CN104027374B - Asarum total lignan extract and extraction method thereof, and application of asarum total lignan extract in preparation of drugs used for mitigating or inhibiting cough - Google Patents
Asarum total lignan extract and extraction method thereof, and application of asarum total lignan extract in preparation of drugs used for mitigating or inhibiting cough Download PDFInfo
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Abstract
The invention discloses an asarum total lignan extract and a preparation method and application thereof, belonging to the technical field of medicines. The preparation method comprises the following steps: extracting the root and/or tuber of asarum with a polar solvent and then carrying out concentration and drying so as to obtain an asarum total extract; and dissolving the asarum total extract with water, injecting the dissolved asarum total extract into a macroporous adsorption resin, carrying out gradient elution by using an aqueous ethanol solution, collecting eluate, recovering the solvent and carrying out drying so as to obtain the asarum total lignan extract with a content of 60 to 80 wt%, wherein yield of the asarum total lignan extract is 8.4 wt%. Through a great number of pharmacodynamic tests, it is proved for the first time that the asarum total lignan extract has substantial cough-inhibiting and cough reflex sensitivity-lowering effects which are close to the effects of codeine, and it is found out that the asarum total lignan extract has effects on inhibiting airway inflammations, reducing coughing frequencies and prolonging the incubation period of cough, exerts good prevention and treatment effects on a variety of coughs and can be used for preparation of drugs used for treating diseases related to cough. The asarum total lignan extract has the advantages of definite activity, usage of original plants as experimental materials and low cost.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, more specifically, it is related to a kind of Herba Asari total lignans extract and its extraction
Method and the purposes in preparation mitigation or suppression cough medicine.
Background technology
Due to factors impacts such as operating pressure, air ambient pollutions, the sickness rate of human respiratory system's disease occupies height
Under not, and coughing is respiratory system commonly encountered diseases, frequently-occurring disease, is the common complication of Pneumology Department outpatient service.Cough is that paging system is subject in itself
To a kind of defense reflex activity producing when stimulating, it is a kind of beneficial action, sometimes also sees healthy population.Ordinary circumstance
Under, need not apply the antitussive just can natural remission for cough slightly and infrequently.Additionally, the impact to health for the cough
Also it is dialectical, is also a kind of pathologic reaction, the cough of long term frequent or excessively fierceness not only can increase the misery of patient,
Impact rest and sleep, increase physical demandss, also can promote advancing of disease, produce other complication when serious.
According to the difference of the course of disease, cough can be divided into acute, subacute and chronic cough.Acute cough<3 weeks, subacute cough
Cough 3-8 week, chronic cough >=8 week.1）Acute cough：At present it is generally acknowledged that acute upper respiratory viral infection is modal disease
Cause, other include rhinitis, sinusitis, acute bronchitiss, acute episode of chronic bronchitis, bronchial asthma etc..Upper breathing
It is rhinovirus, coronavirus, respiratory syncytial virus, parainfluenza viruses, adenoviruss and enteropathy that road infects modal pathogen
Poison etc., wherein most common with rhinovirus.Acute cough is likely to the urgency with asthma, bronchitis and other respiratory system diseases
Property outbreak relevant.Clinically, often visible many infectious disease with acute cough as prodroma or some other illness；2）Sub-
Acute cough：Most common reason is postinfectious cough（Also known as postinfectious cough）, bacterial sinusitis and asthma etc..As cause of disease
The virus of body is common with rhinovirus, accounts for 30%～50%；Secondly it is coronavirus and influenza virus, respectively 10%～15% and 5%
～15%.In addition to Respiroviruses, mycoplasma pneumoniae and Chlamydia pneumoniae etc. also may result in postinfectious cough；3）Chronic cough：
Clinically generally by chronic cough definition with cough for unique symptom or cardinal symptom, time be more than 8 weeks, chest X-ray no
Substantially exception person, referred to as unknown cause chronic cough, abbreviation chronic cough.In special outpatient clinic, the ratio of chronic cough is up to
10%-38%.The questionnaire survey display chronic cough incidence rate of European and American countries is also up to 9%-33%.Commonly encountered diseases are because eosinophilic granulocyte
Property bronchitis（EB）, epithelium healing cough syndrome（UACS, former title postnasal drip symptom（PNDS））, gastroesophageal reflux induced coughs
Cough（GERC）, cough variant asthma（CVA）Deng.
Guangzhou Inst. of Respiratory Diseases's field investigation being directed to 1087 university students' coughs in 2006 shows, this colony
Total incidence rate of coughing is 10.9%, and the wherein total incidence rate of chronic cough is 3.3%, can speculate that the cough incidence of community-based population will
Higher, the average course of disease of this kind of patient is longer, the even up to many decades having.Data is had to show, the whole world there are about 15% every year
Adult and 10% minor are affected by different degrees of cough.Therefore, safely and effectively cough suppressing medicine will be full for exploitation
While sufficient great market demand, create preferable Social benefit and economic benefit.
