U.S. patent application number 15/110100 was filed with the patent office on 2016-11-24 for asarum total polysaccharides extract having antitussive activity and extraction method and use thereof.
This patent application is currently assigned to THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY. The applicant listed for this patent is THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY, GUANGZHOU INSTITUTE OF RESPIRATORY DISEASE. Invention is credited to Kefang LAI, Xiaodong LIU, Yichu NIE, Bonian ZHONG, Shan ZHONG.
Application Number | 20160339054 15/110100 |
Document ID | / |
Family ID | 51458491 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160339054 |
Kind Code |
A1 |
LAI; Kefang ; et
al. |
November 24, 2016 |
ASARUM TOTAL POLYSACCHARIDES EXTRACT HAVING ANTITUSSIVE ACTIVITY
AND EXTRACTION METHOD AND USE THEREOF
Abstract
The present invention discloses an Asarum total polysaccharide
extract with antitussive activity, an extraction method thereof and
an application in the preparation of a medicine for preventing and
treating coughs. The present invention proves that the Asarum total
polysaccharide extract has a significant effect of suppressing
cough for the first time through a large number of pharmacodynamic
tests, has the therapeutic effect approximate to codeine, has the
effect of prolonging the cough latent period better than codeine,
and also finds that the total Asarum polysaccharide extract has the
effects of reducing the cough sensitivity and suppressing the
airway inflammation for the first time, has excellent preventive
and therapeutic effects on various types of coughs, and can be used
for preparing the medicines for preventing and treating
cough-related diseases.
Inventors: |
LAI; Kefang; (Guangzhou
City, CN) ; LIU; Xiaodong; (Guangzhou, CN) ;
ZHONG; Bonian; (Guangzhou, CN) ; ZHONG; Shan;
(Guangzhou, CN) ; NIE; Yichu; (Guangzhou,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
GUANGZHOU INSTITUTE OF RESPIRATORY DISEASE |
Guangdong
Guangdong |
|
CN
CN |
|
|
Assignee: |
THE FIRST AFFILIATED HOSPITAL OF
GUANGZHOU MEDICAL UNIVERSITY
Guangzhou City
CN
GUANGZHOU INSTITUTE OF RESPIRATORY DISEASE
Guangzhou City
CN
|
Family ID: |
51458491 |
Appl. No.: |
15/110100 |
Filed: |
March 25, 2015 |
PCT Filed: |
March 25, 2015 |
PCT NO: |
PCT/CN2015/075054 |
371 Date: |
July 7, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/268 20130101;
A61K 31/715 20130101; A61P 11/14 20180101; A61K 2236/00 20130101;
A61K 2236/30 20130101 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61K 36/268 20060101 A61K036/268 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 25, 2014 |
CN |
CN201410115066.5 |
Claims
1. An application comprising Asarum total polysaccharide extract in
the preparation of a medicine for preventing and treating
coughs.
2. The application according to claim 1, wherein the cough is
chronic cough.
3. The application according to claim 1, wherein the percentage
content of polysaccharide in the Asarum total polysaccharide
extract is 60 wt % to 80 wt %.
4. A pharmaceutical composition for preventing and treating coughs,
wherein the active ingredients comprise Asarum total polysaccharide
extract.
5. The pharmaceutical composition for preventing and treating
coughs according to claim 4, wherein the formulations of the
pharmaceutical composition are granules, tablets, capsules, pills,
dripping pills, effervescent tablets, ointments, syrups,
injections, oral liquid, mixtures, tinctures, sustained-release
preparations, controlled-release preparations or targeting
preparations.
6. The pharmaceutical composition for preventing and treating
coughs according to claim 4, wherein the percentage content of
polysaccharide in the Asarum total polysaccharide extract is 60 wt
% to 80 wt %.
7. A method for extracting the Asarum total polysaccharide extract,
comprising the following steps: using a polar solvent for the heat
reflux extraction of roots and/or rhizomes of Asarum;
decompressing, concentrating and drying the obtained material to
obtain an Asarum total extract; dissolving the Asarum total extract
in water to obtain an Asarum total extract solution; adding
ethanol, wherein the volume percentage of the ethanol in the Asarum
total extract solution is 60%-99%; decompressing and filtering the
solution; using an ethanol solution for washing; and decompressing
and drying to obtain the Asarum total polysaccharide extract.
8. The method according to claim 7, wherein the polar solvent is
water.
9. The method according to claim 7, wherein the weight of the polar
solvent is 5-20 times as much as that of the roots and/or rhizomes
of Asarum.
10. The method according to claim 7, wherein the volume percentage
of the ethanol solution for washing is 60%-100%.
11. The method according to any of claim 7, wherein the Asarum is
Asarum Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.)
Kitag., Asarum sieboldii Miq. var. seoulense Nakai or Asarum
sieboldii Miq.
12. The method according to claim 11, wherein the Asarum is Asarum
Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.) Kitag.
13. The Asarum total polysaccharide extract with antitussive
activity obtained by the method according to claim 7.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a national stage filing under 35 U.S.C.
371 of International Application No. PCT/CN2015/075054, filed Mar.
25, 2015, which claims priority to CN201410115066.5, filed Mar. 25,
2014, the disclosures of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention belongs to the technical field of
medicines, and more particularly relates to an Asarum total
polysaccharide extract with antitussive activity as well as an
extraction method and an application thereof.
BACKGROUND
[0003] Due to the work pressure, air and environment pollution and
many other factors, the incidence of human respiratory diseases
remains high, while cough is a common and frequently-occurring
disease of respiratory system and is a common chief complaint of
respiratory outpatient. The cough itself is a defensive reflex
activity generated when the respiratory system is stimulated, is a
beneficial action, and sometimes is also found in healthy people.
In general, mild and infrequent coughs can be relieved naturally
without applying antitussive medicines. In addition, impact of the
cough on human health is also dialectical and is also a
pathological reaction; and long-term frequent or too intense cough
not only may increase the suffering of patients, affect the rest
and sleep and increase the physical exertion, but also may promote
the development of diseases in severe cases to cause other
complications.
[0004] According to different courses of the disease, the cough can
be divided into acute cough, subacute cough and chronic cough. The
course of acute cough is less than three weeks, that of subacute
cough is three to eight weeks, and that of chronic cough is more
than or equal to eight weeks. 1) The acute cough: at present, acute
viral upper respiratory tract infection is generally considered as
the most common cause, and others include rhinitis, sinusitis,
acute bronchitis, acute exacerbation of chronic bronchitis,
bronchial asthma and the like; the most common pathogens of upper
respiratory tract infection are rhinovirus, coronavirus,
respiratory syncytial virus, parainfluenza virus, adenovirus,
enterovirus and the like, wherein rhinovirus is most common; the
acute cough may also be associated with the acute exacerbation of
asthma, bronchitis and other respiratory diseases; and clinically,
many infectious diseases or some other diseases having the acute
cough as a prodroma can often be seen. 2) The subacute cough: the
most common causes are post-infectious cough (also known as
post-cold cough), bacterial sinusitis, asthma and the like; a virus
as the pathogen is commonly rhinovirus, accounting for about
30%-50%, followed by coronavirus and influenza virus, respectively
accounting for 10%45% and 5%-15%; and in addition to respiratory
viruses, mycoplasma pneumoniae, chlamydia pneumoniae and the like
can also cause post-infectious cough. 3) The chronic cough: the
chronic cough is usually used for defining a patient having cough
as the only symptom or the main symptom, having the course of
disease of more than eight weeks and having no apparent abnormality
in chest X-ray examination in clinical practice, and is known as
unexplained chronic cough, called chronic cough for short; the rate
of chronic cough is up to 10%-38% in specialty clinic;
questionnaires of European and American countries show that the
incidence of chronic cough is also up to 9%-33%; and the common
causes of chronic cough are eosinophilic bronchitis (EB), upper
airway cough syndrome (UACS, formerly known as postnasal drip
syndrome (PNDS)), gastroesophageal reflux cough (GERC), cough
variant asthma (CVA) and the like.
