CN102058828A

CN102058828A - Medicinal composition and detection method for preparation thereof

Info

Publication number: CN102058828A
Application number: CN2009102375631A
Authority: CN
Inventors: 刘丽颖; 郑松涛
Original assignee: Individual
Current assignee: Harbin Pugongying Pharmaceutical Co Ltd
Priority date: 2009-11-12
Filing date: 2009-11-12
Publication date: 2011-05-18
Anticipated expiration: 2029-11-12
Also published as: CN102058828B

Abstract

The invention discloses Chinese medicinal tranquilizing pills and a detection method for a preparation which is prepared from raw materials of a formula of the pills. The detection method comprises an authentication method and/or an assay method, wherein the authentication method is one or more of methods for authenticating cholic acid, hyodeoxycholic acid, golden thread, geniposide and baicalin; and the assay method is one or more of methods for assaying geniposide, baicalin, golden thread, menthol crystal and borneol. The detection method has good linear relationship, precision, stability, repeatability and recovery rate, and has great significance to strict control over quality of the tranquilizing pills and the preparation thereof.

Description

The detection method of a kind of pharmaceutical composition and preparation thereof

Technical field

The present invention relates to a kind of detection method of pharmaceutical composition, particularly the detection method of Annaowan bolus and preparation thereof.

Background technology

Annaowan bolus is the prescriptions of Chinese medicine that is recorded in the 13 77 pages in the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation, standard is numbered WS3-B-2517-97, and the prescription of having put down in writing Annaowan bolus in the standard comprises: artificial Calculus Bovis, Pulvis Fellis Suis, Cinnabaris, Borneolum Syntheticum, Pulvis Cornus Bubali Concentratus, Margarita, Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Realgar, Radix Curcumae, Gypsum Fibrosum, Haematitum, Concha Margaritifera and Mentholum; The patent No. is the prescription consumption proportion relation that 93100258.3 patent applications disclose Annaowan bolus, and conventional preparation technology; The open Annaowan bolus of available data has heat-clearing and toxic substances removing, refreshment and tranquilization, eliminates phlegm for resuscitation, the relieving convulsion effect that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., and nowadays is hyperpyrexia, the delirious clinical application that a kind of treatment acute inflammation commonly used causes.

The method of quality control of having put down in writing Annaowan bolus in the ministry standard is the discrimination method of berberine hydrochloride in the discrimination method of cholic acid, Hyodeoxycholic Acid and the Rhizoma Coptidis.Above-mentioned two kinds of qualitative checking methods can not guarantee the quality of this prescription products comprehensively, lack quantitative detection method or at this prescribing method one cover quality determining method system, could guarantee the quality of this prescription products comprehensively.And contain toxic raw material in this prescription, and as Realgar, Cinnabaris etc., wherein the content of toxic component must have certain limit, and this also will be the key point of this prescription products quality determining method, otherwise with entail dangers to patient's life.Therefore, research one cover will be this prescription problem demanding prompt solution at the quality determining method system of Annaowan bolus and other preparations thereof.

Summary of the invention

The object of the invention is the detection method of the preparation that open Chinese medicine Annaowan bolus and formula material thereof are made.

The present invention seeks to be achieved through the following technical solutions:

Detection method of the present invention comprises one or more in following discriminating and the assay:

Discriminating comprises one or more in the following method:

A. the discriminating of cholic acid, Hyodeoxycholic Acid

The preparation of need testing solution: get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, pulverize, add kieselguhr 0.5-1.5g, grind well, add ethanol 10-30ml, reflux 0.5-1.5 hour, filter, filtrate is as need testing solution;

The preparation of reference substance solution: other gets cholic acid, Hyodeoxycholic Acid reference substance, adds ethanol and makes the mixed solution that every 1ml contains 0.5-1.5mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with isobutyltrimethylmethane .-ethyl acetate-glacial acetic acid is that 10-20: 5-10: 2-8 is developing solvent, launch, take out, dry, spray is with 8-12% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 8-12ml sulphuric acid and 100ml), it is clear to be heated to speckle colour developing at 100-110 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B. the discriminating of Rhizoma Coptidis

The preparation of need testing solution: get the 1/3-2/3 of the daily amount of formulation of preparation of the present invention, pulverize, add methanol 5-15ml, put in the water-bath heating backheat 0.5-1.5 hour, put coldly, filter, filtrate is as for having a try product solution;

The preparation of control medicinal material solution: other gets Rhizoma Coptidis control medicinal material 30-70mg, adds methanol 3-7ml, and supersound process 10-20 minute, filter, filtrate is medical material solution in contrast;

The preparation of reference substance solution: get the berberine hydrochloride reference substance, add methanol and make the solution that every 1ml contains 0.2-0.7mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water is that 8-12: 5-10: 0.5-1.5: 0.5-1.5 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show identical yellow fluorescence speckle;

C. the discriminating of jasminoidin

The preparation of need testing solution: get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, pulverize, add kieselguhr 0.5-1.5g, grind well the 10-30ml that adds diethyl ether, supersound process 3-7 minute, filter, discard ether solution, residue is flung to ether, add ethyl acetate 20-40ml, reflux 0.5-1.5 hour, put cold, filter, filtrate evaporate to dryness, residue add methanol 2-4ml makes dissolving, as need testing solution;

The preparation of reference substance solution: other gets the jasminoidin reference substance, adds methanol and makes the solution that every 1ml contains 0.5-1.5mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 4 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-acetone-formic acid-water is that 8-12: 5-10: 1-3: 0.3-0.7 is developing solvent, launch, take out, dry, spray is with 8-12% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 8-12ml sulphuric acid and 100ml), and it is clear to be heated to the speckle colour developing at 100-110 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

D. the discriminating of baicalin

The preparation of need testing solution: get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, pulverize, add methanol 5-15ml, supersound process 10-30 minute, filter, filtrate is as need testing solution;

The preparation of reference substance solution: other gets the baicalin reference substance, adds methanol and makes the solution that every 1ml contains 0.1-0.5mg, in contrast product solution;

According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005) test, draw each 1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, be developing solvent with acetic acid, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical dark spots;

Assay comprises one or more in the following method:

A. the assay of jasminoidin:

Measuring according to high performance liquid chromatography (appendix VI D), is filler with octadecylsilane chemically bonded silica; With the acetonitrile-water is 10-15: 85-90 or n-butyl alcohol-methanol--water-phosphoric acid is that 10-15: 30-50: 30-40: 0.3-0.5 or n-butanol-water-phosphoric acid are 50--70: 30-40: 0.3-0.5 is as mobile phase; The detection wavelength is 238nm; Number of theoretical plate calculates by the jasminoidin peak should be not less than 1500;

The preparation of reference substance solution: precision takes by weighing the jasminoidin reference substance, adds methanol and makes the solution that every 1ml contains 20-40 μ g, promptly;

The preparation of need testing solution: the 1/6-1/2 that gets the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, adding 20-60% ethanol or acetone-20-60% ethanol are 1: 1 mixed liquor 80-90ml, add 2-3% hydrochloric acid and transfer PH2-3, supersound extraction 10-15 minute, filter; Filtrate is put in the 100ml measuring bottle, and hydro-oxidation sodium is transferred PH=7, filters, and filtrate adds ethanol to scale, shakes up, promptly;

Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; Contain jasminoidin (C with amount of formulation every day ₁₇H ₂₄O ₁₀) 0.6-6mg maybe must not be less than 3.6mg;

B. content of baicalin is measured:

Measuring according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005), is filler with the octadecylsilane chemically bonded silica; With the acetonitrile-water is 10-15: 85-90 or n-butyl alcohol-methanol--water-phosphoric acid is that 10-15: 30-50: 30-40: 0.3-0.5 or n-butanol-water-phosphoric acid are 50--70: 30-40: 0.3-0.5 or methanol-water-phosphoric acid=40-50: 50-60: 0.1-0.3 is as mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500;

The preparation of reference substance solution: precision takes by weighing the baicalin reference substance, adds methanol and makes the solution that every 1ml contains 40-80 μ g, promptly;

The preparation of need testing solution: get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, add 80-90% ethanol 80-90ml, transfer PH=7, supersound extraction 10-15 minute, filter; Filtrate is put in the 100ml measuring bottle, adds 2-3% hydrochloric acid 3-5ml, stirs evenly, and adds sodium hydroxide again and transfers PH=7, leaves standstill 20-30 minute, filters; Filtrate adds 80-90% ethanol to scale, shakes up, promptly;

Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly; This product is pressed dry product and is calculated, and clean Radix Scutellariae, Radix Scutellariae (processed with wine) contain baicalin (C ₂₁H ₁₈O ₁₁) must not be less than 8.0%; Contain baicalin with amount of formulation and must not be less than 0.6-6mg and maybe must not be less than 3.6mg every day of the present invention;

C. the assay of Rhizoma Coptidis:

The preparation of need testing solution: get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, add 1-3% hydrochloric acid 60-80ml, supersound extraction 10-15 minute, filter; Filtrate adds 1-2% ethanol solution hydrochloride (that is: the alcoholic acid mixed liquor of 1-2ml hydrochloric acid and 100ml) to scale, and heating is 10-20 minute in 40-50 ℃ of following water-bath, filters; Filtrate is as need testing solution;

