CN102698083A

CN102698083A - Oldenlandia liver-tonifying and toxicity-eliminating particles and preparation method thereof, as well as detection method

Info

Publication number: CN102698083A
Application number: CN2012102280722A
Authority: CN
Inventors: 王祥培; 靳凤云; 张欣
Original assignee: Guiyang College of Traditional Chinese Medicine
Current assignee: Guiyang College of Traditional Chinese Medicine
Priority date: 2012-07-04
Filing date: 2012-07-04
Publication date: 2012-10-03

Abstract

The invention provides oldenlandia liver-tonifying and toxicity-eliminating particles and a preparation method thereof, as well as a detection method. The particles comprise the following raw materials by weight: 5 to 25kg of oldenlandia, 2 to 15kg of lucid ganoderma, 1 to 10kg of sealwort, 2 to 15kg of astragalus mongholicus, 1 to 10kg of rhizoma alismatis, 1 to 10kg of prepared Chinese rhubarb, 1 to 5kg of cistanche, 2 to 15kg of turtle shell, 1 to 10kg of Chinese magnoliavine fruit, 1 to 10kg of gingko leaves, and 1 to 6kg of liquorice. The preparation method comprises the following steps of: extracting the components, and adding proper soluble starch and Aspartame with 80 percent ethanol serving as a wetting agent. Aiming at the defects in the prior art, the preparation process of the oldenlandia liver-tonifying and toxicity-eliminating particles is optimized, so that a curative effect of the particles to viral hepatitis is remarkable, and a systematic, complete and effective component identification and content determination method is established, and the quality of the medicament can be effectively controlled, so that the clinical curative effect of the particles can be guaranteed.

Description

A kind of the liver benefiting toxin expelling granule and preparation method thereof and detection method spent in vain

Technical field

The present invention relates to a kind of the liver benefiting toxin expelling granule and preparation method thereof and detection method spent in vain, belong to technical field of Chinese medicines.

Background technology

Liver is important detoxifcation organ, and various toxin become nontoxic or low toxicity material through after the series of chemical of liver.In a single day pathological changes takes place in liver, causes hepatopathy, and its hazardness is very big, and common hepatopathy has hepatitis B, hepatitis A, hepatitis C, liver cirrhosis, fattyization, hepatocarcinoma, alcoholic liver etc.Therefore the Chinese medicine preparation that has the liver benefiting toxin expelling effect can be brought into play good action.

Application number discloses a kind of the liver benefiting expellant medicament and preparation method thereof for the patent application of " 200410022730.8 ", and this method for preparing is that 11 flavor medical materials are all used water boiling and extraction; Through further discovering; The a large amount of cyclohexyl biphenyl octene type lignin compositions that contain in the free hydroxyl group anthraquinones that Radix Et Rhizoma Rhei in this prescription contains, the Fructus Schisandrae Chinensis, the effective ingredient in the Rhizoma Alismatis etc. all are the main active substances of enantiopathy virus hepatitis; And water when decocting extraction ratio low; These main active substances are not fully extracted, thereby make the curative effect of this compound recipe be affected.

In addition, at present the liver benefiting toxin expelling medicine also there is not the strict quality inspection standard reliably of a cover.If there is not strict quality standard, the product that obtains can not be guaranteed its quality, and the result will influence the clinical efficacy of this medicine; Think the therapeutical effect that improves the liver benefiting expellant medicament, guarantee medication safe, effectively reach the stable of product quality, formulating a reliable quality standard of strictness becomes the basic demand of ensuring drug quality.Along with the development of Modern Instrument Analytical Technique, analytical methods such as HPLC, tlc identification method have obtained application more and more widely in the quality control of medicine.

Summary of the invention

The objective of the invention is to, a kind of the liver benefiting toxin expelling granule and preparation method thereof and detection method spent in vain is provided.The present invention is directed to the deficiency of prior art; Optimize spending the particulate preparation technology of the liver benefiting toxin expelling in vain; Make its curative effect more remarkable to viral hepatitis; And set up system, complete, effective composition and differentiated and content assaying method, can effectively control the quality of this medicine, thereby guarantee its clinical efficacy.

Technical scheme of the present invention: a kind of the liver benefiting toxin expelling granule of spending in vain; It is to be crude drug with Herba Hedyotidis Diffusae 5 ~ 25kg, Ganoderma 2 ~ 15kg, Rhizoma Polygonati 1 ~ 10kg, the Radix Astragali 2 ~ 15kg, Rhizoma Alismatis 1 ~ 10kg, Radix Et Rhizoma Rhei 1 ~ 10kg, Herba Cistanches 1 ~ 5kg, Carapax Trionycis 2 ~ 15kg, Fructus Schisandrae Chinensis 1 ~ 10kg, Folium Ginkgo 1 ~ 10kg and Radix Glycyrrhizae 1 ~ 6kg; Extract the back and add an amount of soluble starch and aspartame, and be that wetting agent is processed with 80% ethanol.

Preferably spending the liver benefiting toxin expelling granule in vain and be with Herba Hedyotidis Diffusae 20kg, Ganoderma 10kg, Rhizoma Polygonati 8kg, Radix Astragali 10kg, Rhizoma Alismatis 8kg, Radix Et Rhizoma Rhei 8kg, Herba Cistanches 4kg, Carapax Trionycis 10kg, Fructus Schisandrae Chinensis 8kg, Folium Ginkgo 8kg and Radix Glycyrrhizae 6kg is crude drug; Extract the back and add an amount of soluble starch and aspartame, and be that wetting agent is processed with 80% ethanol.

The aforementioned particulate method for preparing of the liver benefiting toxin expelling of spending in vain is: Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis medical material are added 6 times of amount 70% ethanol extractions 3 times, and each 2h filters; Alcohol extract is evaporated to density 1.2 for 70 ℃, and thick extractum gets dry extract in the dry 36h of 80 ℃ of vacuum decompressions; Crushing screening gets the alcohol extraction powder; All the other 8 flavor crude drug mix with the alcohol extraction medicinal residues and add 6 times of water gagings immersion 0.5h, add 6 times of water gagings 0.5h that is decocted first again, add 12 times of amounts of water altogether; Decoct three times, decocting time is respectively: 3h, 3h and 1h, and 85 ℃ are evaporated to density 1.2; 80 ℃ of drying under reduced pressure 48h; Get dry extract, crushing screening gets water and carries powder; Alcohol extraction powder and water are put forward powder mixes, do sweeting agent, do wetting agent with 80% ethanol by 1: 1 part by weight adding adjuvant soluble starch and 0.2% aspartame, wet granulation, 75 ℃ of dry 4-6h of granule, granulate sieves, and packing promptly gets.