[0005] A site investigation for the cough of 1087 college students
of Guangzhou Institute of Respiratory Diseases in 2006 showed that
the overall incidence of cough in groups was 10.9%, wherein the
overall incidence of chronic cough was 3.3%. It can be speculated
that the incidence of cough in community population may be higher,
and the average course of disease of such patients was longer, some
even up to decades of years. Some data showed that about 15% of
adults and 10% of minors were affected by different degrees of
cough all over the world every year. Therefore, the development of
safe and effective antitussive medicines can create better social
benefits and economic benefits while meeting a huge market
demand.
[0006] According to different action mechanisms of medicines, the
antitussive medicines can be divided into two categories as
follows: the first category is centrally acting antitussive
medicines; such medicines can directly and selectively inhibit the
medullary respiratory center to generate an antitussive effect,
such as morphine, codeine, etc.; and such medicines have better
antitussive effect, but have obvious side effects, can lead to
addiction and respiratory depression, and even induce
hallucinations, delusions or other psychotic symptoms. The second
category is peripherally acting antitussive medicines; such
medicines block the afferent impulse of cough reflex by inhibiting
the lung-vagal reflex, thereby playing an antitussive role, such as
benproperine, benproperine phosphate, Moguisteine and the like; and
most of such medicines are alkaloid medicines, and may cause
nausea, palpitation, dizziness or other adverse reactions. Although
various medicines can be used for relieving the cough clinically,
many patients with chronic cough cannot get effective relief after
treatment by using these medicines.
[0007] The traditional Chinese medicine is a crystallization of
rich practical experience of Chinese people in the prevention and
treatment of diseases for thousands of years. Doctors of
traditional Chinese medicine may recognize some diseases unknown by
doctors of western medicine. The traditional Chinese medicines may
be used for treating some diseases refractory and incurable to
western medicines. The traditional Chinese medicine has a long
history and rich experience in the treatment of coughs, and is
complementary with the western medicines used by the doctors of
western medicine. Although there are a lot of traditional Chinese
medicines and Chinese patent medicines for treating coughs in China
at present, the effective components and the indications of these
medicines are not clear. In addition, there is no good treatment
medicine for patients with unexplained chronic cough and airway
inflammation, so the primary task of pharmaceutical workers engaged
in the field of respiratory diseases is to research and develop
pure natural traditional Chinese medicines which have fewer side
effects and can effectively treat coughs.
[0008] Asarum, as a traditional Chinese medicine, is firstly
recorded in the Yellow Emperor's Internal Classic, and is
classified as a top grade. 2010 edition of Chinese Pharmacopoeia
stipulates that the source of Asarum is dried roots and rhizomes of
Asarum Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.)
Kitag., Asarum sieboldii Miq. var. seoulense Nakai, or Asarum
sieboldii Miq. The Asarum is warm in nature, acrid in flavor, and
attributive to heart, lung and kidney meridians, has the effects of
relieving exterior syndrome and dispelling cold, dispelling wind
and relieving pain, opening orifices, and warming the lung to
dissipate phlegm retention, and is used for treating cough and
asthma due to phlegm retention, wind-cold type common cold,
headache, toothache, nasal congestion and discharge, allergic
rhinitis, nasosinusitis, and Rheumatic arthralgia. Some ancient
books record the use of Asarum for treating coughs, for example,
the Yellow Emperor's Internal Classic records, "the Asarum has main
indications for cough with dyspnea, headache during use of brains,
. . . , and has the effects of freeing nine orifices"; Compendium
of Materia Medica records, "acrid in flavor can clear lung, so it
is suitable for use by patients with wind-cold type common cold and
abnormal rising of qi"; Rihuazi Bencao records, "treating coughs, .
. . , chest accumulation"; Changsha Yaojie records, "good at
reducing adverse rising, designed to stop coughing"; and Medicinal
Theory records, "treating cough with dyspnea, . . . ". At present,
reports for literature about the antitussive effect of Asarum
mostly focus on compound methyleugenol of total volatile oil (Zhou
Huiqiu, Study on Pharmacological Effects of Methyleugenol, Acta
Chinese Medicine and Pharmacology, 2000, (2):79-80). Also, the
study on non-volatile components of Asarum is rarely reported, and
only the isolation of non-volatile constituents, including kakuol,
1-asarinin, 1-sesamin, aristololactam I, kaempferol-3-O-glucoside
and the like, from the roots and rhizomes of Asarum is reported
(Cai Shaoqing, Study on Non-Volatile Chemical Constituents of
Asarum Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.)
Kitag., Journal of Beijing Medical University, 1996, 28(3):228-230;
Lv Shuai, Isolation and identification of chemical constituents
from roots of Asarum Heterotropoides Fr. Schmidt var. Mandshuricum
(Maxim.) Kitag. (II), Journal of Shenyang Pharmaceutical
University, 2010, 27(9): 707-710; Wei Qingchun, Isolation and
Identification of Chemical Constituents of Asarum sieboldii Miq.
var. seoulense Nakai, Li Shizhen Medicine and Materia Medica
Research, 2010, 21(3): 676-677).A preparation method of Asarum
polysaccharide has been studied and reported (Li Jingjing, Study on
the Separation, Purification and Immunological Activity of
Polysaccharide from Asarum Heterotropoides, Journal of Changchun
Normal University (Natural Science), 2008, 27(1): 54-58), but this
preparation method needs to extract three times, the extracting
cycle each time is longer (6 hours), and the centrifugation
treatment is required in the preparation process, therefore, this
method is not suitable for industrial mass production; with
adoption of this preparation method, the overall yield of
polysaccharides is lower (9.56 wt %), and the content of
saccharides is also lower (48 wt % to 52 wt %); in addition, it is
reported that Asarum polysaccharide, the non-volatile constituent
of Asarum, has an effect of promoting the proliferation of mouse
spleen lymphocytes T and B (Li Jingjing, Study on the Separation,
Purification and Immunological Activity of Polysaccharide from
Asarum Heterotropoides, Journal of Changchun Normal University
(Natural Science Edition), 2008, 27(1): 54-58). However, the
studies and reports about the antitussive activity of Asarum total
polysaccharide, the non-volatile constituent of Asarum, have not
been seen at present.
SUMMARY
[0009] In view of the above defects of the prior art, the primary
purpose of the present invention is to provide a new application of
an Asarum total polysaccharide extract in medicines.
[0010] The technical solution for achieving the above purpose is as
follows.
[0011] An application of the Asarum total polysaccharide extract in
the preparation of a medicine for preventing and treating coughs is
provided.
[0012] Another purpose of the present invention is to provide a
pharmaceutical composition for preventing and treating coughs.
[0013] The technical solution for achieving the above purpose is as
follows.
[0014] A pharmaceutical composition for preventing and treating
coughs has active ingredients comprising the Asarum total
polysaccharide extract.
[0015] In one embodiment, the dose form of the pharmaceutical
composition is granules, tablets, capsules, pills, dripping pills,
effervescent tablets, ointments, syrups, injections, oral liquid,
mixtures, tinctures, sustained-release preparations,
controlled-release preparations or targeting preparations.
[0016] In one embodiment, the percentage content of polysaccharide
in the Asarum total polysaccharide extract is 60 wt % to 80 wt
%.
[0017] In one embodiment, the above cough is chronic cough.
[0018] Another purpose of the present invention is to provide a
method for extracting the Asarum total polysaccharide extract. The
Asarum total polysaccharide extract obtained by the method has good
antitussive activity.
[0019] The technical solution for achieving the above purpose is as
follows.
[0020] A method for extracting the Asarum total polysaccharide
extract comprises the following steps: using a polar solvent for
the heat reflux extraction of roots and/or rhizomes of Asarum;
decompressing, concentrating and drying the obtained material to
obtain an Asarum total extract; dissolving the Asarum total extract
in water to obtain an Asarum total extract solution; adding
ethanol, wherein the volume percentage of the ethanol in the Asarum
total extract solution is 60%-99%; decompressing and filtering the
solution; using an ethanol solution for washing; and decompressing
and drying to obtain the Asarum total polysaccharide extract.