The preparation of reference substance solution: get the berberine hydrochloride reference substance, add methanol and make the solution that every 1ml contains 0.02-0.06mg, in contrast product solution;

Test according to thin layer chromatography (appendix VI B), draw need testing solution 1 μ l, reference substance solution 1 μ l and 3 μ l, the cross point is on same silica gel g thin-layer plate, with benzene-ethyl acetate-isopropyl alcohol-methanol-water is 5-7: 1-3: 1-2: 1-2: 1 or n-butyl alcohol-glacial acetic acid-methanol-water be that 5-7: 0.8-1: 1-1.5: 0.5-1.5 is developing solvent, groove adds isopyknic strong ammonia solution in addition, launch 8-15 centimetre, take out, volatilize, carry out fluorescent scanning according to thin layer chromatography (appendix VIB thin layer chromatography scanning), wavelength X=356-366nm, the integrated value of measurement test sample and reference substance fluorescence intensity, calculate, promptly; Contain berberine with berberine hydrochloride (C with amount of formulation every day ₂₀H ₁₇NO ₄HCl) meter must not be less than 0.8mg-8mg and maybe must not be less than 3.2mg;

D. the assay of Mentholum and Borneolum Syntheticum

Measure according to gas chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 E); Chromatographic condition and system suitability test chromatographic column: HP-INNOWax capillary chromatographic column (30m*0.32mm*0.25 μ m); 240 ℃ of detector temperatures; 240 ℃ of vaporizer temperature; Column temperature is temperature programming, and initial temperature is 150 ℃, keeps 4 minutes, is warming up to 200 ℃ with the speed of 8 ℃ of per minutes; Number of theoretical plate calculates by the eugenol peak should be not less than 5000;

The preparation of inner mark solution: get eugenol, add ethyl acetate and make the solution that every 1ml contains 0.3-0.7mg, shake up, as inner mark solution;

Correction factor is measured: other gets Borneolum Syntheticum reference substance 10-30mg, Mentholum reference substance 5-15mg, and accurate the title, decide, and puts in the 25ml measuring bottle, adds the inner mark solution dissolving and be diluted to scale, shakes up, and draws 1 μ l, inject gas chromatograph, the calculation correction factor;

Get the 1/6-1/2 of the daily amount of formulation of preparation of the present invention, pulverize, the accurate title, decide, and adds kieselguhr 2-4g, the accurate title, decide, and after grinding well, gets 1-3g, and accurate title is fixed, put in the 25ml measuring bottle, accurate inner mark solution 10-20ml and the water 0.5-1.5ml of adding, close plug shakes up, claim to decide weight, dipping spends the night, and claims to decide weight again, supplies the weight that subtracts mistake with ethyl acetate, shake up, filter, draw 1 μ l, inject gas chromatograph is measured, promptly; Contain Borneolum Syntheticum (C with amount of formulation every day ₁₀H ₁₈O) must not be less than 8-12mg; Contain Mentholum (C ₁₀H ₂₀O) must not be less than 6.4-9.6mg.

Wherein, discriminating preferably includes one or more in the following method:

A. the discriminating of cholic acid, Hyodeoxycholic Acid

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, pulverize, add kieselguhr 1g, grind well, add ethanol 20ml, reflux 1 hour filters, and filtrate is as need testing solution;

The preparation of reference substance solution: other gets cholic acid, Hyodeoxycholic Acid reference substance, adds ethanol and makes the mixed solution that every 1ml contains 1mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with isobutyltrimethylmethane .-ethyl acetate-glacial acetic acid be 15: 7: 5 be developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 10ml sulphuric acid and 100ml), and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B. the discriminating of Rhizoma Coptidis

The preparation of need testing solution: get 1/2 of the daily amount of formulation of preparation of the present invention, pulverize, add methanol 10ml, put in the water-bath heating backheat 1 hour, put coldly, filter, filtrate is as for having a try product solution;

The preparation of control medicinal material solution: other gets Rhizoma Coptidis control medicinal material 50mg, adds methanol 5ml, and supersound process 15 minutes filters, and filtrate is medical material solution in contrast;

The preparation of reference substance solution: get the berberine hydrochloride reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water is 10: 7: 1: 1 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show identical yellow fluorescence speckle;

C. the discriminating of jasminoidin

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, pulverize, add kieselguhr 1g, grind well the 20ml that adds diethyl ether, supersound process 5 minutes, filter, discard ether solution, residue is flung to ether, add ethyl acetate 30ml, reflux 1 hour is put cold, filter, filtrate evaporate to dryness, residue add methanol 3ml makes dissolving, as need testing solution;

The preparation of reference substance solution: other gets the jasminoidin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution;

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 4 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-acetone-formic acid-water is 10: 7: 2: 0.5 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 10ml sulphuric acid and 100ml), 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 chromatograph on, show the speckle of same color;

D. the discriminating of baicalin

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, pulverize, add methanol 10ml, supersound process 20 minutes filters, and filtrate is as need testing solution;

The preparation of reference substance solution: other gets the baicalin reference substance, adds methanol and makes the solution that every 1ml contains 0.3mg, in contrast product solution;

According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005) test, draw each 1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, be developing solvent with acetic acid, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical dark spots.

Wherein, assay preferably includes one or more in the following method:

A. the assay of jasminoidin:

Measuring according to high performance liquid chromatography (appendix VI D), is filler with octadecylsilane chemically bonded silica; With acetonitrile-water is 12: 88 or n-butyl alcohol-methanol--water-phosphoric acid is 12: 32: 35: 0.4 or n-butanol-water-phosphoric acid be 60: 35: 0.4 as mobile phase; The detection wavelength is 238nm; Number of theoretical plate calculates by the jasminoidin peak should be not less than 1500;

The preparation of reference substance solution: precision takes by weighing the jasminoidin reference substance, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly;

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, add 40% ethanol or acetone-40% ethanol and be 1: 1 mixed liquor 85ml, add 3% hydrochloric acid and transfer PH2-3, supersound extraction 12 minutes filters; Filtrate is put in the 100ml measuring bottle, and hydro-oxidation sodium is transferred PH=7, filters, and filtrate adds ethanol to scale, shakes up, promptly;

Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; Contain jasminoidin (C with amount of formulation every day ₁₇H ₂₄O ₁₀) must not be less than 0.6-6mg and maybe must not be less than 3.6mg;

B. content of baicalin is measured:

Measuring according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), is filler with the octadecylsilane chemically bonded silica; With acetonitrile-water is 12: 88 or n-butyl alcohol-methanol--water-phosphoric acid is 12: 40: 35: 0.4 or n-butanol-water-phosphoric acid be that 60: 35: 0.4 or methanol-water-phosphoric acid=45: 55: 0.2 are as mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500;

The preparation of reference substance solution: precision takes by weighing the baicalin reference substance, adds methanol and makes the solution that every 1ml contains 60 μ g, promptly;

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, add 85% ethanol 85ml, transfer PH=7, supersound extraction 12 minutes filters; Filtrate is put in the 100ml measuring bottle, adds 3% hydrochloric acid 4ml, stirs evenly, and adds sodium hydroxide again and transfers PH=7, leaves standstill 25 minutes, filters; Filtrate adds 85% ethanol to scale, shakes up, promptly;

C. the assay of Rhizoma Coptidis:

The preparation of need testing solution: get 1/4 of the daily amount of formulation of preparation of the present invention, put in the 100ml measuring bottle, add 2% hydrochloric acid 70ml, supersound extraction 12 minutes filters; Filtrate adds 1-2% ethanol solution hydrochloride (that is: the alcoholic acid mixed liquor of 1-2ml hydrochloric acid and 100ml) to scale, and heating is 15 minutes in 45 ℃ of following water-baths, filters; Filtrate is as need testing solution;

The preparation of reference substance solution: get the berberine hydrochloride reference substance, add methanol and make the solution that every 1ml contains 0.04mg, in contrast product solution;

Test according to thin layer chromatography (appendix VI B), draw need testing solution 1 μ l, reference substance solution 1 μ l and 3 μ l, the cross point is on same silica gel g thin-layer plate, with benzene-ethyl acetate-isopropyl alcohol-methanol-water is 6: 2: 1.5: 1.5: 1 or n-butyl alcohol-glacial acetic acid-methanol-water are 6: 0.9: 1.2: 1 is developing solvent, groove adds isopyknic strong ammonia solution in addition, launch 12 centimetres, take out, volatilize, carry out fluorescent scanning according to thin layer chromatography (appendix VI B thin layer chromatography scanning), wavelength X=356-366nm, the integrated value of measurement test sample and reference substance fluorescence intensity, calculate, promptly; Contain berberine with berberine hydrochloride (C with amount of formulation every day ₂₀H ₁₇NO ₄HCl) meter must not be less than 0.8mg---8mg and maybe must not be less than 3.2mg;

D. the assay of Mentholum and Borneolum Syntheticum

The preparation of inner mark solution: get eugenol, add ethyl acetate and make the solution that every 1ml contains 0.5mg, shake up, as inner mark solution;

Correction factor is measured: other gets Borneolum Syntheticum reference substance 20mg, Mentholum reference substance 10mg, and accurate the title, decide, and puts in the 25ml measuring bottle, adds the inner mark solution dissolving and be diluted to scale, shakes up, and draws 1 μ l, inject gas chromatograph, the calculation correction factor;

Get 1/4 of the daily amount of formulation of preparation of the present invention, pulverize, the accurate title, decide, and adds kieselguhr 3g, the accurate title, decide, and after grinding well, gets 2g, and accurate title is fixed, put in the 25ml measuring bottle, accurate inner mark solution 15ml and the water 1ml of adding, close plug shakes up, claim to decide weight, dipping spends the night, and claims to decide weight again, supplies the weight that subtracts mistake with ethyl acetate, shake up, filter, draw 1 μ l, inject gas chromatograph is measured, promptly; Contain Borneolum Syntheticum (C with amount of formulation every day ₁₀H ₁₈O) must not be less than 8-12mg; Contain Mentholum (C ₁₀H ₂₀O) must not be less than 6.4-9.6mg.

