CN100504380C

CN100504380C - Detection method for dandengtongnao oral preparation

Info

Publication number: CN100504380C
Application number: CNB2005102008899A
Authority: CN
Inventors: 叶湘武; 张梅
Original assignee: Guizhou Yibai Pharmaceutical Co Ltd
Current assignee: Guizhou Yibai Pharmaceutical Co Ltd
Priority date: 2005-12-29
Filing date: 2005-12-29
Publication date: 2009-06-24
Anticipated expiration: 2025-12-29
Also published as: CN1823899A

Abstract

A quality control method for the orally taken Chinese medicine Dangdeng Tongnao includes such steps as checking its characteristies, descriminating by the thin-layer identification by referring to Chuan-xiong rhizome, kakonein, danshinone IIA and scutelloside, and measuring the content of kakonein.

Description

The detection method of dandengtongnao oral preparation

Technical field: the present invention relates to a kind of method of quality control of dandengtongnao oral preparation, belong to the technical field of medicine being carried out quality control.

Background technology: apoplexy is a kind of common disease in our daily life, and people health in serious threat.The Dandengtongnao preparation mainly is made up of the red sage root, erigeron breviscapus, Ligusticum wallichii and the root of kudzu vine, have activate blood circulation and disperse blood clots, the effect of dispelling wind and removing obstruction in the meridians.Wherein " DANDENG TONGNAO JIAONANG " and " DANDENG TONGNAO RUANJIAONANG " all published and risen at national terrestrial reference that GB Chinese patent drug part channels and collaterals-limbs-brain is a fascicle.This medicine is used for many years clinically, apoplexy due to the treatment hemostasis, apoplex involving the channels and collaterals card aspect obtains satisfied result of treatment, but through discovering, shortcomings such as it is simple that existing Dandengtongnao preparation all exists quality control standard, and product quality is wayward.The red sage root, erigeron breviscapus are the medicine that plays a major role in the Dandengtongnao preparation, and existing preparation quality standard does not carry out the thin layer discriminating to these two kinds of medicinal materials.In addition, in the thin layer discriminating to root of kudzu vine medicinal material in the existing preparation quality standard, the preparation method of test sample soup is not ideal enough, and degree of separation is bad in discrimination process, and the spot colour developing is clear inadequately.So existing method of quality control can not effectively be controlled the quality of this dandengtongnao oral preparation, thereby will influence the clinical efficacy of said preparation.

Summary of the invention:

The objective of the invention is to: the method for quality control that a kind of dandengtongnao oral preparation is provided, this oral formulations comprises granule, capsule and tablet, the present invention is directed to shortcomings such as existing quality control standard is simple, product quality is wayward, increase and improved the thin layer discriminating of the red sage root, erigeron breviscapus and the root of kudzu vine, improve the quality control standard of dandengtongnao oral preparation, thereby guaranteed the clinical efficacy of said preparation.

Dandengtongnao oral preparation of the present invention is to constitute like this: calculate according to composition by weight: it mainly is prepared from according to following method by red sage root 500-600, erigeron breviscapus 500-600, Ligusticum wallichii 500-600 and root of kudzu vine 800-900: Ligusticum wallichii, the root of kudzu vine boiling 1-5 time, filter, merging filtrate, filtrate concentrates, add ethanol, stir evenly, be heated to 60 ℃, leave standstill, filter, decompression filtrate recycling ethanol is condensed into the thick paste shape, and vacuum drying below 85 ℃ becomes dried cream; The red sage root adds the 50-90% alcohol dipping 1-5 time, filters, and decompression filtrate recycling ethanol is condensed into the thick paste shape, and vacuum drying below 85 ℃ becomes dried cream; Erigeron breviscapus adds the 50-90% alcohol dipping 1-5 time, filters decompression filtrate recycling ethanol, put coldly, filter, filtrate is regulated the pH value to 1-3 with sulfuric acid solution, leave standstill, leaching yellow-green precipitate thing, being washed with water to the pH value is 5～7, oven dry below 85 ℃, mix with above-mentioned dried cream, pulverize, sieve, add auxiliary material (also can not add auxiliary material), make capsule, tablet or granule with conventional method again.