[0021] In one embodiment, the polar solvent is water.
[0022] In one embodiment, the weight of the polar solvent is 5-20
times as much as that of the roots and/or rhizomes of Asarum.
[0023] In one embodiment, the Asarum is Asarum Heterotropoides Fr.
Schmidt var. Mandshuricum (Maxim.) Kitag., Asarum sieboldii Miq.
var. seoulense Nakai or Asarum sieboldii Miq., preferably Asarum
Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.) Kitag.
[0024] In one embodiment, the volume percentage of the ethanol
solution for washing is 60%-100%, and the ethanol solution with the
volume percentage of 80%-100% for ethanol washing is preferably
adopted for washing.
[0025] The method for extracting the Asarum total polysaccharide
extract with antitussive activity of the present invention has a
total polysaccharide yield of 15 wt %.
[0026] The use of the above Asarum total polysaccharide extract
with antitussive activity in the preparation of the medicine for
treating coughs is provided. The Asarum total polysaccharide
extract is used for preparing the medicine for treating coughs
individually or in combination with other medicines.
[0027] The Asarum total polysaccharide extract is prepared into
various formulations with available pharmaceutical carriers.
[0028] The formulations are granules, tablets, capsules, pills,
dripping pills, effervescent tablets, ointments, syrups,
injections, oral liquid, mixtures, tinctures, sustained-release
preparations, controlled-release preparations or targeting
preparations.
[0029] The previous study of the inventor of the present invention
finds that: Asarum volatile oil has an obvious antitussive effect
on guinea pig models with acute cough induced by citric acid, but
the antitussive effect on guinea pig models with cough
hypersensitivity under cigarette smoke (CS) exposure is obviously
not better than that of the Asarum total polysaccharide, the
non-volatile constituent of Asarum, and a large dose has a certain
side effect.
[0030] A pharmacological experiment proves that the Asarum total
polysaccharide extract has good activity for alleviating and/or
inhibiting coughs and can be used for preventing or treating
various coughs.
[0031] Pharmacodynamic test results show that, for the guinea pig
models with acute cough, the low-, medium- and high-dosage groups
of Asarum total polysaccharide extracts, compared with the blank
control group, after citric acid excitation can prolong the cough
latent period of the animals and reduce the cough frequency of
guinea pigs; compared with the Asarum total volatile oil, the acute
antitussive activity of Asarum total polysaccharide to the guinea
pigs is relatively weaker, while for the guinea pig models with
cough hypersensitivity and accompanied airway inflammation
(CS-exposure for 14 days), the low-, medium- and high-dosage groups
of Asarum total polysaccharide extracts, compared with the model
group, after citric acid excitation can obviously prolong the cough
latent period of the animals, reduce the cough frequency of guinea
pigs, can also reduce the number of total cells and percentage of
neutrophils in BALF of guinea pigs, and inhibit the airway
inflammation, thereby achieving or approaching the effects of
codeine; compared with the Asarum total extract and the Asarum
total volatile oil, the antitussive rate of the high dose group of
Asarum total polysaccharide extract has a significant difference
(p<0.05), which shows that the Asarum total polysaccharide
extract has better antitussive activity to the guinea pig models
with cough hypersensitivity under long-term CS-exposure.
[0032] The acute toxicity test results show that, after
intragastric administration of the Asarum total polysaccharide
extract, the SPF grade NIH mice are normal in food intake and
weight, and LD.sub.50>10.0 g/kg, thereby indicating that the
Asarum total polysaccharide extract has low toxicity and good
medication safety.
[0033] The present invention has the beneficial effects: [0034] (1)
The present invention provides a new selection and idea for the
current antitussive medicine, extends the selection field of the
antitussive medicine, and makes contributions to the development of
the technical field. The present invention proves that the Asarum
total polysaccharide extract has an obvious antitussive effect for
the first time, and has the functions of reducing cough sensitivity
and inhibiting airway inflammation without addiction. The acute
toxicity test results show that the Asarum total polysaccharide
extract has low toxicity and good medication safety. [0035] (2) The
preparation process of the Asarum total polysaccharide extract
provided by the present invention can effectively enrich the total
polysaccharide and remove impurities. The yield and the content of
the Asarum total polysaccharide are high. In quality control,
content determination analysis can be performed through ultraviolet
spectrophotometry. The method is simple, easy in operation,
controllable, and has the advantages of simplicity, quickness, good
reproducibility; meanwhile, the solvent adopted is cheap and
non-toxic. Especially, the Asarum total polysaccharide obtained by
the extracting method provided by the present invention has better
antitussive activity, and provides more selections of medicines for
preventing and treating coughs clinically.
DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows an influence (Mean+/-s, n=8) of Asarum total
polysaccharide on the cough frequency of guinea pig models with
acute cough, wherein A is the blank control group, B is the solvent
group (mass fraction 0.5% CMC-Na), C is the codeine phosphate 30
mg/kg group, D is the Asarum total polysaccharide 200 mg/kg group,
E is the Asarum total polysaccharide 400 mg/kg group, F is the
Asarum total polysaccharide 800 mg/kg group, G is the total Asarum
extract 1600 mg/kg group, and H is the Asarum total volatile oil
200 mg/kg group; and compared with the blank group, *p is less than
0.05, **p is less than 0.01, and ***p is less than 0.001.
[0037] FIG. 2 shows the influence (Mean+/-s, n =8) of Asarum total
polysaccharide on the cough frequency of guinea pig models with
cough hypersensitivity, wherein A is the blank group, B is the
model group, C is the solvent group (mass fraction 0.5% CMC-Na),D
is the codeine phosphate 30 mg/kg group, E is the Asarum total
polysaccharide 200 mg/kg group, F is the Asarum total
polysaccharide 400 mg/kg group, G is the Asarum total
polysaccharide 800 mg/kg group, H is the total Asarum extract 1600
mg/kg group, and I is the Asarum total volatile oil 200 mg/kg
group; compared with the model group, **p is less than 0.01, and
***p is less than 0.001; compared with the total Asarum extract
group, .tangle-solidup.p is less than 0.05; and compared with the
Asarum total volatile oil group, #p is less than 0.05.
[0038] FIG. 3 shows a representative diagram (HE staining,
.times.200) of the influence of Asarum total polysaccharide on lung
tissue pathology of guinea pig models with cough hypersensitivity,
wherein A is the blank group, B is the model group, C is the
solvent group (mass fraction 0.5% CMC-Na),D is the codeine
phosphate 30 mg/kg group, E is the Asarum total polysaccharide 200
mg/kg group, F is the Asarum total polysaccharide 400 mg/kg group,
G is the Asarum total polysaccharide 800 mg/kg group, H is the
total Asarum extract 1600 mg/kg group, and I is the Asarum total
volatile oil 200 mg/kg group.
[0039] FIG. 4 shows the representative diagram (HE staining,
.times.200) of the influence of Asarum total polysaccharide on
tracheal tissue pathology of guinea pig models with cough
hypersensitivity, wherein A is the blank group, B is the model
group, C is the solvent group (mass fraction 0.5% CMC-Na),D is the
codeine phosphate 30 mg/kg group, E is the Asarum total
polysaccharide 200 mg/kg group, F is the Asarum total
polysaccharide 400 mg/kg group, G is the Asarum total
polysaccharide 800 mg/kg group, H is the total Asarum extract 1600
mg/kg group, and I is the Asarum total volatile oil 200 mg/kg
group.
DETAILED DESCRIPTION
[0040] The present invention is further described in detail below
in combination with the drawings and specific embodiments, but not
regarded as the limitation of the present invention.
[0041] In one embodiment, an application of the Asarum total
polysaccharide extract in the preparation of a medicine for
preventing and treating coughs is provided.
