CN101869595A

CN101869595A - Preparation method and quality detection method of liquorice and gall oral liquid

Info

Publication number: CN101869595A
Application number: CN 201010215585
Authority: CN
Inventors: 江厚生; 王秀敏; 侯晓礁; 李学良
Original assignee: BEIJING CENTRE BIOLOGY Co Ltd
Current assignee: BEIJING CENTRE BIOLOGY Co Ltd
Priority date: 2010-07-02
Filing date: 2010-07-02
Publication date: 2010-10-27

Abstract

The invention relates to liquorice and gallbladder oral liquid mainly treating lung heat cough caused by avian infectious bronchitis and mycoplasma gallisepticum infection, oral, injection and spray preparations containing a liquorice and gallbladder composite and a preparation method and a quality detection method thereof. The liquorice and gallbladder oral liquid comprises the components of isatis root, calculus bovis factitius, liquorice, borneol, porcine gall powder, compound of glauber-salt and liquorice and the like. The invention method has simple and convenient production process and advanced detection means; the product has stable quality, high bioavailability, no toxic and side effects, convenient use and obvious curative effect and has the effects of clearing heat and detoxicating, cooling blood and releasing the lung and relieving cough and asthma.

Description

The preparation of sweet bile oral liquor and quality determining method

Technical field

The present invention relates to a kind of sweet bile oral liquor that cures mainly the cough due to lung-heat that infectious bronchitis of chicken and mycoplasma gallisepticam infection cause and preparation method thereof and quality determining method.

Background technology

Along with the development of China's aviculture, particularly a large amount of rises in intensification, scale chicken farm, the large-scale generation of China various places fowl diseases in recent years and popular, the especially generation of some new fowl diseasess has caused economic loss in various degree to aviculture.In poultry disease, respiratory system disease is that harm is maximum, a class disease the most rambunctious.Through investigation, the sickness rate of poultry infectious respiratory disease can reach 100%, and mortality rate does not coexist between 25～90% with the age.Particularly the appearance of the variant that some pathogen were new in recent years causes the clinical symptoms atypiaization, makes respiratory tract disease become complicated more, is difficult to control.

Infectious bronchitis of chicken (IB) and infectious laryngotracheitis of chicken (ILT) are acute contact respiratory infectious disease, M ﹠ M is all very high, chicken virus mycoplasma (MG) is the cause of disease of chicken chronic respiratory (CRD), can propagate by level and vertical mode, the mortality rate of primary disease is generally not high, but tend to secondary or concurrent other diseases, and case fatality rate is up to more than 40%.More than the disease of three kinds of respiratory systems have a strong impact on health, fertility performance and the economic benefit of poultry, cause heavy losses to poultry husbandry, be the important eqpidemic disease in intensification chicken farm.These three kinds of diseases are mainly puted prevention first with vaccine virus immunization, and also there is defective in vaccination, in case the morbidity Western medicine can not reach the purpose of healing.

Up to now, both at home and abroad the treatment of poultry infectious respiratory disease is all paid special attention to.Chemical drugs treatment at present commonly used, but because the development, the particularly development of broiler export enterprise of the problems affect aviculture of drug residue, therefore, the new medicine of exploitation control poultry respiratory tract diseases has crucial meaning.

Herbal medicine is world-famous for low, the residual green treatment means such as few of its unique traditional Chinese veterinary medicine theoretical system, toxic and side effects and significant curative effect, go through more than 2,000 year lasting.But the most of technologies of herbal medicine conventional dosage forms are simple, dosage form is coarse, use is inconvenient, and effective ingredient can not be utilized fully, can not well adapt to the requirement of modern animal husbandry production intensification and scale.Therefore, utilize modern means of science and technology, the new herbal medicine that research and development taking convenience, dosage can make full use of effective ingredient has again less become urgent problem.

The prescription of sweet bile oral liquor derives from Chinese veterinarian's secret recipe among the people, is made up of Radix Glycyrrhizae, Pulvis Fellis Suis, Radix Isatidis, Calculus Bovis, Matrii Sulfas Exsiccatus, Borneolum Syntheticum.Secular clinical practice proves, this side have good QI invigorating and in, clearing away heat and cooling blood, detoxifcation lung qi dispersing and function of relieving cough and calming asthma, distinguished to multiple animal breath systemic disease curative effect, the principal indication that this research should the side is positioned the poultry respiratory tract infectious disease.

Traditional Chinese veterinary medicine is thought, lung being a delicate viscus, and the table of main the whole body is experienced multiple cause of disease such as six climate exopathogens, epidemic disease easily and is fallen ill.Poultry is all the more so, and respiratory tract communicates with air bag, and huge mucous membrane surface directly contacts with air, just very easily causes infection when rising of cause of disease density or autoimmune function descend in the air.The infectious laryngotracheitis of chicken, infectious bronchitis and virus mycoplasma infect (chronic respiratory tract disease) because of its sickness rate height, spread speed is fast and have a strong impact on fertility performance usually brings the tremendous economic loss to poultry husbandry.Therefrom veterinary's pharmacy angle is captured the basic purpose that this class disease is we's research.

Radix Glycyrrhizae is we's a principal agent, has multiple functions such as invigorating the spleen and replenishing QI, nourishing the lung to arrest cough, heat-clearing and toxic substances removing, the mediation property of medicine; The various active composition has antivirus action, licoflavone can strong inhibition various pathogens such as staphylococcus aureus, Diplococcus pneumoniae and remove active oxygen, the biological effect of the scalable immunocyte of glycyrrhizin and regulate immunologic function;

Pulvis Fellis Suis, Radix Isatidis and Calculus Bovis are we's accessory drugs, have clearing away heat and cooling blood, dampness detoxicating functions, play a role at cause of disease from a plurality of different angles.Antibiotic and the relieving cough and asthma of Fel Sus domestica, Radix Isatidis and Calculus Bovis antiviral and improve immunity, three medicines and Radix Glycyrrhizae are share and bring into play synergism and effect is strengthened greatly.

Matrii Sulfas Exsiccatus is we's a adjuvant drug, by purging heat to relax the bowels, and moistening for dryness and softening hard mass, the putrefaction removing Detoxication excretes pathogenic heat, thereby assists a ruler in governing a country the target that main and auxiliary medicine realizes eliminating cause of disease.

Borneolum Syntheticum is we's a messenger drug, and hot cool liter of its property loose, and except that consciousness and resuscitation restoring own, heat radiation pain relieving and pharynx-clearing throat-benefiting effect, also has the effect of messenger drug, improves the drug effect performance of other drug, accelerates the raising of blood drug level, promotes the barrier permeability of medicine.

In a word, Chinese medicine is having very big potentiality and advantage aspect control ILT, IB, the MG, and we can prevent and treat this three kinds of diseases by the sweet bile oral liquor of development, have more proved the Therapeutic Principle of traditional Chinese veterinary medicine " treating different diseases with the same therapeutic principle ".Prove through clinical trial, sweet bile oral liquor control ILT, IB, MG determined curative effect, no toxicity and drug residue, aspect dosage form, meet the characteristics of " three is little " (using dosage, toxicity and side effect are little), " triple effect " (efficient, quick-acting, long-acting) and " five convenience " (production, storage, transportation, easy to carry and use).Have good market prospect and economic benefit.

