CN102885878A

CN102885878A - Application of effective parts of astragalus mongholicus and hedysarum polybotrys

Info

Publication number: CN102885878A
Application number: CN2012103748231A
Authority: CN
Inventors: 李金田; 李娟�; 刘永琦; 景明; 张毅; 蔺兴遥; 李雪燕; 苏蕴; 孙少伯
Original assignee: GANSU CHINESE OF TRADITIONAL CHINESE MEDICINE
Current assignee: GANSU CHINESE OF TRADITIONAL CHINESE MEDICINE
Priority date: 2012-09-29
Filing date: 2012-09-29
Publication date: 2013-01-23
Anticipated expiration: 2032-09-29
Also published as: CN102885878B

Abstract

The invention belongs to the traditional Chinese medicine field and relates to the preparation of effective parts of astragalus mongholicus and hedysarum polybotrys and selection thereof for pulmonary fibrosis model intervention so as to obtain further study on the effective parts having the optimal curative effect. An interstitial pulmonary fibrosis model of rats is established through modeling; and compared with the normal rats, the rats having interstitial pulmonary fibrosis have obvious difference and pathologic change typical cases in various related detection indexes and pathologic changes. The various effective parts of astragalus mongholicus and hedysarum polybotrys are capable of suppressing various related indexes of the interstitial pulmonary fibrosis model of rats induced by bleomycin and preventing the development of interstitial pulmonary fibrosis, wherein low and medium dose groups of hedysarum polybotrys flavone have the optimal effect. The degree of the effect of the effective parts of astragalus mongholicus and hedysarum polybotrys on stopping the progress of interstitial pulmonary fibrosis is related to appropriate concentrations of equivalent dugs. Through comprehensive assessment, the hedysarum polybotrys flavone is better in influence on the lung function, HA, LN, HYP and the like of the rats having interstitial pulmonary fibrosis than other effective parts.

Description

The application of the Radix Astragali, Radix Hedysari effective site

Technical field

The present invention relates to a kind of application of Chinese medicine extract, particularly, relate to the application of the Radix Astragali, Radix Hedysari effective site.

Background technology

Pulmonary fibrosis, namely the lungs stroma is made of collagen protein, elastoidin and albumen carbohydrate, when fibroblast is subject to chemical or physical property injury, can secretes the repairing that collagen protein carries out interstitial tissue of lung, and then cause the lungs fibrosis; Be after lungs come to harm, the result that the human body reparation produces.

Pulmonary fibrosis is many in the morbidity of 40-50 year, and the male is mainly in the women.Dyspnea is the common sympton of pulmonary fibrosis.During slight pulmonary fibrosis, dyspnea only occurs when aggravating activities, and therefore usually out in the cold or mistaken diagnosis is other diseases.When pulmonary fibrosis makes progress, dyspnea also occurs when tranquillization, carrying out property dyspnea can appear in serious pulmonary fibrosis patients.Other symptoms have dry cough, weak.50% patient has drumstick finger and cyanosis, splits sound in the tiny race of base of lung section audible and air-breathing end.Though dyspnea is arranged, x-ray rabat possibility normal in early days; Netted or the nodular shadow of dispersivity of marching off into political wilderness in two lungs appears in middle and late stage, and accidental pleural effusion thickens or calcification.The serious consequence of lung fibrosis causes normal lung tissue's structural change, afunction.Be exactly not have in a large number the fibrosis tissue of gas exchange function to replace alveolar, cause oxygen can not enter blood.Patient respiratory is not smooth, and anoxia, acidosis, disability, by respirator existence are last depleted, dead.

At present; the method for the treatment of pulmonary fibrosis is generally treatment with western; as; glucocorticoid (prednisone claims again strong Buddhist nun pine); immunosuppressant or cell toxicity medicament (cyclophosphamide; azathioprine); antioxidant (Ambroxol; taurine); anti-fibrosis medicine (pirfenidone; the IT_ receptor antagonist; monoclonal antibody;); acceptor inhibitor (LTRA; inhibitors of tumor necrosis factor-alpha; the shape flower growth factor-beta inhibitor); immunomodulator (bcg-polysaccharides nucleic acid; levamisole); other drug (colchicine; macrolide antibiotics; interferon-γ; angiotensin-convertion enzyme inhibitor) and gene therapy method etc., treatment by Chinese herbs is also more rare.

Summary of the invention

The technical problem to be solved in the present invention is to overcome existing defective, and a kind for the treatment of by Chinese herbs method of using the Radix Astragali, Radix Hedysari effective site treatment pulmonary fibrosis is provided.

In order to solve the problems of the technologies described above, the invention provides following technical scheme:

The Radix Astragali, the application of Radix Hedysari effective site in preparation treatment pulmonary fibrosis medicine.

Particularly, the described Radix Astragali, Radix Hedysari effective site are one or more compositionss in astragalus polysaccharides, Radix Astragali flavone, Radix Astragali saponin, Hedysamn polysaccharide, Radix Hedysari flavone, the Radix Hedysari saponin.

Preferably, the described Radix Astragali, Radix Hedysari effective site are the Radix Hedysari flavone.

The present invention has following beneficial effect:

(1) the lung coefficient results shows: the lung coefficient of model group obviously raises; The lung coefficient of the heavy dose of group of Radix Astragali flavone, the heavy dose of group of Radix Astragali saponin, little, the middle dosage group of Radix Hedysari flavone, Radix Hedysari saponin small dose group obviously reduces.Point out most of effective site groups to make lung tissue weight saving improving in varying degrees bleomycin and cause the lung coefficient of Pulmonary Fibrosis in Rats with Bleomycin-induced, perhaps can improve its morbid state body weight is obviously increased.(2) the pulmonary function experimental result shows: the Radix Hedysari flavone is little, in, heavy dose of group, little, the middle dosage group of Radix Astragali flavone be obvious in the improvement effect of most indexs, these group therapeutic effect are more excellent.(3) HA serum test experience result shows; In little, the heavy dose of group of prednisone group, astragalus polysaccharides, the Radix Astragali flavone, in the heavy dose of group, Radix Astragali saponin, heavy dose of group, the Radix Hedysari flavone is little, in, the heavy dose of group of value that can significantly reduce HA reduce the local ECM level of lung, pulmonary fibrosis alleviates to some extent.(4) LN serum test experience result shows: prednisone group, Radix Astragali flavone be little, in, heavy dose of group, little, the middle dosage group of Radix Astragali saponin, the value that little, the middle dosage group of Radix Hedysari flavone can significantly reduce LN slows down the collagen aggregate velocity among the ECM, and pulmonary fibrosis alleviates to some extent.(5) HYP tissue homogenate test experience result shows: the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone be little, in, heavy dose of group, Radix Hedysari flavone be little, in, heavy dose of group can significantly reduce the collagen fiber deposition pulmonary fibrosis is alleviated to some extent.(6) Comprehensive Assessment, the Radix Hedysari flavone is excellent than other effective site to the influence of lung fibrosis in rats pulmonary function, HA, LN, HYP.

Wherein, hyaluronic acid (HA), laminin，LN (LN) and hydroxyproline (HyP) are when pulmonary fibrosis occurs, and serum and liver organization all raise, and each effective site all has reducing effect to them.

Compare with model group, each effective site group all has some improvement on each index content, illustrates that each effective site group all has certain inhibitory action to the process of pulmonary fibrosis.Treatment difference between the different effective site groups illustrates that drug level is very large to the function influence of disease treatment, thereby makes the grouping of this therapeutic scheme on dosage more meaningful.Illustrate the Radix Astragali, Radix Hedysari effective site little, in, heavy dose of relevant with its drug dose to the process that suppresses pulmonary fibrosis, Comprehensive Assessment, the Radix Hedysari flavone is excellent than other effective site to the influence of lung fibrosis in rats pulmonary function, HA, LN, HYP.

Description of drawings

Accompanying drawing is used to provide a further understanding of the present invention, and consists of the part of description, is used for together with embodiments of the present invention explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:

Fig. 1 is Technology Roadmap of the present invention;

Fig. 2 is the canonical plotting of HA;

Fig. 3 is the canonical plotting of LN.

The specific embodiment

Below in conjunction with accompanying drawing the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, is not intended to limit the present invention.

Embodiment 1

The extraction of red, Radix Astragali effective site

1. experiment material

1.1 medicine and reagent

Astragaloside reference substance, control substance of Rutin, glucose reference substance are all checked institute available from Chinese pharmaceutical biological product.95% ethanol, n-butyl alcohol, ethyl acetate, ether are analytical pure.Radix Hedysari, the Radix Astragali are purchased from medical material market, the west of Gansu Province, Dingxi City, identify through the poplar stannum storehouse professor of pharmacy of Gansu Affiliated Hospital of the college of traditional Chinese medicine, Gansu Province Drug Testing Institute Zhu Jun scholar professor of pharmacy.

1.2 instrument and equipment

UV-3600 type ultraviolet-uisible spectrophotometer (Japanese Shimadzu), AE-240 electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit instrument Shanghai branch company), KQ-100 ultrasonic cleaner (city of Kunshan's ultrasonic technique company limited), R-200 Rotary Evaporators (Switzerland's cloth is strange), DZF6050 vacuum drying oven (Shanghai new talent medical apparatus and instruments Manufacturing Co., Ltd).