The crude drug of Annaowan bolus pharmaceutical formulation of the present invention consists of:

Artificial Calculus Bovis 0.5-2 weight portion Pulvis Fellis Suis 7-20 weight portion Rhizoma Coptidis 5-15 weight portion

Radix Scutellariae 5-15 weight portion Margarita 1-5 weight portion Fructus Gardeniae 5-15 weight portion

Radix Curcumae 5-15 weight portion Haematitum 2-6 weight portion Realgar 3-9 weight portion

Cinnabaris 2-6 weight portion Mentholum 0.5-2 weight portion Gypsum Fibrosum 5-12 weight portion

Borneolum Syntheticum 0.5-4 weight portion Pulvis Cornus Bubali Concentratus 7-20 weight portion

Concha Margaritifera 2-8 weight portion.

The crude drug composition of Annaowan bolus pharmaceutical formulation of the present invention is preferably:

Artificial Calculus Bovis's 1 weight portion Pulvis Fellis Suis 13 weight portion Rhizoma Coptidis 10 weight portions

Radix Scutellariae 10 weight portion Margaritas 3 weight portion Fructus Gardeniaes 10 weight portions

Radix Curcumae 10 weight portion Haematitums 4 weight portion Realgars 6 weight portions

Cinnabaris 4 weight portion Mentholums 1 weight portion Gypsum Fibrosum 8 weight portions

Borneolum Syntheticum 2 weight portion Pulvis Cornus Bubali Concentratuss 13 weight portion Concha Margaritiferas 5 weight portions.

Artificial Calculus Bovis's 0.7 weight portion Pulvis Fellis Suis 18 weight portion Rhizoma Coptidis 6 weight portions

Radix Scutellariae 14 weight portion Margaritas 2 weight portion Fructus Gardeniaes 14 weight portions

Radix Curcumae 6 weight portion Haematitums 5 weight portion Realgars 4 weight portions

Cinnabaris 5 weight portion Mentholums 0.7 weight portion Gypsum Fibrosum 11 weight portions

Borneolum Syntheticum 1 weight portion Pulvis Cornus Bubali Concentratus 18 weight portion Concha Margaritiferas 3 weight portions.

Artificial Calculus Bovis's 1.8 weight portion Pulvis Fellis Suiss 9 weight portion Rhizoma Coptidis 14 weight portions

Radix Scutellariae 6 weight portion Margaritas 4 weight portion Fructus Gardeniaes 6 weight portions

Radix Curcumae 14 weight portion Haematitums 3 weight portion Realgars 8 weight portions

Cinnabaris 3 weight portion Mentholums 1.8 weight portion Gypsum Fibrosum 6 weight portions

Borneolum Syntheticum 3.5 weight portion Pulvis Cornus Bubali Concentratuss 9 weight portion Concha Margaritiferas 7 weight portions.

Annaowan bolus pharmaceutical formulation in the quality determining method of the present invention is to get the above-mentioned raw materials medicine, add conventional adjuvant, according to common process, make the dosage form of clinical acceptance, include but not limited to powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, oral liquid or injection.

Annaowan bolus pharmaceutical formulation in the quality determining method of the present invention also can be prepared from by in the following method one or more:

Technology 1: form by following steps A, B, C, D and E

Steps A: preparation intermediate compound I

A1: crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added hot water reflux, extract, 2-4 time jointly, each 0.5-2.5 hour, merge decocting liquid, be concentrated into thick paste; Add the 30-80% ethanol of 4-10 times of weight in the thick paste, left standstill 12-48 hour, get supernatant and reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A2: crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added hot water reflux, extract, 2-4 time jointly, each 0.5-2.5 hour, merge decocting liquid, be concentrated into relative density 1-1.0～1.4, centrifugal, supernatant is crossed macroporous resin column, the 30-80% ethanol elution, reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A3: Radix Curcumae is added hot water reflux, extract, 2-4 time earlier, and each 0.5-2.5 hour, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 2-4 time, and each 0.5-2.5 hour, merge decocting liquid, be concentrated into thick paste; Add the 30%-80% ethanol of 4-10 times of weight in the thick paste, left standstill 12-48 hour, get supernatant and reclaim ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A4: Radix Curcumae is added hot water reflux, extract, 2-4 time earlier, and each 0.5-2.5 hour, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 2-4 time, each 0.5-2.5 hour, merge decocting liquid, be concentrated into relative density 1-1.0～1.4, centrifugal, supernatant is crossed macroporous resin column, the 30-80% ethanol elution reclaims ethanol, concentrates, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Step B: preparation intermediate II

B1: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add the 3-10% dilute hydrochloric acid, consumption is the 5-15 times of weight, makes into suspension, leaves standstill 12-48 hour, filters, and filtrate transfers pH value to 3-5 with 10-20% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 8-10 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II;

Or B2: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add the 3-10% dilute hydrochloric acid, consumption is the 5-15 times of weight, makes into suspension, leaves standstill 12-48 hour, filters, and filtrate transfers pH value to 3-5 with 10-20% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 8-10 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaim ethanol, be concentrated into relative density 1-1.0～1.4, centrifugal, supernatant is crossed macroporous resin column, the 30-80% ethanol elution reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II;

Or B3: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add the 3-10% dilute hydrochloric acid respectively, consumption is the 5-15 times of weight, make into suspension, left standstill 12-48 hour, and filtered, filtrate transfers pH value to 3-5 with 10-20% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 8-10 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and gets the extract of Pulvis Cornus Bubali Concentratus powder, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly gets intermediate II after the mixing; Or behind the combination drying the powder of intermediate II;

Or B4: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder,, add the 3-10% dilute hydrochloric acid respectively, consumption is the 5-15 times of weight, makes into suspension, leaves standstill 12-48 hour, filter, filtrate transfers pH value to 3-5 with 10-20% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 8-10 times of weight in the concentrated solution, leave standstill, get supernatant, reclaim ethanol, be concentrated into relative density 1-1.0～1.4, centrifugal, supernatant is crossed macroporous resin column, the 30-80% ethanol elution, reclaim ethanol, concentrate, get the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly get intermediate II after the mixing; Or behind the combination drying the powder of intermediate II;

Step C: preparation intermediate III

C1: get Pulvis Fellis Suis and Realgar is broken into fine powder, mix, add 3-5% sodium hydroxide 4-8 times of weight, left standstill 12-48 hour, filter, filtrate transfers pH value to 3-5, concentrates, and adds 30-80% ethanol 8-15 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get intermediate III; Or after the drying the powder of intermediate III;

Or C2: get Pulvis Fellis Suis and be broken into fine powder, add 3-5% sodium hydroxide 4-8 times of weight, left standstill 12-48 hour, filter, filtrate transfers pH value to 3-5, concentrates, and adds 30-80% ethanol 8-15 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get ketone ibuprofen extract, or get the ketone ibuprofen extract powder after the drying; Get Realgar powder and be broken into fine powder, adding 4-8 times of weight 5-10% hydrochloric acid left standstill 8-24 hour, filtered, and filtrate transfers pH value to 3-5, concentrates, and adding is washed to colourless, promptly gets Realgar extract, or gets the Realgar extract powder after the drying; Ketone ibuprofen extract is mixed with Realgar extract, intermediate III or the powder of intermediate III;

Step D: preparation medicated powder

D1: respectively Borneolum Syntheticum, Mentholum, Cinnabaris and Haematitum are prepared impalpable powder, mix medicated powder;

Or D2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 5-8 times of weight water grind to pasty state, again by 1: 60--70 adds entry and stirs, and leaves standstill, and inclining suspension, and sediment grinds again; As above method is 3-6 time repeatedly, merges each time suspension, leaves standstill, and gets to be deposited in 25-45 ℃ of airing, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder;

Step e: preparation

The powder of intermediate compound I, II, III or intermediate compound I, II, III is mixed with medicated powder, technology routinely, add conventional adjuvant and make pharmaceutically acceptable dosage form, as powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, oral liquid or injection.

