CN102106914A - Medicament for treating infectious diseases, preparation method and application thereof - Google Patents

Abstract

The invention provides a medicament for treating infectious diseases, a preparation method and the application thereof. The medicament is prepared from scutellaria baicalensis, jasmine, erigeron breviscapus and cholic acid, and has the following effects: by aiming at the central link--inflammatory reaction of an infectious disease, removing harmful substances such as inflammatory mediators, free radicals and the like, and improving the local microcirculation state, thereby alleviating the pathological damages caused by inflammations; meanwhile, the medicament has the function of regulating the overall internal environment, and can effectively treat lower respiratory tract infection and acute lung injury induced by infection; and the effective substances and functional mechanism of the medicament are relatively clear.

Description

Medicine, the Preparation Method And The Use of treatment infectious disease

Technical field

The invention belongs to the field of Chinese medicines, relate to a kind of medicine, Preparation Method And The Use for the treatment of infectious disease.

Background technology

The lower respiratory infection disease, common chronic bronchitis inflammation, various types of pneumonia, bronchiectasis and infection, lung abscess, pulmonary heart disease and pulmonary infection etc. are respiratory system commonly encountered diseases, frequently-occurring disease.Sickness rate is the highest in all infectious disease, even in today that the antibacterials exploitation is advanced by leaps and bounds, its sickness rate, case fatality rate are still high.China has 2,500,000 routine pneumonia to take place every year approximately, and 12.5 ten thousand people's (5%) are because of pneumonia death.In the U.S., pneumonia accounts for 1/10 of AD, occupies the 5th of the cause of death.Infantile pneumonia is first cause of death of China children's, and its mortality rate accounts for 1/5 of death of child.Add up according to The World Health Organization (WHO), infectious disease accounts for 1/3 in the world population cause of death at present, and acute respiratory infection (being mainly pneumonia) death toll account for wherein 1/4, many chronic disease patients die from protopathy, but die from pulmonary infection.Over nearly 20 years, the variation that the bacterium of pulmonary infection spectrum is taken place is well known, and the secondary of mycete also causes clinicist's attention.A variety of causes causes immunodeficiency patient's increase, makes some seldom morbific pathogen such as Pneumocystis carinii and cytomegaloviruses in the past, threatens patient's life equally, has become the difficult problem in diagnosis and the treatment.Because the transition of pathogen, the rising of bacterial resistance rate, the aging of population, immunity infringement host increases, increasing that the development of critical care medicine and mechanical ventilation are used makes present respiratory tract infection complicated day by day, especially begin in February, 2003 to involve the atypical pneumonia (SARS) of China and Southeast Asia and European and American countries and wreaked havoc bird flu for the moment in 2005, control proposes a new extremely stern challenge to respiratory system infection.

Inflammatory reaction is the core link of lower respiratory infection disease, and the main treatment measure of injury of lung that causes at inflammatory reaction at present has:

(1) antibiotic

Be to use the antibiotic control infection at the topmost Therapeutic Method of pathogen at present, clinical in different pathogen, antibiosis commonly used have beta-lactam (penicillins and cephalosporin two big classes), quinolones, Macrolide, aminoglycosides etc.

(2) glucocorticoid

Glucocorticoid (hereinafter to be referred as hormone) has and alleviates infectious and non-infectious inflammatory reaction, and minimizing is oozed out, the inhibition immunoreation, and shock, pharmacological actions such as antitoxin are applied to treating the injury of lung that inflammatory reaction causes.

(3) vasoactive agent

There is tangible lung microcirculation disturbance in severe pneumonia, and vasoactive drug is obtained obvious effects as adjuvant drug in the treatment of severe pneumonia of infants in recent years.Commonly used have an angiotensin converting enzyme inhibitor, by suppressing Angiotensin-Converting, Angiotensin II and aldosterone are generated reduce, thereby expansion artery reduces water-sodium retention.The activity that suppresses kininase II simultaneously makes the kassinin kinin degraded reduce prostaglandin (PGE ₂, PGI ₂) synthetic increasing, the expansible vein of the latter improves the lung microcirculation.

(4) inflammatory cytokine antagonist

In the cytokine relevant with struvite injury of lung of greatest concern and research more be tumor necrosis factor (TNF), IL-1 β, IL-6 and IL-8.When lung epithelial and endothelium were subjected to directly or indirectly damaging, this type cytokines great expression and secretion by activating neutrophilic granulocyte, made it ooze out, assemble, mediate injury of lung with effects such as peripheral cell take place to adhere to.The antagonist of cytokine antibodies, inhibitor and receptor thereof plays the effect that the retardance inflammation takes place, develops, thereby brings into play tangible clinical efficacy by the above-mentioned effect of blocking-up cytokine.

(5) neutrophil elastase inhibitor

Neutrophil elastase can decompose multiple proteins and comprise extracellular matrix components, elastin laminin, Fibrinogen, proteoglycan and collagen, and can damage capillary endothelium and cause pulmonary edema.Therefore, neutrophil elastase is a target spot of the scorching card damage of treatment pulmonary.

(6) antiprostaglandin class

Experimentation shows that in the acute stage of inflammatory reaction, prostaglandin produces to increase and mainly depends on the synthetic of cyclooxygenase (COX)-2.Macrophage is considered to COX-2 and raises the main source that the back produces prostaglandin, and the expression that can induce IL-6 of the prostaglandin that produces of COX catalysis, so selectivity inhibition COX-2 can reduce the generation of the IL-6 of pulmonary macrophage.In addition, induce under the Corium Mus of generation in the edema due to disorder of QI model and pleuritis model at carrageenin, the reaction that can successfully reduce inflammation of special COX-2 blocker, prompting COX-2 blocker can be applicable to treat various inflammation and does not produce harmful side effect.

(7) associated treatment of active oxygen

Oxidation and antioxidation equilibrium relation are arranged in the organism, and SOD, catalase (CAT), peroxidase etc. are important antioxidants in the body.During inflammatory reaction, a large amount of oxygen-derived free radicals produce and cause oxidation and antioxidation dysequilibrium.The exogenous SOD of giving, CAT can reduce O ₂, H ₂O ₂Generation and correct PGI ₂/ TXA ₂The balance disorder alleviates injury of lung.

(8) traditional Chinese medical herbal treatment

According to the pathological characteristic of pneumonia infection disease, the center of Chinese medicine focuses mostly on the basis of heat-clearing and toxic substances removing, the harm of keypoint control expectorant heat.For example SHUANGHUANGLIAN FENZHENJI is made up of Flos Lonicerae, Fructus Forsythiae, Radix Scutellariae, and modern pharmacological research confirms that this medicine has obviously antibiotic, antivirus action.Be widely used in clinical infectious disease in recent years.Research by etiology and pathology aspect, pointing out this medicine is many-sided to the effect of infectious disease, and not only having showed has certain inhibition and killing action to virus, pathogenic bacterium, also has the effect of enhancing human body immunity power simultaneously, suppressing virus, eliminate more apparent its advantage in symptom aspect.Form from its medicine, be not only applicable to the initial stage of affection due to external wind and heat, pathogenic wind-warm, and all can use the excess-heat syndrome of multiple epidemic febrile disease.But this injection is intravenously administrable not only, and atomizing suction effect same is remarkable, can make medicine directly act on trachea or bronchial mucosa, helps eliminating the inflammatory hyperemia edema, and cough-suppressing phlegm-dispelling functions is preferably arranged.

As-Is analysis about the said medicine treatment is as follows:

Antibiotic has improved the cure rate of people to infectious disease at the beginning of using greatly, has prolonged the human life-span.Yet, because antibiotic long-term unreasonable application, a lot of problems have appearred in present stage, except toxic reaction (as to nervous system, blood system, liver, renal function injury, gastrointestinal reaction), the anaphylaxis of antibiotic itself, easily cause the superinfection, the subject matter that current respiratory tract infection treatment is faced is that the bacterial resistance situation is more and more serious, becomes increasingly complex.No matter be gram positive bacteria or gram negative bacteria, we have run into some ticklish antibacterials, some does not have reliable medicine so far these antibacterials, and the other drug-resistance of bacteria is increasing just with surprising rapidity, and the pathogen transition have brought many new difficult problems for clinical treatment and rehabilitation.

At the SARS epidemic period, the powerful antiinflammatory action of hormone is used to prevent tissue injury and the organ failure due to the excessive defense reaction that body infects SARS virus, be comparatively successful aspect the reduction case fatality rate, but when having saved a lot of patient's life, also brought many-sided side effect, as the convalescent osteonecrosis of SARS, superinfection, tuberculosis send out, electrolyte disturbance and diabetes etc., so application of hormone is still a controversial problem.

New drug at the research and development of inflammation cascade reaction pathology link, as inflammatory cytokine antagonist, neutrophil elastase inhibitor and antiprostaglandin class etc., still be in experiment in vitro and animal experiment stage now mostly, only have the sub-fraction medicine to enter clinical research.In nearest 10 years, the foreign scholar once carried out on a large scale, consumed huge fund, the international perspective clinical trial of multicenter for the treatment of antagonism inflammatory mediator, from the multinomial phase iii clinical trial result who has finished, can not prove aspect relief of symptoms and reduction case fatality rate helpful.

The traditional Chinese medical science adopts traditional decoction to treat such disease and has done many trials, and to having many Chinese medicine granules, oral liquid and vein injection to come out on the ancient prescription sanction basis again, although have typing, by stages, opinion controls the difference of internal organs, heat-clearing and toxic substances removing still is its basic rule of treatment, but the heat-clearing and toxic substances removing decoct can make blood viscosity raise, fibrinolytic reduces, and this shows that cold heat and toxic materials clearing away medicine produces harmful effect and side effect to blood fluidity and microcirculation.In addition, Chinese traditional treatment lacks theoretic breakthrough, the mechanism such as effective ingredient, model of action, approach and target spot of Chinese medicine compound performance curative effect are still not fully aware of, and traditional oral administration mode produces effects slowly, it can only be included in the scope of adjuvant drug in the clinical treatment yet.

The strategy that controls inflammation that need seek renewal is all pointed out in all these aspects.

Summary of the invention

An object of the present invention is to provide a kind of medicine for the treatment of infectious disease, reach therapeutical effect by improving the injury of lung that inflammatory reaction causes.Another object of the present invention provides the Preparation method and use of described medicine.

The objective of the invention is to be achieved through the following technical solutions:

The invention provides a kind of medicine for the treatment of infectious disease, described medicine comprises following composition: Radix Scutellariae, Fructus Gardeniae, Herba Erigerontis and cholic acid.

Further, described medicine only comprises following four flavor effective ingredient: Radix Scutellariae, Fructus Gardeniae, Herba Erigerontis and cholic acid.

Further, described medicine, its main component comprises by weight: 60～120 parts of Radix Scutellariaes (in baicalin), 25～75 parts of Fructus Gardeniaes (in jasminoidin), 2～4 parts of Herba Erigerontiss (in lamp-dish flower acetic), 25～75 parts of cholic acid.

Further again, preferably, described medicine, its main component comprises by weight: 120 parts of Radix Scutellariaes (in baicalin), 25 parts of Fructus Gardeniaes (in jasminoidin), 4 parts of Herba Erigerontiss (in lamp-dish flower acetic), 25 parts of cholic acid.

Described medicine provided by the invention is selected from following preparation: injection, tablet, granule, capsule, soft capsule, drop pill, oral liquid, powder, pill.

The present invention also provides the preparation method of described medicine, may further comprise the steps:

1) get the Radix Scutellariae extracting in water three times, each 60min filters, collect filtrate, merge, be concentrated into original volume about 1/2, add hydrochloric acid and regulate pH value to 1～2, standing over night, centrifugalize, precipitate adds water, adds lime water adjust pH to 7.0, filters, collect filtrate and regulate pH value to 1～2, standing over night, centrifugalize with hydrochloric acid, precipitation is used washing with alcohol, and drying promptly gets baicalin;

2) get the Fructus Gardeniae extracting in water three times, each 60min filters, and filtrate concentrates, and last resin column is used 50% ethanol elution, collects eluent, concentrating under reduced pressure, and drying, promptly;

3) get Herba Erigerontis and add 75% alcohol reflux three times, each 2h filters filtrate recycling ethanol, water precipitating, last resin column, water eluting, collect eluent, concentrate, filter, precipitation leaves standstill with 10% sulfuric acid solution adjust pH to 2.0～2.5, filters, precipitate with ethanol, be washed to neutrality, drying is used ethyl alcohol recrystallization, drying is pulverized, promptly;

(4) get above step 1)-3) three kinds of extracts of gained and cholic acid mix homogeneously, make preparation by the method that this area is commonly used, preferably, described cholic acid is according to " ursodesoxycholic acid of Chinese pharmacopoeia (version was two ones in 2010) preparation or the Hyodeoxycholic Acid for preparing according to " national drug standards ";

5) get above step 1)-4) four kinds of composition mix homogeneously of gained, add right amount of auxiliary materials, with water for injection dissolving, ultrafiltration, injection is made in packing;

6) get above step 1)-4) four kinds of composition mix homogeneously of gained, add right amount of auxiliary materials, make oral formulations respectively.

