CN101708223B

CN101708223B - Preparation method, quality control method and application for Chinese medicinal compound indigowoad leaf preparation

Info

Publication number: CN101708223B
Application number: CN2009102300033A
Authority: CN
Inventors: 郭桂秋; 车蕾; 翟勇; 陈树恩; 李钦刚; 肖金金; 寇丽丽; 王丽庆; 王中成
Original assignee: RONGCHANG PHARMACEUTICAL (ZIBO) CO Ltd
Current assignee: RONGCHANG PHARMACEUTICAL (ZIBO) CO Ltd
Priority date: 2009-11-04
Filing date: 2009-11-04
Publication date: 2011-09-21
Anticipated expiration: 2029-11-04
Also published as: CN101708223A

Abstract

The invention relates to a preparation method and a quality control method for a compound indigowoad leaf preparation. The preparation method comprises a step of preparing a basic remedy and a step of preparing a corresponding preparation. The basic remedy in the basic remedy preparation comprises the following compositions in part by weight: 360 to 440 parts of indigowoad leaves, 180 to 220 parts of lonicera confusa or honeysuckle flower, 90 to 110 parts of incised notopterygium rhizome, 90 to 110 parts of bistort rhizome, and 90 to 110 parts of rhubarb; the medicinal materials are decocted twice with conventional amount of water for 1 hour each time; the decoctions are mixed and filtered; the filtrate is concentrated to the relative density of between 1.08 and 1.32 at 60 DEG C; ethanol is added into the filtrate to ensure that alcohol content reaches 50 to 60 percent, the mixture is stood and is filtered, the filtrate is subjected to ethanol reclamation and is concentrated to an extract with the relative density of between 1.17 and 1.43 at 60 DEG C for later use; and the corresponding preparation is prepared according to a drug specification. The quality control method comprises the following steps of the identification of contents and the content determination of the contained compositions including the identification of the indigowoad leaves, the identification of the lonicera confusa or honeysuckle flower, the identification of the rhubarb, the identification of the incised notopterygium rhizome, the total content determination of emodin and chrysophanol in the rhubarb, and the content determination of chlorogenic acid. The methods can be applied to preparation of medicinal preparations for treating cold, influenza, parotitis and acute viral hepatitis.

Description

The detection method of Chinese medicine compound prescription indigowoad leaf preparation

Technical field

The present invention relates to a kind of detection method of Chinese medicine preparation, be specifically related to the detection method of 'Compound satis Leaf '.

Background technology

'Compound satis Leaf ' is a kind of compound Chinese medicinal preparation of having gone on the market, and has wind and heat dispersing, removing toxicity for detumescence, the effect of cool blood cholagogic.Be mainly used in cold, fever headache clinically, swell and ache under the pharyngitis, ear, diseases such as hypochondriac pain jaundice, and influenza, mumps, acute viral hepatitis are seen the person that has the above-mentioned symptom.But existing 'Compound satis Leaf ' standard falls behind, and contains the sucrose amount up to 46%, and quality control index is less, only has indigo red, chlorogenic acid, rheum officinale to differentiate item, does not have the content of comprehensive assay.The method of quality control that is prior art can not effectively be controlled the quality of 'Compound satis Leaf ', thereby will influence the production of product and ensure the quality of products.

Summary of the invention

Purpose of the present invention is to provide a kind of quality controllable detection method of improved 'Compound satis Leaf '.

Technical scheme of the present invention is:

Develop a kind of detection method of Chinese medicine compound prescription indigowoad leaf preparation, it is characterized in that described detection method mainly is the discriminating of content and the assay that contains composition:

A. the step of the discriminating of content

(1) discriminating of folium isatidis

Sampling amount: oral liquid 10ml; Or the granule of corresponding crude drug amount, tablet, capsule, pill;

Oral liquid is directly used in ethyl acetate or methenyl choloride extracts; Operate with method again after other water 10ml of formulation elder generation dissolvings;

Get oral liquid 10ml, extract 2 times with ethyl acetate or methenyl choloride, each 10ml, combined ethyl acetate or methenyl choloride extract, with 1% sodium hydroxide solution washing 2 times, each 10ml, ethyl acetate or methenyl choloride liquid are put evaporate to dryness in the water-bath, residue adds ethyl acetate or methenyl choloride 1ml makes dissolving, as need testing solution; Other gets the indigo red reference substance, adds ethyl acetate or methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw need testing solution 5～10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, mixed liquor with toluene-ethyl acetate of 5: 4: 1-acetone is a developping agent, launch, take out, dry; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show identical aubergine spot;

(2) discriminating of Flos Lonicerae or honeysuckle

Oral liquid is directly used in by following step and is undertaken; After other water 10ml of formulation elder generation dissolvings, add hydrochloric acid and transfer pH value, operate with method again;

Get oral liquid 10ml, add 2～4 of hydrochloric acid, making the solution pH value is 1～2, extracts 3 times each 20ml with ethyl acetate or methenyl choloride, combined ethyl acetate or methenyl choloride extract, evaporate to dryness, residue add water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, be added on polyamide column, wet method dress post, column length 20cm, internal diameter 15～20mm, transferring pH value with ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, and eluent evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, filter in case of necessity, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5ma, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 1～2 μ l of need testing solution and reference substance solution, putting respectively on same polyamide film, is developping agent with glacial acetic acid-aqueous solution of 1: 4 or 20: 6: 0.8 methenyl choloride-methyl alcohol-formic acid solutions, launches, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Perhaps get oral liquid 10ml, be added on 30～60 orders, on the internal diameter 0.9cm polyamide column, water 50ml wash-out discards eluent, uses 50% ethanol elution again, collects eluent, and evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution;

According to the check of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol of 20: 6: 0.8-formic acid is that developping agent launches, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(3) discriminating of rheum officinale

Oral liquid is directly used in by following step and is undertaken; After other water 10ml of formulation elder generation dissolvings, add hydrochloric acid again and transfer pH value;

Get oral liquid 10ml, add hydrochloric acid 1ml, put and heat 30min in the water-bath, extracted by ether 2 times of cooling back, each 20ml merges ether extracted liquid, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution;

Other gets rheum officinale control medicinal material 0.5g, adds methyl alcohol 10ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add water 10ml makes dissolving, adds hydrochloric acid 1ml, shines medicinal material solution in pairs with legal system; Get archen, Chrysophanol reference substance again, add ethyl acetate and make the mixed solution that every 1ml contains 1mg, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, upper solution with 30～60 ℃ of sherwood oils of 15: 5: 1-formic acid second fat-formic acid is that developping agent launches, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show five identical orange-yellow fluorescence spots; Put in the ammonia and smoke, spot becomes redness;

(4) discriminating of notopterygium root

Oral liquid is directly used in by following step and is undertaken; After other water 10ml of formulation elder generation dissolvings, use ethyl acetate extraction again;

Get oral liquid 10ml, with ethyl acetate or 60～90 ℃ of Petroleum ether extraction 4 times, each 10ml, combined ethyl acetate or 60～90 ℃ of petroleum ether extracts, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution; Other gets notopterygium root control medicinal material 1g, adds ethanol 30ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medicinal material solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5～8 μ l of need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, upper solution with ethyl acetate-methanol-waters of 10: 2: 3 is a developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

Or normal hexane-benzene of 2: 1: 1-ethyl acetate mixed solvent is a developping agent; Or 4: 1 normal hexane-ethyl acetate mixed solvent is a developping agent; Launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

B. the composition that contains is carried out the step of assay according to an appendix VID of Chinese Pharmacopoeia version in 2005 high performance liquid chromatography