Wherein steps A is preferably as follows method:

A1: crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added the hot water reflux, extract, 3 times jointly, each 1.5 hours, merge decocting liquid, be concentrated into thick paste; Add 50% ethanol of 7 times of weight in the thick paste, left standstill 24 hours, get supernatant and reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A2: crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added the hot water reflux, extract, 3 times jointly, each 1.5 hours, merge decocting liquid, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, 50% ethanol elution, reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A3: Radix Curcumae is added the hot water reflux, extract, earlier 3 times, and each 1.5 hours, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 3 times, and each 1.5 hours, merge decocting liquid, be concentrated into thick paste; Add 50% ethanol of 7 times of weight in the thick paste, left standstill 24 hours, get supernatant and reclaim ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Or A4: Radix Curcumae is added the hot water reflux, extract, earlier 3 times, and each 1.5 hours, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 3 times, and each 1.5 hours, merge decocting liquid, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, and 50% ethanol elution reclaims ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I.

Wherein step B is preferably as follows method:

B1: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add 6% dilute hydrochloric acid, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II;

Or B2: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add 6% dilute hydrochloric acid, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, is concentrated into relative density 1-1.2, and centrifugal, supernatant is crossed macroporous resin column, and 50% ethanol elution reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II;

Or B3: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add 6% dilute hydrochloric acid respectively, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and gets the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly gets intermediate II after the mixing; Or behind the combination drying the powder of intermediate II;

Or B4: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add 6% dilute hydrochloric acid respectively, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leave standstill, get supernatant, reclaim ethanol, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, 50% ethanol elution, reclaim ethanol, concentrate, get the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly get intermediate II after the mixing; Or behind the combination drying the powder of intermediate II.

Wherein step C is preferably as follows method:

C1: get Pulvis Fellis Suis and Realgar is broken into fine powder, mix, add 4% sodium hydroxide, 6 times of weight, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adds 50% ethanol, 12 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get intermediate III; Or after the drying the powder of intermediate III;

Or C2: get Pulvis Fellis Suis and be broken into fine powder, add 4% sodium hydroxide, 6 times of weight, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adds 50% ethanol, 12 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get ketone ibuprofen extract, or get the ketone ibuprofen extract powder after the drying; Get Realgar powder and be broken into fine powder, add 6 times of weight, 8% hydrochloric acid, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adding is washed to colourless, promptly gets Realgar extract, or gets the Realgar extract powder after the drying; Ketone ibuprofen extract is mixed with Realgar extract, intermediate III or the powder of intermediate III.

Wherein step D is preferably as follows method:

Or D2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 6 times of weight water grind to pasty state, added entry by 1: 65 again and stir, leave standstill, inclining suspension, and sediment grinds again; As above method is 4 times repeatedly, merges each time suspension, leaves standstill, and gets and is deposited in 35 ℃ of airings, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder.

Wherein, above-mentioned any step all can be substituted by conventional method.

Wherein, above-mentioned macroporous resin column model is: YPR-II, X5, AB-8, HPD-100, DX-5, D101, DA201, DM130, WLD-3 or NKA-9.

The preparation method of Annaowan bolus of the present invention and preparation thereof can also be following method:

Technology 2: form by following steps A, B, C and D

Steps A: preparation intermediate compound I

Step B: preparation intermediate II

B1: crude drug Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita, Concha Margaritifera, Pulvis Fellis Suis and Realgar powder are broken into fine powder, mix, add the 30-80% ethanol of 4-10 times of weight, 40-60 ℃ warm macerating 12-48 hour, filter, get filtrate, reclaim ethanol, concentrate, promptly get intermediate II; Or after the drying the powder of intermediate II;

Step C: preparation medicated powder

C1: respectively Borneolum Syntheticum, Mentholum, Cinnabaris and Haematitum are prepared impalpable powder, mix medicated powder;

Or C2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 5-8 times of weight water grind to pasty state, again by 1: 60--70 adds entry and stirs, and leaves standstill, and inclining suspension, and sediment grinds again; As above method is 3-6 time repeatedly, merges each time suspension, leaves standstill, and gets to be deposited in 25-45 ℃ of airing, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder;

Step D: preparation

The powder of intermediate compound I, II or intermediate compound I, II is mixed with medicated powder, technology routinely, add conventional adjuvant and make pharmaceutically acceptable dosage form, as powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, oral liquid or injection.

Wherein steps A is preferably as follows method:

Wherein step B is preferably as follows method:

B1: crude drug Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita, Concha Margaritifera, Pulvis Fellis Suis and Realgar powder are broken into fine powder, mix, add 50% ethanol of 7 times of weight, 50 ℃ of warm macerating 24 hours filter, and get filtrate, reclaim ethanol, concentrate, and promptly get intermediate II; Or after the drying the powder of intermediate II.

Wherein step C is preferably as follows method:

C1: respectively Borneolum Syntheticum, Mentholum, Cinnabaris and Haematitum are prepared impalpable powder, respectively or mix medicated powder;

Or C2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 6 times of weight water grind to pasty state, added entry by 1: 65 again and stir, leave standstill, inclining suspension, and sediment grinds again; As above method is 4 times repeatedly, merges each time suspension, leaves standstill, and gets and is deposited in 35 ℃ of airings, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder.

Drug regimen object detecting method of the present invention can be applied to the various dosage forms of compositions, as powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, clinical acceptable forms such as oral liquid or injection, because the wherein contained suitable crude drug amount of preparation of different dosage form is identical, therefore each dosage form is when detecting, it is measurement unit for daily amount of formulation that selected sample size can be unified conversion, and the per unit preparation can be for every, every, every or every ball etc.

Description of drawings

Fig. 1: test sample and reference substance chromatographic fractionation figure

Fig. 2: standard curve

Fig. 3: negative control chromatogram

Detection method of the present invention our experiments show that and all shows good linearity relation, precision, stability, reappearance and the rate of recovery.

Following experimental example or embodiment further illustrate but are not limited to the present invention.

The test of experimental example 1 content determination of Baicalin methodology

(1) instrument and reagent

Instrument: the Agilent_1100 high performance liquid chromatograph comprises: vacuum degassing machine, the quaternary gradient pump advances the sample device automatically, diode display detector (DAD detector), Agilent_Chemistation data treatment system.

Reagent: n-butanol, methyl alcohol are that chromatogram is pure, and water is distilled water, and it is pure that other reagent is analysis, scutelloside reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides).

(2) chromatogram condition

Chromatographic column: Diamonsil C18,200 * 4.6mm ID, 5 μ m; Flow mutually respectively take acetonitrile-water as 12: 88 or n-butanol-methyl alcohol--water-phosphoric acid was as 15: 35: 40: 0.4 or n-butanol-water-phosphoric acid as 60: 35: 0.4 or methanol-water-phosphoric acid=45: 58: 0.2 as mobile phase: detection wavelength: 280nm; Post temperature: room temperature; Flow velocity: 1.0ml/min; Sampling volume; 10 μ l; Automatically advance sample, data are processed quantitative approach: mark peak area method outward.

(3) detecting wavelength selectes

By above-mentioned chromatogram condition reference substance is carried out spectrum analysis, scutelloside had absorption maximum at the 280nm place when acetonitrile-water or methanol-water-phosphoric acid flowed phase, at n-butanol-methyl alcohol--water-phosphoric acid or n-butanol-water-phosphoric acid are at the 276nm place absorption maximum to be arranged when flowing phase, are that scutelloside detects wavelength so select 276nm or 280nm. The spectrogram at sample scutelloside peak is consistent with reference substance.

(4) reference substance solution, the preparation of need testing solution and negative sample solution:

The preparation of reference substance solution: precision takes by weighing the scutelloside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 60 μ g, and get final product;

The preparation of need testing solution: get 1/4 of the daily amount of formulation of tablet of the present invention, put in the 100ml measuring bottle, add 85% ethanol 85ml, transfer PH=7, ultrasonic extraction 12 minutes filters; Filtrate is put in the 100ml measuring bottle, adds 3% hydrochloric acid 4ml, stirs evenly, adds NaOH again and transfers PH=7, leaves standstill 25 minutes, filters; Filtrate adds 85% ethanol to scale, shakes evenly, and get final product;

The preparation of negative sample solution: get the negative sample 2g that does not contain the root of large-flowered skullcap, accurately weighed, prepare negative sample solution with the preparation method of need testing solution.

(5) blank test and chromatogram

Draw respectively scutelloside reference substance solution, tablet need testing solution of the present invention and negative sample solution, inject high performance liquid chromatograph and analyze, experimental result shows that negative sample solution does not have interference to the assay of scutelloside.

(6) linear relation is investigated

Precision takes by weighing at dry 4 hours scutelloside reference substance of 60 ℃ of decompressions, add methyl alcohol and make the solution that every 1ml contains 0.1248mg, be diluted to respectively 0.04352mg/ml with methyl alcohol again, 0.07252mg/ml, 0.08703mg/ml, 0.11604mg/ml solution. get respectively 10 μ l and inject high performance liquid chromatograph, measure by above-mentioned chromatogram condition, take concentration (mg/ml) as abscissa, the integrated value of peak area (Mau) is ordinate, the drawing standard curve, Y=2.689 * 104X+2.3264 * 10-1, V=0.99996. show that Determination of baicalin has good linearity relation in 0.04352～0.14500mg/ml scope, the results are shown in Table 1.

