Journal of Ethnopharmacology 139 (2012) 189–193

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

Antitussive, expectorant and anti-inflammatory activities of four alkaloids

isolated from Bulbus of Fritillaria wabuensis
Dongdong Wang a , Shu Wang a,∗ , Xiong Chen a , Xiaolong Xu a , Jingyi Zhu b , Lihuan Nie b , Xia Long a
a
Department of Pharmacogonosy, West China College of Pharmacy, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, PR China
b
West China School of Stomatology, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Bulbus Fritillaria Cirrhosae (BFC), known by the Chinese name “Chuan-
Received 18 May 2011 Bei-Mu”, is used as an antitussive, antiasthmatic and expectorant drug for more than 2000 years in
Received in revised form 22 October 2011 China, and Bulbus of Fritillaria wabuensis S. Y. Tang & S. C. Yueh (BFW) was recorded in the 2010 edition
Accepted 31 October 2011
of China Pharmacopoeia as one of sources for BFC. The primary objective of this study was to evaluate the
Available online 10 November 2011
antitussive, expectorant and anti-inflammatory effects of alkaloids – imperialine, imperialine-␤-N-oxide,
isoverticine, and isoverticine-␤-N-oxide, which were isolated from BFW, and to provide experimental
Keywords:
evidence for its traditional use.
Antitussive
Expectorant
Materials and methods: The alkaloids were isolated using phytochemical methods, and their structures
Anti-inflammatory were determined by spectroscopic analysis. Their antitussive effects were measured using murine model
Alkaloid of ammonia induced cough, the expectorant effects were evaluated by measuring mice’s tracheal phenol
Bulbus Fritillaria Cirrhosae red output, and the anti-inflammatory effects were assessed by using the murine model of xylene induced
Fritillaria wabuensis ear edema.
Results: The structures of the four alkaloids – imperialine, imperialine-␤-N-oxide, isoverticine, and
isoverticine-␤-N-oxide isolated from BFW were confirmed. The four alkaloids significantly inhibited
cough frequency and increased latent period of cough in mice induced by ammonia. Imperialine and
isoverticine showed obviously antitussive activities in a dose-dependent manner. Besides, the four alka-
loids markedly enhanced mice’s tracheal phenol red output in expectorant assessment and significantly
inhibited the development of ear edema in anti-inflammatory evaluation assay. Moreover, significant
differences were found between the structure–activity relationships of the four alkaloids.
Conclusions: The four alkaloids exhibited significant antitussive, expectorant and anti-inflammatory activ-
ities. We suggest that they may be the active ingredients of BFW. The results of the present study provided
evidence for BFW to be used as an antitussive and expectorant Traditional Chinese Medicine (TCM).
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Coughing is one of common symptoms associated with many
respiratory diseases such as asthma, chronic bronchitis, pneumo-
nia and so on (Irwing and Madison, 2000; Ge et al., 2009). Presently
cough can be regulated by usual drugs such as codeine but real
treatment concerns the pathology that induces cough. The prob-
lem is not to only use an efficient therapy against coughing, which
will inevitably bring side effects. Therefore, there is an increasing
demand for searching medicine in the therapy of cough and its
pathology. In Traditional Chinese Medicine (TCM), many medicine
herbs are used for hundreds of years to treat respiratory com-
plaints such as bronchial inflammatory, pneumonia, expectoration
∗ Corresponding author. Tel.: +86 28 85503950; fax: +86 28 85503950.
E-mail address: wddong1988@hotmail.com (S. Wang).
and cough, and have shown less or no side effects as compared to
synthetic drugs (Jiangsu New Medical College, 1977; Shang et al.,
2010). So it is valuable to search for effective medicines for treat-
ment of cough in the rich resources of TCM (Akah et al., 2003; Chu
et al., 2007; Yang et al., 2008).
Bulbus Fritillaria Cirrhosae (BFC) can be derived from six plants –
Fritillaria cirrhosa, Fritillaria wabuensis, Fritillaria unibracteata, Frit-
illaria przewalskii, Fritillaria delavayi and Fritillaria taipaiensis. The
Bulbus of Fritillaria wabuensis S. Y. Tang & S. C. Yueh (BFW) is
one of the sources for BFC. It is powdered, then taken orally at
3–10 g/day, 1–2 g/time to treat cough in traditional medicine. BFW
has been widely used in clinic to treat cough and asthma for a
long time in China due to its positive therapeutic effects and few
side effects. Nowadays, it has been recorded in the Pharmacopoeia
of the People’s Republic of China (2010). The pharmacological
studies demonstrated that the crude alkaloid extracts of BFC/BFW
had remarkable antitussive, expectorant and antiasthmatic