The present invention also provides described medicine to be used for the treatment of application in the medicine of lower respiratory infection disease in preparation.

Further, described lower respiratory infection disease is selected from: chronic bronchitis, pneumonia, bronchiectasis and/or infection, lung abscess, pulmonary heart disease and/or pulmonary infection.

The application that the present invention also provides described medicine to be used for antimicrobial, antiinflammatory/antiallergic action in preparation, to remove the medicine of oxygen-derived free radicals and anti-oxidative damage, adjusting immunologic function, anticoagulation and antithrombotic formation, microcirculation improvement and/or protection vascular endothelial cell.

The present invention also provides described medicine to be used for the application of the medicine of antibiotic, resisiting influenza virus, antitussive and/or anti-inflammation detumescence in preparation.

Discover through a large amount of modern pharmacologies, composition in the medicine provided by the invention is all influential to a plurality of pathology links of infectious disease, not only pathogenic microorganism had direct inhibition and lethal effect, the more important thing is that medicine is inhibited to over-drastic inflammatory reaction, the realization of this effect is embodied in two aspects: the one, and anti-damaging action, be antimicrobial, antiinflammatory, antiallergic action, removing free radical and antioxidation etc., the just traditional Chinese medical science so-called " getting rid of evils "; The 2nd, plerosis function; promptly by regulating the anti-damage ability of enhancing body such as the impaired vascular endothelial cell of immunologic function, protection, microcirculation improvement state; promote tissue repair, just the traditional Chinese medical science so-called " setting upright " has embodied the unification of " getting rid of evils " and " setting upright ".Chinese medicine is nothing like the Western medicine antibiotic at antibiotic, anti-virus aspect, but the function of the antiendotoxin of Chinese medicine, antiinflammatory, antioxidation and enhancing human body immunity power is that antibiotic is not available, and this is Chinese medicine anti-infective advantage place just also.

The medicine of treatment infectious disease provided by the invention, the compatibility effect of main component is analyzed as follows:

The lower respiratory infection disease shows as heating, cough, expectoration, chest pain or uncomfortable in chest, out of breath or asthma or companion's frequent micturition, urgent micturition, dysurea etc. clinically mostly, show as resembling of excessive noxious heat, therefore heat-clearing and toxic substances removing is the classical method of treatment of such disease, yet, similar according to pathological changes such as the local consolidation of pathological changes, edema, erosion, ulcer, ischemia, blood fortune obstacle, tissue degeneratiaon, hypertrophy with the modern disease Neo-Confucianism notion of blood network stasis of blood resistance, the method for activating blood circulation to dissipate blood stasis and dredge the collateral is incorporated in the treatment of respiratory infectious disease.The promoting blood circulation to remove obstruction in the collateral medicine has protection impaired endotheliocyte, blood viscosity lowering, and the effect of microcirculation improvement helps the absorption of transudate and the discharge of expectorant, promotes the recovery of broncho-pulmonary function of organization, can apply to the treatment of respiratory tract infectious disease.

The heat-clearing and toxic substances removing decoct and decoct compatibility the acting as of invigorating blood circulation to endotoxemia and nonspecific inflammation animal model, two class medicine compatibilities can make PGE2, endotoxin blood concentration, whole blood reduced viscosity reduce, the azovan blue seepage discharge reduces, foot pawl swelling percentage rate descends, blood plasma cortisol content increases, and fibrinolytic strengthens.Wherein most measurement results all are better than the animal model group of the independent medication of two classes.Proof thus, the heat-clearing and toxic substances removing decoct with invigorate blood circulation behind the decoct compatibility, more help detoxifying, the treatment of antiinflammatory and inflammation ^[43]Simultaneously its side effect has antagonism to heat and toxic materials clearing away medicine owing to blood circulation promoting medicine, and the heat-clearing and toxic substances removing decoct of invigorating blood circulation can make the blood viscosity reduction, and fibrinolytic obviously strengthens, therefore, but two class medicines share the accelerate blood circulation, in order to the removing of toxin, reduce the side effect of heat and toxic materials clearing away medicine.Heat and toxic materials clearing away medicine and blood circulation promoting and blood stasis dispelling compatibility are used can increase under the physiological conditions abdominal cavity to the absorbability under antibacterial absorbability and the peritonitis situation ^[44]Heat and toxic materials clearing away medicine and drug for invigorating blood circulation and eliminating stasis also show with the synergism that produces with the back and suppress the exponential rising of viral pneumonia mouse lung, alleviating of pulmonary lesion, and can resist the heating effect of viral pneumonia rabbit, the control inflammatory lesions, reducing blood viscosity, also there is synergism the vigor aspect that improves superoxide dismutase.

Therefore, medicine provided by the invention is followed the Chinese medical theory compatibility and is formed, and simultaneously based on research experiment and clinical practice, heat-clearing and toxic substances removing and removing heat from blood collateral dredging is combined, and can effectively prevent lower respiratory infection disease inflammation damnification cascade reaction.

Through the inventor's lot of experiment validation, the pharmacological action of the anti-inflammatory damage of each main component of medicine of the present invention is specific as follows:

(1) anti-microbial effect

The baicalin that Radix Scutellariae contains has broad spectrum antibacterial function, as gram positive bacterias such as staphylococcus aureus, Hemolytic streptococcus and streptococcus pneumoniae and Gram-negative coccus such as Neisseria meningitidis, Neisseria gonorrhoeae are had inhibitory action.Baicalin and beta-lactam penicillin such as amoxicillin share the antibacterial potency that can effectively improve penicillin resistant enzyme staphylococcus aureus.Baicalin also has anti-Chlamydia pneumoniae and protection cytosis.In addition, baicalin has anti-preferably Candida albicans to act on external

Fructus Gardeniae has the moderate strength antibacterial action to staphylococcus aureus, Hemolytic streptococcus, micrococcus catarrhalis, diphtheria corynebacterium, Bacillus tuberculosis etc.Its infusion can suppress various dermatophytess external, and decocting liquid can kill leptospira and schistosomicide external, and the effect of anti-echovirus is arranged.

Cholic acid has also shown the bacteriostatic activity stronger to gram-positive cocci.

(2) antiinflammatory, anti-allergy action

Baicalin, baicalin and other flavone compound have in various degree inhibitory action to many types of allergy, are strong to the I allergic reaction type especially.Radix Scutellariae suppresses antigen and combines with IgE, suppresses mastocyte and discharges histamine and become clinical preferably antiabnormal reaction agent.Modal with the allergy diseases associated is bronchial asthma, and baicalin is shunk by bronchus anaphylaxis and allergic asthma all has mitigation, and with ephedrine synergism is arranged.

The antiinflammatory action that Fructus Gardeniae gives birth to product is the strongest, and has the effect of antianaphylaxis.

Cholic acid also has antiinflammatory, anti-allergy action.

(3) remove oxygen-derived free radicals and anti-oxidative damage

Baicalin has stronger free radical scavenging activity.

Breviscapine has the effect of removing oxygen-derived free radicals and anti-oxidative damage simultaneously.

Calculus Bovis has the effect of anti-oxidative damage.

(4) regulate immunologic function

Baicalin has facilitation to the erythrocyte immune adhesion function.

Fructus Gardeniae is effective with oral mucosa pathological changes and external genitalia ulcer that the relevant Behcet of disfunction when the compound recipe that be grouped into pair and cell immune system is showed, and inhibition IV type anaphylactic reaction and pair cell immunity have inhibitory action.

(5) anticoagulation and antithrombotic form

The generation of baicalin energy Trombin inhibiting and the inductive plasminogen agonist of thrombin receptor tonin inhibitive factor-1 (PAI-1).

Gardenoside has anti thrombotic action.

Herba Erigerontis total flavones all has obvious inhibitory action to the platelet aggregation that ADP, arachidonic acid (AA) or platelet activating factor (PAF) cause in vitro and in vivo.Breviscapine can influence the intrinsic coagulation approach, can act on exogenous cruor pathway again, and it has stronger inhibitory action to platelet the 3rd factor, can reduce thrombinogen and be converted into thrombin, and can significantly increase fibrinous fibrinolytic.

(6) microcirculation improvement

Breviscapine has the effect of microcirculation improvement.

(7) protection vascular endothelial cell

Though baicalin can not be eliminated the generation of the endothelial denudation phenomenon due to the bacterial endotoxin (ET), protect the cell between endotheliocyte to connect to a great extent, the endochylema that alleviates due to the ET is loose, mitochondrial swelling and reticulum dilatation.

The external propagation that can strengthen endotheliocyte of cape jasmine fruit extract (GFE).The cape jasmine fruit water extract can significantly increase DNA and proteinic synthetic in the cell.

Breviscapine causes the PMEC (Pulmonary Microvascular Endothelial Cells) damage to platelet activating factor (PAF) and has protective effect.Breviscapine can suppress the inductive PMEC (Pulmonary Microvascular Endothelial Cells) damage of PAF.

In sum, beneficial effect of the present invention is as follows:

The medicine of treatment infectious disease provided by the invention, from traditional Chinese medical science network ens morbi, combine with the differential diagnosis of diseases of doctor trained in Western medicine microcosmic traditional Chinese medical science macroscopic view is dialectical, the theory of Chinese medical science that forms on the basis according to clinical practice-" poison decreases the lung network " pathogenesis theory, and in conjunction with the achievement in research of modern medicine relevant lower respiratory infection disease pathological process and treatment, by Radix Scutellariae, Fructus Gardeniae, Herba Erigerontis and cholic acid compatibility form, key link-inflammatory reaction at infectious disease, harmful substances such as eliminate the inflammation medium and free radical, improve the local microcirculation state, thereby reduce inflammation pathology damage, has the effect of regulating whole interior environment simultaneously, can effectively treat lower respiratory infection and by the acute lung injury of infection-induced, and effective substance is relative with the mechanism of action clear.

Description of drawings

Below, describe embodiment of the present invention in conjunction with the accompanying drawings in detail, wherein:

Fig. 1 is a RT-PCR amplified production electrophoretogram;

Fig. 2 wherein, from left to right is respectively: TLR7, β-actin, Myd88, IRAK4, P65, TRAF6 for amplification melting curve figure.

The specific embodiment

Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.

Following examples are intended to illustrate that the present invention treats concrete prescription composition, preparation method and the function and the effect of the medicine of infectious disease by way of example.

Embodiment 1

GetRadix Scutellariae (in baicalin) 120mg, Fructus Gardeniae (in jasminoidin) 25mg, Herba Erigerontis (in lamp-dish flower acetic) 4mg, cholic acid 25mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, and each aggregative indicator and major influence factors lung index, whole blood total white blood cells, survival rate, lung coefficient are optimum level.

Embodiment 2

GetRadix Scutellariae (in baicalin) 60mg, Fructus Gardeniae (in jasminoidin) 75mg, Herba Erigerontis (in lamp-dish flower acetic) 2mg, cholic acid 25mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, and whole blood leukocyte count, bronchoalveolar lavage fluid lymphocyte percentage ratio are significantly reduced.

Embodiment 3

GetRadix Scutellariae (in baicalin) 60mg, Fructus Gardeniae (in jasminoidin) 50mg, Herba Erigerontis (in lamp-dish flower acetic) 2mg, cholic acid 50mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, can make that leukocyte count significantly reduces in the lung-douching fluid.

Embodiment 4

GetRadix Scutellariae (in baicalin) 60mg, Fructus Gardeniae (in jasminoidin) 25mg, Herba Erigerontis (in lamp-dish flower acetic) 2mg, cholic acid 50mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, and full bronchoalveolar lavage fluid tumor necrosis factor is significantly reduced.

Embodiment 5

GetRadix Scutellariae (in baicalin) 120mg, Fructus Gardeniae (in jasminoidin) 50mg, Herba Erigerontis (in lamp-dish flower acetic) 4mg, cholic acid 75mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, and the animals survived number is the highest.

Embodiment 6

GetRadix Scutellariae (in baicalin) 120mg, Fructus Gardeniae (in jasminoidin) 25mg, Herba Erigerontis (in lamp-dish flower acetic) 4mg, cholic acid 75mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, and the lung index is significantly reduced.

