CN101991832A

CN101991832A - Medicament for treating idiopathic pulmonary fibrosis and preparation method thereof

Info

Publication number: CN101991832A
Application number: CN 201010531346
Authority: CN
Inventors: 支开叶
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-10-30
Filing date: 2010-10-30
Publication date: 2011-03-30
Anticipated expiration: 2030-10-30
Also published as: CN101991832B

Abstract

The invention relates to a medicament for treating idiopathic pulmonary fibrosis. Dried ginger, schisandra, seed of snakegourd, cassia twig, bupleurum, pinellia, codonopsis pilosula, radix glycyrrhizae preparata, ginger and jujube are used as raw material medicaments and can be prepared into any common oral preparation. The medicament of the invention has the efficacy of removing evil diseases, conditioning cold and heat, relating lung qi and relieving cough and asthma, and can be used for treating the idiopathic pulmonary fibrosis. Animal test results show that the medicament of the invention can relieve abnormal change of PF I rat lung tissue, regulate free radical metabolism in a model body of rat pulmonary fibrosis, enhance the activity of GSH-Px and S0D, reduce the content of MDA and NO and effectively reduce PDGF, TGF-beta 1 and TNF-a levels in blood serum, and has the functions of preventing and treating pulmonary damage and fibrosis caused by bleomycin. Clinical tests prove that the total effective rate of the medicament for treating the idiopathic pulmonary fibrosis is up to 88.5%.

Description

A kind of medicine for the treatment of idiopathic pulmonary fibrosis and preparation method thereof

Technical field

The present invention relates to a kind of medicine for the treatment of interstitial lung disease, particularly relating to a kind of is the medicine of the treatment idiopathic pulmonary fibrosis of crude drug preparation with Chinese medicine.The invention still further relates to the preparation method of this medicine.

Background technology

(interstitial lung disease ILD) is otherwise known as that (diffuse parenchymal lung disease DPLD), is meant big group pulmonary disease of different nature to the diffuse interstitial lung disease to interstitial lung disease.Its histological characteristic is that pathological changes is filled the air and is distributed in two lungs, mainly invades peripheral lung tissue, as alveolar, alveolar septum, little air flue and little blood vessel and interstitial lung structure.Acute stage, ooze out based on the infiltration of alveolar wall various kinds of cell, alveolar space albumen, and based on granuloma, chronic phase presents diffuse interstitial fibrosis at subacute stage, and " honeycomb lung " appears in severe patient.

Interstitial lung disease can be divided into two kinds of constitutional and Secondary cases, and idiopathic pulmonary fibrosis (IPF) belongs to constitutional, is the common type of diffuse interstitial lung disease.It is meant agnogenio and is a kind of chronic inflammation interstitial lung disease of characteristic pathological change with plain edition interstitial pneumonia (UIP), mainly show as diffusivity alveolitis, the disorder of alveolar unit structure and pulmonary fibrosis (UIP is different from other type of idiopathic interstitial pneumonia (IIP), as special inflammation desquamative interstitial pneumonia/respiratory bronchioles companion interstitial lung disease (DIP/RBILD), the special property sent out nonspecific interstitial pneumonia (NSIP) and acute interstitial pneumonia (AIP)).The clinical manifestation of IPF based on carrying out property dyspnea, pant, breathe hard, dry cough, breathe heavily and suppress etc., with pulmonary dysfunctions such as restrictive ventilatory functional disturbance, hypoxemia is feature, even that HRCT shows is streak with twice lungs, lung periphery grid, ground glass shape, the cellular feature of changing into of nodositas.

Foreign data shows that idiopathic pulmonary fibrosis (IPF) prevalence is male's 20.2/10 ten thousand populations, women's 7.4/10 ten thousand populations, and the prevalence of Europe and Japan's report is 3～8,/10 ten thousand populations, China does not still have definite prevalence statistics.Men and women's morbidity ratio of idiopathic pulmonary fibrosis is 2: 1, and sending out the age well is 40～70 years old, the deterioration of carrying out property of disease, and be 2～5 years life cycle.China's M ﹠ M obviously rises in recent years.

In recent years, by basis and the clinical research to interstitial lung disease, scholars more as one man thought, no matter were 17-hydroxy-11-dehydrocorticosterone or immunosuppressant and other treatment, and the curative effect of its treatment interstitial lung disease all is unsatisfied with.At acute stage, subacute stage, doctor trained in Western medicine is treated with the application of antiinflammatory, hormone etc., and Zhong Nanshan academician points out, " formed Fibrotic case for chronic, the glucocorticoid curative effect is relatively poor." " special fibrosis diagnosis of property lung (matter) and the treatment guide (draft) sent out " (in July, 2002) treatment to interstitial lung disease mainly contains glucocorticoid, immunosuppressant, non-treatment certainly has erythromycin, N-acetylcystein, interferon, adjoins medicines such as non-Buddhist nun's ketone, colchicine.But above all treatments all can not improve interstitial lung disease patient's prognosis and life cycle.

Be widely used in the medicine of the various fibrotic diseases of treatment at present, comprise that specific cell factor antagonist, therapeutic monoclonal antibodies that various participation fibrosiss take place all can not stop Fibrotic development effectively, more can not reduce tissue fibering disease death rate.Genome and proteomics research are recognized us, and the tissue fibers proliferative disease cause of disease is numerous, and mechanism is complicated, belongs to typical polygenes, multilist type disease, and this is at present just in the main cause of the various anti-fibrosis medicine poor effect of clinical use.Therefore think to have only those medicines that can intervene multichannel, many target spots just can obtain satisfactory therapeutic effects.

The idiopathic pulmonary fibrosis cause of disease so far is not bright.According to clinical observation for many years, find that the patient mostly results from being invaded by exogenous pathogen.The exopathogen invasion is at first violated table, in traditional Chinese medical science differentiation of febrile diseases in accordance with the theory of the six meridians, at first invading Taiyang channel; The treatment of exterior syndrome of TAI YANG should be used diaphoresis.In modern society, most of people mostly adopt the intravenous drip medicine or use the OTC Drug therapy of not verifying for pathogen criminal table, and diaphoresis is all failed to use in actual clinical or failed and correctly uses, thereby the pathogen generation change of disease of invasion.Follow through saequential transmission and then import few sun into, treatment then should treating SHAO YANG disease by mediation.Yet also fail complete introducer, exterior syndrome of TAI YANG also simultaneously and.Go up most lung position in the human five internal organs, and again for tender dirty, the pathogen invasion sun does not have not and sees that lungs are subjected to the shower of heresy.Lungs are subjected to heresy, and cough, asthma can occur surely.Hope, hear, ask, cut comprehensive analysis of the data gained by the four methods of diagnosis according to the traditional Chinese medical science, idiopathic pulmonary fibrosis clinically show as basically that cough, expectorating thick saliva, asthma are uncomfortable in chest, emotion light vexation, bitter taste, dry pharynx, dizziness headache, hyperhidrosis, poor appetite, have with heating, have with nausea and vomiting, red tongue thick fur, stringy pulse or stringy and thready pulse.Anatomize these symptoms, find the performance of existing shaoyang disease, have the performance of sun card again concurrently.

Summary of the invention

The purpose of this invention is to provide a kind of medicine for the treatment of idiopathic pulmonary fibrosis, this medicine has the exopathogen of dispeling, mildly regulating cold and heat, and dispersing obstructed lung-QI, the effect of relieving cough and asthma can be used for treating idiopathic pulmonary fibrosis.

A kind of manufacturing method for above mentioned medicine is provided, and is another goal of the invention of the present invention.

The traditional Chinese medical science is thought, lung governing qi, and department breathes.The course of disease of pulmonary fibrosis presents chronic performance, and this type of patient has resembling of the deficiency of vital energy mostly.Prolonged illness must be empty, so should be the task of top priority to set upright in treatment.Secondly, this type of patient is in very long lysis, because the unable imperial heresy of the deficiency of vital energy, eliminating evil, often multiple pathogen is stayed for a long time, coldheat complex, and deficiency and excess is also seen." element is asked the numbness opinion " points out: " wet three gas of wind and cold are assorted extremely, close and be numbness also ... BI-syndrome related to skin is endlessly felt again in heresy, the interior house in lung ... the objective the five internal organs person of all numbness, lung-QI stagnation syndrome person, stuffy sensation with restlessness is breathed heavily and is vomitted." pathogen numbness hinders in lung, Lung Qi obstraction, the lung network is obstructed, and the change of all diseases gives birth to.So in treatment, should dispel exopathogen, mildly regulating cold and heat, the double gas of setting upright.Lung qi must be declared, and the lung network must be logical, and all diseases must be removed, and the negative and positive runback, then disease is from removing.

Based on above-mentioned mechanism, medicine of the present invention is that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 5～10 Fructus Schisandrae Chinensis 12～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 10～15

Radix Bupleuri 20～30 Rhizoma Pinelliaes 8～12

Radix Codonopsis 12～18 Radix Glycyrrhizae Preparatas 8～12

Rhizoma Zingiberis Recens 5～15 Fructus Jujubaes 15～20.

The preferred raw material medicines in portions by weight number of said medicine is:

Rhizoma Zingiberis 6～8 Fructus Schisandrae Chinensis 15～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 12～15

Radix Bupleuri 20～25 Rhizoma Pinelliaes 9～12

Radix Codonopsis 12～15 Radix Glycyrrhizae Preparatas 9～10

Rhizoma Zingiberis Recens 6～12 Fructus Jujubaes 16～18.

More than the consumption of various crude drug be that the inventor draws through groping in a large number to sum up, facts have proved that various crude drug all have curative effect preferably in above-mentioned weight range.

The conventional method of Chinese medicine preparation can be adopted, said medicine of the present invention is prepared into conventional peroral dosage form on any pharmaceutics, but these dosage forms protection scope of the present invention can not be used to limit.It is emphasized that before any dosage form of preparation, all need to prepare earlier the active component of medicine of the present invention.

The concrete preparation method of above-mentioned each crude drug being made medicine activity component of the present invention may further comprise the steps:

A) Rhizoma Pinelliae powder with described parts by weight is broken into fine powder, and is standby;

B) extract the Fructus Schisandrae Chinensis three times of described parts by weight with the alcoholic solution of 80wt%, it is standby to collect medicinal residues, merges ethanol extract, reclaim ethanol and also be concentrated into and do not have the alcohol flavor, dry extract 1;

C) behind the Radix Bupleuri and Rhizoma Zingiberis pulverizing with described parts by weight, collect volatile oil with the vapor extraction method, it is standby to collect the water extract, and gained volatile oil is made the volatile oil beta cyclodextrin clathrate with saturated water solution method;

D) get Semen Trichosanthis, Ramulus Cinnamomi, Radix Codonopsis, Radix Glycyrrhizae Preparata, Rhizoma Zingiberis Recens and the Fructus Jujubae of described parts by weight, add the medicinal residues of step b), the water extract of step c), decoct with water twice, merge decocting liquid twice, filter, be concentrated into 60 ℃ of relative densities 1.05, centrifugal, get supernatant, be evaporated to 60 ℃ of relative densities 1.25～1.28, drying is pulverized, and gets dry extract 2;

E) merge dry extract 1 and dry extract 2, add above-mentioned Rhizoma Pinelliae fine powder and volatile oil beta cyclodextrin clathrate, mix homogeneously obtains the active component of medicine of the present invention.