Table 1 scutelloside is linear to be investigated

(7), precision experiment

Accurate scutelloside reference substance solution 10 μ l that draw respectively repeat to record the scutelloside peak area into sample 9 times continuously, the results are shown in Table 2, show that precision is good.

Table 2 precision test

(8) stability test

Get need testing solution, measured respectively the content of scutelloside in 0,6,12,18,24 hour after preparation, measurement result sees Table 3. The result shows that sample is basicly stable in 24 hours.

Table 3 stability test result

(9) reappearance experiment

Get same lot number (0801020) sample tablet 2g, totally 5 parts, prepare need testing solution according to aforementioned method, the accurate need testing solution 10 μ l that draw carry out HPLC and analyze, and the results are shown in Table 4, show that reappearance is good.

The test of table 4 reappearance

(10) recovery test

Adopt the application of sample absorption method, get the same batch sample (lot number 0801020 of known content, content of baicalin 1.83486mg/g), precision takes by weighing 5 parts in sample, each 1g adds respectively scutelloside 0.04618mg/ml, measures by aforementioned content assaying method, calculate recovery rate, the average rate of recovery of result is 101.6%.

(11) sample determination

Respectively three batch samples are measured by content determination of Baicalin method of the present invention, be the results are shown in Table 5, according to measurement result, stipulate that every contains the root of large-flowered skullcap with scutelloside (C₂₁H ₁₈O ₁₁) meter, must not be less than 0.6mg.

The assay of table 53 batch sample scutellosides

Above-mentioned experiment is the data of n-butanol-water-phosphoric acid for flowing mutually, investigate respectively acetonitrile-water or n-butanol-methyl alcohol with same test method--when phase was flowed in water-phosphoric acid or methanol-water-phosphoric acid conduct, each flows all can desirable linearity relation, precision, stability, reappearance and the rate of recovery mutually.

Experimental example 2 cape jasmine glycosides content assaying methods are learned test

1, instrument and reagent

2, chromatogram condition

Chromatographic column: Diamonsil C18,200 * 4.6mm ID, 5 μ m; Take acetonitrile-water as 12: 88 or n-butanol-methyl alcohol--water-phosphoric acid was as 14: 36: 37: 0.4 when flowing phase the cape jasmine glycosides at the 280nm place absorption maximum is arranged, at 276nm place absorption maximum being arranged as 65: 32: 0.4 when flowing phase take n-butanol-water-phosphoric acid, is cape jasmine glycosides detection wavelength so select 276nm or 280nm. The spectrogram at sample cape jasmine glycosides peak is consistent with reference substance.

3, reference substance solution, the preparation of need testing solution and negative sample solution

The preparation of reference substance solution: precision takes by weighing cape jasmine glycosides reference substance, adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, and get final product;

The preparation of need testing solution: get 1/4 of the daily amount of formulation of tablet of the present invention, put in the 100ml measuring bottle, add 40% ethanol or acetone-40% ethanol and be 1: 1 mixed liquor 85ml, add 3% hydrochloric acid and transfer PH2-3, ultrasonic extraction 12 minutes filters; Filtrate is put in the 100ml measuring bottle, and hydro-oxidation sodium is transferred PH=7, filters, and filtrate adds ethanol to scale, shakes evenly, and get final product;

4, blank test and chromatogram

Draw respectively cape jasmine glycosides reference substance solution, tablet need testing solution of the present invention and negative sample solution, inject high performance liquid chromatograph and analyze, experimental result shows that negative sample solution does not have interference to the assay of cape jasmine glycosides.

5, linear relation is investigated

Precision takes by weighing at dry 4 hours cape jasmine glycosides reference substance of 60 ℃ of decompressions, add methyl alcohol and make the solution that every 1ml contains 0.1248mg, be diluted to respectively 0.04352mg/ml with methyl alcohol again, 0.07252mg/ml, 0.08703mg/ml, 0.11604mg/ml solution. get respectively 10 μ l and inject high performance liquid chromatograph, measure by above-mentioned chromatogram condition, take concentration (mg/ml) as abscissa, the integrated value of peak area (Mau) is ordinate, the drawing standard curve, cape jasmine glycosides concentration has good linearity relation in 0.02342～0.14500mg/ml scope, the results are shown in Table 6.

Table 6 cape jasmine glycosides is linear to be investigated

6, precision test

Accurate cape jasmine glycosides reference substance solution 10 μ l that draw respectively repeat to record peak area into sample 9 times continuously, the results are shown in Table 7, show that precision is good.

Table 7 precision test

7, stability test

Get need testing solution, measured respectively the content of cape jasmine glycosides in 0,6,12,18,24 hour after preparation, measurement result sees Table 8. The result shows that sample is basicly stable in 24 hours.

Table 8 stability test result

8, reappearance experiment

Get same lot number (0801020) sample tablet 2g, totally 5 parts, prepare need testing solution according to aforementioned method, the accurate need testing solution 10 μ l that draw carry out HPLC and analyze, and the results are shown in Table 9, show that reappearance is good.

The test of table 9 reappearance

9, recovery test

Adopt the application of sample absorption method, get the same batch sample (lot number 0801020 of known content, cape jasmine glycosides content 1.7335mg/g), precision takes by weighing 5 parts in sample, each 1g adds respectively cape jasmine glycosides 0.04618mg/ml, measures by aforementioned content assaying method, calculate recovery rate, the average rate of recovery of result is 101.6%.

10, sample determination

Respectively three batch samples are measured by text cape jasmine glycosides content assaying method, be the results are shown in Table 10, according to measurement result, stipulate that every contains cape jasmine with cape jasmine glycosides (C₁₇H ₂₄O ₁₀) meter, must not be less than 0.6mg.

The assay of table 10 3 batch sample cape jasmine glycosides

Above-mentioned experiment is n-butanol-methyl alcohol--the data of water-phosphoric acid all can have desirable linearity relation, precision, stability, reappearance and the rate of recovery with same test method investigation acetonitrile-water or n-butanol-water-phosphoric acid mutually for flowing.

Experimental example 3 borneols, the test of Determination of menthol mensuration methodology

1. instrument and material

1.1 instrument

U.S. VARIAN CP-3800; Chromatographic column: HP-INNOWax capillary chromatographic column (30m*0.32mm*0.25 μ m); The BP211D electronic balance.

1.2 material

Borneol reference substance (Chinese medicine is examined and determine institute, lot number 110743-200504); Menthol reference substance (Chinese medicine is examined and determine institute, lot number 0728-200005); Eugenol reference substance (Chinese medicine is examined and determine institute, lot number 725-200209); Ethyl acetate (Tianjin chemical reagent one factory); Sample is tablet of the present invention.

1.3 chromatogram condition

Chromatographic column: HP-INNOWax capillary chromatographic column (30m*0.32mm*0.25 μ m); 240 ℃ of detector temperature; Gasification room temperature: 240 ℃; The intensification program: 150 ℃ (4min)-(8 ℃/min)-200 ℃-(200 ℃/min)-240 ℃ (4min); Carrier gas: High Purity Nitrogen; Carrier gas flow velocity 2.0ml/min; Split ratio is about: 20: 1; Advance the sample amount: 1 μ l. Chromatographic isolation is all right, sees Fig. 1.

2. the preparation of inner mark solution: it is an amount of to get eugenol, adds ethyl acetate and makes the solution that every 1ml contains 0.5mg, shake even, as inner mark solution.

3. the preparation of reference substance solution: get borneol reference substance 20mg, menthol reference substance 10mg, accurately weighed, put in the 25ml measuring bottle, add the inner mark solution dissolving and be diluted to scale.

4. the preparation of need testing solution: get the tablet 1.5g of the present invention under the weight difference item, accurately weighed, put in the 25ml measuring bottle, the accurate inner mark solution 10ml that adds, close plug shakes even, weighed weight, ultrasonic processing 10 minutes, more weighed weight, supply the weight that subtracts mistake with ethanol, shake evenly, filter, draw 1 μ l, inject gas chromatograph is measured, and be get final product.

5. the degree of accuracy

Precision takes by weighing 6 parts of 0.75g test samples (lot number 060307), and accurate a certain amount of borneol, the menthol reference substance of adding measured by the method under the preparation of need testing solution respectively, the results are shown in Table 11, table 12. The borneol rate of recovery is that 102.55%, RSD is 1.8%; The menthol rate of recovery is that 101.01%, RSD is 2.0%, shows that the rate of recovery is good.

Table 11 borneol degree of accuracy test result

Table 12 menthol degree of accuracy test result

6. precision

6.1 instrument precision

Get reference substance solution, repeat into sample 5 times, the RSD that calculates the chromatographic peak area ratio of borneol and internal standard compound, menthol and internal standard compound is respectively 0.7% and 1.4%, the results are shown in following table 13. Show that instrument precision is good.