Embodiment 7

GetRadix Scutellariae (in baicalin) 60mg, Fructus Gardeniae (in jasminoidin) 25mg, Herba Erigerontis (in lamp-dish flower acetic) 4mg, cholic acid 75mg press medicine preparation method preparation commonly used.The preparation of making is used for pharmacodynamic study, can make that NO concentration significantly reduces in the lung homogenate.

Embodiment 8 and 9 has studied the resisting pathogenic microbes effect of the medicine of treatment infectious disease provided by the invention.Lower respiratory infection is referred to as pulmonary infection usually, pathogenic microorganism is the principal element that causes various pulmonary infections, diseases associated with inflammation, mainly contains antibacterial, secondly is virus, mycoplasma, idol has chlamydia, rickettsia etc., still belongs to bacterial infection disease country occurred frequently in China.The growth and breeding of pathogenic microorganism in lung can cause the change of multiple tissue injury and functional activity, causes various respiratory symptoms, so but pathogenic microorganism is significant to the treatment of pulmonary infection extremely.

Embodiment 8: vitro antibacterial activity

(1) experiment purpose

Observe the external inhibitory or killing effect of medicine of the present invention, and adopt tube dilution method to measure its minimal inhibitory concentration (MIC) the respiratory tract infection common bacteria.

(2) experiment material

1, experiment bacterial strain: the experiment bacterial strain uses therefor is Basic Medical Institute, Beijing University of Chinese Medicine's medical science cause of disease laboratory to be provided.

Staphylococcus aureus (being called for short " golden Portugal ") (26102)

Group B streptococcus (being called for short " second chain ") (32204)

Bacillus subtilis (being called for short " hay ") (44813)

Escherichia coli (being called for short " large intestine ") (44813)

Pseudomonas aeruginosa (being called for short " green pus ") (10104)

Candida albicans (being called for short " the white thought ")

2, culture medium: nutrient broth, contain serum nutrient broth, Nutrient agar, fungi culture medium, being strong power garden pharmaceuticals provides.

3, the medicine that medicine: embodiment 1 provides (hereinafter to be referred as " example 1 medicine "), color: outward appearance is pale brown color, content: 17.4mg/ml, lot number: 20060410;

SHUANGHUANLIAN, 600mg, lot number 0501004, No. 2 TCM Factory;

Penicillin G sodium, lot number 0503032, China industry pharmacy Group Plc;

Streptomycin sulfate, lot number 05111102, Shangdong Ruiyang Pharmaceutical Co., Ltd.

(3) experimental technique

1, bacterium liquid preparation:

Staphylococcus aureus, bacillus subtilis, escherichia coli, Pseudomonas aeruginosa are inoculated in the nutrient broth respectively; Group B streptococcus is inoculated in the serum broth; Candida albicans is inoculated in the fungi culture medium, and 37 ℃, after 18～24h cultivates, the antibacterial of cultivating 18h is mixed with the bacterium liquid of 1.5 hundred million/ml with turbidimetry, 4 ℃ of preservation (use in 3 days) are standby.

2, medicinal liquid preparation:

With the nutrient broth preparation, the medicine original liquid concentration is: example 1 medicine, stock solution; SHUANGHUANLIAN, 40mg/ml; It is standby to prepare 4 ℃ of preservations of medicine stock solution.The ultimate density of medicine: example 1 medicine (ml/ml): 0.5,0.25,0.125,0.0625,0.0313,0; SHUANGHUANLIAN is (mg/ml): 20,10,5,2.5,1.25,0.625,0.

3, determination of tube method example 1 medicine, SHUANGHUANLIAN and antibiotic are to the minimal inhibitory concentration (MIC) of different bacterium:

Every kind of medicine to be measured is got 8 test tubes, and every test tube adds nutrient broth 1ml, and the 1st test tube adds medicine stock solution 1ml to be measured, takes out 1ml behind the mixing and adds the 2nd, and successively continuously to doubly rare, the 7th is blank, and the 8th is the experimental bacteria contrast.Total amount of liquid is the 1ml/ pipe.The medicine ultimate density with 2, the medicinal liquid preparation.Every pipe adds each 0.1ml of bacterium liquid of 1.5 hundred million/ml.37 ℃, 24h cultivates, observed result, and with culture fluid transferred species (golden Portugal, hay, large intestine, green pus are inoculated in ordinary flat, and the second chain is inoculated in blood plate, and white the thought is inoculated in brave red flat board) to solid medium, observation has or not bacterial growth, measures its MIC again.

4, determination of tube method example 1 medicine and antibiotic compatibility are to the MIC of antibacterial:

Experiment divides 2 groups to carry out, and the independent group detection of antibiotics is separately to the MIC of different bacterium; 5 usefulness group test example 1 medicine and antibiotic 5 usefulness back are to the MIC of different bacterium.Method adopts tube dilution method.

Every kind of medicine to be measured of independent group is got 11 test tubes, and every test tube adds fluid medium 1ml, and the 1st adds medicine stock solution 1ml to be measured, takes out 1ml behind the mixing and adds the 2nd test tube, continuous successively two-fold dilution, and the 10th is blank, the 11st is the bacterial strain contrast.Total amount of liquid is the 1ml/ pipe.The medicine ultimate density sees Table 1.Every pipe adds each 0.1ml of bacterium liquid of 1.5 hundred million/ml.37 ℃, 24h cultivates, observed result.

The different antibiotic MIC that 5 usefulness group draws according to independent group adds 0.5ml example 1 medicine, the same independent group of antibiotic ultimate density in every test tube except that control tube.Method is the same.

The different antibiotic of table 1 ultimate density in vitro

(3) experimental result and analysis

1, example 1 medicine and SHUANGHUANLIAN are to the MIC (seeing Table 2) of different bacterium:

Table 2 example 1 medicine and SHUANGHUANLIAN are to the MIC of different bacterium

The result shows: example 1 medicine has certain inhibitory or killing effect to group B streptococcus, but little than the SHUANGHUANLIAN effect.

2, behind example 1 medicine and the antibiotic compatibility to the influence (seeing Table 3) of antibacterial MIC:

The variation (μ g/ml) of table 3 antibiotic and 0.5ml/ml example 1 medicine 5 usefulness front and back MIC

The result shows: behind penicillin G sodium and example 1 compatibility of drugs, and its MIC no change.Behind streptomycin sulfate and example 1 compatibility of drugs, the MIC of staphylococcus aureus and bacillus subtilis 4～8 times have been reduced; To escherichia coli and Pseudomonas aeruginosa, no change before and after the compatibility.

Conclusion: scorchingly hot flat extracorporeal antivirus effect studies show that, scorchingly hot 48h antiviral of putting down behind viral infection is most effective, basically can suppress virus multiplication fully, and high concentration (〉=antivirus action of medicine does not increase in time and significant change is arranged 0.27mg/ml) time.Under close concentration, compare its antiviral efficient with several experimental concentrations of RHL and preponderate with Rib.

Embodiment 9: external resisiting influenza virus effect

(1) experiment purpose

It is external to infective inhibitory action of influenza virus A-prime Mus lung adapted strain (FM1) and time-effect relationship to observe medicine of the present invention, for this medicine antivirus action and mechanism thereof provide experimental basis.

(2) experiment material

1, virus: influenza virus A-prime Mus lung adapted strain, the Chinese Chinese medicine academy traditional Chinese medical science provides, and this laboratory-76 ℃ preservation is standby.After twice of nine age in days chick embryo allantoic cavity continuous passage, surveying the blood clotting titre is 1: 512, the LD of mice ₅₀Be 10 ^-2.2

2, cell: Madin-Darby canine kidney(cell line) (MDCK) strain (MDCK) is available from institute of section of army Cytology Lab, used by the back of going down to posterity, this chamber.

3, medicine: example 1 medicine, brown liquid, 17.4mg/ml; Positive control drug: ZHUSHEYONG SHUANGHUANGLIAN (lyophilized powder): 600mg dry powder/, it is standby to be mixed with 40mg/ml with serum-free DMEM culture fluid before the experiment, lot number: 0501004 (No. 2 TCM Factory); Ribavirin: 100mg/ml, lot number: 0506251 (Ou Yi Shi Pharmaceutical Group Pharmaceutical Co).

4, reagent: Dulbecco ' s Modified Eagle medium culture medium (DMEM culture medium), GBICO company product; New-born calf serum: people's marine growth company product.Tetramethyl azo azoles salt (MTT) and dimethyl sulfoxide (DMSO) are the Amresco product.Growth-promoting media: the DMEM culture fluid that contains 10% new-born calf serum; Keep liquid: do not contain serum and contain the tryptic DMEM culture fluid of 2 μ g/ml.1% crystal violet: take by weighing 1g crystal violet and add the aseptic tri-distilled water of 20ml earlier, add the waterside in the mortar limit and be ground to smalls, be settled to 100ml at last, coarse filter paper filters back 4 ℃ and deposits standby.33% glacial acetic acid: prepare with deionized water.

(3) experimental technique

1, drug cell toxicity test (MTT colorimetry)

When treating that the mdck cell growth conditions preferably, with 0.02%EDTA and 0.5% trypsin digestion cell, adjust cell to desired concn with growth-promoting media, this Cell sap is added in the 96 porocyte culture plates, 100 μ l/r are in 37 ℃ 5%CO ₂Be cultured to fine and close cell monolayer in the incubator.With keeping liquid example 1 medicine (DRP), ribavirin (Rib) and SHUANGHUANLIAN (RHL) dilution are variable concentrations, add in the Tissue Culture Plate, 100 μ l/r, each drug dilution concentration is parallel does 3 multiple holes, establishes the normal cell contrast simultaneously.5%CO in 37 ℃ ₂After cultivating 72h in the incubator,, press the Reed-Muench method and calculate medicine maximal non-toxic concentration (TC with the toxicity of MTT (final concentration is 0.5mg/ml) colorimetry detection of drugs to mdck cell ₀) and median toxic concentration (TC ₅₀).

2, viral infection is measured (TCID ₅₀Mensuration)

With serum-free DMEM culture fluid influenza virus FM1 is carried out continuous 10 times of successives dilution, with each dilution virus inoculation in 3 * 10 ⁵In the mdck cell of/ml, each dilution factor is parallel does 6 multiple holes, establishes the normal cell contrast simultaneously.Inhale behind 37 ℃ of absorption 2h and abandon viral liquid and use instead and keep liquid and continue to cultivate, every day is observation of cell pathological changes effect (CPE) under inverted microscope, and record lesion degree and hole count, with the no obvious regression of cell contrast, virus infected cell pathological changes rate reach 50% and above cell hole be the pathological changes hole, the cytopathy variability less than 50% be non-pathological changes hole, press the TCID that the Reed-Muench method is calculated virus ₅₀(median tissue culture infective dosage).

3, the time-effect relationship of medicine antivirus action of the present invention

Use 100TCID ₅₀FM1 influenza virus 50 μ l/r behind 37 ℃ of absorption monolayer mdck cell 2h, suction is abandoned viral liquid and is used example 1 medicine of serial dilution concentration instead as experiment medicine group, if ribavirin (Rib) and the positive medicine matched group of SHUANGHUANLIAN (RHL), be provided with the normal cell matched group simultaneously, 3 multiple holes of every concentration.5%CO at 37 ℃ ₂Continue to cultivate 24h, 36h, 48h and 72h in the incubator respectively, abandon culture fluid with fixing 1% violet staining behind the 20min of cold methanol, room temperature 20min post rinse 96 porocyte culture plates, dissolve with 33% glacial acetic acid 37 ℃ of dry backs, 100 μ l/r, purple to be crystallized dissolve the back fully and measure absorbance A value (OD down in the 570nm wavelength ₅₇₀).

Medicine antiviral effective percentage (effective rate, ER%) calculate by following formula:

Calculate the half-inhibition concentration (IC of medicine simultaneously to virus ₅₀) and therapeutic index (TI).

TI＝TC ₅₀/IC ₅₀

4, the antivirus action of the different dosing methods of medicine of the present invention

According to the virus replication cycle, 3 medicine antivirus actions of this experimental design target spot, and establish a chemoprophylaxis group, have 4 processed group, the parallel 3 multiple holes of doing of each drug level.Establish positive drug matched group and normal cell matched group simultaneously.Virus attack concentration is 100TCID ₅₀, virus is incorporated as 50 μ l/r.Dosing method is respectively: 1. antiviral biosynthesis group: virus is changed pastille earlier and is kept liquid behind 37 ℃ of adherent cell 2h; 2. antiviral absorption group: virus is kept liquid with pastille and is added simultaneously in the mdck cell that has become monolayer; 3. direct effect group: pastille is kept liquid and is mixed with virus, in 37 ℃ hatch 1.5h jointly after infection cell again, the viral liquid of keeping that changes pastille not behind the 2h that adsorbs; 4. chemoprophylaxis group: pastille is kept liquid add after 37 ℃ in cell hatches 24h, abandon and keep liquid and change virus absorption 2h, change not pastille again and keep liquid.Respectively organize cell later on and continue to cultivate 3d, when the cytopathy in virus control hole reaches 75%～100%,, use 1% violet staining, survey OD with experiment 3 at 37 ℃ ₅₇₀(A value), and the antiviral effective percentage (ER%) of calculating medicine.