Described method of quality control mainly comprise in proterties, inspection, discriminating, the assay project partly or entirely; Wherein differentiate comprise with the Ligusticum wallichii control medicinal material differentiate Ligusticum wallichii medicinal material in the preparation, with the Puerarin reference substance differentiate root of kudzu vine medicinal material in the preparation, with Tanshinone I I _AReference substance is differentiated red rooted salvia in the preparation, is differentiated the thin layer discriminating of erigeron breviscapus medicinal material in the preparation with the scutellarin reference substance; Assay is that content of puerarin in the preparation is measured.

The discrimination method of Ligusticum wallichii is to be contrast with the Ligusticum wallichii control medicinal material in this preparation, is the thin layer discrimination method of developping agent with normal hexane: ethyl acetate=1-9:9-1.

The discrimination method of the root of kudzu vine is to be contrast with the Puerarin reference substance in this preparation, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is the thin layer discrimination method of developping agent.

The discrimination method of the red sage root is with Tanshinone I I in this preparation _AReference substance is the thin layer discrimination method of developping agent for contrast with toluene: ethyl acetate=10-30:0.5-5.

The discrimination method of erigeron breviscapus is to be contrast with the scutellarin reference substance in this preparation, is the thin layer discrimination method of developping agent with glacial acetic acid: ethanol=1-9:9-1.

Discrimination method comprises the part or all of of following project:

(1) gets content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor, heating and refluxing extraction, put coldly, filter, filter residue washs with above-mentioned mixed liquor, merge cleansing solution, evaporate to dryness, residue add chloroform makes dissolving, as need testing solution; Other gets the Ligusticum wallichii control medicinal material, adds diethyl ether, and heating and refluxing extraction filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol, and close plug is placed, and filters, and filtrate evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds ether, close plug, and shake well is placed, and filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds the ethyl acetate dissolving, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol, heating and refluxing extraction filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of entry low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same polyamide membrane, is developping agent with glacial acetic acid: ethanol=1-9:9-1, launches, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot.

Discrimination method comprises the part or all of of following project more specifically:

(1) gets each 1-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor 10-50ml, reflux 10-60 minute, put coldly, filter, filter residue washs with above-mentioned mixed liquor 2-10ml, merge cleansing solution, evaporate to dryness, residue add chloroform 0.5-5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.2-1g, the 5-50ml that adds diethyl ether, and reflux 0.5-5 hour, filter, filtrate evaporate to dryness, residue add ethyl acetate 0.5-5ml makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 0.5-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol 10-50ml, and close plug was placed 0.5-5 hour, filters, and filtrate evaporate to dryness, residue add methyl alcohol 0.5-3ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get each 1-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, and 10-50ml adds diethyl ether, close plug, shake well was placed 0.5-5 hour, filtered, filtrate evaporate to dryness, residue add ethyl acetate 0.5-3ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 1-5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 5-25 μ l of above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get each 1-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol 10-100ml, reflux 10-60 minute, filter, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 10-30ml low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 0.5-5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing each 2-20 μ l of above-mentioned need testing solution and reference substance solution puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=1-9:9-1 is developping agent, launch, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot.

The content of puerarin assay method is to be contrast with the Puerarin reference substance in this preparation, is the high performance liquid chromatography of moving phase with methyl alcohol or acetonitrile: water=10-90:90-10.

Content assaying method is:

Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90:90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds the 10-80% dissolve with ethanol, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize is put in the tool plug conical flask, the accurate 10-80% ethanolic solution that adds, claim to decide weight, heating and refluxing extraction is put cold, claim to decide weight again, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Content assaying method is more specifically:

Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90:90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 10-80% ethanol and makes the solution that every 1ml contains 10-100 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 10-80% ethanolic solution 10-100ml, claims to decide weight, reflux 10-60 minute, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5-25 μ l of need testing solution of drawing injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Described method of quality control comprises:

Proterties:

For capsule: the product content thing shows light yellowish brown to dark brown brown, bitter, puckery;

For tablet: medicine shows light yellowish brown to dark brown brown, bitter, puckery;

For granule: product is the particle of light yellowish brown to dark brown brown; It is sweet to distinguish the flavor of, little hardship, puckery;