Table 13 instrument Precision test result

6.2 repeatability

Get 6 parts of about 1.5g test samples, accurately weighed, measure by the method under the preparation of need testing solution, the results are shown in following table 14. RSD is respectively 2.4% and 2.7% as a result, shows that repeatability is good.

Table 14 repeatability test result

7. linear

7.1 the preparation of borneol and menthol reference substance solution: get borneol reference substance 20mg, menthol reference substance 10mg, accurately weighed, put in the 25ml measuring bottle, add the inner mark solution dissolving and be diluted to scale.

7.2 inner mark solution preparation: it is an amount of to get eugenol, adds ethyl acetate and makes the solution that every 1ml contains 0.5mg, shake even, as inner mark solution.

7.3 linear scope: accurate reference substance solution 0.3 μ l, 0.5 μ l, 1 μ l, 2 μ l, the 3 μ l of drawing, inject gas chromatograph, the record peak area, take the peak area of reference substance as abscissa, the sample amount of advancing of reference substance is ordinate, calculate respectively the equation of linear regression of borneol, menthol, see accompanying drawing 2. Borneol: Y=0.00000115x-0.0197, r=0.9995, linear scope (0.26436～2.6435) μ g; Menthol: Y=0.00000116x-0.02, r=0.9996, linear scope (0.1152～1.152) μ g; Show the linearity relation that in the concentration scope, is good.

8. sample stability

Get about 1.5g test sample, accurately weighed, be prepared by the method under the preparation of need testing solution, place room temperature, respectively at 2h, 4h, 6h, 8h, 24h, measure, calculate, the results are shown in following table 15. RSD is respectively 0.4% and 0.8% as a result, shows to have good stability.

Table 15 stability test result

9. the specificity test is got respectively in the prescription ratio and with same process and is prepared the feminine gender contrast that does not contain borneol and menthol, makes respectively negative control solution by the need testing solution method for making. Under above-mentioned chromatogram condition, precision is drawn reference substance solution, negative control solution, each 1 μ l of need testing solution respectively, the difference inject gas chromatograph, recording the result is: in the negative contrast color spectrogram, do not have the chromatogram peak occurs at the retention time place corresponding with borneol, menthol reference substance and test sample chromatogram, show that other composition is noiseless to the mensuration of borneol, menthol, see Fig. 3.

10. measurement result

Press the method for setting up, the sample of 10 lot numbers has been carried out assay, see Table 16. The result shows that the content range of borneol is at 1.2mg/ sheet～1.7mg/ sheet; The content range of menthol is at 1.0mg/ sheet～1.7mg/ sheet. Consider that in conjunction with ten batch sample measured values and large-scale production process the content limit of tentative tablet borneol of the present invention must not be and is less than the 1.0mg/ sheet; The content limit of menthol must not be and is less than the 0.8mg/ sheet.

Table 16 assay result

Following embodiment all can realize the described effect of above-mentioned experimental example.

The specific embodiment

Crude drug among the following embodiment is formed 1 and is:

Artificial Calculus Bovis 1kg Pulvis Fellis Suis 13kg Rhizoma Coptidis 10kg

Radix Scutellariae 10kg Margarita 3kg Fructus Gardeniae 10kg

Radix Curcumae 10kg Haematitum 4kg Realgar 6kg

Cinnabaris 4kg Mentholum 1kg Gypsum Fibrosum 8kg

Borneolum Syntheticum 2kg Pulvis Cornus Bubali Concentratus 13kg Concha Margaritifera 5kg.

Crude drug is formed 2 and is:

Artificial Calculus Bovis 0.7kg Pulvis Fellis Suis 18kg Rhizoma Coptidis 6kg

Radix Scutellariae 14kg Margarita 2kg Fructus Gardeniae 14kg

Radix Curcumae 6kg Haematitum 5kg Realgar 4kg

Cinnabaris 5kg Mentholum 0.7kg Gypsum Fibrosum 11kg

Borneolum Syntheticum 1kg Pulvis Cornus Bubali Concentratus 18kg Concha Margaritifera 3kg.

Crude drug is formed 3 and is:

Artificial Calculus Bovis 1.8kg Pulvis Fellis Suis 9kg Rhizoma Coptidis 14kg

Radix Scutellariae 6kg Margarita 4kg Fructus Gardeniae 6kg

Radix Curcumae 14kg Haematitum 3kg Realgar 8kg

Cinnabaris 3kg Mentholum 1.8kg Gypsum Fibrosum 6kg

Borneolum Syntheticum 3.5kg Pulvis Cornus Bubali Concentratus 9kg Concha Margaritifera 7kg.

Discrimination method A is: the discriminating of cholic acid, Hyodeoxycholic Acid

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with isobutyltrimethylmethane .-ethyl acetate-glacial acetic acid be 15: 7: 5 be developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 10ml sulphuric acid and 100ml), and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Discrimination method B is: the discriminating of Rhizoma Coptidis

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water is 10: 7: 1: 1 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show identical yellow fluorescence speckle.

Discrimination method C is: the discriminating of jasminoidin

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 4 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-acetone-formic acid-water is 10: 7: 2: 0.5 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid (that is: the alcoholic acid mixed liquor of 10ml sulphuric acid and 100ml), 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 chromatograph on, show the speckle of same color.

Discrimination method D is: the discriminating of baicalin

Content assaying method A is: the assay of jasminoidin:

Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; Preparation of the present invention every day contains jasminoidin (C17H24O10) with amount of formulation and must not be less than 0.6-6mg and maybe must not be less than 3.6mg;

Content assaying method B is: content of baicalin is measured:

Measuring according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005), is filler with the octadecylsilane chemically bonded silica; With acetonitrile-water is 12: 88 or n-butyl alcohol-methanol--water-phosphoric acid is 12: 40: 35: 0.4 or n-butanol-water-phosphoric acid be that 60: 35: 0.4 or methanol-water-phosphoric acid=45: 55: 0.2 are as mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500;

Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly; This product is pressed dry product and is calculated, and clean Radix Scutellariae, Radix Scutellariae (processed with wine) contain baicalin (C ₂₁H ₁₈O ₁₁) must not be less than 8.0%; Preparation of the present invention every day contains baicalin with amount of formulation and must not be less than 0.6-6mg and maybe must not be less than 3.6mg;

Content assaying method C is: the assay of Rhizoma Coptidis:

Test according to thin layer chromatography (appendix VI B), draw need testing solution 1 μ l, reference substance solution 1 μ l and 3 μ l, the cross point is on same silica gel g thin-layer plate, with benzene-ethyl acetate-isopropyl alcohol-methanol-water is 6: 2: 1.5: 1.5: 1 or n-butyl alcohol-glacial acetic acid-methanol-water are 6: 0.9: 1.2: 1 is developing solvent, groove adds isopyknic strong ammonia solution in addition, launch 12 centimetres, take out, volatilize, carry out fluorescent scanning according to thin layer chromatography (appendix VI B thin layer chromatography scanning), wavelength X=356-366nm, the integrated value of measurement test sample and reference substance fluorescence intensity, calculate, promptly; Preparation of the present invention contains berberine in berberine hydrochloride (C20H17NO4HCl) with amount of formulation every day, must not be less than 0.8mg---8mg and maybe must not be less than 3.2mg;

Content assaying method D is: the assay of Mentholum and Borneolum Syntheticum

Get 1/4 of the daily amount of formulation of preparation of the present invention, pulverize, the accurate title, decide, and adds kieselguhr 3g, the accurate title, decide, and after grinding well, gets 2g, and accurate title is fixed, put in the 25ml measuring bottle, accurate inner mark solution 15ml and the water 1ml of adding, close plug shakes up, claim to decide weight, dipping spends the night, and claims to decide weight again, supplies the weight that subtracts mistake with ethyl acetate, shake up, filter, draw 1 μ l, inject gas chromatograph is measured, promptly; Contain Borneolum Syntheticum (C with amount of formulation every day ₁₀H ₁₈0) must not be less than 8-12mg; Contain Mentholum (C ₁₀H ₂₀O) must not be less than 6.4-9.6mg.

Described process 1 A1 is:

A1: crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added the hot water reflux, extract, 3 times jointly, each 1.5 hours, merge decocting liquid, be concentrated into thick paste; Add 50% ethanol of 7 times of weight in the thick paste, left standstill 24 hours, get supernatant and reclaim ethanol, concentrate, get the powder of intermediate compound I after the drying.

Described process 1 A2 is:

Crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added the hot water reflux, extract, 3 times jointly, each 1.5 hours, merge decocting liquid, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, 50% ethanol elution, reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I.

Described process 1 A3 is:

Radix Curcumae is added the hot water reflux, extract, earlier 3 times, and each 1.5 hours, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 3 times, and each 1.5 hours, merge decocting liquid, be concentrated into thick paste; Add 50% ethanol of 7 times of weight in the thick paste, left standstill 24 hours, get supernatant and reclaim ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I.

Described process 1 A4 is:

Radix Curcumae is added the hot water reflux, extract, earlier 3 times, and each 1.5 hours, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 3 times, and each 1.5 hours, merge decocting liquid, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, and 50% ethanol elution reclaims ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I.