(4) experimental result

1, drug cell toxicity test (seeing Table 4)

Table 4 example 1 medicine is to the TC of mdck cell ₀And TC ₅₀(mg/ml)

2, viral infection is measured

According to experimental result, press the TCID of the influenza virus FM1 of Reed-Muench method calculating ₅₀=10 ^-3.3/0.1ml

3, the time-effect relationship of medicine antivirus action of the present invention (seeing Table 5,6,7)

Table 5 example 1 medicine is to the IC50 (μ g/ml) and the therapeutic index (TI) of virus

From therapeutic index, the safety of example 1 medicine is sharp high than SHUANGHUANLIAN and ribavirin.

The time-effect relationship (n=4) of table 6 example 1 medicine resisiting influenza virus A-prime Mus lung adapted strain FM1 effect

The time-effect relationship (n=4) of table 7 example 1 medicine resisiting influenza virus A-prime Mus lung adapted strain FM1 effect

According to table 6,7 result, viral infection peaks at 36h, and the antiviral efficient of DRP is the highest at 48h, and along with the dense reduction of medicine, antiviral efficient descends gradually.Low concentration is with prolonging action time, and efficient reduces, high concentration (〉=0.27mg/ml) to prolong antivirus action in time more stable.DRP and Rib ratio seemingly have protective effect at its pair cell of 24h that acts on, and Rib does not have inhibitory action to viral infection this moment, but strengthen with prolonging its antivirus action action time, at 72h downward trend are arranged again.The antivirus action of DRP is all obvious than RHL and Rib effect 4 periods.

4, the antivirus action result (seeing Table 8,9) of the different dosing methods of medicine of the present invention

The effect (n=4) of the different dosing method resisiting influenza virus of table 8 example 1 medicine A-prime Mus lung adapted strain FM1

Annotate: mode 1.: drug effect is viral infection behind cell 24h; Mode is 2.: virus is through medicine pretreatment postoperative infection cell; Mode is 3.: medicine and virus add simultaneously; Mode is 4.: virus absorption back dosing earlier.

The effect (n=4) of the different dosing method resisiting influenza virus of table 9 example 1 medicine A-prime Mus lung adapted strain FM1

Annotate: mode 1.: drug effect is viral infection behind cell 24h; Mode is 2.: virus is through medicine pretreatment postoperative infection cell; Mode is 3.: medicine and virus add simultaneously; Mode is 4.: virus absorption back dosing earlier.

From table 8,9 as seen, example 1 medicine can obviously suppress virus at the intracellular i.e. biosynthetic process of virus that duplicates, and can stop virus to be adsorbed in cell, and the effect of directly killing the virus is arranged when high concentration (0.14mg/ml).Ribavirin can obviously stop virus absorption, but does not have preventive effect, directly kill virus.With the SHUANGHUANLIAN ratio, example 1 medicine antivirus action under experimental concentration than obviously.

Conclusion: the extracorporeal antivirus effect of medicine of the present invention studies show that, the 48h antiviral of this medicine behind viral infection is most effective, basically can suppress virus multiplication fully, and high concentration (〉=antivirus action of medicine does not increase in time and significant change is arranged 0.27mg/ml) time.Under close concentration, compare its antiviral efficient with several experimental concentrations of RHL and preponderate with Rib.

Embodiment 10 and 11 has studied the resisting pathogenic microbes relieving cough and reducing sputum effect of the medicine of treatment infectious disease provided by the invention.Acute bronchus pulmonary infection, or chronic infection acute attack often cause symptoms such as cough, expectoration.Slight cough helps getting rid of the secretions or the foreign body of respiratory tract, keeps the respiratory tract cleaning unimpeded, and still frequent play is coughed, and can bring to the patient and not accommodate misery, maybe might cause complication, as pneumothorax, spitting of blood, hernia etc., but the severe patient threat to life.This research adopts classical mice ammonia to cause the method for coughing, the mensuration relieving cough and reducing sputum effect of having observed medicine of the present invention of the phenol red excretion of mice trachea.

Embodiment 10: the influence cough caused to strong aqua ammonia

(1) experiment purpose

Observe the influence of medicine of the present invention, study the pharmacological action of its antitussive the cough of strong aqua ammonia induced mice.

(2) experiment material

1. medicine and reagent

Be subjected to reagent: example 1 medicine, color: outward appearance is pale brown color, content: 17.4mg/ml.Lot number: 20060410.Clinical plan consumption: 348mg/d, intravenous injection.

Positive control drug: the pentoxyverine citrate sheet, produce lot number: 0511023 by Tianjin Lisheng Pharmaceutical Co., Ltd..Tanreqin injection is provided by Shanghai Kaibao Pharmaceutical Co., Ltd, lot number: 060301.

Reagent: strong aqua ammonia

2. animal

Kunming (KM) mice, the male and female dual-purpose, body weight 18～22g, is provided the quality certification number: SCXK (capital) 2002-2006 by 60 by Inst. of Genetics and Development Biology, CAS's animal center.

(3) experimental technique

1, grouping and administration

Get 50 of healthy Kunming mouses, body weight 18～22g is divided into 6 groups at random, 10 every group, is respectively blank group, the basic, normal, high dosage group of example 1 medicine, positive drug control group.Except that positive drug pentoxyverine group, intraperitoneal injection is all adopted in administration, once a day, and shared 3d.The blank group gives normal saline, drug component does not give example 1 medicine 13.05mg/kg, 52.2mg/kg, 91.35mg/kg, and the positive drug group gives the pentoxyverine citrate sheet, and dosage is 50mg/kg, adopt the administration by gavage administration, tanreqin injection dosage is 3ml/kg.

2. modeling and detection method

Behind administration 1h, mice is placed in the 500ml wide mouthed bottle of 200 μ l strong aqua ammonia, cough number of times in the record mice 2min (typical case's cough is that the mice abdominal muscle shrinks, and magnifies mouth simultaneously, can have and cough sound) and coughre flex incubation period (beginning to producing the required time of cough) by spraying into the ammonia mist.

(3) experimental result and analysis

Respectively organize mice 2min cough number of times and cause and cough incubation period, relatively adopt the T check between group.The results are shown in Table 10.

The influence that table 10 example 1 medicine is cough caused to strong aqua ammonia

Annotate: each group is compared with model group ^*P＜0.05, ^*P＜0.01.

The result shows: each dosage of example 1 medicine, expectorant heat are clear, pentoxyverine all can significantly reduce the mouse cough number of times due to the strong aqua ammonia spraying, compared significant difference (P＜0.01) with the matched group group, and can prolong to cause and cough incubation period, wherein this medicine high dose group is compared with matched group and is had diversity (P＜0.05), illustrates that medicine of the present invention has antitussive effect.

Embodiment 11: to the influence of the phenol red excretion amount of trachea

(1) experiment purpose

Observe the influence of medicine of the present invention, study its expectorant pharmacological action the phenol red excretion amount of mice trachea.

(2) experiment material

1. medicine and reagent

Be subjected to reagent: example 1 medicine.

Positive control drug: ammonium chloride, analytical pure, Beijing northization fine chemicals Co., Ltd, lot number: 20060220; Tanreqin injection is provided by Shanghai Kaibao Pharmaceutical Co., Ltd, lot number: 060301.

Reagent: phenol red

2. animal

The KM mice, the male and female dual-purpose, body weight 18～22g, is provided the quality certification number: SCXK (capital) 2002-2006 by 60 by Inst. of Genetics and Development Biology, CAS's animal center.

3. instrument

752 type UV, visible light grating spectrophotometers, Beijing Optical Instrument Factory's product.

(3) experimental technique

1, grouping and administration

Get 60 of healthy Kunming mouses, body weight 18～22g is divided into 6 groups at random, 10 every group, is respectively blank group, the basic, normal, high dosage group of example 1 medicine, 2 positive drug control group.Except that the positive drug group, intraperitoneal injection is all adopted in administration, once a day, and shared 3d.The blank group gives normal saline, and drug component does not give example 1 medicine 13.05mg/kg, 52.2mg/kg, 91.35mg/kg, and the positive drug group gives ammonium chloride 50mg/kg, adopts the administration by gavage administration, and tanreqin injection 3ml/kg.

2, modeling and detection method

Behind each medicamental pulverata time administration 30min, lumbar injection 5% phenol red 0.1ml/10g, behind 30min, put to death mice, peel off the trachea surrounding tissue, cut one section trachea to the trachea crotch from thyroid cartilage, put into the 2ml normal saline, 2 of the sodium hydroxide of adding 1mol/L are surveyed absorbance (A) value with 721 spectrophotometers in wavelength 546nm place.

(4) experimental result and analysis

Each is organized the phenol red excretion amount of mice trachea and the results are shown in Table 11.

Table 11 example 1 medicine is to the influence of the phenol red excretion amount of trachea

Annotate: each group is compared with model group (normal saline group), ^*P＜0.01.

The result shows: example 1 medicine, ammonium chloride all can make the phenol red excretion of trachea increase, and wherein the middle and high dosage of example 1 medicine, ammonium chloride group and normal saline group relatively have significant difference (P＜0.01).The phenol red excretion amount of the big more expression of absorbance is many more, illustrates that the phlegm-dispelling functions of medicine is strong more.Results suggest medicine of the present invention has good phlegm-dispelling functions.

Embodiment 12～14 has studied the resist inflammation on repercussive function of the medicine of treatment infectious disease provided by the invention.In various pulmonary infections, inflammatory reaction is the important step that causes the histopathology damage.Inflammatory reaction has three phases when tangible: acute stage, increase to feature with partial vasodilation and capillary permeability; Subacute stage is a feature with leukocyte and cytophagous infiltration; The chronic propagation phase turns to feature with tissue degeneratiaon and fiber.This research has been adopted the inflammatory swelling experiment of reacting acute, subacute pathological changes feature and has been the chronic inflammation model of feature with the granuloma, has observed the antiinflammatory action of medicine of the present invention.

Embodiment 12: the influence of rat paw edema due to the on Carrageenan

(1) experiment purpose

By the acute inflammation model of rat paw edema due to the carrageenin, observe the antiinflammatory action of medicine of the present invention.

(2) experiment material

1, medicine and reagent

Be subjected to reagent: example 1 medicine.

Positive control drug: tanreqin injection is provided by Shanghai Kaibao Pharmaceutical Co., Ltd, and it is brown that outward appearance is, lot number: 060301.Dexamethasone is produced lot number: 0508051 by Tianjin gold credit aminoacid company limited.

Reagent: carrageenin is available from sigma company, is mixed with 0.1% suspension with the injection normal saline.

2, laboratory animal

The SD rat, the male and female dual-purpose, body weight 180～200g is provided by Inst. of Genetics and Development Biology, CAS's animal center, the quality certification number: SCXK (capital) 2002-2006.

3, instrument

Slide gauge, 0.02mm, Harbin Co., Ltd of measurer cutter group.

(3) experimental technique

1, grouping and administration

The SD rat is divided into 6 groups at random, every group 10, example 1 medicine is divided into three dosage group: high dose group 71.05mg/kg, middle dosage group 40.6mg/kg, low dose group 10.15mg/kg, positive drug is dexamethasone 10mg/kg, tanreqin injection 2.33ml/kg, model control group gives the equivalent normal saline, and each medicine all adopts lumbar injection, once a day, continuous 3d.

2, modeling method and measurement

After the last administration, use the left back toes joint of vernier caliper measurement rat thickness, it is subcutaneous so that scorching that each group of while is injected in left back toes with 1% carrageenin 0.1mL immediately, after administration 1,2,4,6h measures once left back toes thickness respectively so that thickness deducts that to cause scorching back thickness be swelling degree (cm) before scorching.

(4) experimental result and analysis

Each is organized the rat paw edema degree and the results are shown in Table 12.

The influence of rat paw edema degree due to the table 12 example 1 medicine on Carrageenan

Annotate: each group is compared * P＜0.05, * * P＜0.01 with model group.