[0042] In one embodiment, the Asarum total polysaccharide extract
has a good preventive and therapeutic effect on the acute cough or
the chronic cough. The percentage content of effective
polysaccharide in the Asarum total polysaccharide extract is
measured by using an ultraviolet spectrophotometry, using anhydrous
glucose as a reference substance, and using a phenol-sulfuric acid
method at 490 nm wavelength or using an anthrone method at 620 nm
wavelength. The percentage content of polysaccharide is 60 wt % to
80 wt %; and a polysaccharide sample is hydrolyzed and derived, and
then is measured to contain glucose, mannose, arabinose, galactose,
xylose, rhamnose, ribose and galacturonic acid through GC-MS
measurement.
[0043] In one embodiment, a pharmaceutical composition for
preventing and treating coughs has active ingredients comprising
Asarum total polysaccharide extract with the polysaccharide
percentage content of 60 wt % to 80 wt %, and can also be combined
with other medicines with antitussive activity. The active
ingredients further comprise a pharmaceutically acceptable carrier,
such as fillers, adhesives, lubricants, disintegrants or wetting
agents, wherein the fillers comprise lactose, sucrose, corn starch
and sorbitol; the adhesives comprise syrup, arabic gum, gelatin,
sorbitol, hydroxypropyl methylcellulose or polyvinylpyrrolidone;
the lubricants comprise magnesium stearate; the disintegrants
comprise starch, polyvinylpyrrolidone, crospovidone and
microcrystalline cellulose; and the wetting agents comprise sodium
dodecyl sulfate.
[0044] In one embodiment, the formulations of the pharmaceutical
composition are granules, tablets, capsules, pills, dripping pills,
effervescent tablets, ointments, syrups, injections, oral liquid,
mixtures, tinctures, sustained-release preparations,
controlled-release preparations or targeting preparations.
[0045] In one embodiment, the effective dose of the Asarum total
polysaccharide extract with antitussive activity, calculated
according to the weight of a person of 60 kg, is 2.5 g-11.0 g per
person per day.
[0046] A method for extracting the Asarum total polysaccharide
extract with antitussive activity of the present embodiment
comprises the following steps: using a polar solvent for the heat
reflux extraction of roots and/or rhizomes of Asarum, wherein the
polar solvent is preferably water, and the weight of the polar
solvent is 5-20 times as much as that of the roots and/or rhizomes
of Asarum; then, decompressing, concentrating and drying the
obtained material to obtain an Asarum total extract; next,
dissolving the Asarum total extract in water; using an ethanol
solution with the ethanol volume percentage of 40%-99% (preferably
60%-90% or preferably 60%-99%) for precipitation; decompressing and
filtering the solution; using the ethanol solution (with the
ethanol volume percentage of 60%-100%, the ethanol solution or
ethanol with the ethanol volume percentage of 80%-100% is more
preferably used in the present embodiment) for washing precipitates
for 1-3 times, preferably 2 times; and then decompressing and
drying the precipitates to obtain the Asarum total polysaccharide
extract.
[0047] The Asarum is Asarum Heterotropoides Fr. Schmidt var.
Mandshuricum (Maxim.) Kitag., Asarum sieboldii Miq. var. seoulense
Nakai or Asarum sieboldii Miq., preferably Asarum Heterotropoides
Fr. Schmidt var. Mandshuricum (Maxim.)Kitag. in the present
embodiment.
[0048] The method for extracting the Asarum total polysaccharide
extract with antitussive activity of the present invention has a
total polysaccharide yield of 15 wt %. The Asarum total
polysaccharide extract obtained by the above method has a better
antitussive effect.
[0049] The following is the part of the specific embodiments, for
illustrating the present invention in more detail, but not limiting
the protection scope of the present invention.
Embodiment 1
[0050] A method for preparing the Asarum total polysaccharide
extract of the present embodiment comprises the following
steps:
[0051] Crushing 1kg of dried roots and/or rhizomes of Asarum
Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.) Kitag. into
coarse powder; screening the coarse powder with a sieve of 80
meshes; using water being 10 times as much as the weight of
medicinal materials for slight-boiling heating and reflux
extraction for 5 hours; filtering an extracting solution with 6
layers of gauze; decompressing, concentrating and drying the
filtrate to obtain the Asarum total extract;
[0052] Using water for heating and dissolving the Asarum total
extract to be 1 L; adding 9 times volume of anhydrous ethanol until
the volume percentage concentration of ethanol is 90% while
stirring; stewing the mixture in a refrigerator at 4.degree. C.
after full precipitation; pouring supernatant; decompressing and
filtering the precipitates; using the same amount of ethanol
aqueous solution with the volume percentage of 90% for washing
twice; drying the obtained material conventionally to obtain the
Asarum total polysaccharide extract, wherein the yield is 15 wt %.
The contents of Asarum total polysaccharide measured by ultraviolet
spectrophotometry using the phenol-sulfuric acid method and the
anthrone method respectively are 68 wt % and 60 wt %.
Embodiment 2
[0053] A method for preparing the Asarum total polysaccharide
extract of the present embodiment comprises the following
steps:
[0054] Crushing 1 kg of dried roots and/or rhizomes of Asarum
Heterotropoides Fr. Schmidt var. Mandshuricum (Maxim.) Kitag. into
coarse powder; screening the coarse powder with a sieve of 80
meshes; using water being 10 times as much as the weight of
medicinal materials for slight-boiling heating and reflux
extraction for 5 hours; filtering the extracting solution with 6
layers of gauze; and decompressing, concentrating and drying the
filtrate to obtain the Asarum total extract;
[0055] Using water for heating and dissolving the Asarum total
extract to be 1 L; adding 15 times volume of anhydrous ethanol
until the volume percentage concentration of ethanol is 94% while
stirring; stewing the mixture in the refrigerator at 4.degree. C.
after full precipitation; pouring supernatant; decompressing and
filtering the precipitates; using the same amount of ethanol
aqueous solution for washing three times; and drying the obtained
material conventionally to obtain the Asarum total polysaccharide
extract, wherein the yield is 13 wt %. The contents of Asarum total
polysaccharide measured by ultraviolet spectrophotometry using the
phenol-sulfuric acid method and the anthrone method respectively
are 75 wt % and 70 wt %. A polysaccharide sample is hydrolyzed and
derived, and then is measured to contain glucose, mannose,
arabinose, galactose, xylose, rhamnose, ribose and galacturonic
acid through GC-MS measurement.
Embodiment 3
[0056] Test for acute toxicity of Asarum total polysaccharide
extract orally and intragastrically administered to mice.
[0057] 1. Animals
[0058] SPF grade NIH mice, including half males and half females,
weighing 18-22 g, being provided by the Guangdong Medical
Laboratory Animal Center, having an animal certificate number of
SOCK (Guangdong province, China) 2008-0002.
[0059] 2. Method
[0060] The acute toxicity of Asarum total polysaccharide extract
orally and intragastrically administered to mice is investigated by
referring to the Experimental Methodology of Pharmacology and the
Experimental Methodology of TCM Pharmacology and using a normal
Bliss method. Firstly, the maximum concentration, the maximum
administration dose and the maximum intragastric administration
volume are used for testing. Before the test, the mice are
subjected to food-fasting but not water-fasting for 12 hours; and
when the test is carried out, 10.0 g/kg of Asarum total
polysaccharide extract (obtained according to the preparation
method described in the Embodiment 1) is intragastrically
administered to the mice (equivalent to 24.4 g/kg of crude
medicine, equivalent to 12 times as much as the clinical dose). The
laboratory temperature is 25+/-2.degree. C.; the observation is
kept for 14 days after once administration; each animal is observed
carefully, and the appearing and disappearing time of various
toxicities are recorded in detail. Observed and recorded contents
include behavioral expressions of skin, mucosa, hair color, eyes,
respiration, circulation, locomotor activity, central nervous
system and the like. The animals should be weighed within a week
before and after administering a test substance, when the animals
die and the test is ended. The autopsy should be conducted on all
animals, including animals which die or are killed; and the
histopathological examination should be conducted on organs
abnormal in autopsy.