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.10.036
190 D. Wang et al. / Journal of Ethnopharmacology 139 (2012) 189–193
activities (Mo et al., 1998; Yan, 2005; Chen, 2008). In addition, the
prior in vitro studies suggested that verticinone, imperialine and
imperialine-3-␤-glucoside, 3␤-acetylimperialine and sinpeinine A
may have effective anti-asthmatic activities (Zhang et al., 2003;
Zhou et al., 2003; Lin et al., 2006). Alkaloids were found to be the
major biologically active ingredients in genus of Fritillaria.
BFW was considered as a valuable species in genus of Fritil-
laria, however, further pharmacological studies on monomers of
alkaloids isolated from BFW in vivo were absent. So we isolated
alkaloids – imperialine, imperialine-␤-N-oxide, isoverticine, and
isoverticine-␤-N-oxide from BFW and evaluated their antitussive,
expectorant, and anti-inflammatory effects in mice.
2. Materials and methods
2.1. Materials
BFW was collected by D. Wang in April 2010 in Songpan County
(Sichuan Province, China) and identified by Dr. Wang (Professor
in Department of Pharmacogonosy, West China College of Phar-
macy, Sichuan University). A voucher specimen (Wang 20100420)
has been deposited in the Herbarium of West China College of Phar-
macy, Sichuan University.
2.2. Extraction and isolation
Dried bulbs (9.5 kg) of BFW were powdered and immersed
in ammonia (1000 ml) for 24 h, and then extracted thrice with
CHCl3 –MeOH (4:1) solvent to obtain crude extracts (35 g). The con-
centrated extracts were dissolved three times in 200 ml of 3% HCl
and partitioned twice with 200 ml CHCl3 . The pH of the aque-
ous solution was readjusted with NH4 OH to 10.0 and extracted
seven times with 100 ml CHCl3 . The CHCl3 extracts (crude alkaloid,
8.5 g) were purified repeatedly by silica gel column chromatogra-
phy using petroleum ether–acetone–diethylamine (6:1:1–1:1:1) of
increasing polarity as eluent as reported (Chen, 2005; Jiang et al.,
2005). 20-ml fractions were collected in the elution and pooled
into groups according to the composition, as visualized by Thin
Layer Chromatography (TLC). The alkaloids isolated from BFW were
determined by comparing the samples’ melting point (m.p.) and
spectral data (1 H, 13 C NMR spectra) with those reported in liter-
ature, and by direct comparison of TLC with authentic reference
compounds.
2.3. Animal and drug administration
Kunming mice of either sex (18–22 g) were purchased from
Experimental Animal Center (West China College of Pharmacy,
Sichuan University, Chengdu, China). All mice were housed at
room temperature (22–24 ◦ C) and constant humidity (40–70%)
in laboratory, and provided food and water ad libitum. All ani-
mal experiments were conducted according to the “Regulation for
the Administration of Affairs Concerning Experimental Animals”
(State Science and Technology Commission in China, 1988), the
international rules considering animal experiments and the inter-
nationally accepted ethical principles for laboratory animal use
and care. The procedures were approved by the Sichuan University
Committee on Animal Care and Use.
After 3–5 days of adaptation, the eligible animals were
randomly divided into six groups of 10 mice each, includ-
ing group 1 (control) (0.5% carboxyl methylcellulose solution,
0.2 ml/20 g body weight), group 2 (the positive control) (codeine
phosphate/30 mg/kg for antitussive experiment, ammonium
chloride/1500 mg/kg for expectorant experiment, dexametha-
sone/5.25 mg or 10.50 mg/kg for anti-inflammatory experiment),
groups 3–6 (imperialine, imperialine-␤-N-oxide, isoverticine,
isoverticine-␤-N-oxide, respectively). The low, medium and high
dose of the test compounds were 1.5, 3.0, 4.5 mg/kg body weight,
respectively. The mice were orally administered positive control
drugs and test compounds which were suspended in 0.5% carboxyl
methylcellulose solution. The experimental mice were deprived of
food for 12 h before experimentation but still allowed free access
to water.
2.4. Antitussive activity evaluation
Kunming mice of either sex were randomly divided into 6
groups of 10 mice each and treated as in Section 2.3. Antitussive
effects were investigated by using a classical mouse cough model
induced by ammonia liquor (Xu et al., 1991; Zhang et al., 2009).
Briefly, 60 min after oral administration of the test compounds and
ammonium chloride, each mouse was placed in a 1000 ml special
glass chamber and exposed to 0.3 ml 25% NH4 OH for 45 s. The fre-
quency and latent period of cough were observed for 6 min. During
6 min observation period, the animals were continuously moni-
tored by a trained observer with a stethoscope at the tip of the
glass chamber. The criteria to define cough in mice is that open-
ing the mouth accompanying sound of coughing, contraction of