Cough suppressing medicine can be divided into two big class by the difference according to drug mechanism：The first kind is central antitussive, this
Class medicine can the suppression medullary respiratory center of directly selecting property and produce antitussive effect, such as morphine, codeine etc., this kind of medicine town
Cough effect preferably, but side effect is substantially, may result in additive and respiration inhibition, or even induction hallucination, vain hope or other psychosiss
Symptom etc.；Equations of The Second Kind is peripheral antitussive drugs, and this kind of medicine blocks the biography of coughre flex by suppressing lung-vagus reflex
Enter impulsion, play antitussive effect, such as benproperine, benproperine phosphate, moguisteine etc., most is alkaloids medicament, can
Can produce the untoward reaction such as nausea, cardiopalmus, dizziness.Although the medicine being clinically used for antitussive is various, a lot of chronic coughs
Patient can not obtain effective alleviation with these Drug therapys.
Chinese medicine and pharmacy is the thousand of crystallization come abundant practical experience of preventing and curing diseases of the Chinese nation, and the traditional Chinese medical science can distinguish that doctor trained in Western medicine fails to understand it
Disease, Chinese medicine can control Western medicine refractory and incurable disease, and Chinese medicine has long history and rich experience to the treatment of cough,
Can complement one another with western medicine and medical practitioners.Although the domestic at present Chinese medicine having a lot for the treatment of coughs and Chinese patent medicine, these medicines
Active component, indication are not known.In addition, the Chronic Cough Patients for unknown cause and with airway inflammation, also do not have
Well medicine, thus research and development side effect is less and can the natural Chinese medicines of effectively treatment cough be to be engaged in respiratory system disease
The top priority of sick field pharmacy worker.
Chinese medicine Herba Asari, begins to be loaded in《Sheng Nong's herbal classic》, it is classified as top grade.2010 editions《Chinese Pharmacopoeia》Regulation Herba Asari is originated as horse
Semen Oroxyli section plant Herba Asari Asarum Heterotropoids Fr.Schmidt var.mandshuricum（Maxim.）
Kitag., Seoul Herba Asari Asarum sieboldii Miq.var.seoulense Nakai or asarum sieboldii Asarum
The dry root of sieboldii Miq. and rhizome.Warm in nature, acrid in the mouth, GUIXIN, lung, kidney channel.There is relieving exterior syndrome and dispelling cold, wind-expelling pain-stopping, lead to
Key, effect of warming the lung to resolve fluid-retention, for phlegm retention cough with asthma, anemofrigid cold, headache, toothache, nasal obstruction watery nasal discharge, allergic rhinitis, nasal sinusitis, rheumatic arthralgia
Treatment.Ancient books has Herba Asari to control the record coughed,《This warp》Meaning：" Herba Asari, main cough with dyspnea, headache brain move ... sharp nine orifices "；《Herbal guiding principle
Mesh》：" pungent can let out lung, therefore the upper gas person of cough due to wind and cold preferably uses it "；《Japan hanako materia medica》：" control and cough ... to tie in the heart and gather "；《Changsha medicine
Solution》：" kind fall rush inverse, special cough-relieving is coughed "；《Haigoushen》：" controlling cough with dyspnea ... " etc..At present about Herba Asari cough-relieving document report
Great majority concentrate on the methyleugenol composition in volatile oil（Zhou Huiqiu. methyleugenol pharmacological research. Chinese medicine and pharmacy
Report .2000, (2):79-80）.We find early-stage Study：Acute cough's guinea pig model that Herba Asari volatile oil excites to citric acid
There is obvious antitussive effect, but lower high quick guinea pig model antitussive effect of coughing is exposed for medicated cigarette and then waves apparently without Herba Asari is non-
The property sent out composition Herba Asari total lignans are good, and heavy dose has certain side effect.Additionally, for the research of Herba Asari involatile constituent
Report is also less, only has been reported that separation obtains involatile constituent kakuol, l- asarinin, l- sesame from Herba Asari root and rhizome
Numb fat element, Fructus Aristolochiae amide I, kaempferol -3-O- glucoside etc.（Cai Shaoqing. the research of Heterotropoides Var. Mandshuricum chemical composition.
Journal of Beijing Medical University .1996,28 (3):228-230；Lv Shuai. the chemical composition of Herba Asari under ground portion（Ⅱ）. Shenyang medicine
Section college journal .2010,27 (9):707-710；Wei Qingchun. the separation of Seoul Herba Asari chemical composition and identification. Shi Zhen traditional Chinese medical science state
Medicine .2010,21 (3):676-677）.Have not yet to see the report of preparation method about Herba Asari total lignans and assay,
More have no the research report about Herba Asari total lignans antitussive activity.
Content of the invention
For above the deficiencies in the prior art, the primary and foremost purpose of the present invention is to provide a kind of Herba Asari with antitussive activity
The extracting method of total lignans extract.
A further object of the present invention is the Herba Asari total lignans extract providing said extracted method to obtain.
It is still another object of the present invention to provide above-mentioned Herba Asari total lignans extract mitigates or suppression cough medicine in preparation
Purposes in thing.