Differentiate: (1) gets content, tablet or the granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor, heating and refluxing extraction, put coldly, filter, filter residue washs with above-mentioned mixed liquor, merge cleansing solution, evaporate to dryness, residue add chloroform makes dissolving, as need testing solution; Other gets the Ligusticum wallichii control medicinal material, adds diethyl ether, and heating and refluxing extraction filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(4) get content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol, heating and refluxing extraction filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of entry low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same polyamide membrane, is developping agent with glacial acetic acid: ethanol=1-9:9-1, launches, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Capsule of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item;

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90:90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds the 10-80% dissolve with ethanol, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize is put in the tool plug conical flask, the accurate 10-80% ethanolic solution that adds, claim to decide weight, heating and refluxing extraction is put cold, claim to decide weight again, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Find after deliberation, adopt the quality of following method of quality control, be more conducive to guarantee the clinical efficacy of this preparation easier control dandengtongnao oral preparation.So described method of quality control also can comprise:

Proterties:

Differentiate: (1) gets each 1-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor 10-50ml, reflux 10-60 minute, put coldly, filter, filter residue washs with above-mentioned mixed liquor 2-10ml, merge cleansing solution, evaporate to dryness, residue add chloroform 0.5-5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.2-1g, the 5-50ml that adds diethyl ether, and reflux 0.5-5 hour, filter, filtrate evaporate to dryness, residue add ethyl acetate 0.5-5ml makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(4) get each 1-5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol 10-100ml, reflux 10-60 minute, filter, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 10-30ml low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 0.5-5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing each 2-20 μ l of above-mentioned need testing solution and reference substance solution puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=1-9:9-1 is developping agent, launch, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90:90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 10-80% ethanol and makes the solution that every 1ml contains 10-100 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 10-80% ethanolic solution 10-100ml, claims to decide weight, reflux 10-60 minute, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5-25 μ l of need testing solution of drawing injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Method of quality control of the present invention is the preferred plan that just obtains through a large amount of experiment screenings, and following experimental study is a preferred process of the present invention.

One, puerarin content study on determination method

1, need testing solution preparation method research:

Method 1: the product of getting it filled, porphyrize is put in the tool plug conical flask, and accurate 30% ethanolic solution that adds claims decide weight, and reflux 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 30% ethanol, shakes up, and filtration is got subsequent filtrate, promptly.

Method 2: the product of getting it filled, porphyrize is put in the tool plug conical flask, and the accurate methanol solution that adds claims decide weight, and reflux 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up, and filtration is got subsequent filtrate, promptly.

Sample	Method 1 (mg/g)	Method 2 (mg/g)
Sample	Method 1 (mg/g)	Method 2 (mg/g)	1	5.104	4.841
2	5.195	4.990	1	5.104	4.841
2	5.195	4.990	3	5.049	4.852

According to test findings as can be known, employing method 1 preparation need testing solution, Puerarin extracts more complete.

2, the selection of moving phase:

Moving phase 1: the mixed solution with methyl alcohol and 2.5% acetic acid different proportion is a moving phase.

Moving phase 2: the mixed solution with methyl alcohol and 3% acetic acid different proportion is a moving phase.

Moving phase 3: the mixed solution with acetonitrile, methyl alcohol and Chinese holly Citron acid solution different proportion is a moving phase.

Moving phase 4: the mixed solution with first alcohol and water different proportion is a moving phase.

Moving phase 5: the mixed solution with acetonitrile and water different proportion is a moving phase.

The result: with methyl alcohol or acetonitrile: water=10-90:90-10 is moving phase; The negative sample chromatogram is at non-false positive peak, Puerarin position, and Puerarin separates fully (degree of separation〉1.5) with close impurity peaks, and promptly Puerarin separates with other components fully under this condition.Optimal flow is mutually: methyl alcohol: water=25:75.

3, repeated experiment:

The product of getting it filled prepare 5 parts of test liquids by the preparation method of test liquid under the method for quality control assay item of the present invention, and sample introduction is measured peak area, and result of calculation is listed following table in, and the Puerarin average content is 5.0275mg/g, and RSD is 0.72%.

The reappearance of Puerarin test in the preparation test sample

The sample introduction number of times	1	2	3	4	5	Mean value	RSD(％)
The sample introduction number of times	1	2	3	4	5	Mean value	RSD(％)	Content (mg/g)	5.0613	5.0422	4.9676	5.0441	5.0223	5.0275	0.72

According to the result as seen, this method reappearance is good.