Described process 1 B1 is:

Respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add 6% dilute hydrochloric acid, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II.

Described process 1 B2 is:

Respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, mix; Add 6% dilute hydrochloric acid, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, is concentrated into relative density 1-1.2, and centrifugal, supernatant is crossed macroporous resin column, and 50% ethanol elution reclaims ethanol, concentrates, and promptly gets intermediate II; Or after the drying the powder of intermediate II.

Described process 1 B3 is:

Respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add 6% dilute hydrochloric acid respectively, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leaves standstill, and gets supernatant, reclaims ethanol, concentrates, and gets the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly gets intermediate II after the mixing; Or behind the combination drying the powder of intermediate II.

Described process 1 B4 is:

Respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add 6% dilute hydrochloric acid respectively, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leave standstill, get supernatant, reclaim ethanol, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, 50% ethanol elution, reclaim ethanol, concentrate, get the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly get intermediate II after the mixing; Or behind the combination drying the powder of intermediate II.

Described process 1 C1 is:

Get Pulvis Fellis Suis and Realgar is broken into fine powder, mix, add 4% sodium hydroxide, 6 times of weight, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adds 50% ethanol, 12 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get intermediate III; Or after the drying the powder of intermediate III.

Described process 1 C2 is:

Get Pulvis Fellis Suis and be broken into fine powder, add 4% sodium hydroxide, 6 times of weight, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adds 50% ethanol, 12 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get ketone ibuprofen extract, or get the ketone ibuprofen extract powder after the drying; Get Realgar powder and be broken into fine powder, add 6 times of weight, 8% hydrochloric acid, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adding is washed to colourless, promptly gets Realgar extract, or gets the Realgar extract powder after the drying; Ketone ibuprofen extract is mixed with Realgar extract, intermediate III or the powder of intermediate III.

Described process 1 D1 is:

Respectively Borneolum Syntheticum, Mentholum, Cinnabaris and Haematitum are prepared impalpable powder, mix medicated powder.

Described process 1 D2 is:

Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 6 times of weight water grind to pasty state, added entry by 1: 65 again and stir, leave standstill, inclining suspension, and sediment grinds again; As above method is 4 times repeatedly, merges each time suspension, leaves standstill, and gets and is deposited in 35 ℃ of airings, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder.

Described process 2 A1 are:

Crude drug Radix Scutellariae, Rhizoma Coptidis, Fructus Gardeniae, Radix Curcumae and Gypsum Fibrosum are added the hot water reflux, extract, 3 times jointly, each 1.5 hours, merge decocting liquid, be concentrated into thick paste; Add 50% ethanol of 7 times of weight in the thick paste, left standstill 24 hours, get supernatant and reclaim ethanol, concentrate, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I.

Described process 2 A2 are:

Described process 2 A3 are:

Described process 2 A4 are:

Described process 2 B1 are:

Crude drug Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita, Concha Margaritifera, Pulvis Fellis Suis and Realgar powder are broken into fine powder, mix, add 50% ethanol of 7 times of weight, 50 ℃ of warm macerating 24 hours filter, and get filtrate, reclaim ethanol, concentrate, and promptly get intermediate II; Or after the drying the powder of intermediate II.

Described process 2 C1 are:

Respectively Borneolum Syntheticum, Mentholum, Cinnabaris and Haematitum are prepared impalpable powder, respectively or mix medicated powder.

Described process 2 C2 are:

Embodiment 1: the detection method of medicinal composition powders of the present invention

Crude drug forms 1, gets crude drug, adds conventional adjuvant, makes powder according to common process, takes 6g every day.

Detection method is: the combination of discrimination method A, B, C, D, content assaying method A, B, C and D.

Embodiment 2: the detection method of medicinal composition powders of the present invention

Crude drug forms 2;

Processing step is: the steps A 3 in the technology 1, B2, C1, D2, the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes powder routinely, takes 6g every day.

Detection method is: the combination of discrimination method A, B, content assaying method A and B.

Embodiment 3: the detection method of pharmaceutical composition tablet of the present invention

Crude drug forms 3, gets crude drug, adds conventional adjuvant, makes tablet according to common process, and is oral, one time 4,2-3 time on the one.

Detection method is: the combination of discrimination method A and content assaying method A.

Embodiment 4: the detection method of pharmaceutical composition tablet of the present invention

Crude drug forms 1;

Processing step is steps A 4, B2, C1, the D2 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes powder routinely, takes 6g every day.

Detection method is: the combination of discrimination method A, content assaying method B, C and D.

Embodiment 5: the detection method of medicament composition granule agent of the present invention

Crude drug forms 2, gets crude drug, adds conventional adjuvant, makes granule according to common process, takes 6g every day.

Detection method is: the combination of discrimination method A, B, C and content assaying method D.

Embodiment 6: the detection method of medicament composition granule agent of the present invention

Crude drug forms 3;

Processing step is steps A 3, B3, C1, the D2 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes granule routinely, takes 6g every day.

Detection method is: the combination of content assaying method A, B, C and D.

Embodiment 7: the detection method of medicament composition capsule agent of the present invention

Crude drug forms 1, gets crude drug, adds conventional adjuvant, makes capsule according to common process, and is oral, one time 4,2-3 time on the one.

Detection method is: the combination of content assaying method A and C.

Embodiment 8: the detection method of medicament composition capsule agent of the present invention

Crude drug forms 2;

Processing step is steps A 4, B3, C1, the D2 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes capsule routinely, and is oral, one time 4,2-3 time on the one.

Detection method is: the combination of discrimination method A, C, content assaying method A, B, C and D.

Embodiment 9: the detection method of medicament combination dispersible tablet of the present invention

Crude drug forms 3;

Processing step is steps A 1, B1, the C1 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes dispersible tablet routinely, and is oral, one time 4,2-3 time on the one.

Detection method is: the combination of discrimination method C, content assaying method A, B, C and D.

Embodiment 10: the detection method of medicament combination dispersible tablet of the present invention

Crude drug forms 1;

Processing step is steps A 2, B1, the C1 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes dispersible tablet routinely, and is oral, one time 4,2-3 time on the one.

Detection method is: the combination of discrimination method A, B, C, D and content assaying method A.

Embodiment 11: the detection method of medicament composition dropping pills of the present invention

Crude drug forms 2;

Processing step is steps A 3, B1, the C1 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes drop pill routinely, and is oral, one time 2 ball, 2-3 time on the one.

Detection method is: the combination of discrimination method A, D, content assaying method A and B.

Embodiment 12: the detection method of medicament composition dropping pills of the present invention

Crude drug forms 3;

Processing step is steps A 4, B1, the C1 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes drop pill routinely, and is oral, one time 2 ball, 2-3 time on the one.

Detection method is: the combination of discrimination method C, D, content assaying method A, B and C.

Embodiment 13: the detection method of the pharmaceutical composition watered pill of the present invention

Crude drug forms 1;

Processing step is steps A 1, B1, the C2 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes the watered pill routinely, and is oral, one time 2 ball, 2-3 time on the one.

Detection method is: the combination of content assaying method A and C.

Embodiment 14: the detection method of the pharmaceutical composition watered pill of the present invention

Crude drug forms 2;

Processing step is steps A 2, B1, the C2 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes the watered pill routinely, and is oral, one time 2 ball, 2-3 time on the one.

Detection method is: the combination of discrimination method D, content assaying method A, B, C and D.

Embodiment 15: the detection method of pharmaceutical composition honeyed pill of the present invention

Crude drug forms 3, gets crude drug, adds conventional adjuvant, makes honeyed pill according to common process, and is oral, one time 1 ball, 2-3 time on the one.

Detection method is: content assaying method D.

Embodiment 16: the detection method of pharmaceutical composition honeyed pill of the present invention

Crude drug forms 1;

Processing step is steps A 3, B1, the C2 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes honeyed pill routinely, and is oral, one time 1 ball, 2-3 time on the one.

Detection method is: the combination of content assaying method A, B and C.

Embodiment 17: the detection method of pharmaceutical composition slow releasing agent of the present invention

Crude drug forms 2;

Processing step is steps A 4, B1, the C2 in the technology 2, and the powder of intermediate compound I, II is mixed with medicated powder, and technology adds conventional adjuvant and makes slow releasing agent routinely, and is oral, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method A, B, C, D, content assaying method A and D.

Embodiment 18: the detection method of pharmaceutical composition slow releasing agent of the present invention

Crude drug forms 3;

Processing step is steps A 1, B1, C1, the D1 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes slow releasing agent routinely, and is oral, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method B, C, D, content assaying method B, C and D.

Embodiment 19: the detection method of drug composition oral liquid of the present invention

Crude drug forms 1;

Processing step is steps A 3, B1, C1, the D1 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes oral liquid routinely, and is oral, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method B, content assaying method A, B and C.

Embodiment 20: the detection method of drug composition oral liquid of the present invention

Crude drug forms 2;

Processing step is steps A 2, B2, C2, the D2 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes oral liquid routinely, and is oral, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method A, B, D, content assaying method C and D.

