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Introduction
Cough is one of the most common complaints from
patients[1]. The present therapies were limited for lack of
effective medications. Moreover, most existed medicine for
treatment could bring inevitable or intolerable side-effects.
The most pressing and unmet current need is to seek more
effective and safer antitussive
agents[2]. Traditional medicines represent a promising new trend for developing new
antitussive drugs.
Shedan_Chuanbei powder, a complex of traditional
Chinese medicine preparation, which consists of snake bile
(Chinese name "Shedan") and Fritillariae cirrhosae
(Chinese name "Chuanbei"), is the most popular antitussive
and expectorant formulation in Chinese communities. It has
been officially listed in the Chinese Pharmacopoeia (1995,
2000, and 2005) due to the positive potent therapeutic effects,
low toxicity, and minimal side-effects. Snake biles are the
bile acids of Naja naja Linnaeus,
Krait, zaocys, and Fritillariae cirrhosae
among others, and are the bulbs of Fritillariae
cirrhosae, Fritillariae unibracteata,
Fritillariae przewalskii, and Fritillariae
delavayi. However, the clinical application of Shedan_Chuanbei powder is now
stringently limited because of the shortage of the 2 crude
medicinal materials, especially for the sake of animal
protection, therefore, it prompts us to search for new bioactive
substitutes for Shedan_Chuanbei powder.
Most of the complex of traditional Chinese medicine
formulations have sound scientific basis through modern
pharmacological evaluation[3]. For example, many
combination formulations showed significantly better pharmacological
effects than individual herbal medicines in the formulations
through synergistic interaction and attenuation of their
toxicity. Significant chemical changes may occur during the
preparation (decoction) process of a prescribed herbal
formulation[4]. Verticinone and cholic acid were identified as
major bioactive components in Fritillariae and snake
bile[5_7], both of which can be determined with high concentrations
by advanced analytical technology in Shedan_Chuanbei
powder[8,9]. Enlightened with "combination principles" in
drug discovery and hypothesizing that the 2 ingredients may
have a synergism, in the present study, we made a salifying
reaction of the 2 ingredients considering their distinct
properties in acidity or alkalinity. We then obtained
verticinone_cholic acid salt from the reaction. Furthermore, we also
elucidated the antitussive activity, the acute toxicity, and
the antitussive mechanism involved in guinea pigs.
Materials and methods
Equipment Melting points were measured with a Fisher
Scientific instrument (New Jersey, USA) and were uncorrected. Infra-red (IR) spectra were recorded on a
Shimadzu IR2460 spectrometer. The NMR spectra in
D2O were recorded with Bruker AM-500 spectrometers (Bruker,
Germany). FAB-MS was measured on a VG Auto Spec-3000
mass spectrometer (Micromass, England). An elemental
analysis were performed on a CarloErba-1106 autoanalyzer
(CarloErba, Milan, Italy). The compressor nebulizer (Medel
ap112U, Polo di Torrile, Italy) was used to produce an
aerosol with a particle median diameter of 1.87_3.54 µm. The
cough sound was amplified via a clip-on microphone (Takstar
DA-141, Guangdong Takstar Electronic, Dongguan,
Guang-dong province, China).
Herbal materials, antitussive agents and
drugs Verticinone was isolated from Fritillariae hupehensis
Hsiao et KC Hsia, which is commercially available from the Hubei
Institute of Chinese Materia Medica (Wuhan, China) and
was identified by Prof De-tai PENG (Lichuan Institute of
Chinese Materia Medica, Lichuan, Hubei province, China).
A voucher specimen (F030823) was deposited at the Faculty
of Pharmaceutical Science, Tongji Medical College, Huazhong
University of Science and Technology (Wuhan, China).
Cholic acid was purchased from Asia Talent Enterprise
(Shenzhen, Guangdong province, China). Its identity was
confirmed with IR, 1H-NMR,
13C-NMR, and MS analyses. Codeine phosphate was purchased from Qinghai
Pharmaceutical Factory (Qinghai, China). Glybenclamide was
purchased from ABCR Gmbh (Karlsruhe, Germany). The
naloxone hydrochloride injection was purchased from Beijing
Four-Ring Pharmaceutical Factory (Beijing, China). Moguisteine
was purchased from Boehringer Mannheim Italia (Monza,
Italy).
Synthesis and identification of Ver_CA The powdered
bulbs of Fritillariae hupehensis Hsiao
et K C. Hsia (3.5 kg) were extracted with 95% EtOH (ethanol) and the
solvent was removed to give an EtOH extract. Then the
extract was dissolved in 2% HCl and filtered. After filtration,
the water layer was basified with ammonia (pH=12) and
continuously extracted with chloroform to give a crude alkaloid
fraction (20 g, 0.6% yield). The crude alkaloid fraction was
then subjected to silica gel column chromatography with a
discontinuous gradient elusion using the solvent system
consisting of petroleum ethyl acetate-ethylene diamine. The
elution was monitored by TLC and the fractions only
containing verticinone were combined and dried. Then the
residue was crystallized from ethyl acetate to give verticinone
(10 g, 0.3% yield), and its identify was further confirmed by
1H-NMR, 13C-NMR, and MS analyses (Figure 1).