The aforementioned particulate detection method of the liver benefiting toxin expelling of spending in vain comprises character, discriminating, inspection and assay project; Wherein differentiate it is thin layer chromatography discriminating to the Folium Ginkgo in the preparation, Radix Glycyrrhizae, Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis; Assay is a content of measuring the contained total free anthraquinone constituents of Radix Et Rhizoma Rhei and the contained schisandrin of Fructus Schisandrae Chinensis in the preparation with HPLC respectively.

The discrimination method of Folium Ginkgo is to be contrast with the Folium Ginkgo control medicinal material, is the thin layer chromatography of developing solvent with ethyl acetate-butanone-formic acid-water=5:3:1:1; The discrimination method of Radix Glycyrrhizae is to be contrast with Radix Glycyrrhizae control medicinal material and ammonium glycyrrhizinate reference substance, is the thin layer chromatography of developing solvent with ethyl acetate-formic acid-glacial acetic acid-water=15:1:1:2; The discrimination method of Radix Et Rhizoma Rhei is to be contrast with the Radix Et Rhizoma Rhei control medicinal material, is the thin layer chromatography of developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; The discrimination method of Fructus Schisandrae Chinensis is to be contrast with Fructus Schisandrae Chinensis control medicinal material and deoxyschizandrin reference substance, is the thin layer chromatography of developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1.

Concrete discrimination method is:

(1) thin layer chromatography of Folium Ginkgo is differentiated: get this granule 4g, add 40% methanol 20mL, reflux 30min is put coldly, filters, and gets filtrating 10mL and is concentrated into 2mL, as need testing solution; Other gets Folium Ginkgo control medicinal material 1g, shines medical material solution in pairs with legal system; According to " an appendix VI of Chinese pharmacopoeia B thin layer chromatography test is drawn above-mentioned need testing solution 10 μ L and control medicinal material solution 6 μ L and put in the same carboxymethylcellulose sodium solution that contains 4% sodium acetate respectively to be on the silica gel g thin-layer plate of adhesive preparation, to be developing solvent with ethyl acetate-butanone-formic acid-water=5:3:1:1; Launch; Take out, dry, spray is with 3% aluminum chloride alcoholic solution; Hot blast drying is put under the 365nm ultra-violet lamp and is inspected; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence principal spot of same color;

(2) thin layer chromatography of Radix Glycyrrhizae is differentiated: get this granule 4g, and the 40mL that adds diethyl ether, reflux 1 h filters, and medicinal residues add methanol 30mL; Reflux 1h filters, the filtrating evaporate to dryness, and residue adds water 40mL makes dissolving; With n-butanol extraction 3 times, each 20mL merges n-butyl alcohol liquid, with water washing 3 times; Discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue add methanol 5mL makes dissolving, as need testing solution; Extracting liquorice control medicinal material 1g shines medical material solution in pairs with legal system in addition; Extracting liquorice acid ammonium reference substance adds methanol and processes the solution that every 1mL contains 2mg, as reference substance solution again; According to " need testing solution 8 μ L are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography, and other draws above-mentioned reference substance solution, each 1～2 μ L of control medicinal material solution; Putting respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, is developing solvent with ethyl acetate-formic acid-glacial acetic acid-water=15:1:1:2, launches; Take out, dry, spray is with 10% ethanol solution of sulfuric acid; It is clear to be heated to speckle colour developing at 105 ℃, puts under the 365nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color; With the corresponding position of reference substance chromatograph on, show identical orange-yellow fluorescence speckle;

(3) thin layer chromatography of Radix Et Rhizoma Rhei is differentiated: get this granule 4g, add methanol 20mL, soak 1h, filter; Get filtrating 5mL, evaporate to dryness, residue add water 10mL makes dissolving, adds hydrochloric acid 1mL again; Reflux 30min, cooling divides 2 extractions with ether immediately, each 20mL; Merge ether solution, evaporate to dryness, residue add chloroform 1mL makes dissolving, as need testing solution; Other gets Radix Et Rhizoma Rhei control medicinal material 0.1g, shines medical material solution in pairs with legal system; According to " appendix a VI of Chinese pharmacopoeia B thin layer chromatography test; Draw each 4 μ L of above-mentioned need testing solution, control medicinal material solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, be developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; Launch; Take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show five identical orange-yellow fluorescence principal spots;

(4) thin layer chromatography of Fructus Schisandrae Chinensis is differentiated: get this granule 3g, add chloroform 20mL, reflux 30min filters, the filtrating evaporate to dryness, and residue adds chloroform 1mL makes dissolving, as need testing solution; Other gets Fructus Schisandrae Chinensis control medicinal material 1g, shines medical material solution in pairs with legal system; Get the deoxyschizandrin reference substance again, add chloroform and process the solution that every 1mL contains 1mg, as reference substance solution; According to " test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography is drawn Fructus Schisandrae Chinensis control medicinal material solution, each 2 μ L of deoxyschizandrin reference substance solution and need testing solution 6 μ L and put in same silica gel G F respectively ₂₅₄On the lamellae, be developing solvent, launch, take out, dry, put under the 254nm ultra-violet lamp and inspect with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color.

The content assaying method of total free anthraquinone constituents is to be contrast with aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance and chrysophanol reference substance in the Radix Et Rhizoma Rhei, and with acetonitrile: 0.1% phosphoric acid=58%-85%:42%-15% is the HPLC of mobile phase; The content assaying method of schisandrin is to be contrast with the schisandrin reference substance in the Fructus Schisandrae Chinensis, is the HPLC of mobile phase with acetonitrile: water=45%:55%.

Concrete content assaying method is:

(1) total free anthraquinone constituents assay: with reference to 2010 editions " an appendix VI of Chinese pharmacopoeia D high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler, Diamonsil C18 (250mm * 4.6mm, 5 μ m); With acetonitrile: 0.1% phosphoric acid=58%-85%:42%-15% is a mobile phase; Flow velocity 1.0 mLmin ^-1Detecting wavelength is 254 nm; Column temperature is 25 ℃; Theoretical cam curve is calculated with the chrysophanol peak and is not less than 6000;

The preparation of reference substance solution: precision takes by weighing aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance and chrysophanol reference substance respectively, adds methanol and processes the reference substance solution that every 1mL contains 0.0145mg, 0.0277mg, 0.0283mg, 0.0655mg;

The preparation of need testing solution: get this granule 0.6g under the content uniformity item, the accurate title, decide, and puts in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution 10mL, supersound process 5min; Add chloroform 10mL again, reflux 1 h is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges chloroform liquid; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get need testing solution;

Algoscopy: accurate respectively reference substance solution 5 μ L and the need testing solution 10 μ L of drawing, inject high performance liquid chromatograph, measure, promptly get;

This granule contains Radix Et Rhizoma Rhei to comprise the total free anthraquinone constituents of aloe-emodin, chrysophanic acid, emodin, chrysophanol for every bag, must not be lower than 4.9mg;