4, recovery experiment:

Adopt the application of sample absorption method, it is an amount of that precision takes by weighing the preparation (average content 5.0275mg/g) of measuring content, porphyrize, put in the tool plug conical flask, accurate Puerarin reference substance solution (0.0505mg/ml) 25.00ml that adds adds 30% ethanol 25.00ml again, claim to decide weight, refluxing extraction 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with 30% ethanol, shake up, get supernatant and filter (0.45 μ m), make need testing solution.Prepare 5 parts respectively, measure, average recovery rate is 96.35%, and RSD is 0.60%.

Puerarin is measured recovery test in the need testing solution

Experiment number	Sample size (g)	Contain Puerarin (mg)	Add Puerarin (mg)	The amount of recording (mg)	The recovery (%)	Average recovery rate (%)	RSD(％)
Experiment number	Sample size (g)	Contain Puerarin (mg)	Add Puerarin (mg)	The amount of recording (mg)	The recovery (%)	Average recovery rate (%)	RSD(％)	1 2 3 4 5	0.2518 0.2503 0.2570 0.2543 0.2517	1.2659 1.2583 1.2920 1.2784 1.2654	1.2625	2.4706 2.4789 2.5083 2.4950 2.4893	95.42 96.68 96.34 96.36 96.94	96.35	0.60

Two, Ligusticum wallichii thin layer Study on Identification

With the Ligusticum wallichii medicinal material in the Ligusticum wallichii control medicinal material discriminating preparation

Need testing solution preparation method one: the product of getting it filled, porphyrize is put in the conical flask, adds chloroform-methanol (2:1) mixed liquor, reflux 30 minutes is put coldly, filters, and filter residue washs with above-mentioned mixed liquor, merge cleansing solution, evaporate to dryness, residue add the chloroform dissolving, as need testing solution; Lack the negative test liquid of Ligusticum wallichii with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, accurate claim surely, to put in the tool plug triangular flask, the accurate methyl alcohol that adds is weighed, and ultrasonic Extraction is put coldly, replenishes the weight that loses, and filters, and precision is measured subsequent filtrate, adds the methyl alcohol dilution, filters, promptly; Lack the negative test liquid of Ligusticum wallichii with the method preparation.

Developping agent is selected: respectively with the mixed solution of sherwood oil and ethyl acetate different proportion; The mixed solution of normal hexane, ethyl acetate different proportion is a developping agent.

The result: employing method one preparation need testing solution is a developping agent with normal hexane: ethyl acetate=1-9:9-1, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.Best developping agent is: normal hexane: ethyl acetate=9:1.

Three, root of kudzu vine thin layer Study on Identification

With the root of kudzu vine medicinal material in the Puerarin reference substance discriminating preparation

Need testing solution preparation method one: the thing of getting it filled, porphyrize is put in the conical flask, adds ethanol, and close plug was placed 2 hours, filters, the filtrate evaporate to dryness, residue adds dissolve with ethanol, as need testing solution; Lack the negative test liquid of the root of kudzu vine with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, porphyrize is put in the conical flask, adds methyl alcohol, and close plug was placed 2 hours, filters, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Lack the negative test liquid of the root of kudzu vine with the method preparation.

Need testing solution preparation method three: the product of getting it filled, put in the conical flask, add chloroform-methanol (2:1) mixed liquor, reflux 30 minutes is put coldly, filters, filter residue washs with above-mentioned mixed liquor, adds methyl alcohol, jolting, it is fully dissolved, filter the residue methanol wash, washing lotion and filtrate merge, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Lack the negative test liquid of the root of kudzu vine with the method preparation.

Need testing solution preparation method four: the product of getting it filled, put in the conical flask, add chloroform-methanol (2:1) mixed liquor, reflux 30 minutes is put coldly, filters, and filter residue washs with above-mentioned mixed liquor, the merging cleansing solution, evaporate to dryness, residue add chloroform makes dissolving, as need testing solution; Lack the negative test liquid of the root of kudzu vine with the method preparation.

Developping agent is selected: respectively with the mixed solution of chloroform, acetonitrile, water different proportion; The mixed solution of chloroform, methyl alcohol, water different proportion; The mixed solution of chloroform, methyl alcohol, water, glacial acetic acid different proportion is a developping agent.

The result: adopt method two to prepare need testing solution, with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.Best developping agent is: chloroform: methyl alcohol: water=5:2.5:0.25.