Summary of the invention

The purpose of this invention is to provide a kind of sweet bile oral liquor that is used for the cough due to lung-heat that infectious bronchitis of chicken and mycoplasma gallisepticam infection cause, this drug quality is stable, determined curative effect.Another object of the present invention provides a kind of technology and quality determining method thereof of producing sweet bile oral liquor.

Sweet bile oral liquor of the present invention prepares by following steps:

Radix Isatidis, Radix Glycyrrhizae, Matrii Sulfas Exsiccatus are decocted with water 2～3 times, and collecting decoction filters, and filtrate concentrates at 30～60 ℃, is concentrated into relative density and is 1.10～1.35 extractum, puts coldly, and it is an amount of to add ethanol, leaves standstill, filter, and filtrate recycling ethanol, it is an amount of to add water, leaves standstill; An amount of dissolve with ethanol of Pulvis Fellis Suis, artificial Calculus Bovis, Borneolum Syntheticum adds said extracted liquid, adds water to 500～1800ml, shakes up, and leaves standstill, and filters, and promptly gets sweet bile oral liquor.

The quality determining method of the sweet bile oral liquor of the present invention is:

[character] this product is tan liquid, the precipitation that has a small amount of jog easily to loose;

This product 0.5ml is got in [discriminating] (1), adds Diluted Alcohol 0.5ml, as need testing solution; Other gets the arginine reference substance, adds Diluted Alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 1～2 μ l of above-mentioned solution, put respectively in same be on the silica gel G plate of adhesive with the sodium carboxymethyl cellulose, with the ratio of n-butyl alcohol-glacial acetic acid-water be 19: 5: 5 be developing solvent, launch, take out, dry, spray is with ninhydrin solution, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance on, show the speckle of same color;

(2) get this product 10ml, water bath method, residue add 10% sodium hydroxide solution 5ml, and 120 ℃ of heating 4 hours are put coldly, drip dilute hydrochloric acid and regulate pH value to 2～3; With ethyl acetate extraction 4 times, each 10ml, merge extractive liquid,, evaporate to dryness, residue add ethanol 10ml makes dissolving, as need testing solution; It is an amount of that other gets the Hyodeoxycholic Acid reference substance, adds ethanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel G thin plate of adhesive with the sodium carboxymethyl cellulose, ratio with the isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-water of new preparation is 10: 5: 5: 3: 1 upper solution is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts daylight and ultra-violet lamp respectively and inspects under the 365nm wavelength; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle or the fluorescence speckle of same color;

[inspection] amount of alcohol should be 10%～20%;

Relative density should be not less than 1.01;

Other should meet relevant every regulation under 10 pages of mixture items of " People's Republic of China's veterinary drug allusion quotation 2005 editions " appendix;

[assay] is according to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-0.5% potassium dihydrogen phosphate is mobile phase, and ratio is 60: 40, and wherein 0.5% potassium dihydrogen phosphate is 2.6 with phosphoric acid adjusting pH value; The detection wavelength is 254nm; Theoretical cam curve is calculated by glycyrrhizic acid should be not less than 1000;

The preparation of reference substance solution: precision takes by weighing monoammonium glycyrrhizinate reference substance 10mg, places the volumetric flask of 100ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is made the solution that every 1ml contains 100 μ g, shakes up, promptly;

The preparation of need testing solution: precision is drawn this product 5ml, places the volumetric flask of 50ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is placed to room temperature, and precision is drawn 2ml, places the volumetric flask of 10ml, adds methanol to scale, shakes up, and filters standby;

Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly;

Among the present invention, the sweet bile oral liquor of every 1ml contains Radix Glycyrrhizae with glycyrrhizic acid (C ₄₂H ₆₂O ₁₆) meter, must not be less than 2.0mg.

By the present invention, we have verified the cough due to lung-heat's that sweet bile oral liquor causes at treatment infectious bronchitis of chicken and mycoplasma gallisepticam infection effect, and occupation mode and dosage are: mix drink, the every 1.5L drinking-water of chicken use this product 1ml, free drink-service, logotype 3～5 days.

Drug quality of the present invention is stable, drug effect height, good effect.For show medicine technology of the present invention rationally, steady quality, good effect, we have done great deal of experimental.

Description of drawings

Accompanying drawing 1 is for the arginine being the sweet bile oral liquor thin-layer chromatogram of contrast

Accompanying drawing 2 is for the Hyodeoxycholic Acid being the sweet bile oral liquor thin-layer chromatogram of contrast

Accompanying drawing 3 is the liquid chromatogram of glycyrrhizic acid standard substance

Accompanying drawing 4 is the liquid chromatogram of sweet bile oral liquor

Accompanying drawing 5 is the glycyrrhizic acid canonical plotting

Specific embodiment:

Embodiment 1: the preparation of the sweet bile oral liquor of the present invention

Take by weighing Radix Isatidis 180g, Radix Glycyrrhizae 60g, Matrii Sulfas Exsiccatus 30g, add the 2700ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 2160ml decocting and boil, collect decocting liquid; To remain medicinal residues again and add the 1890ml decocting and boil, collect decocting liquid; Three decocting liquids are merged, filter, filtrate is concentrated into relative density 1.18 (50 ℃), puts coldly, adds 900ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 240ml of water, leaves standstill.Take by weighing Pulvis Fellis Suis 20g, artificial Calculus Bovis 20g, Borneolum Syntheticum 20g, add ethanol 300ml and make dissolving, add said extracted liquid, add water to 1800ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 1.

Take by weighing Radix Isatidis 100g, Radix Glycyrrhizae 30g, Matrii Sulfas Exsiccatus 10g, add the 1400ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 1120ml decocting and boil, collect decocting liquid; To remain medicinal residues again and add the 980ml decocting and boil, collect decocting liquid; Three decocting liquids are merged, filter, filtrate is concentrated into relative density 1.35 (30 ℃), puts coldly, adds 600ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 200ml of water, leaves standstill.Take by weighing Pulvis Fellis Suis 20g, artificial Calculus Bovis 60g, Borneolum Syntheticum 10g, add ethanol 110ml and make dissolving, add said extracted liquid, add water to 1000ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 2.

Take by weighing Radix Isatidis 50g, Radix Glycyrrhizae 40g, Matrii Sulfas Exsiccatus 40g, add the 1300ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 1040ml decocting and boil, collect decocting liquid; The secondary decocting liquid is merged, filter, filtrate is concentrated into relative density 1.10 (60 ℃), puts coldly, adds 500ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 200ml of water, leaves standstill.Take by weighing Pulvis Fellis Suis 40g, artificial Calculus Bovis 34g, Borneolum Syntheticum 30g, add ethanol 120ml and make dissolving, add said extracted liquid, add water to 800ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 3.

Take by weighing Radix Isatidis 120g, Radix Glycyrrhizae 45g, add the 1650ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 1320ml decocting and boil, collect decocting liquid; To remain medicinal residues again and add the 1155ml decocting and boil, collect decocting liquid; Three decocting liquids are merged, filter, filtrate is concentrated into relative density 1.15 (50 ℃), puts coldly, adds 500ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 200ml of water, leaves standstill.Take by weighing Pulvis Fellis Suis 29g, artificial Calculus Bovis 50g, add ethanol 130ml and make dissolving, add said extracted liquid, add water to 1000ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 4.