(2) schisandrin assay: with reference to 2010 editions " an appendix VI of Chinese pharmacopoeia D high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler, Diamonsil C18 (250mm * 4.6mm, 5 μ m); Acetonitrile: water=45%:55% is a mobile phase; Flow velocity is 1.0 mLmin ^-125 ℃ of column temperatures; Detecting wavelength is 250 nm; Theoretical cam curve is calculated with the schisandrin peak and is not less than 2000;

The preparation of reference substance solution: precision takes by weighing the schisandrin reference substance, adds methanol and processes the reference substance solution that every 1mL contains schisandrin 7.8 μ g;

The preparation of need testing solution: get this granule 0.6g under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask; Add methanol 20mL,, add methanol to scale taking out behind the supersound process 45min under power 250W, the frequency 20HZ condition; Shake up, get subsequent filtrate, promptly get need testing solution;

Algoscopy: accurate respectively reference substance solution 10 μ L and the need testing solution 10 μ L of drawing, inject high performance liquid chromatograph, measure, promptly get;

This granule contains Fructus Schisandrae Chinensis in schisandrin for every bag, must not be lower than 1.56mg.

Detection method of the present invention comprises:

Character: this preparation is a brown granular, gas fragrance, little sweet acid that has a little of distinguishing the flavor of;

Differentiate: the thin layer chromatography of (1) Folium Ginkgo is differentiated: get this granule 4g, add 40% methanol 20mL, reflux 30min is put coldly, filters, and gets filtrating 10mL and is concentrated into 2mL, as need testing solution; Other gets Folium Ginkgo control medicinal material 1g, shines medical material solution in pairs with legal system; According to " above-mentioned need testing solution 10 μ L and control medicinal material solution 6 μ L are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography, and putting in the same carboxymethylcellulose sodium solution that contains 4% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive preparation; With ethyl acetate-butanone-formic acid-water=5:3:1:1 is developing solvent, launches, and takes out; Dry; Spray is with 3% aluminum chloride alcoholic solution, and hot blast drying is put under the 365nm ultra-violet lamp and inspected; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence principal spot of same color;

(4) thin layer chromatography of Fructus Schisandrae Chinensis is differentiated: get this granule 3g, add chloroform 20mL, reflux 30min filters, the filtrating evaporate to dryness, and residue adds chloroform 1mL makes dissolving, as need testing solution; Other gets Fructus Schisandrae Chinensis control medicinal material 1g, shines medical material solution in pairs with legal system; Get the deoxyschizandrin reference substance again, add chloroform and process the solution that every 1mL contains 1mg, as reference substance solution; According to " test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography is drawn Fructus Schisandrae Chinensis control medicinal material solution, each 2 μ L of deoxyschizandrin reference substance solution and need testing solution 6 μ L and put in same silica gel G F respectively ₂₅₄On the lamellae, be developing solvent, launch, take out, dry, put under the 254nm ultra-violet lamp and inspect with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;

Inspection: should meet " relevant each item regulation under an appendix I of Chinese pharmacopoeia version in 2010 the C granule item;

Assay: (1) total free anthraquinone constituents assay: with reference to 2010 editions " an appendix VI of Chinese pharmacopoeia D high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With acetonitrile: 0.1% phosphoric acid=58%-85%:42%-15% is a mobile phase; Flow velocity 1.0 mLmin ^-1Detecting wavelength is 254 nm; Column temperature is 25 ℃; Theoretical cam curve is calculated with the chrysophanol peak and is not less than 6000;

The preparation of reference substance solution: precision takes by weighing aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance and chrysophanol reference substance reference substance respectively, adds methanol and processes the reference substance solution that every 1mL contains 0.0145mg, 0.0277mg, 0.0283mg, 0.0655mg;

Spend the liver benefiting toxin expelling side in vain and form, have heat-clearing and toxic substances removing, nourishing YIN for suppressing the hyperactive YANG by 11 flavor medicines such as Folium Ginkgo, Fructus Schisandrae Chinensis, Radix Et Rhizoma Rhei, Radix Glycyrrhizae; Hard masses softening and resolving, the supplementing QI for promoting the production of body fluid function, this is write out a prescription on the proved recipe basis; Combine with dialectical with differential diagnosis of diseases; Through tonification liver body, hard masses softening and resolving, the smooth liver of accent with, prevent a plurality of links such as the change of disease, promoting the function of the gallbladder to alleviate jaundice, be used to treat the damp and hot one-tenth poison of a specified duration that accumulates, tie in the various hepatitis of hepatobiliary; Like viral hepatitis, chronic viral hepatitis B, hepatitis C, icterohepatitis etc., evident in efficacy.

Spend the liver benefiting toxin expelling side in vain and be made up of 11 flavor medical materials, medicine is formed more, and chemical constituent is complicated.Lot of documents shows; Effective ingredient etc. all is the main active substances of enantiopathy virus hepatitis in a large amount of cyclohexyl biphenyl octene type lignin compositions (schizandrin, deoxyschizandrin, schisandrin B, schisandrin C, schisandrin, schisantherin B, schizantherin the second grade) that contain in the free hydroxyl group anthraquinones that Radix Et Rhizoma Rhei in the prescription contains, the Fructus Schisandrae Chinensis, the Rhizoma Alismatis; Extraction ratio is low during with decocting, so consider to adopt alcohol extraction through consulting documentation integrators.According to the situation of this prescription, consult document and find that the contained effective ingredient of all the other 8 flavor medical materials of this side all has better water solubility, should adopt water to carry in clinical use.

The inventor combines with clinical efficacy according to the chemical constituent of 11 flavor medical materials, has designed 4 kinds of different extraction schemes.Scheme one: 11 flavor medical materials are made up with distilled water decoction three times, add 6 times of amount pure water by total amount for the first time and soak 30min, heated and boiled 2h, it is subsequent use to get decoction liquor; Add for the second time 4 times of amount pure water, it is subsequent use that heated and boiled 1.5h gets decoction liquor; Add 2 times of amount pure water for the third time, it is subsequent use that heated and boiled 45min gets decoction liquor; Merge three times decoction liquor, leave standstill 4-6h, cross 200 mesh sieves, the solution heating evaporation is concentrated into the thick paste shape, and 60 ℃ of drying under reduced pressure get dry extract, and pulverizes, and promptly gets.

Scheme two: Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis three flavor medicines are adopted ethanol refluxing process, add 9 times of amount 70% soak with ethanol 30min by total amount for the first time, reflux, extract, 1.5h, it is subsequent use to get alcohol extract; Add for the second time 7 times of amount 70% ethanol, it is subsequent use that reflux, extract, 1h gets alcohol extract; Add 5 times of amount 70% ethanol for the third time, it is subsequent use that reflux, extract, 0.5h gets alcohol extract; Merge three times alcohol extract, concentrate, drying, pulverize powder.All the other 8 flavor medical materials mix with the alcohol extraction medicinal residues, and by " scheme one " decocte with water, 60 ℃ of drying under reduced pressure get dry extract, and pulverize, and with the alcohol extraction powder mixes, promptly get.