Adopt additive method to prepare need testing solution, the identification result degree of separation is bad, and the spot colour developing is unintelligible.

Four, red sage root Study on Identification

With Tanshinone I I _AReference substance is differentiated the red rooted salvia in the preparation

Need testing solution preparation method one: the thing of getting it filled, grind well, add chloroform dipping 1 hour, filter, filtrate evaporate to dryness, residue add the chloroform dissolving, filter, promptly; Lack the negative test liquid of the red sage root with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, porphyrize is put in the conical flask, adds diethyl ether, close plug, shake well was placed 1 hour, filtered, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, as need testing solution; Lack the negative test liquid of the red sage root with the method preparation.

Developping agent is selected: respectively with the mixed solution of benzene, ethyl acetate different proportion; The mixed solution of toluene, ethyl acetate different proportion; The mixed solution of normal hexane, ethyl acetate different proportion is a developping agent.

The result: adopting method two to prepare need testing solution, is developping agent with toluene: ethyl acetate=10-30:0.5-5, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.Best developping agent is: toluene: ethyl acetate=20:1.

Five, erigeron breviscapus Study on Identification

With the erigeron breviscapus medicinal material in the scutellarin reference substance discriminating preparation

Need testing solution preparation method one: the thing of getting it filled, porphyrize adds 50% ethanol, and sonicated filters, the filtrate evaporate to dryness, residue is dissolved in water, and aqueous solution is extracted 2 times with saturated normal butyl alcohol, merges normal butyl alcohol, evaporate to dryness, residue adds methyl alcohol, promptly; Lack the negative test liquid of erigeron breviscapus with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, porphyrize is put in the conical flask, add 50% ethanol, heating and refluxing extraction filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water low-grade fever, filters while hot, cooling, filtrate, are extracted 2 times with ethyl acetate to 1-2 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Lack the negative test liquid of erigeron breviscapus with the method preparation.

Developping agent is selected: respectively with the mixed solution of glacial acetic acid, ethanol different proportion; The mixed solution of ethyl acetate, butanone, methyl alcohol, water different proportion is a developping agent.

The result: adopting method two to prepare need testing solution, is developping agent with glacial acetic acid: ethanol=1-9:9-1, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.Best developping agent is: glacial acetic acid: ethanol=4:1.

Compared with prior art, the present invention is directed to shortcomings such as existing quality control standard is simple, product quality is wayward, increase and improved the thin layer discriminating of the red sage root, erigeron breviscapus and the root of kudzu vine, selected method degree of separation is good, favorable reproducibility, recovery height, measurement result is accurate, improve the quality control standard of dandengtongnao oral preparation, thereby guaranteed the clinical efficacy of said preparation.

Embodiment:

Further specify the present invention by the following examples, but not as limitation of the present invention.

Embodiments of the invention 1:

Proterties:

Differentiate: (1) gets each 2g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol=2:1 mixed liquor 25ml, reflux 30 minutes, put coldly, filter, filter residue washs with above-mentioned mixed liquor 5ml, merge cleansing solution, evaporate to dryness, residue add chloroform 1ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.5g, the 10ml that adds diethyl ether, and reflux 1 hour filters, and filtrate evaporate to dryness, residue add ethyl acetate 2ml makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2 μ l of above-mentioned need testing solution 15 μ l control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=9:1, launches, take out, dry, put under the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 1g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol 20ml, and close plug was placed 2 hours, filters, and filtrate evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution 5 μ l and reference substance solution 2 μ l difference ribbon point on same silica gel g thin-layer plate, with chloroform: methyl alcohol: water=5:2.5:0.25 is a developping agent, launch, take out, dry, put under the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get each 2g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, the 20ml that adds diethyl ether, and close plug, shake well was placed 1 hour, filtered, and filtrate evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 15 μ l and reference substance solution 5 μ l puts respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate=20:1 is developping agent, launch, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get each 2g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 50% ethanol 30ml, reflux 30 minutes filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 20ml low-grade fever, filters while hot, cooling, filtrate, are extracted 2 times with ethyl acetate to 1-2 with the hydrochloric acid adjust pH, each 15ml merges the ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 5 μ l and reference substance solution 2 μ l puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=4:1 is developping agent, launch, take out, dry, spray is with 2% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is the C18 post, is moving phase with methyl alcohol: water=25:75; The detection wavelength is 250nm; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 4000; Precision takes by weighing the Puerarin reference substance, adds 30% ethanol and makes the solution that every 1ml contains 40 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 30% ethanolic solution 50ml, claims to decide weight, reflux 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 30% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Embodiments of the invention 2:

Proterties:

Differentiate: (1) gets each 1g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: acetonitrile=1:9 mixed liquor 10ml, reflux 10 minutes, put coldly, filter, filter residue washs with above-mentioned mixed liquor 2ml, merge cleansing solution, evaporate to dryness, residue add chloroform 0.5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.2g, the 5ml that adds diethyl ether, and reflux 0.5 hour filters, and filtrate evaporate to dryness, residue add ethyl acetate 0.51 makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2 μ l of above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1:9, launches, take out, dry, put under the ultraviolet lamp 200nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 1g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 20% ethanol 10ml, reflux 10 minutes filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 10ml low-grade fever, filters while hot, cooling, filtrate, are extracted 1 time with ethyl acetate to 0.5-2 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 20 μ l and reference substance solution 10 μ l puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=1:9 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is the C8 post, is moving phase with acetonitrile: water=10:90; The detection wavelength is 200nm; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 10% ethanol and makes the solution that every 1ml contains 10 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 10% ethanolic solution 10ml, claims to decide weight, reflux 10 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 10% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Embodiments of the invention 3:

Proterties:

Differentiate: (1) gets each 0.5g of content, tablet or granule in the capsule respectively, and porphyrize is put in the conical flask, adds methyl alcohol 10ml, and close plug was placed 0.5 hour, filters, and filtrate evaporate to dryness, residue add methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution 20 μ l and reference substance solution 10 μ l difference ribbon point on same silica gel g thin-layer plate, with chloroform: acetonitrile: water=1:10:0.1 is a developping agent, launch, take out, dry, put under the ultraviolet lamp 200nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(2) get each 1g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, the 10ml that adds diethyl ether, and close plug, shake well was placed 0.5 hour, filtered, and filtrate evaporate to dryness, residue add ethyl acetate 0.5ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 25 μ l and reference substance solution 15 μ l puts respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate=10:5 is developping agent, launch, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is the C4 post, is moving phase with methyl alcohol: water=90:10; The detection wavelength is 500nm; Flow velocity: 1.2ml/min; Column temperature: 60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 80% ethanol and makes the solution that every 1ml contains 100 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 80% ethanolic solution 100ml, claims to decide weight, reflux 60 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 25 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Embodiments of the invention 4:

Proterties:

Differentiate: (1) gets each 5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol=9:1 mixed liquor 50ml, reflux 60 minutes, put coldly, filter, filter residue washs with above-mentioned mixed liquor 10ml, merge cleansing solution, evaporate to dryness, residue add chloroform 5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 1g, the 50ml that adds diethyl ether, and reflux 5 hours filters, and filtrate evaporate to dryness, residue add ethyl acetate 5ml makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 20 μ l of above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=6:4, launches, take out, dry, put under the ultraviolet lamp 500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol 50ml, and close plug was placed 5 hours, filters, and filtrate evaporate to dryness, residue add methyl alcohol 3ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution 15 μ l and reference substance solution 8 μ l difference ribbon point on same silica gel g thin-layer plate, with chloroform: methyl alcohol: water=10:1:1 is a developping agent, launch, take out, dry, put under the ultraviolet lamp 500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get each 5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, add 85% ethanol 100ml, reflux 60 minutes filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 30ml low-grade fever, filters while hot, cooling, filtrate, are extracted 5 times with ethyl acetate to 1-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 15 μ l and reference substance solution 8 μ l puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=9:1 is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Capsule of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item.

Embodiments of the invention 5:

Proterties:

Differentiate: get each 5g of content, tablet or granule in the capsule respectively, porphyrize is put in the conical flask, the 50ml that adds diethyl ether, and close plug, shake well was placed 5 hours, filtered, and filtrate evaporate to dryness, residue add ethyl acetate 3ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing above-mentioned need testing solution 20 μ l and reference substance solution 10 μ l puts respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate=30:0.5 is developping agent, launch, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is the C18 post, is moving phase with acetonitrile: water=70:30; The detection wavelength is 365nm; Flow velocity: 1.0ml/min; Column temperature: 50 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 50% ethanol and makes the solution that every 1ml contains 70 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or granule in the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 50% ethanolic solution 80ml, claims to decide weight, reflux 40 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 15 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.