1, the assay method of archen and Chrysophanol total content in the rheum officinale

(1) chromatographic condition and system suitability test

With octadecylsilane chemically bonded silica is filling agent; 85: 15 methyl alcohol-0.1% phosphoric acid solution is a moving phase; Detect wavelength 254nm; Number of theoretical plate calculates by archen, Chrysophanol peak all must not be lower than 4000;

(2) preparation of reference substance solution

Precision takes by weighing archen, the Chrysophanol reference substance is an amount of, adds moving phase and makes the mixing reference substance solution that every 1ml contains archen 30 μ g, contains Chrysophanol 50 μ g, and is standby;

(3) preparation of need testing solution

Accurate this product 5ml that draws adds hydrochloric acid 1ml, chloroform 30ml, and reflux 30min is put cold, divide and get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform extract, water bath method, residue adds the mutual-assistance dissolving of flowing, and is settled in the 10ml measuring bottle, shakes up, and filters, and is standby;

(4) determination method

Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;

The every 1ml of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅) and Chrysophanol (C ₁₅H ₁₀O ₄) the sum meter, must not be less than 0.1mg;

2, chlorogenic acid contents assay method

(1) chromatographic condition and system suitability test

With octadecylsilane chemically bonded silica is filling agent; 15: 85 methyl alcohol-0.4% phosphoric acid solution is a moving phase; Detect wavelength 324nm; Number of theoretical plate is pressed the chlorogenic acid peak and is calculated, and must not be lower than 3000;

(2) preparation of reference substance solution

It is an amount of that precision takes by weighing the chlorogenic acid reference substance, adds 50% methyl alcohol and make the reference substance solution that every 1ml contains 20 μ g, preserves below 10 ℃;

(3) preparation of need testing solution

Accurate this product 1ml that draws puts in the brown measuring bottle of 50ml, adds 50% methyl alcohol and is diluted to scale, shakes up, and is standby;

(4) determination method

Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly.

The every 1ml of this product contains chlorogenic acid (C ₁₆H ₁₈O ₉) must not be less than 0.6mg.

In the detection method of above-mentioned Chinese medicine compound prescription indigowoad leaf preparation, the preparation method of described Chinese medicine compound prescription indigowoad leaf preparation comprises main ingredient preparation and preparation two steps of corresponding preparations, it is characterized in that:

(1) main ingredient preparation

Main ingredient components by weight percent proportioning is 360～440 parts of folium isatidis, Flos Lonicerae or honeysuckle 180～220g part, 90～110 parts of notopterygium roots, 90～110 parts of adder-worts, 90～110 parts of rheum officinales;

More than all flavor medicinal materials, add the convention amount decocting and boil twice, each 1h; Merge decoction liquor, filter, relative density 1.08～1.32 when filtrate was concentrated into 60 ℃; Add ethanol and make and contain alcohol amount and reach 50～60%, leave standstill, filter, the filtrate recycling ethanol and the clear cream of relative density 1.17～1.43 when being concentrated into 60 ℃, standby;

(2) preparation corresponding preparations

1. prepare oral liquid

Upwards go on foot clear cream and add 2～3 times of water, stir evenly, refrigeration 〉=12h filters, and filtrate heated and boiled 0.5h when treating that temperature is reduced to 60 ℃, adds antiseptic, and refrigeration 〉=12h filters; Add flavouring, stir evenly, make medicine stoste, standby;

The weight proportion of adjuvant is: main ingredient total amount: antiseptic: flavouring=1: 0.003～0.004: 0.14～0.16;

In the agent of tender flavor, sucrose: non-nutritive or net heat value flavouring=1: 0.04～0.05,

Non-nutritive or net heat value flavouring be from xylitol, antierythrite, and glycyrrhizic acid is selected in honey element or the stevioside;

Dosage sucrose adds water boil and makes syrup, adds non-nutritive again or the net heat value flavouring makes dissolving, merges with medicine stoste, stirs evenly; Add water to 1000 parts of cumulative volumes, by the drug specifications can, sterilization, promptly;

2. prepare granule

Last clear cream of step≤100 ℃ of dryings are also made fine powder, add suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, and above-mentioned dosage can make 1000 parts of particles, by the drug specifications packing, and sealing, promptly;

3. prepare tablet

Last clear cream of step≤100 ℃ of dryings are also made fine powder, add suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, compressing tablet, and sugar coating or film-coating, above-mentioned dosage can make 1000 in tablet, by the drug specifications packing, sealing, promptly;

4. prepare capsule

Last clear cream of step is also made fine powder, adds suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, is filled in the capsulae vacuus, and above-mentioned dosage can make 1000 of capsules, by the drug specifications packing, and sealing, promptly;

5. prepare pill

Last clear cream of step≤100 ℃ of dryings are also made fine powder, add suitable pharmaceutic adjuvant, and to add water or honey and water be the binder mixing and make sphere or the class spherical pellets ,≤80 ℃ of dryings by the pharmacy routine; Sugar coating or film-coating, above-mentioned dosage can make 1000 of pills, by the drug specifications packing, and sealing, promptly.

The preparation method of above-mentioned Chinese medicine compound prescription indigowoad leaf preparation is characterized in that main ingredient components by weight percent proportioning is, 380～420 parts of folium isatidis, Flos Lonicerae or honeysuckle 190～210g part, 95～105 parts of notopterygium roots, 95～105 parts of adder-worts, 95～105 parts of rheum officinales.

The application of above-mentioned Chinese medicine compound prescription indigowoad leaf preparation is characterized in that the application in preparation treatment flu, influenza, mumps, acute viral hepatitis pharmaceutical preparation.

For effectively controlling product quality, we have set up the detection method of new 'Compound satis Leaf ', and this method adopts thin-layered chromatography that composition is wherein differentiated, the employing high performance liquid chromatography is carried out assay to composition wherein; And reduced the sugar content of 'Compound satis Leaf '.

Up to now, do not find report that this target level of product quality is studied comprehensively as yet, only have indivedual researchers that wherein chlorogenic acid or rheum officinale were done research separately.

Advantage of the present invention is:

1. reduce cane sugar content in the method for making, reduce to 15% by 46%, both economized on resources, reduce production costs, meet the requirement of modern's healthy living again.

2. this detection method is comprehensive, and precision, sensitivity, stability are all good, and method is simpler and more direct, can guarantee that constant product quality is controlled.

3. function cures mainly with usage, consumption and is equal to prior art, and is easy to utilize.

Above compound indigowoad leaf prescription is formed and is that large-scale production can be unit with the kilogram by weight ratio, or is unit with the ton.

More than composition can be made into 1000 doses of pharmaceutical preparations, described 1000 doses of fingers, and the final drug preparation of making, as make liquid preparation 1000ml, and 1000 of capsule preparations or pills, 1000 in tablet, granule 1000g etc.,

Description of drawings

Fig. 1 is the archen reference substance area normalization high performance liquid chromatogram collection of illustrative plates of embodiment 1;

Fig. 2 is the Chrysophanol reference substance area normalization high performance liquid chromatogram collection of illustrative plates of embodiment 1;

Fig. 3 is the archen typical curve of embodiment 1;

Fig. 4 is the Chrysophanol typical curve of embodiment 1;

Fig. 5 is negative control test-archen, the Chrysophanol reference substance high-efficient liquid phase chromatogram of embodiment 1;

Fig. 6 is the omnidistance high-efficient liquid phase chromatogram of the negative control test-negative sample of embodiment 1;

Fig. 7 is the omnidistance high-efficient liquid phase chromatogram of the negative control test-rhubarb medicinal material of embodiment 1;

Fig. 8 is the omnidistance high-efficient liquid phase chromatogram of the negative control test-sample of embodiment 1;

Fig. 9 is the negative control test-chlorogenic acid reference substance high-efficient liquid phase chromatogram of embodiment 1;

Figure 10 is the negative control test-negative sample high-efficient liquid phase chromatogram of embodiment 1;

Figure 11 is the negative control test-sample high-efficient liquid phase chromatogram of embodiment 1;

Figure 12 is embodiment 1 a chlorogenic acid reference substance typical curve.