[0061] 3. Results
[0062] The test results show that 10.0 g/kg of Asarum total
polysaccharide extract is orally and intragastrically administered
to the mice, no animal dies within 14 days after administration,
the mental and behavioral activities of all animals are in good
condition, the skin and the hair are clean, the defecation and the
increases of weight and food intake are normal, and no other
symptom of toxicity is found. No obvious organ abnormality is found
in gross anatomy after the end of observation. LD.sub.50 of the
Asarum total polysaccharide is more than 10.0 g/kg.
[0063] The above results indicate that the Asarum total
polysaccharide has high safety and has no risk of severe acute
poisoning.
Embodiment 4
[0064] Acute antitussive effect of Asarum total polysaccharide
extract on cough of guinea pigs induced by citric acid
[0065] 1. Materials
[0066] Animals: ordinary grade Hartley guinea pigs, including half
males and half females, weighing 250-350 g, being provided by the
Guangdong Medical Laboratory Animal Center, having the animal
certificate number of SOCK (Guangdong province, China)
2008-0002.
[0067] Reagent and Instruments: citric acid monohydrate (Guangzhou
Chemical Reagent Factory, Batch Number: 20121001-2), which is
prepared into a normal saline solution of 0.8 M when use; a Buxco
noninvasive animal pulmonary function testing system (US Buxco
Company); an Aeroneb Pro nebulizer (Aerogen (Ireland) Co., Ltd.);
and an intragastric administration needle (Guangdong Medical
Laboratory Animal Center).
[0068] 2. Method
[0069] A guinea pig acute cough model induced by citric acid is
used for the efficacy evaluation. 128 Hartley guinea pigs are taken
and are placed in a 6 L body plethysmograph of the Buxco
noninvasive animal pulmonary function testing system one by one;
the air flow rate of a bias meter is 2.5 L/min; the Aeroneb Pro
nebulizer is used for feeding nebulized citric acid ultrapure
aqueous solution aerosol of 0.8 M for 1 min, wherein the average
diameter of nebulized particles is 2.5 .mu.m; the observation is
kept for 5 min, to monitor the cough frequency within 6 min; the
sound of cough is recorded and amplified by a microphone placed
indoors; sound waves are analyzed through Biosystem XA software;
the sound data are processed by the computer software to record the
cough frequency; and meanwhile, a trained observer carries out
supplementary observation. The guinea pigs with the cough frequency
of more than 10 times are selected as qualified animals for
testing.
[0070] 64 screened and qualified Hartley guinea pigs, including
half males and half females, are taken and randomly divided into
eight groups; the blank group is administered with 1 mL/kg of
normal saline; the solvent group is administered with the same
volume of corresponding solvent (a CMC-Na aqueous solution with the
mass percentage concentration of 0.5%); a positive medicine group
is administered with the same volume of 30 mg/kg of codeine
phosphate; and the administration groups are administered with the
same volume of Asarum total polysaccharide extract (200 mg/kg, 400
mg/kg and 800 mg/kg of samples in the Embodiment 1), Asarum total
extract (1600 mg/kg), Asarum total volatile oil (200 mg/kg,
prepared by conventional steam distillation), once a day for
consecutive 3 days. A citric acid nebulization excitation test
(nebulization conditions are the same as above) is conducted in the
Buxco noninvasive animal pulmonary function testing system within 2
hours after the last administration; the nebulization excitation is
conducted for 1 min to induce the guinea pigs to have acute cough;
the observation is kept for 5 min; the cough frequency (N) within 6
min and a cough latent period are calculated; and the antitussive
rate of each administration group is calculated by using the
formula "the antitussive rate=(N.sub.blank control
group-N.sub.administration group)/N.sub.blank control
group.times.100%".
[0071] Data are represented by mean +/-s. The statistical software
IBM SPSS Statistics 19.0 is used for the one-way analysis of
variance of each group of parameters. In the comparison between
groups, if p is less than 0.05, there is a significant
difference.
[0072] 3. Results
[0073] (1) Influence of Asarum Total Polysaccharide Extract on the
Cough Frequency of Guinea Pig Models with Acute Cough
[0074] The cough frequency and the cough suppression rate of
animals in each group within 6 min after the citric acid
nebulization excitation are shown in Table 1 and FIG. 1. Table 1
and FIG. 1 show that, compared with the blank group, the medium-
and the high-dosage groups of Asarum total polysaccharide extract
both can significantly reduce the cough frequency of guinea pigs
(p<0.05), and have the antitussive rates up to 28.5% and 32.3%,
respectively. When the dose of Asarum total extract is 1600 mg/kg,
the antitussive rate is 28.8%, while the antitussive rate of the
high-dosage group of Asarum total polysaccharide extract reaches a
considerable level when the dose is 800 mg/kg, thereby indicating
that the antitussive activity of the Asarum total polysaccharide
extract is higher than that of the Asarum total extract, and
further explaining that the Asarum total polysaccharide is one of
the main active ingredients of the Asarum for relieving cough, and
there is no significant difference between the Asarum total
polysaccharide extract and the Asarum total extract. The acute
antitussive activity (the antitussive rate is 53.8%) of Asarum
total volatile oil on the guinea pigs is better than that of the
Asarum total extract and the Asarum total polysaccharide, but there
is no significant difference among the three.
[0075] The above results indicate that the Asarum total
polysaccharide extract has a good acute antitussive effect, has a
dose-effect relationship, and is relatively safe in medication.
TABLE-US-00001 TABLE 1 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on the cough frequency of guinea pig
models with acute cough Cough frequency Antitussive Group Dose
(mg/kg) (times) rate (%) Blank control -- 28.8 +/- 3.2 -- Solvent
control -- 26.8 +/- 4.4 -- Codeine 30 8.5 +/- 2.6*** 70.5 phosphate
Low dose of 200 22.0 +/- 0.9 21.5 Asarum total polysaccharide
Medium dose of 400 20.6 +/- 2.3* 28.5 Asarum total polysaccharide
High dose of 800 19.5 +/- 2.5* 32.3 Asarum total polysaccharide
Asarum total 1600 20.5 +/- 3.4* 28.8 extract Asarum total 200 13.3
+/- 3.3** 53.8 volatile oil Note: compared with the blank group, *p
< 0.05, **p < 0.01, ***p < 0.001.
[0076] (2) Influence of Asarum Total Polysaccharide Extract on the
Cough Latent Period of Guinea Pig Models with Acute Cough
[0077] The cough latent period of animals in each group within 6
min after the citric acid nebulization excitation is shown in Table
2. Table 2 shows that, compared with the blank control group,
different dose groups of Asarum total polysaccharide extract can
significantly prolong the cough latent period of the guinea pigs,
wherein p is less than 0.05 in the low-dosage group; p is less than
0.01 in the medium- and the high-dosage groups; and the Asarum
total polysaccharide extract has the dose-effect relationship.
Effects of different dose groups of Asarum total polysaccharide
extract for prolonging the cough latent period are better than the
effect of codeine phosphate. The Asarum total extract and the
Asarum total volatile oil may also prolong the cough latent period,
but compared with the blank group, there is no statistical
significance (p>0.05).
TABLE-US-00002 TABLE 2 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on cough latent period of guinea pig
models with acute cough Cough latent period Group Dose (mg/kg)
(seconds) Blank control -- 45.8 +/- 6.2 Solvent control -- 43.7 +/-
16.8 Codeine phosphate 30 95.3 +/- 18.3 Low dose of Asarum total
200 118.9 +/- 24.7* polysaccharide Medium dose of Asarum 400 133.3
+/- 29.5** total polysaccharide High dose of Asarum total 800 140.9
+/- 19.7** polysaccharide Asarum total extract 1600 106.4 +/- 21.3
Asarum total volatile oil 200 100.2 +/- 6.4 Note: compared with the
blank group, *p < 0.05, **p < 0.01.