The result shows: dexamethasone, expectorant heat all can significantly suppress rat toes swelling due to the carrageenin at 1h, 2h, 4h, 6h clearly, and the middle and high dosage of example 1 medicine also can obviously suppress toes swelling due to the carrageenin at 2h, 4h, 6h.(P＜0.01，P＜0.05)。

Embodiment 13: the influence of xylol induced mice ear swelling

(1) experiment purpose

By the inflammatory model of dimethylbenzene induced mice ear swelling, observe the anti-acute inflammatory reaction effect of medicine of the present invention.

(2) experiment material

1, medicine and reagent

Be subjected to reagent, positive control drug with embodiment 12.

2, animal

The KM mice, the male and female dual-purpose, body weight 18～22g, is provided the quality certification number: SCXK (capital) 2002-2006 by 71 by Inst. of Genetics and Development Biology, CAS's animal center.

3, instrument

Slide gauge, 0.02mm, Harbin Co., Ltd of measurer cutter group; AEG-220 type electronic analytical balance, day island proper Tianjin instrument company product.

(2) experimental technique

1, grouping and administration

60 of KM mices, male and female half and half, body weight 18g～22g is divided into 6 groups at random.Be respectively model group (normal saline group), the high, medium and low dosage group of example 1 medicine (dosage is respectively 91.35mg/kg, 2.2mg/kg, 13.05mg/kg) and positive drug control group, positive drug is dexamethasone and tanreqin injection, dexamethasone dosage is 10mg/kg, and the clear dosage of expectorant heat is that 3ml/kg respectively organizes mice intraperitoneal injection of drugs respectively.

2. modeling method and measurement

Accurately drip dimethylbenzene 0.05mL to every mouse right ear behind the last administration 30min and duplicate the ear swelling model, 1h puts to death mice after the last administration, lays left and right corresponding site auricle with the 8mm card punch, weighs respectively, represents the swelling degree with the difference of two ear weight.

(3) experimental result and analysis

What each organized the mice ear degree the results are shown in Table 13.

The influence of table 13 example 1 medicine xylol induced mice ear swelling

Annotate: compare with model group ^*P＜0.05.

The result shows: the model group mice ear is obvious, and the middle and high dosage group of example 1 medicine ear swelling situation is compared obviously with model group and alleviated, and has significant difference (P＜0.05).

Embodiment 14: to the outgrowth influence of rat granuloma due to the cotton balls

(1) experiment purpose

By rat granuloma model of hyperplasia due to the cotton balls, observe the anti-inflammatory proliferative effect of medicine of the present invention.

(2) experiment material

1, medicine and reagent

Be subjected to reagent, positive control drug with embodiment 12.

2, animal

The SD rat, the male and female dual-purpose, body weight 180～220g is provided by Inst. of Genetics and Development Biology, CAS's animal center, the quality certification number: SCXK (capital) 2002-2006.

3, instrument

AEG-220 type electronic analytical balance, day island proper Tianjin instrument company product.

(3) experimental technique

Cotton balls is prepared: preparation weight is the cotton balls of 30mg, and big or small degree of tightness is close, adds ampicillin 1mg/0.1ml in each cotton balls behind the autoclaving, dries standby at 50 ℃ of baking boxs.

Operation process: get 60 of healthy SD rats, male, body weight 180～220g, etherization, hypogastric region unhairing, skin routine disinfection.The rat dorsal position is fixed, the hypogastric region otch, it is subcutaneous that two cotton balls through the said process preparation are implanted rat both sides groin respectively, sew up wound, in operation back 8d, put to death rat with the cervical vertebra dislocation method, take out cotton balls precision after 60 ℃ of baking boxs are placed 12 hours and weigh, it is heavy that this weight is deducted former cotton balls, is the granuloma net weight.

The animal grouping: rat is divided into 5 groups after implanting cotton balls at random, every group 10, example 1 medicine is divided into three dosage group: high dose group 71.05mg/kg, middle dosage group 40.6mg/kg, low dose group 10.15mg/kg, positive control drug is 10mg/kg for dexamethasone dosage, tanreqin injection matched group dosage is 2.33ml/kg, matched group gives the equivalent normal saline, below respectively organize equal intraperitoneal administration, once-a-day, continuous 7d, 4h puts to death animal after the last administration, and granuloma weight, granuloma suppression ratio (%)=[(treatment group granuloma nt wt net weight-blank group granuloma nt wt net weight)/blank group granuloma nt wt net weight] * 100% respectively organized in record.

(4) experimental result and analysis

Each is organized rat granuloma net weight and the results are shown in Table 14.

Table 14 example 1 medicine is to the outgrowth influence of rat granuloma due to the cotton balls

Annotate: compare with model group ^*P＜0.01.

The result shows: dexamethasone can obviously suppress rat granuloma hypertrophy due to the cotton balls, with model group significant difference (P＜0.01) is arranged relatively.Each dosage group of example 1 medicine, expectorant heat all demonstrate certain outgrowth effect of inhibition granuloma clearly, but compare there was no significant difference with model group.

The conclusion of embodiment 12～14: the middle and high dosage of medicine of the present invention can obviously suppress toes swelling and the dimethylbenzene induced mice ear swelling due to the carrageenin, showing that this medicine can effectively reduce in the acute subacute inflammation phase oozes out, alleviate acutely inflamed swelling degree, the granulation tissue hyperplasia of inflammation chronic phase is had certain inhibitory action.Point out this medicine to have certain antiinflammatory action, especially the acute stage effect to inflammatory reaction is obvious.

Embodiment 15～18 is the medicine of treatment infectious disease provided by the invention adapts to FM1 strain induced mice injury of lung to influenza virus A-prime mouse lung a pharmacodynamic study, by observing the therapeutical effect of this medicine to influenza virus induced mice pneumonia, determining antiviral, antiinflammatory action and the related mechanism thereof of this medicine, for the clinical practice of this medicine provides foundation.

Embodiment 15: influenza virus A-prime mouse lung is adapted to the experiment of FM1 strain infecting mouse protective rate

(1) experimental technique

1, modeling method:

Get virus stock solution used 90 μ l, be added in the normal saline of 3510 μ l, mixing places ice bath;

With mice with the slight anesthesia of ether after, normal group is with normal saline 50 μ l collunariums, other infect every 50 μ l with viral liquid (virus stock solution used dilution in 1: the 40) collunarium for preparing.Treat to weigh after the mice recovery, other are divided into 6 groups at random except that normal group (20), are respectively: the heavy dose of group of dosage group (20) and example 1 medicine (20) in model group (24), ribavirin positive controls (20), SHUANGHUANLIAN positive controls (20), example 1 medicine small dose group (20), example 1 medicine.

2, medication:

Normal group: normal saline lumbar injection, every 0.3ml;

Model group: normal saline lumbar injection, every 0.3ml;

Ribavirin group: ribavirin diluent (stock solution dilution in 1: 12) lumbar injection, every 0.3ml;

SHUANGHUANLIAN group: SHUANGHUANLIAN diluent (45mg/ml) lumbar injection, every 0.3ml;

Example 1 medicine small dose group: 1: 8 example 1 drug dilution liquid lumbar injection, every 0.3ml;

Dosage group in example 1 medicine: 1: 4 example 1 drug dilution liquid lumbar injection, every 0.3ml;

The heavy dose of group of example 1 medicine: 1: 2 example 1 drug dilution liquid lumbar injection, every 0.3ml.

Dead protective rate=viral infection matched group mortality rate * experimental group mortality rate/viral infection matched group mortality rate * 100%

(2) experimental result and analysis

Example 1 medicine adapts to FM1 strain infecting mouse protective rate experimental result to influenza virus A-prime mouse lung and sees Table 15,16.

The dead protective rate laboratory observation of table 15 data (natural law of surviving after the mouse infection virus)

Table 16 is respectively organized infected mice influenza virus dead mouse protective rate experimental result

Annotate: compare with normal group, ^#P＜0.01; Compare * P＜0.01 with model group.

The result shows: the dead protective rate of the large, medium and small dosage of example 1 medicine is respectively 29.1%, 29.1%, 12.8%; compare with model group; through chi-square criterion, show that the big or middle dosage group of this medicine has significant protective effect (p＜0.05) to influenza virus FM1 infecting mouse.

Embodiment 16: to the exponential influence of infected mice influenza virus mouse lung

(1) experimental technique

Modeling: with mice with etherization after, normal group and model group N.S. collunarium, every 50 μ l, other each group is with 1: 400 influenza virus Mus lung adapted strain diluent collunarium, each 50 μ l/.Medication is the same.Weighed in the 7th day, and plucked the eyeball blood-letting, put to death the aseptic lungs of winning in back, inhale after removing unnecessary liquid with aseptic filter paper that the weighing lung is heavy respectively.

Lung index=lungs weight (mg)/mice weight (g)

The lungs index suppresses percentage rate=(matched group lungs index mean-test group lungs index mean)/matched group lungs index mean

(2) experimental result and analysis

Each is organized the mouse lung assessment of indices and the results are shown in Table 17.

Table 17 example 1 medicine is to the exponential influence of infected mice influenza virus mouse lung

Annotate: compare with normal group, ^###P＜0.001; Compare * * P＜0.01 with model group.

The result shows: the lung index of model group obviously increases (P＜0.001), illustrates that influenza infection causes mouse lung addle swollen, and weight increases; Compare with model group, the lung index of the large, medium and small dosage group of example 1 medicine all obviously descends (P＜0.01), points out the large, medium and small dosage of this medicine all can alleviate the edema state of influenza infection mice lungs.

Embodiment 17: to the influence of infected mice influenza virus mouse lung disease of ZANG-organs reason

(1) experimental technique

Modeling, administration and method of drawing material are the same.

Get 5 lung tissues for every group, after 10% formalin fixed, lung tissue pathology section HE dyeing, and under optical microscope, observe the variation of lung tissue pathology.Concrete steps are:

1) fixing: as to get 5 lung tissues for every group, use 10% formalin fixed, change fixative within one month;

2) piece is repaiied in washing: with fixative to the greatest extent, soak 24h with the tap water flushing, every 1h changes water once, and tissue is repaired smooth tangent plane;

3) dehydration: with water to the greatest extent, 50%, 70%, 80%, 95% alcoholic solution at different levels each 60min that dewaters, put into 95%, 100% alcoholic solution each twice, each 90min blots residual liquid with filter paper before immersing;

4) transparent: after immersing xylene solution, cover tight lid, in case air admission, the dimethylbenzene that more renews behind the 35min soaks 35min;

5) saturating wax: twice, each 90min keeps the temperature inside the box about 55～60 ℃;

6) embedding: the paraffin of fusing is poured in the container, will be put into the paraffin of fusing, note tangent plane down, drop into immediately in the cold water then, make it be frozen into wax stone at once through the tissue of saturating wax;

7) section: wax stone is fixed on the microtome cuts into slices, slice thickness is 6 μ m, and it is a bit of to cut wax disk(-sc) with single-edge blade, is placed on to carry on the glass to add one in water, and whether good, the selected part paster if placing magnifier or microscopically to observe section;

8) dewaxing rehydration: paraffin section is through each 5～10min of dimethylbenzene I, II dewaxing, puts into each 3～5min of alcoholic solutions at different levels such as 100%, 95%, 90%, 80%, 70% then, puts into distilled water 3min again;

9) brazilwood extract dyeing: about 15min, with the about 15min of tap water flushing, see color change basket with microscopy at any time till;

10) return blue color separation: 1% hydrochloride ethanol liquid (100 parts of hydrochloric acid 1 part+70% ethanol) is put in section faded, see that section reddens, color is more shallow to get final product, approximately several seconds to tens of seconds; Tap water flowing water is put in section again makes it recover blue.The low power lens inspection sees that nucleus is blue, structure is clear; Cytoplasm or connective fiber composition are colourless to be standard.Put into the distilled water rinsing then once, each 3～5min in 50% ethanol → 70% ethanol → 80% ethanol is gone in section;

11) Yihong dyeing: with 0.5% Yihong ethanol liquid counterstaining 2～5min;

12) dehydration of alcohol: put into the unnecessary redness of 95% ethanol flush away, put into dehydrated alcohol 3～5min then.Blot unnecessary ethanol with absorbent paper at last;

13) mounting: optics natural gum is sealed up for safekeeping;

14) microscopic examination is taken a picture.