Wherein, when extracting medicine with alcoholic solution in the step b), extract 1.5h for the first time, 1h is extracted in second and third time.

When decocting with water medicine in the step d), add water is 8 times of weight of medicine at every turn, decocts 1.5h.

The vapor extraction of described volatile oil is that medicine is pulverized, and crosses 24～35 sieves, adds the water logging bubble 1h of 8 times of drug weight, extracts 8h, collects volatile oil.

The preparation of described volatile oil beta cyclodextrin clathrate is according to beta cyclodextrin: volatile oil=8: 1, beta cyclodextrin: the weight proportion of water=1: 6, volatile oil and beta cyclodextrin, water are stirred 1h down at 35 ℃, and separate obtaining the volatile oil beta cyclodextrin clathrate.

Further, medicine of the present invention can also be that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 5～10 Fructus Schisandrae Chinensis 12～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 10～15

Radix Bupleuri 20～30 Radix Scutellariaes 8～15

The Rhizoma Pinelliae 8～12 Radix Codonopsis 12～18

Radix Glycyrrhizae Preparata 8～12 Radix Trichosanthis 10～15

Radix Platycodonis 10～15 Rhizoma Cyperis 6～15

Rhizoma Zingiberis Recens 5～15 Fructus Jujubaes 15～20.

Rhizoma Zingiberis 6～8 Fructus Schisandrae Chinensis 15～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 12～15

Radix Bupleuri 20～25 Radix Scutellariaes 9～12

The Rhizoma Pinelliae 9～12 Radix Codonopsis 12～15

Radix Glycyrrhizae Preparata 9～10 pollen 12～15

Radix Platycodonis 10～12 Rhizoma Cyperis 8～12

Rhizoma Zingiberis Recens 6～12 Fructus Jujubaes 16～18.

B) Fructus Schisandrae Chinensis of described parts by weight and the Radix Platycodonis alcoholic solution with 80wt% is extracted three times, it is standby to collect medicinal residues, merges ethanol extract, reclaim ethanol and also be concentrated into and do not have the alcohol flavor, dry extract 1;

D) get Semen Trichosanthis, Ramulus Cinnamomi, Radix Scutellariae, Radix Codonopsis, Radix Glycyrrhizae Preparata, Radix Trichosanthis, Rhizoma Cyperi, Rhizoma Zingiberis Recens and the Fructus Jujubae of described parts by weight, add the medicinal residues of step b), the water extract of step c), decoct with water twice, merge decocting liquid twice, filter, be concentrated into 60 ℃ of relative densities 1.05, centrifugal, get supernatant, be evaporated to 60 ℃ of relative densities 1.25～1.28, drying is pulverized, and gets dry extract 2;

Wherein, when extracting medicine with alcoholic solution in the step b), extracted 1.5 hours for the first time, second and third time extracted 1 hour.

When decocting with water medicine in the step d), add water is 8 times of weight of medicine at every turn, decocts 1.5 hours.

The vapor extraction of described volatile oil is that medicine is pulverized, and crosses 24～35 mesh sieves, adds the water logging bubble 1h of 8 times of drug weight, extracts 8h, collects volatile oil.

The active component of medicine of the present invention can add various conventional adjuvant required when preparing different dosage form, as disintegrating agent, lubricant, binding agent etc., method of Chinese medicinal with routine is prepared into any peroral dosage form commonly used, as pill, powder, tablet, capsule, oral liquid etc.

In the medicine of the present invention, the Radix Bupleuri bitter in the mouth is flat, goes into the liver and gall warp, and the heresy of the few sun of expelling heat from the exterior, and the stasis of energy catharsis mechanism of qi are evacuated the heresy of few sun half table, are monarch drug.The Radix Scutellariae bitter cold, the heat of clearing away the heat in SHAO YANG half table is ministerial drug.The liter of Radix Bupleuri looses, and gets falling of Radix Scutellariae and lets out, and the two phase 5 is the basic structure for the treatment of SHAO YANG disease by mediation.Gallbladder-QI invading the stomach, stomach-QI being unable to descend normally is with the Rhizoma Pinelliae, Rhizoma Zingiberis Recens regulating the stomach and sending down the adverse rising QI to arrest nausea; Evil import few sun into, come from the healthy energy deficiency in origin from the sun, so with the dispel heresy of the sun of Ramulus Cinnamomi, with Radix Codonopsis, Fructus Jujubae replenishing QI to invigorate the spleen, the latter two one can be set upright getting rid of evils, but two QI invigorating pass to resist in the pathogen, make healthy energy vigorous, the then evil machine that does not have interior biography.Fructus Schisandrae Chinensis, Rhizoma Zingiberis warming lung for dispelling cold, then cough with asthma is from ending, and sputum is from changing; The Semen Trichosanthis the chest stuffiness relieving is regulated the flow of vital energy, then uncomfortable in chest must removing; Radix Trichosanthis, Radix Platycodonis sore-throat relieving, then dry pharynx must be separated; Radix Glycyrrhizae Preparata helps Radix Codonopsis, Fructus Jujubae to set upright, and can coordinating the actions of various ingredients in a prescription.All diseases are share, and have the exopathogen of dispeling, mildly regulating cold and heat, and dispersing obstructed lung-QI, the effect of relieving cough and asthma can be used for treating idiopathic pulmonary fibrosis.

Description of drawings

Fig. 1 is blank group lung tissue of rats HE colored light sem observation figure;

Fig. 2 is the 7th day lung tissue HE colored light of model control group rat sem observation figure;

Fig. 3 is the 14th day lung tissue HE colored light of model control group rat sem observation figure;

Fig. 4 is the 21st day lung tissue HE colored light of model control group rat sem observation figure;

Fig. 5 is the 7th day lung tissue HE colored light of medicine of the present invention heavy dose of group rat sem observation figure;

Fig. 6 is the 14th day lung tissue HE colored light of medicine of the present invention heavy dose of group rat sem observation figure;

Fig. 7 is the 21st day lung tissue HE colored light of medicine of the present invention heavy dose of group rat sem observation figure;

Fig. 8 is the 7th day lung tissue HE colored light of positive controls rat sem observation figure;

Fig. 9 is the 14th day lung tissue HE colored light of positive controls rat sem observation figure;

Figure 10 is the 21st day lung tissue HE colored light of positive controls rat sem observation figure.

The specific embodiment

Embodiment 1

A) get the 2Kg Rhizoma Pinelliae, it is standby to be ground into fine powder;

B) get the 3Kg Fructus Schisandrae Chinensis, add in 80% alcoholic solution of 30L heating extraction 1.5h, filter, collect filtrate, add 80% alcoholic solution 25L in the medicinal residues, heating extraction 1h filters, and collects filtrate, add 80% alcoholic solution 25L in the medicinal residues again, heating extraction 1h filters, it is standby to stay medicinal residues, merge three times ethanol extract, reclaim ethanol and be concentrated into and do not have the alcohol flavor, get dry extract 1;

C) get Radix Bupleuri 6.5Kg, Rhizoma Zingiberis 2.5Kg, pulverized 24～35 mesh sieves, add in the 75L water, soak 1h earlier, extract 8h with the vapor extraction method again, collect volatile oil, it is standby to leave and take the water extract; According to beta cyclodextrin: volatile oil=8: 1, beta cyclodextrin: the weight ratio of water=1: 6, volatile oil and beta cyclodextrin added in 35 ℃ the water, continue to stir 1h, standing demix, behind the branch vibration layer, drying obtains the volatile oil beta cyclodextrin clathrate;

D) get Semen Trichosanthis 4Kg, Ramulus Cinnamomi 3Kg, Radix Codonopsis 4.5Kg, Radix Glycyrrhizae Preparata 3Kg, Rhizoma Zingiberis Recens 3Kg and Fructus Jujubae 4Kg and mix, add the medicinal residues of step b) and the water extract of step c), moisturizing 150L, heating decocts 1.5h, filters, and adds 150L water in the medicinal residues again, heating decocts 1.5h, filters, and merges decocting liquid twice, be concentrated into 60 ℃ of relative densities 1.05, centrifugal, get supernatant, be evaporated to 60 ℃ of relative densities 1.25～1.28, after the dry also pulverizing, get dry extract 2;

E) merge dry extract 1 and dry extract 2, add above-mentioned Rhizoma Pinelliae fine powder and volatile oil beta cyclodextrin clathrate again, mix homogeneously gets medicine activity component;

F) get it filled 1 part of thing active component, 1 part of sucrose, 0.5 part in dextrin, it is an amount of to add 80% ethanol, and drying is made granule.

Embodiment 2

A) get the 2.5Kg Rhizoma Pinelliae, it is standby to be ground into fine powder;

B) get 4Kg Fructus Schisandrae Chinensis, 3Kg Radix Platycodonis, heating extraction 1.5h in 80% alcoholic solution of adding 60L filters, collect filtrate, add 80% alcoholic solution 50L in the medicinal residues, heating extraction 1h, filter, collect filtrate, add 80% alcoholic solution 50L in the medicinal residues again, heating extraction 1h, filter, it is standby to stay medicinal residues, merges three times ethanol extract, reclaiming ethanol and being concentrated into does not have the alcohol flavor, gets dry extract 1;

C) get Radix Bupleuri 6Kg, Rhizoma Zingiberis 1.5Kg, pulverized 24～35 mesh sieves, add in the 60L water, soak 1h earlier, extract 8h with the vapor extraction method again, collect volatile oil, it is standby to leave and take the water extract; Press beta cyclodextrin: volatile oil=8: 1, beta cyclodextrin: the weight ratio of water=1: 6, volatile oil and beta cyclodextrin added in 35 ℃ the water, continue to stir 1h, standing demix, behind the branch vibration layer, drying obtains the volatile oil beta cyclodextrin clathrate;

D) get Semen Trichosanthis 4.5Kg, Ramulus Cinnamomi 3Kg, Radix Scutellariae 2.5Kg, Radix Codonopsis 4Kg, Radix Glycyrrhizae Preparata 2Kg, Radix Trichosanthis 3Kg, Rhizoma Cyperi 2Kg, Rhizoma Zingiberis Recens 2Kg and Fructus Jujubae 4.5Kg and mix, add the medicinal residues of step b) and the water extract of step c), moisturizing 200L, heating decocts 1.5h, filters, and adds the heating of 200L water in the medicinal residues and decocts 1.5h, filter, merge decocting liquid twice, be concentrated into 60 ℃ of relative densities 1.05, centrifugal, get supernatant, be evaporated to 60 ℃ of relative densities 1.25～1.28, after the dry also pulverizing, get dry extract 2;

E) merge dry extract 1 and dry extract 2, add above-mentioned Rhizoma Pinelliae fine powder and volatile oil beta cyclodextrin clathrate again, mix homogeneously gets medicine activity component.