1.2 experimental technique

1.2.1 the extraction of Radix Hedysari effective site separates

1.2.1.1 Radix Hedysari total polysaccharides

Decoct with water 3 times after Radix Hedysari medicinal residues behind the ethanol extraction are volatilized ethanol, each 1h adds the water of 8 times of amounts for the first time, add 6 times of water gagings rear twice, merge three times decocting liquid, filter, it is 1.15(20 ℃ that filtrate decompression is concentrated into relative density) extractum, centrifugalize discards precipitation, supernatant slowly adds 95% ethanol under stirring, and makes the alcohol amount that contains of solution reach 70%, fully stirs evenly, airtight, leave standstill 24 h, filter, precipitation precipitates 2 times with an amount of 70% washing with alcohol, the precipitation dissolved in distilled water, centrifugalize (2500r/min, 15min) discards precipitation, supernatant stirs lower 95% ethanol that slowly adds, make the alcohol amount that contains of solution reach 70%, fully stir evenly, airtight, leave standstill 24 h, filter centrifugally, precipitation is with 70% washing with alcohol precipitation 2 times, drying below 50 ℃; With the dry thing of water dissolution, get solution after dry, the volume of chloroform-n-butyl alcohol (5:1) by 0.2 times added in the solution, the albumen in the polysaccharide is removed in extraction (repeated multiple times); After removing Deproteinization, the polysaccharide decolouring is pressed 3 ﹪ with the aqueous solution adding heating activated carbon backflow 0.5h(active carbon of polysaccharide and is added), filtered while hot gets the Radix Hedysari total polysaccharides behind the filtrate evaporate to dryness.

1.2.1.2 Radix Hedysari total flavones

Get the Radix Hedysari medical material, cut into the decoction pieces that thickness is 2～3 mm after the moistening, dry, take by weighing 6.5 kg, add people's 70% ethanol, reflux, extract, 3 times, each 1h adds the ethanol of 8 times of amounts for the first time, adds 6 times of amount ethanol rear twice, merge three times extracting solution, filter, filtrate is left standstill 24 h, the elimination precipitation, decompression filtrate recycling ethanol lets cool to without alcohol flavor, with distilled water diluting to 1:2, then go up polyamide column, upper column liquid concentration: 1g/ml, upper prop speed: 2BV/h, then washing, then wash with 70% ethanol, wash with 95% ethanol at last, behind the eluent Recycled ethanol, use the water saturated ethyl acetate extraction of equal-volume 5 times, the combined ethyl acetate extract, reclaim ethyl acetate, dry in the residue water-bath, get the Radix Hedysari total flavones.

1.2.1.3 Radix Hedysari total saponins

The effluent of upper complete polyamide column is continued upper D-101 macroporous resin, upper column liquid concentration: 1g/ml, upper prop speed: 2BV/h, then washing, then wash with 70% ethanol, wash with 95% ethanol at last, behind the eluent Recycled ethanol, use the water saturated n-butanol extraction of equal-volume 5 times, merge butanol extraction liquid, reclaim n-butyl alcohol, dry in the residue water-bath, get the Radix Hedysari total saponins.

1.2.2 the assay of Radix Hedysari effective site

1.2.2.1 the preparation of reference substance solution

The preparation of glucose reference substance solution: precision takes by weighing the glucose reference substance 10.68mg that is dried to constant weight under 105 ℃, places 100 mL volumetric flasks, with dissolved in distilled water and be diluted to scale, shakes up, and is for subsequent use.Getting concentration of glucose is 0.1068mg/ml.

The preparation of control substance of Rutin solution: precision takes by weighing control substance of Rutin 50 mg that are dried to constant weight under 105 ℃, place 25 dry mL volumetric flasks, with dissolve with methanol and be diluted to scale, shake up, precision is measured 10ml, places the 100ml volumetric flask, adds water to scale, shake up, namely get (every 1ml contains anhydrous rutin 0.2mg) for subsequent use.Joining concentration in fact is 0.2198mg/ml.

The preparation of astragaloside reference substance solution: precision takes by weighing astragaloside reference substance 12.5 mg that are dried to constant weight under 105 ℃, places 25 dry mL volumetric flasks, with anhydrous alcohol solution and be diluted to scale, shakes up, and is for subsequent use.Joining concentration in fact is 0.532mg/ml.

1.2.2.2 the preparation of sample solution

Precision takes by weighing Radix Hedysari total polysaccharides 0.5002g,, shakes up in the 50mL volumetric flask with the distilled water standardize solution, and precision pipettes 10ml and is settled in the 100ml volumetric flask, shakes up, and draws 5ml again and is settled in the 25ml volumetric flask, gets 2ml and measures; Precision takes by weighing Radix Hedysari total flavones 1.521g, is settled in the 100ml volumetric flask with 70% ethanol, and the accurate 2ml that draws draws in 1ml to the 25ml volumetric flask in the 25ml volumetric flask again, and the method under the sighting target directrix curve from " adding water to 8.0ml ", is measured in accordance with the law; Precision takes by weighing Radix Hedysari total saponins 1.536g,, filters in the 25mL volumetric flask with the dehydrated alcohol standardize solution, and precision is got filtrate 0.5ml, adds the vanillin reagent of 0.5ml, slowly adds 5ml72% sulphuric acid to ice-water bath, shakes up placement, measures.

1.2.2.3 the drafting of standard curve

The drafting of glucose standard curve: accurate above-mentioned dextrose standard sample solution 0.1,0.2,0.4,0.6,0.8,1.0 mL of drawing, place the 10mL test tube, it is 1.0ml that each adding distil water makes cumulative volume, shakes up.Add respectively 5% phenol solution 1.5mL, shake up, add rapidly 5 mL concentrated sulphuric acids, jolting 5 minutes was put in the boiling water bath heating 15 minutes, then put in the psychrolusia cooling 30 minutes, measured absorbance in wavelength 490 nm places, the retinue blank.Data get regression equation through regression treatment: A=57.2482C+0.0695, r=0.9984.

The drafting of rutin standard curve: precision is measured above-mentioned control substance of Rutin solution 0,1,2,3,4,6,8mL, to the 25mL volumetric flask, respectively add water to 8ml respectively, add respectively people 5% sodium nitrite solution 1mL, shake up and placed 6 minutes, add people 10% aluminum nitrate solution 1mL, placed 6 minutes, add people 1N(4%) sodium hydroxide solution 10mL, add water to again scale, placed 15 minutes, and put in the cuvette, measure absorbance at wavelength 500 nm places.Data get regression equation through regression treatment: A=11.66035C+0.0062, r ²=0.99839.

The drafting of astragaloside standard curve: accurate above-mentioned astragaloside reference substance solution 0.15,0.30,0.45,0.60,0.75 mL of drawing, place respectively tool plug test tube, add respectively dehydrated alcohol 0.75 mL, add respectively again 0.75 mL8% vanillin-concentrated sulphuric acid reagent, placing ice bath to add the 7.5ml volume fraction is that 72% sulphuric acid shakes up, and puts into 62 ℃ water bath with thermostatic control insulation 20 minutes, takes out, flowing water cools off 5 min, shakes up.In 30 minutes, measure absorbance in wavelength 544 nm places, retinue is blank.Data get regression equation through regression treatment: A=21.102C+0.0298, r=0.9993.

1.2.2.4 precision test

Accurate respectively 5 parts of Radix Hedysari total flavones sample solution, Radix Hedysari total saponins sample solution, Radix Hedysari total polysaccharides sample solutions, every part of 0.5 mL of drawing; Measure absorbance, calculate with absorbance, Radix Hedysari total polysaccharides RSD is 2.92%, and Radix Hedysari total flavones RSD is 2.52%, and Radix Hedysari total saponins RSD is 3.08%.

1.2.2.5 stability test

Get the total flavones sample solution, surveyed an absorbance in per 15 minutes, 2h investigates its stability continuously, and the result shows that colour developing is all stable in the 2h, can carry out assay.Get the total saponins sample solution, surveyed an absorbance in per 10 minutes, 1h investigates its stability continuously, and the result shows that colour developing is all stable in the 1h, can carry out assay.Get the total polysaccharides sample solution, surveyed an absorbance in per 10 minutes, 1h investigates its stability continuously, and 1h shows that interior colour developing is all stable as a result, can carry out assay.

1.2.2.6 determination of recovery rates

Precision takes by weighing Radix Hedysari total polysaccharides 125mg, adds a certain amount of glucose reference substance,, shakes up in the 100mL volumetric flask with the distilled water standardize solution; Precision takes by weighing Radix Hedysari total flavones 40mg, adds a certain amount of control substance of Rutin,, shakes up in the 25mL volumetric flask with methanol constant volume; Precision takes by weighing Radix Hedysari total saponins 25mg, adds a certain amount of astragaloside reference substance,, shakes up in the 25mL volumetric flask with the dehydrated alcohol standardize solution.Press the method for assay, measure calculate recovery rate.Measurement result: the glucose average recovery rate is that 98.3%, RSD is 2.19%(n=5); The rutin average recovery rate is that 95.8%, RSD is 2.96%(n=5); The astragaloside average recovery rate is that 92.5%, RSD is 3.21%(n=5).

1.2.2.7 sample size is measured

The assay of Radix Hedysari total polysaccharides: the accurate Radix Hedysari total polysaccharides sample solution 2ml that draws measures, and presses the standard curve method operation, surveys its content according to regression equation, gets 0.718g/g.

The assay of Radix Hedysari total flavones: the accurate Radix Hedysari total flavones sample solution of drawing, press the standard curve method operation, survey its content according to regression equation, get 0.526g/g.

The assay of Radix Hedysari total saponins: the accurate Radix Hedysari total saponins sample solution of drawing, press the standard curve method operation, survey its content according to regression equation, get 0.517g/g.