The object of the invention is achieved through the following technical solutions：
A kind of extracting method of Herba Asari total lignans extract, including following operating procedure：By the root of Herba Asari and/or rhizome
Extracted with polar solvent, concentrate drying obtains Herba Asari total extract；After Herba Asari total extract is suspended with water dissolution, upper macroporous absorption tree
Fat, carries out gradient elution with ethanol water, collects eluent, recycling design, is dried, obtains final product Herba Asari total lignans extract；
Described gradient elution is to carry out eluting using the ethanol water that ethanol concentration of volume percent is 10%-50%.
Described polar solvent is water or concentration of volume percent is the ethanol solution of 10%-100%, more preferred volume basis
Specific concentration is the ethanol solution of 50%-90%.
The weight of described polar solvent is 5-20 times of the root of Herba Asari and/or rhizome weight.
Described Herba Asari is Herba Asari, Seoul Herba Asari or asarum sieboldii, more preferably Herba Asari.
Described macroporous resin is LS-300, LS-300B, AB-8, D101, NKA-9, X-5, HPD-100 or HPD-400.
A kind of Herba Asari total lignans extract being prepared by said method, wood in described Herba Asari total lignans extract
Fat cellulose content is 60wt%-80wt%, wherein asarinin（asarinin）Content be 40wt%-50wt%.
Above-mentioned Herba Asari total lignans extract is in the purposes that preparation mitigates or suppression is coughed in medicine, described Herba Asari hammer butt fat
Extract combines preparation treatment cough medicine individually or with other drugs.
Described Herba Asari total lignans extract and available pharmaceutical carrier make various dosage forms.
Described dosage form be granule, tablet, capsule, pill, drop pill, effervescent tablet, ointment, syrup, injection,
Oral liquid, mixture, tincture, slow releasing agent, controlled release agent or targeting preparation.
Pharmaceutically acceptable carrier is such as：Filler, binding agent, lubricant, disintegrating agent or wetting agent.Filler includes breast
Sugar, sucrose, corn starch, Sorbitol；Binding agent includes syrup, arabic gum, gelatin, Sorbitol, hydroxypropyl methyl cellulose
Or Polyvinylpyrrolidone；Lubricant includes magnesium stearate；Disintegrating agent includes starch, Polyvinylpyrrolidone, crospovidone, micro-
Crystalline cellulose；Wetting agent such as sodium lauryl sulphate.
Described asarinin（asarinin）, that is, structural formula is C20H18O6, chemical formula is 2,6-bis (3,4-
Methylenedioxyphenyl) the compound of -3,7-dioxabicyclo [3.3.0] octane.
The present invention can adopt ultraviolet spectrophotometry, with asarinin as reference substance, measures Herba Asari total at 280nm wavelength
Herba Asari effective total lignans content in lignans extract.
When chromatographic condition is：Chromatographic column is Agilent Zorbax SB-C18 post（4.6mm × 250mm, 5 μm）；Mobile phase
A phase is water, and B phase is acetonitrile, gradient elution, and elution program is：0-30min, 45%-60%B, 30-40min, 60%-100%B；Fortune
The row time：40min, equilibration time：10min；Volume flow：1.0mL/min；Detection wavelength：285nm；Column temperature：30℃；Sample introduction
Amount：10 μ L, can measure the content of asarinin.
The effective dose that the present invention has the Herba Asari total lignans extract of antitussive activity to be counted according to body weight 60kg of people
Calculate, be the 2.5g-11.0g/ man day.
The principle of the present invention is：
Pharmacological evaluation proves, the Herba Asari total lignans extract of the present invention has good mitigation and/or suppresses cough
Activity, can be used for preventing or treat various cough.
Results of pharmacodynamic test shows, for guinea pig acute cough model, citric acid excites rear Herba Asari total lignans to extract
The basic, normal, high dosage group of thing can extend the cough latent period of animal compared with blank control group, reduces the cough number of times of Cavia porcelluss；With
It is relatively weak to the acute antitussive activity of Cavia porcelluss that Herba Asari total volatile oil compares Herba Asari total lignans；And for chronic cough sensitivity
Increase and the guinea pig model with airway inflammation（Medicated cigarette exposes 14 days）, citric acid excite rear Herba Asari total lignans extract low,
Middle and high dosage group can be obviously prolonged the cough latent period of animal compared with model group, reduces the cough number of times of Cavia porcelluss, simultaneously permissible
Inflammatory cell sum in reduction Cavia porcelluss BALF and the ratio of neutrophilic granulocyte, suppress airway inflammation, reach or close to codeine
Effect；Compared with Herba Asari total extract and Herba Asari total volatile oil, Herba Asari total lignans extract middle and high dosage group antitussive rate is equal
There is significant difference（p<0.05）, display Herba Asari total lignans extract long-term medicated cigarette is exposed under the cough quick Cavia porcelluss mould of height
Type has more preferable antitussive activity.
The invention has the beneficial effects as follows：
（1）The present invention is that current antitussive medicine provides a kind of brand-new selection and thinking, has expanded the choosing of antitussive medicine
Select field, be also made that contribution for the development of this technical field.
（2）First demonstration that Herba Asari total lignans extract has significant antitussive effect, cough can be reduced quick
Perception and the effect of suppression airway inflammation, and no additive.