Scheme three: Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis three flavor medicines adopt ethanol refluxing processes, and method merges three times alcohol extract with " scheme two ", and is concentrated, drying, pulverize powder.All the other 8 flavor medical materials mix with the alcohol extraction medicinal residues, carry by " scheme one " water, merge three times decoction liquor, are concentrated into certain density, add 6 times of amount 80% ethanol; Leave standstill 24h, cross 200 mesh sieves, solution heating evaporation to thick paste shape, 60 ℃ of drying under reduced pressure; Get dry extract, pulverize,, promptly get with the alcohol extraction powder mixes.

Four: 11 flavors of scheme medical material makes up with distilled water and decocts three times, and add 9 times of amount pure water by total amount for the first time and soak 30min, heated and boiled 2h, it is subsequent use to get decoction liquor; Add for the second time 7 times of amount pure water, it is subsequent use that heated and boiled 1.5h gets decoction liquor; Add 5 times of amount pure water for the third time, it is subsequent use that heated and boiled 45min gets decoction liquor; Merge three times decoction liquor, be concentrated into certain density, add 6 times of amount 80% ethanol, leave standstill 24h, filter with 200 mesh sieves, solution heating evaporation to thick paste shape, 60 ℃ of drying under reduced pressure get dry extract, and pulverize, and promptly get.

Spend the liver benefiting toxin expelling compound recipe in vain to CCl through what four kinds of different extraction schemes were extracted ₄Due to acute liver damage mice serum AST, ALT influence and normal control group relatively, preferred optimum extraction process.Experimental result shows raise to suppressing AST and the ALT degree different (scheme two ﹥ schemes three ﹥ schemes one ﹥ scheme four) of (P < 0.05) of the liver benefiting toxin expelling compound recipe of spending in vain of 4 different extraction schemes.Prescription precipitate with ethanol front and back have been compared to 0.5%CCL ₄Cause the influence of chmice acute hepatic injury Serum ALT, AST content; Scheme four these prescription 11 flavor medicines do not suppress the effect of AST, ALT basically through the compound recipe of water extract-alcohol precipitation; And scheme 33 flavor medical material alcohol extractions; Medicinal residues and other 8 flavor medical material water are proposed the influence of back precipitate with ethanol compound recipe to chmice acute hepatic injury Serum ALT, AST, though can find out that high dose has significant inhibitory effect, the undiminished trend of low dosage.Protect the liver according to having in this prescription of bibliographical information 11 flavor medical materials, the effective ingredient such as polysaccharide of anti-immunization are removed in the process of precipitate with ethanol; Thereby cause the curative effect of this compound recipe influenced, prove again that also decoction and alcohol sedimentation technique is not the optimal extraction separation method of Chinese medicine.And the chemical constituent of this prescription medical material of this experimental basis and combine two couples of 0.5%CCL of scheme of clinical efficacy ₄The ALT, the AST content that cause chmice acute hepatic injury serum show very remarkable influence.

Therefore,, filter out the optimum extraction scheme and be in conjunction with above-mentioned pharmacological experiment study: Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis three flavor medical material alcohol extractions, medicinal residues are carried with all the other 8 flavor medical materials merging water; Concentrate, drying is pulverized the powder that gets dry extract, and adds adjuvant and granulates; Drying, granulate promptly gets and spends the liver benefiting toxin expelling granule in vain.

In addition, in order to ensure detection method science of the present invention, reasonable, feasible, the applicant has carried out series of experimental research and investigation.

(1) differentiates

1, instrument and reagent

Instrument adopts ultrasonic cleaner (Ningbo new sesame biotechnology share), electric drying oven with forced convection (going up the grand experimental facilities company limited of Nereid DHG9070A) and ZF ultraviolet analysis instrument for three purposed; Reagent is analytical pure, and thin layer chromatography uses silica gel to be chemical pure.

2, discrimination method and result

(1) the thin layer chromatography discrimination method of Radix Et Rhizoma Rhei

This test is basic with reference to " the thin layer chromatography discrimination method of 2010 editions middle Radix Et Rhizoma Rhei of Chinese pharmacopoeia is got each 4g of three lot sample article granules (lot number 20111201,20111202,20111203), lacks the about 2g of powder of Radix Et Rhizoma Rhei, respectively adds methanol 20mL; Soak 1h, filter, get filtrating 5mL, evaporate to dryness; Residue adds water 10mL makes dissolving, adds hydrochloric acid 1mL again, reflux 30min, cooling immediately; Divide 2 extractions with ether, each 20mL merges ether solution; Evaporate to dryness, residue add chloroform 1mL makes dissolving, as 3 parts of need testing solutions and 1 part of negative solution; Other gets Radix Et Rhizoma Rhei control medicinal material 0.1g, processes 1 part of control medicinal material solution with method; According to " appendix a VI of Chinese pharmacopoeia B thin layer chromatography test; Draw each 4 μ L of above-mentioned need testing solution, negative solution, control medicinal material solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, be developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; Launch; Take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show five identical orange-yellow fluorescence principal spots; With the corresponding position of reference substance chromatograph on, show identical orange-yellow fluorescence speckle.Through test of many times, method is sensitive, good reproducibility; Convenient feasible, negative fluid is noiseless, and the result sees Fig. 1; Wherein, 1: the Radix Et Rhizoma Rhei control medicinal material, 2: lot number is 20111201 need testing solution; 3: lot number is 20111202 need testing solution, and 4: lot number is 20111203 need testing solution, 5: lack the negative solution of Radix Et Rhizoma Rhei.So list it in test item of the present invention.

(2) the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis

This test is basic with reference to " the thin layer chromatography discrimination method of 2010 editions middle Fructus Schisandrae Chinensis of Chinese pharmacopoeia is got three lot sample article (lot number 20111201,20111202,20111203) each 3g of granule, lacks the about 2g of negative powder of Fructus Schisandrae Chinensis; Respectively add chloroform 20mL; Reflux 30min filters, the filtrating evaporate to dryness; Residue adds chloroform 1mL makes dissolving, as 3 parts of need testing solutions and 1 part of negative solution; Other gets Fructus Schisandrae Chinensis control medicinal material 1g, shines medical material solution in pairs with legal system; Get the deoxyschizandrin reference substance again, add chloroform and process the solution that every 1mL contains 1mg, as reference substance solution; According to " appendix a VI of Chinese pharmacopoeia B thin layer chromatography test; Absorption Fructus Schisandrae Chinensis control medicinal material solution, each 2 μ L of deoxyschizandrin reference substance solution and need testing solution, each 6 μ L of negative solution put respectively on same silica GF254 lamellae; Upper solution with 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1 is developing solvent, launches, and takes out; Dry, put under the 254nm ultra-violet lamp and inspect; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color.Through test of many times, method is sensitive, and good reproducibility is convenient feasible; Negative fluid is noiseless, and the result sees Fig. 2, wherein; 1: the deoxyschizandrin reference substance solution, 2: Fructus Schisandrae Chinensis control medicinal material solution, 3: lot number is 20111201 need testing solution; 4: lot number is 20111202 need testing solution, and 5: lot number is 20111203 need testing solution, 6: lack the negative solution of Fructus Schisandrae Chinensis.So list it in test item of the present invention.