Embodiment

Below in conjunction with accompanying drawing and example the present invention is further set forth.

Among Fig. 1, reference substance: the archen reference substance is packed 20mg, lot number: 110756-200110) available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Area normalization method is measured, and this product purity is 100%.

Among Fig. 2, reference substance: the Chrysophanol reference substance is packed 20mg, lot number: 110796-200110) available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Area normalization method is measured, and this product purity is 100%.

Fig. 3, Fig. 4 are respectively the typical curve of archen, Chrysophanol.Ordinate is a peak area, and horizontal ordinate is a sample size, and the result shows archen in 0.340 μ g～1.700 μ g scopes, and Chrysophanol is in 0.380 μ g～1.900 μ g scopes, and peak area value and sample size have good linear relationship.

Fig. 5 is negative control test-archen, Chrysophanol reference substance high-efficient liquid phase chromatogram;

Fig. 6 is the omnidistance high-efficient liquid phase chromatogram of negative sample of removing rhubarb medicinal material;

Fig. 7 is the omnidistance high-efficient liquid phase chromatogram of rhubarb medicinal material;

Fig. 8 is the omnidistance high-efficient liquid phase chromatogram of sample;

Fig. 9 is a chlorogenic acid reference substance high-efficient liquid phase chromatogram;

Figure 10 is the negative sample high-efficient liquid phase chromatogram that does not contain Flos Lonicerae;

Figure 11 is the sample high-efficient liquid phase chromatogram;

Figure 12 is the typical curve of chlorogenic acid.Ordinate is a peak area, and horizontal ordinate is a sample size, and the result shows chlorogenic acid in 0.20 μ g～0.80 μ g scope, and peak area value and sample size have good linear relationship.

Embodiment 1

(1) main ingredient preparation

Main ingredient proportioning: folium isatidis 360g, Flos Lonicerae 180g, notopterygium root 100g, adder-wort 90g, rheum officinale 100g.

(2) preparation oral liquid

Upwards go on foot clear cream and add 2～3 times of water, stir evenly, refrigeration 〉=12h filters, and filtrate heated and boiled 0.5h when treating that temperature is reduced to 60 ℃, adds antiseptic, and refrigeration 〉=12h filters; Add flavouring and syrup, stir evenly, make medicine stoste, standby;

The weight proportion of adjuvant is: main ingredient total amount: antiseptic: sweetener=1: 0.003～0.004: 0.14～0.16;

In the sweetener, sucrose: non-nutritive or net heat value flavouring=1: 0.04～0.05,

Dosage sucrose adds water boil and makes syrup, adds non-nutritive again or the net heat value flavouring makes dissolving, merge with medicine stoste,

Stir evenly; Add water to 1000 parts of cumulative volumes, by the drug specifications can, sterilization, promptly;

(3) discriminating of content and contain the assay of composition:

A1. the step of the discriminating of content

(1) discriminating of folium isatidis

(2) discriminating of Flos Lonicerae

Get oral liquid 10ml, add 2～4 of hydrochloric acid, making the solution pH value is 1～2, extracts 3 times each 20ml with ethyl acetate or methenyl choloride, combined ethyl acetate or methenyl choloride extract, evaporate to dryness, residue add water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, be added on polyamide column, wet method dress post, column length 20cm, internal diameter 15～20mm, transferring pH value with ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, and eluent evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, filter in case of necessity, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution;

Perhaps get this product 10ml, be added on the polyamide column (30～60 orders, 3g, internal diameter 0.9cm), water 50ml wash-out discards eluent, uses 50% ethanol elution again, collects eluent, and evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Check according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8) is 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 the fluorescence spot of same color.

(3) discriminating of rheum officinale

Oral liquid is directly used in by following step and is undertaken; After other water 10ml of formulation elder generation dissolvings, add hydrochloric acid again and transfer pH value.

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, be developping agent with the upper solution of 30～60 ℃ of sherwood oils of 15: 5: 1-formic acid second fat-formic acid, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show five identical orange-yellow fluorescence spots; Put in the ammonia and smoke, spot becomes redness;

(4) discriminating of notopterygium root

Oral liquid is directly used in by following step and is undertaken; After other water 10ml of formulation elder generation dissolvings, use ethyl acetate extraction again.

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5～8 μ l of need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, be developping agent with the upper solution of ethyl acetate-methanol-waters of 10: 2: 3, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

Or be developping agent with normal hexane-benzene-ethyl acetate mixed solvent of 2: 1: 1; Or be developping agent with normal hexane-ethyl acetate mixed solvent of 4: 1; Launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

Or the A2. content is differentiated the screening of step

(1) discriminating of folium isatidis

Method 1

Oral liquid 10ml is got in the preparation of need testing solution, uses ethyl acetate extraction 2 times, each 10ml, the combined ethyl acetate extract, with 1% sodium hydroxide solution washing 2 times, each 10ml, acetic acid ethyl fluid is put evaporate to dryness in the water-bath, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution;

The indigo red reference substance is got in the preparation of reference substance solution, adds ethyl acetate and makes the solution that every 1ml contains 0.05mg, in contrast product solution.

Folium isatidis during the preparation of negative solution will be write out a prescription is removed, and other four flavors are prepared the negative control sample by the method for making item among the embodiment 1; Preparation method according to need testing solution prepares with method then.

Developping agent: toluene-ethyl acetate-acetone (5: 4: 1)

Developing the color and inspecting does not need developer, dries under the daylight and inspects.

As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show identical aubergine spot, negative control is noiseless.

Method 2

Oral liquid 10ml is got in the preparation of need testing solution, extracts 2 times with methenyl choloride, each 10ml, merge the methenyl choloride extract, with 1% sodium hydroxide solution washing 2 times, each 10ml, methenyl choloride liquid is put evaporate to dryness in the water-bath, and residue adds methenyl choloride 1ml makes dissolving, as need testing solution;

The indigo red reference substance is got in the preparation of reference substance solution, adds methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution.

Other operations are with method 1

(2) discriminating of Flos Lonicerae or honeysuckle

Method 1

Oral liquid 10ml is got in the preparation of need testing solution, add 2～4 of hydrochloric acid, making the solution pH value is 1-2, with ethyl acetate extraction 3 times, each 20ml, combined ethyl acetate liquid evaporate to dryness, residue adds water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, be added on the polyamide column (wet method dress post, column length 20cm, internal diameter 15-20mm), transferring pH value in order to ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, eluent evaporate to dryness, residue add methyl alcohol 1ml makes dissolving (filtering in case of necessity), as need testing solution.

The chlorogenic acid reference substance is got in the preparation of reference substance solution, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.

Flos Lonicerae during the preparation of negative solution will be write out a prescription is removed, and other four flavors are prepared the negative control sample by the method for making item among the embodiment 1; Preparation method according to need testing solution prepares with method then.

Developping agent: glacial acetic acid-aqueous solution (1: 4)

Developing the color and inspecting does not need developer, dries, and inspects under the uviol lamp (365nm); Place after 2 hours and observe, effect is better.

As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, negative control is noiseless.

