%
by treatments with ALK at dosages of 10, 30, and 90 mg/kg
(P o0.05), compared with data before administration. The latent
period is only prolonged for 41.77% by codeine phosphate treat-
ment at dosage of 30 mg/kg. In addition, all mice treated with EXT
and ALK exhibit signicantly differences compared with control
mice (Fig. 2B).
The effects of different extract and fractions from APP in mice
challenged with capsaicin are given in Fig. 3. The frequency of
cough within 2 min have been strongly decreased for 16.38%,
30.38% and 49.36% (Po0.05) by treatments with EXT at dosages
of 183.3, 550, and 1650 mg/kg, for 26.64%, 36.70% and 52.93%
(Po0.05) by treatments with ALK at dosages of 10, 30, and
90 mg/kg, respectively, compared with that of before drug admin-
istration. However, the frequency of cough within 2 min is not
obviously decreased (P40.05) by treatments with FLA at dosages of
10, 30, and 90 mg/kg, compared with that of before drug adminis-
tration. Given the fact that codeine phosphate (30 mg/kg) is only
decreased of frequency of cough for 47.84%, it can be concluded that
EXT and ALK are more effective than codeine phosphate at high
dosage. In addition, compared with the cough frequency of control
group, the cough frequency in all the EXT and ALK treatment groups
are signicantly decreased (Po0.05, Fig. 3A). Also, the latent period
are signicantly prolonged for 34.90%, 55.69% and 90.36% by EXT at
dosages of 183.3, 550, and 1650 mg/kg (Po0.05), for 35.87%, 36.60%
and 78.91% by ALK at dosages of 10, 30, and 90 mg/kg (Po0.05),
compared with data before administration. However, the latent
period is only prolonged 82.96% by codeine phosphate (30 mg/kg).
In addition, all mice treated by EXT and ALK have signicantly
differences compared with control mice (Po0.05, Fig. 3).
The antitussive effects of different extract and fractions from
APP in guinea pigs are shown in Fig. 4. EXT and ALK have been
demonstrated strong inhibition effects on cough induced by citric
acid. Compared with that of before drug administration, cough
frequency within 5 min have been decreased for 11.94%, 26.84%,
72.97% and 25.02%, 45.86%, 78.75% after treatment by EXT at 183.3,

Fig. 2. Effects of extract (EXT), alkaloids fraction (ALK) and avonoids fraction (FLA) of aerial parts of Peganum harmala (APP) administration on the frequency of cough
within 2 min (A) and latent period of cough (B) in ammonia liquor induced mice cough. Control mice were treated with 0.5% CMC-Na, codeine phosphate (30 mg/kg) was
taken as positive control. Values are expressed as mean 7 S.E.M. (n 10). nPo 0.05, nn P o 0:01; nnnP o 0.001, compared with data of negative control (0.5% CMC-Na) after drug
administration. #Po 0.05, ##Po 0.01, ###Po 0.001, compared with data before drug administration.

Fig. 3. Effects of extract (EXT), alkaloids fraction (ALK) and avonoids fraction (FLA) of aerial parts of Peganum harmala (APP) administration on the frequency of cough
within 2 min (A) and latent period of cough (B) in capsaicin induced mice cough. Control mice were treated with 0.5% CMC-Na, codeine phosphate (30 mg/kg) was taken as
positive control. Values are expressed as mean 7S.E.M. (n10). nPo 0.05, nnPo 0.01, nnnP o 0.001, compared with data of negative control (0.5% CMC-Na) after drug
administration. #Po 0.05, ##Po 0.01, ###Po 0.001, compared with data before drug administration.
84 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986

Fig. 4. Effects of extract (EXT), alkaloids fraction (ALK) and avonoids fraction (FLA) of aerial parts of Peganum harmala (APP) administration on the frequency of cough
within 5 min (A) and latent period of cough (B) in citric acid induced guinea pigs cough. Control mice were treated with 0.5% CMC-Na, codeine phosphate (30 mg/kg) was
taken as positive control. Values are expressed as mean 7 S.E.M. (n 10). nPo 0.05, nnP o0.01, nnnP o 0.001, compared with data of negative control (0.5% CMC-Na) after drug
administration. #Po 0.05, ##Po 0.01, ###Po 0.001, compared with data before drug administration.