（3）The preparation technology of the Herba Asari total lignans extract that the present invention provides first can effectively be enriched with and go remove impurity
Matter, its Herba Asari total lignans yield and content are high, and quality control can pass through ultraviolet spectrophotometry and high performance liquid chromatography
Carry out assay analysis, the method is simple controlled.
（4）The method for preparing extractive that the present invention provides is simple, quick, favorable reproducibility；Selected solvent is cheap, no
Poison.
Brief description
The impact to acute cough's guinea pig model cough number of times for Fig. 1 Herba Asari total lignans extract（n=8）, wherein
A：Blank group；B：Solvent group（Mass fraction 0.5%CMC-Na）；C：Codeine sulfate 30mg/kg group；D Herba Asari total lignans
200mg/kg group；E：Herba Asari total lignans 400mg/kg group；F：Herba Asari total lignans 800mg/kg group；G：Herba Asari total extract
1600mg/kg group；H：Herba Asari total volatile oil 200mg/kg group；Compared with blank group：*p<0.05,**p<0.01,***p<0.001.
The impact of Fig. 2 Herba Asari total lignans extract quick guinea pig model cough number of times high to chronic cough（n=8）,
Wherein A：Blank group；B：Model group；C：Solvent group（Mass fraction 0.5%CMC-Na）；D：Codeine sulfate 30mg/kg group；E is thin
Pungent total lignans 200mg/kg group；F:Herba Asari total lignans 400mg/kg group；G：Herba Asari total lignans 800mg/kg group；H：Herba Asari
Total extract 1600mg/kg group；I：Herba Asari total volatile oil 200mg/kg group；Compared with model group：***p<0.001；Total with Herba Asari
Extract group is compared：▲p<0.05；Compared with Herba Asari total volatile oil group#p<0.05.
Fig. 3 Herba Asari total lignans extract high quick guinea pig model pathologic representative graph to chronic cough（HE dyes, ×
200）, wherein A：Blank group；B：Model group；C：Solvent group（Mass fraction 0.5%CMC-Na）；D：Codeine sulfate 30mg/kg
Group；E Herba Asari total lignans 200mg/kg group；F:Herba Asari total lignans 400mg/kg group；G：Herba Asari total lignans 800mg/kg group；
H：Herba Asari total extract 1600mg/kg group；I：Herba Asari total volatile oil 200mg/kg group.
Fig. 4 Herba Asari total lignans extract is to chronic cough Gao Min guinea pig model tracheal tissue pathology representative graph（HE dyes,
×200）, wherein A：Blank group；B：Model group；C：Solvent group（Mass fraction 0.5%CMC-Na）；D：Codeine sulfate 30mg/kg
Group；E Herba Asari total lignans 200mg/kg group；F:Herba Asari total lignans 400mg/kg group；G：Herba Asari total lignans 800mg/kg group；
H：Herba Asari total extract 1600mg/kg group；I：Herba Asari total volatile oil 200mg/kg group.
Specific embodiment
With specific embodiment, the present invention is described in further detail below in conjunction with the accompanying drawings, but not as the limit to the present invention
Fixed.
Embodiment 1
Prepared by Herba Asari total lignans extract
The Radix Asari being dried and/or rhizome 5kg, the ethanol with the concentration of volume percent 70% of medical material weight 10 times amount is molten
Liquid, heating and refluxing extraction three times, each 2h, extracting solution drying under reduced pressure, obtain Herba Asari total extract, yield is 12.3wt%；It is suspended with water
After Herba Asari total extract, upper LS-300B macroporous adsorptive resins, the water elution of 3BV removes impurity, then the percent by volume with 10BV
Concentration 50% ethanol elution, collects eluent, concentrating under reduced pressure, drying, obtains final product Herba Asari total lignans extract 421g, yield is
8.4wt%, Herba Asari total lignans content is 65wt%.
Embodiment 2
The acute antitussive effect to guinea pig cough caused by citric acid for the Herba Asari total lignans extract
1 experiment material
Laboratory animal：Regular grade Hartley Cavia porcelluss, male and female half and half, body weight 250-350g, by Guangdong Province medical experiment animal
Center provides, animal quality certification number：SCXK（Guangdong）2008-0002.
Reagent and instrument：Monohydrate potassium（Guangzhou Chemical Reagent Factory, lot number：20121001-2）, the used time is configured to
The normal saline solution of 0.8M；Animal noinvasive Buxco lung function system（Buxco company of the U.S.）；Aeroneb Pro is atomized
Device（Aerogen（Ireland）Company limited）；Gastric perfusion needle（Guangdong Medical Lab Animal Center）.
2 experimental techniques
Evaluating drug effect is carried out using citric acid induction guinea pig acute cough model.Take 128 Hartley Cavia porcelluss, by its by
One is placed in animal noinvasive Buxco lung function system 6L body retouches in instrument, drift meter air velocity 2.5L/min, uses
Aeroneb Pro nebulizer is passed through the citric acid ultra-pure water solution aerosol 1min of the 0.8M of atomization, and the particle of atomization is averagely straight
2.5 μm of footpath, observes 5min, the cough number of times in monitoring 6min, mike record and amplification that the sound of cough is placed by interior,
Sound wave is analyzed through Biosystem XA software, then processes voice data record cough number of times through computer software, simultaneously by warp
The observer crossing training supplements observation.Select the Cavia porcelluss that cough number of times is more than 10 times to be tested for qualified animal.