1.2.2 the extraction of Radix Astragali effective site separates

1.2.2.1 Radix Astragali Mongolici total polysaccharide

Decoct with water 3 times after astragalus root dregs behind the ethanol extraction volatilized ethanol, each 1h adds the water of 8 times of amounts for the first time, add 6 times of water gagings rear twice, merge three times decocting liquid, filter, it is 1.15(20 ℃ that filtrate decompression is concentrated into relative density) extractum, centrifugalize discards precipitation, supernatant slowly adds 95% ethanol under stirring, and makes the alcohol amount that contains of solution reach 70%, fully stirs evenly, airtight, leave standstill 24 h, filter, precipitation precipitates 2 times with an amount of 70% washing with alcohol, the precipitation dissolved in distilled water, centrifugalize (2500r/min, 15min) discards precipitation, supernatant stirs lower 95% ethanol that slowly adds, make the alcohol amount that contains of solution reach 70%, fully stir evenly, airtight, leave standstill 24 h, filter centrifugally, precipitation is with 70% washing with alcohol precipitation 2 times, drying below 50 ℃; With the dry thing of water dissolution, get solution after dry, the volume of chloroform-n-butyl alcohol (5:1) by 0.2 times added in the solution, the albumen in the polysaccharide is removed in extraction (repeated multiple times); After removing Deproteinization, the polysaccharide decolouring is pressed 3 ﹪ with the aqueous solution adding heating activated carbon backflow 0.5h(active carbon of polysaccharide and is added), filtered while hot gets Radix Astragali Mongolici total polysaccharide behind the filtrate evaporate to dryness.

1.2.2.2 Radix Astragali total flavones

Get Milkvetch Root, cut into the decoction pieces that thickness is 2～3 mm after the moistening, dry, take by weighing 6.5 kg, add people's 70% ethanol, reflux, extract, 3 times, each 1h adds the ethanol of 8 times of amounts for the first time, adds 6 times of amount ethanol rear twice, merge three times extracting solution, filter, filtrate is left standstill 24 h, the elimination precipitation, decompression filtrate recycling ethanol lets cool to without alcohol flavor, with distilled water diluting to 1:1, then go up polyamide column, upper column liquid concentration: 1g/ml, upper prop speed: 2BV/h, then washing, then wash with 70% ethanol, wash with 95% ethanol at last, behind the eluent Recycled ethanol, use the water saturated ethyl acetate extraction of equal-volume 5 times, the combined ethyl acetate extract, reclaim ethyl acetate, dry in the residue water-bath, get Radix Astragali total flavones.

1.2.2.3 Radix Astragali total saponins

The effluent of upper complete polyamide column is continued upper D-101 macroporous resin, upper column liquid concentration: 1g/ml, upper prop speed: 2BV/h, then washing, then wash with 70% ethanol, wash with 95% ethanol at last, behind the eluent Recycled ethanol, use the water saturated n-butanol extraction of equal-volume 5 times, merge butanol extraction liquid, reclaim n-butyl alcohol, dry in the residue water-bath, get Radix Astragali total saponins.

2.2.3 the assay of Radix Astragali effective site

2.2.3.1 the preparation of reference substance solution

2.2.3.2 the preparation of sample solution

Precision takes by weighing Radix Astragali Mongolici total polysaccharide 0.5005g,, shakes up in the 50mL volumetric flask with the distilled water standardize solution, and precision pipettes 10ml and is settled in the 100ml volumetric flask, shakes up, and draws 5ml again and is settled in the 25ml volumetric flask, gets 2ml and measures; Precision takes by weighing Radix Astragali total flavones 1.501g, is settled in the 100ml volumetric flask with 70% ethanol, and the accurate 2ml that draws draws in 1ml to the 25ml volumetric flask in the 25ml volumetric flask again, and the method under the sighting target directrix curve from " adding water to 8.0ml ", is measured in accordance with the law; Precision takes by weighing Radix Astragali total saponins 1.582g,, filters in the 25mL volumetric flask with the dehydrated alcohol standardize solution, and precision is got filtrate 0.5ml, adds the vanillin reagent of 0.5ml, slowly adds 5ml72% sulphuric acid to ice-water bath, shakes up placement, measures.

2.2.3.3 the drafting of standard curve

2.2.3.4 precision test

Accurate respectively 5 parts of Radix Astragali total flavones sample solution, Radix Astragali total saponins sample solution, Radix Astragali Mongolici total polysaccharide sample solutions, every part of 0.5 mL of drawing; Measure absorbance, calculate with absorbance, Radix Astragali Mongolici total polysaccharide RSD is 3.05%, and Radix Astragali total flavones RSD is 2.63%, and Radix Astragali total saponins RSD is 3.16%.

2.2.3.5 stability test

2.2.3.6 determination of recovery rates

Precision takes by weighing Radix Astragali Mongolici total polysaccharide 125mg, adds a certain amount of glucose reference substance,, shakes up in the 100mL volumetric flask with the distilled water standardize solution; Precision takes by weighing Radix Astragali total flavones 40mg, adds a certain amount of control substance of Rutin,, shakes up in the 25mL volumetric flask with methanol constant volume; Precision takes by weighing Radix Astragali total saponins 25mg, adds a certain amount of astragaloside reference substance,, shakes up in the 25mL volumetric flask with the dehydrated alcohol standardize solution.Press the method for assay, measure calculate recovery rate.Measurement result: the glucose average recovery rate is that 98.3%, RSD is 2.18%(n=5); The rutin average recovery rate is that 96.8%, RSD is 2.85%(n=5); The astragaloside average recovery rate is that 92.8%, RSD is 3.12%(n=5).

2.2.3.7 sample size is measured

The assay of Radix Astragali Mongolici total polysaccharide: the accurate Radix Hedysari total polysaccharides sample solution 2ml that draws measures, and presses the standard curve method operation, surveys its content according to regression equation, gets 0.721g/g.

The assay of Radix Astragali total flavones: the accurate Radix Hedysari total flavones sample solution of drawing, press the standard curve method operation, survey its content according to regression equation, get 0.558g/g.

The assay of Radix Astragali total saponins: the accurate Radix Hedysari total saponins sample solution of drawing, press the standard curve method operation, survey its content according to regression equation, get 0.531g/g.

Embodiment 2

One, experiment

1 materials and methods

1.1 material

1.1.1 laboratory animal and raising condition

The healthy Wistar rat of SPF level, male and female half and half, body weight (200 ± 10) g is provided by Gansu college of traditional Chinese medicine scientific experiment center.Quality of Experimental Animals quality certification numbering: SCXK(is sweet) 2011-0001-0001059(sees appendix 1) SPF level feedstuff (being supplied with by Beijing section Austria feed corporation,Ltd that pulls together) feeds, and freely drink water, prepare raising and test after 3 days.Animal facility use certificate numbering: SCSY(is sweet) 20011-0001-0002753(sees appendix 2).

1.1.2 main agents

The hydrochloride for injection Bleomycin A5	Be purchased from Harbin bleomycin company limited	Lot number: 110424
			Pentobarbital sodium	Be purchased from Beijing chemical reagents corporation	Lot number: 101021
HA immune radiating immunoassay medicine box	Be purchased from Beijing North biotechnology research institute	Lot number: 111220
			LN radioimmunoassay, RIA medicine box	Be purchased from Beijing North biotechnology research institute	Lot number: 111220
The hydroxyl dried meat is by sour testing cassete	Be purchased from Nanjing and build up bio-engineering research institute	Lot number: 20111210

1.1.3 Experimental agents

Tested medicine: the polysaccharide of the Radix Astragali, Radix Hedysari, flavone, saponin are provided by Gansu college of traditional Chinese medicine scientific experiment center pharmaceutical preparation laboratory, and check reaches more than 50%.Control drug: prednisone acetate tablets, 5mg/ sheet, Zhejiang Province XianJu Pharmacy stock Co., Ltd, the accurate word H33021207 of traditional Chinese medicines, lot number 110810.

1.1.4 key instrument

GC-9111 type radioimmunity r enumerator	Good branch company in the Keda Innovation Co., Ltd
		The KDC-2044 low speed refrigerated centrifuge	Good company in the Keda Innovation Co., Ltd
The full-automatic fluorescence microscope of OLYMPUS AX80	Japan
		The AE200 electronic analytical balance	Prunus mume (sieb.) sieb.et zucc. Teller---holder benefit instrument Shanghai company limited
SFC-182 type biological microscope	Maike Aodi Industry Group Co Ltd
		EMKA animal lung function detection system	France
Ultraviolet spectrometry Density Measuring Instrument UV-2601	Beijing North divides Rayleigh analytical tool (group) company

1.2 experimental technique

1.2.1 laboratory animal grouping

252 of SPF level Wistar rats, body weight 200 ± 20g, male and female half and half, adaptability were fed after 3 days, according to the table of random number method tell first blank group, model group, prednisone group, the Radix Astragali, Hedysamn polysaccharide little, in, heavy dose of group, the Radix Astragali, Radix Hedysari flavone be little, in, heavy dose of group, the Radix Astragali; The Radix Hedysari saponin is little, in, heavy dose of group, 12 every group, divide cage to place by sex, 6 in every cage, all rats are unified modeling, number with pre-configured 3% the picric acid solution dyeing row labels of going forward side by side.