thoracic and abdomen muscles, and a jerking of the front body.
The antitussive activities were assessed as the percentage of inhi-
bition of the number of coughs in terms of that in control groups
by using the following equation: %Inhibition = [(C0 − Ct )/C0 × 100%]
(C0 : the number of coughs of control, Ct : the number of coughs of
the treatment group).
2.5. Expectorant activity evaluation
Kunming mice of either sex were randomly divided into 6 groups
of 10 mice each and treated as in Section 2.3. The test was per-
formed as described previously (Engler and Szelenyi, 1984; Zhang
et al., 2009). Briefly, each mouse was treated with a single dose
of the test drugs for 30 min before intraperitoneal injection of
phenol red solution (5% in saline solution, w/v, 0.2 ml/20 g body
weight). Then 30 min after application of phenol red solution, the
mice were sacrificed by cervical dislocation without damaging the
trachea. After dissected free from adjacent organs, the trachea
was removed from the thyroid cartilage to the main stem bronchi
and put into 1 ml normal saline immediately. After ultrasonic for
15 min, 1 ml NaHCO3 solution (5%, w/v) was added to the nor-
mal saline, and optical density of the mixture was measured at
558.5 nm using Alpha-1900PC UV–Vis spectrophotometer (Shang-
hai Lab-Spectrum Instruments Co., Ltd., China). The standard curve
of phenol red was performed as described in Pharmacology Exper-
iment Methodology.
2.6. Anti-inflammatory activity evaluation
The mice of either sex were randomly divided into 6 groups
of 10 mice each and treated as in Section 2.3. The experi-
ment was carried out as described previously (Akindele and
Adeyemi, 2007; Li et al., 2010). Briefly, 30 min after oral admin-
istration of the test compounds and dexamethasone, 0.05 ml
xylene was applied to both surfaces of the right ear lobe
evenly. The left ear was considered as a control. Thirty min-
utes later, the mice were sacrificed by cervical dislocation and
both ears were removed. Circular sections were taken using a
cork borer with a diameter of 6.0 mm and weighed. The anti-
inflammatory effects were assessed as percentage of extent of ear
edema by using the following equation: S = [(Wr − Wl )/Wl × 100%]
(Wr : weight of right ear, Wl : weight of left ear), and the
percentage of inhibition of extent of ear edema was evalu-
ated: %Inhibition = [(S0 − St )/S0 × 100%] (S0 : extent of ear edema
 D. Wang et al. / Journal of Ethnopharmacology 139 (2012) 189–193
of control group, St : extent of ear edema of the treatment
group).
2.7. Statistical analysis
The experimental results were expressed as mean ± standard
error of mean (S.E.M.) and analyzed by the non-parametric test with
Wilcoxon rank sum test by the software of SPSS statistics 17.0. Val-
ues of P < 0.05 imply significance of the pharmacological effects in
the experiments.
3. Results
3.1. Identification of alkaloids isolated from BFW
Imperialine, imperialine-␤-N-oxide, isoverticine, and
isoverticine-␤-N-oxide were isolated from BFW and identi-
fied on the basis of spectral data (1 H, 13 C NMR spectra) and
melting point (m.p.). After identification of their structure, we
obtained imperialine (25.3 mg), imperialine-␤-N-oxide (5.0 mg),
isoverticine (4.8 mg) and isoverticine-␤-N-oxide (3.0 mg), their
purities were above 95%. The spectral data of them were as
follows.
Imperialine, m.p., 266–268 ◦ C, 1 H NMR (400 MHz, CDCl3 ), ı:
0.72(3H, s, 19-CH3 ), 1.04(3H, s, 21-CH3 ), 1.07(3H, d, J = 5.4 Hz, 27-
CH3 ), 3.55(1H, m, W1/2 = 24 Hz, 3-␣H). 13 C NMR (50 MHz, CDCl3 ),
ı: 37.4(C-1), 30.4(C-2), 70.8(C-3), 30.4(C-4), 56.4(C-5), 210.8(C-
6, C O), 46.8(C-7), 42.0(C-8), 54.7(C-9), 38.1(C-10), 29.8(C-11),
46.5(C-12), 34.1(C-13), 40.1(C-14), 26.7(C-15), 18.7(C-16), 39.0(C-
17), 59.7(C-18), 12.6(C-19), 72.0(C-20), 22.3(C-21), 63.4(C-22),
19.5(C-23), 29.4(C-24), 27.7(C-25), 61.3(C-26), 17.2(C-27). The
melting point, 1 H NMR, 13 C NMR data were consistent with those
reported in the literature (Itô et al., 1976; Kaneko et al., 1986; Zhang
et al., 1998; Chen et al., 2004).
Imperialine-␤-N-oxide, m.p., 231–234 ◦ C, 1 H NMR (200 MHz,
CDCl3 ), ı: 0.81(3H, s, 19-CH3 ), 1.12(3H, s, 21-CH3 ), 3.53(3-␣H),
1.46(d, 27-CH3 ). 13 C NMR (50 MHz, CDCl3 ), ı: 37.5(C-1), 30.5(C-
2), 70.3(C-3), 29.5(C-4), 56.7(C-5), 210.4(C-6, C O), 46.7(C-7),
38.1(C-8), 56.7(C-9), 38.0(C-10), 30.1(C-11), 46.6(C-12), 28.5(C-13),
39.5(C-14), 26.8(C-15), 19.7(C-16), 42.0(C-17), 70.4(C-18), 12.6(C-
19), 72.7(C-20), 24.5(C-21), 67.5(C-22), 15.5(C-23), 28.8(C-24),
27.6(C-25), 72.1(C-26), 19.5(C-27). The melting point, 1 H NMR, 13 C
NMR data were consistent with those reported in the literature
(Zhang et al., 1995, 1998; Chen et al., 2004).
Isoverticine, m.p., 134–136 ◦ C, 1 H NMR (200 MHz, CDCl3 ), ı:
1.03(6H, s, 19-CH3 , 21-CH3 ), 1.09(3H, d, 27-CH3 ), 3.63(IH, m, 3␣-H),
3.85(1H, m, 6␣-H). 13 C NMR (200 MHz, CDCl3 ), ı: 38.7(C-1), 31.2(C-
2), 71.8(C-3), 34.8(C-4), 48.2(C-5), 72.7(C-6, C O), 39.3(C-7),
35.7(C-8), 57.5(C-9), 35.4(C-10), 29.5(C-11), 40.9(C-12), 39.0(C-13),
43.7(C-14), 24.8(C-15), 20.7(C-16), 48.9(C-17), 61.7(C-18), 14.9(C-
19), 71.0(C-20), 20.8(C-21), 70.5(C-22), 19.0(C-23), 29.5(C-24),
27.7(C-25), 62.5(C-26), 17.3(C-27). The melting point, 1 H NMR,
13 C NMR data were consistent with those reported in the liter-
ature (Itô et al., 1968; Kaneko et al., 1979; Zhang et al., 1993,
1998).
Isoverticine-␤-N-oxide, m.p., 207–210 ◦ C, 1 H NMR (200 MHz,
CDCl3 ), ı: 3.85(6␣-H), 3.59(3␣-H). 13 C NMR (400 MHz, CDCl3 ),
ı: 39.5(C-1), 32.3(C-2), 72.5(C-3), 35.7(C-4), 49.5(C-5), 73.4(C-
6, C O), 40.3(C-7), 35.7(C-8), 58.8(C-9), 35.5(C-10), 31.9(C-11),
40.8(C-12), 36.8(C-13), 45.1(C-14), 25.8(C-15), 21.4(C-16), 49.7(C-
17), 73.5(C-18), 15.2(C-19), 72.9(C-20), 18.6(C-21), 73.4(C-22),
21.1(C-23), 30.5(C-24), 26.7(C-25), 75.5(C-26), 16.8(C-27). The
melting point, 1 H NMR, 13 C NMR data were consistent with those
reported in the literature (Chen et al., 2004).
191
3.2. Effects of alkaloids on the ammonia liquor induced cough in
mice
The effects of the alkaloids at high, medium and low dose on the
ammonia liquor induced cough in mice are shown in Table 1. Four
alkaloids showed potent antitussive effects. At dose of 4.5 mg/kg
(high dose) and 3.0 mg/kg (medium dose), test compounds signif-
icantly enhanced latent period of cough as compared with that of
control group. At dose of 1.5 mg/kg (low dose), only isoverticine
and isoverticine-␤-N-oxide markedly enhanced the latent period of
cough as compared with control. At dose of 4.5 mg/kg (high dose),
3.0 mg/kg (medium dose) and 1.5 mg/kg (low dose), all alkaloids
inhibited the cough frequency significantly. Especially, at medium
dose, four alkaloids had nearly equivalent antitussive activities in
comparison with decuple dose of codeine phosphate. In addition,
imperialine and isoverticine inhibited cough frequency in a dose-
dependent manner.
3.3. Effects of alkaloids on the amount of phenol red output in
mice
The results of expectorant test are shown in Table 2. The
regression equation of the standard curve was Y = 6.9984X + 0.1952
(X = absorbance, Y = the amount of phenol red secretion, r = 0.9998).
At dose of 3.0 mg/kg, imperialine, imperialine-␤-N-oxide,
isoverticine-␤-N-oxide significantly enhanced tracheal phe-
nol red output, compared with that of control group. Moreover,
effects of isoverticine-␤-N-oxide on increasing phenol red output
were better than those of the positive control.
3.4. Effects of alkaloids on xylene induced ear edema in mice
The effects of the alkaloids at high, medium and low dose on
the xylene induced ear edema in mice are shown in Table 3. At
dose of 4.5 mg/kg (high dose) and 3.0 mg/kg (medium dose), the
four alkaloids significantly inhibited the xylene-induced mice ear
edema, compared with that of control group. It was worth noting
that at medium dose their effects on inhibiting the xylene-induced
ear edema in mice were markedly better than that of the posi-
tive control drug dexamethasone. However, at dose of 1.5 mg/kg,
only the isoverticine presented significant inhibition of the xylene-
induced mice ear edema. In addition, the four alkaloids inhibited
the xylene-induced mice ear edema in a dose-dependent manner.
4. Discussion and conclusions
BFW, which is one main source for BFC, is one of the most
widely used antitussive and expectorant medications in TCM. The
crude alkaloid extracts were considered as the major biologically
active ingredients in genus of Fritillaria. However, further phar-
macological studies on monomers of alkaloids isolated from BFW
in vivo were absent. In this present study, imperialine-␤-N-oxide,
isoverticine and isoverticine-␤-N-oxide isolated from BFW, which
were very rare in natural products, were the characteristic con-
stituents of BFW. So we reported their antitussive, expectorant and
anti-inflammatory activities in this paper. Besides, we also isolated
alkaloids verticinone and verticine and their activities have been
reported elsewhere (Wang et al., 2011).
The results of antitussive test showed that all four alkaloids had
potent antitussive activities. It is interesting that imperialine and
isoverticine inhabited cough frequency in a dose-dependent man-
ner, but isoverticine-␤-N-oxide and imperialine-␤-N-oxide did not
show the dose-dependent manner on inhibition of cough fre-
quency. One reason may be that two oxidized alkaloids, whose
nitrogenous position combined with oxygen, are less sensitive to
dosage; another reason may be that antitussive effects of two
192 D. Wang et al. / Journal of Ethnopharmacology 139 (2012) 189–193