(3) the thin layer chromatography discrimination method of Radix Glycyrrhizae

This test is basic with reference to " the thin layer chromatography discrimination method of 2010 editions middle Radix Glycyrrhizaes of Chinese pharmacopoeia is got three lot sample article (lot number 20111201,20111202,20111203) each 4g of granule, lacks the about 2g of powder of Radix Glycyrrhizae, and 40mL respectively adds diethyl ether; Reflux 1 h filters, and medicinal residues add methanol 30mL, reflux 1h; Filter, the filtrating evaporate to dryness, residue adds water 40mL makes dissolving, with n-butanol extraction 3 times; Each 20mL merges n-butyl alcohol liquid, with water washing 3 times, discards water liquid; N-butyl alcohol evaporate to dryness, residue add methanol 5mL makes dissolving, as 3 parts of need testing solutions and 1 part of negative solution; Extracting liquorice control medicinal material 1g shines medical material solution in pairs with legal system in addition; Extracting liquorice acid ammonium reference substance adds methanol and processes the solution that every 1mL contains 2mg, as reference substance solution again; According to " need testing solution, each 8 μ L of negative solution are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography, and other draws above-mentioned reference substance solution, each 1～2 μ L of control medicinal material solution; Putting respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, is developing solvent with ethyl acetate-formic acid-glacial acetic acid-water=15:1:1:2, launches; Take out, dry, spray is with 10% ethanol solution of sulfuric acid; It is clear to be heated to speckle colour developing at 105 ℃, puts under the 365nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color; With the corresponding position of reference substance chromatograph on, show identical orange-yellow fluorescence speckle, negative noiseless.Through test of many times, method is sensitive, and good reproducibility is convenient feasible; Negative fluid is noiseless, and the result sees Fig. 3, wherein; 1: the ammonium glycyrrhizinate reference substance solution, 2: Radix Glycyrrhizae control medicinal material solution, 3: lot number is 20111201 need testing solution; 4: lot number is 20111202 need testing solution, and 5: lot number is 20111203 need testing solution, 6: lack the negative solution of Radix Glycyrrhizae.So list it in test item of the present invention.

(4) the thin layer chromatography discrimination method of Folium Ginkgo

This test is basic with reference to " the thin layer chromatography discrimination method of 2010 editions middle Folium Ginkgos of Chinese pharmacopoeia is got three lot sample article (lot number 20111201,20111202,20111203) granule 4g, lacks the about 2g of powder of Folium Ginkgo; Respectively add 40% methanol 20mL; Reflux 30min is put coldly, filters; Get filtrating 10mL and be concentrated into 2mL, as 3 parts of need testing solutions and 1 part of negative solution; Other gets Folium Ginkgo control medicinal material 1g, shines medical material solution in pairs with legal system; According to " above-mentioned need testing solution, negative solution each 10 μ L and control medicinal material solution 6 μ L are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography, and putting in the same carboxymethylcellulose sodium solution that contains 4% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive preparation; With ethyl acetate-butanone-formic acid-water=5:3:1:1 is developing solvent, launches, and takes out; Dry; Spray is with 3% aluminum chloride alcoholic solution, and hot blast drying is put under the 365nm ultra-violet lamp and inspected; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence principal spot of same color, negative noiseless.Through test of many times, method is sensitive, good reproducibility; Convenient feasible, negative fluid is noiseless, and the result sees Fig. 4; Wherein, 1: Folium Ginkgo control medicinal material solution, 2: lot number is 20111201 need testing solution; 3: lot number is 20111202 need testing solution, and 4: lot number is 20111203 need testing solution, 5: lack the negative solution of Folium Ginkgo.So list it in test item of the present invention.

(2) inspection

1, granularity inspection

Granulometry is with reference to " an appendix XI of Chinese pharmacopoeia version in 2010 B second method is measured.5 bags of these article of getting granules are claimed decide weight, put in the medicine sieve of regulation and sieve, and keep level, about the round 3min that sieves gently.Get and through a sieve and can claim to decide weight, calculate shared percentage ratio through the summation of No. five sieves.All be less than 15% through detecting three lot sample article, up to specification, the result sees table 1.

The inspection of table 1 three lot sample article granularities

2, content uniformity inspection

10 bags of these article of getting granules, the weight of dividing the fixed every bag of content of another name, every packed amount is compared with labelled amount, must not exceed labelled amount ± 10%, all up to specification through mensuration three lot sample article, the result sees table 2.

The inspection of table 2 three lot sample article content uniformity

3, melting

These article of getting test sample 10g adds 20 times of hot water, stirs 5min, observes immediately, and sol particle should all be melted, and allows slight haze, and 3 crowdes of test sample results see table 3.

Table 3 three lot sample article melting check results

4, moisture inspection

Aquametry is with reference to " an appendix IX of Chinese pharmacopoeia version in 2010 H first method is measured.These article of getting test sample 3g, the accurate title, decide, and is tiled in the flat weighing botle that is dried to constant weight, and thickness is no more than 5mm; The accurate title, decide, and opens bottle cap at 100-105 ℃ of dry 5h, bottle cap built, in the dislocation exsiccator; Cooling 30min, the accurate title, decide, again in the dry 1h of said temperature, cooling; Weigh, extremely double difference of weighing is no more than till the 5mg, according to the weight that subtracts mistake, calculates water content in the test sample.Standard code moisture is no more than 6.0%, and through detecting three lot sample article all less than 6.0%, up to specification, the result sees table 4.

Table 4 three lot sample article moisture check results

5, heavy metal and arsenic salt inspection

(1) heavy metal inspection

These article of getting granule 2g, porphyrize is put in the crucible, and is slowly blazing to carbonization fully, takes out; Put coldly, add sulphuric acid 1.0m1, make its proper moistening, low temperature is flung to sulphuric acid; Put coldly, add nitric acid 0.5mL, evaporate to dryness is after eliminating to the nitrogen oxide steam; Put cold, 500-600 ℃ blazing to fully ashing, by " an appendix IX of Chinese pharmacopoeia version in 2010 B heavy metal inspection technique second method checks that the result of three batches of test samples sees table 5.