1.2.2 experimental animal model preparation

Conventional letting animals feed copied pulmonary fibrosis model, with 3% pentobarbital sodium (40～50mgkg after 3 days ^-1) intraperitoneal injection anesthesia, wait press from both sides tail without pain reaction after, the rat dorsal position is fixed on the Mus plate, fixing limbs, fixing head makes head slightly to layback.The assistant strangles tooth on rat with a rubber band, to the front lower place tractive, fasten lower tooth to back upper place tractive (angle is advisable with 60o) with another root rubber band, patient's left hand is held the special-purpose laryngoscope of toy, stretch in the rat oral cavity, opening is to the bottom right, dorsal segment is lifted the gently jack-up root of the tongue on upwards slightly, exposes glottis, takes advantage of animal air-breathing moment, ((22G * 25mm/Y-G) cuts off the part of syringe needle with disposable use venous detaining needle with homemade tracheal casing pipe, the length of remaining plastics sebific duct is advisable with 6-7cm, with thin wire tail end is made in the circular insertion sebific duct as nook closing member, and length equates with the plastics sebific duct, can not exceed sebific duct, in order to avoid stab rat throat.) take advantage of a situation and insert in the rat trachea, in sleeve pipe, inject in advance 2-3 and save little water column, if motionless or fiercely ejection or the suction of water column in the sleeve pipe illustrates that intubate enters esophagus, need again intubate; If rhythmical the moving up and down of water column follower respiratory frequency in the sleeve pipe illustrates and inserts trachea.At this moment, extract rapidly nook closing member, will take out in advance bleomycin (5mgkg ^-1, 5mgml ^-1) the lml syringe be connected on the tracheal casing pipe the disposable trachea that pushes.After the injection, extract immediately sleeve pipe, animal is upright, and then left rotation and right rotation is about 1 minute, and medicine is evenly distributed in lung.Inject isopyknic normal saline in the blank group trachea.After animal was naturally clear-headed, conventional SPF raised.

1.2.3 dosage regimen

The 2nd day beginning gastric infusion after the modeling.Every day 1 time, the gavage treatment is 28 days continuously, the fetching mark.The theory that each treatment group dosage converts according to clinical kg body weight dosage multiple, simultaneously determine that in conjunction with document and principle of comparability the dosage of this experiment is as follows: Radix Astragali 6500g extracts and isolates polysaccharide: 53.6g, saponin: 32.36g, flavone: 32.4g, converts every gram Radix Astragali and contains polysaccharide: 8.25mg, saponin: 4.98mg, flavone: 4.98mg; Radix Hedysari 6500g extracts and isolates polysaccharide: 53.6g, saponin: 32.36g, flavone: 32.4g, converts every gram Radix Hedysari and contains polysaccharide: 5.72mg, saponin: 4.23mg, flavone: 2.92mg.Adult's the Radix Astragali, the dosage of Radix Hedysari are pressed 30g(pharmacopeia regulation adult one consumption per day 9～30g) and are calculated, and then a consumption per day is 0.5gkg ^-1Crude drug.Pressing the direct scaling method of body surface area or man and animal conversion dosage multiple calculates.6 times of behaving of one day dosage of rat, namely one day crude drug consumption of rat is 3.0gkg ^-1The amount that goes out effective site according to every gram crude drug actual extracting converses dosage such as the following table of each effective site: (seeing Table 1,2)

The actual dosage of table 1 Radix Astragali, Hedysamn polysaccharide, saponin, flavone

The actual dosage of table 2 Radix Astragali, Hedysamn polysaccharide, saponin, flavone

The Radix Astragali, Hedysamn polysaccharide, flavone, saponin are mixed with respectively the solution of respective concentration before use with cosolvent 0.5% carboxymethylcellulose sodium solution, every day gastric infusion, gavage volume 1mL100g ^-1Body weight.The prednisone group gives prednisone acetate tablets, and reference literature determines that dosage is 3mgKg ^-1D ^-1, prepare with 0.5% carboxymethylcellulose sodium solution; Model group and blank group give equal-volume 0.5% carboxymethylcellulose sodium solution gavage.

1.2.4 laboratory animal collection of specimens and detection

1.2.4.1 the assay method of pulmonary function and drawing materials

Start computer, operation IOX Data Collection Software based, commissioning device is guaranteed system's normal operation.Open animal respirator, signal conditioner.Set up a new experiment, the log-on data collection.Rat is put into the animal plethysmography box in batches, trace case each puts one for four, each four, do pulmonary function test (pft) under noinvasive and the waking state (rf).Test index has: the Ti(inspiratory duration), the Te(expiratory duration), EIP (air-breathing latter stage pause), EEP (EEP pauses), RT (persistent period), PIF(inspiratory airflow peak), PEF(expiratory airflow peak), the flow velocity when EF50(breathes out 50% tolerance), MV (per minute ventilation), TV(tidal volume), the EV(forced volume,expiratory), AV (alveolar ventilation), Penh (bronchoconstriction degree), F(frequency) etc., test finish after saving result.Behind the pulmonary function of finishing under the noinvasive state, just anaesthetize and have the pulmonary function test (pft) of (rcv) under the wound state.Adopt the isoflurane inhalation anesthesia, under the small animal laryngoscope guiding, carry out tracheal intubation, then the rat clinostatism is placed the airtight plethysmography box of toy respirator.Trace first one section eupnea, then survey the Ti(inspiratory duration), the Te(expiratory duration), EIP (pause air-breathing latter stage), EEP (EEP pause), RT (persistent period), PIF(inspiratory airflow peak), PEF(expiratory airflow peak), flow velocity when EF50(breathes out 50% tolerance), MV (per minute ventilation), the TV(tidal volume), the EV(forced volume,expiratory), the cdyn(Cdgn dyanamic compliance), Penh (bronchoconstriction degree), the LR(lung resistance), the dppl(pleura is pressed and is changed), EEW (EEP merit), pau(pauses), the F(frequency) etc.After lung function tests is complete, carries out femoral artery and get blood.Then cut off the rat thoracic cavity, the careful separation lung tissue, the complete pair lungs of isolating, connective tissue around carefully removing blots with filter paper after the normal saline washing, electronic balance weighing lung weight in wet base, calculating lungs coefficient.Win inferior lobe of right lung, do tissue homogenate in order to the detection of lung tissue HYP.

1.2.4.2 HA detects

0,25,50,100,200,400,800ng/mL adopt radio immunoassay, carry out according to the test kit description, determination step is as follows: 7 bottles of concentration of (1) HA standard substance are followed successively by:.(2) HA quality controlled serum: use the 0.5mL dissolved in distilled water before using, at least 15 minutes ability is used for application of sample after the dissolving.(3) BALF gets 100 μ L and measures with 0.2mLPBS dilution lung tissue homogenate dried frozen aquatic products.(4) it is some to get the polystyrene test tube, carries out application of sample by the application of sample sequence list after the numbering, and all reagent comprise that testing sample is careful and shake up before the application of sample, avoid producing foam as far as possible.For application of sample is accurate, various reagent and sample should equilibrate to first room temperature before application of sample.Such as table 3, Fig. 1.

Table 3 application of sample sequence list (unit: μ L)

1.2.4.3 LN detects

Adopt radio immunoassay, carry out according to the test kit description, determination step is as follows: (1) LN standard substance: with 2.0mL dissolved in distilled water S0, with 1.0ml dissolved in distilled water S1～S5,0,40,80,160,320,640ng/mL fully after the dissolving, its accurate concentration is respectively:.(2) with 10ml distilled water dissolving 125I-LN.(3) with l1mL distilled water dissolving LN antibody.(4) BALF respectively gets 100 μ L and measures with 0.2mLPBS dilution lung tissue homogenate dried frozen aquatic products.(5) it is some to get round bottom polystyrene test tube, and with marking pen or special pencil numbering NSB, then S0-S5 and testing sample pipe etc. use micro sample-adding according to the form below application of sample.All reagent (especially separating medium) comprise that testing sample will shake up before the application of sample, avoid producing foam as far as possible, and preferably equilibrate to room temperature.(such as table 4, Fig. 2)

Table 4LN application of sample sequence list (unit: μ L)

Table 4LN application of sample sequence list (unit: μ L)

1.2.4.4HYP detection

The oxidation product that hydroxyproline produces under the effect of oxidant and dimethylaminobenzaldehyde effect present aubergine, can extrapolate its content according to the depth of its colour generation.Carry out according to the test kit description, determination step is as follows: (1) accurately takes by weighing tissue wet 30-100ml and puts into test tube, adds hydrolyzed solution 1ml.Mixing.Add a cover rear 95 degrees centigrade or boiling water bath hydrolysis 20 minutes.(when being hydrolyzed 10 minutes mixing once, purpose is to make the hydrolysis more abundant).(2) transfer pH value to the 6.0-6.8; 1. each pipe after each test tube flowing water cooling is added one of indicator, shake up; 2. each pipe adds accent PH first liquid 1.0ml, mixing (this moment, solution should be red); 3. draw the second liquid of transferring pH value with the sample injector of 200 μ L, want mixing after whenever being added dropwise to, until the color of liquid indicating agent becomes yellow green.This moment, pH value (added about the second liquid 100 μ L-500 μ L that transfer pH value) about 6.0～6.8 approximately; 4. then adding distil water to 10ml, mixing; 5. get an amount of active carbon (about about 20-30mg, with colourless being as the criterion of the centrifugal rear clarification of supernatant) of hydrolyzed solution man of 34ml dilution, mixing, 3500 turn ∕ divided centrifugal 10 minutes, got supernatant 1ml and did check.(3) add mixing behind the reagent, 60 ℃ of water-baths 15 minutes, after the cooling, 3500 turn ∕ divided centrifugal 10 minutes, got supernatant at the 550nm place, the 1cm optical path, the distilled water zeroing is surveyed and is respectively managed absorbance.

Accurately draw normal rat serum 0.5ml, detect by operating procedure.

Surveying the blank tube absorbance is 0.075, standard pipe absorbance 0.61.