Table 1
Effects of alkaloids at low, medium and high dose on the ammonia liquor induced cough in mice.

Group Dose (mg/kg) No. of animals Latent period of cough(s) No. of coughs Inhibition (%)

0.5%CMC-Na – (for low dose) 10 16.85 ± 2.19 62.50 ± 5.31 –

– (for medium dose) 10 15.61 ± 3.28 60.50 ± 6.09 –
– (for high dose) 10 16.20 ± 3.96 61.40 ± 11.70 –

Codeine phosphate 30 (for low dose) 10 39.83 ± 3.76* 24.60 ± 4.86*** 60.64
30 (for medium dose) 10 33.61 ± 4.13* 18.20 ± 3.54*** 69.92
30 (for high dose) 10 36.80 ± 10.75* 20.40 ± 9.15*** 66.78

Imperialine 1.5 10 29.17 ± 3.39 46.30 ± 5.98* 25.92

3 10 32.30 ± 6.93* 20.90 ± 4.89** 65.45
4.5 10 38.60 ± 10.01** 14.60 ± 10.21*** 76.22

Imperialine-␤-N-oxide 1.5 10 23.34 ± 3.06 23.30 ± 3.06** 62.72

3 10 29.44 ± 5.05* 24.80 ± 5.27** 59.01
4.5 10 32.20 ± 11.52* 20.40 ± 12.66** 66.78

Isoverticine 1.5 10 59.07 ± 8.65* 35.30 ± 5.24* 43.52

3 10 52.47 ± 9.75** 25.90 ± 5.83** 57.19
4.5 10 58.40 ± 13.46** 19.40 ± 9.50*** 68.40

Isoverticine-␤-N-oxide 1.5 10 45.79 ± 3.79* 20.10 ± 1.15* 67.84

3 10 45.70 ± 8.40** 23.10 ± 3.85*** 61.82
4.5 10 44.40 ± 11.72** 20.80 ± 9.88*** 66.12

Values expressed as mean ± S.E.M. (n = 10).