(2) arsenic salt inspection

These article of getting 1g, porphyrize adds no arsenic calcium hydroxide 0.5g mix homogeneously; It is moistening to add water; Slowly blazingly eliminate to smog, again 500-600 ℃ blazing to ashing fully, put cold; According to " a Chinese pharmacopoeia appendix of version (IXB) in 2010 arsenic salt inspection technique first method from " adding hydrochloric acid 5m1 and water 21m1 ... " Operation, the result of three batches of test samples sees table 5.

Table 5 three lot sample article heavy metals, arsenic salt check result

The result shows: the heavy metal of three batches of test samples, the inspection of arsenic salt all meet 2010 editions " regulations of Chinese pharmacopoeia.

(2) assay

Because the Study on mechanism of monarch drug Herba Hedyotidis Diffusae is not deep enough; The effective substance research of shortage system; Do not recorded at present in 2010 editions that " Chinese pharmacopoeia, its quality of no unified standard control is not therefore as assay index components of the present invention.This granule is ministerial drug with the Radix Et Rhizoma Rhei; Effect with purging heat and dredging bowels, removing pathogenic heat from blood and toxic substance from the body, eliminating blood stasis and inducing menstruation; And the main component of its hepatic cholagogic is the free anthraquinones extracted from rheum constituents; So with aloe-emodin, chrysophanic acid, emodin, chrysophanol in the Radix Et Rhizoma Rhei is the assay index components, controls the quality of granule, to guarantee its clinical efficacy; In addition, Fructus Schisandrae Chinensis has supplementing QI for promoting the production of body fluid, kidney calming, the astringent or styptic treatment for spontaneous sweating effect of convergence, and its effective ingredient schisandrin can be controlled to quality better as assay index components of the present invention.

1, total free anthraquinone constituents in the Radix Et Rhizoma Rhei

(1) instrument and reagent

Instrument: high performance liquid chromatograph (Agilent1100), ultrasonic washing unit (HS20500D), balance (AB204-S), B μ chi Rotary Evaporators.

Reagent: acetonitrile (Tianjin Ke Miou) chromatograph alcohol, WAHAHA pure water.

Reference substance: emodin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number is 0756-200110); Chrysophanic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number is 0757-200206); Aloe-emodin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number is 110795-200504); Chrysophanol reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number is 110796-201017);

Sample: granule of the present invention (spend the liver benefiting toxin expelling granule in vain, Guiyang College of Traditional Chinese Medicine develops the pharmaceutical analysis experimental center lot number 20111118,20111119,20111120 voluntarily).

(2) preparation of reference substance solution

Precision takes by weighing aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance, chrysophanol reference substance respectively, adds methanol and processes the reference substance solution that every 1mL contains 0.0145mg, 0.0277mg, 0.0283mg, 0.0655mg.

(3) chromatographic condition is preferred

Chromatograph wavelength preferred: carrying out free anthraquinones extracted from rheum assay chromatographic condition when selecting, with reference to " the assay chromatographic condition of free anthraquinones extracted from rheum composition has carried out the screening of chromatograph wavelength under Herba Ephedrae item of Chinese pharmacopoeia version in 2010.Compare separating degree and the theoretical cam curve and the peak area thereof of free anthraquinones extracted from rheum under 240nm, 250nm, 254nm, the 260nm equiwavelength respectively, the result proves that free anthraquinones extracted from rheum component separating degree is best at wavelength 250nm place, and peak area is maximum, and interference factor is minimum.

Chromatogram column temperature preferred: this experiment has been compared 20 ℃, 25 ℃, 30 ℃ column temperature to the influence of free anthraquinones extracted from rheum composition, and experimental result shows that separating degree is good under 25 ℃ of column temperature conditions, and retention time is short, and theoretical cam curve is high.

The selection of mobile phase: " the Chinese pharmacopoeia method is methanol-0.1% phosphoric acid water (85%:15% relatively in this experiment reference 2010 editions; 70%:30%; 60%:40%; 50%:50%) the mobile phase of different proportion such as grade, the composition of free anthraquinones extracted from rheum all can not reach fully and separate.Designed methanol-0.1% phosphoric acid water gradient elution in the experiment and found that the retention time of 5 chemical constituents is prone to drift, so the gradient elution of this experimental design variable concentrations acetonitrile-0.1% phosphoric acid water reaches the separation fully between each composition.Final definite best chromatographic condition is: acetonitrile: 0.1% phosphoric acid=58%-85%:42%-15% is a mobile phase, and flow velocity is 1.0mLmin-1, and detecting wavelength is 254 nm, and column temperature is 25 ℃.

(4) system suitability test

The preparation of need testing solution: get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and puts in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution 10mL, supersound process 5min; Add chloroform 10mL again, reflux 1 h is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.

The preparation of negative solution: get the about 0.6g of negative sample that lacks Radix Et Rhizoma Rhei, the accurate title, decide, and puts in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution 10mL, supersound process 5min; Add chloroform 10mL again, reflux 1h is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.

The preparation of blank solution: in the 50mL round-bottomed flask, add 8% hydrochloric acid solution 10mL, supersound process 5min adds chloroform 10mL, reflux 1h again; Put coldly, put in the separatory funnel,, incorporate in the separatory funnel with a small amount of chloroform washing container; Obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.

Assay method: accurate respectively reference substance solution 5 μ L, need testing solution 10 μ L, blank solution 10 μ L, the negative solution 10 μ L of drawing, inject high performance liquid chromatograph, measure, promptly get.

Can know by collection of illustrative plates, sample introduction analysis under above-mentioned chromatographic condition, the separating degree of aloe-emodin, chrysophanic acid, emodin, chrysophanol and adjacent chromatographic peak is all greater than 1.5.Theoretical cam curve is calculated with the chrysophanol peak and is not less than 6000, and in the retention time identical with aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance, chrysophanol reference substance, negative control is noiseless.Its HPLC figure is respectively like Fig. 5, Fig. 6, Fig. 7, shown in Figure 8.

(5) method for preparing of need testing solution is selected

A.8% the ratio of hydrochloric acid and chloroform is selected

Get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution XmL (pressing table 6 operation), supersound process 5min; Add chloroform YmL (press table 6 operation) again, reflux 1 h is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.The result sees table 6.

8% hydrochloric acid of table 6 different proportion and the extraction efficiency of chloroform

Conclusion: ratio is that the total content of aloe-emodin in the Radix Et Rhizoma Rhei of 8% hydrochloric acid and chloroform extraction of different proportions such as 8% hydrochloric acid and chloroform and 5:10,10:5, the 10:10 of 10:15, chrysophanic acid, emodin, chrysophanol exists significant difference; Ratio is the total content there was no significant difference of aloe-emodin in the Radix Et Rhizoma Rhei of 8% hydrochloric acid and chloroform extraction of 10:15,15:10,15:15, chrysophanic acid, emodin, chrysophanol.Taking all factors into consideration from the solvent angle, is the optimum extraction ratio so preferred proportion is 8% hydrochloric acid and the chloroform of 10:15.