Method 2

Oral liquid 10ml is got in the preparation of need testing solution, add 2～4 of hydrochloric acid, making the solution pH value is 1-2, extract 3 times with methenyl choloride, each 20ml, methenyl choloride liquid, evaporate to dryness, residue adds water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, is added on polyamide column (wet method dress post, column length 20cm, internal diameter 15-20mm) on, transferring pH value in order to ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, the eluent evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving (filtering in case of necessity), as need testing solution.

Other operations are with method 1.

Method 3

Oral liquid 10ml is got in the preparation of need testing solution, is added on the polyamide column (30～60 orders, 3g, internal diameter 0.9cm), water 50ml wash-out discards eluent, uses 50% ethanol elution again, collects eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.

The chlorogenic acid reference substance is got in the preparation of reference substance solution in addition, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8) is 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 the fluorescence spot of same color.

Developping agent: methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8).

Other operations are with method 1.

(3) discriminating of rheum officinale

The preparation sampling amount of need testing solution: oral liquid 10ml; Or the granule of corresponding crude drug amount, tablet, capsule, pill;

Get this product 10ml, add hydrochloric acid 1ml, put and heat 30min in the water-bath, extracted by ether 2 times of cooling back, each 20ml merges ether solution, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution.

Rheum officinale control medicinal material 0.5g is got in the preparation of control medicinal material solution, adds methyl alcohol 10ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add water 10ml makes dissolving, adds hydrochloric acid 1ml, makes control medicinal material solution with the need testing solution preparation method.

Archen reference substance, Chrysophanol reference substance are got in the preparation of reference substance solution, add ethyl acetate and make the mixed solution that every 1ml contains 1mg, in contrast product solution.

Rheum officinale during the preparation of negative solution will be write out a prescription is removed, and other four flavors are prepared the negative control sample by the method for making item in the quality standard; Preparation method according to need testing solution prepares with method then.

The upper solution of developping agent sherwood oil (30～60 ℃)-formic acid second fat-formic acid (15: 5: 1)

Developing the color and inspecting does not need developer, dries, and inspects under the uviol lamp (365nm); Put in the ammonia and inspect under the daylight of smoked back.

As a result in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show five identical orange-yellow fluorescent spots, with the corresponding position of reference substance chromatogram on, show identical orange-yellow fluorescence spot; Put in the ammonia and smoke, spot becomes redness.Negative control is noiseless.

(4) discriminating of notopterygium root

Method 1

This product 10ml is got in the preparation of need testing solution, uses ethyl acetate extraction 4 times, each 10ml, and combined ethyl acetate liquid, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution.

Notopterygium root control medicinal material 1g is got in the preparation of control medicinal material solution, adds ethanol 30ml, floods 1 hour, filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medicinal material solution.

Notopterygium root during the preparation of negative solution will be write out a prescription is removed, and other four flavors are prepared the negative control sample by the method for making item in the quality standard; Preparation method according to need testing solution prepares with method then.

The upper solution of developping agent ethyl acetate-methanol-water (10: 2: 3).

Developing the color and inspecting does not need developer, dries, and inspects under the uviol lamp (365nm).

As a result in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical fluorescence spot.Negative control is noiseless.

Method 2

This product 10ml is got in the preparation of need testing solution, and with 60～90 ℃ of Petroleum ether extraction 4 times, each 10ml merges 60～90 ℃ of sherwood oil liquid, and evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution.

Other operations are with method 1.

B1. the composition that contains is carried out the step of assay

1, the mensuration of archen and Chrysophanol total content in the rheum officinale

(1) instrument and reagent

Instrument: the U.S. wears the peace high performance liquid chromatograph, the auspicious Venus-C of CREATE section ₁₈-5 μ chromatographic column P680 pump UVD170 UV-detector CHROMELEON data processing software Startorius BP211D electronic analytical balance d=0.01mg;

Reagent: methyl alcohol is chromatographically pure, and water is redistilled water, and it is pure that all the other reagent are analysis;

Reference substance: the archen reference substance is packed 20mg, lot number 110756～200110 available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

(2) assay method

Measure according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).

Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; Methyl alcohol-0.1% phosphoric acid solution (85: 15) is a moving phase; Detect wavelength 254nm.Number of theoretical plate calculates by archen, Chrysophanol peak all should be lower than 4000.

The preparation precision of reference substance solution takes by weighing archen, the Chrysophanol reference substance is an amount of, adds moving phase and makes the mixing reference substance solution that every 1ml contains archen 30 μ g, contains Chrysophanol 50 μ g.

The accurate this product 5ml that draws of the preparation of need testing solution adds hydrochloric acid 1ml, chloroform 30ml, reflux 30min is put coldly, divides and to get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform liquid, water bath method, residue adds the mutual-assistance dissolving of flowing, and is settled in the 10ml measuring bottle, shakes up, filter, promptly.

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.

The every 1ml of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅) and Chrysophanol (C ₁₅H ₁₀O ₄) the sum meter, must not be less than 0.1mg.

2, chlorogenic acid contents is measured:

(1) instrument and reagent

Reference substance: chlorogenic acid is packed 20mg, lot number 110753～200413 available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

(2) assay method

Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; Methyl alcohol-0.4% phosphoric acid solution (15: 85) is a moving phase; Detect wavelength 324nm.Number of theoretical plate is pressed the chlorogenic acid peak and is calculated, and should be lower than 3000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the chlorogenic acid reference substance, adds 50% methyl alcohol and make the reference substance solution (preserving below 10 ℃) that every 1ml contains 20 μ g.

The accurate this product 1ml that draws of the preparation of need testing solution puts in the brown measuring bottle of 50ml, adds 50% methyl alcohol and is diluted to scale, shakes up, promptly.

Method of the present invention obtains through screening, and screening process is described as follows:

The screening of B2 archen and Chrysophanol total content and determination of chlorogenic acid method

(1) instrument and reagent

Instrument: the U.S. wears the peace high performance liquid chromatograph, the auspicious Venus-C of CREATE section ₁₈-5 μ chromatographic column P680 pump UVD170 UV-detector CHROMELEON data processing software startorius BP211D electronic analytical balance d=0.01mg

Reagent: methyl alcohol is chromatographically pure, and water is redistilled water, and it is pure that all the other reagent are analysis.

Reference substance: archen reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 20mg, 110756～200110).Normalization method is measured, and this product purity is 100%.(referring to accompanying drawing 1)

Chrysophanol reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 20mg, 110796～200412).Normalization method is measured, and this product purity is 100%.(referring to accompanying drawing 2)

(2) selection of chromatographic condition

1) detects wavelength determination: according to " regulation under 2005 editions one one 17 pages rhubarb medicinal material assay items of Chinese pharmacopoeia determines that the detection wavelength is: 254nm.

2) selection of moving phase: adopt the U.S. to wear the peace high performance liquid chromatograph, P680 pump UVD170 UV-detector, flow velocity 1ml/min.Moving phase following system once on probation:

Methyl alcohol-0.1% phosphoric acid solution (85: 15)

The result: the archen retention time is 10.0min, and the Chrysophanol retention time is 15.6min, and reference substance and sample peak shape are good;

Methanol-water (92: 8)

The result: the archen retention time is 4.4min, and the Chrysophanol retention time is 5.9min, and hangover all appears in reference substance and sample, and the peak shape evaluation is general.

By above test findings as can be known: reference substance and sample peak shape are good during with methyl alcohol-0.1% phosphoric acid solution (85: 15) system, so select moving phase to be: methyl alcohol-0.1% phosphoric acid solution (85: 15).