Take by weighing Radix Isatidis 100g, Radix Glycyrrhizae 50g, Matrii Sulfas Exsiccatus 25g, Indigo Naturalis 25g, add the 200ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 1600ml decocting and boil, collect decocting liquid; To remain medicinal residues again and add the 1400ml decocting and boil, collect decocting liquid; Three decocting liquids are merged, filter, filtrate is concentrated into relative density 1.16 (50 ℃), puts coldly, adds 700ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 200ml of water, leaves standstill.Take by weighing Pulvis Fellis Suis 22g, artificial Calculus Bovis 25g, Venenum Bufonis 40g, Borneolum Syntheticum 20g, add ethanol 280ml and make dissolving, add said extracted liquid, add water to 1500ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 5.

Take by weighing Radix Isatidis 50g, Radix Glycyrrhizae 40g, Indigo Naturalis 20g, add the 1100ml decocting and boil, collect decocting liquid; To remain medicinal residues and add the 900ml decocting and boil, collect decocting liquid; The secondary decocting liquid is merged, filter, filtrate is concentrated into relative density 1.13 (60 ℃), puts coldly, adds 350ml ethanol, leaves standstill, and filters, and filtrate recycling ethanol adds the about 220ml of water, leaves standstill.Take by weighing bilein 40g, Venenum Bufonis 35g, Borneolum Syntheticum 25g, add ethanol 110ml and make dissolving, add said extracted liquid, add water to 1000ml, shake up, leave standstill, filter, promptly get sweet bile oral liquor 6.

Embodiment 2: the quality standard research of the sweet bile oral liquor of the present invention

The main component of the sweet bile oral liquor of the present invention is the tan liquid of water extract-alcohol precipitations such as Radix Glycyrrhizae, Radix Isatidis, Calculus Bovis, Borneolum Syntheticum, Pulvis Fellis Suis, Matrii Sulfas Exsiccatus; be pure Chinese medicinal preparation; be mainly used in the treatment of infectious bronchitis of chicken, infectious laryngotracheitis of chicken and chicken virus mycoplasma, have the function of heat-clearing and toxic substances removing, preventing phlegm from forming and stopping coughing.Main bioactive ingredients in the Radix Glycyrrhizae is a glycyrrhizic acid, and its topmost pharmacological action is an antiinflammatory, and antiinflammatory mechanism is relevant with the effect that suppresses prostaglandin media such as (PGs).It is generally acknowledged that glycyrrhizic acid can be by suppressing the synthetic of PGs and discharging to the required phospholipase A2 inhibitory action of AA hydrolysis.Another main pharmacological action of glycyrrhizic acid is an antiviral, as hepatitis virus resisting, HIV (human immunodeficiency virus) etc.In addition, glycyrrhizic acid has the nonspecific immunity regulating action, and it mainly is to strengthen immunization of cell, can strengthen

Phagocytic function is eliminated inhibition

The inhibition activity, also optionally strengthen the multiplication capacity and the activity of helper T cell.Therefore with glycyrrhizic acid as index components, carried out Determination on content.

1 instrument and reagent

1.1 instrument

SHIMADZU LC-10ATVP high performance liquid chromatograph; SPD-10AVP UV, visible light detector; Chromatographic column: SHIMADZU ODSC18 post (150 * 4.6mm, 5 μ m)

1.2 reagent

Monoammonium glycyrrhizinate reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute); Potassium dihydrogen phosphate (analytical pure); Phosphoric acid (analytical pure); Methanol (chromatographically pure); The mobile phase water is a distilled water; All the other reagent are analytical pure.

2 test methods and result

2.1 chromatographic condition

Chromatographic column: ODSC18 post (150 * 4.6mm, 5 μ m); Mobile phase: methanol-0.5% potassium dihydrogen phosphate (60: 40), it is 2.6 that 0.5% potassium dihydrogen phosphate is regulated pH value with phosphoric acid; Detect wavelength: 254nm; Column temperature: 40 ℃; Flow velocity: 1ml/min.

2.2 the preparation of need testing solution

2.2.1 the preparation of glycyrrhizic acid standard solution

Precision takes by weighing monoammonium glycyrrhizinate standard substance (content is 96.6%) 2.5mg, places the volumetric flask of 25ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is made the solution that concentration is 96.6 μ g/ml, filters standby.

2.2.2 the preparation of sample solution

Precision is drawn the sweet bile oral liquor 5ml of the present invention, places the volumetric flask of 100ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is cool to room temperature, filters standby.

2.2.3 the preparation of sample negative control solution

After removing the Radix Glycyrrhizae in the prescription, prepare the negative control solution of sweet bile oral liquor, prepare negative controls by the method for " 2.2.2 " according to production technology.

2.3 linear relationship is investigated

Accurate monoammonium glycyrrhizinate reference substance solution 4,6,8,10, the 12 μ l that draw, measure the peak area integrated value by above-mentioned chromatographic condition, with the peak area is vertical coordinate, time is abscissa, get regression equation through linear regression treatment, regression equation is Y=1.577123e-004X+8.714488 (r=0.9948438), shows that the sample size of monoammonium glycyrrhizinate is good linear relationship in 19.32～115.92 μ g scopes.

2.4 precision test

Precision is drawn monoammonium glycyrrhizinate reference substance solution 10 μ l, repeats sample introduction 5 times, measures peak area by above-mentioned chromatographic condition, and according to peak area value, getting average peak area is 606222.2, RSD=0.63%.

2.5 repeatability test

Get 5 bottles of same batch of sweet bile oral liquors of the present invention, be prepared according to the preparation method of above-mentioned sample solution, and under above-mentioned chromatographic condition, measure peak area, according to peak area value, average peak area be 577092.3, RSD=1.85%.

2.6 average recovery test

Get 5 parts of the sweet bile oral liquors of the present invention of known content respectively, per minute 5ml adds the monoammonium glycyrrhizinate reference substance respectively, measures peak area by above-mentioned sample preparation methods and chromatographic condition, and the average recovery rate of glycyrrhizic acid is 99.68%, RSD=0.59% (n=5).

Table 1 determination of recovery rates result (n=5)

2.7 sample determination

Get the different sweet bile oral liquor of the present invention respectively, the accurate absorption measured peak area, each sample feeding 3 times by above-mentioned sample solution preparation method and chromatographic condition.The results are shown in Table 2.

The sweet bile oral liquor assay of table 2 result (n=5)

3 discuss

We once adopted acetonitrile-0.5% potassium dihydrogen phosphate (55: 45), the acetonitrile-water system of different proportion, the methanol-water system of different proportion, the methanol of different proportion-0.5% potassium dihydrogen phosphate system, the result shows: during methanol-0.5% potassium dihydrogen phosphate (60: 40), potassium dihydrogen phosphate is regulated pH to 2.6 with phosphoric acid, glycyrrhizic acid peak shape symmetry is separated fully, and negative control does not disturb, we also find simultaneously, the retention time of the minor variations meeting remarkable influence glycyrrhizic acid of methanol ratio.

4 conclusions

The high performance liquid chromatography specificity is strong, can measure the glycyrrhizic acid content in the sweet bile oral liquor of the present invention fast and accurately, when glycyrrhizic acid is measured in the sweet bile oral liquor different to the present invention, find that its content all reaches requirement, therefore, can be used as the method for quality control of the sweet bile oral liquor of the present invention.

Embodiment 3: the study on the stability of the sweet bile oral liquor of the present invention

Purpose: with the character of sweet bile oral liquor, discriminating, content be changed to the detection index, the stability of the sweet bile oral liquor of the present invention is investigated, for clinical application provides scientific basis.