F) medicine activity component is pulverized, added 10% microcrystalline Cellulose, incapsulate behind the mixing and make capsule in the shell as lubricant.

Embodiment 3

Get the 3Kg Rhizoma Pinelliae powder and be broken into fine powder; Get 4.5Kg Fructus Schisandrae Chinensis, 3.5Kg Radix Platycodonis, obtain dry extract 1 and medicinal residues according to embodiment 1 step b) method; Get Radix Bupleuri 6.5Kg, Rhizoma Zingiberis 1.5Kg, obtain water extract and volatile oil beta cyclodextrin clathrate according to embodiment 2 step c) methods; Get Semen Trichosanthis 5Kg, Ramulus Cinnamomi 2.5Kg, Radix Scutellariae 3Kg, Radix Codonopsis 3Kg, Radix Glycyrrhizae Preparata 2Kg, Radix Trichosanthis 3.5Kg, Rhizoma Cyperi 3Kg, Rhizoma Zingiberis Recens 1.5Kg and Fructus Jujubae 5Kg and mix, add above-mentioned medicinal residues and water extract, obtain dry extract 2 according to embodiment 2 step d) methods; Merge dry extract 1 and dry extract 2, add above-mentioned Rhizoma Pinelliae fine powder and volatile oil beta cyclodextrin clathrate again, mix homogeneously gets medicine activity component.

Medicine activity component is pulverized, add 5% microcrystalline Cellulose, mixing, make granule with 70% ethanol as wetting agent, the magnesium stearate of adding 1%, mixing, tabletting, bag film-coat (the film-coat prescription is 70% hydroxypropyl emthylcellulose, 15% plasticizer Polyethylene Glycol-6000,15% lubricant Pulvis Talci) is made tablet.

Embodiment 4

Get the 2.5Kg Rhizoma Pinelliae powder and be broken into fine powder; Get the 4Kg Fructus Schisandrae Chinensis, obtain dry extract 1 and medicinal residues according to embodiment 1 step b) method; Get Radix Bupleuri 5Kg, Rhizoma Zingiberis 2Kg, obtain water extract and volatile oil beta cyclodextrin clathrate according to embodiment 2 step c) methods; Get Semen Trichosanthis 4Kg, Ramulus Cinnamomi 3.5Kg, Radix Codonopsis 4Kg, Radix Glycyrrhizae Preparata 2.5Kg, Rhizoma Zingiberis Recens 2.5Kg and Fructus Jujubae 4Kg and mix, add above-mentioned medicinal residues and water extract, obtain dry extract 2 according to embodiment 1 step d) method; Merge dry extract 1 and dry extract 2, add above-mentioned Rhizoma Pinelliae fine powder and volatile oil beta cyclodextrin clathrate again, mix homogeneously gets medicine activity component.

The thing active component of getting it filled adds 20% sodium carboxymethyl cellulose, mixing, with polyethylene glycol 6000: the mixed-matrix of Macrogol 4000=3: 1 mixes, and behind the heating and melting, splashes in the liquid paraffin condensed fluid and is condensed into drop pill, take out, make drop pill after draw in cold lime set and the drying.

Test example 1

Pharmaceutical intervention bleomycin of the present invention causes the morphologic experimentation of lung fibrosis in rats lung tissue

Idiopathic pulmonary fibrosis shows as matter pneumonia between homeliness type on lung tissue disease's Neo-Confucianism, be based on diffusivity alveolitis and alveolar structure disorder, and finally causing pulmonary fibrosis is the carrying out property lower respiratory tract systemic disease of feature.Still there is not specific Therapeutic Method at present.This experiment is investigated medicine of the present invention to the situation of change of the influence of experimental pulmonary fibrosis lung tissue of rats pathomorphism and bronchoalveolar lavage fluid cell component classification and respectively organize the induced lung coefficient by zoopery, for objective evaluation medicine of the present invention provides the pathomorphology foundation to the preventive and therapeutic effect of pulmonary fibrosis.

1, experiment material

The embodiment of the invention 2 medicine activity components.With the medicine activity component cubage, the heavy dose of group of medicine of the present invention concentration is 0.32g/ml, and middle dosage group is 0.16g/ml, and small dose group is 0.08g/ml (is equivalent to the clinical daily dosage body weight ratio of people 15,7.5 and 3.75 times).

Bleomycin A5 (BLM), trade name: Bleomycin A5; Harbin Bolai Pharmaceutical Co., Ltd. produces, lot number: 090607, and specification: 8mg/ props up, and the time spent adds the 1.6ml physiological saline solution.

Prednisone acetate tablets: Tianjin Pacific Pharmaceutical Co., Ltd. produces, lot number: 090707, and specification: 5mg/ sheet.Time spent is the prednisone acetate tablets pulverize, the adding distil water dissolving, and it is standby to be mixed with the 0.6mg/ml suspension, and dosage 10ml/kgbw is equivalent to 7.5 times of the clinical daily dosage 48mg body weight ratio of people.

Phosphate buffered solution (pH6.8): 1/15mol/L disodium phosphate soln 49.60ml and 1/15mol/L potassium dihydrogen phosphate 50.40ml mix homogeneously are formulated.Be used for the preparation of GiemSa dyeing liquor.Face with preceding and now join.

Ji's nurse Sa dyeing stock solution (Giemsa): get Giemsa 1g, glycerol 66ml, methanol 66ml.Giemsa is put into mortar, add earlier a small amount of glycerol and grind dissolving, remaining glycerol is poured in gradation into, is transferred in the beaker, puts 2h in 56 ℃ of water-baths, and it is even to add the 66ml methanol mixed after the cooling again, leaves standstill for 1～2 week in room temperature.Use filter paper filtering, be stored in the brown bottle standby.

Ji's nurse Sa dyeing liquor: get 1 part of mixing of 9 parts of phosphate buffered solution (pH6.8) and Ji's nurse Sa dyeing stock solution and be made into.Matching while using.

2, experimental technique

2.1 laboratory animal and grouping

144 of the healthy Wistar rats of cleaning level, male and female half and half, body weight 200 ± 20g, provide by Shanxi Province institute of traditional Chinese medicine Experimental Animal Center, animal quality certification SCXK (Shanxi) 2009-10, in disposable buying experiment the last week, raise in quiet, warm and photophygous environment the feed of freely drinking water by the cleaning grade standard.

With 144 rats be divided at random blank group (A group), model control group (B group), positive controls (C group), medicine of the present invention big (D group), in (E group), little (F group) dosage group, every group respectively 24.

2.2 modelling

Set up model by the Szapiel method: 20% urethane 5ml/kgbw intraperitoneal injection of anesthesia rat, lie on the back and be fixed on the rat experiment platform, skin of neck is cut in the sterilization back, successively separate, fully expose trachea, remove other each groups of blank group and slowly inject the bleomycin solution of dilution with the 1ml syringe, metering is 5ml/kg, capacity normal saline such as blank group injection, the upright and rotation with animal immediately after the injection, medicine is dispersed evenly in two lungs, skin suture then, the wound sterilization places the experiment indoor heat insulating to feed, treat that it revives voluntarily, the experimental session animal freely drinks water and takes food.

2.3 medication

Second day beginning gastric infusion after the modeling, the large, medium and small dosage group of medicine of the present invention gives the medicinal liquid gastric infusion of respective concentration (0.32g/ml, 0.16g/ml, 0.08g/ml) respectively, dosage is 10ml/kgbw, is equivalent to 15,7.5 and 3.5 times of the clinical daily dosage body weight ratio of people; Positive controls prednisolone acetate suspension (concentration 0.6mg/ml) gastric infusion, dosage is 10ml/kgbw, is equivalent to 7.5 times of the clinical daily dosage 48mg body weight ratio of people; Blank group and model control group all give the equivalent normal saline and irritate stomach, 1 time/day.21 days administration cycles.

2.4 method of drawing material

Rat is in modeling 7,14, divide behind the 21d and draw materials for three times, the front and back phase, each examined 10 at random extremely, examine 4 contrast checkings mid-term extremely, weigh, be fixed to the rat dissecting table behind 20% urethane solution (5ml/kg) intraperitoneal injection of anesthesia, open abdominal cavity and thoracic cavity, abdominal aortic blood, room temperature leaves standstill 2h, and freezing low speed centrifuge separation of serum (3000r/min, 10min), fully peel off trachea and lung, win two lungs rapidly, quick flush away blood in the normal saline of pre-cooling, the fixedly HE dyeing of left lung, right lung is weighed and is calculated the lung coefficient, and prepare 10% lung tissue homogenate, centrifugalize supernatant homogenate ,-70 ℃ of freezing preservations, to be checked.

2.5 ordinary circumstance is observed

The rat feed situation of every day, body weight change, breathing and appearance sign etc. are respectively organized in experiment after the observed and recorded modeling.

2.6 cell component analysis in the bronchoalveolar lavage fluid (BALF)

Lavation left side lung, folder closes the right lung main bronchus, to the capable bronchoalveolar lavage of left lung, once injects cold saline 2ml, quadruplication is collected bronchoalveolar lavage fluid, 4 ℃ of centrifugal (1500r/min, 15min), get sedimentation cell and wash twice, then the 1mlPBS re-suspended cell with PBS.45ul cell suspension adds the 5ul4% trypan blue, according to a conventional method the counting cells sum; 100ul cell suspension, cell get rid of the sheet machine and do the cell section, Giemas dyeing back differential counting.

2.7 light microscopy specimen is made

After getting rat left side lung and being soaked in 4% paraformaldehyde solution fixedly 24h, organize crosscut to become the thick thin slice of 3mm apex pulmonis to base of lung portion, 4% paraformaldehyde is 72h fixedly, ethanol gradient dehydration (50% ethanol 2min, 75% ethanol 2min, 80% ethanol 2min, 95% ethanol 2min, 100% ethanol 2min), dimethylbenzene is transparent, the directed embedding of paraffin, serial section, thickness 6um selects the 5th in the every specimen serial section, HE dyes (specifically being finished by Chinese Radiation Protection Research Inst pathology chamber), carries out qualitative and the sxemiquantitative observation.

2.8 light microscopic pathological examination

Determine alveolitis (～+++level) degree and pulmonary fibrosis (～+++level) degree by the Szapiel method.

Alveolitis is divided into level Four:

0 grade: no alveolitis;

1 grade: slight alveolitis, show as alveolar septum because of monocyte infiltration broadening, only limit to local and near pleura portion, area is less than 20% of full lung, and alveolar structure is normal;

2 grades: the moderate alveolitis, show as extent of disease and account for 20～50% of full lung, nearly pleura portion is heavier;

3 grades: the severe alveolitis, be diffusivity and distribute, extent of disease has mononuclear cell and the hemorrhage consolidation that causes greater than 50% in the accidental alveolar space.

Pulmonary fibrosis is divided into level Four:

0 grade: no fibrosis;

1 grade: mild fibrosis, extent of disease are less than 20%, and fibrosis is involved pleura and subpleural interstitial lung, and the structure of alveolar gets muddled;

2 grades: moderate fibrosis, extent of disease account for 20～50% of full lung, and extend from pleura in the fibrosis zone, still belong to local patholoic change;

3 grades: the severe pulmonary fibrosis: extent of disease is greater than 50%, the capsule air cavity that as seen the alveolar fuse damaged differs in size, pulmonary parenchyma structure disturbance.