(3) selection of extraction conditions

1) selection of extracting method: draw 060302 batch sample, extract according to following method respectively:

Method one: accurate this product 5ml that draws, add ethanol 50ml refluxing extraction 30min, incline and get ethanol liquid, water bath method, residue add water 10ml makes dissolving, adds hydrochloric acid 1ml, the water-bath temperature is soaked (80 ℃) 30min, put cold, with extracted with diethyl ether 3 times, each 30ml merges ether solution, water bath method, residue adds the mutual-assistance dissolving of flowing, and is settled in the 10ml measuring bottle, shakes up, filter, promptly.

Method two: accurate this product 5ml that draws adds hydrochloric acid 1ml, chloroform 30ml, reflux 30min is put coldly, divides and to get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform liquid, water bath method, residue adds the mutual-assistance dissolving of flowing, and is settled in the 10ml measuring bottle, shakes up, filter, promptly.

Method three: accurate this product 5ml that draws, add hydrochloric acid 1ml, extract 3 times with the powerful jolting of ether, each 50ml merges ether solution, and water bath method, residue add the mutual-assistance dissolving of flowing, and are settled in the 10ml measuring bottle, shake up, and filter, promptly.

Draw each 20 μ l injection liquid chromatograph of above sample liquid respectively and survey its content, the results are shown in following table:

Table-1 extracting method selection result table

The result shows: total content is the highest when extracting sample with method two, so choosing method two is an extracting method, but still needs further refinement with preferred to each step in the method.

2) selection of hydrolysis time: the active component in the rheum officinale is mainly the anthraquinone class, often the form with glucoside exists, glycoside compound easily heats hydrolyzable and becomes glucoside unit under acid condition, whether complete for investigating in the rheum officinale glucoside hydrolysis, get three parts of 060301 batch samples, respectively hydrolysis time is decided to be 20min, 30min, 40min, all the other operations are constant, the preparation test liquid.Respectively test liquid is injected liquid chromatograph and survey its content, following table as a result.

Table-2 extraction time selection result table

The result shows: hydrolysis is complete for sample hydrolysis 30min, and effective constituent does not have significant change behind 30min, so the hydrolysis time of sample is decided to be 30min.

3) selection of extraction time: extraction time is one of principal element that influences extraction effect, is maximum effective component extracting, saves reagent simultaneously, and this test has been carried out preferably extraction time.Get three parts of 060301 batch samples, respectively hydrolysis is put the cold water liquid of getting behind the chloroform that inclines and extract 2 times, 3 times, 4 times, all the other operations are constant, prepare test liquid.Respectively test liquid is injected liquid chromatograph and survey its content, following table as a result.

Table-3 extraction time selection result tables

The result shows: during sample extraction 3 times, effective constituent has been extracted fully, so determine that extraction time is three times.

(4) range of linearity is investigated: get archen, the Chrysophanol reference substance is an amount of, the accurate title, decide, add moving phase and make the mixing contrast solution that every 1ml contains archen 85 μ g, Chrysophanol 95 μ g, accurate respectively 20 μ l, 15 μ l, 10 μ l, 8 μ l, the 4 μ l sample introductions drawn are measured its peak area.The results are shown in Table-4, show-5.

Table-4 archen reference substance measurement results

Table-5 Chrysophanol reference substance measurement results

With peak area (Y) sample size (X) is returned, get the typical curve equation.Getting archen typical curve equation is: Y=50.797X-0.4352 (r=0.9998), Chrysophanol typical curve equation is: Y=72.116X+0.4771 (r=0.9998).Above result shows archen in 0.340 μ g～1.700 μ g scopes, and Chrysophanol is in 0.380 μ g～1.900 μ g scopes, and peak area value and sample size have good linear relationship (referring to accompanying drawing-3, figure-4).

(5) negative control test: get the negative pilot sample 5ml that does not contain rheum officinale, with " need testing solution preparation " item down, after the method processing, make negative controls, get reference substance solution, negative controls, rhubarb medicinal material solution, compound Folium Isatidis mixture need testing solution respectively and respectively advance 20 μ l, the record chromatogram.

The result shows: in the negative control collection of illustrative plates archen, the Chrysophanol peak is noiseless.(referring to figure-5, figure-6, figure-7, figure-8)

(6) precision test: draw reference substance solution (archen 22.8 μ g/ml, Chrysophanol 50.8 μ g/ml) and 060302 batch of same sample test liquid and repeat sample introduction respectively 6 times, each each 20 μ l, calculate the RSD of both peak areas respectively, the results are shown in Table-6, show-7.

Table-6 reference substance Precision test result

Table-7 test sample Precision test result

The result shows: reference substance archen peak area RSD is 0.45%, and Chrysophanol peak area RSD is 0.39%, and test sample archen peak area RSD is 0.63%, and Chrysophanol peak area RSD is 0.41%, and the precision of reference substance and test sample is good.

(7) reappearance test: get 060303 batch 5 parts in sample, press respectively under the test sample preparation prepare, measurement result, total content sees Table-8.

Table-8 reproducible test results

The result shows: the average content of 5 parts of test samples is 1.74mg/10ml, and RSD is 0.70%, and the reappearance of test method is good.

(8) stability test: get with a need testing solution, respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, sample introduction.The results are shown in Table-9, show-10.

Table-9 archen stability test results

Table-10 Chrysophanol stability test results

The result shows: test sample archen RSD in 8 hours is 1.16%, and Chrysophanol RSD in 8 hours is 0.23%, and need testing solution is good at 8 hours internal stabilities.

(9) recovery test: (known emodin content is 0.65mg/ml in 060301 batch of (adopting the application of sample absorption method) accurate absorption, Chrysophanol content is 0.110mg/ml) sample 2.5ml, nominal is got 5 parts, adds identical archen, Chrysophanol reference substance respectively, measure total content, calculate recovery rate.The recovery=(measuring in total amount-sample)/pure product amount of adding.The results are shown in Table-11, show-12.

Table-11 archen recovery test results

Table-12 Chrysophanol recovery test results

The result shows: the archen average recovery rate is 98.9%, and RSD is 1.79%, and the Chrysophanol average recovery rate is 8.1%, and RSD is 0.46%, and average recovery is good.

2, the screening of determination of chlorogenic acid method

(1) instrument

The U.S. wears the peace high performance liquid chromatograph, the auspicious Venus-C of CREATE section ₁₈-5 μ chromatographic column P680 pump UVD170 UV-detector CHROMELEON data processing software startorius BP211D electronic analytical balance d=0.01mg

(2) reagent

Methyl alcohol (chromatographically pure), phosphoric acid (analyzing pure), water (double distilled water); Other reagent are pure for analyzing.

The chlorogenic acid reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 20mg, 10753-200212).

(3) chromatographic condition and basic parameter moving phase: methyl alcohol-0.4% phosphoric acid solution (15: 85) flow velocity: 1.000ml/min; Column temperature: room temperature; Detect wavelength: 324nm.Sample size: 20 μ l (quantitatively encircling sample introduction); Number of theoretical plate: 3000～6000.

(4) detect the selection of wavelength according to " Chinese pharmacopoeia 2005 editions an one 405～406 pages " shuanghuanglian mixture, shuanghuanglian koufuyes " is measured the item assay of honeysuckle down, determines to detect wavelength and is: 324nm.

(5) the negative control test is by the not extracting honeysuckle of writing out a prescription, and the simulation production technology prepares compound Folium Isatidis mixture blank product.Measure in accordance with the law, in the blank product chromatogram, do not seeing that with chlorogenic acid reference substance chromatogram corresponding position significant Interference Peaks is arranged.Referring to accompanying drawing-9, figure-10, figure-11.