(2) experimental result and analysis

Each is organized mouse lung and organizes the HE coloration result as seen:

Normal group visible tissue structural integrity, cell takes on a red color, and it is blue that nuclear is, and the alveolar size is well-balanced, and cell wall thickness is normal; Model group mainly shows as interstitial inflammation, the congestion of blood vessel in the visible interstitial lung, and edema and a large amount of inflammatory cells (lymphocyte, mononuclear cell) soak into, and alveolar wall thickens, and alveolar is compressed, and proliferation of fibrous tissue is oozed out in the alveolar space; The SHUANGHUANLIAN group is compared with normal group, and cell wall thickens slightly, and a spot of inflammatory cell infiltration is arranged, but structural integrity, the alveolar size is normally well-balanced; The as seen a small amount of lymphocytic infiltration of example 1 medicine small dose group, alveolar size are near normal, with oozing out on a small quantity and hyperemia.Dosage group visible tissue structural integrity in example 1 medicine, alveolar septum is normal substantially; The heavy dose of group of example 1 medicine visible tissue structural integrity, cell wall is basic near normal, and a spot of inflammatory cell infiltration is arranged.The result shows: this medicine can obviously alleviate hyperemia, edema and the inflammatory exudation of lung tissue, the structural integrity of protection lung tissue.

Embodiment 18: to infected mice influenza virus effect of immunologic function

(1) experimental technique

Animal modeling, administration and method of drawing material are the same.

1, the mensuration of splenic T lymphopoiesis ability

Adopt mtt assay.Experiment mice is plucked the aseptic spleen (operate in the ice bath and carry out) of winning after the eyeball sacrificed by exsanguination, and conventional preparation splenocyte suspension adds 10～15mlTris-NH ₄Cl, room temperature 5～10 minutes is washed (1500rpm, 10min, 4 ℃) twice with 1640 of serum-free, and the complete RPMI1640 culture medium of reuse is regulated cell concentration to 5 * 10 ⁶/ ml adds in 96 orifice plates, 100 μ l/ holes.Add respectively and contain the complete RPMI640 solution of ConA 100 μ l/ holes (the ConA final concentration is 5 μ g/ml), place 37 ℃ 5%CO ₂Incubator is hatched 68h, takes out, and each hole sucking-off 100 μ l supernatant adds MTT10 μ l/ hole (final concentration 0.5mg/ml), continues to hatch 4～6h, adds 0.01NHCl-10%SDS100 μ l/ hole, vibration, and 4 ℃ are spent the night, and next day, microplate reader 570nm measured each hole A value down.

2, the mensuration of spleen bone-marrow-derived lymphocyte multiplication capacity

Adopt mtt assay.Experiment mice is plucked the aseptic spleen (operate in the ice bath and carry out) of winning after the eyeball sacrificed by exsanguination, and conventional preparation splenocyte suspension adds 10～15mlTris-NH ₄Cl, room temperature 5～10min washes (1500rpm, 10min, 4 ℃) twice with 1640 of serum-free, and the complete RPMI1640 culture medium of reuse is regulated cell concentration to 5 * 10 ⁶/ ml adds in 96 orifice plates, 100 μ l/ holes.Add again and contain the complete RPMI1640 solution of LPS 100 μ l/ holes (the LPS final concentration is 10 μ g/ml), place 37 ℃ 5%CO ₂Incubator is hatched 68h, takes out, and each hole sucking-off 100 μ l/ hole, vibration, 4 ℃ are spent the night, and next day, microplate reader 570nm measured each hole A value down.

3, INF-γ's induces and determination of activity

Adopt the ELISA method.After experiment mice after handling plucked the eyeball sacrificed by exsanguination, the aseptic spleen of winning, conventional preparation splenocyte suspension is with complete RPMI1640 culture fluid adjusting cell concentration to 5 * 10 ⁶/ ml adds in 24 orifice plates, the 1ml/ hole, and it is 5 μ g/ml that adding ConA makes final concentration, places 37 ℃ 5%CO ₂Incubator is hatched 72h, takes out, and the sucking-off supernatant, the centrifugal 10min of 3000rpm draws supernatant and promptly gets INF-γ semifinished product, is sub-packed in the effendoff pipe-75 ℃ of preservations.Detect INF-γ content with the ELISA test kit.Trace routine is as follows:

1) dosing: PBST:0.5mlTween-20 is dissolved among the PBS of 1L; PBST-B:2mlBSA solution (10%)+38mlPBST;

2) wash plate: PBST300 μ l/ hole, wash plate 3 times, on filter paper, buckle for the last time and do;

3) add sample and cytokine standard substance: 100 μ l/ holes add sample and cytokine, cover the shrouding film.Hatch 2h for 37 ℃, blank well is set, replace sample and standard substance with PBST;

4) wash plate: deduct liquid in the hole, PBST300 μ l/ washes plate 6 times in the hole, buckles on filter paper for the last time and does;

5) add detection antibody: 100 μ l/ holes add Biotinylated detector antibodies working solution, cover the shrouding film, hatch 1h for 37 ℃;

6) wash plate: deduct liquid in the hole, PBST300 μ l/ washes plate 6 times in the hole.On filter paper, buckle for the last time and do;

7) add SPP:100 μ l/ hole and add the SPP working solution, cover the shrouding film, hatch 1h for 37 ℃;

8) wash plate: deduct liquid in the hole, PBST300 μ l/ washes plate 6 times in the hole, buckles on filter paper for the last time and does;

9) add TMB (zymolyte): add TMB working solution 100 μ l/ holes, color development at room temperature 10-30min;

10) cessation reaction: treat aerial liquid colour developing appropriateness, add the H of 2M immediately ₂SO ₄50 μ l/ holes, cessation reaction;

11) read plate: λ=450nm reading.

4, the peritoneal macrophage phagocytic function is measured

Adopt the dimethyl diaminophenazine chloride colorimetry.After experiment mice plucked the eyeball sacrificed by exsanguination,, draw the about 6ml of peritoneal fluid for every, wash cell twice (1500rpm, 10min, 4 ℃), with complete RPMI1640 adjusting cell concentration to 4 * 10 with PBS to contain the cold PBS lavation abdominal cavity of heparin ⁶/ ml adds in 96 orifice plates, 100 μ l/ holes, every group six hole, 37 ℃, 5%CO ₂Hatch 4h, abandon supernatant, add 0.075% neutral red solution, 200 μ l/ holes, wash 1～2 time with warm PBS behind the 30min, add macrophage lysate (50% dehydrated alcohol+50%0.1M acetic acid) 200 μ l/ holes, 4 ℃ are spent the night, and next day, microplate reader 530nm measured each hole A value down.

5, lung homogenate NO assay

After experiment mice plucked eyeball and put to death, the aseptic lungs of winning added cold normal saline, centrifugal (1500rpm after the homogenate according to 1: 9 ratio, 10min) get supernatant, add in 96 orifice plates, 100 μ l/ holes, the Griess liquid that adds equivalent, room temperature is placed 10min, and microplate reader 530nm surveys the A value down.

6, serum NO levels is measured

Pluck eyeball after mice is weighed and put to death and to get blood 2ml, treat coagulation after centrifuge centrifugal, 3000rpm, 10min gets serum and adds in 96 orifice plates, 50 μ l/ holes add the Griess liquid of equivalent, room temperature is placed 10min, microplate reader 530nm surveys the A value down.

7, lung homogenate IL-1 assay

Adopt the method for ELISA.Mice is plucked the eyeball sacrificed by exsanguination, win lungs, add cold normal saline according to 1: 9 ratio, centrifugal after the homogenate (1500rpm, 10min), get supernatant and detect by the test kit operating procedure:

1) bag quilt: 10 * bag is cushioned liquid places room temperature, get the DI H that 2.5ml adds 22.5ml ₂O; Get the above-mentioned bag for preparing of coated antibody (Capture Ab) 48 μ l+12ml and be cushioned liquid; The coated antibody that above-mentioned dilution is good is added in 96 orifice plates, every hole 100 μ l, and shrouding spends the night for 4 ℃;

2) washing: wash each hole 3 times with PBS-Tween20, every hole must not be less than 300 μ l, and 96 orifice plates that overturn are inhaled in absorbent paper and removed remaining buffer;

3) the DI H of 5 * chemical dilution liquid of 10ml (Assay diluent)+40ml ₂O → 1 * chemical dilution liquid; Add 96 orifice plates, the every hole of 200 μ l, incubated at room 1 hour;

4) as step 2) described in wash plate;

5) application of sample: get the standard substance of 10 μ l standard substance+10ml chemical dilution liquid (Assay diluent) → highly diluted concentration, get 100 μ l to be added in the suitable hole, carry out the two-fold dilution then, to make standard curve, add testing sample, every hole 100 μ l, shrouding incubated at room 2 hours;

6) described in step 2, wash plate, wash altogether 5 times.

7) add enzyme labelled antibody: get 1 * chemical dilution liquid (Assay diluent) splice of 48 μ l enzyme labelled antibody (Detection Ab)+12ml, every hole 100 μ l, shrouding, incubated at room 1 hour;

8) described in step 2, wash plate, wash altogether 5 times.

9) 48 μ l enzymes (Avidin-HRP)+12ml chemical dilution liquid; Splice, 100 μ l/ holes, shrouding, incubated at room 30min;

10) described in step 2, wash plate, in this step, abandon liquid and soak plate hole 1～2min with washing liquid before, wash plate altogether 7 times;

11) add substrate solution (Substrate Solution), 100 μ l/ holes, incubated at room 15min;

12) add 50 μ l/ hole stop buffers;

13) reading under the 450nm.

8, lung homogenate TNF-alpha content is measured

Adopt the method for ELISA, detect by the test kit operating procedure.

9, lung homogenate IL-4 assay

Adopt the method for ELISA, detect by the test kit operating procedure.

10, IL-10 assay in the lung homogenate

Adopt the method for ELISA, detect by the test kit operating procedure.

(2) experimental result and analysis

1, the measurement result of splenic T lymphopoiesis ability sees Table 18.

Table 18 example 1 medicine is to the influence of infected mice influenza virus mouse T lymphocyte multiplication capacity

Annotate: compare with normal group, ^#P＜0.05; Compare * * P＜0.01, * * * P＜0.001 with model group.

The result shows: with normal group relatively, model group A value decline (P＜0.05) illustrates behind the mouse infection influenza virus FM1 inhibitedly to immunologic function, can cause T lymphopoiesis ability drop; Compare with model group, example 1 medicine is little, in, heavy dose of group A value all increases (P＜0.01, P＜0.001), points out this medicine can improve influenza virus infection mouse T lymphocyte multiplication capacity, strengthens its immunologic function.

2, the measurement result of spleen bone-marrow-derived lymphocyte multiplication capacity sees Table 19.

Table 19 example 1 medicine is to the influence of infected mice influenza virus mice bone-marrow-derived lymphocyte multiplication capacity

Annotate: compare with normal group, ^###P＜0.001; Compare * * * P＜0.001 with model group.

The result shows: compare with normal group, model group A value significantly descends (P＜0.001), illustrates behind the mouse infection influenza virus FM1 that immunologic function is had the obvious suppression effect, can cause the bone-marrow-derived lymphocyte multiplication capacity significantly to descend; Compare with model group, example 1 medicine is little, in, heavy dose of group A value all significantly increases (P＜0.001), points out this medicine can improve influenza virus infection mice bone-marrow-derived lymphocyte multiplication capacity, strengthens its immunologic function.

3, INF-γ induce and determination of activity the results are shown in Table 20.

Table 20 example 1 medicine is to the influence of the content of infected mice influenza virus mice INF-γ

Annotate: compare with normal group, ^###P＜0.001; Compare * * P＜0.01, * * * P＜0.001 with model group.

The result shows: with normal group relatively, model group INF-γ content significantly descend (P＜0.001); Compare with model group, example 1 medicine is little, in, heavy dose of group INF-γ content all significantly increases (P＜0.01, P＜0.001), points out this medicine can improve the anti-virus ability of influenza virus infection mice.

4, peritoneal macrophage phagocytic function measurement result sees Table 21

Table 21 example 1 medicine is to the influence of infected mice influenza virus Turnover of Mouse Peritoneal Macrophages phagocytic function

Annotate: compare with normal group, ^###P＜0.001; Compare * * P＜0.01, * * * P＜0.001 with model group; Compare with the SHUANGHUANLIAN group, ^△P＜0.05, ^{△ △}P＜0.01.

The result shows: compare with normal group, model group A value significantly descends (P＜0.001), illustrates that mouse infection influenza virus FM1 pneumoretroperitoneum macrophage phagocytic function significantly descends; Compare with model group, example 1 medicine is little, in, heavy dose of group A value all significantly increases (P＜0.01, P＜0.001), and compare with positive drug SHUANGHUANLIAN group, in this medicine, heavy dose of group A value also significantly increases (P＜0.05, P＜0.01), point out this medicine can significantly improve influenza virus infection Turnover of Mouse Peritoneal Macrophages phagocytic function, this effect is better than SHUANGHUANLIAN.

5, lung homogenate NO assay the results are shown in Table 22.

