Application of Asarum total polysaccharides in preparation of medicine for treating cough

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.

Claims

1. A method of treating cough, comprising: administering an effective amount of a composition comprising Asarum total polysaccharides to a subject in need thereof, wherein the Asarum total polysaccharides are prepared by the following steps: a. subjecting roots and/or rhizomes of Asarum to a heat reflux using water; b. concentrating and drying under vacuum to produce an Asarum total extract; c. dissolving the Asarum total extract in water to produce an Asarum total extract solution and adding 60-99% ethanol by volume into the Asarum total extract solution to completely precipitate to produce a supernate and a precipitate; d. discarding the supernate and filtering the precipitate under vacuum; e. washing the filtered precipitate with 60-100% ethanol by volume and drying under vacuum to obtain the Asarum total polysaccharides.

2. The method of claim 1, wherein the cough is chronic cough.

3. The method of claim 1, wherein the content of polysaccharides in the Asarum total polysaccharides is 60-80 wt %.

4. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

5. The method of claim 4, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a filler, a binder, a lubricant, a disintegrant and a wetting agent.

6. The method of claim 4, wherein a formulation of the composition is selected from the group consisting of granule, tablet, capsule, pill, dripping pill, effervescent tablet, ointment, syrup, injection, oral liquid, mixture, tincture, sustained-release preparation, controlled-release preparation and targeting preparation.

7. The method of claim 1, wherein the Asarum is selected from Asarum Heterotropoides Fr. Schmidt var. Mandshricum (Maxim.) Kitag., Asarum sieboldii Miq. var. seoulense Nakai or Asarum sieboldii Miq.

Description

DESCRIPTION OF THE DRAWINGS

(1) 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.

(2) 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, .sup..box-tangle-solidup.p is less than 0.05; and compared with the Asarum total volatile oil group, #p is less than 0.05.

(3) FIG. 3 shows a representative diagram (HE staining, 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.

(4) FIG. 4 shows the representative diagram (HE staining, 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

(5) 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.

(6) In one embodiment, an application of the Asarum total polysaccharide extract in the preparation of a medicine for preventing and treating coughs is provided.

(7) 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) 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.

(13) 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.

(14) 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

(15) A method for preparing the Asarum total polysaccharide extract of the present embodiment comprises the following steps:

(16) 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 an extracting solution with 6 layers of gauze; decompressing, concentrating and drying the filtrate to obtain the Asarum total extract;

(17) 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 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

(18) A method for preparing the Asarum total polysaccharide extract of the present embodiment comprises the following steps:

(19) 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;

(20) 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 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

(21) Test for acute toxicity of Asarum total polysaccharide extract orally and intragastrically administered to mice.

(22) 1. Animals

(23) 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.

(24) 2. Method

(25) 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 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.

(26) 3. Results

(27) 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. LD50 of the Asarum total polysaccharide is more than 10.0 g/kg.

(28) The above results indicate that the Asarum total polysaccharide has high safety and has no risk of severe acute poisoning.

Embodiment 4

(29) Acute antitussive effect of Asarum total polysaccharide extract on cough of guinea pigs induced by citric acid

(30) 1. Materials

(31) 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.

(32) 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).

(33) 2. Method

(34) 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 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.

(35) 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=(Nblank control groupNadministration group)/Nblank control group100%.

(36) 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.

(37) 3. Results

(38) (1) Influence of Asarum Total Polysaccharide Extract on the Cough Frequency of Guinea Pig Models with Acute Cough

(39) 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.

(40) 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.

(41) 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 Dose Cough frequency Antitussive rate Group (mg/kg) (times) (%) 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 400 20.6 +/ 2.3* 28.5 of 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.

(42) (2) Influence of Asarum Total Polysaccharide Extract on the Cough Latent Period of Guinea Pig Models with Acute Cough

(43) 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).

(44) 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 Dose Cough latent period Group (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 200 118.9 +/ 24.7* total polysaccharide Medium dose of Asarum 400 133.3 +/ 29.5** total polysaccharide High dose of Asarum 800 140.9 +/ 19.7** total 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

(45) Antitussive effect of Asarum total polysaccharide on guinea pig models with cough hypersensitivity

(46) 1. Materials

(47) 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.

(48) 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).

(49) 2. Method

(50) 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.

(51) Observation Method and Indicators:

(52) (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=(Nmodel groupNadministration group)/Nmodel group100%.

(53) (2) Calculating the cough latent period of each administration group within 6 min after citric acid excitation;

(54) (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 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.