1 instrument and medicine

1.1 instrument

SHIMADZU LC-10ATVP high performance liquid chromatograph; SPD-10AVP UV, visible light detector; Chromatographic column: SHIMADZU ODSC18 post (150 * 4.6mm, 5 μ m); Thunder magnetic PHS-25 type digital display pH meter (Shanghai Precision Scientific Apparatus Co., Ltd).

1.2 reagent

Monoammonium glycyrrhizinate reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute); Potassium dihydrogen phosphate (analytical pure); Phosphoric acid (analytical pure); Methanol (chromatographically pure); The mobile phase water is a distilled water; Sodium hydroxide (analytical pure); Chloroform (analytical pure); Ethanol (analytical pure); Paradime thylaminobenzaldehyde (analytical pure); Sulphuric acid (analytical pure); The sweet bile oral liquor of the present invention 3,4,6 is provided by Beijing Centre Biology Co., Ltd..

2 experimental techniques

2.1 accelerated tests

Accelerated test by People's Republic of China's veterinary drug allusion quotation regulation, three batch samples are at 40 ± 2 ℃, placed six months under the condition of relative humidity 75 ± 5%, the every month of sampling regularly once, measure the content of glycyrrhizic acid in the sweet bile oral liquor then, and observe outward appearance, color and luster (character), according to prediction of result effect duration.

2.2 illumination accelerated tests

Get and supply three batches of test agents, place the light kitchen, under the condition of illumination 4500 ± 500Lx, placed 10 days, the regularly sampling in the 5th, 10 day, the content of glycyrrhizic acid in the working sample, and observe color and luster, abnormal smells from the patient variation.

2.3 reserved sample observing method

2.3.1 three batches of test samples 25 ± 2 ℃ of temperature, were placed 12 months under the condition of relative humidity 60 ± 10%.Took a sample once in per 3 months, respectively at 0,3,6,9,12 month, to the content of glycyrrhizic acid in the sweet bile oral liquor, outward appearance, color and luster (character) etc. detected then.

2.3.212 continued later in individual month to observe, detect respectively at sampling in 18,24 months.With result and comparison in 0 month, determine the effect duration of medicine.Because the dispersibility of measured data carries out statistical analysis by 95% fiducial limit, draws rational effect duration.Less as three batches of statistic analysis result difference, then getting its meansigma methods is expiry date; If difference is bigger, then gets it and the shortest be effect duration.Data show very stable medicine, and analysis does not take statistics.

2.4 identification experiment

Get this product 2ml, put in the separatory funnel, add water 20ml, 10% sodium hydroxide solution 10ml, chloroform 10ml shakes up, and extracts; Divide and to get chloroform solution, put in the water-bath behind the evaporate to dryness, residue adds ethanol 2ml makes dissolving, adds paradime thylaminobenzaldehyde crystallization number, slowly adds sulphuric acid 1ml along wall, observes change color.

3 experimental results

3.1 sweet bile oral liquor accelerated test result

Sweet bile oral liquor was placed six months under the condition of relative humidity 75 ± 5% at 40 ± 2 ℃, and its appearance luster and clarity do not have significant change, and sweet bile oral liquor content difference is not remarkable.The results are shown in Table 1.

3.2 sweet bile oral liquor acceleration by light result of the test

Sweet bile oral liquor was placed 10 days under the condition of illumination 4500 ± 500Lx, the regularly sampling in the 5th, 10 day, and its outer character, glycyrrhizic acid content change all not remarkable.The results are shown in Table 2.

3.3 reserved sample observing result

3.3.1 sweet bile oral liquor was kept somewhere 12 months under approaching actual storage requirement, its outward appearance and color and luster do not have significant change, and the variation difference of glycyrrhizic acid content is not remarkable, and the quality index difference is very little, can determine that effect duration is 2 years.The results are shown in Table 3.

Table 1 accelerated test result

Annotate: lilac or purple+character changes not obvious ++ and character changes obviously-

Table 2 acceleration by light result of the test

Annotate: lilac or purple+character changes not obvious ++

Table 3 reserved sample observing result

Annotate: lilac or purple+character changes not obvious ++

3.3.2 sweet bile oral liquor was kept somewhere 2 years at normal temperatures, its outward appearance and color and luster do not have significant change, and the variation difference of glycyrrhizic acid content is not remarkable, and the quality index difference is very little, and effect duration can reach 2 years.

The results are shown in Table 4.

36 months result of table 4 storage at normal temperature

Annotate: lilac or purple+character changes not obvious ++

4 conclusions

The sweet bile oral liquor of the present invention quickens in temperature, character and changes of contents are not obvious under the acceleration by light experimental condition.According to the standard that requires of Food and Drug Administration (FDA), can determine tentatively that its stability is 2 years.By the stability of the sweet bile oral liquor of reserved sample observing method investigation the present invention, to place 2 years, each index changes very little, so it has good stability.We are according to demand of practical production, and the effect duration of determining the sweet bile oral liquor of the present invention is 2 years.

Embodiment 4: the sweet bile oral liquor of the present invention is to the therapeutic effect of infectious bronchitis of chicken

Infectious bronchitis of chicken (Infectious bronchitis, IB) be a kind of acute, the height contagious disease that causes by infectious bronchitis virus (IBV), this disease pathogen exists numerous serotype and variant, bring great difficulty to vaccine immunity, be still one of disease of chicken farm pilosity at present.Therefore, development effectively preventing medicine is with significant.The sweet bile oral liquor of the present invention is by prescriptions such as Radix Isatidis, Radix Glycyrrhizaes, and the Chinese medicine preparation through extraction processes has heat-clearing and toxic substances removing, cough and asthma relieving efficacy.In order to observe the effectiveness of sweet bile oral liquor treatment infectious bronchitis of chicken, this test adopts sweet bile oral liquor that the sick chicken of 7340 spontaneous generation infectious bronchitiss is treated, and has observed the effect of this medicine treatment.

1 materials and methods

1.1 material

1.1.1 the sweet bile oral liquor 1,2,3 of the present invention

Provide by Beijing Centre Biology Co., Ltd.; Composition: Radix Isatidis, Radix Glycyrrhizae etc.; Specification: 100ml/ bottle.

1.1.2 other medicines

Other conventional medicines such as penicillin, streptomycin are available from Jilin Province pharmaceuticals.

1.1.3 test the disease chicken

Select 7340 abiogenous sick chickens in 2 chicken farms, town, grew sheet sea, Dehui, Jilin Province, differentiate through Epidemiological study, clinical symptoms, pathology cuts open inspection to be changed and lab testing, is diagnosed as the sick chicken of infectious bronchitis.

1.1.4 medicine and reagent preparation

The IBV standard positive serum is purchased to China Veterinary Drugs Supervisory Inst..

Sucrose: purchase Beijing Yili Fine Chemicals Co., Ltd..

TNE buffer: 10mmol Tris-HC1pH7.4,100mmol NaCl, 5mmolEDTA.

A Shi liquid: glucose 20.5g, sodium chloride 4.2g, citric acid 0.55g, sodium citrate 8.0g are dissolved in 1000mL.

0.5% chicken erythrocyte suspension: take the blood of 4 chickens to mix, with normal saline washing 3～4 times, with the centrifugal 5～10min of 1500r/min, sedimentary erythrocyte is mixed with 0.5% chicken erythrocyte suspension with normal saline then at every turn with equivalent A Shi liquid.

1.2 method

1.2.1 set up diagnosis

According to Epidemiological study, clinical symptoms differentiates that pathology cuts open the inspection variation and lab testing is set up diagnosis.