Table 22 example 1 medicine is to the influence of infected mice influenza virus mouse lung homogenate NO content

Annotate: compare with normal group, ^###P＜0.001; Compare * * * P＜0.001 with model group.

The result shows: lung homogenate NO burst size significantly raises behind the mouse infection influenza virus FM1, with normal group comparing difference remarkable (P＜0.001); Example 1 medicine is little, in, heavy dose of it is had remarkable downward modulation effect, with model comparing difference significantly (P＜0.001).

6, the serum NO levels measurement result sees Table 23.

Table 23 example 1 medicine is to the influence of infected mice influenza virus mice serum NO content

Annotate: compare with normal group, ^#P＜0.05; Compare * P＜0.05, * * P＜0.01 with model group.

The result shows: serum NO levels raises behind the mouse infection influenza virus FM1, with normal group comparing difference remarkable (P＜0.05); Example 1 medicine is little, in, heavy dose and positive drug all have remarkable downward modulation effect to it, with model comparing difference significantly (P＜0.05, P＜0.01), especially the effect of dosage group is more obvious in example 1 medicine.

7, lung homogenate IL-1 Determination on content the results are shown in Table 24.

Table 24 example 1 medicine is to the influence of infective virus mouse lung homogenate IL-1 content

Annotate: compare with normal group, ^###P＜0.001; Compare * * * P＜0.001 with model group.

The result shows: with normal group relatively, model group lung homogenate IL-1 content significantly raise (P＜0.001); Compare with model group, example 1 medicine is little, in, heavy dose and positive drug all can reduce influenza infection mouse lung homogenate IL-1 content (P＜0.001).

8, the measurement result of lung homogenate TNF-alpha content sees Table 25.

Table 25 example 1 medicine is to the influence of infective virus mouse lung homogenate TNF-alpha content

Annotate: compare with normal group, ^###P＜0.001; Compare * * * P＜0.001 with model group.

The result shows: with normal group relatively, model group lung homogenate TNF-alpha content significantly raise (P＜0.001); Compare with model group, example 1 medicine is little, in, heavy dose and positive drug all can reduce influenza infection mouse lung homogenate TNF-alpha content (P＜0.001).

9, lung homogenate IL-4 Determination on content the results are shown in Table 26.

Table 26 example 1 medicine is to the influence of infective virus mouse lung homogenate IL-4 content

Annotate: compare with normal group, ^###P＜0.001; Compare * * P＜0.01, * * * P＜0.001 with model group; Compare with the SHUANGHUANLIAN group, ^△P＜0.05.

The result shows: lung homogenate IL-4 content significantly raises behind the mouse infection influenza virus FM1, with normal group comparing difference remarkable (P＜0.001); Example 1 medicine is little, in, heavy dose and positive drug all have remarkable downward modulation effect to it, with remarkable (P＜0.01 of model group comparing difference, P＜0.001), and compare with positive drug SHUANGHUANLIAN group, in example 1 medicine, heavy dose of group is to the downward modulation effect of lung homogenate IL-4 content more obvious (P＜0.05), points out this medicine being better than SHUANGHUANLIAN aspect the reduction influenza virus infection mouse lung homogenate IL-4 content.

10, the IL-10 Determination on content the results are shown in Table 27 in the lung homogenate.

Table 27 example 1 medicine is to the influence of infective virus mouse lung homogenate IL-10 content

Annotate: compare with normal group, ^###P＜0.001; Compare * P＜0.05, * * * P＜0.001 with model group; Compare with the SHUANGHUANLIAN group, ^△P＜0.05.

The result shows: lung homogenate IL-10 content significantly raises behind the mouse infection influenza virus FM1, with normal group comparing difference remarkable (P＜0.001); Example 1 medicine is little, in, heavy dose and SHUANGHUANLIAN all have remarkable downward modulation effect to it, with remarkable (P＜0.05 of model group comparing difference, P＜0.001), and compare with positive drug SHUANGHUANLIAN group, the downward modulation effect of dosage group more obvious (P＜0.05) in example 1 medicine, dosage is being better than SHUANGHUANLIAN aspect the reduction influenza virus infection mouse lung homogenate IL-10 content in prompting example 1 medicine.

Embodiment 19: to the pharmacodynamic study of Acute Lung Injury due to the endotoxin (LPS)

(1) experiment purpose

Observe the protective effect of medicine of the present invention, and deeply inquire into its related mechanism, for the clinical practice of this medicine provides foundation Acute Lung Injury due to the endotoxin (LPS).

(2) experiment material

1, laboratory animal

120 of healthy cleaning level SD rats, male and female half and half, body weight 210-240g (Beijing Vital River Experimental Animals Technology Co., Ltd.).

2, medicine and reagent

Example 1 medicine, and tanreqin injection (Shanghai Kaibao Pharmaceutical Co., Ltd, lot number: 040607), and LPS (EcoliO55B5, sigma company), other reagent in the experiment is analytical pure (available from Beijing chemical reagents corporation).Bradford protein quantification test kit, P-selectin ELISA test kit (cat.NO.3R262RB company), sICAM-1 ELISA test kit (cat.NO.3R110 RB company).

(3) experimental technique

1, the preparation of animal model

The etherization rat, sublingual vein injection LPS, 6mg/kg (LPS of 10mg is dissolved among 0.9% the NS of 4ml, actual administration 0.24ml/100g).

2, grouping and administration

Zoopery prospective adaptation nursing 2 days.The animal random packet is a normal group, model control group, the heavy dose of group of example 1 medicine, dosage group in example 1 medicine, example 1 medicine small dose group, positive controls (tanreqin injection), 10 every group.

Adopt the administration of lumbar injection mode.Normal group injecting normal saline, each administration group behind Sublingual injection LPS immediately intraperitoneal injection once, the corresponding volumetrical normal saline of model control group lumbar injection, every afterwards 12h is administered once, and puts to death animal behind the 24h, draws materials.

Each administration group consumption following (rat dosage is 6 times of people's clinical application amount), example 1 medicine: heavy dose of group 0.3ml/100g, middle dosage group 0.2ml/100g, small dose group 0.1ml/100g; Tanreqin injection: rat clinical equivalent amount 0.2ml/100g.

3, draw materials and handle

(1) get blood: the about 5ml of ventral aorta blood sampling, wherein 2ml joins in the pipe that has added EDTA, and mixing places and is used to survey routine blood test on ice gently, has surveyed to remain centrifugal blood behind the routine blood test and get supernatant and get blood plasma; 3ml add leave standstill in the other pipe serum to be had separate out the back low-temperature centrifugation get serum.

(2) get the bilateral lungs and collect bronchoalveolar lavage fluid:

Separate trachea, inverted T shape otch (avoid blood to enter trachea when drawing materials, influence the composition of irrigating solution) is done with knife blade in the cricoid cartilage below, will make plastic flexible pipe by oneself and insert trachea, fix (avoiding gas leakage) with stitching thread, separate cardiopulmonary (being sure not to touch lungs) in order to avoid influence lavation.Folder closes the right side lobar bronchi, extracts 1.6mlPBS liquid with syringe and slowly injects the slowly extraction again of left lung, carries out continuously 3 times, and total amount is 4.8ml altogether, requires the response rate greater than 75% (softly pushing lung tissue with gauze in case of necessity).

(3) right lung lobus diaphragmaticus, filter paper blots, and is used to measure lung tissue and does (wetting) anharmonic ratio and moisture content of lung.The right lung remaining tissue is done morphological observation through formaldehyde fixed.

4, index detects and analyzes

(1) moisture content of lung and lung weight in wet base/dry weight ratio: get the right lung lobus diaphragmaticus, filter paper blots, and accurately claims weight in wet base on analytical balance, and 80 ℃ of constant temperature toasted 48 hours then, treated that quality no longer changes accurate weighing dry weight, calculated lung tissue and did (wetting) anharmonic ratio and moisture content of lung.Moisture content of lung=(wet lung heavy-dried lung is heavy)/wet lung heavy * 100%.

(2) bronchoalveolar lavage fluid cell counting: bronchoalveolar lavage fluid through refrigerated centrifuge centrifugal (3000r/m, 15min), the settled cell in centrifugal back hangs again with the PBS of 200 μ L, add two glacial acetic acid after, carry out cell counting.

(3) bronchoalveolar lavage fluid total protein and TNF-alpha content are measured: centrifugal (3000r/m 15min), gets supernatant to bronchoalveolar lavage fluid, is used to measure the content and the TNF-α of total protein through refrigerated centrifuge.The bronchoalveolar lavage fluid protein quantification adopts Bradford protein quantification test kit hypersensitization scheme, and the TNF-alpha content adopts to put and exempts from method mensuration in the bronchoalveolar lavage fluid.

(4) lung tissue pathological observation: the right lung tissue is repaiied and is got the middle period through 10% formalin fixed, routine paraffin wax embedding, the thick section of 5 μ m, HE dyeing, om observation injury of lung degree.

(5) calculate the lung permeability coefficient: the plasma protein assay adopts Bradford protein quantification test kit.Lung permeability coefficient=bronchoalveolar lavage fluid protein content/plasma protein content * 100%.

(6) serum il-1 β, IL-8 assay: adopt the method for exempting from of putting.

(7) blood-serum P-selectin and sICAM-1 assay: adopt the ELISA method.

5, data statistics processing method

Each is organized experimental data and represents that with mean ± standard deviation the group difference significance adopts variance analysis (Oneway-Anova).

(4) experimental result and analysis

1, the moisture content of lung measurement result sees Table 28.

Table 28 example 1 medicine causes the influence of acute lung injury lung tissue of rats water content to LPS

Annotate: compare with normal group, ^##P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group; Compare with the clear positive drug group of expectorant heat, ^△P＜0.05.

The result shows: LPS causes acute lung injury, moisture content of lung increases behind the 24h, with normal group significant difference (P＜0.01) is arranged relatively, in example 1 medicine, heavy dose of group can effectively alleviate moisture content of lung, with remarkable (P＜0.05 of model group comparing difference, and be better than the clear group of expectorant heat (P＜0.05) P＜0.01).

2, lung weight in wet base/dry weight ratio the results are shown in Table 29.

Table 29 example 1 medicine to LPS cause the acute lung injury induced lung wet/influence of dry weight ratio

Annotate: compare with normal group, ^##P＜0.01; Compare * P＜0.05 with model group; Compare with the clear positive drug group of expectorant heat, ^△P＜0.05.

The result shows: lung weight in wet base/dry weight ratio increased after LPS caused Acute Lung Injury, with normal group significant difference (P＜0.01) was arranged relatively, illustrated that LPS causes pulmonary edema, and the lungs water content is obviously increased; Example 1 medicine is little, in, lung weight in wet base/dry weight of heavy dose of group is than all descending, with model group comparing difference remarkable (P＜0.05), prompting example 1 medicine can effectively reduce moisture content of lung, alleviates the pulmonary edema state, the effect of especially middle dosage group is more obvious, is better than the expectorant clear group of heat (P＜0.05).

3, lung permeability coefficient testing result sees Table 30.

Table 30 example 1 medicine causes the influence of acute lung injury induced lung permeability coefficient to LPS

Annotate: compare * P＜0.05, * * P＜0.01 with model group

The result shows: with normal group relatively, the lung permeability coefficient of model group increases, and the permeability increase of pulmonary capillary after the acute lung injury be described, albumen oozes out increase; Example 1 medicine is little, in, the lung permeability coefficient of heavy dose of group and the clear group of expectorant heat all significantly reduces (P＜0.05, P＜0.01), prompting example 1 medicine can reduce the permeability of pulmonary capillary after the acute lung injury, alleviates and oozes out.

4, cell counting the results are shown in Table 31 in the bronchoalveolar lavage fluid.

Table 31 example 1 medicine causes Cytometric influence in the acute lung injury rat bronchoalveolar lavage fluid to LPS

Annotate: compare with normal group, ^##P＜0.01; Compare * * P＜0.01 with model group;

The result shows: compare with normal group, cell counting increases (P＜0.01) in the bronchoalveolar lavage fluid of model group; With model group relatively, example 1 medicine is little, in, the cytometer number average significantly reduces (P＜0.01) in the bronchoalveolar lavage fluid of heavy dose of group and the clear group of expectorant heat, points out the inflammatory cell infiltration after this medicine can alleviate acute lung injury.

5, TNF-alpha content testing result sees Table 32 in the bronchoalveolar lavage fluid.

Table 32 example 1 medicine causes the influence of acute lung injury rat bronchoalveolar lavage fluid TNF-alpha content to LPS

Annotate: compare with normal group, ^##P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group.