Then the result of calculation formula is:

Hydroxyproline content (μ g ∕ ml)=mensuration pipe absorbance-blank tube Xi Guang Du ∕ standard pipe absorbance-blank tube absorbance * standard pipe content (5 μ g ∕ ml) * hydrolyzed solution cumulative volume (ml) ∕ sampling amount (ml) (seeing Table 5)

Table 5HYP operation table

2 experimental results

2.1 respectively organize the variation of rat general status and lung coefficient

Without dead, feed and the mental status are good, are quick on the draw in the blank group rat experiment process, and fur gloss is shinny, and body weight increases gradually, breathes steadily.The model group rat mental status is poor, appetite and movable the minimizing, and fur is sparse, and its colour changed into yellow tarnishes, and commonly falls perpendicular phenomenon, and hogback is rolled up, bradykinesia, rapid breathing, health are become thin etc.The situation of all the other each treated animals after administration falls between.

(mg) ∕ body weight (g) * 100% is calculated the lung coefficient, as can be seen from Table 6, compares in model group, the astragalus polysaccharides dosage group induced lung coefficient with the blank group and significantly raises notable difference (P ﹤ 0.01) is arranged according to formula lung coefficient=lung weight in wet base; Dosage group, little, the middle dosage group of Hedysamn polysaccharide induced lung coefficient rising variant (P ﹤ 0.05) in the Radix Astragali flavone; Compare with model group, the heavy dose of group of Radix Astragali flavone, the heavy dose of group of Radix Astragali saponin, little, the middle dosage group of Radix Hedysari flavone, Radix Hedysari saponin small dose group induced lung coefficient significantly reduction have notable difference (P ﹤ 0.01); Astragalus polysaccharides is little, in the heavy dose of group, Radix Hedysari saponin, heavy dose of group induced lung coefficient reduces variant (P ﹤ 0.05); (seeing Table 6)

2.2 respectively organize the pulmonary function testing result

Compare the lung function index of model group rat with the blank group: under noinvasive (rf) state, TV, EV, MV, PIF, PEF, EF50 significantly reduce, difference has statistical significance (P ﹤ 0.01), and AV also reduces, and difference has statistical significance (P ﹤ 0.05).Have under wound (rcv) state, TV, EV, cdyn significantly reduce, and difference has statistical significance (P ﹤ 0.01), and EF50 reduces, and difference has statistical significance (P ﹤ 0.05).All in various degree the risings of the numerical value of comparing index TV, EV, MV, AV, PIF, PEF under each effective site treatment group pulmonary function rf state with model group.Have under the wound rcv state, the numerical value of EF50, TV, EV, cdyn also has rising in various degree.(seeing Table 7,8,9,10,11,12,13,14)

Wherein (rf) TV of astragalus polysaccharides small dose group compares variant (P ﹤ 0.05) with model group.(rf) MV of dosage compares variant (P ﹤ 0.05) in the astragalus polysaccharides with model group.(rf) MV, (rcv) cdyn, the EV of the heavy dose of group of astragalus polysaccharides have compared significant difference (P ﹤ 0.01) with model group.(rf) AV of Radix Astragali flavone small dose group, (rcv) TV, cdyn have compared significant difference (P ﹤ 0.01) with model group, (rf) EV, MV compare variant (P ﹤ 0.05) with model group.(rf) AV of dosage group, (rcv) TV, cdyn have compared significant difference (P ﹤ 0.01) in the Radix Astragali flavone with model group, and (rf) EV compares variant (P ﹤ 0.05) with model group.(rcv) EV, the cdyn of the heavy dose of group of Radix Astragali flavone have compared significant difference (P ﹤ 0.01) with model group.(rcv) EV of Radix Astragali saponin small dose group has compared significant difference (P ﹤ 0.01) with model group, (rf) TV, AV compare variant (P ﹤ 0.05) with model group.

Table 6 is on respectively organizing the result that affects of induced lung coefficient

Annotate: compare with the blank group: ^△P＜0.05, ^{△ △}P＜0.01; Compare with model group: ^▲P＜0.05, ^{▲ ▲}P＜0.01;

Table 7 pair is respectively organized the result of pulmonary function Index Influence

Annotate: compare with the blank group: ^△P＜0.05, ^{△ △}P＜0.01; Compare with model group: ^▲P＜0.05, ^{▲ ▲}P＜0.01

(rcv) EV of Hedysamn polysaccharide small dose group has compared significant difference (P ﹤ 0.01) with model group.(rcv) EV, the cdyn of dosage group compares variant (P ﹤ 0.05) in the Hedysamn polysaccharide with model group.(rcv) cdyn of the heavy dose of group of Hedysamn polysaccharide has compared significant difference (P ﹤ 0.01) with model group, (rf) TV compares variant (P ﹤ 0.05) with model group.(rf) TV of Radix Hedysari flavone small dose group, MV, AV, (rcv) TV, EV, cdyn have compared significant difference (P ﹤ 0.01) with model group.(rf) EV compares variant (P ﹤ 0.05) with model group.(rf) TV, the MV of dosage group, (rcv) EV, cdyn have compared significant difference (P ﹤ 0.01) in the Radix Hedysari flavone with model group, and (rf) EV, AV, (rcv) TV compare variant (P ﹤ 0.05) with model group.(rcv) TV, EV, the cdyn of the heavy dose of group of Radix Hedysari flavone have compared significant difference (P ﹤ 0.01) with model group, (rf) EV compares variant (P ﹤ 0.05) with model group.(rcv) TV of Radix Hedysari saponin small dose group compares variant (P ﹤ 0.05) with model group.(rcv) TV of dosage group compares variant (P ﹤ 0.05) in the Radix Hedysari saponin with model group.The LR of dosage group has compared significant difference (P ﹤ 0.01) in the Radix Hedysari saponin with model group.(rf) MV, (rcv) TV of the heavy dose of group of Radix Hedysari saponin compare variant (P ﹤ 0.05) with model group.

Little, the middle dosage group of Radix Hedysari flavone and model group comparison numerical value in the impact of (rf) TV (aspirated volume) are obviously raise, difference has statistical significance (P ﹤ 0.01), Radix Astragali saponin small dose group, the heavy dose of group of Hedysamn polysaccharide and relatively numerical value rising of model group, difference has statistical significance (P ﹤ 0.05).

The heavy dose of group of Hedysamn polysaccharide in the impact of (rf) EV (forced volume,expiratory) is obviously raise with model group comparison numerical value, difference has statistical significance (P ﹤ 0.01), little, the middle dosage group of Radix Astragali flavone, Radix Hedysari flavone be little, in, heavy dose of group and model group relatively numerical value raise, difference has statistical significance (P ﹤ 0.05).

Prednisone group, the heavy dose of group of astragalus polysaccharides, little, the middle dosage group of Radix Hedysari flavone and model group comparison numerical value in the impact of (rf) MV (per minute ventilation) are obviously raise, difference has statistical significance (P ﹤ 0.01), relatively numerical value rising of dosage group, Radix Astragali flavone small dose group and model group in the astragalus polysaccharides, difference has statistical significance (P ﹤ 0.05).

Table 8 pair is respectively organized the result of pulmonary function Index Influence

Table 9 pair is respectively organized the result of pulmonary function Index Influence

On (rf) AV(alveolar ventilation) impact in little, the middle dosage group of Radix Astragali flavone, Radix Hedysari flavone small dose group and model group comparison numerical value obviously raise, difference has statistical significance (P ﹤ 0.01), relatively numerical value rising of dosage group and model group in Radix Astragali saponin small dose group, the Radix Hedysari flavone, difference has statistical significance (P ﹤ 0.05).

Little on Radix Astragali flavone in the impact of (rcv) TV (tidal volume), in, heavy dose of group, little, the heavy dose of group of Radix Hedysari flavone obviously raise with model group comparison numerical value, difference has statistical significance (P ﹤ 0.01), dosage group in the Radix Hedysari flavone, Radix Hedysari saponin be little, in, heavy dose of group and model group relatively numerical value raise, difference has statistical significance (P ﹤ 0.05).

On in dosage group, the Radix Hedysari flavone in Radix Astragali flavone small dose group, Radix Astragali saponin small dose group, the Hedysamn polysaccharide in the impact of (rcv) EV (forced volume,expiratory), heavy dose of group, Radix Hedysari saponin small dose group and model group comparison numerical value obviously raises, difference has statistical significance (P ﹤ 0.01), dosage group, the heavy dose of group of Hedysamn polysaccharide and relatively numerical value rising of model group in the astragalus polysaccharides, difference has statistical significance (P ﹤ 0.05).

On (rcv) cdyn(lung compliance) impact in the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone little, in, heavy dose of group, the heavy dose of group of Hedysamn polysaccharide, Radix Hedysari flavone be little, in, heavy dose of group obviously raises with model group comparison numerical value, difference has statistical significance (P ﹤ 0.01).Relatively numerical value rising of dosage group and model group in prednisone group, the Hedysamn polysaccharide, difference has statistical significance (P ﹤ 0.05).

In addition, interstitial pulmonary fibrosis is little on the impact variation dependency of other lung function index, just no longer carries out statistics and analysis.