1.2.1.1 Epidemiological study is carried out according to a conventional method.

1.2.1.2 cuing open the inspection variation, pathology carries out according to a conventional method.

1.2.1.3 lab testing

1.2.1.3.1 the preparation of infectious bronchitis of chicken antigen hemagglutinating antigen

Aseptic collection is cutd open the pathological material of disease of examining the sick chicken of tentative diagnosis through clinical and pathology, cultivates propagation through chick embryo allantoic cavity, the collection allantoic fluid, and 4 ℃ of centrifugal 15min of 8000 * g get supernatant, and 4 ℃ of centrifugal 3h of 25000 * g remove supernatant, and precipitation suspends with the TNE buffer.Slightly carry viral suspension and carry out that the sucrose linear gradient is centrifugal to be further purified, its method is: get 50% sucrose 6ml, add in 10cm * transparent centrifuge tube of 1.5cm thin-walled, go up overlapping 20% sucrose 6ml, freezing 24h takes out the back and melts, on add viral crude extract 1ml, be full of centrifuge tube with 20% sucrose, 4 ℃ of centrifugal 3h of 25000 * g collect the virus band, with 5 times of TNE buffer dilutions, with 30000 * g4 ℃ of centrifugal 1h, precipitation adds 1ml TNE buffer and suspends, and is standby as antigen hemagglutinating antigen.1.2.1.3.2 Microhemagglutination and hemagglutination inhibition test (HA-HI)

HA: in " V " shape micro-reaction plate, carry out.Every hole adds 25 μ l normal saline.Dip in the dilution rod and to get 1 of antigen hemagglutinating antigen (doing 5 times of dilutions in advance) (25 μ 1),, make doubling dilution successively,, abandon or adopt in the dilution rod to last hole from first hole.Every hole adds 0.5% chicken erythrocyte suspension, 25 μ l, and establishes the erythrocyte control wells that does not add virus, shakes up on agitator immediately, puts 37 ℃ of sense works in the wet box, 30min left and right sides observed result.With the high dilution of the complete agglutinative viral liquid of erythrocyte is the terminal point of judging the blood clotting valency.

HI: with the blood clotting valency titre in the above-mentioned HA test divided by 4 value as the antigen hemagglutinating antigen (i.e. 4 HAUs) of this test for example the blood clotting titre be 320 times, 4 HAUs are 80 times so, with 80 times of above-mentioned antigen hemagglutinating antigen dilutions, use after the neutralization of infectious bronchitis of chicken standard positive serum, do hemagglutination test again, can reach the highly diluted multiple of the serum that suppresses hemagglutinative function fully and be blood clotting and suppress valency.

1.2.2 experimental animal grouping and processing

7340 of sick chickens of 2 chicken house spontaneous generation infectious bronchitiss are divided into 5 groups separately at random by feed lot, and testing 1 group is matched group, does not process; Test 2 groups and be the sweet bile oral liquor 1 treatment group of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Test 3 groups and be the sweet bile oral liquor 2 treatment groups of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Test 4 groups and be the sweet bile oral liquor 3 treatment groups of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Testing 5 groups is enrofloxacin treatment group, converts water 1.0kg with the 75mg enrofloxacin, and the free drink-service of chicken, successive administration 5d are suffered from order.

1.2.3 observation index

Write down the number of dying of illness, the healing number of each test group, calculate case fatality rate and cure rate, the difference between relatively each is organized with statistical method.

2 results

2.1 sick chicken diagnostic result

According to Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection to be changed and lab testing, 7340 sick chickens of making a definite diagnosis 2 chicken houses, and the disease that takes a disease is an infectious bronchitis of chicken.

2.1.1 main clinic symptoms

Chicken group sudden onset, sick chicken asthma, cough, spirit is depressed, appetite impairment or useless exhausted, each house all finds to die of illness chicken.Employing ofloxacin drinking-water, the state of an illness are not seen and are alleviated, and involve full group rapidly thereupon.Sick young fear of cold is afraid of cold, and the feather pine is random, and the wing that hangs down is slow-witted upright, and photophobia is shed tears, tunica mucosa nasi swelling, snivel.Then dyspnea, straight of peak with heading, tachypnea is breathed heavily slightly, and tangible trachea hello sound appears in the sneeze of coughing, and especially night is clearly audible.

2.1.2 cuing open inspection, pathology changes

Nasal cavity, throat, trachea and bronchus have serosity, Catarrhal and caseous exudate.Air bag presents muddiness or contains yellow cheesy exudate, in trachea back segment or the bronchus, can see caseous embolus.Around big bronchus, can see the special mess pneumonia.The sick Testis et penis Gallus domesticus enlargement of part is hemorrhage, most show class " flower kidneys " of refuting shape, and renal tubules and fallopian tube are expanded because of urate deposition.

2.1.3 laboratory diagnosis

Using micro-HA-HI result of the test shows, after the 37 ℃ of processing of phosphatase of sick fowl disease material viral isolates through sucrose density gradient centrifugation purification, 1 μ g/ml, has typical blood clotting, can coagulation chicken 0.5% erythrocyte, hemagglutinative titer all reaches more than 27, and this blood clotting can neutralize by infected property bronchitis specific corrosioning anteserum, proves and contains infectious bronchitis virus in the pathological material of disease, makes a definite diagnosis disease chicken trouble in view of the above and is infectious bronchitis.

2.2 sweet bile oral liquor is to the therapeutic effect of infectious bronchitis

Make the state of an illness very fast controlled after taking sweet bile oral liquor, untreated matched group spontaneous recovery rate is 72.77% and 76.06%, though fatality rate is not high, glucose recovery is slow; The cure rate of 3 sweet bile oral liquor groups is all more than 96.94%, and case fatality rate significantly is lower than matched group (P＜0.01), and glucose recovery is very fast; The results are shown in Table 1.

The sweet bile oral liquor of table 1 is to the therapeutic effect of No. 1 chicken house infectious bronchitis of chicken

Annotate: * represents to compare p＜0.05, significant difference with matched group; * represents to compare p＜0.01 with matched group, and difference is extremely remarkable.

The sweet bile oral liquor of table 2 is to the therapeutic effect of No. 2 chicken house infectious bronchitis of chickens

3 analyze

3.1 sick chicken is made a definite diagnosis

Epidemiological study finds that respiratory symptom appears in the chicken group sudden onset of 2 chicken houses, and the incidence that involves full group rapidly meets the infectious bronchitis of chicken principal character; Sick chicken has occurred should typical clinical symptoms of disease and pathological change; Trace HA-HI test has typical blood clotting, can coagulation chicken 0.5% erythrocyte, and hemagglutinative titer all reaches more than 27, and this blood clotting can neutralize by infected property bronchitis specific corrosioning anteserum, makes a definite diagnosis disease chicken trouble in view of the above and is infectious bronchitis.

3.2 sweet bile oral liquor is to the therapeutical effect of infectious bronchitis of chicken

Primary disease is by due to the infectious bronchitis virus, so general antibiotic or other antimicrobial drugs are all invalid.2 matched groups of this test are tested disease chicken nature case fatality rate and are respectively 27.23% and 23.94%.Adopt 3 kinds of different sweet bile oral liquors by the drinking-water disease chicken of feeding, make the natural infection infectious bronchitis state of an illness very fast controlled, 1 group of cure rate of sweet bile oral liquor is 97.08% and 96.94%, and 2 groups of cure rates of sweet bile oral liquor reach 99.08% and 99.06%, 3 groups of cure rates of sweet bile oral liquor are 98.92% and 98.82%.Show that sweet bile oral liquor is the active drug of treatment infectious bronchitis of chicken.