Verticinone (1 g) and cholic acid (1 g) were dissolved
together in acetone and stirred for 48 h at room temperature
under reflux. The organic solvent was evaporated to
dryness under reduced pressure at 40 oC to afford a white
residue. The residue was then further purified with distilled
water. The processes of purification were as follows: first,
the residue was dissolved in distilled water and then filtered
in order to remove the insoluble substances; second, the
water was evaporated to dryness under reduced pressure at
80 oC. We then got a white compound (1.8 g, 90% yield),
which was confirmed as a salifying derivative of verticinone
and cholic acid by comparing its IR, 1H-NMR,
13C-NMR, and MS with that of verticinone and cholic acid. As shown in
Table 1, the difference between Ver_CA and the admixture of
verticinone and cholic acid also proved that it was a
salifying derivative.
Animals Dunkin_Hartley guinea pigs of both sexes
(Experimental Animal Center, Tongji Medical College,
Huazhong University of Science and Technology, China),
weighing about 250_350 g, were used. The animals were
housed in groups of 6 per cage under a 12 h light_dark cycle
with food and water available continuously. Kunming mice
of both sexes were used in acute toxicity experiment. This
study was carried out in accordance with the "Regulation
for the Administration of Affairs Concerning Experimental
Animals" (State Council of China, 1988).
Drug administration Codeine phosphate and Ver_CA
were dissolved in 0.9% saline. Verticinone and cholic acid
were suspended in 0.5% carboxyl methylcellulose solution.
Glybenclamide (3 mg/kg), also suspended in 0.5% carboxyl
methylcellulose solution, was injected 30 min after the
administration of the antitussive agents. Naloxone
hydrochloride (0.8 mg/kg) was injected 45 min after the administration
of the antitussive agents.
Assay of antitussive activity in guinea pigs
The unanesthetized and unrestrained animals were placed individually
in a transparent perspex chamber (dimensions: 16 cm×16 cm×
16 cm) and exposed to a nebulized aqueous solution of
17.5% citric acid saline solution for 5 min with a flow rate of
0.5 mL/min. The same nebulizer was used throughout the
experiment. During the 5 min observation period, the
animals were continuously watched by a trained observer and
the frequency of coughs was recorded. The coughs could
easily be distinguished from sneezes since there is a clear
difference in sound as well as in the behavior of the animals[10]. The animals were selected by the number of
outbursts of coughing observed during an aerosol exposure
24 h before the test; animals with more than 30 or fewer than
10 outbursts in 5 min were not used for the studies. After
24 h recovery, the selected guinea pigs were randomly
divided into several groups with at least 6 animals in each
group for different treatments. The number of coughs
produced 60 min after the administration of antitussive agents
(Ct) was compared with the number of control coughs (Cc)
which was tested at the first day. The antitussive effect was
expressed as the percentage of inhibition of the number of
control coughs [(Cc_Ct)/Cc×100%].
Assay of acute toxicity of Ver_CA and verticinone in
mice Test solutions of various concentrations were
administrated (po) to mice. The animals were observed
continuously for gross effects and then at 24 h intervals for up to
7 d. Behavioral, toxic effects, and mortality response were
recorded.
Statistical analysis The LD50
values and their 95% confidence intervals were determined by the Bliss
method[11] using Lanzhou Pharmaceutical software (version 1.01,
Lan-zhou Biopharmaceutics, Shenyang, Liaoning Province,
China). Data of antitussive effect were expressed as mean±
SEM. The statistical significance of difference was assessed
by t-test. A P-value of 0.05 or less was considered significant.
Results
Antitussive effect of Ver_CA, verticinone, and cholic
acid The guinea pigs produced 23.20±4.44 coughs in 5 min
of exposure to the aerosol of 17.5% citric acid. On the next
day, 60 min after administration of the vehicle, the guinea
pigs were treated with the same aerosol again and produced
19.00±4.06 coughs in 5 min, which suggested that the effect
of the vehicle on the number of citric acid-induced coughs
was not significant (P>0.05).
The antitussive effects of Ver_CA, verticinone, and cholic
acid are shown in Figure 2. At the same dose, Ver_CA, but
not cholic acid and verticinone, showed potent antitussive
effects in the guinea pigs. Furthermore, compared with
verticinone and cholic acid, the cough reduction of the
effects produced by Ver_CA were significantly different
(P<0.01 vs verticinone and cholic acid).
Moreover, as shown in Figure 3, the dose_response
relationship studies suggest that at dosages ranging from 2 to
4.5 mg/kg (po), Ver-CA showed potent antitussive activity in
a dose-dependent manner. In a parallel study, codeine
phosphate, one of the most commonly used potent
antitussive agents[12], was used as the positive control. A
dose_response inhibitory effect of codeine on the citric
acid-induced cough was also monitored at dosages ranging from 2
to 10 mg/kg (po). It was found to be less effective in
comparison to Ver_CA. The values for the 50% inhibition of
cough (ID50) of Ver_CA and codeine phosphate were
determined as 2.9 and 10 mg/kg, respectively.