3, statistical procedures

The experimental data measurement data is with mean ± standard deviation

Expression adopts SPSS13.0 software to carry out statistical analysis, and data relatively adopt one factor analysis of variance and LSD-t check between group, and the ranked data result adopts Radit to analyze inspection level α=0.05.

4, result

4.1 general state

4.1.1 mode of appearance changes

Blank group rat is vivaciously active, and diet drinking-water is normal, fur gloss, and body weight increases gradually, breathes steadily.Lethargy in one week of model control group modeling, diet drinking-water is few, is slow in action, fur tarnish, rapid breathing, weight increase is not obvious.Two week back diet drinking-water increases gradually, body weight begins slow rising.The early stage effect of positive controls is better, and it is hard perpendicular that fur appears in the later stage, and lazy moving, state is not so good as early stage, but is better than model control group.The heavy dose of group of medicine of the present invention fur still can, diet drinking-water better, weight increase is obvious, breathes well-balanced.

4.1.2 respectively organize the rat body weight situation of change before and after the modeling

Observe rat the 0th, 7,14,21d body weight, the results are shown in Table 1-1.Compare the weight loss of model control group rat the most remarkable (P＜0.01) with the blank group; All the other respectively organize rat body weight also in various degree decline; Compare with model control group, the big-and-middle dosage group of medicine of the present invention rat body weight ascendant trend is obvious, has remarkable significant difference (P＜0.05).

Respectively organize the rat body weight situation of change before and after the table 1-1 modeling

Annotate: compare △ P＜0.05, ▲ P＜0.01 with the blank group; Compare ☆ P＜0.05, ★ P＜0.01 with model group

4.1.3 respectively organize the lung coefficient ratio

According to formula: lung coefficient=lung weight (g)/body wt (kg) calculates the lung coefficient, by table 1-2 as can be seen, each phase lung coefficient of blank group does not have significant change, each phase lung coefficient of model control group rat is all apparently higher than the blank group same period (P＜0.01 or 0.05), positive controls, each phase lung coefficient of each dosage group rat of medicine of the present invention all is higher than the blank group rat same period, but be starkly lower than the model control group rat same period (P＜0.01 or 0.05), wherein, positive controls and the big-and-middle dosage group of medicine of the present invention lung coefficient all have improvement trend, with small dose group significant difference (P＜0.05) is arranged relatively, but compare no difference of science of statistics (P＞0.05) between three groups.

Each experimental group induced lung coefficient ratio of table 1-2

Annotate: compare ▲ P＜0.01, △ P＜0.01 with the blank group; Compare ★ P＜0.01, ☆ P＜0.05 with model group.

4.1.4 total cellular score and classification in the bronchoalveolar lavage fluid (BALF)

Compare with the blank group, 7, during 14d in the model control group BALF of Rats total cellular score all obviously raise (P＜0.01), peak during with 7d, begin during 14d to descend, recover normal during 21d substantially.Compare with model control group, 7, the reduction effect comparatively significantly (P＜0.01) of each dosage group of medicine of the present invention during 14d, the reduction effect of dosage group significantly (P＜0.01) in the medicine of the present invention during 14d, it is normal that 21d recovers substantially.

The cell divide results suggest, cell divide is based on pulmonary alveolar macrophage (AM) among the blank group BALF.Compare 7, model control group rat AM percentage ratio all obviously descends (P＜0.01 or 0.05) during 14d, returns to normal during 21d gradually with the blank group.Compare with model control group, 7, each dosage group of medicine of the present invention all can improve IPF rat AM percentage ratio during 14d, the wherein effect of medicine of the present invention senior middle school dosage group comparatively significantly (P＜0.01 or P＜0.05) during 7d, the effect of dosage group significantly (P＜0.05) in the medicine of the present invention returns to normal substantially during 14d during 21d.Compare 7, model control group rat neutrophilic granulocyte (PMN) and lymphocyte (LYM) percentage ratio all obviously rise (P＜0.01) during 14d, return to normal during 21d gradually with the blank group.Compare with model control group, 7, each dosage group of medicine of the present invention all can reduce IPF rat PMN and LYM percentage ratio during 14d, wherein during 7d medicine of the present invention senior middle school dosage group to the reduction effect of PMN percentage ratio comparatively significantly (P＜0.01), during 14d in the medicine of the present invention the dosage group to the reduction effect of PMN and LYM percentage ratio significantly (P＜0.05), substantially recover normal during 21d, see Table 1-3.

Table 1-3 respectively organizes total cellular score and classification comparison in the BALF of Rats

4.2 lung tissue pathology morphological change

4.2.1 gross examination of skeletal muscle

Two lung pinkiness when blank group 7,14,21d, smooth surface, good springiness.Visible intensive petechial hemorrhage kitchen range and the black ecchymosis of two lungs during model control group 7d, lung volume increases; Two lungs are canescence during 14d, and the local nodal-like that as seen differs in size changes and the remote hemorrhage point, and lung tissue is harder; Two lungs are pale during 21d, and volume-diminished is touched firmlyer, and surperficial nodal-like changes and streak chase.With model control group relatively, the big-and-middle small dose group two lung outward appearance pathological changes of positive controls and medicine of the present invention are significantly better than model control group during 7d, two lungs are dark red slightly, elasticity still can, wherein big-and-middle dosage group is significantly better than positive controls, small dose group; During 14～21d, the most of animal two lung colors of big-and-middle dosage group are dark slightly, two elastance of lung still can, the even visible part lobe of the lung has the point-like Lycoperdon polymorphum Vitt speckle that is dispersed in, but is better than positive controls and small dose group, and positive controls and small dose group slightly are better than model control group.

4.2.2HE colored light sem observation

The blank group: lung tissue structure is clear, and alveolar septum is not seen and thickened, no congestion and edema, inflammation and fibrosis performance, and nothing is obviously oozed out (see figure 1) in the alveolar space.

Model control group: alveolitis is obvious during 7d, all visible massive inflammatory cells infiltrated of alveolar space and interstitial lung, and based on neutrophilic granulocyte, lymphocyte, alveolar septum edema, slight broadening (see figure 2).The alveolitis degree alleviates to some extent during 14d, and alveolar septum thickens based on macrophage, lymphocytic infiltration, fibroblast proliferation, and alveolar space narrows down, and it is banded that interstitial collagen fibroplasia is, and the fibrosis (see figure 3) takes place.Fibrosis further increases the weight of during 21d, and alveolar structure destroys or disappears, a large amount of inflammatory cell infiltrations, and lung tissue is changed into main (see figure 4) with collagen deposition, pulmonary fibrosis.

Positive controls: alveolitis and pulmonary fibrosis degree alleviate than model control group, and inflammatory cell infiltration obviously reduces (see figure 8) than model control group during 7d.Be light poisoning alveolitis during 14d and change, alveolar septum thickens, lesion degree than the same period model control group light, the complete substantially (see figure 9) of lung tissue structure.Be slight pulmonary fibrosis during 21d and change, extent of disease is limitation, and pleura and alveolar septum collagen fiber deposition are the pencil (see figure 10).

Each dosage group of medicine of the present invention: big-and-middle dosage group alveolitis and pulmonary fibrosis degree all obviously alleviate than model control group.(see figure 5) is reduced than model group in lung tissue inflammatory cell infiltration zone during 7d; Be light moderate alveolitis during 14d and change, lung tissue structure is complete than model group, and fibroblast proliferation alleviates, and lesion degree is than the light (see figure 6) of the model control group same period; Light moderate fibrosis changes during 21d, the extent of disease limitation, and pleura and alveolar septum collagen fiber deposition are the pencil (see figure 7).The alveolitis of small dose group and pulmonary fibrosis degree and model control group alleviate more slightly, but not statistically significant (P＞0.05).

Alveolar structure destruction of five groups of rats (removing the blank group) and lesion degree are by heavily the order to light is as follows:

7d: model control group, small dose group, middle dosage group, positive controls, heavy dose of group.

21d: model control group, small dose group, positive controls, middle dosage group, heavy dose of group.

4.3 pathomorphology semi-quantitative analysis result

Om observation, the pathological change of alveolitis, pulmonary fibrosis does not appear in the blank group.The pathological change of alveolitis to pulmonary fibrosis obviously appears in model control group.The big-and-middle dosage group of medicine of the present invention is significantly better than small dose group and positive controls.Determine the degree of the degree of alveolitis (～+++level) and pulmonary fibrosis (～+++level), concrete outcome analysis in table 1-4 according to the Szapiel method.

Table 1-4 respectively organizes rat alveolitis and interstitial pulmonary fibrosis pathological analysis result (n=10)

Analyze through Radit, the alveolitis degree compares, and by being model control group, small dose group, middle dosage group, positive controls, heavy dose of group, blank group to light order heavily, comparing difference has statistical significance (P＜0.05) between group.The pulmonary fibrosis degree compares, and by being model control group, small dose group, positive controls, middle dosage group, heavy dose of group, blank group to light order heavily, comparing difference has statistical significance (P＜0.05) between group.

5, conclusion

This experiment is by to induced lung PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM after the modeling, and BLM causes lung fibrosis in rats the 1st～7d mainly based on the inflammatory cell infiltration of AM and PMN, with blood capillary proliferation, and fibroblast showed increased, alveolar septum broadening; The 8th～21d alveolar structure destroys, and interstitial pulmonary fibrosis forms, the blood capillary obturation.Its pathological characters changes consistent with bibliographical information.

5.1 medicine of the present invention is to the influence of IPF rat body weight and lung index variation

In the IPF rat pathogenic process, because the lung tissue respiratory dysfunction, secondary causes that serious hindrance appears in body nutrient digestion, absorption, and catabolism is greater than anabolism in the body, and poor growth appears in body.The rat body weight result shows: blank group body weight normally increases, the model control group rat follows the self-regeneration of lung tissue wound, body weight gain is slow, medicine group rat body weight of the present invention significantly increases in various degree than model control group, improve the inflammation hypertrophy situation after lung tissue is subjected to the BLM damage simultaneously, alleviate the lung coefficient, improve the lungs function of experimental rat.The result shows: medicine of the present invention has the certain protection effect to the infringement by BLM inductive airframe systems visceral organ injury reparation and general utility functions state.

5.2 medicine of the present invention is to the intervention of cell component in the IPF BALF of Rats

Compare with the blank group, 7, during 14d in the model control group BALF of Rats total cellular score all obviously raise (P＜0.01), peak during with 7d, cell percentage constituent ratio changes, the downward modulation of AM cell proportion, PMN rises; Total cellular score begins to descend during 14d, and cellularity strengthens than changeing with LYM and AM ratio; Total cellular score has returned to normal level among the 21dBALF, visible a small amount of LYM and AM.Compare with model control group, medicine senior middle school's dosage group of the present invention and positive controls reduce PMN recovering cell percentage ratio, recover on the AM percentage ratio trend significantly (P＜0.01 or P＜0.05).