Take 64 qualified Hartley Cavia porcelluss of screening, male and female half and half, be randomly divided into 8 groups, blank group gives the life of 1mL/kg
Reason saline, solvent group gives isopyknic coordinative solvent（The CMC-Na aqueous solution of mass percent concentration 0.5%）, positive drug group
Give isopyknic 30mg/kg codeine phosphate, administration group gives isopyknic Herba Asari lignin extract（Embodiment 1 sample
200mg/kg、400mg/kg、800mg/kg）, Herba Asari total extract（1600mg/kg）, Herba Asari total volatile oil（200mg/kg, often
Rule steam distillation prepares gained）, once a day, successive administration 3 days.After last dose, 2h examines in animal noinvasive Buxco pulmonary function
Examining system carries out citric acid atomization provocative test（Atomization condition is the same）, it is atomized and excites 1min, induction Cavia porcelluss produce acute cough,
Observe 5min, calculate the cough number of times in 6min（N）, cough latent period, and with formula " antitussive rate=（NBlank-NAdministration group）/
NBlank× 100% " calculate the antitussive rate of each administration group.
Data is usedRepresent, Dan Yin is carried out to each group parameter using IBM SPSS Statistics19.0 statistical software
Plain variance analyses.Compare between being organized, p<0.05 is to have significant difference.
3 experimental results
（1）The impact to acute cough's guinea pig model cough number of times for the Herba Asari total lignans extract
Cough number of times in each group animal 6min after citric acid atomization excites and cough suppression ratio are shown in Table 1 and Fig. 1.By
Table 1 and Fig. 1 understand, compared with blank group, the basic, normal, high dosage group of Herba Asari total lignans extract all can significantly reduce Cavia porcelluss
Cough number of times, wherein low dose group has significant difference（p<0.05）, middle and high dosage has pole significant difference（p<
0.01）, antitussive rate respectively reaches 30%, 39.6% and 44.4%.Herba Asari total extract antitussive rate in 1600mg/kg dosage is
39.2%, and the middle and high dosage group of Herba Asari lignin extract antitussive activity in 400mg/kg, 800mg/kg dosage more always extracts
Thing be height, further relating to Herba Asari total lignans is one of main active component of Herba Asari antitussive, Herba Asari total lignans extract with
Between Herba Asari total extract, there was no significant difference.Herba Asari total volatile oil is all more total extraction than Herba Asari for the acute antitussive activity of Cavia porcelluss
Thing and Herba Asari total lignans are good（Antitussive rate 53.8%）, but between three, there are no significant difference.
Result above shows, Herba Asari total lignans extract has good acute antitussive effect, and has dose-effect relationship, and
Medication is comparatively safe.
The impact to acute cough's guinea pig model cough number of times for the table 1 Herba Asari total lignans extract（n=8）
Note：Compared with blank group：*p<0.05,**p<0.01,***p<0.001.
（2）The impact to acute cough's guinea pig model cough latent period for the Herba Asari total lignans extract
Cough latent period in each group animal 6min after citric acid atomization excites is shown in Table 2.As shown in Table 2, with blank
Group is compared, Herba Asari total lignans extract various dose group all can significant prolonged guinea pig cough latent period, wherein middle dosage
Group p<0.05, high dose group p<0.01, and have dose-effect relationship.It is latent that the middle and high dosage group of Herba Asari total lignans extract extends cough
The effect of volt phase is all better than codeine phosphate.Herba Asari total extract and Herba Asari total volatile oil can also be obviously prolonged cough latent period
（p<0.05）, and effect is also better than codeine phosphate.
The impact to acute cough's guinea pig model cough latent period for the table 2 Herba Asari total lignans extract（n=8）
Note：Compared with blank group：*p<0.05,**p<0.01.
Embodiment 3
Herba Asari total lignans extract increases the antitussive effect of guinea pig model to chronic cough sensitivity
1 experiment material
Laboratory animal：Regular grade Hartley Cavia porcelluss, male and female half and half, body weight 250-350g, by Guangdong Province medical experiment animal
Center provides, animal quality certification number：SCXK（Guangdong）2008-0002.
Reagent and instrument：Monohydrate potassium（Guangzhou Chemical Reagent Factory, lot number：20121001-2）, the used time is configured to
The normal saline solution of 0.8M；Animal noinvasive Buxco lung function system（Buxco company of the U.S.）；Aeroneb Pro is atomized
Device（Aerogen（Ireland）Company limited）；Cell counting count board（Zhejiang Yuhuan county Chu Men medical apparatus factory）；Gastric perfusion needle（Guangdong
Save medical experiment animal center）.