Claims

The detection method of [claim 1] a kind of dandengtongnao oral preparation, this oral formulations comprises granule, capsule and tablet, is made up of the red sage root, erigeron breviscapus, Ligusticum wallichii and the root of kudzu vine, described detection method is proterties, inspection, discriminating and assay; It is characterized in that: discrimination method is:

(1) gets content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor, heating and refluxing extraction, put coldly, filter, filter residue washs with above-mentioned mixed liquor, merge cleansing solution, evaporate to dryness, residue add chloroform makes dissolving, as need testing solution; Other gets the Ligusticum wallichii control medicinal material, adds diethyl ether, and heating and refluxing extraction filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol, and close plug is placed, and filters, and filtrate evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds ether, close plug, and shake well is placed, and filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds the ethyl acetate dissolving, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol, heating and refluxing extraction filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of entry low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same polyamide membrane, is developping agent with glacial acetic acid: ethanol=1-9:9-1, launches, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot.
[claim 2] is characterized in that according to the detection method of the described dandengtongnao oral preparation of claim 1: discrimination method is:

(1) gets each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor 10-50ml, reflux 10-60 minute, put coldly, filter, filter residue washs with above-mentioned mixed liquor 2-10ml, merge cleansing solution, evaporate to dryness, residue add chloroform 0.5-5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.2-1g, the 5-50ml that adds diethyl ether, and reflux 0.5-5 hour, filter, filtrate evaporate to dryness, residue add ethyl acetate 0.5-5ml makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 0.5-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol 10-50ml, and close plug was placed 0.5-5 hour, filters, and filtrate evaporate to dryness, residue add methyl alcohol 0.5-3ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, and 10-50ml adds diethyl ether, close plug, shake well was placed 0.5-5 hour, filtered, filtrate evaporate to dryness, residue add ethyl acetate 0.5-3ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 1-5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 5-25 μ l of above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol 10-100ml, reflux 10-60 minute, filter, filtrate is waved to there not being the alcohol flavor, residue adds the dissolving of water 10-30ml low-grade fever, filter while hot, cooling, filtrate uses the hydrochloric acid adjust pH to 0.5-3, extract 1-5 time with ethyl acetate, merge ethyl acetate and carry

Get liquid, evaporate to dryness, residue add methyl alcohol 0.5-5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing each 2-20 μ l of above-mentioned need testing solution and reference substance solution puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=1-9:9-1 is developping agent, launch, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot.
[claim 3] is characterized in that according to the detection method of the described dandengtongnao oral preparation of claim 1: described detection method is:

Proterties:

For capsule: the product content thing shows light yellowish brown to dark brown brown, bitter, puckery;

For tablet: medicine shows light yellowish brown to dark brown brown, bitter, puckery;

For granule: product is the particle of light yellowish brown to dark brown brown; It is sweet to distinguish the flavor of, little hardship, puckery;

Differentiate: (1) gets content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor, heating and refluxing extraction, put coldly, filter, filter residue washs with above-mentioned mixed liquor, merge cleansing solution, evaporate to dryness, residue add chloroform makes dissolving, as need testing solution; Other gets the Ligusticum wallichii control medicinal material, adds diethyl ether, and heating and refluxing extraction filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, in contrast medicinal material solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and control medicinal material solution, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=1-9:9-1, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol, and close plug is placed, and filters, and filtrate evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, adds ether, close plug, and shake well is placed, and filters, and filtrate evaporate to dryness, residue add ethyl acetate makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds the ethyl acetate dissolving, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get content, tablet or the granule of capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol, heating and refluxing extraction filters, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of entry low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned need testing solution and reference substance solution, putting respectively on same polyamide membrane, is developping agent with glacial acetic acid: ethanol=1-9:9-1, launches, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Capsule of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item;