(6) 060301 crowd of compound Folium Isatidis mixture sample 1ml of the accurate absorption of the preparation of need testing solution, with methyl alcohol, 50% methyl alcohol and moving phase dilution and be settled to 50ml, filter with miillpore filter respectively, respectively as test liquid, inject liquid chromatograph and survey its content, the results are shown in Table-13.

Table-13 extracts solvent selection result table

The result indicates: three extract solvent gained sample chlorogenic acid content is more or less the same, but 50% methyl alcohol peak shape is better, so select 50% methyl alcohol for extracting solvent.

(7) it is an amount of that the regulation of the range of linearity is got the chlorogenic acid reference substance, and accurate the title decides, and adds 50% methyl alcohol and make the solution that every 1ml contains 40 μ g, and accurate respectively 20 μ l, 15 μ l, 10 μ l, 8 μ l, the 5 μ l sample introductions drawn are measured its peak area.The results are shown in Table 14, accompanying drawing-12.With peak area (y) sample size (x) is returned, get the typical curve equation.y＝57.496x-0.0979(r＝0.9999)。Above result shows that chlorogenic acid peak area value and sample size have good linear relationship in 0.20 μ g～0.80 μ g scope.

Table-14 chlorogenic acid reference substance measurement results

(8) precision test is drawn reference substance solution and 060301 batch of same duplicate samples solution repeats sample introduction respectively 6 times, and each each 20 μ l calculate the RSD of both peak areas respectively, the results are shown in Table-15.

Table-15 Precision test result

The result shows: reference substance peak area RSD is 0.27%, and test sample peak area RSD is 0.57%, and precision is good.

(9) 060301 batch of compound Folium Isatidis mixture is got in the reappearance test, prepares test sample according to selected method respectively, measures wherein chlorogenic acid content.Test findings sees Table-16:

Table-16 reproducible test results

The result indicates: the average content of sample is 0.7275mg/ml, and RSD is 1.68%, and the reappearance of test method is good.

(10) average recovery test (adopting the application of sample absorption method) precision takes by weighing 060301 crowd of (known content is 0.7275mg/ml) 1ml, and nominal is got 5 parts, adds identical chlorogenic acid reference substance respectively, measures total content, calculate recovery rate.

The recovery=(measuring in total amount-sample)/pure product amount of adding.The results are shown in Table-17.

Table-17 recovery test results

The result shows: average recovery rate is 98.68%, and RSD is 0.68%, and average recovery is good.

3, sample determination: get the compound Folium Isatidis mixture sample that our company produces, measure by the content method of working out, archen, Chrysophanol total content, chlorogenic acid content see Table-18.

Table-18 compound Folium Isatidis mixture assays are table as a result

The application of above-mentioned Chinese medicine compound prescription indigowoad leaf preparation in preparation treatment flu, influenza, mumps, acute viral hepatitis pharmaceutical preparation.

[function cures mainly] wind and heat dispersing, removing toxicity for detumescence, cool blood cholagogic.Be used for cold, fever headache, swell and ache under the pharyngitis, ear, diseases such as hypochondriac pain jaundice, and influenza, mumps, acute viral hepatitis are seen the person that has the above-mentioned symptom.

[usage and consumption] is oral, a 10～20ml, 2～3 times on the one.Be used for acute viral hepatitis, a 30ml, 3 times on the one.

Embodiment 2 preparation granules

The main ingredient proportioning:

Folium isatidis 360g, honeysuckle 180g, notopterygium root 90g, adder-wort 90g, rheum officinale 90g.

More than all flavor medicinal materials, add the convention amount decocting and boil twice, each 1h; Merge decoction liquor, filter, relative density 1.08～1.32 when filtrate was concentrated into 60 ℃; Add ethanol and make and contain alcohol amount and reach 50～60%, leave standstill, filter, the filtrate recycling ethanol and the clear cream of relative density 1.25～1.30 when being concentrated into 60 ℃, standby;

(2) preparation granule

Last clear cream of step adds suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, and dry below 60 ℃, above-mentioned dosage can make 1000 parts of particles, by the drug specifications packing, and sealing, promptly.

The discriminating of content and contain the assay of composition:

A. the step of the discriminating of content

(1) discriminating of folium isatidis

After getting granule 9g and adding that 10ml is water-soluble and separate, extract 2 times with ethyl acetate or methenyl choloride, each 10ml, combined ethyl acetate or methenyl choloride extract, with 1% sodium hydroxide solution washing 2 times, each 10ml, ethyl acetate or methenyl choloride liquid are put evaporate to dryness in the water-bath, residue adds ethyl acetate or methenyl choloride 1ml makes dissolving, as need testing solution; Other gets the indigo red reference substance, adds ethyl acetate or methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution; Other gets the indigo red reference substance, adds methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw need testing solution 10～15 μ l, reference substance solution 6 μ l, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, mixed liquor with toluene-ethyl acetate of 5: 4: 1-acetone is a developping agent, launch, take out, dry; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show identical aubergine spot;

(2) discriminating of honeysuckle

Get granule 10g, after adding that 10ml is water-soluble and separating, add 2～4 of hydrochloric acid, making the solution pH value is 1～2, extract 3 times with ethyl acetate or methenyl choloride, each 20ml, combined ethyl acetate or methenyl choloride extract, evaporate to dryness, residue adds water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, is added on polyamide column, wet method dress post, column length 20cm, internal diameter 15～20mm, transferring pH value with ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, the eluent evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, filters in case of necessity, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 1～2 μ l of need testing solution and reference substance solution, putting respectively on same polyamide film, is developping agent with methenyl choloride-methyl alcohol of 20: 6: 0.8-formic acid solution, launches, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Or granule 10g, after adding that 10ml is water-soluble and separating, being added on the polyamide column (30～60 orders, 3g, internal diameter 0.9cm), water 50ml wash-out discards eluent, uses 50% ethanol elution again, collects eluent, and evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8) is 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 the fluorescence spot of same color.

(3) discriminating of rheum officinale

After getting granule 10g and adding that 10ml is water-soluble and separate, add hydrochloric acid 1ml, put and heat 30min in the water-bath, the cooling back is with extracted by ether 2 times, and each 20ml merges ether extracted liquid, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with 30～60 ℃ of sherwood oils of 15: 5: 1-formic acid second fat-formic acid preparation mixed solvent, getting its upper solution is developping agent, launch, take out, dry, put and inspect under the 365nm ultraviolet lamp and ammonia is inspected after smoked, in the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) discriminating of notopterygium root

After getting granule 10g and adding that 10ml is water-soluble and separate, with ethyl acetate or 60～90 ℃ of Petroleum ether extraction 4 times, each 10ml, combined ethyl acetate or 60～90 ℃ of petroleum ether extracts, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution; Other gets notopterygium root control medicinal material 1g, adds ethanol 30ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medicinal material solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5～8 μ l of need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, be developping agent with normal hexane-benzene of 2: 1: 1-ethyl acetate mixed solvent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

B. the composition that contains is carried out the step of assay

[assay]

1, the mensuration of archen and Chrysophanol total content

(1) instrument and reagent

Reference substance: the archen reference substance is packed 20mg, lot number 110756-200110 available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

(2) assay method

The preparation precision of need testing solution takes by weighing this product 5g, adds to add hydrochloric acid 1ml, chloroform 30ml after water 10ml dissolves, reflux 30min is put coldly, divides and to get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform liquid, water bath method, residue adds the mutual-assistance dissolving of flowing, and is settled in the 10ml measuring bottle, shakes up, filter, promptly.