*
P < 0.05 for comparison of treated groups with control.
**
P < 0.01 for comparison of treated groups with control.
***
P < 0.001 for comparison of treated groups with control.

Table 2
Effects of alkaloids on the tracheal phenol red output in mice.

Group Dose (mg/kg) No. of animals Absorbance (A) Phenol red secretion (␮g/ml) Increasing (%)

0.5%CMC-Na – 10 0.08 ± 0.01 0.76 ± 0.06 –

NH3 Cl 1500 10 0.17 ± 0.01 1.38 ± 0.02*** 83.41
Imperialine 3 10 0.14 ± 0.02 1.18 ± 0.12* 55.61
Imperialine-␤-N-oxide 3 10 0.13 ± 0.02 1.11 ± 0.11* 46.34
Isoverticine 3 10 0.11 ± 0.02 0.97 ± 0.14 27.80
Isoverticine-␤-N-oxide 3 10 0.18 ± 0.04 1.45 ± 0.18** 92.68

Values expressed as mean ± S.E.M. (n = 10).

*
P < 0.05 for comparison of treated groups with control.
**
P < 0.01 for comparison of treated groups with control.
***
P < 0.001 for comparison of treated groups with control.

Table 3
Effects of alkaloids at low, medium and high dose on the xylene induced ear edema in mice.

Group Dose (mg/kg) No. of animals Degree of edema Inhibition (%)

0.5%CMC-Na – (for low dose) 10 0.99 ± 0.06 –

– (for medium dose) 10 1.35 ± 0.07 –
– (for high dose) 10 1.33 ± 0.18 –

Dexamethasone 5.25 (for low dose) 10 0.78 ± 0.14* 21.21

5.25 (for medium dose) 10 0.96 ± 0.11* 28.89
10.5 (for high dose) 10 0.60 ± 0.05** 54.49

Imperialine 1.5 10 0.91 ± 0.16 8.08

3 10 0.70 ± 0.21* 48.15
4.5 10 0.55 ± 0.25** 59.01

Imperialine-␤-N-oxide 1.5 10 0.86 ± 0.08 13.13

3 10 0.75 ± 0.11** 44.44
4.5 10 0.58 ± 0.19*** 56.63

Isoverticine 1.5 10 0.86 ± 0.03* 13.13

3 10 0.62 ± 0.18* 54.07
4.5 10 0.48 ± 0.14*** 63.27

Isoverticine-␤-N-oxide 1.5 10 0.87 ± 0.04 12.12

3 10 0.91 ± 0.04** 32.59
4.5 10 0.71 ± 0.19** 46.63

Values expressed as mean ± S.E.M. (n = 10).