550, 1650 mg/kg and by ALK at 10, 30, 90 mg/kg (Po 0.05). It
shows that the effect of EXT at dose of 1650 mg/kg is even better
than that of codeine phosphate (75.47%) (Fig. 4A). In addition,
compared with the control group, the cough frequency is signi-
cantly decreased by treatment with EXT and ALK administrations
(P o0.05). Meanwhile, the latent period is prolonged for 20.69%,
43.13% and 140.66% by treatments with EXT at dosages of 183.3,
550, and 1650 mg/kg (P o0.05), and for 41.66%, 116.85% and
238.74% by treatments with ALK at dosages of 10, 30, and
90 mg/kg (Po0.05), compared with data before administration.
However, the latent period is prolonged for 119.82% after treat-
ment by codeine phosphate (30 mg/kg). It indicates that the
antitussive effects of EXT and ALK at high dose are much better
than that of codeine phosphate, especially the high ALK dose
group. In addition, the latent period is signicantly prolonged after
middle and high dose EXT and ALK treatments (P o0.05, Fig. 4B),
compared with control mice.
Fig. 5. Effects of extract (EXT), alkaloids fraction (ALK) and avonoids fraction (FLA)
of aerial parts of Peganum harmala (APP) treatment on the amount of phenol red
3.3. Expectorant activity secretion in mice. Values are expressed as mean 7 S.E.M. (n10). Control mice
were treated with 0.5% CMC-Na, NH4Cl (1500 mg/kg) was taken as positive
As showing in Fig. 5, the expectorant activities are studied with control.nPo 0.05, nnPo 0.01, nnnPo 0.001, compared with data of negative control
the method of measuring the amount of phenol red secretion in (0.5% CMC-Na) after drug administration.