Embodiment 5
[0078] Antitussive effect of Asarum total polysaccharide on guinea
pig models with cough hypersensitivity
[0079] 1. Materials
[0080] Animals: ordinary grade Hartley guinea pigs, including half
males and half females, weighing 250-350 g, being provided by the
Guangdong Medical Laboratory Animal Center, having the animal
certificate number of SOCK (Guangdong province, China)
2008-0002.
[0081] Reagent and Instruments: citric acid monohydrate (Guangzhou
Chemical Reagent Factory, Batch Number: 20121001-2), which is
prepared into the normal saline solution of 0.8 M when use; the
Buxco noninvasive animal pulmonary function testing system (US
Buxco Company); the Aeroneb Pro nebulizer (Aerogen (Ireland) Co.,
Ltd.); a cell counting chamber (Zhejiang Yuhuan County Chumen
Medical Instrument Factory); and the intragastric administration
needle (Guangdong Medical Laboratory Animal Center).
[0082] 2. Method
[0083] The method for screening qualified animals is the same as
that described in the Embodiment 3. 72 screened and qualified
animals are taken and are randomly divided into nine groups; the
blank group and the model group are administered with 1 mL/kg of
normal saline; the solvent group is administered with the same
volume of corresponding solvent (the CMC-Na aqueous solution with
the mass percentage concentration of 0.5%); the positive medicine
group is administered with the same volume of 30 mg/kg of codeine
phosphate; the administration groups are administered with the same
volume of Asarum total polysaccharide extract (200 mg/kg, 400 mg/kg
and 800 mg/kg of samples in the Embodiment 1), Asarum total extract
(1600 mg/kg), Asarum total volatile oil (200 mg/kg, prepared by
conventional steam distillation), and are intragastrically
administered once a day for consecutive 14 days. Except the blank
control group, each group is smoked by 10 cigarettes Hongmei (China
Tobacco Guangdong Industrial Co., Ltd., tar 13 mg, nicotine 1.3 mg,
carbon monoxide 15 mg) for 20 min every time, twice a day for
consecutive 14 days; and an efficacy evaluation model for guinea
pigs with chronic cough is built. The citric acid nebulization
excitation is conducted for 1 min (the Buxco noninvasive animal
pulmonary function testing system, the nebulization conditions is
the same as those in the Embodiment 3) within 24 hours after the
last smoking exposure, to induce the guinea pigs to have a cough;
the observation is kept for 5 min; and the cough frequency (N)
within 6 min, the antitussive rate and the cough latent period are
calculated. Sodium pentobarbital with the mass percentage
concentration of 3% is used for anesthetization within 24 hours
after the animals are excited; the blood is collected from the
heart; 6 mL of ice PBS is used for the bronchoalveolar lavage
(BALF) of guinea pigs; and meanwhile, the lung tissues and the
tracheal tissues are taken and are fixed with 10% of
formaldehyde.
[0084] Observation Method and Indicators:
[0085] (1) Calculating the cough frequency within 6 min after
citric acid excitation, and calculating the antitussive rate of
each administration group by using the formula "the antitussive
rate=(N.sub.model group-N.sub.administration group)/N.sub.model
group.times.100%".
[0086] (2) Calculating the cough latent period of each
administration group within 6 min after citric acid excitation;
[0087] (3) Airway inflammation indicators: <1> total cell
count (TCC) in BALF: taking 0.5 mL of BALF fluid after 24 hours
following the end of nebulization excitation to centrifuge at 3000
r/min for 10 min, adding 0.3 mL of erythrocyte lysate to the
precipitates, shaking and mixing the mixture uniformly, stewing the
mixture for 10 min, centrifuging the mixture at 3000 r/min for 10
min, using 0.5 mL of PBS for suspending the precipitates, and
taking the cell counting chamber under 10 .mu.L optical microscope
to count the TCC; <2> classification of cells in BALF:
washing fixed BALF cell smears with running water for 5 min, using
hematoxylin for staining 20 seconds, washing the stained cell
smears with running water for 5 min, using 1% of hydrochloric acid
alcohol for destaining 5 seconds, washing the destained cell smears
with running water for 5 min, using eosin for staining 5 seconds,
washing the stained cell smears with running water for 5 min, using
neutral balsam for mounting after drying the cell smears, and
classifying the cells under the optical microscope; and <3>
histopathological observation of the lung tissues and the tracheal
tissues: conducting HE staining on the lung tissues and the
tracheal tissues, wherein for the lung tissues, it is mainly
observed that whether bronchi, alveoli and accompanying arterioles
are complete in structure, whether ciliated epithelia of the airway
are arranged in order, and whether there are inflammatory cells for
infiltration around blood vessels, hyperemia and edema and the
like; for the tracheal tissues, it is mainly observed that whether
the epithelia are complete, whether the lumina are neat and clean,
whether the tracheal wall is thickened, and whether the cilia are
arranged regularly, fall off, are infiltrated by inflammatory cells
and the like.
[0088] 3. Results
[0089] (1) Influence of Asarum Total Polysaccharide Extract on the
Cough Frequency of Guinea Pig Models with Cough
Hypersensitivity
[0090] Compared with the blank group, the cough frequency of guinea
pigs in the model group within 6 min after citric acid nebulization
excitation and CS-exposure for 14 days is significantly increased
(p<0.01), so the guinea pigs with cough hypersensitivity are
modeled successfully. Compared with the model group, the low-,
medium- and high-dosage groups of Asarum total polysaccharide
extract may significantly reduce the cough frequency of guinea pigs
(p<0.001), and have the antitussive rates up to 54.0%, 62.6% and
70.9%, respectively. Compared with the Asarum total extract and the
Asarum total volatile oil, the antitussive rate of the high-dosage
group of Asarum total polysaccharide extract has a significant
difference (p<0.05). Compared with the codeine phosphate, the
antitussive rate of the high-dosage group of Asarum total
polysaccharide extract has no significant difference, thereby
showing that the Asarum total polysaccharide extract has better
antitussive activity to the guinea pig models with cough
hypersensitivity (the results are shown in Table 3 and FIG. 3).
[0091] The above results indicate that the Asarum total
polysaccharide extract has the effect of treating the guinea pigs
with chronic cough hypersensitivity, and has the dose-effect
relationship, wherein the treatment effect of the high-dosage group
of Asarum total polysaccharide extract is approximate to that of
codeine.
TABLE-US-00003 TABLE 3 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on the cough frequency of guinea pig
models with cough hypersensitivity Dose Cough frequency Antitussive
Group (mg/kg) (times) rate (%) Blank control -- 19.4 +/- 2.6** --
Model group -- 30.2 +/- 2.7 -- Solvent control -- 32.0 +/- 3.2 --
Codeine phosphate 30 5.7 +/- 1.9*** 81.1 Low dose of Asarum 200
13.9 +/- 1.7*** 54.0 total polysaccharide Medium dose of 400 11.3
+/- 0.9*** 62.6 Asarum total polysaccharide High dose of Asarum 800
8.8 +/- 3.6***.sup..tangle-solidup.# 70.9 total polysaccharide
Asarum total extract 1600 16.2 +/- 2.3*** 46.4 Asarum total
volatile 200 16.2 +/- 1.4*** 46.4 oil Note: compared with the model
group, **p < 0.01, ***p < 0.001; compared with the Asarum
total extract group, .sup..tangle-solidup.p < 0.05; and compared
with the Asarum total volatile oil group, .sup.#p < 0.05.