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90: 90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds the 10-80% dissolve with ethanol, shakes up, and promptly gets reference substance solution; Get content, tablet or the granule of the capsule under the content uniformity item, porphyrize is put in the tool plug conical flask, the accurate 10-80% ethanolic solution that adds, claim to decide weight, heating and refluxing extraction is put cold, claim to decide weight again, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.
[claim 4] is characterized in that according to the detection method of the described dandengtongnao oral preparation of claim 3: described detection method is:

Proterties:

For capsule: the product content thing shows light yellowish brown to dark brown brown, bitter, puckery;

For tablet: medicine shows light yellowish brown to dark brown brown, bitter, puckery;

For granule: product is the particle of light yellowish brown to dark brown brown; It is sweet to distinguish the flavor of, little hardship, puckery;

Differentiate: (1) gets each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, adds chloroform: methyl alcohol or acetonitrile=1-9:9-1 mixed liquor 10-50ml, reflux 10-60 minute, put coldly, filter, filter residue washs with above-mentioned mixed liquor 2-10ml, merge cleansing solution, evaporate to dryness, residue add chloroform 0.5-5ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.2-1g, the 5-50ml that adds diethyl ether, and reflux 0.5-5 hour, filter, filtrate evaporate to dryness, residue add ethyl acetate 0.5-5ml makes dissolving, in contrast medicinal material solution; According to the test of Chinese Pharmacopoeia thin-layered chromatography, draw above-mentioned test sample

Each 2-20 μ l of solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with normal hexane: ethyl acetate=1-9:9-1 is developping agent, launch, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot;

(2) get each 0.5-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, adds methyl alcohol 10-50ml, and close plug was placed 0.5-5 hour, filters, and filtrate evaporate to dryness, residue add methyl alcohol 0.5-3ml makes dissolving, as need testing solution; Other gets the Puerarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 2-20 μ l of above-mentioned need testing solution and reference substance solution, ribbon is put on same silica gel g thin-layer plate respectively, and with chloroform: methyl alcohol or acetonitrile: water=1-10:1-10:0.1-1 is a developping agent, launches, take out, dry, put under the ultraviolet lamp 200-500nm and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show same color fluorescence spot;

(3) get each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, and 10-50ml adds diethyl ether, close plug, shake well was placed 0.5-5 hour, filtered, filtrate evaporate to dryness, residue add ethyl acetate 0.5-3ml makes dissolving, as need testing solution; Other gets Tanshinone I I _AReference substance adds ethyl acetate and makes the solution that every 1ml contains 1-5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw each 5-25 μ l of above-mentioned need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developping agent with toluene: ethyl acetate=10-30:0.5-5, launches, take out, dry, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

(4) get each 1-5g of content, tablet or granule of capsule respectively, porphyrize is put in the conical flask, add 20-85% ethanol 10-100ml, reflux 10-60 minute, filter, filtrate is waved to there not being the alcohol flavor, and residue adds the dissolving of water 10-30ml low-grade fever, filters while hot, cooling, filtrate, are extracted 1-5 time with ethyl acetate to 0.5-3 with the hydrochloric acid adjust pH, merge the ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 0.5-5ml makes dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-3mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, drawing each 2-20 μ l of above-mentioned need testing solution and reference substance solution puts respectively on same polyamide membrane, with glacial acetic acid: ethanol=1-9:9-1 is developping agent, launch, take out, dry, spray is with 1-5% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

Check:

Capsule of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item;

Assay: Puerarin shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with methyl alcohol or acetonitrile: water=10-90:90-10; The detection wavelength is 200-500nm; Flow velocity: 0.8-1.2ml/min; Column temperature: 30-60 ℃; Number of theoretical plate calculates by puerarin peak should be not less than 3000; Precision takes by weighing the Puerarin reference substance, adds 10-80% ethanol and makes the solution that every 1ml contains 10-100 μ g, shakes up, and promptly gets reference substance solution; Get content, tablet or the granule of the capsule under the content uniformity item, porphyrize, precision takes by weighing 0.5g, put in the tool plug conical flask, precision adds 10-80% ethanolic solution 10-100ml, claims to decide weight, reflux 10-60 minute, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 10-80% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5-25 μ l of need testing solution of drawing injects hplc determination content; In this oral formulations, contain the root of kudzu vine in the capsule, must not be less than 35mg/g in Puerarin; Contain the root of kudzu vine in the tablet in Puerarin, must not be less than 30mg/g; Contain the root of kudzu vine in the granule in Puerarin, must not be less than 4mg/g.