4 conclusions

4.1 according to Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection mutation analysis and lab testing, making a definite diagnosis 2 batches of breathings is that infectious disease chicken trouble is infectious bronchitis of chicken.

4.23 plant different sweet bile oral liquors by the drinking-water disease chicken of feeding, can effectively treat the natural infection infectious bronchitis, its cure rate is all more than 96.94%.

Embodiment 5 sweet bile oral liquors are to the therapeutic effect of infectious laryngotracheitis of chicken

A kind of respiratory infectious disease takes place suddenly in the chicken group in two chicken farms, Kuancheng District Lan Jia town, Changchun, Jilin Province, and involves full group rapidly.Its basic Clinical symptoms is the secretions that dyspnea, cough and expectoration contain blood, and larynx and tracheal mucosa swelling are hemorrhage, the formation erosion that has.Through Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection to be changed, and pathogen separation is identified, is diagnosed as infectious laryngotracheitis of chicken.The sweet bile oral liquor of Chinese medicine preparation that adopts Beijing Centre Biology Co., Ltd. to provide is added in the drinking-water throws something and feeds, and has obtained excellent curative.

1 materials and methods

1.1 material

1.1.1 the sweet bile oral liquor 4,5,6 of the present invention: provide by Beijing Centre Biology Co., Ltd.; Composition: Radix Isatidis, Radix Glycyrrhizae etc.; Specification: 100mL/ bottle.

1.1.2 other medicines: other conventional medicines such as penicillin, streptomycin are available from Jilin Province pharmaceuticals.

1.1.3 diagnostic reagent: ILT standard agp antigen and ILT standard positive serum, negative serum are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture.

1.1.4 experimental animal: select 4361 of 2 batches of infectious laryngotracheitis chickens of natural infection morbidity, wherein 5 age in week 3679 of AA broiler, 682 of the blue brown laying hens in 28 seas in ages in week.The SPF Embryo Gallus domesticus is provided by Liaoning Yi Kang biological factory.

1.1.5 feedstuff and raising: the complete feed that uses antibiotic-free to add, provide by institute of animal husbandry and veterinary medicine of Jilin University experiment pasture, test chicken is all raised at the standard hen house, free choice feeding and drinking-water.

1.2 method

1.2.1 set up diagnosis

According to Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection to be changed, and methods such as pathogen separation evaluation are set up diagnosis.

1.2.1.3 pathogen separation and evaluation

Aseptic disease chicken larynx and the tracheal tissue of taking shreds, made 20% homogenate suspension with grinder by 1: 4 behind the adding normal saline,, get supernatant and inoculate 10 age in days SPF chick chorioallantoic membranes again through the centrifugal 10min of 1000r/min, 0.2mL/ piece, postvaccinal Embryo Gallus domesticus is hatched at 37 ℃, observes Embryo Gallus domesticus every day 2 times, observes 5d continuously, discard Embryo Gallus domesticus dead in the 24h, dead Embryo Gallus domesticus in 24～120h, put 4 ℃ of coolings after, observe and have or not the pox speckle to form on the chick chorioallantoic membrane; The Embryo Gallus domesticus that 120h is still not dead also takes out, put 4 ℃ of coolings after, observe and have or not the pox speckle to form on the chick chorioallantoic membrane.Pox speckle person is arranged, take out chick chorioallantoic membrane and allantoic fluid, after the aseptic grinding, the centrifuging and taking supernatant after concentrating, as antigen to be checked, carries out virus with immune agar gel diffusion test and identifies.

Behind the conventional making agar plate, drawing antigen to be checked with micropipettor drips in periphery hole, add portion every a hole, central authorities add standard positive serum, and all the other holes add standard male antigen, and every Kong Jun does not overflow degree of being to fill it up with, after application of sample finishes, leave standstill 10min, put into 37 ℃ of wet boxes with cover and react, respectively 12,24,36h observes and the record result.

Decision method: agar plate is put dark background or strong illumination observation down.Occur a precipitation line clearly between standard positive serum hole and the standard positive antigen hole, then test is set up.

Criterion: form precipitation line clearly between sample well to be checked and the medium pore, and with the precipitation line in standard positive serum hole fusion person mutually, positive; Do not have precipitation line between antigen to be checked hole and the standard positive serum, be judged to feminine gender.

1.2.2 experimental animal grouping and processing

4361 of the infectious laryngotracheitis chickens of 2 batches of natural occurrences are divided into 5 groups separately at random by feed lot, and testing 1 group is matched group, does not process; Test 2 groups and be the sweet bile oral liquor 4 treatment groups of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Test 3 groups and be the sweet bile oral liquor 5 treatment groups of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Test 4 groups and be the sweet bile oral liquor 6 treatment groups of the present invention, convert water 1.5kg with the sweet bile oral liquor of 1mL; Testing 5 groups is enrofloxacin treatment group, converts water 1.0kg with the 75mg enrofloxacin, and the free drink-service of chicken, successive administration 5d are suffered from order.

1.2.3 observation index

Write down the sick chicken of each test group clinical symptoms, pathological change, the number of dying of illness, cure number, the course of treatment, growth promoter and the laying hen situation etc. of laying eggs, calculate case fatality rate, cure rate, body weight gain rate and laying rate of laying hen etc., the difference between relatively each is organized with statistical method.

2 results

2.1 sick chicken diagnostic result

According to Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection to be changed, and pathogen separation identifies that prove 2 batches of (4361) sick chickens, the disease that takes a disease is an infectious laryngotracheitis of chicken.

Main clinic symptoms:

Chicken group sudden onset, and involve full group rapidly.Sick chicken essence god is depressed, sheds tears, and normally closed order is slow-witted upright, the minimizing of laying eggs, and anorexia flows clear rare nose liquid, head cyanosis afterwards, dyspnea, severe patient height dyspnea, the peak with heading of dehiscing is breathed, and sends the sound of stridulating during breathing; The part pertussis is coughed frequently, even spasmodic cough, the mucus of the normal expectoration band blood streak.

Pathology cuts open inspection to be changed:

Most cases are seen has catarrh rhinitis and serosity conjunctivitis to change.Sick chicken nasal cavity, oral cavity, larynx are full of the mucus of band blood, larynx severe haemorrhage, edema, the generation mucosa degeneration that has, downright bad, be coated with yellow cheesy thing, the hyperemia of trachea front end mucosa height, hemorrhage, slightly thicken, kermesinus clot and yellow exudate grumeleuse are arranged in the trachea.Alluvial has volume band bleeding blister foam sample mucus in the pulmonary venous pleonaemia, alveolar.

Pathogen separation and evaluation:

Sick chicken larynx and the homogenate supernatant inoculation SPF of tracheal tissue Embryo Gallus domesticus have typical pox speckle to form on its allantocherion; The immunity agar gel diffusion test positive shows that 2 batches of sick chicken institute pathogenic infections are avian infectious laryngotracheitis virus.