2 experimental techniques
Qualified Animal screening procedure is with embodiment 3.Take the qualified animal 72 of screening, be randomly divided into 9 groups, blank group and mould
Type group gives the normal saline of 1mL/kg, and solvent group gives isopyknic coordinative solvent（The CMC- of mass percent concentration 0.5%
Na aqueous solution）, positive drug group gives isopyknic 30mg/kg codeine phosphate, and administration group gives isopyknic Herba Asari hammer butt fat
Extract（Embodiment 1 sample 200mg/kg, 400mg/kg, 800mg/kg）, Herba Asari total extract（1600mg/kg）, Herba Asari
Total volatile oil（200mg/kg, conventional steam distillation prepares gained）, daily gastric infusion 1 time, successive administration 14 days.Except blank
Group is outer, every group of sootiness 20min, 10 red rose medicated cigarette every time（Guangdong China Tobacco Industry Co., Ltd, tar content 13mg, nicotine
Amount 1.3mg, CO content 15mg）, daily 2 times, continuous 14 days, set up Cavia porcelluss chronic cough evaluating drug effect model.Last cigarette
24h after smoked, excites 1min using citric acid atomization（Animal noinvasive Buxco lung function system, the same embodiment of atomization condition
3）, induction Cavia porcelluss produce cough, observe 5min, calculate the cough number of times in 6min（N）, antitussive rate and cough latent period.Animal
Excite rear 24h, 3% pentobarbital sodium anesthesia, heart extracting blood, the PBS row guinea pig bronchial alveolar wass of 6mL ice（BALF）, simultaneously
Take lung and tracheal tissue, 10% formaldehyde is fixed.
Observation procedure and index：
（1）Calculate citric acid and excite cough number of times in 6min, and with formula " antitussive rate=（NModel group-NAdministration group）/NModel group×
100% " calculate the antitussive rate of each administration group；
（2）Calculate the cough latent period that citric acid excites each administration group in 6min；
（3）Airway inflammation index：1. BALF total cellular score（TCC）：Atomization is taken to excite BALF irrigating solution after end 24h
0.5mL, 3000r/min are centrifuged 10min, precipitation plus 0.3mL erythrocyte cracked liquid, and vibration mixes, and stands 10min, 3000r/min
Centrifugation 10min, precipitation is suspended with 0.5mL PBS, takes cell counting count board row total cellular score under 10 μ L optical microscopes to count
（TCC）；2. BALF cell divide：Fixing BALF cell smear, flowing water rinses 5min, haematoxylin dyeing 20sec, and flowing water rinses
5min, 1% hydrochloride alcohol decolouring 5sec, flowing water rinses 5min, eosin stains 5sec, and flowing water rinses 5min, neutral gum after drying
Mounting, carries out cell divide under optical microscope；3. lung, tracheal tissue's pathological observation：Lung, tracheal tissue HE dyeing；Lung tissue
Whether main detection bronchus, alveolar, companion's row small artery structure are complete, and whether air flue Ciliated epithelium arrangement is neat, bronchus, blood
Have or not the infiltration of inflammatory cell around pipe, have or not congestion and edema etc.；Whether tracheal tissue's main detection epithelium is complete, and whether tube chamber
Regular, tube wall has or not and thickens, and whether cilium arrangement is neat, has or not and comes off, has or not inflammatory cell infiltration etc..
3 experimental results
（1）Herba Asari total lignans extract increases the impact of guinea pig model cough number of times to chronic cough sensitivity
Compared with blank group, citric acid atomization excites the model group Cavia porcelluss after medicated cigarette exposure 14d, the cough number of times in 6min
Significantly increase（p<0.001）, the high quick guinea pig model modeling success of cough.Compared with model group, Herba Asari total lignans extract is low,
Middle and high dosage group all extremely can significantly reduce the cough number of times of Cavia porcelluss（p<0.001）, antitussive rate respectively reaches 51.7%,
69.2% and 74.8%.Compared with Herba Asari total extract and Herba Asari total volatile oil, Herba Asari total lignans extract middle and high dosage group town
The rate of coughing is respectively provided with significant difference（p<0.05）, display Herba Asari total lignans extract long-term medicated cigarette is exposed under cough Gao Min
Guinea pig model has more preferable antitussive activity（The results are shown in Table 3 and Fig. 3）.
Result above shows, Herba Asari total lignans extract increases Cavia porcelluss to chronic cough sensitivity and has therapeutical effect,
And have dose-effect relationship, wherein Herba Asari total lignans extract high dose group is close to the therapeutic effect of codeine.
Table 3 Herba Asari total lignans extract increases the impact of guinea pig model cough number of times to chronic cough sensitivity（
n=8）
Note：Compared with model group：***p<0.001；Compared with Herba Asari total extract group：▲p<0.05；With Herba Asari total volatile oil
Group is compared#p<0.05.
（2）Herba Asari total lignans extract increases the impact of guinea pig model cough latent period to chronic cough sensitivity
After each group animal medicated cigarette exposes 14d, the cough latent period in 6min after citric acid atomization excites is shown in Table 4.By
Table 4 understands, compared with model group, the cough that Herba Asari total lignans extract various dose group all can be obviously prolonged Cavia porcelluss is hidden
Phase, wherein basic, normal, high dosage group p<0.001, and have dose-effect relationship.Compared with Herba Asari total extract and Herba Asari total volatile oil, carefully
Pungent total lignans extract high dose group cough latent period is respectively provided with significant difference（p<0.05）.