Table 10 pair is respectively organized the result of pulmonary function Index Influence

Table 11 pair is respectively organized the result of pulmonary function Index Influence

Table 12 pair is respectively organized the result of pulmonary function Index Influence

Table 13 pair is respectively organized the result of pulmonary function Index Influence

Table 14 pair is respectively organized the result of pulmonary function Index Influence

2.3 respectively organize the impact of HA in the rat blood serum, LN content

2.3.1 HA testing result in the serum

Compare with the blank group, in dosage group in model group, the astragalus polysaccharides, Radix Astragali saponin small dose group, little, the heavy dose of group of Hedysamn polysaccharide, the Radix Hedysari saponin, the value of heavy dose of group rat HA significantly raises, difference has statistical significance (P ﹤ 0.01), the value of little, the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone small dose group, little, the heavy dose of group rat of Radix Hedysari flavone HA raises, and difference has statistical significance (P ﹤ 0.05);

Compare with model group, dosage group in little, the heavy dose of group of prednisone group, astragalus polysaccharides, the Radix Astragali flavone, in the heavy dose of group, Radix Astragali saponin, in the heavy dose of group, Hedysamn polysaccharide, the Radix Hedysari flavone is little, in, the value of heavy dose of group, rat HA significantly reduces, difference has statistical significance (P ﹤ 0.01), the value of dosage group, Radix Astragali flavone small dose group, the heavy dose of group of Hedysamn polysaccharide rat HA reduces in the astragalus polysaccharides, and difference has statistical significance (P ﹤ 0.05); (seeing Table 15)

2.3.2 LN testing result in the serum

Compare with the blank group, model group, astragalus polysaccharides be little, in, heavy dose of group, Radix Astragali saponin be little, in, heavy dose of group, Hedysamn polysaccharide be little, in, heavy dose of group, the heavy dose of group of Radix Hedysari flavone, Radix Hedysari saponin be little, in, the value of heavy dose of group rat LN significantly raises, difference has statistical significance (P ﹤ 0.01), the value of the heavy dose of group of Radix Astragali flavone rat LN obviously raises, and difference has statistical significance (P ﹤ 0.05).

With the model group ratio, prednisone group, Radix Astragali flavone be little, in, heavy dose of group, little, the middle dosage group of Radix Astragali saponin, the value of little, the middle dosage group of Radix Hedysari flavone rat LN significantly reduces, difference has statistical significance (P ﹤ 0.01); The value of dosage group rat LN reduces in the astragalus polysaccharides, and difference has statistical significance (P ﹤ 0.05); (seeing Table 15)

Table 15 is on the result of the HA in the serum, LN impact

2.4 respectively organize the HYP content detection result in the lung tissue of rats

Compare with the blank group, model group, prednisone group, astragalus polysaccharides be little, in, heavy dose of group, Radix Astragali saponin be little, in, heavy dose of group, Hedysamn polysaccharide be little, in, the value of heavy dose of group, little, the middle dosage group of Radix Hedysari saponin rat HYP significantly raises, difference has statistical significance (P ﹤ 0.01); The value of little, the heavy dose of group rat of Radix Hedysari flavone HYP raises, and difference has statistical significance (P ﹤ 0.05);

With the model group ratio, the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone be little, in, heavy dose of group, Radix Hedysari flavone be little, in, the value of heavy dose of group rat HYP significantly reduces, difference has statistical significance (P ﹤ 0.01); The value of the heavy dose of group of Hedysamn polysaccharide rat HYP reduces, and difference has statistical significance (P ﹤ 0.05).(seeing Table 16)

The result of the HYP content influence in the table 16 pair lung tissue

Discuss

The research of 1 model

Bleomycin (BLM) is a peptide species class antineoplastic agent, and because it has the side effect that obviously causes pulmonary fibrosis, thereby development has worked the animal model that utilizes BLM to induce and set up pulmonary fibrosis.Zoopery has proved in the trachea that disposable injection BLM is reproducible and has gone out the animal model similar to people's interstitial pulmonary fibrosis pathological process.At present, the method has become the classical animal model that carries out in the world pulmonary fibrosis research.It is easy and simple to handle because of it that bleomycin copies the interstitial pulmonary fibrosis model, and modeling is stable, and the characteristics that success rate is high are widely used.This experiment adopts the method for small animal laryngoscope guidance tracheal intubation to copy pulmonary fibrosis model, this method has avoided the skin of neck of incision rat successively to separate and expose the method for trachea, has eliminated the incision skin postoperative local infection of rat and the inducement of some complication.In the modeling process, use small animal laryngoscope and guide the sewing process that can reduce in the classical modeling process, and can reduce the consumption of anaesthetic, in the process of a large amount of animal models, can greatly improve efficient.Having avoided secondary insult is improvement to classical modeling method.Modeling method after the improvement, have easy, without wound, speed are fast, the modeling success rate is high characteristics.Have the difference that significantly increases the weight of trend by the blank group of the rising of lung coefficient, pulmonary function (rf) TV, EV, MV, PIF, PEF, EF50, (rcv) TV, EV, cdyn, HA, LN, HYP and the relatively demonstration of model group, difference has statistical significance (P ﹤ 0.01).It is more successful that the result can show that this tests improved modeling method.

The selection of 2 control drug

Glucocorticoid is the medicine that a class has very strong antiinflammatory action and immunosuppressive action, once once is being used as the choice drug for the treatment of pulmonary fibrosis.At present, the animal experiment study of most IPF all selects glucocorticoid to do positive control drug.There are some researches prove that dexamethasone can promote the pulmonary macrophage apoptosis of lung fibrosis in rats; Reduce the generation of inflammatory cytokine, suppress gathering, the infiltration of pulmonary's inflammatory cell in lung tissue; Inhibition apoptosis of vascular endothelial cell has protective effect to pulmonary's capillary endothelial cell; The collagen secretion increase that minimizing fibrocyte propagation causes etc.Because durative action preparation is more obvious to immune inhibitory action, life-time service can disturb fat and protein metabolism, affect growth promoter, although have report to find that the early application Low-dose dexamethasone can alleviate the generation of experimental rat pulmonary fibrosis, its body weight on rat has obvious impact.Choose the positive control drug of dexamethasone in the experiment in early stage, rat body weight occurred and obviously reduce, and had the phenomenon of rats death to occur.And prednisone is middle effect preparation, and its half-life and half effect phase are all short than dexamethasone.Therefore in a large amount of long-term application processes, prednisone is compared with dexamethasone, brings out or increases the weight of in prevention and infect, and alleviates the side effect successful of the complication of digestive system.

Based on above reason, so this experiment is intended selecting prednisone as positive control drug.Experimental result shows, the prednisone group is less on the general status impact of experimental rat, and the lung coefficient is close with the other medicines treatment group, the body weight phenomenon such as obviously become thin do not occur.And the lung function index of the pulmonary fibrosis model rat that BLM is induced makes moderate progress, and compares with model group also HA, LN content in the serum, and the content of the HYP in the lung tissue has certain reducing effect (P ﹤ 0.01).

The selection foundation of 3 indexs

3.1 integral status

Observe from traditional Chinese medical science angle, reactivity, fur color and luster, diet etc. are the important signs of the spirited prosperity and decline of reflection.Be quick on the draw, fur is moist, honey stomach, body contain strong, muscle enriches that god is then arranged; Otherwise bradykinesia, fur are withered, diet difficultly advances, emaciated physique, withered bone with deficient medulla, extreme emaciation be then without refreshing.Lung governing qi, department breathes, and main Xuan Fasu falls, kidney governing storage, main improving inspiration by invigorating kidney-QI is so respiratory movement is the direct sign of reflection lung renal function.Lung qi is abundant, and kidney qi is filled Sheng, then breathes normal; Otherwise the lung qi virtual loss is declared respectful mistake department, and kidney loses envelope and hides, and takes the photograph to receive and haves no right, and then Function of Respiratory Movement is not normal.Observe from the modern medicine angle: the variation of Organism of Rats integral status general state Observable, the judgement interstitial pulmonary fibrosis disease;

3.2 lung coefficient

In the Pulmonary Fibrosis in Rats with Bleomycin-induced pathogenic process, because the body respiratory dysfunction, secondary causes digestion and the absorption of body nutrient substance, serious hindrance occurs, makes the interior catabolism of body greater than anabolism, makes body poky symptom occur.Interstitial pulmonary fibrosis patient's lung tissue volume reduces, and the collagen deposition that inflammation, fibrosis cause and hardening, weight increases, then the lung coefficient increases relatively, in addition lung tissue inflammatory exudation, edema, hyperemia, hemorrhage etc. also be the reason that the weight of lung increases, the lung coefficient is one of index of reaction interstitial pulmonary fibrosis degree.Therefore the bleomycin reason that causes Pulmonary Fibrosis in Rats with Bleomycin-induced lung tissue weight and increase comprises above-mentioned two aspects.

3.3 detect in the respiratory function

The indexs such as PIF, PEF, EF50, TV, EV, MV, AV, cdyn, LR, penh can be judged airway patency in the interstitial pulmonary fibrosis disease, breathe the state of muscle strength, lung tissue elasticity, ventilatory function and storage capacity.

3.4 the impact on the relevant cell epimatrix

But the synthetic and precipitation situation of HA content reacting cells epimatrix collagen fiber.LN content can react the situation of the attraction of pulmonary epithelial cells, fibroblast and inflammatory cell and adhesion and T lymphocyte and macrophage secretion lymphokine and be suppressed to the situation of fibrocyte and the synthetic collagen of epithelial cell.HYP is the representative index of collagen fiber deposition.

4 Radixs Astragali, Radix Hedysari effective site are on the impact of Pulmonary Fibrosis in Rats with Bleomycin-induced lung coefficient

The mensuration of lung coefficient helps assess inflammation and fibrosis in this experiment.The lung coefficient results shows: the lung coefficient of comparing model group with the blank group obviously raises; The lung coefficient of comparing the heavy dose of group of Radix Astragali flavone, the heavy dose of group of Radix Astragali saponin, little, the middle dosage group of Radix Hedysari flavone, Radix Hedysari saponin small dose group with model group obviously reduces, in little, the heavy dose of group of astragalus polysaccharides, the Radix Hedysari saponin, the reduction of heavy dose of group induced lung coefficient.Point out most of effective site groups to make lung tissue weight saving improving in varying degrees bleomycin and cause the lung coefficient of Pulmonary Fibrosis in Rats with Bleomycin-induced, perhaps can improve its morbid state body weight is obviously increased.