Effects of naloxone and glybenclamide on the
antitussive effect of Ver_CA As shown in Figure 4, pretreatment
with naloxone (0.8 mg/kg, ip), a non-selective opioid
receptor antagonist, significantly (P<0.05) reduced the
antitussive effect of codeine phosphate, a centrally-acting, narcotic
antitussive drug (with saline, 49.04%±7.32%,
n=6; with naloxone, 11.12%±3.80%,
n=6). The antitussive effect of Ver_CA, but not moguisteine, was partially antagonized by
naloxone. On the other hand, pretreatment with
glyben-clamide (3 mg/kg, ip), an ATP-sensitive
K+ channel blocker, significantly reduced the antitussive effect both of
Ver_CA and moguisteine (a peripheral non-narcotic antitussive
drug), but not that of codeine phosphate.
Acute toxicity of Ver_CA and verticinone
After oral administration of more than 22.8 mg/kg of Ver_CA, fidgety and
tachypnea were observed within a few minutes and the belly
of the mouse touched the floor of the cage. One or 2 min
before death, sudden jumps and convulsions were observed.
All deaths occurred within 60 min after drug administration
(po). Similar behavioral changes were observed after the
administration of lethal doses of verticinone. Table 2 shows
the LD50 values (po) of Ver_CA and verticinone. The toxicity
of Ver_CA was only one-third that of verticinone.
Discussion
Shedan_Chuanbei powder, a complex traditional Chinese
medicine formulation, is used for coughs,
expectorant, and asthma therapy in clinics in China. Its potent antitussive
effect was previously reported in experimental models of
coughs[13]. Substitutes for Shedan_Chuanbei powder are
already available in markets, such as an artificial snake bile
substitute of natural snake bile or other Fritillariae
species to substitute Fritillariae
cirrhosae[13,14], but all of these
substitutes still confront many daunting challenges, such as
standardization problems due to the natural variability of the
crude materials and the chemical complexity of the
prepara-tion. However, Ver_CA, a salifying derivative of verticinone
and cholic acid, would make standardization and
preparation problems simpler because it is a single compound.
Further bioactivity studies showed that Ver_CA had much
more potent antitussive effects than the monomer verticinone
or cholic acid at the same doses. We compared the effects of
the free state and inorganic salty state of the total alkaloids
from Fritillariae hupehensis in a previous study and found
that the antitussive effect of free-state alkaloids was a little
more potent than the alkaloids' hydrochloride and much more
potent than the alkaloids' sulfate[15]. So the antitussive
effect of the alkaloids from Fritillariae hupehensis
may not be increased because of the increased solubility in water.
Therefore, the much more potent antitussive effect of
Ver_CA salt rather than verticinone may be due to the synergistic
effect between verticinone and cholic acid.
Moreover, the antitussive effect of Ver_CA was also
higher than codeine phosphate, which could be proven by
comparison of their antitussive ID50
values. In addition, the antitussive effect of Ver_CA was only partially antagonized
by pretreatment with naloxone, although naloxone completely
antagonized the antitussive effect of codeine phosphate,
which indicated that the potent antitussive effect of Ver_CA
was partially through the opioid receptor.
Animal studies have demonstrated that the activation of
K+ channels can inhibit airway sensory nerve activity, and
the modulation of these channels can attenuate
experimentally-induced coughs[2]. Morita and
Kamei[18] studied the antitussive effect of moguisteine, a peripherally_acting,
non-narcotic antitussive drug, and had proposed that
ATP-sensitive K+ channels may be involved in the antitussive effect
of peripherally-acting antitussive
drugs[16_18]. In the present study, moguisteine was also used as a control. Similar to
moguisteine, the antitussive effect of Ver_CA was
significantly reduced by pretreatment of glybenclamide,
suggesting that Ver_CA may exert its antitussive effect partially
through ATP-sensitive K+ channels. Thus, the antitussive
effect of Ver_CA may depend on both the peripheral
(modulated by ATP-sensitive K+ channels) and central
mechanisms (modulated by the opioid receptor). Moreover, the
acute toxicity of Ver_CA was only one-third that of
verticin-one. Thus, the salifying derivative appeared to have a
synergism and attenuated toxic effects compared to single
verticinone and cholic acid. Therefore, our present data, if
confirmed for other animal species and for humans, may
suggest a novel approach to the symptomatic treatment of
coughing derived from a traditional Chinese formulation. In
addition, further attention should be paid to the water
solubility of the compound which would be a versatile choice in
future preparations.
We had elucidated that the antitussive mechanism of the
total alkaloids from Fritillariae hupehensis
was central through the experimental cough model elicited in
pentobarbital-anesthetized cats by electrical stimulation of the
superior laryngeal nerve, which had been used to determine the
site of antitussive drugs[19, 20]. So the central and peripheral
mechanisms of Ver_CA may be a result of the synergism of
verticinone and cholic acid. Our present study could also
provide evidence that Fritillariae combined with snake bile
in Shedan_Chuanbei powder has a sound scientific base.
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