mice tracheas. The amount of phenol red secretion is signicantly

increased in mice for 0.64, 1.08 and 1.29 fold averagely by
treatment with EXT at dosages of 183.3, 550, and 1650 mg/kg
(P o0.05), and for 0.63, 0.96, 1.06 fold by treatment with ALK at
dosages of 10, 30, and 90 mg/kg (P o0.05), compared with control
group. The amount of phenol red secretion is increased for 0.97
fold averagely by treatment with standard expectorant drug
ammonium chloride (1500 mg/kg), compared with control group.
However, the amount of phenol red secretion is not inuenced by
treatment with FLA at any dosages (P 40.05). The expectorant
activities created by 1650 mg/kg of EXT and 90 mg/kg of ALK are
even better than that of ammonium chloride at the dosages of
1500 mg/kg. It indicates that the expectorant effects of EXT and
ALK from APP are quite effective.
3.4. Bronchodilating activity
The bronchodilating effects of different extract and fractions from
APP in guinea pigs are shown in Fig. 6. Aminophylline can drama-
tically prolong the preconvulsive time in guinea pigs for 162.28%. All
of EXT, ALK, even FLA show signicantly and dose-dependently
bronchodilating effects by prolonging the preconvulsive time in
guinea pigs. It is notable that FLA has not shown antitussive and
expectorant activity. The preconvulsive time is prolonged for 67.34%,
101.96% and 138.00% after treatment by EXT at dosages of 183.3, 550,
and 1650 mg/kg, for 55.47%, 97.74% and 126.77% after treatment by
ALK at dosages of 10, 30, and 90 mg/kg, and for 84.69%, 95.94% and
154.52% after treatment by FLA at dosages of 10, 30, and 90 mg/kg,
compared with data before administration.
4. Discussion and conclusions
Peganum harmala, a perennial plant in family Zygophyllaceae,
mainly grows in Xinjiang Uygur Autonomous Region, Ningxia Hui
Autonomous Region, Inner Mongolia Autonomous Region, Tibet
Autonomous Region, Gansu Province, and other places in China. It
has been historically used in Uighur medicine to treat chronic
respiratory diseases for several hundred years. The traditional
knowledge led us to evaluate antitussive, expectorant and bronch-
odilating effects of APP. In present study, the ethanol extract from
APP was separated into alkaloids and avonoids fractions by
macroporous resin. Through the pharmacological evaluation on
 W. Liu et al. / Journal of Ethnopharmacology 162 (2015) 7986
Fig. 6. Effects of extract (EXT), alkaloids fraction (ALK) and avonoids fraction (FLA)
of aerial parts of Peganum harmala (APP) administration on the preconvulsive time
of guinea pigs challenged with the solution mixture of 2% acetylcholine chloride
and 0.1% histamine. Control guinea pigs were treated with 0.5% CMC-Na, amino-
phylline (50 mg/kg) was taken as positive control. Values are expressed as
mean 7 S.E.M. (n 10). nP o0.05, nnPo 0.01, nnnPo 0.001, compared with data of
negative control (0.5% CMC-Na) after drug administration. #Po 0.05, ##Po 0.01,
###
P o0.001, compared with data before drug administration.
ammonia and capsaicin induced mice coughing, citric acid induced
guinea pigs coughing, and phenol red secretion experiments in
mice, ALK and EXT but FLA have shown signicant antitussive and
expectorant activity (Figs. 25). Therefore, the alkaloids are to be
believed as the predominant active fractions of APP for treatment
cough. In addition, FLA, ALK and EXT have signicantly prolonged
the preconvulsive time in acetylcholine chloride and histamine
induced guinea pigs bronchoconstrictive challenge model. The ALK
and FLA in APP may be play a common effect on bronchodilating
activities (Fig. 6).
Previous studies have proved that the quinazoline and beta-
carboline alkaloids and avonoids are the main chemical ingredients
in APP (Zhao et al., 2010; Wen et al., 2014). The main quinazoline
alkaloids are vasicine, deoxyvasicine and their analogs, and the main
beta-carboline alkaloids are harmine and harmaline. The majority of
avonoids separated from Peganum harmala are deacetylpeganetin
and peganetin, as conrmed by our previous works and literature
(Duan et al., 1998, Wen et al., 2014). It has been reported that the
alkaloids mainly hamaline, harmine and vasicine from seeds of
Peganum harmala could inhibit the contraction of isolated guinea-
pig tracheal smooth muscle induced by acetylcholine, KCl and
histamine in vitro and show potential bronchodilatory activity
(Hider et al., 1981; Nie et al., 2004). Vasicine, harmaline, harmine
and other alkaloids from Peganum harmala are strong cholinesterases
inhibitors, and they could elevate the acetylcholine level by inhibiting
cholinesterases in vivo (Zheng et al., 2009, 2011; Zhao et al., 2011,
2013). Acetylcholine plays an important role both in the peripheral
nervous system (PNS) and in the central nervous system (CNS) as a
neuromodulator. In the PNS, acetylcholine is a major neurotransmit-
ter in the autonomic nervous system and can increase glandular
secretion. ALK shows more antitussue, expectorant and bronchodi-
lating activities than that of EXT and FLA. It is noticeable that FLA
shows some bronchodilating activity, this may be related to the main
constituents (deacetylpeganetin and peganetin) or more likely rele-
vant to the effects of the residual alkaloids (harmaline and harmine)
(Hider et al., 1981; Nie et al., 2004) in FLA (Fig. 1). It is needed to
investigate further by other experiment design. To the best of our
knowledge, the antitussive and expectorant effects of the main
alkaloid compounds in APP or in seeds of Peganum harmala are not
been investigated. Thus, further investigation needs to be conducted.
85
In conclusion, the results indicate that EXT and ALK of APP have
potent antitussive, expectorant and bronchodilating activities that
make them effective alternative remedy for the treatment of
respiratory diseases. The mechanism for the observed activities
has not been established and it is very urgent to be investigated
further.
Acknowledgment
The authors gratefully acknowledge the award from the Key
Projects of Joint Funds of the National Natural Science Foundation
of China and Xinjiang Uygur Autonomous Region of China (no.
U1130303), the National Natural Science Foundation of China
(Grant no. 81173119), the National Science & Technology Major
Project Key New Drug Creation and Manufacturing Program,
China (Grant nos. 2012ZX0910320-051), and the Program of
Shanghai Subject Chief Scientist (13XD1403500) awarded to Pro-
fessor Chang-hong Wang for nancial support of this study.
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