Table 4 Herba Asari total lignans extract increases the impact of guinea pig model cough latent period to chronic cough sensitivity（n=8）
Note：Compared with model group：**p<0.01,***p<0.001；Compared with Herba Asari total extract group：▲p<0.05；With Herba Asari
Total volatile oil group is compared#p<0.05.
（3）Herba Asari total lignans extract increases the impact of guinea pig model BALF total cellular score to chronic cough sensitivity
Compared with blank group, in model group BALF after medicated cigarette exposure 14d, total cellular score has and significantly increases（p<0.05）,
Modeling success.Compared with model group, Herba Asari total lignans extract various dose group all can significantly reduce inflammation in Cavia porcelluss BALF
Total cellular score（p<0.001）, and have dose-effect relationship；Compared with Herba Asari total extract, the middle and high dosage of Herba Asari total lignans extract
Group total cellular score counts and is respectively provided with significant difference（p<0.05）；Compared with Herba Asari total volatile oil, Herba Asari total lignans extract
High dose group total cellular score counts has significant difference（p<0.05）；Codeine phosphate can not reduce air flue hypersensitivity air flue
Inflammatory cell sum in inflammation Cavia porcelluss BALF（It is shown in Table 5）.
Table 5 Herba Asari total lignans extract increases the impact of guinea pig model BALF total cellular score to chronic cough sensitivity（n=8）
Compared with model group：*p<0.05,***p<0.001；Compared with Herba Asari total extract group：▲p<0.05；Total with Herba Asari
Volatilization line of oils is compared#p<0.05.
（4）Herba Asari total lignans extract increases the impact of guinea pig model BALF cell divide to chronic cough sensitivity
Compared with blank group, medicated cigarette expose 14d after model group and solvent control group BALF in macrophage（Mac）Ratio
Significantly reduce and neutrophilic granulocyte（Neu）Ratio significantly raises, and is respectively provided with significant differences（p<0.001）, modeling one-tenth
Work(.Compared with model group, Herba Asari total lignans extract various dose group all can reduce the ratio of Neu in Cavia porcelluss BALF, and has
Dose-effect relationship, wherein middle and high dosage group has significant differences（p<0.001）.The each dosage group of Herba Asari total lignans simultaneously
The ratio of Mac all can be raised, wherein low dose group has significant difference（p<0.05）, middle and high dosage group has extraordinary
Significant difference（p<0.01）；Compared with Herba Asari total extract and Herba Asari total volatile oil, Herba Asari total lignans extract can be notable
Reduction Neu ratio；Codeine phosphate can not reduce the inflammatory cell ratio in air flue hypersensitivity airway inflammation Cavia porcelluss BALF
Example（It is shown in Table 6）.
Table 6 Herba Asari total lignans extract increases the impact of guinea pig model BALF cell divide to chronic cough sensitivity（n=8）
Note：Compared with model group：*p<0.05,**p<0.01,***p<0.001；Compared with Herba Asari total extract group：▲p<
0.05,▲▲p<0.01；Compared with Herba Asari total volatile oil group#p<0.05,##p<0.01.
（5）Herba Asari total lignans extract increases the sight of guinea pig model lung, tracheal tissue's pathology to chronic cough sensitivity
Examine
After the exposure of each group animal medicated cigarette and pharmaceutical intervention, pathologic result is as shown in Figure 3.As seen from the figure, normal group
（Fig. 3 A）Guinea pig bronchial, alveolar, companion's row small artery structural integrity, air flue Ciliated epithelium's marshalling, around bronchus, blood vessel
The no infiltration of inflammatory cell, no congestion and edema；Compared with Normal group, model group（Fig. 3 B）Guinea pig lung tissue assumes typical case's disease
Reason inflammation changes, and bronchial mucosa epithelial cilia lodges, exuviation, official jargon inflammatory exudate, a large amount of inflammation of peribronchial
Cellular infiltration, alveolar septum is broadening, and alveolar structure is disorderly, and part alveolar septum ruptures；Solvent group（Fig. 3 C）Pathologic changes
Become substantially same model group；Compare with model group, Herba Asari total lignans are high, middle dose group（Fig. 3 E, F）Guinea pig lung tissue's pathology all has
Improve in various degree, wherein improved with high dose group the most obvious, bronchus, alveolar structure are substantially complete, and bronchus cilium is no
Substantially lodge, skin no substantially comes off, and bronchus, perivascular inflammation cellular infiltration significantly reduce；Middle dose group alveolar septum is slightly
Micro- broadening, visible a small amount of inflammatory cell infiltration around trachea, blood vessel；Low dose group（Fig. 3 G）Bronchus, alveolar structure destroy, gas
Road cilium lodging, bronchus, perivascular inflammatory cell infiltration are substantially；Herba Asari total extract group（Fig. 3 H）And Herba Asari total volatile oil
Group（Fig. 3 I）Pathologic relatively model group also has largely to be improved, and bronchial wall structure is substantially complete, wherein total extract
The group a small amount of inflammatory exudation of bronchial lumen, total volatile oil group bronchial lumen has no inflammatory exudation, a small amount of inflammatory around bronchus, blood vessel
Cellular infiltration, both pathologics improve suitable with Herba Asari total lignans middle dose group；Codeine phosphate group（Fig. 3 D）Lung group
Knit pathology also to make moderate progress compared with model group, alveolar septum is broadening, bronchus, perivascular inflammatory cell infiltration reduce.