B. the selection of reflux extracting time

Get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution 10mL, supersound process 5min; Add chloroform 10mL again, reflux (time press table 7 operation) is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction 4 times, each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.The result sees table 7.

The extraction efficiency of the different reflux extracting times of table 7 (mg/g)

Conclusion: backflow 60min, the 90min 45min that refluxes, Radix Et Rhizoma Rhei total free anthraquinones content obviously increases; And the relative 90min of backflow 60min, Radix Et Rhizoma Rhei total free anthraquinones content and there was no significant difference are taken all factors into consideration selection, and extracting 60min is the optimum extraction time.

C. extraction times is selected

Get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and in the 50mL round-bottomed flask, adds 8% hydrochloric acid solution 10mL, supersound process 5min; Add chloroform 10mL again, reflux 1h is put coldly, puts in the separatory funnel, with a small amount of chloroform washing container; Incorporate in the separatory funnel, obtain the chloroform layer, sour water layer reuse chloroform extraction (number of times is pressed table 8 operation), each 10mL merges the chloroform filtrate; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 10mL measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get.The result sees table 8.

The extraction efficiency of the different extraction times of table 8

Conclusion: according to the color of solution extraction, sample is colourless when extracting for the third time.Through relatively extracting there was no significant difference 3 times, 4 times, 5 times, confirm extraction 4 times so take all factors into consideration.

(6) investigation of linear relationship

Down each precision of each reference substance solution (being aloe-emodin, chrysophanic acid, emodin, chrysophanol reference substance solution) is drawn 2 μ L, 4 μ L, 6 μ L, 8 μ L, 10 μ L, 12 μ L by (2) item respectively; Inject instrument; By the chromatographic condition sample introduction under (3) item, measuring peak area respectively, is abscissa with concentration; The peak area integrated value is a vertical coordinate, carries out linear regression and obtains linear equation (n=6):

Aloe-emodin y=3.7654x+5.6267 (R=0.9999);

Chrysophanic acid y=3.5933x+7.367 (R=0.9998);

Emodin y=3.2078x+3.9133 (R=0.9999);

Chrysophanol y=4.1528x+10.653 (R=0.9999).

The mass concentration of proof aloe-emodin, chrysophanic acid, emodin, chrysophanol reference substance has good linear relationship respectively in 29 μ g/mL-174 μ g/mL, 55.4 μ g/mL-332.4 μ g/mL, 56.6 μ g/mL-339.6 μ g/mL, 131 μ g/mL-786 μ g/mL scopes.

(7) precision test

Get each the 5 μ L of reference substance solution under (2) item respectively; Inject high performance liquid chromatograph; Measure peak area by (3) down described chromatographic condition, repetition sample introduction 6 times calculates with aloe-emodin, chrysophanic acid, emodin, chrysophanol area; Obtain relative standard deviation RSD (%), the result sees table 9, table 10, table 11, table 12.

Table 9 aloe-emodin precision is investigated the result

Table 10 chrysophanic acid precision is investigated the result

Table 11 emodin precision is investigated the result

Table 12 chrysophanol precision is investigated the result

The result: < 3% (n=6) shows that precision is good to the precision relative standard deviation RSD (%) of aloe-emodin, chrysophanic acid, emodin, chrysophanol.

(8) stability test

Get same lot number sample; By (4) need testing solution method for preparing operation down, absorption need testing solution 10 μ L, respectively 0,2,4,8,12h injects high performance liquid chromatograph; Chromatographic condition by under (3) item is measured peak area; Calculate with free anthraquinones extracted from rheum constituents area, obtain relative standard deviation RSD (%), the result sees table 13, table 14, table 15, table 16.

Table 13 aloe-emodin study on the stability result

Table 14 chrysophanic acid study on the stability result

Table 15 emodin study on the stability result

Table 16 chrysophanol study on the stability result

Conclusion: aforementioned stable property is the result show, aloe-emodin, chrysophanic acid, emodin, chrysophanol are good at the 12h internal stability, therefore meets the requirements.

(9) replica test

Get same lot number sample,, draw need testing solution 10 μ L by the need testing solution method for preparing operation under (4) item; Inject high performance liquid chromatograph; Chromatographic condition by under (3) item is measured peak area, calculates free anthraquinones extracted from rheum total content in each sample by one point external standard method, and the result sees table 17.

Table 17 replica test is table as a result

The result shows: the RSD of the aloe-emodin in this sample, chrysophanic acid, emodin, chrysophanol is all less than 3%, so repeatability is good.

(10) average recovery test

Get same this sample of lot number, mixing is got about 0.3g; 9 parts, be divided into three groups, 3 parts every group; The accurate title, decide; Put in the tool plug conical flask, per three parts of accurate respectively reference substance storing solutions (volume of concentration and adding thereof is seen table 18) that pipette, add aloe-emodin, chrysophanic acid, emodin, chrysophanol mix water bath method in reference substance and sample mix to the 50mL round-bottomed flask.By (4) item down, the need testing solution method for preparing is operated, and draws reference substance solution and need testing solution each 5 μ L and 10 μ L, and the injection high performance liquid chromatograph is measured peak area by the chromatographic condition under (3) item, is calculated as follows out the response rate, and calculating formula is following:

The response rate=(C-A)/B * 100%;

Wherein, A: the amount (mg) that contains corresponding reference substance in the sample;

B: reference substance addition (mg);

C: the amount of recording (mg).

The result that application of sample reclaims sees table 19.

Table 18 adds reference substance concentration and volume thereof

The result that table 19 application of sample reclaims

Conclusion: the average recovery rate of aloe-emodin, chrysophanic acid, emodin, chrysophanol all is respectively between 95%-105%, and RSD is respectively less than 3% (n=9), and this result shows that the response rate is high.

(12) sample determination

Get three lot sample article,, prepare need testing solution, measure the content of the aloe-emodin in the Radix Et Rhizoma Rhei, chrysophanic acid, emodin, chrysophanol in the three lot sample article, measure the result and see table 20 by (a 5) need testing solution method for preparing operation down.

Table 20 three lot sample article assay results

Mensuration result shows, on the basis of average content, total content is floated downward and 20% formulated this granule content limit, limits in every bag granule, and the total free anthraquinone constituents content of aloe-emodin, chrysophanic acid, emodin, chrysophanol must not be lower than 4.9mg in the Radix Et Rhizoma Rhei.

2, schisandrin in the Fructus Schisandrae Chinensis

(1) instrument and reagent

Instrument: high performance liquid chromatograph (Agilent1100), ultrasonic washing unit (HS20500D), balance (AB204-S).

Reference substance: schisandrin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110857-200709).

Sample: granule of the present invention (spend the liver benefiting toxin expelling granule in vain, Guiyang College of Traditional Chinese Medicine develops the pharmaceutical analysis experimental center voluntarily, lot number 20111201,20111202,20111203).