5.3 medicine of the present invention is to the morphological observation of IPF rat

Function and structure are the pair of interacting entities, and unusual structure will inevitably cause the undesired of function, and function is in abnormal state for a long time, can cause serious structural change again.During pulmonary fibrosis organizational structure show as inflammatory cell infiltration unusually, alveolar structure destroys or disappears alveolar septum broadening, fibroblast proliferation.This experiment is observed comparison from lungs organizational structure and view of morphology thereof to the therapeutic effect of medicine of the present invention.The result shows that the inflammation degree of rat model pulmonary alleviates after the Drug therapy of the present invention, and alveolar structure is complete, and the interstitial collagen precipitation reduces, and confirms that medicine of the present invention can improve the unusual lung tissue structure of IPF rat, thereby makes the improvement of function become possibility.And from lung tissue irrigating solution cell component change aspect, medicine of the present invention can improve cell component poised state unusual in the IPF lung tissue of rats, also confirmed this point, illustrated that medicine of the present invention all can alleviate the abnormal change of the lung tissue of I PF rat from function and structure two aspects.

Experimental result shows that at the commitment that pulmonary fibrosis takes place, prednisone and medicine of the present invention (big-and-middle dosage) all have the effect that improves the alveolar inflammation preferably, and the normal configuration of keeping alveolar and the structure of alveolar parenchyma are had good effect.But along with advancing of disease, to middle and late stage, the curative effect of medicine of the present invention (big-and-middle dosage) is significantly better than positive controls, the long-term efficacy that medicine fibrosis of the present invention is described is better than positive controls, and this may can reduce immunity of organisms with the life-time service prednisone, hinder the side reactions such as reparation of body tissue relevant.As seen medicine of the present invention is a kind of active drug of control pulmonary fibrosis, and presents certain dose-effect relationship.

Test example 2

The experimentation of pharmaceutical intervention lung fibrosis in rats free radicals in lung tissue damage of the present invention

Oxygen-derived free radicals plays an important role in the pathogenic process of pulmonary fibrosis.Under the physiological condition, antioxidase system can in time regulate and remove oxygen-derived free radicals as superoxide dismutase (SOD), glutathion oxide enzyme (GSH-Px) and nitric oxide synthetase (i NOS) in the body.Wherein malonaldehyde (MDA) is the snperoxiaized main metabolites of cytolipin, but the unsaturated fatty acid in the direct aggression tissue biological film causes tissue oxidizing damage and unusual reparation.The extracellular matrix metabolism disorder is to cause the main diseases of pulmonary fibrosis pathological changes therefore, and during lungs generation disease, body is removed the function generation obstacle of reactive oxygen free radical, and the activity in vivo oxygen-derived free radicals increases unusually, thereby influences the reconstruct of substrate such as extracellular collagen protein.

For the mechanism of action of further inquiring into medicine pulmonary fibrosis resistant of the present invention and may approach, this experimentation medicine of the present invention Pingyangmycin is induced the influence of GSH-Px, SOD in the lung fibrosis in rats lung tissue, MDA and NO content.

1, experiment material

GSH-Px, SOD, MDA, NO and Coomassie brilliant blue protein determination kit build up bio-engineering research by Nanjing and provide that (lot number: GSH-Px is 20090721, SOD is 20090721, MDA is 20090722, and NO is 20090722, and Coomassie brilliant blue is 20090725).

2, experimental technique

2.1 collection of specimens

Laboratory animal and grouping, modelling, medication are with test example 1.

Respectively at after the modeling the 7th, 21d, each group is 10 rats of picked at random respectively, 20% urethane intraperitoneal injection of anesthesia, be fixed on the rat experiment dissecting table, cut off skin of neck and open the thoracic cavity, peel off trachea and lungs, win two lungs, put into the cold saline rinsing and remove floating blood, filter paper blots, and gets inferior lobe of right lung and organizes in right amount, is cut into fragment on ice bath, the normal saline that adds 9 times of pre-coolings, grind fast in the ice bath and make 10% tissue homogenate, at 4 ℃ of centrifugal 10min, rotating speed is 3000r/min, get supernatant and measure ,-70 ℃ of freezing preservations.

2.2 detection method

In strict accordance with test kit description step, carry out GSH-Px and SOD activity, MDA and NO Determination on content.

3, statistical procedures

Each is organized data and all adopts

Expression adopts the SPSS13.0 statistical software to handle, and data relatively adopt one factor analysis of variance between many groups, relatively check inspection level a=0.05 between group in twos with LSD-t.

4, result

4.1 glutathion peroxidase in the lung tissue of rats (GSH-Px) assay

Glutathion peroxidase (GSH-Px) stable content in blank group rat mid-term and the late period lung tissue; Compare with the blank group, GSH-Px content all obviously reduces (P＜0.01) in the model control group lung tissue of rats, and all the other each groups also have reduction in various degree, and the model copy success is described; Compare the big-and-middle dosage group of the medicine of the present invention GSH-Px content (P＜0.05) in the lung fibrosis in rats lung tissue that all can significantly raise with model control group.The results are shown in Table 2-1.

Table 2-1 medicine of the present invention is to the active influence of GSH-Px in each group lung tissue of rats

Annotate: compare with the blank group: ▲ P＜0.01, △ P＜0.05; Compare with model control group: ★ P＜0.01, ☆ P＜0.05;

4.2 superoxide dismutase in the lung tissue of rats (SOD) assay

Compare with the blank group, 7, during 21d in the model control group lung tissue of rats SOD content all obviously reduce (P＜0.01), all the other respectively organize rat also has in various degree and reduces, and illustrate that model copy is successfully.Compare with model control group, the big-and-middle dosage group of the medicine of the present invention SOD content (P＜0.05) in the IPF lung tissue of rats that all can significantly raise, during 21d, the rising effect of the heavy dose of group of medicine of the present invention is (P＜0.01) significantly.The results are shown in Table 2-2.

Table 2-2 medicine of the present invention is to the active influence of SOD in each group lung tissue of rats

Annotate: compare ★ P＜0.01, ☆ P＜0.05 with normal group; Compare ▲ P＜0.01, △ P＜0.05 with model group;

4.3 malonaldehyde in the lung tissue of rats (MDA) assay

Compare with the blank group, 7, during 21d in the model control group lung tissue of rats MDA content all obviously raise (P＜0.01), all the other each groups also have in various degree and raise, and illustrates that model copy is successful.Compare with model control group, the big-and-middle dosage group of medicine of the present invention all can significantly reduce MDA content in the PF lung tissue of rats (P＜0.05).The results are shown in Table 2-3.

Table 2-3 medicine of the present invention is respectively organized the influence of MDA content in the lung tissue of rats

4.4 nitric oxide in the lung tissue of rats (NO) assay

Compare with the blank group, 7, during 21d in the model control group lung tissue of rats NO content all obviously raise (P＜0.01), all the other each groups also have in various degree and increase, and illustrates that model copy is successfully; Compare with model control group, the big-and-middle dosage group of medicine of the present invention can significantly reduce the content (P＜0.05) of NO in the lung fibrosis in rats lung tissue, and during 21d, the reduction effect of the heavy dose of group of medicine of the present invention is (P＜0.01) significantly.The results are shown in Table 2-4.

The content of NO in the homogenate of table 2-4 lung tissue of rats

Annotate: compare ★ P＜0.01 with the blank group; ☆ P＜0.05; Compare ▲ P＜0.01, △ P＜0.05 with model control group;

5, conclusion

In the formation mechanism of pulmonary fibrosis, oxygen free radical injury and Its Mechanisms thereof are subjected to numerous scholars' attention gradually.Under the physiological condition, antioxidase system can in time regulate and remove free radical as SOD, GSH-Px and iNOS in the body.If oxygen-derived free radicals discharges too much, or body removes the oxygen-derived free radicals ability drop, and oxygen-derived free radicals will the damaged tissue cell, and lungs are one of easily impaired target organs.Therefore, in the research of the I PF active drug that seeks treatment, the oxidation resistance of enhancing body is especially removed the ability of oxygen-derived free radicals, has become and has weighed one of efficacy criteria.

The pulmonary fibrosis pathogenesis it be unclear that so far, and many experiments show that it is unbalance to exist lower respiratory tract oxidation/antioxidation in the pulmonary fibrosis pathogenic process.Active oxygen such as superoxide anion and hydrogen peroxide are the important medium of multiple disease, comprise the necrosis of alveolar inflammation, lung tissue fibrosis lesion etc.Interrelated data shows: the unusual increase of reactive oxygen free radical can cause that hydrogen chain interruption, base degraded and the main chain of DNA in the cell untwist, all nucleic acid compositions all can be subjected to the attack of free radical, this damage can be repaired by some special mechanisms, but also can cause permanent damage.So, when the cell DNA damaged, can cause activity of cell biology to change, even cause gene mutation, tumor and cell death.Free radical can cause chain interruption when attacking deoxyribose, but this class damage can be repaired by the repair system of DNA, yet the dna mutation rate of repairing is but much larger than the mutation rate of normal DNA.

There are some researches prove that reactive oxygen free radical has the obvious impairment effect to protein, under pathologic condition, the proteinic damaging action of radical pair mainly is the modified amino acid residue, causes that structure and space conformation change, and cause peptide chain interruption, polymerization and crosslinked.Old and feeble in recent years, cancer exists that non-activity SOD is existing to be reported during with radiation damage in the blood samples of patients, may be due to the endogenous oxygen free radical injury SOD structure.Studies show that free radical is to make its inactivation by the specific action to the methionine residues in protease inhibitor and the alpha1-antitrypsin structure.Active group on many enzymes and the acceptor molecule---sulfydryl is the target gene that oxygen-derived free radicals is attacked, and free radical and sulfydryl reaction can make the biologic activity of enzyme and receptor etc. reduce, and function is impaired.Damaging action also can take place to biological polyoses, hyaluronic acid, unsaturated fatty acid etc. in active oxygen, and oxygen-derived free radicals makes monosaccharide generation autoxidation, thereby promotes protein cross, causes the albumen polymerization, can make basement membrane thickened, causes that a series of diseases take place.Unsaturated fatty acid is the important component in the biological film phospholipid, if its receptor 1 activity oxygen is attacked, then membranous structures such as erythrocyte, mitochondrion, microsome, lysosome all can be destroyed.

Reactive oxygen free radical damage formation to pulmonary fibrosis after the bleomycin modeling plays key effect.Under normal physiological condition, the tissue injury that free radical causes can be suppressed by the endogenous antioxidant.But, be oxidation antioxidation system when unbalance when these polyphenoils are not enough to resist a large amount of oxides, irreversible damage will take place in tissue.Think that at present the generation that lower respiratory tract oxidation/antioxidation is unbalance may be assembled at lower respiratory tract with inflammatory cell such as neutrophilic granulocyte confidential relation.When lung tissue sustains damage when being inflamed, inflammatory cell such as neutrophilic granulocyte, pulmonary alveolar macrophage can be assembled at lower respiratory tract, causes lungs oxygen overload, and body is removed the function generation obstacle of free radical, interior free yl increases, thereby causes the normal reconstruct of extracellular matrix.Neutrophilic granulocyte can discharge a large amount of oxygen-derived free radicals, arachidonic acid metabolite and proteolytic enzyme etc., causes inflammation to continue and the damage of lung tissue.