[0092] (2) Influence of Asarum Total Polysaccharide Extract on the
Cough Latent Period of Guinea Pig Models with Cough
Hypersensitivity
[0093] The cough latent period of animals in each group within 6
min after the citric acid nebulization excitation and after the
cigarette exposure for 14 days is shown in Table 4. Table 4 shows
that, compared with the model group, different dose groups of
Asarum total polysaccharide extract can significantly prolong the
cough latent period of the guinea pigs, wherein p is less than
0.001 in the medium- and high-dosage groups, the Asarum total
polysaccharide extract has the dose-effect relationship, and
compared with the codeine phosphate group, the medium- and
high-dosage groups have no significant difference, thereby showing
that the influence of Asarum total polysaccharide extract on the
cough latent period is approximate to the effect of codeine
phosphate. Compared with the Asarum total extract and the Asarum
total volatile oil, the cough latent period of the high-dosage
group of Asarum total polysaccharide extract has a significant
difference (p>0.05).
TABLE-US-00004 TABLE 4 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on the cough latent period of guinea
pig models with cough hypersensitivity Cough latent Group Dose
(mg/kg) period (seconds) Blank control -- 61.2 +/- 6.0 Model group
-- 45.2 +/- 4.0 Solvent control -- 33.6 +/- 3.9 Codeine phosphate
30 199.7 +/- 37.7*** Low dose of Asarum total 200 100.3 +/- 17.8
polysaccharide Medium dose of Asarum total 400 156.1 +/- 25.0***
polysaccharide High dose of Asarum total 800 189.0 +/-
20.4***.sup..tangle-solidup.# polysaccharide Asarum total extract
1600 117.7 +/- 16.0* Asarum total volatile oil 200 112.0 +/- 7.0*
Note: compared with the model group, *p < 0.05, ***p < 0.001;
compared with the Asarum total extract group,
.sup..tangle-solidup.p < 0.05; and compared with the Asarum
total volatile oil group, .sup.#p < 0.05.
[0094] (3) Influence of Asarum Total Polysaccharide Extract on TCC
in BALF of Guinea Pig Models with Cough Hypersensitivity
[0095] Compared with the blank group, the TCC in BALF of the model
group after CS-exposure for 14 days is significantly increased
(p<0.05), thereby modeling successfully. Compared with the model
group, different dose groups of Asarum total polysaccharide extract
can reduce the number of total cells in BALF of guinea pigs,
wherein the low-dosage group has a better significant difference
(p<0.01); the medium- and high-dosage groups have significant
differences (p<0.001) and have the dose-effect relationship.
Compared with the Asarum total extract and the Asarum total
volatile oil, the TCC of the medium- and high-dosage groups of
Asarum total polysaccharide extract has a significant difference
(p<0.05). Codeine phosphate cannot reduce the total number of
inflammatory cells in BALF of airway hypersensitivity guinea pigs
with airway inflammation (see Table 5).
TABLE-US-00005 TABLE 5 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on TCC in BALF of guinea pig models
with cough hypersensitivity Group Dose (mg/kg) TCC
(.times.10.sup.5, cells/mL) Blank control -- 10.8 +/- 2.7*** Model
group -- 27.8 +/- 1.2 Solvent control -- 26.1 +/- 1.3 Codeine
phosphate 30 26.5 +/- 5.9 Low dose of Asarum total 200 16.5 +/-
5.2** polysaccharide Medium dose of Asarum total 400 8.1 +/-
0.6***.sup..tangle-solidup.# polysaccharide High dose of Asarum
total 800 7.8 +/- 0.6***.sup..tangle-solidup.# polysaccharide
Asarum total extract 1600 16.1 +/- 1.2** Asarum total volatile oil
200 17.6 +/- 2.3* Note: compared with the model group, *p <
0.05, **p < 0.01, ***p < 0.001; compared with the Asarum
total extract group, .sup..tangle-solidup.p < 0.05; and compared
with the Asarum total volatile oil group, .sup.#p < 0.05.
[0096] (4) Influence of Asarum Total Polysaccharide Extract on the
Classification of Cells in BALF of Guinea Pig Models with Cough
Hypersensitivity
[0097] Compared with the blank group, the ratios of macrophages
(Mac) in BALF of the model group and the solvent control group
after CS-exposure for 14 days are significantly reduced, while the
ratios of neutrophils (Neu) are significantly increased, and the
ratios have a significant difference (p<0.001), which indicates
the successful modeling. Compared with the model group, different
dose groups of Asarum total polysaccharide extract can reduce the
ratio of Neu in BALF of the guinea pigs, and have the dose-effect
relationship, wherein the high-dosage group has a better
significant difference (p<0.01); meanwhile, all dose groups of
Asarum total polysaccharide extract can increase the ratio of Mac,
wherein the high-dosage group has a better significant difference
(p<0.01); compared with the Asarum total extract and the Asarum
total volatile oil, the high-dosage group of Asarum total
polysaccharide extract can significantly reduce the ratio of Neu;
and codeine phosphate cannot reduce the ratio of inflammatory cells
in BALF of airway hypersensitivity guinea pigs with airway
inflammation (see Table 6).
TABLE-US-00006 TABLE 6 Influence (mean +/- s, n = 8) of Asarum
total polysaccharide extract on the classification of cells in BALF
of guinea pig models with cough hypersensitivity Group Dose (mg/kg)
Mac % Neu % Lym % Eos % Blank control -- 77.0 +/- 1.0*** 13.3 +/-
0.7*** 8.4 +/- 1.1 1.3 +/- 0.3 Model group -- 46.5 +/- 1.7 44.4 +/-
1.7 7.1 +/- 0.8 2.1 +/- 0.7 Solvent control -- 46.9 +/- 3.4 46.4
+/- 2.7 4.7 +/- 0.7 2.0 +/- 0.7 Codeine phosphate 30 39.4 +/- 7.1
51.8 +/- 8.4 5.3 +/- 1.1 3.6 +/- 3.1 Low dose of 200 48.0 +/- 2.7
42.2 +/- 2.5 6.1 +/- 1.3 3.7 +/- 0.8 Asarum total polysaccharide
Medium dose of 400 51.6 +/- 4.5.sup..tangle-solidup. 37.2 +/- 2.9
7.6 +/- 1.8 3.5 +/- 0.7 Asarum total polysaccharide High dose of
800 61.9 +/-
1.5**.sup..tangle-solidup..tangle-solidup..tangle-solidup.# 29.4
+/- 0.9**.sup..tangle-solidup.# 7.1 +/- 2.1 1.6 +/- 0.4 Asarum
total polysaccharide Asarum total 1600 42.6 +/- 0.9 41.6 +/- 2.1
10.8 +/- 1.8 5.0 +/- 0.9 extract Asarum total 200 50.9 +/- 4.0 40.7
+/- 5.4 6.3 +/- 1.5 2.1 +/- 1.1 volatile oil Note: compared with
the model group, **p < 0.01, ***p < 0.001; compared with the
Asarum total extract group, .sup..tangle-solidup.p < 0.05,
.sup..tangle-solidup..tangle-solidup..tangle-solidup.p < 0.001;
and compared with the Asarum total volatile oil group, .sup.#p <
0.05.
[0098] (5) Observation on Influence of Asarum Total Polysaccharide
Extract on Pathology of Lung Tissues and Tracheal Tissues of Guinea
Pig Models with Cough Hypersensitivity
[0099] The pathological results of lung tissues of animals in each
group after CS-exposure and medicine intervention are as shown in
FIG. 3. The figures show that bronchi, alveoli, accompanying
arterioles of guinea pigs in a normal group (FIG. 3A) are complete
in structure; the ciliated epithelia of the airway are arranged in
order; there is no inflammatory cell for infiltration around
bronchi and blood vessels, and no hyperemia and edema. Compared
with the normal control group, the lung tissues of guinea pigs in
the model group (FIG. 3B) show typical pathological inflammatory
changes that the bronchial mucosal epithelium cilia are in a
lodging state, the bronchial walls are infiltrated by a large
number of inflammatory cells, the alveolar septa are widened, the
alveolar structure is disordered, and some alveolar septa are
fractured. Pathological changes of the lung tissues in the solvent
group (FIG. 3C) are almost the same as those in the model group.