The result shows: compare with normal group, the TNF-alpha content significantly increases (P＜0.01) in the bronchoalveolar lavage fluid of model group; Compare with model group, the TNF-alpha content all significantly reduces (P＜0.05, P＜0.01) in little, heavy dose of group of example 1 medicine and the clear bronchoalveolar lavage fluid of organizing of expectorant heat.

6, serum il-1 content detection the results are shown in Table 33.

Table 33 example 1 medicine causes the influence of acute lung injury rat blood serum IL-1 content to LPS

7, serum il-8 content detection the results are shown in Table 34.

Table 34 example 1 medicine causes the influence of acute lung injury rat blood serum IL-8 content to LPS

8, blood-serum P-selectin content detection the results are shown in Table 35.

Table 35 example 1 medicine causes the influence of acute lung injury rat blood serum P-selectin content to LPS

Annotate: compare with normal group, ^##P＜0.01.

9, serum sICAM-1 content detection the results are shown in Table 36.

Table 36 example 1 medicine causes the influence of acute lung injury rat blood serum sICAM-1 content to LPS

Annotate: compare ##P＜0.01 with normal group; Compare * P＜0.05, * * P＜0.01 with model group.

The result shows: compare with normal group, model group serum sICAM-1 content significantly increases (P＜0.01); Compare with model group, heavy dose of group of example 1 medicine and expectorant heat can effectively reduce acute lung injury rat blood serum sICAM-1 content (P＜0.05, P＜0.01) clearly.

Embodiment 20～22 is that the medicine of treatment infectious disease provided by the invention is to influenza infection macrophage antivirus action Study on Mechanism, by observe this medicine to influenza infection after the influence of the excretory various kinds of cell factor content of macrophage and coherent signal pathway thereof, inquire into the antiviral amynologic mechanism of this medicine.

Embodiment 20: to the influence of NO content in the influenza infection macrophage supernatant

(1) experimental technique

Recovery Turnover of Mouse Peritoneal Macrophages Ana-1 is inoculated in 96 porocyte culture plates when being cultured to optimum state, behind most of cell attachment, use 100TCID ₅₀Influenza virus FM1 strain attack cells 1.5h, use the pastille of variable concentrations then instead and keep liquid.Get cell conditioned medium in the different set time, add in 96 orifice plates, 100 μ l/ holes, the Griess liquid of adding equivalent, room temperature is placed 10min, and microplate reader 530nm surveys the A value down.

(2) experimental result and analysis

Table 37 example 1 medicine is to the influence of NO content in the influenza infection macrophage supernatant

The result shows: compare with the normal cell matched group, the NO content that macrophage produces behind the influenza infection obviously increases, and increases along with the prolongation of time; With the viral infection matched group relatively, each dosage group of example 1 medicine all can reduce behind the influenza infection NO content in the macrophage supernatant, and demonstrates tangible dose-effect relationship, and is promptly along with the increase of drug level, more obvious to the reduction of NO content.

Conclusion: 1, macrophage discharges NO after by influenza infection increases.2, medicine of the present invention can suppress the release of viral infection macrophage NO, and raises with drug level, and its inhibitory action is more obvious, and clear and definite dose-effect relationship is arranged.3, medicine inhibition NO of the present invention discharges with the drug treating time prolongation and strengthens.

Embodiment 21: to the influence of cytokine content in the influenza infection macrophage supernatant

(1) experimental technique

Recovery Turnover of Mouse Peritoneal Macrophages Ana-1 treats to be inoculated in when growth conditions is good 24 porocyte culture plates, and incubator spends the night, and the influenza viruse attack cell 1.5h with 100TCID50 establishes virus control hole and cell control well simultaneously.After use variable concentrations instead pastille keep liquid and continue to cultivate 6h, 12h and 24h, get supernatant respectively at different time points, detect the content of IFN-beta, IL-6, MIP-1a with test kit ELISA method.

(2) experimental result

Table 38 example 1 medicine is to the influence of IFN-beta content in the influenza infection macrophage supernatant

Annotate: virus control and cell are compared, * P＜0.05, * * P＜0.01; Example 1 drug study group and virus control group compare, ^##P＜0.01.

The result shows: compare with the normal cell matched group, behind the influenza infection in the macrophage supernatant IFN-beta content obviously increase (P＜0.05, P＜0.01); With the viral infection matched group relatively, each dosage group of example 1 medicine IFN-beta content (P＜0.01) in the macrophage supernatant that all can obviously raise behind the influenza infection, and along with the increase of drug level, effect strengthens; In pharmaceutically-active three time points, the strongest with effect in 12 hours.

Table 39 example 1 medicine is to the influence of IL-6 content in the influenza infection macrophage supernatant

Annotate: virus control and cell are compared, * * P＜0.01; Example 1 drug study group and virus control group compare, ^##P＜0.01.

The result shows: compare with the normal cell matched group, behind the influenza infection in the macrophage supernatant IL-6 content obviously increase (P＜0.01); With the viral infection matched group relatively, each dosage group of example 1 medicine all can obviously reduce behind the influenza infection IL-6 content (P＜0.01) in the macrophage supernatant, and along with the increase of drug level, effect strengthens; In pharmaceutically-active three time points, the strongest with effect in 12 hours.

Table 40 example 1 medicine is to the influence of MIP-1a content in the influenza infection macrophage supernatant

Annotate: virus control and cell are compared, * * P＜0.01; Example 1 drug study group and virus control group compare, ^##P＜0.01.

The result shows: compare with the normal cell matched group, behind the influenza infection in the macrophage supernatant MIP-1a content obviously increase (P＜0.01); With the viral infection matched group relatively, each dosage group of example 1 medicine all can obviously reduce behind the influenza infection MIP-1a content (P＜0.01) in the macrophage supernatant, and along with the increase of drug level, effect strengthens; In pharmaceutically-active three time points, the strongest with the 12h effect.

Embodiment 22: to the influence of the serial mark of TLR7 Mediated Signal Transduction path

(1) experimental technique

By the external pertinent literature of a large amount of readings, design the primer of each label, and at experiment synthetic relevant primer the last week.Carry out the RT-PCR experiment then.RNA extracts by Trizol extraction test kit operating instruction and is undertaken, and preparation cDNA carries out the real-time quantitative PCR reaction at the PRISM7700 of ABI type quantitative real time PCR Instrument.

(2) experimental result

The quantitative fluorescent PCR product is through sepharose electrophoresis, and scanning analysis the results are shown in Figure 1 under the uviol lamp, and the mrna length of amplification meets design length, and the result is consistent with quantitative fluorescent PCR.

Melting curve is seen Fig. 2, does not see other assorted peak, no non-specific amplification.

Amplification slope of standard curve with β-actin of trying to achieve after the sample cDNA dilution is-3.273, and correlation coefficient is-0.998, is good linear relationship between template concentrations logarithm value and the Ct value.Obtain the relative copy number of sample to be tested genes of interest mRNA according to standard curve, with the relative copy number of corresponding β-actin relatively, obtain its mRNA expression, shown in table 41.

Table 41 example 1 drug effect 12h is to the influence of the mRNA expression of the signal transduction molecule of TLR7 mediation

Annotate: virus control group and cell matched group compare, * * P＜0.01.Experimental group and virus control group compare, ##P＜0.01.

Table 42 example 1 drug effect 24h is to the influence of the mRNA expression of the signal transduction molecule of TLR7 mediation

Annotate: virus control group and cell matched group compare, ^*P＜0.01.Experimental group and virus control group compare, ^##P＜0.01.

The result shows: the high expressed of virus control group macrophage TLR7mRNA, the 12h expression is higher than 24h, simultaneously P65, MyD88, IRAK4, the TRAF6 of virus control group are significantly higher than the normal cell matched group, have proved immune active responding behind the viral infection.After use-case 1 drug treating, in the signal path that MyD88 relies on the way each developed by molecule level all descend, shown the regulating action of example 1 medicine to the macrophage immunity function.

Embodiment 20～22 conclusions:

1. behind the influenza infection macrophage, activatory macrophage discharges a large amount of proinflammatory factor IL-6, MIP-1a, help body in early days in infection and remove virus, but continuous release can cause inflammatory cell and Th1 type cell to produce serial response in the gathering of focus, comprises the further release of inflammatory factor and direct tissue injury.Medicine of the present invention can significantly reduce the content of IL-6, MIP-1a, thus the tissue injury that the reaction that reduces inflammation causes, the effect of performance organization protection.

2. in the signal conductive process, the macrophage TLR7 behind the viral infection expresses apparently higher than normal cell contrast with MyD88.Behind the prompting influenza infection, immune system is that main product is given birth to serial reaction with the TLR7 signal transduction pathway.Behind the drug effect of the present invention, the multiple molecular marker of this path is obviously downward modulation all, points out this medicine to suppress macrophage activation by the joint albumen that suppresses this path, thus the performance immunoregulation effect.

3. medicine of the present invention is subjected to the inflammation damnification except that the protection body, also influenza virus is had initiatively lethal effect, and the macrophage of this drug treating viral infection produces high-caliber IFN-interferon-, and this interference have clear and definite antivirus action.

Claims (10)

1. a medicine for the treatment of infectious disease is characterized in that, described medicine comprises following composition: Radix Scutellariae, Fructus Gardeniae, Herba Erigerontis and cholic acid.

2. medicine according to claim 1 is characterized in that, described medicine only comprises following four flavor effective ingredient: Radix Scutellariae, Fructus Gardeniae, Herba Erigerontis and cholic acid.

3. medicine according to claim 1 and 2, it is characterized in that described main component comprises by weight: 60～120 parts of Radix Scutellariaes (in baicalin), 25～75 parts of Fructus Gardeniaes (in jasminoidin), 2～4 parts of Herba Erigerontiss (in lamp-dish flower acetic), 25～75 parts of cholic acid.

4. medicine according to claim 3 is characterized in that, described main component comprises by weight: 120 parts of Radix Scutellariaes (in baicalin), 25 parts of Fructus Gardeniaes (in jasminoidin), 4 parts of Herba Erigerontiss (in lamp-dish flower acetic), 25 parts of cholic acid.

5. according to each described medicine of claim 1-4, it is characterized in that described medicine is to be selected from following preparation: injection, tablet, granule, capsule, soft capsule, drop pill, oral liquid, powder and pill.

6. according to the preparation method of each described medicine of claim 1-5, it is characterized in that, said method comprising the steps of:

1) get the Radix Scutellariae extracting in water three times, each 60min filters, collect filtrate, merge, be concentrated into 1/2 of original volume, add hydrochloric acid and regulate pH value to 1～2, standing over night, centrifugalize, precipitate adds water, adds lime water adjust pH to 7.0, filters, collect filtrate and regulate pH value to 1～2, standing over night, centrifugalize with hydrochloric acid, precipitation is used washing with alcohol, and drying promptly gets baicalin;

2) get the Fructus Gardeniae extracting in water three times, each 60min filters, and filtrate concentrates, and last resin column is used 50% ethanol elution, collects eluent, concentrating under reduced pressure, and drying, promptly;

3) get Herba Erigerontis and add 75% alcohol reflux three times, each 2h filters filtrate recycling ethanol, water precipitating, last resin column, water eluting, collect eluent, concentrate, filter, precipitation leaves standstill with 10% sulfuric acid solution adjust pH to 2.0～2.5, filters, precipitate with ethanol, be washed to neutrality, drying is used ethyl alcohol recrystallization, drying is pulverized, promptly;

4) get above step 1)-3) three kinds of extracts of gained and cholic acid mix homogeneously make preparation, and preferably, described cholic acid is ursodesoxycholic acid or Hyodeoxycholic Acid;

5) get above step 1)-4) four kinds of composition mix homogeneously of gained, add right amount of auxiliary materials, with water for injection dissolving, ultrafiltration, injection is made in packing;

6) get above step 1)-4) four kinds of composition mix homogeneously of gained, add right amount of auxiliary materials, make oral formulations respectively.

7. be used for the treatment of application in the medicine of lower respiratory infection disease according to each described medicine of claim 1-5 in preparation.

8. application according to claim 7 is characterized in that, described lower respiratory infection disease is selected from: chronic bronchitis, pneumonia, bronchiectasis and/or infection, lung abscess, pulmonary heart disease and/or pulmonary infection.

9. the application that is used for antimicrobial, antiinflammatory/antiallergic action in preparation, removes the medicine of oxygen-derived free radicals and anti-oxidative damage, adjusting immunologic function, anticoagulation and antithrombotic formation, microcirculation improvement and/or protection vascular endothelial cell according to each described medicine of claim 1-5.

10. be used for the application of the medicine of antibiotic, resisiting influenza virus, antitussive and/or anti-inflammation detumescence in preparation according to each described medicine of claim 1-5.

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