The every 1g of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅) and Chrysophanol (C ₁₅H ₁₀O ₄) the sum meter, must not be less than 0.1mg.

2, chlorogenic acid contents is measured:

(1) instrument and reagent

Instrument: the U.S. wears the peace high performance liquid chromatograph, the auspicious Venus-C18-5 μ of CREATE section chromatographic column P680 pump UVD170 UV-detector CHROMELEON data processing software Startorius BP211D electronic analytical balance d=0.01mg;

Reference substance: chlorogenic acid is packed 20mg, lot number 110753-200413 available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

(2) assay method

Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; Methyl alcohol-0.4% phosphoric acid solution (15: 85); Or acetonitrile-0.4% phosphoric acid solution (10: 90) or methanol-water-glacial acetic acid (20: 80: 1) or methanol-water-glacial acetic acid-triethylamine (18: 85: 1: 0.3) be moving phase; Detect wavelength 324nm.Number of theoretical plate is pressed the chlorogenic acid peak and is calculated, and should be lower than 3000.

The preparation precision of need testing solution takes by weighing this product 1.0g, puts in the tool plug triangular flask, adds 50% methyl alcohol 50ml sonicated 30 minutes, supplies to subtract weight loss, shakes up, and filters, promptly.

The every 1g of this product contains chlorogenic acid (C ₁₆H ₁₈O ₉) must not be less than 0.6mg.

According to above method ten batch samples have been carried out assay, result such as following table-19:

Table-19 compound indigowoad leaf particle content measurings are table as a result

All the other are with embodiment 1.

Embodiment 3 preparation capsules

The main ingredient proportioning:

Folium isatidis 300g, Flos Lonicerae 200g, notopterygium root 100g, adder-wort 100g, rheum officinale 100g.

The preparation capsule

Clear cream of last step vacuum drying or microwave drying below 80 ℃ are ground into fine powder, add suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, and drying is filled in the capsulae vacuus, and above-mentioned dosage can make 1000 of capsules, by the drug specifications packing, and sealing, promptly.

The discriminating of content and contain the assay of composition:

A. the step of the discriminating of content

(1) discriminating of folium isatidis

Get the about 3g of content in the capsule, after adding that 10ml is water-soluble and separating, extract 2 times with ethyl acetate or methenyl choloride, each 10ml, combined ethyl acetate or methenyl choloride extract are with 1% sodium hydroxide solution washing 2 times, each 10ml, ethyl acetate or methenyl choloride liquid are put evaporate to dryness in the water-bath, and residue adds ethyl acetate or methenyl choloride 1ml makes dissolving, as need testing solution; Other gets the indigo red reference substance, adds ethyl acetate or methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution; Other gets the indigo red reference substance, adds methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution;

(2) discriminating of Flos Lonicerae

Get the content 3g in the capsule, after adding that 10ml is water-soluble and separating, add 2～4 of hydrochloric acid, making the solution pH value is 1～2, extract 3 times with ethyl acetate or methenyl choloride, each 20ml, combined ethyl acetate or methenyl choloride extract, evaporate to dryness, residue adds water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, is added on polyamide column, wet method dress post, column length 20cm, internal diameter 15～20mm, transferring pH value with ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, the eluent evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, filters in case of necessity, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 1～2 μ l of need testing solution and reference substance solution, putting respectively on same polyamide film, is developping agent with 20: 6: 0.8 methenyl choloride-methyl alcohol-formic acid solutions, launches, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Or get content 3g in the capsule, after adding that 10ml is water-soluble and separating, be added on polyamide column (30～60 orders, 3g, internal diameter 0.9cm) on, water 50ml wash-out discards eluent, uses 50% ethanol elution again, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8) is 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 the fluorescence spot of same color.

(3) discriminating of rheum officinale

After getting content 3g in the capsule and adding that 10ml is water-soluble and separate, add hydrochloric acid 1ml, put and heat 30min in the water-bath, the cooling back is with extracted by ether 2 times, and each 20ml merges ether extracted liquid, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution;

(4) discriminating of notopterygium root

After getting content 3g in the capsule and adding that 10ml is water-soluble and separate, with ethyl acetate or 60～90 ℃ of Petroleum ether extraction 4 times, each 10ml, combined ethyl acetate or 60～90 ℃ of petroleum ether extracts, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution; Other gets notopterygium root control medicinal material 1g, adds ethanol 30ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medicinal material solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5～8 μ l of need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with 10: 2: 3 ethyl acetate-methanol-water preparation mixed solvent, getting its upper solution is developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;

B. the composition that contains is carried out the step of assay

[assay]

1, the mensuration of archen and Chrysophanol total content

(1) instrument and reagent

(2) assay method

Capsule 's content 3g is got in the preparation of need testing solution, and accurate the title decides, and adds to add hydrochloric acid 1ml after water 10ml dissolves, chloroform 30ml, reflux 30min is put cold, divide and get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform liquid, water bath method, residue add the mutual-assistance dissolving of flowing, be settled in the 10ml measuring bottle, shake up, filter, promptly.

The every 1g of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅) and Chrysophanol (C ₁₅H ₁₀O ₄) the sum meter, must not be less than 0.30mg.

2, chlorogenic acid contents is measured:

(1) instrument and reagent

(2) assay method

Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; Methyl alcohol-0.4% phosphoric acid solution (15: 85); Acetonitrile-0.4% phosphoric acid solution (10: 90) or methanol-water-glacial acetic acid (20: 80: 1) or methanol-water-glacial acetic acid-triethylamine (18: 85: 1: 0.3) be moving phase; Detect wavelength 324nm.Number of theoretical plate is pressed the chlorogenic acid peak and is calculated, and should be lower than 3000.

This product 0.3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug triangular flask, adds 50% methyl alcohol 50ml sonicated 30 minutes, and deficiency subtracts weight loss, shakes up, and filters, promptly.

The every 1g of this product contains chlorogenic acid (C ₁₆H ₁₈O ₉) must not be less than 1.8mg.

According to above method ten batch samples have been carried out assay, result such as following table-20:

Table-20 compound indigowoad leaf capsule content measurement result tables

All the other are with embodiment 1.

Embodiment 4 preparation tablets

Main ingredient proportioning: folium isatidis 380g, Flos Lonicerae 190g, notopterygium root 95g, adder-wort 95g, rheum officinale 95g.

The preparation tablet

Clear cream of last step vacuum drying or microwave drying below 80 ℃ are ground into fine powder, add suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, compressing tablet, and sugar coating or film-coating, above-mentioned dosage can make 1000 in tablet, by the drug specifications packing, sealing, promptly.

The discriminating of content and containing in the assay of composition:

A. the discriminating step of content

(1) discriminating of folium isatidis

Getting tablet 3.0g grinds, after adding that 10ml is water-soluble and separating, extract 2 times with ethyl acetate or methenyl choloride, each 10ml, combined ethyl acetate or methenyl choloride extract are with 1% sodium hydroxide solution washing 2 times, each 10ml, ethyl acetate or methenyl choloride liquid are put evaporate to dryness in the water-bath, and residue adds ethyl acetate or methenyl choloride 1ml makes dissolving, as need testing solution; Other gets the indigo red reference substance, adds ethyl acetate or methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution; Other gets the indigo red reference substance, adds methenyl choloride and makes the solution that every 1ml contains 0.05mg, in contrast product solution;

(2) discriminating of Flos Lonicerae

Getting tablet 3.0g grinds, after adding that 10ml is water-soluble and separating, add 2～4 of hydrochloric acid, making the solution pH value is 1～2, extract 3 times with ethyl acetate or methenyl choloride, each 20ml, combined ethyl acetate or methenyl choloride extract, evaporate to dryness, residue adds water 10ml, the gradation dissolving, transferring pH value with ammonia solution is 7.0～7.5, is added on polyamide column, wet method dress post, column length 20cm, internal diameter 15～20mm, transferring pH value with ammonia solution is 7.0～7.5 aqueous solution 40ml wash-out, the eluent evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, filters in case of necessity, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution;

Or tablet 3.0g grinds, and after adding that 10ml is water-soluble and separating, is added on polyamide column (30～60 orders, 3g, internal diameter 0.9cm) on, water 50ml wash-out discards eluent, uses 50% ethanol elution again, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 0.5～1 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with methenyl choloride-methyl alcohol-formic acid (20: 6: 0.8) is 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 the fluorescence spot of same color.