Compared with the model group, the pathology of the lung tissues of
guinea pig in the high-, medium- and low-dosage groups of Asarum
total polysaccharide (FIG. 3E, F, G) is improved to different
degrees, wherein the improvement of the high-dosage group is the
most obvious; the bronchial and alveolar structures are almost
complete; the bronchial cilia have no obvious lodging; the airway
epithelial structure is almost complete; and the infiltration of
inflammatory cells around the lung tissues and the blood vessels is
reduced obviously. In the medium-dosage group, the alveolar septa
are slightly widened; and there are a small amount of inflammatory
cells for infiltration around the bronchi and the blood vessels. In
the low-dosage group, the bronchial and alveolar structures are
damaged; the airway cilia are in lodging state; and the
infiltration of inflammatory cells around the bronchi and the blood
vessels is obvious. Compared with the model group, the pathology of
the lung tissues in the Asarum total extract group (FIG. 3H) and
the Asarum total volatile oil group (FIG. 3I) is also improved
greatly; and the bronchial wall structure is almost complete. In
the total extract group, there are only a very small amount of
inflammatory exudates in the bronchial lumina; in the total
volatile oil group, there is almost no inflammatory exudate in the
bronchial luma; there are a small amount of inflammatory cells for
infiltration around the bronchi and the blood vessels; the
improvement degrees of the pathology of the lung tissues in the
both are substantially the same as that of the medium-dosage group
of Asarum total polysaccharide. Compared with the model group, the
pathology of the lung tissues in the codeine phosphate group (FIG.
3D) is also improved; the alveolar septa are widened; and the
infiltration of inflammatory cells around the bronchi and the blood
vessels is reduced.
[0100] The observation on the tracheal pathology of animals in each
group after CS-exposure and medicine intervention is as shown in
FIG. 4. The figures show that the tracheal epithelia of guinea pigs
in the normal group (FIG. 4A) are complete; the tracheal lumina are
regular; the tracheal walls are not thickened; cilia are arranged
in order; and there is almost no infiltration of inflammatory
cells. Compared with the normal control group, tracheae of the
guinea pigs in the model group (FIG. 4B) show typical pathological
inflammatory changes that tracheal mucosal epithelium cilia are in
the lodging state, the mucosal epithelial cells fall off, the
tracheal walls are infiltrated by a large number of inflammatory
cells, and the basement membranes are thickened in different
degrees. The tracheal pathological changes of the solvent group
(FIG. 4C) are almost the same as those of the model group. Compared
with the model group, the tracheal pathology of guinea pigs in the
high-, medium- and low-dosage groups of Asarum total polysaccharide
(FIG. 4E, F, G) is improved in different degrees; the tracheal
epithelium structure is almost complete; the basement membrane has
no obvious hyperplasia; the exudates in the lumina are reduced; the
cilia are arranged regularly; and there is no obvious infiltration
of inflammatory cells, wherein the improvement of the high-dosage
group is the most obvious. Compared with the model group, the
pathology of the tracheal tissues in the Asarum total extract group
(FIG. 4H) and the Asarum total volatile oil group (FIG. 4I) is also
improved; the airway epithelial cilia are in a slight lodging
state; the basement membrane is not obviously thickened; the
tracheal walls are infiltrated by a small amount of inflammatory
cells; and the improvement degrees of tracheal tissue pathology of
the both are slightly worse than that of the medium-dosage group of
Asarum total polysaccharide. The tracheal pathology in the codeine
phosphate group (FIG. 4D) is almost the same as that in the model
group, and is not improved obviously.
Embodiment 6
[0101] Taking the Asarum total polysaccharide extract prepared
according to the method of the Embodiment 1, adding distilled water
for dissolution, adding simple syrup to be more than 50% of the
sugar content, adding 0.3% of sodium benzoate and ethylparaben,
mixing the mixture uniformly, boiling and filtering the mixture
immediately, adding distilled water to a predetermined amount, and
sub-packaging the mixture to obtain Asarum total polysaccharide
cough syrup.
Embodiment 7
[0102] Taking 800 g of Asarum total polysaccharide extract prepared
according to the method of the Embodiment 1 and 40 g of aerosil;
screening the materials with the sieve of 60 meshes; mixing the
screened materials fully and uniformly; adding 24 g of magnesium
stearate; mixing; conducting dry granulation; screening the
obtained granules with the sieve of 40 meshes; and filling the
screened granules to form capsules.
Embodiment 8
[0103] Antitussive Effect of Different Asarum Polysaccharide
Extracts on Guinea Pig Models with Cough Hypersensitivity
[0104] Experimental materials are same as those of the Embodiment
5. The animals are grouped as follows: the blank group and the
model group (1 mL/kg of saline), the solvent group (CMC-Na aqueous
solution with the mass percentage concentration of 0.5%), the
positive medicine group (30 mg/kg of codeine phosphate), a group of
the Asarum total polysaccharide extract prepared in the prior art
(800 mg/kg, the preparation method comprises the following steps:
cutting the roots of Asarum Heterotropoides Fr. Schmidt var.
Mandshuricum (Maxim.) Kitag. into pieces, adding 20 times amount of
distilled water to soak the cut roots overnight, extracting
decoction from boiling water for 6 hours, filtering the decoction
with four layers of gauze, repeatedly decocting filter residues
twice, 6 hours each time, combining the filtrates, concentrating
the filtrate to 1000 ml, precipitating with 80% of ethanol,
centrifuging (4500 r/m, 10 min), and conventionally drying the
precipitates (washing the precipitates three times with each of
ethanol and ether) to obtain coarse Asarum polysaccharides (Li
Jingjing, Study on the Separation, Purification and Immunological
Activity of Polysaccharide from Asarum heterotropoides, Journal of
Changchun Normal University (Natural Science), 2008, 27(1):
54-58)), a group of the Asarum total polysaccharides extract in the
Embodiment 1 (800 mg/kg), and a group of the Asarum total
polysaccharide extract in the Embodiment 2 (800 mg/kg).The rest of
experimental methods are same as the Embodiment 5. Experimental
results show that the Asarum total polysaccharides prepared in the
prior art, the Embodiment 1 and the Embodiment 2 can significantly
reduce the cough frequency of guinea pigs (see Table 7), prolong
the cough latent period of guinea pigs, and also reduce the ratio
of Neu in BALF of the guinea pigs, wherein the Asarum total
polysaccharide prepared in the Embodiment 2 has the best effect,
and the Asarum total polysaccharides prepared in the prior art also
have antitussive activity, but do not get effect much better than
the total polysaccharides prepared in the present invention.
TABLE-US-00007 TABLE 7 Influence (mean +/- s, n = 8) of different
Asarum total polysaccharide extracts on the cough frequency of
guinea pig models with cough hypersensitivity Dose Cough frequency
Antitussive Group (mg/kg) (times) rate (%) Blank control -- 19.4
+/- 2.6** -- Model group -- 30.2 +/- 2.7 -- Solvent control -- 32.0
+/- 3.2 -- Codeine phosphate 30 5.7 +/- 1.9*** 81.1 Asarum total
800 10.7 +/- 1.4*** 64.6 polysaccharide prepared in the prior art
Asarum total 800 8.8 +/- 3.6*** 70.9 polysaccharide in the
Embodiment 1 Asarum total 800 8.2 +/- 1.4*** 72.8 polysaccharide in
the Embodiment 2 Note: compared with the model group, **p <
0.01, ***p < 0.001.
[0105] The technical features of all the above embodiments may be
combined arbitrarily. In order to make the description concise, all
possible combinations of all the technical features in the above
embodiments are not described one by one. However, as long as there
is no contradiction in the combinations of these technical
features, all the combinations should be considered within the
scope of the disclosure contained in the present description.
[0106] The above embodiments only express some embodiments of the
present invention, are described more specifically and in detail,
but shall not be understood as a limitation to the scope of the
invention patent. It should be noted that those skilled in the art
can also make several modifications and improvements, which belong
to the protection scope of the present invention, without departing
from the concept of the present invention. Therefore, appended
claims shall prevail in the protection scope of the invention
patent.
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