*
P < 0.05 for comparison of treated groups with control.
**
P < 0.01 for comparison of treated groups with control.
***
P < 0.001 for comparison of treated groups with control.
 D. Wang et al. / Journal of Ethnopharmacology 139 (2012) 189–193
oxidized alkaloids have reached maximum efficacy at this low
dose. In addition, the model of ammonia liquor induced cough
in mice, whose cough reflex is not easy to be observed, required
trained observers to monitor carefully. The generally antitussive
animal model is cough induced by citric acid aerosol in guinea
pigs, which has obvious cough reflex (Widdicombe, 1995; Mazzone,
2005).
In expectorant assay, we suggest that imperialine, imperialine-
␤-N-oxide and isoverticine-␤-N-oxide may have significant
expectorant effects. The sputum secreting was profited for dephlo-
gistication of trachea and relieving cough. Most expectorant drugs
could increase the secretion and dilute the sputamentum in the
respiratory tract so that it could be expectorated easily with the
ciliary movement (Han et al., 2010). These results indicated that
the alkaloids may have potent eliminating phlegm effects. How-
ever, we were not able to observe the expectorant activities at the
high and low dose because of a limited amount of the compounds.
Whether these alkaloids have dose dependant manner needs fur-
ther investigation.
The results of anti-inflammatory test indicated that the four
alkaloids had significant anti-inflammatory effects. Chronic inflam-
matory disease of the respiratory tract is one of origins of cough,
then anti-inflammatory effects of alkaloids can contribute to treat
cough. The inflammatory model of edema, which indicated early
symptoms of the inflammation, is commonly used by investiga-
tors (Xu et al., 1991; Li et al., 2010). Anti-inflammatory activities
of BFW have rarely been evaluated, so, we made a preliminary
investigation on their anti-inflammatory effects. The evaluation
of anti-inflammatory effects will serve as the basis for further
investigation on treating lung inflammation induced by all kinds
of factors.
In conclusion, the data of the present study would provide some
convincing evidence for BFW to be used as one antitussive and
expectorant medicine in the clinic. Through the pharmacologi-
cal evaluation of antitussive, expectorant and anti-inflammatory
effects of the four alkaloids, imperialine, imperialine-␤-N-oxide,
isoverticine and isoverticine-␤-N-oxide were confirmed as active
ingredients of the crude alkaloids extracted from BFW. However,
the exact mechanism under the observed effects is still elusive,
which needs our further in-depth studies.
Acknowledgement
The authors are grateful to National Science and Technology
Support Program (2009BAI84B02) from The Ministry of Science and
Technology of China for financial support. The funding agency did
not have any role in collection, analysis, interpretation of data, writ-
ing of the report or the decision to submit the paper for publication.
References
Akah, P.A., Ezike, A.C., Nwafor, S.V., Okoli, C.O., Enwerem, N.M., 2003. Evaluation
of the anti-asthmatic property of Asystasia gangetica leaf extracts. Journal of
Ethnopharmacology 89, 25–36.
Akindele, A.J., Adeyemi, O.O., 2007. Antiinflammatory activity of the aqueous leaf
extract of Byrsocarpus coccineus. Fitoterapia 78, 25–28.
Chen, M.H., 2008. Studies on Fritillaria Cirrhosa D. Don and its Effects of Antitussive
and Antiasthma. Qinghai Normal University, Xining.
Chen, Q., 2005. Study on Alkaloids of Fritillaria wabuensia. Sichuan University,
Chengdu.
193
Chen, Q., Zhu, L.H., Xu, Y.F., Fan, J.Z., 2004. A new steroidal alkaloid from the bulbus
of Fritillaria wabuensia. Acta Pharmaceutica Sinica 39, 348–350.
Chu, X.P., Xu, Z.H., Wu, D.Z., Zhao, A.H., Zhou, M.M., Qiu, M.F., Jia, W., 2007. In vitro and
in vivo evaluation of the anti-asthmatic activities of fractions from Pheretima.
Journal of Ethnopharmacology 111, 490–495.
Engler, H., Szelenyi, I., 1984. Tracheal phenol red secretion, a new method for
reening mucosecretolytic compounds. Journal of Pharmacological Methods 319,
151–157.
Ge, Y.B., Liu, J.Q., Su, D.F., 2009. In vivo evaluation of the anti-asthmatic, antitussive
and expectorant activities of extract and fractions from Elaeagnus pungens leaf.
Journal of Ethnopharmacology 126, 538–542.
Han, N., Chang, C.L., Wang, Y.C., Huang, T., Liu, Z.H., Yin, J., 2010. The in vivo expec-