5 Radixs Astragali, Radix Hedysari effective site are on the impact of Pulmonary Fibrosis in Rats with Bleomycin-induced pulmonary function

The pulmonary function method of testing has a variety of, and pulmonary function test (pft) method is in the past carried out the mode of tracheal intubation mainly with tracheotomy, connects toy respiratory function test macro, test each side index.Leading indicator comprises that FVC, FEV0.2, Cydn, MVV, LR, Re etc. react the variation of pulmonary function.

In order to promote quality of experiments, transform experiment condition, the French Biotech EMKA of the company pulmonary function equipment of the special introduction of my special interest group pulmonary function laboratory, IOX Data Collection Software based and equipment, can do the noinvasive animal lung function under the waking state, by tracheal intubation under the aerosol narcotism wound animal lung function be arranged.This method is surveyed pulmonary function under the waking state, and animal freely breathes, and by the pressure receptor sensing on the plethysmography box, each index is unaffected.Anesthesia method adopts the isoflurane inhalation anesthesia, and anesthesia is fast, and is clear-headed also fast, only needs the time in several seconds; By tracheo-laryngoscope mode intubate, animal is without wound.Consider, this method has been avoided to greatest extent anesthetics and has been performed the operation on the impact of animal lung function index.Main test index is: TI (inspiratory duration), TE (expiratory duration), the length of reaction breathing time; PIF (inspiratory airflow peak), PEF (expiratory airflow peak) judges the intensity that reaction is breathed by crest (trough) height of respiratory curve waveform; TV (aspirated volume), EV (forced volume,expiratory), the cubical content that reaction is breathed; RT (intermission) is the middle down-time period of twice breathing; MV (per minute ventilation), the efficient that reaction is breathed; The amount of gas exchange is effectively carried out in AV (alveolar ventilation) reaction; F (respiratory frequency), the speed that reaction is breathed; EIP (pause air-breathing latter stage), EEP (EEP pause), Penh (bronchoconstriction degree).Behind the pulmonary function of finishing under the noinvasive state, just anaesthetize and have the pulmonary function test (pft) of (rcv) under the wound state.Adopt the isoflurane inhalation anesthesia, insert tracheal intubation, then the rat clinostatism is placed the airtight plethysmography box of toy respirator.Trace first one section eupnea, then survey TI (inspiratory duration), TE (expiratory duration), PIF (inspiratory airflow peak), PEF (expiratory airflow peak), TV (aspirated volume), EV (forced volume,expiratory), RT (intermission), MV (per minute ventilation); F (respiratory frequency), EIP (air-breathing latter stage pause) EEP (EEP pauses), Penh (bronchoconstriction degree) cdyn(Cdgn dyanamic compliance), Penh (bronchoconstriction degree), LR(lung resistance), the dppl(pleura presses and changes), EEW (EEP merit), pau(pause) etc.By wound pulmonary function and noinvasive pulmonary function test are arranged, carry out lung capacity, the mensuration of ventilatory function not only can be found to have or not unusually to distinguish restricted or obstructive dysfunction of pulmonary ventilation.

Little, the middle dosage group of Radix Hedysari flavone and model group comparison numerical value in the impact of (rf) TV (aspirated volume) are obviously raise Radix Astragali saponin small dose group, the heavy dose of group of Hedysamn polysaccharide and relatively numerical value rising of model group.The heavy dose of group of Hedysamn polysaccharide in the impact of (rf) EV (forced volume,expiratory) is obviously raise with model group comparison numerical value, little, the middle dosage group of Radix Astragali flavone, Radix Hedysari flavone be little, in, heavy dose of group and model group relatively numerical value raise.Little on Radix Astragali flavone in the impact of (rcv) TV (tidal volume), in, heavy dose of group, little, the heavy dose of group of Radix Hedysari flavone obviously raise with model group comparison numerical value, dosage group in the Radix Hedysari flavone, Radix Hedysari saponin be little, in, heavy dose of group and model group relatively numerical value raise.On in dosage group, the Radix Hedysari flavone in Radix Astragali flavone small dose group, Radix Astragali saponin small dose group, the Hedysamn polysaccharide in the impact of (rcv) EV (forced volume,expiratory), heavy dose of group, Radix Hedysari saponin small dose group obviously raise dosage group, the heavy dose of group of Hedysamn polysaccharide and relatively numerical value rising of model group in the astragalus polysaccharides with model group comparison numerical value.These two indexs have reacted above-mentioned treatment group aspirated volume and the expiration volume all increases to some extent than model group, have met restrictive ventilatory disorder and have made moderate progress through treatment, and pulmonary fibrosis alleviates to some extent.

Prednisone group, the heavy dose of group of astragalus polysaccharides, little, the middle dosage group of Radix Hedysari flavone and model group comparison numerical value in the impact of (rf) MV (per minute ventilation) are obviously raise, and Radix Astragali flavone small dose group and model group relatively numerical value raise.This index has reflected elasticity and the airway resistance of breathing muscle strength, thorax and lung tissue, is the reliability index of an overall merit pulmonary ventilation function storage level.This makes moderate progress after having met the treatment of restrictive ventilatory disorder process, and pulmonary fibrosis alleviates to some extent.

On (rf) AV(alveolar ventilation) impact in little, the middle dosage group of Radix Astragali flavone, Radix Hedysari flavone small dose group and model group comparison numerical value obviously raise, in Radix Astragali saponin small dose group, the Radix Hedysari flavone dosage group and model group relatively numerical value raise.This index refers to that per minute sucks the amount that the amount of fresh air of alveolar or per minute and blood are carried out gas exchange.The ventilation that only enters alveolar just has the chance of the ventilation of participating in, and this is the important indicator of reaction pulmonary ventilation function.This makes moderate progress after having met the treatment of restrictive ventilatory disorder process, and pulmonary fibrosis alleviates to some extent.

On (rcv) cdyn(lung compliance) impact in the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone little, in, heavy dose of group, the heavy dose of group of astragalus polysaccharides, Radix Hedysari flavone be little, in, heavy dose of group obviously raises with model group comparison numerical value, prednisone group, the heavy dose of group of Hedysamn polysaccharide and model group relatively numerical value raise.Caused volume when lung compliance refers to the unit pressure change, in order to reflect the elasticity of lung tissue, generally include lung compliance, chest wall compliance and total compliance, and Cdgn dyanamic compliance is wherein a kind of, it is the compliance of the lung that records when air-flow is not blocked in the breathing cycle, it is subjected to the impact of airway resistance, indirectly can reflect the volume-variation of lung; The elastic resistance of lung increases during pulmonary fibrosis, and the compliance of lung reduces, and expiratory dyspnea appears in the patient.The cdyn(Cdgn dyanamic compliance) be a sensitive indicator of reflection pulmonary fibrosis degree, when its value reduced, the indication lung compliance was relatively poor, and pulmonary fibrosis this moment degree increases the weight of; When its value raise, indication pulmonary fibrosis degree alleviated.The lung compliance attenuating is mainly seen in Fei Xian Weiization ﹑ Fei Shui Zhong ﹑ pulmonary atelectasis and pneumonia etc. makes the limited Pulmonary Diseases of lung expansion.During pulmonary fibrosis, because interstitial lung inflammation, fibrosis or honeycomb sample change and be mutually mixed with normal structure, elastance of lung lowers, so lung compliance reduces.This makes moderate progress after having met the treatment of restrictive ventilatory disorder process, and pulmonary fibrosis alleviates to some extent.

This experiment will be measured in the evaluation procedure that the pulmonary function technology is applied to rats with pulmonary interstitial fibrosis model and curative effect of medication.The main pathological change of IPF is the interstitial lung fibroblast proliferation, and can form honeycomb lung late period.Because there are a large amount of fibroblasts in interstitial lung, restrictive ventilatory functional disturbance occurs, the compliance of lung capacity and lung reduces.In the dosage group contrast that there were significant differences from each index, just can find several effective site dosage groups of curative effect optimum in this test.Experimental result shows: compare with model group the Radix Hedysari flavone little, in, heavy dose of group, little, the middle dosage group of Radix Astragali flavone improve obviously most indexs, these group therapeutic effect are more excellent.

6 Radixs Astragali, Radix Hedysari effective site are on the impact of Pulmonary Fibrosis in Rats with Bleomycin-induced serum HA, LN

Someone finds that Serum Laminin (Laminin, LN) and hyaluronic acid (Hyaluronic, HA) can be used as a kind of mark in the research Interstitial Lung Disease.The discovery HA such as Li accumulate in the interstitial lung inhibition air of damage and the gas exchange between blood.The lung tissue cell injury, the release cells factor, cytokine activation fibroblast and synthetic a large amount of HA also increase the synthetic of HA behind the Interstitial cell irriate.HA is a kind of polysaccharide, and it also links to each other with other matrix components as a kind of intermediary that links to each other with cell surface, structure that can stable matrix, and the effect of modified cells is arranged at cell surface.HA is the simplest a kind of glycosaminoglycan of structure in the extracellular matrix, and the someone thinks that matrix components mainly contains collagen protein, proteoglycan and adhesion glycoprotein, these compositions mix interweave make be organized into as a whole, to finish certain physiological function.Under pathological state, its content, distribution and serum-concentration can change.In the research of in the past pulmonary fibrosis, just there is the scholar to find that the variation of lung local cells epimatrix can cause that the level of extracellular matrix increases in the serum, and thinks that these compositions that increase derive from lung.