2.2 sweet bile oral liquor is to the therapeutic effect of AA broiler infectious laryngotracheitis

Make the state of an illness very fast controlled after taking sweet bile oral liquor, case fatality rate significantly is lower than matched group.Behind the 12h, the state of an illness is tending towards alleviating gradually, and spirit is faded in vivaciously, movable increasing, and appetite takes a turn for the better; Dyspnea improves behind the 24h, and moist rales weakens with the sound of stridulating, and cough alleviates; Sick chicken stops death behind the 48h, and every symptom greatly alleviates; Sick chicken symptom disappears substantially behind the 72h, and it is normal that the diet desire is recovered.Statistical result shows that untreated matched group spontaneous recovery rate is 85.18%, though fatality rate is not high, glucose recovery is slow; The cure rate of 3 sweet bile oral liquor groups is all more than 97%, and case fatality rate extremely significantly is lower than matched group (P＜0.01), and glucose recovery is very fast; The results are shown in Table 1.

The sweet bile oral liquor of table 1 is to the therapeutic effect of AA broiler infectious laryngotracheitis

2.3 sweet bile oral liquor is to the therapeutic effect of the blue brown laying hen infectious laryngotracheitis in sea

Also obtained excellent curative with the blue brown laying hen infectious laryngotracheitis in Chinese medicine preparation sweet bile oral liquor treatment sea.After sick chicken was taken sweet bile oral liquor by drinking-water, the state of an illness was very fast controlled, and from beginning in the 2nd day, spirit was faded in vivaciously, movable increasing, appetite takes a turn for the better, and dyspnea improves, and moist rales weakens with the sound of stridulating, cough alleviates, and the 3rd day symptom disappears substantially after the medication, and it is normal that the diet desire is recovered.Statistical result shows, 3 different sweet bile oral liquor treatment groups and enrofloxacin treatment group case fatality rate extremely significantly are lower than matched group (P＜0.01) and the results are shown in Table 2.

The sweet bile oral liquor of table 2 is to the therapeutic effect of the blue brown laying hen infectious laryngotracheitis in sea

2.4 the variation of sick laying rate of chicken before and after the treatment

Chicken group premorbid laying rate 90.67%, the beginning in the 4th day of morbidity back sharply descends, and drops to about 60% when serious.Drinking-water is taken sweet bile oral liquor treatment back laying rate and is able to rapid recovery, promptly return to the preceding level of disease the 3rd week substantially, and matched group returns to 77.12% until the 5th Zhou Houcai, the results are shown in Table 3.

Respectively organize the disease laying rate of chicken after table 3 morbidity and change (%)

3 analyze

3.1 the effectiveness of sweet bile oral liquor treatment infectious laryngotracheitis of chicken

The infectious laryngotracheitis chicken of selected 2 batches of natural occurrences makes the state of an illness very fast controlled after taking sweet bile oral liquor, and case fatality rate significantly is lower than matched group.After the medication, behind the 12h, the state of an illness is tending towards alleviating gradually, and spirit is faded in vivaciously, movable increasing, and appetite takes a turn for the better; Dyspnea improves behind the 24h, and moist rales weakens with the sound of stridulating, and cough alleviates; Sick chicken stops death behind the 48h, and every symptom greatly alleviates; Sick chicken symptom disappears substantially behind the 72h, and it is normal that the diet desire is recovered.The infectious laryngotracheitis of chicken that shows sweet bile oral liquor treatment natural infection morbidity is effective.

The constituent of sweet bile oral liquor is Radix Isatidis, Radix Glycyrrhizae etc., nearly all Chinese medicine all the experiment proved that and has antivirus action in the prescription, various bacteria, mycoplasma and other cause of diseases also there is positive effect, especially Radix Isatidis, be the antiviral key medicine of veterinary clinic, after composition of prescription and refining processing, its effect is more remarkable.Infectious laryngotracheitis of chicken is a kind of acute contact upper respiratory infectious disease that is caused by avian infectious laryngotracheitis virus, we are directly at cause of disease, and design and produce according to characteristics biology of chicken, has heat-clearing and toxic substances removing on the whole, relieving cough and asthma, the expectorant dryness-transformation is wet, and lung qi dispersing sore-throat relieving effect is so received good curative effect in the infectious laryngotracheitis of chicken of treatment natural infection morbidity.

3.2 sweet bile oral liquor is to recovering the effect of disease chicken egg laying performance

Infectious laryngotracheitis of chicken is serious to the production performance of layer chicken influence, generally can make laying rate descend 12%～62%, after the sweet bile oral liquor treatment of sick chicken drink-service, laying rate is able to rapid recovery, promptly returns to the preceding level of disease the 3rd week substantially, exceeds matched group about 17%, show that sweet bile oral liquor can not only effectively treat infectious laryngotracheitis of chicken, respiratory disease is rehabilitated, and can recover egg laying performance rapidly, effectively reduce the economic loss that disease is brought.

4 conclusions

4.1 according to Epidemiological study, clinical symptoms differentiates that pathology cuts open inspection to be changed, and pathogen separation is identified, make a definite diagnosis the secretions that 2 batches of dyspnea, cough and expectorations contain blood, larynx and tracheal mucosa swelling, hemorrhage or rotten to the corn sick chicken by Clinical symptoms are taken a disease disease for infectious laryngotracheitis of chicken.

4.23 plant different sweet bile oral liquors through the drinking-water administration, can effectively treat the infectious laryngotracheitis of chicken of natural infection morbidity.

4.3 after the sweet bile oral liquor treatment of infectious laryngotracheitis chicken drink-service, can recover egg laying performance rapidly, can make laying rate of laying hen in 3 weeks, return to the preceding level of disease substantially.

Embodiment 6 sweet bile oral liquor acute toxicity test researchs

1 materials and methods

1.1 material

Bile oral liquor is provided by Beijing Centre Biology Co., Ltd. 1.1.1 trial drug the present invention is sweet.Chinese drug-treated group becomes Radix Isatidis, Radix Glycyrrhizae etc.; Packing specification is the 100ml/ bottle.

1.1.2 50 of the healthy Kunming white mouses of experimental animal, body weight 18～22g, male and female half and half are purchased the Experimental Animal Center in military supplies university of PLA.

1.2 method

Mice is divided into 5 groups at random, and 10 every group, male and female half and half gavage administration according to 27.90g/kg, 22.88g/kg, 18.75g/kg, 15.37g/kg, 12.60g/kg (the geometric ratio coefficient is 1.22) per os respectively.The ergasia that continuous 7d observes white mice respectively after the administration, diet desire and death condition.Dead white mice is cutd open inspection, the pathological change of each histoorgan of perusal.Calculate LD with improving karber's method ₅₀

2 result of the tests

After taking, it is uneasy that white mice shows slightly, and then quiet gradually, the diet desire reduces.Behind the 10h, the white mice of high dose group is prostrate motionless, occurs dead in second day.White mice is cutd open inspection, find that liver has the black speckle, little intestinal segment is hemorrhage, and red thick content is arranged, and the results are shown in Table 1.Calculate LD with the improvement karber's method ₅₀Be 9.02527g/kg, 95% the credible 7.6394～12.9735g/kg that is limited to.

The The acute toxicity tests of the sweet bile oral liquor of table 1

3 conclusions

3.1 acute toxicity test shows that its mouse oral chamber gavages the LD of sweet bile oral liquor ₅₀Be 9025.27mg/kg, LD ₅₀95% the credible 7639.4～12973.5mg/kg that is limited to, considerably beyond clinical using dosage (0.15g/kg).So clinical using dosage is safe.