(3) discriminating of rheum officinale

Get tablet 3.0g and grind, after adding that 10ml is water-soluble and separating, add hydrochloric acid 1ml, put and heat 30min in the water-bath, the cooling back is with extracted by ether 2 times, and each 20ml merges ether extracted liquid, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution;

Check according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with 30～60 ℃ of sherwood oils of 15: 5: 1-formic acid second fat one formic acid preparation mixed solvent, getting its upper solution is developping agent, launch, take out, dry, put and inspect under the 365nm ultraviolet lamp and ammonia is inspected after smoked, in the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) discriminating of notopterygium root

Get tablet 3.0g and grind, after adding that 10ml is water-soluble and separating, with ethyl acetate or 60～90 ℃ of Petroleum ether extraction 4 times, each 10ml, combined ethyl acetate or 60～90 ℃ of petroleum ether extracts, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution; Other gets notopterygium root control medicinal material 1g, adds ethanol 30ml, and dipping 1h filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medicinal material solution;

B. the composition that contains is carried out the step of assay

1, the mensuration of archen and Chrysophanol total content

(1) instrument and reagent

(2) assay method

The preparation of need testing solution is got tablet 3.0g and is ground, and accurate the title decides, and adds to add hydrochloric acid 1ml after water 10ml dissolves, chloroform 30ml, reflux 30min is put cold, divide and get chloroform layer, water layer extracts 3 times with the powerful jolting of chloroform, each 30ml, combined chloroform liquid, water bath method, residue add the mutual-assistance dissolving of flowing, be settled in the 10ml measuring bottle, shake up, filter, promptly.

2, chlorogenic acid contents is measured:

(1) instrument and reagent

(2) assay method

The preparation of need testing solution is got tablet 0.3g and is ground, and accurate the title decides, and puts in the tool plug triangular flask, adds 50% methyl alcohol 50ml sonicated 30 minutes, and deficiency subtracts weight loss, shakes up, and filters, promptly.

According to above method ten batch samples have been carried out assay, result such as following table-21:

Table-21 compound indigowoad leaf sheet assays are table as a result

All the other are with embodiment 1.

Embodiment 5 preparation pills

Main ingredient proportioning: folium isatidis 420g, Flos Lonicerae 210g, notopterygium root 105g, adder-wort 105g, rheum officinale 105g.

The preparation pill

Clear cream of last step vacuum drying or microwave drying below 80 ℃ are ground into fine powder, and to add water or honey and water be the binder mixing and make sphere or the class spherical pellets by the pharmacy routine, and is dry below 80 ℃; Sugar coating or film-coating, above-mentioned dosage can make 1000 of pills, by the drug specifications packing, and sealing, promptly.

The discriminating of content and containing in the assay of composition:

A. the discriminating step of content

(1) sampling amount of sample is 5.0g in the discriminating of folium isatidis, and all the other are with embodiment 4.

(2) sampling amount of sample is 5.0g in the discriminating of Flos Lonicerae, and all the other are with embodiment 4.

(3) sampling amount of sample is 5.0g in the discriminating of rheum officinale, and all the other are with embodiment 4.

(4) sampling amount of sample is 5.0g in the discriminating of notopterygium root, and all the other are with embodiment 4.

B. the composition that contains is carried out the step of assay

1, the assay of archen and Chrysophanol

(1) instrument and reagent are with embodiment 4.

(2) in the assay method, under the preparation of test sample, the sampling amount of sample is 5.0g, and all the other are with embodiment 4.

The every 1g of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅) and Chrysophanol (C ₁₅H ₁₀O ₄) the sum meter, must not be less than 0.15mg.

2, chlorogenic acid contents is measured

(1) instrument and reagent are with embodiment 4.

The every 1g of this product contains chlorogenic acid (C ₁₆H ₁₈O ₉) must not be less than 0.9mg.

According to above method ten batch samples have been carried out assay, result such as following table-22:

Table-22 compound indigowoad leaf ball assays are table as a result

All the other are with embodiment 1

Embodiment 6

The main ingredient proportioning is folium isatidis 420g, honeysuckle 190g, notopterygium root 105g, adder-wort 105g, rheum officinale 105g.

The preparation oral liquid, all the other are with embodiment 1.

Embodiment 7

The main ingredient proportioning is folium isatidis 380g, Flos Lonicerae 210g, notopterygium root 95g, adder-wort 95g, rheum officinale 95g.

The preparation tablet, all the other are with embodiment 4.

Embodiment 8

The main ingredient proportioning is folium isatidis 400g, honeysuckle 210g, notopterygium root 105g, adder-wort 95g, rheum officinale 105g.

The preparation particle, all the other are with embodiment 2.

Embodiment 9

The preparation capsule, all the other are with embodiment 3.

Embodiment 10

The main ingredient proportioning is folium isatidis 400g, Flos Lonicerae 210g, notopterygium root 105g, adder-wort 95g, rheum officinale 105g.

The preparation pill, all the other are with embodiment 5.

Embodiment 11

The main ingredient proportioning is folium isatidis 400g, Flos Lonicerae 200g, notopterygium root 100g, adder-wort 100g, rheum officinale 100g.

The preparation oral liquid, all the other are with embodiment 1.

Embodiment 12

The preparation granule, all the other are with embodiment 2.

Embodiment 13

The preparation capsule, all the other are with embodiment 3.

Embodiment 14

The preparation tablet, all the other are with embodiment 4.

Embodiment 15

The preparation pill, all the other are with embodiment 5.

Claims

1. the detection method of a Chinese medicine compound prescription indigowoad leaf preparation is characterized in that described detection method mainly comprises the discriminating of content and contains the assay of composition:

A. the step of the discriminating of content

(1) discriminating of folium isatidis

(2) discriminating of Flos Lonicerae or honeysuckle

(3) discriminating of rheum officinale

(4) discriminating of notopterygium root

(1) chromatographic condition and system suitability test

(2) preparation of reference substance solution

(3) preparation of need testing solution

(4) determination method

2, chlorogenic acid contents assay method

(1) chromatographic condition and system suitability test

(2) preparation of reference substance solution

(3) preparation of need testing solution

(4) determination method

2. in accordance with the method for claim 1, it is characterized in that: the method for making of described Chinese medicine compound prescription indigowoad leaf preparation comprises main ingredient preparation and preparation two steps of corresponding preparations,

(1) main ingredient preparation

(2) preparation corresponding preparations

1. prepare oral liquid

2. prepare granule

3. prepare tablet

4. prepare capsule

Last clear cream of step≤100 ℃ of dryings are also made fine powder, add suitable pharmaceutic adjuvant by the pharmacy routine, and mixing is also made particle, is filled in the capsulae vacuus, and above-mentioned dosage can make 1000 of capsules, by the drug specifications packing, and sealing, promptly;

5. prepare pill