A lot of researchs point out that HA is relevant with the generation of a lot of lungs illness.Especially in the generation of pulmonary fibrosis, play an important role.HA also participates in the anabolic effect of collagen fiber, can make static fibrocyte activation, forms to have fibroblast synthetic and secretion collagen protein function.Have the HA receptor on the fibroblast film, HA can by membrane receptor and fibroblast with covalency be connected dual mode and be connected.HA is after cell-membrane receptor is combined, and activated protein kinase promotes the synthetic of DNA and protein.Therefore the HA increased content prompting lung local organization Extracellular Matrix Levels in the serum increases, and the prompting of the HA content in serum lung local cells epimatrix level reduces.

HA serum test experience is the result show; Compare in little, the heavy dose of group of prednisone group, astragalus polysaccharides, the Radix Astragali flavone with model group, in the heavy dose of group, Radix Astragali saponin, heavy dose of group, the Radix Hedysari flavone is little, in, heavy dose of group is by above-mentioned mechanism, the value that significantly reduces HA reduces the local ECM level of lung, and pulmonary fibrosis alleviates (P ﹤ 0.01) to some extent.

ECM comprises collagen, laminin，LN, fibronectin, proteoglycan and elastin laminin etc.And ECM reconstruction disorder is the main manifestations of pulmonary fibrosis.LN is produced by various kinds of cell, comprise I type alveolar epithelial cells and vascular endothelial cell etc., LN has the various biological effect, comprise inducing cell adhesion, Growth and Differentiation, also attract, adhere to chrotoplast, inflammatory cell, and stimulate T lymphocyte and macrophage secretion lymphokine, promote the synthetic of collagen, someone thinks that matrix components mainly contains collagen protein, proteoglycan and adhesion glycoprotein, and these compositions mix to interweave with making and are organized into as a wholely, and nationality is to finish certain physiological function.Under pathological state, its content, distribution and serum-concentration can change.LN belongs to the macromole glycoprotein in the cellular matrix, is distributed widely in the clear layer of basement membrane, consists of the basement membrane skeleton with collagen, and LN is mainly synthetic by the endotheliocyte that is positioned at basement membrane and epithelial cell under normal circumstances, and content is less in the serum.Many results of study prove that LN has participated in the endothelial cell damage process, have brought into play important function especially in the pulmonary fibrosis process, become one of monitoring pulmonary fibrosis pathological changes than sensitive indicator.The numerical value increase of LN shows the epithelial cell damage, constantly has new Growth of Cells reparation and hypertrophy to cause epithelium to thicken.LN increases and also can cause inflammatory cells increased and accumulate in basement membrane, and the damage lung tissue causes fibrosis.

LN serum test experience is the result show: compare with model group prednisone group, Radix Astragali flavone little, in, heavy dose of group, little, the middle dosage group of Radix Astragali saponin, little, the middle dosage group of Radix Hedysari flavone is by above-mentioned mechanism, the value that significantly reduces LN slows down the collagen aggregate velocity among the ECM, and pulmonary fibrosis alleviates (P ﹤ 0.01) to some extent.

7 Radixs Astragali, Radix Hedysari effective site are on the impact of Pulmonary Fibrosis in Rats with Bleomycin-induced lung tissue homogenate HYP

Pulmonary fibrosis is that collagen fiber increase gradually, finally cause collagen to accumulate gradually because due to the over-deposit of ECM, the speed that collagen protein forms in the lung is greater than the speed of degraded.HYP be the speed that forms of collagen fiber collagen protein greater than the speed of degraded, collagen fiber increase gradually, finally cause collagen to accumulate gradually.HYP is the representative index of collagen fiber deposition.HYP is distinctive class of amino acid in the collagen fiber, and content accounts for 12%～14% in collagen protein, and HYP mainly is present in the collagen protein, and its hetero-organization contains HYP hardly.A synthetic significant process of collagen is exactly, proline is converted into HYP through the hydroxylase effect, at least to there be 100 HYP residues just can make the triple-helix structure of collagen stable in every a peptide chain in each tropocollagen molecule, this stable proportionate relationship of peptide chain in HYP and the tropocollagen molecule makes the HYP assay become the obvious index of indirect reflection collagen content and is widely used in the pulmonary fibrosis diagnosis.HYP is a kind of peculiar aminoacid that forms the body collagen protein, except elastin laminin contains a small amount of hydroxyproline (about 1%), nearly all hydroxyproline all is present in the collagen, the main component of ECM when collagen is organ fibrosis, hydroxyproline in tissue can the reaction member Fibrotic degree, organize the mensuration of hydroxyproline content to become the important method of research organization's collagenic supersession.

HYP tissue homogenate test experience is the result show: compare with model group the heavy dose of group of astragalus polysaccharides, Radix Astragali flavone little, in, heavy dose of group, Radix Hedysari flavone be little, in, heavy dose of group is by above-mentioned mechanism, significantly reduce collagen fiber and deposit and make pulmonary fibrosis alleviate to some extent (P ﹤ 0.01).

This experiment is from the ordinary circumstance of rat, the content of the content of HA, LN and lung tissue HYP is observed in lung coefficient, pulmonary function, the serum, experimental result shows, compare with model group, each effective site group all is improved effect in majority parameters, illustrate that each effective site group all has certain inhibitory action to the process of pulmonary fibrosis, the progress that all can suppress pulmonary fibrosis, this explanation drug level is very large to the function influence of disease treatment, thus make this therapeutic scheme little, in, grouping on the heavy dose is more meaningful.Illustrate the Radix Astragali, Radix Hedysari effective site little, in, heavy dose of relevant with its drug dose to the process that suppresses pulmonary fibrosis.

The innovative point of 8 tests

8.1 the improvement of pulmonary fibrosis in rats preparation method

In the preparation process of in the past pulmonary fibrosis model, the modeling method that splashes into bleomycin and intratracheal instillation bleomycin through the nostril is arranged.But its shortcoming is both arranged, and the latter is classical modeling method.The former modeling success rate is not high, and classical modeling method wound is difficult for more greatly recovering.Therefore this subject group has been improved classical modeling method.

This test copies pulmonary fibrosis model, carries out under the small animal laryngoscope guiding, and this is further Improvement and perfection in the classical pulmonary fibrosis model preparation.Adopt the small animal laryngoscope guidance tracheal intubation can avoid the skin of neck of incision rat successively to separate and expose the method for trachea, eliminated the incision skin postoperative local infection of rat and the inducement of some complication.In the modeling process, use small animal laryngoscope and guide the sewing process that can reduce in the classical modeling process, and can reduce the consumption of anaesthetic, in the process of a large amount of animal models, can greatly improve efficient.Having avoided secondary insult is improvement to classical modeling method.Modeling method after the improvement, have easy, without wound, speed are fast, the modeling success rate is high characteristics.This test is obtained a result: the induced lung coefficient of comparing model group with the blank group significantly raises, and the lung function index TV of rat, EV, MV, AV, cdyn significantly reduce.The value of HA, LN significantly raises this, and just to point out this improved pulmonary fibrosis in rats preparation method be successfully.

8.2 the application of novel animal lung function system

My subject is for the accuracy of the lung function index that promotes the lung pattern disease experiment and detect and comprehensive, improves the animal lung function detection system that experiment equipment has been introduced French emka company, and uses AniRes animal lung function analytical system.Using Scientific Articles that this system experimentation achievement delivers has had many pieces to be published on the magazine that external SCI etc. includes.In the world, the method for now animal lung function analysis roughly is divided into has the wound body to retouch the case method; The noinvasive body is retouched the case method.

8.2.1 being arranged, the wound body retouches the case method

Animal is put into airtight body retouches in the case, implement tracheal intubation, animal breath need to have accurate animal respirator Aided Machine ventilation, detects simultaneously the airway pressure of animal and the variation of lung capacity, and then calculates airway resistance and the lung compliance of toy.This method can very accurately detect the minor variations of toy tidal volume, and data result also is the most accurate and the most stable, is mainly used in requiring very high scientific research.Because its higher precision and sensitivity, good stability and repeated, use also the most extensively, be the first-selected system of toy pulmonary function scientific research.Because it can obtain extraordinary experimental result, the scientific research institution more than 95% adopts this type systematic in the world.

8.2.2 the noinvasive body is retouched the case method

Animal is put into body retouch case, external air source is retouched highly stable air of input in the case to body, guarantees that animal has normal oxygen supply, is unlikely to be choked to death, and measures simultaneously the gas flow that animal turnover body is retouched case.The index that detects comprises tidal volume, inspiratory duration and the expiratory duration etc. of animal, and then calculates the penh value, and being used for indirectly, the pulmonary function of assessment animal changes.

Conclusion

This experiment modeling method is slight to the integral animal damage, and every detection index and pathological change all have significant difference with normal group, and the pathological change typical case.

The process of the Pulmonary Fibrosis in Rats with Bleomycin-induced model that the Radix Astragali, each effective site group of Radix Hedysari can be induced by the establishment bleomycin stops the development of interstitial pulmonary fibrosis, and wherein the Radix Astragali, little, the middle dosage group of Radix Hedysari flavone effect are more excellent.

The Radix Astragali, Radix Hedysari effective site stop the action intensity of interstitial pulmonary fibrosis process relevant with suitable equivalent drug level.

Comprehensive Assessment, the Radix Hedysari flavone is excellent than other effective site to the influence of lung fibrosis in rats pulmonary function, HA, LN, HYP.

It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.

Claims

1. the Radix Astragali, the application of Radix Hedysari effective site in preparation treatment pulmonary fibrosis medicine.

2. application according to claim 1 is characterized in that, the described Radix Astragali, Radix Hedysari effective site are one or more compositionss in astragalus polysaccharides, Radix Astragali flavone, Radix Astragali saponin, Hedysamn polysaccharide, Radix Hedysari flavone, the Radix Hedysari saponin.

3. application according to claim 1 is characterized in that, the described Radix Astragali, Radix Hedysari effective site are the Radix Hedysari flavone.