3.2 new veterinary drug general toxicity test specification regulation, LD ₅₀＞5000mg/kg person is actual nontoxic, is low toxicity at 500～5000mg/kg.According to the rules, sweet bile oral liquor belongs to innocuous substance.

Embodiment 7 sweet bile oral liquor long term toxicity test researchs

1 materials and methods

1.1 material

1.1.1 trial drug: the sweet bile oral liquor of the present invention, provide by Beijing Centre Biology Co., Ltd., Chinese drug-treated group becomes Radix Isatidis, Radix Glycyrrhizae etc.; Packing specification is the 100ml/ bottle.

1.1.2 experimental animal: healthy Wistar is 80 of rats, male and female half and half, and average weight 90.42 ± 10.21g is provided by military supplies university of PLA experimental animal center.The animal sub-cage rearing is in cleaning on the cage automatic water-supply, pellet nursing.

1.2 test method

Getting Wistar is 80 of rats, and male and female half and half are divided into 4 groups at random by the sex body weight, 20 every group, and respectively as the administration group and the blank group of the high, medium and low three kinds of dosage of sweet bile oral liquor.Be respectively LD by high, medium and low three dosage groups (1805.05mg/kg, 902.53mg/kg, 601.68mg/kg) ₅₀1/5,1/10,1/15 administration.In every morning animal gastric infusion 1 time on an empty stomach, successive administration 30 days.The outward appearance, behavior, appetite, two of observing each treated animal during the administration just, situations such as body weight, death.Weigh in weekly 1 time.24h gets 14 rats for every group after the drug withdrawal, gets blood from left iliac vein.Check blood parameters with the test kit method.Cut open the liver spleen thymus kidney of coring and weigh, be converted into organ index by the contained organ weights of every 100g body weight.Internal organs are done perusal and are done optical microscope undertissue and learn inspection, observe to have or not pathological change, and medication group testing result and blank group are compared, and do the t check.Handle with method remaining two week of the animal drug withdrawal back of each group.

2 result of the tests

2.1 influence to the rat ordinary circumstance

In 30 days of successive administration, to each administration group and matched group comparative observation, the situation of the activity of animal, diet, stool is similar, and the situation that shows no obvious abnormalities takes place.

2.2 influence to the growth of Wistar rat body weight

Experimental result sees Table 1.During administration, it is normal that each measures the treated animal body weight gain, compares there was no significant difference with matched group.Experimental result shows that sweet bile oral liquor is heavy dose of, and the long period medication is to the growth promoter avirulence influence of rat.

The influence that the sweet bile oral liquor of table 1 increases the Wistar rat body weight (g,

N=20)

2.3 influence to blood and biochemical indicator

14 animals are respectively got in administration after 30 days, measure following index: erythrocyte (RBC), hemoglobin (Hb), leukocyte (WBC), serum globulin (Glob), albumin (ALB), flesh liver (Cr), glutamate pyruvate transaminase (ALT), blood urea nitrogen (BUN), platelet count (PLT).The results are shown in Table 2, administration group and matched group compare, and all difference is not remarkable for hepatic and renal function, red blood cell count(RBC), numeration of leukocyte etc.

The sweet bile oral liquor of table 2 is to the result that influences of blood and biochemical indicator

2.4 influence to organ index

Each organize the rat medication phase and convalescent period body weight, organ weights difference not remarkable, its organ coefficient (g/100g body weight) does not have significant difference yet.See Table 3.

The sweet bile oral liquor of table 3 is to influence (the g/100g body weight of organ index

2.5 dissect and pathological examination

After putting to death rat, core, liver, spleen, lung, kidney, thymus, adrenal gland etc. carry out the tectology inspection, the above equal color and luster of each internal organs of medicine group and control rats is normal, quality softness, abnormal phenomenas such as surface nothing necrosis, sclerosis, enlargement, atrophy; Get above-mentioned organs and tissues and fix, section, H.E dyeing back histological examination there is no solid lesion and pathological change.

3 conclusions

The result shows through the rat long term toxicity test, with 1805.05mg/kg, 902.53mg/kg, three kinds of dosage of 601.68mg/kg, once a day, continuous 30 days rat gastric infusions, compare with the blank group, each dosed administration all finds no the overt toxicity effect to general state, growth promoter, hemopoietic function, liver function, renal function and the vitals weight coefficient of rat; And prove that through pathological examination organ-tissues such as the heart of high dose group and blank group comparative observation rat, liver, spleen, lung, kidney, thymus, adrenal gland all find no tangible toxic damages to be changed.So think heavy dose of and take sweet bile oral liquor toxicity the longer course of treatment very low, so sweet bile oral liquor clinical application is safe.

Claims

1. sweet bile oral liquor that cures mainly the cough due to lung-heat that infectious bronchitis of chicken and mycoplasma gallisepticam infection cause, its raw material is formed and is comprised:

Radix Isatidis 50～180g

Artificial Calculus Bovis 0～60g

Radix Glycyrrhizae 30～60g

Borneolum Syntheticum 0～30g

Pulvis Fellis Suis 20～40g

Matrii Sulfas Exsiccatus 0～40g

2. according to the described sweet bile oral liquor of claim 1, it is characterized in that preparation method is:

Radix Isatidis, Radix Glycyrrhizae, Matrii Sulfas Exsiccatus are decocted with water 2～3 times, and collecting decoction filters, and filtrate concentrates at 30～60 ℃, is concentrated into relative density and is 1.10～1.35 extractum, puts coldly, and it is an amount of to add ethanol, leaves standstill, filter, and filtrate recycling ethanol, it is an amount of to add water, leaves standstill; An amount of dissolve with ethanol of Pulvis Fellis Suis, artificial Calculus Bovis, Borneolum Syntheticum adds said extracted liquid, adds water to 500～1800ml, shakes up, and leaves standstill, and filters, promptly.

3. sweet bile oral liquor according to claim 1 is characterized in that raw material also adds Venenum Bufonis, Indigo Naturalis.

4. sweet bile oral liquor according to claim 1 is characterized in that the Pulvis Fellis Suis in the raw material can also be gallbladder powder or the gallbladder cream that derives from cattle, sheep, chicken, duck, goose.

5. sweet bile oral liquor according to claim 1 is characterized in that quality determining method is:

[inspection] amount of alcohol should be 10%～20%;

Relative density should be not less than 1.01;

[assay] is according to high effective liquid chromatography for measuring;

The every 1ml of this product contains Radix Glycyrrhizae with glycyrrhizic acid C ₄₂H ₆₂O ₁₆Meter must not lack 2.0mg.

6. state sweet bile oral liquor according to claim 1～5 is arbitrary, it is characterized in that it also can be prepared into ordinary tablet, dispersible tablet, effervescent tablet, oral liquid, granule, powder, powder, electuary, spray, injection.

7. state sweet bile oral liquor according to claim 1～6 is arbitrary, it is characterized in that having heat-clearing and toxic substances removing, removing heat from blood lung qi dispersing, drying dampness to eliminate phlegm, the function of relieving cough and asthma; Have effects such as antiviral, antibiotic, antitoxin, antiinflammatory, enhancing immunity and anti-stress; Cure mainly acute and chronic respiratory system diseases such as infectious bronchitis of chicken, infectious laryngotracheitis, mycoplasma gallisepticam infection, can eliminate cough and asthma, mouth breathing, head cyanosis rapidly, get rid of head, respiratory symptoms such as snore stridulates, throat and tracheorrhagia swelling.