SOD can remove the ultra-oxygen anion free radical that produces in the biological oxidation process; it is one of body important antioxidant reductase of effectively removing active oxygen; the first line of defence that is called as the organism antioxidant system; its activity increases; can stop the chain reaction of oxygen-derived free radicals; remove oxygen-derived free radicals, the protection cell is avoided damage, so it can reflect the ability of body removing oxygen-derived free radicals.GSH-Px is a kind of important catalyzing hydrogen peroxide catabolic enzyme that extensively exists in the body, and its special reproducibility polypeptide such as catalytic reduction type glutathion can play the effect of protection membrane structure and telotism to the reduction reaction of hydrogen peroxide.Body all can produce oxygen-derived free radicals by enzyme system and non-enzyme system, the latter can attack polyunsaturated acid and the fatty acid in the biomembrane, cause lipid peroxidation, and therefore form lipid peroxide, as: aldehyde radical, ketone group, hydroxyl or new oxygen-derived free radicals etc.Oxygen-derived free radicals not only causes cell injury by the peroxidating of polyunsaturated acid in the biomembrane and fatty acid, and can also cause cell injury by the catabolite of fat hydroperoxides.MDA is the lipid peroxide catabolite, and its content often can reflect the snperoxiaized degree of body inner lipid.Thereby the amount of measuring MDA can reflect the snperoxiaized degree of body inner lipid, reflects the degree of cell injury indirectly.Simultaneously, when the excessive generation of active oxygen, intravital defense function often also is suppressed, and the consumption of enzymes such as SOD is increased, and vigor reduces in the tissue.

Nitricoxide synthase iNOS can generate citrulline and NO by the catalyzing levorotatory arginine, is the key enzyme that generates NO.The NO of low concentration can strengthen mitochondrial reducing activity.Higher concentration NO then can increase pulmonary's nitrous acid accumulation of salts.This experimental result shows that the active of iNOS obviously raises in the IPF lung tissue of rats, and this explanation BLM acts on rat and can produce NO and participate in the oxidative damage lung tissue.The fibrotic disease human fibroblasts can be synthesized NO, and the normal person fibroblast then can not be synthesized NO.This also explains the oxidative damage of pulmonary fibrosis pathological process from another point of view.After Drug therapy lung fibrosis in rats of the present invention, lung tissue i NOS decrease to some degree points out this medicine can reduce the generation of NO to a certain extent.

Though, free radical is necessary by the many important reaction of normal activities, and it participates in synthetic (as the arachidonic acid synthesis of prostaglandins) of bioactive substance, the detoxifcation reaction, the activity of phagocyte kill bacteria etc., but excessive oxygen-derived free radicals has damage effect widely to body.

This experimental result shows, medicine of the present invention can be regulated the intravital Radical Metabolism of Pulmonary Fibrosis in Rats model in various degree, improve the activity of interior antioxidase GSH-Px of rat model body and SOD, reduce the content of MDA and NO, wherein better with the heavy dose of group of medicine of the present invention effect, present certain dose-effect relationship.But relatively with the model matched group, GSH-Px, SOD increased activity all have difference (P＜0.01 or 0.05) in various degree in each dosage group lung tissue liquid of positive controls and medicine of the present invention, illustrate that medicine of the present invention can improve intravital Radical Metabolism, but still there be in various degree unusual in the rat model free radical metabolism.The active of GSH-Px and SOD significantly reduces in the pulmonary fibrosis model rat body, and MDA and NO content significantly raise, and this may be one of pathogenesis that brings out pulmonary fibrosis.Through after the Drug therapy of the present invention; intravital GSH-Px of pulmonary fibrosis model rat and SOD are active significantly to raise; MDA and NO content significantly descend; illustrate that medicine of the present invention can increase the content of oxygen free radical scavenger; reduce the infringement of radical metabolism product, reach the equilibrated effect of adjusting oxidation/antioxidant system, bleomycin is caused the induced lung damage have the certain protection effect; thereby alleviate the alveolitis degree, alleviate the Pulmonary Fibrosis in Rats pathological changes.

Test example 3

The experimentation that pharmaceutical intervention lung fibrosis in rats serum cytokines of the present invention is expressed

In the pathogenesis research of pulmonary fibrosis, various effect of cytokines are also more and more paid attention to by the scholar, and then have proposed the notion of cytokine network.There are some researches show, multiple somatomedin participates in pulmonary inflammatory reaction and pulmonary fibrosis process, and wherein transforming growth factor-beta 1 (TGF-β 1) and platelet-derived growth factor (PDGF) etc. are to the synthetic and crucial effect of having degraded of the division of regulation and control lung fibroblast, propagation and collagen protein.The research report is arranged simultaneously, and tumor necrosis factor-a (TNF-a) is for the proinflammatory factor important in the pulmonary fibrosis and cause the fibrosis factor, and they make alveolar fibroblast hypertrophy by different links, and a large amount of collagens of justacrine cause the formation of pulmonary fibrosis.

TGF-β 1 is one of the strongest extrtacellular matrix deposition promoter of finding so far.In the pulmonary fibrosis pathogenic process, activation and expression that inflammation and damage can be induced TGF-β 1.Activated T GF-β 1 increases the formation of extracellular matrix by its receptor and Smad approach, thereby further brings out pulmonary fibrosis.TGF-β 1 is considered to one of cytokine that plays an important role in the pulmonary fibrosis pathological process, occupies critical role in the cytokine network that pulmonary fibrosis takes place.Studies show that TGF-β 1 is its important function in the generation of pulmonary fibrosis and evolution.

Medicine of the present invention has the pulmonary fibrosis effect that anti-bleomycin brings out, the content in the classical sandwich assay enzyme-linked immunosorbent assay experimental model rat blood serum is adopted in this experiment, observe the intervention situation that medicine of the present invention is expressed the lung fibrosis in rats cytokine network, further inquire into the mechanism of medicine pulmonary fibrosis resistant of the present invention effect from the angle of cytokine.

1, experiment material

TNF-a ELISA test kit: lot number: 20091205; Wuhan doctor's moral Bioisystech Co., Ltd;

Rat PDGF ELISA test kit: lot number: 20091205; Wuhan doctor's moral Bioisystech Co., Ltd;

TGF-β 1 ELISA test kit: lot number: 20091205; Wuhan doctor's moral Bioisystech Co., Ltd;

The automatic microplate reader of Bio-Tek ELX8000.

2, experimental technique

2.1 collection of specimens

Laboratory animal and grouping, modelling, medication are with test example 1.

Respectively at after the modeling the 7th, the 21d picked at random respectively organizes 10 rats, with 20% urethane intraperitoneal injection of anesthesia, fixing, expose the abdominal cavity, use the 5ml syringe in abdominal aortic blood 4ml, room temperature leaves standstill 2h, 4 ℃ of centrifuge 3000r/min, time 15min, separation of serum, deposit in-70 ℃ of refrigerators, to be measured.

2.2 detection method

TNF-a adopts the ABC-ELISA method to detect, and the method step that provides according to test kit reacts, and surveys light absorption value (OD) at the 450nm place with microplate reader after the cessation reaction, and result and standard curve are relatively determined TNF-a content.

PDGF adopts the ELISA method to detect, and with 5000 times of sample serum dilutions, reacts in strict accordance with test kit description operating procedure, surveys light absorption value (OD) at the 450nm place with microplate reader after the cessation reaction, and result and standard curve are relatively determined PDGF content.

TGF-β 1 adopts the ABC-ELISA method to detect, and the method step that provides according to test kit reacts, and surveys light absorption value (OD) at the 450nm place with microplate reader after the cessation reaction, and result and standard curve are relatively determined TGF-β 1 content.

3, statistical procedures

Each is organized data and all adopts

Expression adopts the SPSS13.0 statistical software to handle, and relatively adopts one factor analysis of variance between many groups, relatively adopts the LSD-t check between each group in twos, inspection level a=0.05, and promptly P＜0.05 thinks that two group differences have statistical significance.

4, result

4.1 each experimental group rat blood serum is at the TNF-a of different time points content

TNF-a content remains unchanged substantially in blank group mid-term and the late period serum; Compare with the blank group, model control group TNF-a content in each time point serum all significantly increases (P＜0.01), and the big-and-middle dosage group of medicine of the present invention increases in various degree, and the modeling success is described; Compare with model control group, the level of TNF-a significantly reduces (P＜0.05) in the big-and-middle dosage group of the medicine of the present invention rat blood serum; The 8th～21d, the TNF-a level has lasting reduction trend in the serum, has significant difference (P＜0.01).See table 3-1 for details.

Each time point of table 3-1 is respectively organized the level of TNF-a in the rat blood serum

Annotate: compare ★ P＜0.01, ☆ P＜0.05 with the blank group; Compare ▲ P＜0.01, △ P＜0.05 with model group;

4.2 each experimental group rat blood serum is at the PDGF of different time points content

The fluctuation of PDGF content is little in blank group mid-term and the late period serum; Compare with the blank group, PDGF content all significantly increases (P＜0.01) at each time point in the model control group serum, and positive controls, the big-and-middle small dose group of medicine of the present invention blood-serum P DGF content on each time point all increases in various degree, and the modeling success is described.Compare with model control group, the big-and-middle dosage group of medicine of the present invention blood-serum P DGF content reduces significant difference (P＜0.05).See table 3-2 for details.

Each experimental group rat blood serum of table 3-2 is at the PDGF of different time points content

4.3 each experimental group rat blood serum is at the TGF-of different time points β 1 content

The content change of blank group TGF-β 1 in mid-term and late period serum is little; Compare with the blank group, TGF-β 1 content all significantly increases (P＜0.01) in model control group mid-term and the late period serum, TGF-β 1 content slightly increases (P＞0.05) in positive controls, medicine of the present invention big-and-middle small dose group mid-term and the late period serum, and the model copy success is described.Compare with model control group, medicine of the present invention big-and-middle small dose group mid-term and late period, serum TG F-β 1 content had significant difference (P＜0.05).See table 3-3 for details.

Each experimental group rat blood serum of table 3-3 is at the TGF-of different time points β 1 content

5, conclusion

5.1 cytokine and interstitial pulmonary fibrosis

Cytokine is being brought into play important effect in human body, can mediate natural immunity, regulate specific immune response, apoptosis-induced and hemopoietic etc.Studies show that cytokine network is being brought into play important effect in the generation evolution of I PF, these cytokine media can influence the deposition of fibroblastic activation, propagation and collagen in the lysis.

TNF-a is called the proinflammatory cytokine again, is the key factor that starts antimicrobial antiphlogistic, plays an important role in the generation evolution of pulmonary fibrosis.TNF-a has multiple biological activity, after the tissue injury, macrophage is raised damaged tissue and is activated on every side, discharge a large amount of TNF-a, increase the weight of inflammatory reaction on the one hand, can promote the fibroblast hypertrophy on the other hand, the collagen that justacrine is a large amount of, under the combined effect of fibronectin, form the alveolar inflammation at initial stage, cause the formation of pulmonary fibrosis at last.