Embodiment 21: the detection method of medicine composition injection of the present invention

Crude drug forms 3;

Processing step is steps A 4, B2, C2, the D2 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes injection routinely, injection, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method A, D, content assaying method B, C and D.

Embodiment 22: the detection method of medicine composition injection of the present invention

Crude drug forms 1;

Processing step is steps A 2, B3, C1, the D1 in the technology 1, and the powder of intermediate compound I, II, III is mixed with medicated powder, and technology adds conventional adjuvant and makes injection routinely, injection, one time 1,2-3 time on the one.

Detection method is: the combination of discrimination method A, D and content assaying method D.

Claims

1. the detection method of a pharmaceutical composition is characterized in that this method comprises one or more in the following content assaying method:

A. the assay of jasminoidin:

B. content of baicalin is measured:

Measuring according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), is filler with the octadecylsilane chemically bonded silica; With the acetonitrile-water is 10-15: 85-90 or n-butyl alcohol-methanol--water-phosphoric acid is that 10-15: 30-50: 30-40: 0.3-0.5 or n-butanol-water-phosphoric acid are 50--70: 30-40: 0.3-0.5 or methanol-water-phosphoric acid=40-50: 50-60: 0.1-0.3 is as mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500;

C. the assay of Rhizoma Coptidis:

Wherein, the crude drug of this pharmaceutical composition consists of:

Concha Margaritifera 2-8 weight portion;

This preparation of drug combination method is:

Get the above-mentioned raw materials medicine, add conventional adjuvant, according to common process, make the dosage form of clinical acceptance, include but not limited to powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, oral liquid or injection.

2. detection method as claimed in claim 1 is characterized in that this method comprises one or more in the following content assaying method:

A. the assay of jasminoidin:

B. content of baicalin is measured:

C. the assay of Rhizoma Coptidis:

Test according to thin layer chromatography (appendix VI B), draw need testing solution 1 μ l, reference substance solution 1 μ l and 3 μ l, the cross point is on same silica gel g thin-layer plate, with benzene-ethyl acetate-isopropyl alcohol-methanol-water is 6: 2: 1.5: 1.5: 1 or n-butyl alcohol-glacial acetic acid-methanol-water are 6: 0.9: 1.2: 1 is developing solvent, groove adds isopyknic strong ammonia solution in addition, launch 12 centimetres, take out, volatilize, carry out fluorescent scanning according to thin layer chromatography (appendix VI B thin layer chromatography scanning), wavelength X=356-366nm, the integrated value of measurement test sample and reference substance fluorescence intensity, calculate, promptly; Contain berberine with berberine hydrochloride (C with amount of formulation every day ₂₀H ₁₇NO ₄HCl) meter must not be less than 0.8mg---8mg and maybe must not be less than 3.2mg.

3. as the arbitrary described detection method of claim 1-2, it is characterized in that this method also comprises one or more in the following discrimination method:

A. the discriminating of cholic acid, Hyodeoxycholic Acid

B. the discriminating of Rhizoma Coptidis

C. the discriminating of jasminoidin

D. the discriminating of baicalin

4. detection method as claimed in claim 1 or 2 is characterized in that this method also comprises one or more in the following discrimination method:

A. the discriminating of cholic acid, Hyodeoxycholic Acid

B. the discriminating of Rhizoma Coptidis

Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2 μ l of above-mentioned three kinds of solution, put respectively in-be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water is 10: 7: 1: 1 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show identical yellow fluorescence speckle;

C. the discriminating of jasminoidin

D. the discriminating of baicalin

5. as the arbitrary described detection method of claim 1-4, it is characterized in that this method also comprises following content assaying method:

The assay of Mentholum and Borneolum Syntheticum

6. as the arbitrary described detection method of claim 1-4, it is characterized in that this method comprises following content assaying method:

The assay of Mentholum and Borneolum Syntheticum

7. as the described detection method of claim 1-6, it is characterized in that the crude drug of the pharmaceutical composition described in this method consists of:

Borneolum Syntheticum 2 weight portion Pulvis Cornus Bubali Concentratuss 13 weight portion Concha Margaritiferas 5 weight portions;

Or

Borneolum Syntheticum 1 weight portion Pulvis Cornus Bubali Concentratus 18 weight portion Concha Margaritiferas 3 weight portions;

Or

8. as the arbitrary described detection method of claim 1-7, it is characterized in that the preparation of drug combination method described in this method is a kind of in the following method:

Method 1: form by following steps A, B, C, D and E

Steps A: preparation intermediate compound I

Step B: preparation intermediate II

Step C: preparation intermediate III

Step D: preparation medicated powder

Step e: preparation

The powder of intermediate compound I, II, III or intermediate compound I, II, III is mixed with medicated powder, technology routinely, add conventional adjuvant and make pharmaceutically acceptable dosage form, as powder, granule, tablet, capsule, dispersible tablet, drop pill, watered pill, honey pill agent, pellet, concentrated pill, soft capsule, slow releasing agent, oral liquid or injection;

Method 2: form by following steps A, B, C and D

Steps A: preparation intermediate compound I

Step B: preparation intermediate II

Step C: preparation medicated powder

Step D: preparation

9. detection method as claimed in claim 8 is characterized in that the preparation of drug combination method described in this method is a kind of in the following method:

Method 1: form by following steps A, B, C, D and E

Steps A: preparation intermediate compound I

Or A4: Radix Curcumae is added the hot water reflux, extract, earlier 3 times, and each 1.5 hours, volatile oil was standby; Radix Curcumae medicinal residues and the common decocting of crude drug Radix Scutellariae, Rhizoma Coptidis, Gypsum Fibrosum and Fructus Gardeniae boil 3 times, and each 1.5 hours, merge decocting liquid, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, and 50% ethanol elution reclaims ethanol, concentrate, spray into volatile oil, promptly get intermediate compound I; Or after the drying the powder of intermediate compound I;

Step B: preparation intermediate II

Or B4: respectively Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera are ground into fine powder, add 6% dilute hydrochloric acid respectively, consumption is 10 times of weight, makes into suspension, leaves standstill 24 hours, filters, and filtrate is transferred pH value to 4 with 15% sodium hydroxide (or other alkali liquor); Filter, filtrate is concentrated into thick paste, adds the ethanol of 9 times of weight in the concentrated solution, leave standstill, get supernatant, reclaim ethanol, be concentrated into relative density 1-1.2, centrifugal, supernatant is crossed macroporous resin column, 50% ethanol elution, reclaim ethanol, concentrate, get the extract of Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita and Concha Margaritifera respectively, promptly get intermediate II after the mixing; Or behind the combination drying the powder of intermediate II;

Step C: preparation intermediate III

Or C2: get Pulvis Fellis Suis and be broken into fine powder, add 4% sodium hydroxide, 6 times of weight, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adds 50% ethanol, 12 times of weight; Get supernatant, reclaim ethanol, concentrate, promptly get ketone ibuprofen extract, or get the ketone ibuprofen extract powder after the drying; Get Realgar powder and be broken into fine powder, add 6 times of weight, 8% hydrochloric acid, left standstill 24 hours, filter, filtrate is transferred pH value to 4, concentrates, and adding is washed to colourless, promptly gets Realgar extract, or gets the Realgar extract powder after the drying; Ketone ibuprofen extract is mixed with Realgar extract, intermediate III or the powder of intermediate III;

Step D: preparation medicated powder

Or D2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 6 times of weight water grind to pasty state, added entry by 1: 65 again and stir, leave standstill, inclining suspension, and sediment grinds again; As above method is 4 times repeatedly, merges each time suspension, leaves standstill, and gets and is deposited in 35 ℃ of airings, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder;

Step e: preparation

Method 2: form by following steps A, B, C and D

Steps A: preparation intermediate compound I

Step B: preparation intermediate II

B1: crude drug Pulvis Cornus Bubali Concentratus, artificial Calculus Bovis, Margarita, Concha Margaritifera, Pulvis Fellis Suis and Realgar powder are broken into fine powder, mix, add 50% ethanol of 7 times of weight, 50 ℃ of warm macerating 24 hours filter, and get filtrate, reclaim ethanol, concentrate, and promptly get intermediate II; Or after the drying the powder of intermediate II;

Step C: preparation medicated powder

Or C2: Borneolum Syntheticum, Mentholum and Haematitum are prepared impalpable powder, Cinnabaris is added 6 times of weight water grind to pasty state, added entry by 1: 65 again and stir, leave standstill, inclining suspension, and sediment grinds again; As above method is 4 times repeatedly, merges each time suspension, leaves standstill, and gets and is deposited in 35 ℃ of airings, and levigation promptly gets the Cinnabaris impalpable powder; With the impalpable powder of above-mentioned four kinds of crude drug mix medicated powder;

Step D: preparation

10. as the arbitrary described detection method of claim 8-9, it is characterized in that the arbitrary steps in the preparation of drug combination technology described in this method is substituted by conventional method;

Described macroporous resin column model is: YPR-II, X5, AB-8, HPD-100, DX-5, D101, DA201, DM130, WLD-3 or NKA-9.