After the exposure of each group animal medicated cigarette and pharmaceutical intervention, trachea pathological observation is as shown in Figure 4.As seen from the figure, normal group（Figure
4A）Guinea pig tracheal epithelia is complete, and tube chamber is regular, and tube wall no thickens, cilium marshalling, basic NIP cellular infiltration；With just
Often matched group is compared, model group（Fig. 4 B）Guinea pig trachea present typical pathologic inflammation change, tunica mucosa tracheae epithelial cilia lodging, on
Chrotoplast comes off, a large amount of inflammatory cell infiltration of Submucosa, and basement membrane thickens in various degree；Solvent group（Fig. 4 C）Trachea pathology changes
Become substantially same model group；Compare with model group, Herba Asari total lignans are high, middle dose group（Fig. 4 E, F）Guinea pig trachea pathology difference journey
Degree improves, and is wherein improved the most obvious with high dose group, tracheal epithelium structure is substantially complete, and cilium arranges fundamental rule, substrate
The no obvious hypertrophy of film, in tube chamber, exudate reduces, no obvious inflammatory cell infiltration.Middle dose group still visible cilium lodging, epithelium
Come off, tube wall inflammatory cell infiltration；And low dose group（Fig. 4 G）Tracheal tissue's pathology improves very unobvious；Herba Asari total extract group
（Fig. 4 H）Tracheal tissue's pathology is improved very substantially compared with model group, and cilium comes off seriously, a small amount of inflammatory cell of Submucosa
Infiltration；Herba Asari total volatile oil group（Fig. 4 I）Improve more obvious, substantially with Herba Asari total lignans middle dose group；Codeine phosphate group
（Fig. 4 D）The substantially same model group of trachea pathology, has no and is obviously improved.
Embodiment 4
The Herba Asari total lignans extract of Example 1 method preparation, plus distillation water dissolution, add simple syrup to containing saccharic
Amount content more than 50%, adds 3/1000ths sodium benzoate and ethyl hydroxybenzoate, mix homogeneously, boils, filtered while hot, plus distillation
Water is Herba Asari total lignans cough syrup to ormal weight, subpackage.
Embodiment 5
The Herba Asari total lignans extract 800g and micropowder silica gel 40g of Example 1 method preparation, crosses 60 mesh sieves fully mixed
Even, add magnesium stearate 24g, mixing, dry granulation.Cross 40 mesh sieves, fill becomes capsule.
Above-mentioned specific embodiment is the preferred embodiments of the present invention, can not limit the invention, other
Change or other equivalent substitute mode that what is made without departing from technical scheme, are included in the protection of the present invention
Within the scope of.
Claims (10)
1. a kind of extracting method of Herba Asari total lignans extract is it is characterised in that include following operating procedure：Root by Herba Asari
And/or rhizome is extracted with polar solvent, concentrate drying obtains Herba Asari total extract；After Herba Asari total extract is suspended with water dissolution, on
LS-300B macroporous adsorbent resin, removes impurity with the water elution of 3BV, then is washed with concentration of volume percent 50% ethanol of 10BV
De-, collect eluent, concentrating under reduced pressure, be dried, obtain final product Herba Asari total lignans extract, wherein lignanoid's content is 60wt%-
80wt%；Described polar solvent is the ethanol solution of 50%-90% for concentration of volume percent.
2. extracting method according to claim 1 it is characterised in that：The weight of described polar solvent be Herba Asari root with/
Or 5-20 times of rhizome weight.
3. extracting method according to claim 1 it is characterised in that：Described Herba Asari is Herba Asari, Seoul Herba Asari or China are thin
Pungent.
4. extracting method according to claim 3 it is characterised in that：Described Herba Asari is Herba Asari.
5. a kind of Herba Asari total lignans extract being prepared by claim 1-4 any one methods described it is characterised in that：
In described lignanoid, the content of asarinin is 40wt%-50wt%.
6. a kind of Herba Asari total lignans extract according to claim 5 mitigates in preparation or suppresses in chronic cough medicine
Purposes it is characterised in that：Described Herba Asari total lignans extract combines preparation treatment chronic cough individually or with other drugs
Medicine.
7. purposes according to claim 6 it is characterised in that：Described Herba Asari total lignans extract and available pharmaceutical carrier
Make various dosage forms.
8. purposes according to claim 7 it is characterised in that：Described dosage form is granule, tablet, capsule, pill, soft
Unguentum, injection, oral liquid, tincture, slow releasing agent, controlled release agent or targeting preparation.
9. purposes according to claim 7 it is characterised in that：Described dosage form is drop pill, effervescent tablet or syrup.
10. purposes according to claim 7 it is characterised in that：Described dosage form is mixture.
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