(55) 3. Results

(56) (1) Influence of Asarum Total Polysaccharide Extract on the Cough Frequency of Guinea Pig Models with Cough Hypersensitivity

(57) 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).

(58) 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.

(59) 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 rate Group (mg/kg) (times) (%) Blank control 19.4 +/ 2.6** Model group 30.2 +/ 2.7 Solvent control 32.0 +/ 3.2 Codeine 30 5.7 +/ 1.9*** 81.1 phosphate Low dose of 200 13.9 +/ 1.7*** 54.0 Asarum total polysaccharide Medium dose 400 11.3 +/ 0.9*** 62.6 of Asarum total polysaccharide High dose of 800 8.8 +/ 3.6***.sup.<.box-tangle-solidup. #> 70.9 Asarum total polysaccharide Asarum total 1600 16.2 +/ 2.3*** 46.4 extract Asarum total 200 16.2 +/ 1.4*** 46.4 volatile oil Note: compared with the model group, **p < 0.01, ***p < 0.001; compared with the Asarum total extract group, .sup.<.box-tangle-solidup.>p < 0.05; andcompared with the Asarum total volatile oil group, .sup.<#>p < 0.05.

(60) (2) Influence of Asarum Total Polysaccharide Extract on the Cough Latent Period of Guinea Pig Models with Cough Hypersensitivity

(61) 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).

(62) 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 Dose Cough latent period Group (mg/kg) (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 200 100.3 +/ 17.8 total polysaccharide Medium dose of Asarum 400 156.1 +/ 25.0*** total polysaccharide High dose of Asarum 800 189.0 +/ 20.4***.sup.<.box-tangle-solidup. #> total 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.<.box-tangle-solidup.>p < 0.05; and compared with the Asarum total volatile oil group, .sup.<#>p < 0.05.

(63) (3) Influence of Asarum Total Polysaccharide Extract on TCC in BALF of Guinea Pig Models with Cough Hypersensitivity

(64) 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).

(65) 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 Dose TCC Group (mg/kg) (10<5>, cells/mL) Blank control 10.8 +/ 2.7*** Model group 27.8 +/ 1.2 Solvent control 26.1 +/ 1.3 Codeine 30 26.5 +/ 5.9 phosphate Low dose of 200 16.5 +/ 5.2** Asarum total polysaccharide Medium dose 400 8.1 +/ 0.6***.sup.<.box-tangle-solidup. #> of Asarum total polysaccharide High dose of 800 .sup.7.8+/0.6***.sup.<.box-tangle-solidup. #> Asarum total polysaccharide Asarum total 1600 16.1 +/ 1.2** extract Asarum total 200 17.6 +/ 2.3* volatile oil Note: compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the Asarum total extract group, .sup.<.box-tangle-solidup.>p < 0.05; and compared with the Asarum total volatile oil group, .sup.<#>p < 0.05.

(66) (4) Influence of Asarum Total Polysaccharide Extract on the Classification of Cells in BALF of Guinea Pig Models with Cough Hypersensitivity

(67) 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).

(68) 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 Dose Group (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 30 39.4 +/ 7.1 51.8 +/ 8.4 5.3 +/ 1.1 3.6 +/ 3.1 phosphate 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 400 .sup.51.6 +/ 4.5.sup.<.box-tangle-solidup.> 37.2 +/ 2.9 7.6 +/ 1.8 3.5 +/ 0.7 of Asarum total polysaccharide High dose of 800 .sup.61.9 +/ 1.5**.sup.<.box-tangle-solidup. .box-tangle-solidup. .box-tangle-solidup. #> 29.4 +/ 0.9**.sup.<.box-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.<.box-tangle-solidup.>p < 0.05, .sup.<.box-tangle-solidup. .box-tangle-solidup. .box-tangle-solidup.>p < 0.001; and compared with the Asarum total volatile oil group, .sup.<#>p < 0.05.

(69) (5) Observation on Influence of Asarum Total Polysaccharide Extract on Pathology of Lung Tissues and Tracheal Tissues of Guinea Pig Models with Cough Hypersensitivity

(70) 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.

(71) 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

(72) 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

(73) 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

(74) Antitussive Effect of Different Asarum Polysaccharide Extracts on Guinea Pig Models with Cough Hypersensitivity

(75) 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.

(76) 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 rate Group (mg/kg) (times) (%) Blank control 19.4 +/ 2.6** Model group 30.2 +/ 2.7 Solvent control 32.0 +/ 3.2 Codeine 30 5.7 +/ 1.9*** 81.1 phosphate 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.

(77) 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.

(78) 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.