torant and antitussive activity of extract and fractions from Reineckia carnea.
Journal of Ethnopharmacology 131, 220–223.
Irwing, R.S., Madison, J.M., 2000. The diagnosis and treatment of cough. New England
Journal of Medicine 34, 1715–1721.
Itô, S., Fukazawa, Y., Miyashita, M., 1976. Structure of imperialine. Tetrahedron Let-
ters 36, 3161–3164.
Itô, S., Fukazawa, Y., Okuda, T., Iitaka, Y., 1968. The structure of verticinone metho-
bromide. Tetrahedron Letters 9, 5373–5375.
Jiangsu New Medical College (Ed.), 1977. Encyclopedia of Chinese Materia Medica
(Appendix). Shanghai People’s Press, Shanghai, pp. 595–596.
Jiang, Y., Li, H., Li, P., Cai, Z., Ye, W., 2005. Steroidal alkaloids from the bulbs of
Fritillaria puqiensis. Journal of Natural Products 68, 264–267.
Kaneko, K., Katsuhara, T., Mitsuhashi, H., Chen, Y.P., Hsu, H.Y., Shiro, M., 1986. Chuan-
beinone, a novel D/E cis-(22R, 25S)-5␣-cevanine alkaloid from Chinese herbal
drug, chuan-bei-mu. Tetrahedron Letters 27, 2387–2390.
Kaneko, K., Tanaka, M., Haruki, K., Naruse, N., Mitsuhashi, H., 1979. 13 C NMR studies
on the cevanine alkaloids: the application of 13 C NMR spectrum for structure
elucidation of new alkaloids, baimonidine and isoverticine. Tetrahedron Letters
20, 3737–3740.
Lin, B.Q., Ji, H., Li, P., Jiang, Y., Fang, W., 2006. Selective antagonism activity of alka-
loids from bulbs Fritillariae at muscarinic receptors: functional studies. Journal
of Ethnopharmacology 551, 125–130.
Li, Y.W., Sun, Z.R., Li, Z.Y., Jing, J.X., Wang, W.Q., Sun, J.N., Yan, Y.N., 2010. Study
of antitussive, expectorant and anti-inflammatory pharmacological activities of
Xiaoyerong. Lishizhen Medicine and Materia Medica Research 11, 3041–3042.
Mazzone, S.B., 2005. An overview of the sensory receptors regulating cough. Cough
1:2, 2–9.
Mo, Z.J., Tang, X.Y., Sun, Z., Li, W., 1998. Comparison of Pharmacological Effects
Between Cultispecies Sichuan Fritillary Bulb (F. wabueasis, F. mellea) and Wild
Sichuan Fritillary Bulb (F. unibracteata). China Journal of Chinese Materia Medica
23, 14–16.
Shang, J.H., Cai, X.H., Zhao, Y.L., Feng, T., Luo, X.D., 2010. Pharmacological evalua-
tion of Alstonia scholaris: anti-tussive, anti-asthmatic and expectorant activities.
Journal of Ethnopharmacology 129, 293–298.
State Science and Technology Commission in China, 1988. Regulation for the Admin-
istration of Affairs Concerning Experimental Animals. The Ministry of Science
and Technology of the People’s Republic of China, Beijing.
Wang, D.D., Zhu, J.Y., Wang, S., Wang, X.X., Ou, Y., Wei, D.D., Li, X.P., 2011. Antitussive,
expectorant and anti-inflammatory alkaloids from Bulbus Fritillariae Cirrhosae.
Fitoterapia 82, 1290–1294.
Widdicombe, J.G., 1995. Neurophysiology of the cough reflex. European Respiratory
Journal 8, 1193–1202.
Xu, S.Y., Bian, R.L., Chen, X., 1991. Pharmacological Experiment Methodology Peo-
ple’s. Medical Publishing House, Beijing, China, 1167 pp.
Yan, X.Y., 2005. Effects of Ethanol Extract of Three Kinds of Bulb Fritillariae Cirrhosae
on Guinea Pigs with Allergic Asthma. Sichuan University, Chengdu.
Yang, E.J., Lee, J.S., Yun, C.Y., Kim, J.H., Kim, J.S., Kim, D.H., Kim, I.S., 2008. Inhibitory
effects of Duchesnea chrysantha extract on ovalbumin-induced lung inflamma-
tion in a mouse model of asthma. Journal of Ethnopharmacology 118, 102–107.
Zhang, A.J., Wang, H.Y., Tang, X.Y., Zheng, Y., Yi, X.H., Yu, K.B., 1998. Isolation and
structure elucidation of alkaloids from the bulb of Fritillaria wabuensis. Planta
Medica 64, 448–450.
Zhang, J.A., Wang, H.Y., Tang, X.Y., 1995. 2D-NMR studies on an alkaloid from Fritil-
laria wabuensis. West China Journal of Pharmaceutical Sciences 10, 69–72.
Zhang, J.L., Wang, H., Pi, H.F., Ruan, H.L., Zhang, P., Wu, J.Z., 2009. Structural analy-
sis and antitussive evaluation of five novel esters of verticinone and bile acids.
Steroids 74, 424–434.
Zhang, J.X., Lao, A., Xu, R.S., 1993. Steroidal alkaloids from Fritillaria thunbergii var.
chekiangensis. Phytochemistry 33, 946–947.
Zhang, Y.H., Ruan, H.L., Zeng, F.B., Pi, H.F., Zhao, W., Wu, J.Z., 2003. Effective part
screening on antitussive, expectorant and antiasthmatic activities of Fritillaria
hupehensis. Zhongcaoyao 34, 1016–1018.
Zhou, Y., Ji, H., Li, P., Jiang, Y., 2003. Antimuscarinic function of five alkaloids from Frit-
illaria on guinea-pig tracheal strips. Zhongguo Yaoke Daxue Xuebao 34, 58–60.