TNF-a also can induce the alveolar type II epithelial cell to secrete multiple transforming growth factor, increases the weight of the lesion degree of lung tissue damage and interstitial lung inflammation, can induce lung to become propagation and the differentiation of target cells such as fibrosis cell.Intravenous injection TNF-a can cause diffusivity alveolar inflammation damnification, based on alveolar epithelial cells and endotheliocyte necrosis.TNF-a can promote fibroblast proliferation, differentiation and collagen to transcribe by TGF-β 1 or PDGF ways of regeneration indirectly, a large amount of TNF-a can induce the alveolar epithelial cells apoptosis, and alveolar and bronchioles epithelial cell apoptosis may cause the pulmonary fibrosis reaction, and Kuwano etc. find in bronchioles and the alveolar epithelial cells apoptosis marker is arranged in the biopsy specimen of IPF patient's lung tissue.As a kind of potential proinflammatory cytokine, TNF-a is considered to play an important role in pulmonary fibrosis always.In pulmonary fibrosis patient's serum, the expression of TNF-a is apparently higher than the normal person, and with after prednisone/prednisolone treatment, can obviously reduce the level of TNF-a, significantly improves pulmonary fibrosis patient's life quality.The close ties that are formed with of damage of the unusual increase of TNF-a and lung tissue and pulmonary fibrosis are described.

The dimer that PDGF is made up of A chain and two polypeptide chains of B chain, play a role by the pdgf receptor on the target cell membrane, one of its main biological effect is short cell division, PDGF can stimulate the division and proliferation of various kinds of cell such as vascular smooth muscle cell, fibroblast, glial cell, synthesize the renewal of regulating extracellular matrix with the activation of collagenase by stimulation collagen, final DNA synthesizes and lysis, propagation.Experiment is early stage, and bleomycin also stimulates cytokines such as its synthesis secretion PDGF to epithelial cell, endothelial cell damage the time, and the appearance of these cytokines in early days may help epithelial cell, endotheliocyte reparation.The PDGF that intralesional pulmonary alveolar macrophage etc. produced can pass through the epithelial cell of damaged and envelope barrier thereof and enter matter between alveolar, the PDGF that produces from macrophage in alveolar space and the alveolar septum etc. is the chemotactic Interstitial cell jointly, promotes its propagation, differentiation.PDGF has intensive chemotaxis and mitogenesis activity to fibroblast, and it can not only promote isolating fibroblast proliferation, and makes its I, III, the synthetic increase of IV procollagen type.Experiment in vivo and vitro confirms that also PDGF can promote lung fibroblast hypertrophy and collagen protein synthesis.

The TGF-beta superfamily is the polypeptide that a class has biologic activity, important function in cell growth and atomization.In the cytokine that participates in pulmonary fibrosis.TGF-β be study the most deep, act on most important cytokine, and the strongest extrtacellular matrix deposition promoter that is considered to find so far.It can act on a plurality of links, stimulates the synthetic and deposition of various extracellular matrix components, the activity that suppresses the active of extracellular matrix degrading enzyme and increase the substrate degradation enzyme inhibitor.In mammal, TGF-β has three kinds of hypotypes, and wherein TGF-β 1 is that it is separated the earliest, also is most important hypotype simultaneously.High-caliber TGF-β 1 is arranged in the experimental pulmonary fibrosis pathological changes, increase with the mRNA expression of I, III Collagen Type VI simultaneously.Anti-TGF-beta 1 serum can make hydroxyproline content minimizing in the fibroid lung tissue, and the pulmonary fibrosis degree alleviates.Studies show that TGF-β 1 mainly acts on transcribing of collagen and translation process, can induce precollagen mRNA to produce, thereby promote the formation and the deposition of collagen protein.Prompting is in the interstitial pulmonary fibrosis forming process, and TGF-β 1 has important function, reduces TGF-β 1 level by treatment, helps the control of interstitial pulmonary fibrosis.

At present, Most scholars is all thought has numerous cytokines to participate in the generating process of pulmonary fibrosis, and PDGF and TGF-β 1 participate in two important factors in the cytokine network of pulmonary fibrosis, and the relation between the two is very close.Huang Qunhua points out that the PDGF-a receptor of dissimilar cells may produce rise and reduce two kinds of different results the inducing action of the cytokines such as TGF-β 1, IL-1 of macrophage source property; The PDGF-beta receptor then is not subjected to the influence of above two kinds of cytokines.Ludwica separation and Culture from systemic sclerosis companion patients with interstitial pneumonia lung goes out myofibroblast, earlier with TGF-β 1 pretreatment, stimulate through PDGF again, the mitosis ability that found that myofibroblast strengthens, and he thinks that this may be to induce the result who has produced the PDGF-a receptor through TGF-β 1.In addition, Gleen etc. find when inquiring into the mechanism of tissue injury's reparation, PDGF is as a kind of effective chemotactic factor, injury region macrophage, fibroblast there is stronger chemotaxis, and can stimulate these cells to produce the endogenous somatomedin, comprising TGF-β 1, stimulate the synthetic of new collagen, thereby promote wound healing.Think that thus the effect of PDGF realizes by TGF-β 1.Wu Hao etc. studies show that, the main source of PDGF is a macrophage, play a significant role in morning, the mid-term of pulmonary fibrosis, and TGF-β 1 is mainly produced by Interstitial cell self, intermediary and later stages in pulmonary fibrosis play a significant role, and short extracellular matrix is synthetic, makes carrying out property of pulmonary fibrosis progress.

5.2 the influence that medicine of the present invention changes lung fibrosis in rats model serum cytokines network

Under the physiological status, rat blood serum PDGF and TGF-β 1 to be expressed in the change of each time point little, after the bleomycin modeling, PDGF content reaches peak value during 7d, though the 8th～21d decreases, but still is in high level; The expression of TGF-β 1 begins remarkable rising during 7d, the 8th～21d still rises to some extent, prompting PDGF and TGF-β 1 cause in the pulmonary fibrosis process at bleomycin has important function, and the expression that suppresses PDGF and TGF-β 1 is possible be one of approach of control pulmonary fibrosis.This experiment is observed simultaneously, TNF-a in each time point model control group rat blood serum expresses and is significantly higher than the blank group, and at 7d, positive controls, each group of medicine of the present invention all have the effect of TNF-a level in remarkable reduction (P＜the 0.01 or 0.05) serum, during 8～21d, TNF-a still has obvious reduction (P＜0.01 or 0.05) in the big-and-middle dosage group of the medicine of the present invention serum, and positive controls and medicine small dose group of the present invention do not have remarkable reduction (P＞0.05).Illustrate that bleomycin causes in the Pulmonary Fibrosis in Rats process, the rising of the level of TNF-a plays an important role in alveolitis, pulmonary fibrosis generating process; The alveolitis stage has and the positive similar excretory effect of inhibition TNF-a medicine of the present invention in early days, along with further developing of disease, the inhibition pulmonary fibrosis pathological changes effect of the big-and-middle dosage group of medicine of the present invention obviously is better than positive controls, and presents certain dose-effect relationship.Infer that thus medicine of the present invention is to the protective effect of lung fibrosis in rats lung tissue, may can reduce in the serum TNF-a etc. with it and cause inflammation, cause Fibrotic cytokine-expressing level close association is arranged.

Each group of medicine of the present invention all can effectively reduce the level of PDGF in the serum, TGF-β 1 and TNF-a on each time point, injury of lung due to the bleomycin and fibrosis are had preventive and therapeutic effect.Therefore, cytokine levels such as PDGF, TGF-β 1 and TNF-a may be one of mechanism of medical treatment lung fibrosis in rats lung tissue pathological changes of the present invention in the reduction lung fibrosis in rats serum.And we also observe, and the effect of heavy dose of group is better than small dose group, are reducing aspect TGF-β 1 horizontal, as if dosage group during the effect of heavy dose of group also is better than is certain dose-effect relationship, awaits later further research.

Test example 4

For further investigating the clinical efficacy and the safety of medicine of the present invention, selected 44 routine idiopathic pulmonary fibrosis patients that the curative effect of medicine of the present invention is carried out clinical observation.

Clinical data

Among the 44 routine patients, man's 25 examples, woman's 19 examples, 48～82 years old age, on average (65.1 ± 8.99) year, the diagnosis of spy's property sent out lung (matter) fibrosis and the treatment guide (draft) that all meet Chinese Medical Association's respiratory disease credit meeting in April, 2002 formulation: 1) concealment onset or carrying out property dyspnea are primary symptom, or with dry cough; 2) the air-breathing property of pulmonary's characteristic velcro sound; 3) drumstick finger and/or cyanosis; 4) to show as under two lungs down and the pleura be that main netted change or companion's honeycomb sample changes to the chest high resolution ct, can be with minute quantity ground glass sample shade.5) pulmonary function shows restrictive ventilatory disorder and/or diffusing capacity obstacle, and arterial blood gas is shown hypoxemia or motion back hypoxemia; 6) the interstitial lung pathological changes is shown in the lung biopsy.Have 1), 2), 4), 5) can make clinical diagnosis.Wherein make a definite diagnosis 1 example by fibre bronchus mirror lung biopsy (TBLB), all the other are clinical diagnosis.The idiopathy is the shortest 3 months, the longest 5 years to consultation time.The patient who enters test is clearly but not regular treatment of first prescription on individual diagnosis or diagnosis.

Patient is divided into treatment group and matched group at random, and 26 examples are organized in treatment, male 15 examples, women 11 examples, 66.1 years old mean age; Matched group 18 examples, male 10 examples, women 8 examples, 64.6 years old mean age; Age, sex, the balanced comparing difference of the state of an illness do not have significance (P＞0.05) before two groups of treatments.

Therapeutic Method

The treatment group: give the embodiment of the invention 2 capsules, every day 3 times, each 4, oral, took continuously two months, altogether 60d.

Matched group: give prednisone 0.5mgkg (ideal body weight) oral 4 weeks; Then oral 8 weeks of 0.25mgkg every day (ideal body weight), decrement is oral to 0.125mgkg every day (ideal body weight) afterwards, keeps 1～1.5 year, but experimental observation two months.

Two groups of patients have hypoxemia person to give oxygen uptake, and the complicated with pulmonary infection person uses the antibiotic control infection.

Therapeutic evaluation

Reach back 3 months symptoms such as main monitoring dyspnea of treatment before two groups of patient treatments, with chest imaging evaluation clinical therapeutic efficacy.Criterion of therapeutical effect evaluation curative effect with reference to " the special property sent out lung (matter) fibrosis diagnosis and treatment guide (draft) ".

1, produce effects treatment is after 3 months, if meet following 2 or 2 above conditions then think produce effects.

1) sx (cough, dyspnea); 2) X line or HRCT show that the interstitial lung change alleviates; 3) pulmonary function improves: meet following 2 or 2 above conditions, TLC or VC increase 〉=10%, SaO ₂Rise 〉=4%, PaO ₂Rising 〉=4mmHg during motion.

2, effectively treat and meet following 2 or 2 above conditions after 3 months, think that then treatment effectively.

1) TLC or VC increase＜10%, SaO ₂Rise＜4%, PaO ₂Rising＜4mmHg during motion.

3, futile treatment is after March, as following situation occurs, then thinks and fails to respond to any medical treatment or fail.

1) sx (cough, dyspnea); 2) chest x-ray or HRCT show that the interstitial lung change increases the weight of, and honeycomb lung or pulmonary hypertension sign especially occur; 3) pulmonary function worsens, and meets following 2 or 2 above conditions, and TLC or VC descend 〉=10%, SaO ₂Decline 〉=4% or AaDO ₂〉=4mmHg.

Observation of curative effect

Curative effect behind two groups of patient treatments is compared, the results are shown in Table 4-1,4-2.

Two groups of patient's symptom curative effects relatively before and after the table 4-1 treatment

Annotate: 1) relatively (adopt Ridit to analyze), u=3.3678, P=0.0009 with matched group.

By table 4-1 as can be known, treatment group produce effects 15 examples, effective 8 examples, total effective rate 88.5%, 95%CI (95% credibility interval)=63.8%～98.7%; Matched group produce effects 3 examples, effective 3 examples, total effective rate 33.3%, 95%CI=11.5%～55.1%; Two groups of comprehensive therapeutic effects compare u=3.3678, P=0.0009, and difference has the highly significant meaning, and the 95%C I of two groups of total effective rates is not overlapping.The relative risk that invalid patient is organized in treatment is 0.07 times (OR can see relative risk as, its 95%CI=0.01～0.31 for than several ratios) of matched group; Every treatment 2 people are organized in treatment, can reduce 1 routine invalid patient (NNT is clean treatment number) than matched group, and its 95% credibility interval is 1.20～3.75.

Two groups of patient's erythrocyte sedimentation rates, liver functions change before and after the table 4-2 treatment

Annotate: with comparison after the treatment of control group, 1) t=-2.1815, P＜0.05; 2) t=-3.1722 ,-2.7579, P＜0.01.

Erythrocyte sedimentation rate changes before and after the treatment of treatment group significant difference (t '=5.7493, P＜0.001), and does not show significant difference (t '=0.5094, P＞0.05) before and after the treatment of control group; There is significant difference (t=-2.1815, P＜0.05) the treatment back for two groups.The disease that can cause the erythrocyte sedimentation rate quickening in clinical is a lot, as rheumatic fever, acute infectious disease, pneumonia, cholecystitis, leukemia, myocardial infarction and some malignant tumor or the like, so esr detection is not the specificity method of inspection of disease, but can be used for roughly inferring advancing of disease and observe therapeutic effect, and data shows according to the study, and erythrocyte sedimentation rate and immunoreation have dependency.This observes demonstration, and the treatment group has significant inhibitory effect to immunoreation, points out the mechanism of this disease may be relevant with immunoreation, and the mechanism of action of medicine also may be with to regulate immunoreation relevant.In the process of Drug therapy idiopathic pulmonary fibrosis of the present invention, the variation of erythrocyte sedimentation rate has reflected the variation and the therapeutic effect of disease really, and this mentions in the diagnosis and treatment guide.

In two of the liver functions, there was no significant difference (t '=0.6599, P＞0.05) before and after the treatment group ALT treatment, before and after the AST treatment significant difference (t=2.1478 is arranged, P＜0.05), and clinical meaning and little, illustrate: 1) medicine of the present invention has good safety.2) ALT and AST all reflect the integrity of cell membrane, and the former is relevant with hepatocyte, and the latter is relevant with the outer cell of liver.ALT and AST are isolating state in this observation, illustrate that medicine of the present invention is harmless to hepatocyte, and disease and hepatocyte are irrelevant, AST significantly descends, and pathological changes is described outside hepatocyte, and pathological changes is relevant with cell membrane stability, also explanation of these reactions, disease is relevant with immunoreation.Immunoreation had both destroyed cell membrane stability, and erythrocyte sedimentation rate is accelerated, and after Drug therapy of the present invention, erythrocyte sedimentation rate and AST significantly descend, and was all relevant with stabilizing cell membrane, inhibition immunoreation.This observation shows that the effect link of medicine of the present invention is that stabilizing cell membrane has concurrently with suppressing immunoreation, is promptly intervened disease at multilevel, multi-level, many target spot.

In addition, chest x-ray or HRCT show, bilateral enhanced lung markings, disorder before the treatment, the bright degree of two lung fields obviously lowers, even be the ground glass sample, the cloud honeycomb lung changes, have also with emphysema, bilateral pleural thickening and a small amount of hydrothorax.After treatment, though two lungs see that still texture increases, disorder, even ground glass sample, the cellular change of cloud alleviating of obviously alleviating, have is fairly obvious, and all toward two lungs down, near pleura place limitation, the variation of pleural thickening improves obviously, and hydrothorax all absorbs; Emophysematous improvement is for short can all the loseing of the course of disease, and the course of disease does not obviously change than the elder.

The clinical observation on the therapeutic effect result shows, medicine of the present invention has the obvious treatment effect for interstitial lung disease especially idiopathic pulmonary fibrosis, total effective rate reaches 88.5%, compared significant difference with matched group, improved symptom, improve patient's life quality, prolonging and demonstrate special advantages aspect the life.In addition, medicine of the present invention and glucocorticoid all can improve erythrocyte sedimentation rate, and medicine of the present invention is more remarkable, and significant difference is arranged between the two.Medicine of the present invention occurs uncomfortable unusually during medication, and blood before and after the medication, urine, just reaches the no significant change of hepatic and renal function chemical examination, and the clinical observation on the therapeutic effect result shows safety of medicine of the present invention, effectively, worth clinical application.

English breviary vocabulary.

Abbreviation	Full name	Chinese
			IPF?	Idiopathic?pulmonary?fibrosis	Idiopathic pulmonary fibrosis
BLM?	Bleomycin?	Bleomycin
			ELISA?	Enzyme-linked?immunoadsordent?assay	Enzyme Linked Immunoadsorbent Assay
TNF-a?	Tumor?necrosis?factor-a	Tumor necrosis factor-a
			IL?	Inerlenkin?	Interleukin
SOD?	Superoxide?dismutase	Superoxide dismutase
			GSH-Px?	Glutathione?peroidase	Glutathion peroxidase
MDA?	Maleic?dialdehyde	Malonaldehyde
			PDGF?	Platelet-derived?growth?factor	Platelet-derived growth factor
TGF?	Transforming?growth?factor	Transforming growth factor
			PF?	Pulmonary?fibrosis	Pulmonary fibrosis
AT-II?	Alveolar?type?II?cell	The alveolar type II epithelial cell
			PBS?	Phosphate?buffered?saline	Phosphate buffer normal saline
AM?	Alveolar?macrophase	Pulmonary alveolar macrophage
			LMY?	Lymphocyte?	Lymphocyte
IFN?	Interferon?	Interferon
			HE?	Haematoxylin-Eosin?	Haematoxylin-Yihong staining
iNOS?	nitric?oxide?synthetase	Nitric oxide synthetase
			PMN?	polymorphonuclear?	Neutrophilic granulocyte

Claims

1. medicine for the treatment of idiopathic pulmonary fibrosis is to be that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 5～10 Fructus Schisandrae Chinensis 12～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 10～15

Radix Bupleuri 20～30 Rhizoma Pinelliaes 8～12

Radix Codonopsis 12～18 Radix Glycyrrhizae Preparatas 8～12

Rhizoma Zingiberis Recens 5～15 Fructus Jujubaes 15～20.

2. the medicine of treatment idiopathic pulmonary fibrosis according to claim 1 is to be that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 6～8 Fructus Schisandrae Chinensis 15～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 12～15

Radix Bupleuri 20～25 Rhizoma Pinelliaes 9～12

Radix Codonopsis 12～15 Radix Glycyrrhizae Preparatas 9～10

Rhizoma Zingiberis Recens 6～12 Fructus Jujubaes 16～18.

3. the preparation method of the medicine of claim 1 or 2 described treatment idiopathic pulmonary fibrosises may further comprise the steps:

E) merge dry extract 1 and dry extract 2, add the beta cyclodextrin clathrate of described Rhizoma Pinelliae fine powder and volatile oil, mix homogeneously obtains the active component of medicine of the present invention.

4. medicine for the treatment of idiopathic pulmonary fibrosis is to be that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 5～10 Fructus Schisandrae Chinensis 12～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 10～15

Radix Bupleuri 20～30 Radix Scutellariaes 8～15

The Rhizoma Pinelliae 8～12 Radix Codonopsis 12～18

Radix Glycyrrhizae Preparata 8～12 pollen 10～15

Radix Platycodonis 10～15 Rhizoma Cyperis 6～15

Rhizoma Zingiberis Recens 5～15 Fructus Jujubaes 15～20.

5. the medicine of treatment idiopathic pulmonary fibrosis according to claim 4 is to be that crude drug prepares by following parts by weight of traditional Chinese medicines:

Rhizoma Zingiberis 6～8 Fructus Schisandrae Chinensis 15～18

Semen Trichosanthis 15～20 Ramulus Cinnamomi 12～15

Radix Bupleuri 20～25 Radix Scutellariaes 9～12

The Rhizoma Pinelliae 9～12 Radix Codonopsis 12～15

Radix Glycyrrhizae Preparata 9～10 pollen 12～15

Radix Platycodonis 10～12 Rhizoma Cyperis 8～12

Rhizoma Zingiberis Recens 6～12 Fructus Jujubaes 16～18.

6. the preparation method of the medicine of claim 4 or 5 described treatment idiopathic pulmonary fibrosises may further comprise the steps:

7. according to the preparation method of the medicine of claim 3 or 6 described treatment idiopathic pulmonary fibrosises, when it is characterized in that wherein in the step b) extracting medicine, to extract 1.5 hours for the first time with alcoholic solution, second and third time extracted 1 hour.

8. according to the preparation method of the medicine of claim 3 or 6 described treatment idiopathic pulmonary fibrosises, when it is characterized in that decocting with water medicine in the step d), add water is 8 times of weight of medicine at every turn, decocts 1.5 hours.

9. according to the preparation method of the medicine of claim 3 or 6 described treatment idiopathic pulmonary fibrosises, the vapor extraction that it is characterized in that described volatile oil is that medicine is pulverized, and crosses 24～35 mesh sieves, adds the water logging bubble 1h of 8 times of drug weight, extract 8h, collect volatile oil.

10. according to the preparation method of the medicine of claim 3 or 6 described treatment idiopathic pulmonary fibrosises, the preparation that it is characterized in that described volatile oil beta cyclodextrin clathrate is according to beta cyclodextrin: volatile oil=8: 1, beta cyclodextrin: the weight proportion of water=1: 6, volatile oil and beta cyclodextrin, water are stirred 1h down at 35 ℃, separate obtaining the volatile oil beta cyclodextrin clathrate.