(2) reference substance solution preparation

Precision takes by weighing the schisandrin reference substance, adds methanol and processes the reference substance solution that every 1mL contains 7.8 μ g.

(3) selection of chromatographic condition

Chromatograph wavelength preferred: when selecting schisandrin to measure chromatographic condition, with reference to " the assay chromatographic condition of schisandrol carries out the screening of chromatograph wavelength under five tastes subitem of Chinese pharmacopoeia version in 2010.The separating degree, theoretical cam curve and the peak area thereof that compare schisandrin peak under 240nm, 250nm, 254nm, the 260nm equiwavelength respectively; Result's proof is at wavelength 250nm place; Free anthraquinone component separating degree is best in the Radix Et Rhizoma Rhei, and peak area is maximum, and interference factor is minimum.

Chromatogram column temperature preferred: this experiment has been compared 20 ℃, 25 ℃, 30 ℃ column temperature to the influence of free anthraquinone component in the Radix Et Rhizoma Rhei, and experimental result shows that under 25 ℃ of column temperature conditions, separating degree is good, and retention time is short, and theoretical cam curve is high.

The selection of mobile phase: this experiment is " the mobile example of comparing of difference such as the mobile phase comparison methanol-water of Chinese pharmacopoeia with reference to 2010; The composition of schisandrin all can not reach fully and separate; And drift about with methanol-water flow phase system retention time, main cause is that methanol is easy to generate bubble when mixing with water, and the gradient elution ratio that this experiment optimizes causes the chromatographic peak retention time of Radix Et Rhizoma Rhei to be drifted about; And unstability of base line, can not reach the related request that flows kind new medicine.Because the eluting power of acetonitrile is strong, separating degree good, so this experiment employing acetonitrile-water is mobile phase, compared the different proportion (45:55 of acetonitrile-water; 40:60; 55:45; 50:50), thus the schisandrin peak is arrived with other chromatographic peaks to be separated fully.

(4) system suitability test

The preparation of negative solution: get the about 0.6g of negative sample that lacks Fructus Schisandrae Chinensis, the accurate title, decide, and puts in the 25mL volumetric flask, adds the about 20mL of methanol, and supersound process (power 250W, frequency 20HZ) 45min takes out, and adds methanol to scale, shakes up, and gets subsequent filtrate, promptly gets.

The preparation of blank solution: get the 25mL volumetric flask, add the about 20mL of methanol, supersound process (power 250W, frequency 20HZ) 45min takes out, and adds methanol to scale, shakes up, and gets subsequent filtrate, promptly gets.

The preparation of need testing solution: get the about 0.6g of these article under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask, adds the about 20mL of methanol, and supersound process (power 250W, frequency 20HZ) 45min takes out, and adds methanol to scale, shakes up, and gets subsequent filtrate, promptly gets.

Assay method: the accurate respectively reference substance solution 5 μ l that draw, blank solution, negative solution, each 10 μ L of need testing solution inject high performance liquid chromatograph, measure, and promptly get.

Can know by collection of illustrative plates, sample introduction analysis under the above-mentioned chromatographic condition, the separating degree of schisandrin and adjacent chromatographic peak is all greater than 1.5.The sample number of theoretical plate is in schisandrin, so confirm that theoretical cam curve is not less than 3000 in schisandrin, in the retention time identical with schisandrin, negative control is noiseless.Result such as Fig. 9, Figure 10, Figure 11, shown in Figure 12.

(5) selection of the method for preparing of need testing solution

A. extract choice of Solvent:

Get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask, adds the about 20mL of different extraction solvent (specifically seeing table 20); Supersound process (power 250W, frequency 20HZ) 45min takes out, and adds different solvents to scale; Shake up, get subsequent filtrate, promptly get.The result sees table 21.

Table 21 different solvents is to the influence of the extraction ratio of schisandrin

Conclusion: the chemical property of considering schisandrin; With reference to 2010 editions " extraction solvents of Chinese pharmacopoeia schisandrin; Investigation is with the influence of different solvents such as 50% methanol, 70% ethanol, 100% methanol to the extraction ratio of schisandrin; The result shows that the extraction ratio of 50% methanol is the highest, so select 50% methanol as the optimum extraction solvent.

B. the selection of method for distilling:

Supersound extraction: get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask, adds the about 20mL of 50% methanol, and supersound process (power 250W, frequency 20HZ) 45min takes out, and adds 50% methanol to scale, shakes up, and gets subsequent filtrate, promptly gets.

Reflux, extract: get the about 0.6g of this granule under the content uniformity item, accurate claim surely, put the 50mL conical flask, precision is measured 25mL 50% methanol, and precision is weighed, and reflux 45min is put coldly, mends heavyly, shakes up, and filters, and gets subsequent filtrate, promptly gets.The result sees table 22.

Table 22 Different Extraction Method is to the influence of the extraction ratio of schisandrin

Conclusion: the efficient of these two kinds of method for distilling of reflux and supersound extraction is suitable, but ultrasonic extraction extraction time weak point, method is simple, therefore selects ultrasonic extraction for use.

C. the selection of extraction time

Supersound extraction: get the about 0.6g of this granule under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask, adds the about 20mL of 50% methanol; Supersound process (power 250W, frequency 20HZ) different time takes out, and adds 50% methanol to scale; Shake up, get subsequent filtrate, promptly get.The result sees table 23.

Table 23 different extraction times are to the influence of the extraction ratio of schisandrin

Conclusion: after the supersound extraction 45min, schisandrin content no longer increases, and schisandrin had extracted fully when supersound extraction 45min was described, therefore selecting the supersound extraction time is 45min.

(6) investigation of linear relationship

Accurate respectively (2) item reference substance solution 4 μ L, 8 μ L, 12 μ L, the 16 μ L down of drawing; Measure peak area by chromatographic condition sample introduction under (3) item; With concentration is abscissa; The peak area integrated value is a vertical coordinate, carries out linear regression and obtains linear equation Y=3.9032X-18.93 (n=5), proves that the mass concentration of schisandrin has good linear relationship in 31.2 μ g/mL-124.8 μ g/mL (r=1) scopes.

(7) precision test

Get reference substance solution 5 μ L, inject high performance liquid chromatograph, measure peak area by chromatographic condition under (3) item, repeat sample introduction 6 times, calculate with the schisandrin area, obtain relative standard deviation RSD (%), the result sees table 24.

Table 24 schisandrin precision is investigated the experimental result table

< 3% (n=6) shows that precision is good to conclusion: relative standard deviation RSD.

(8) stability test

Get same lot number sample; By (4) preparation need testing solution down, draw need testing solution 10 μ L, respectively 0,2,4,8,12h injects high performance liquid chromatograph; Measure peak area by chromatographic condition under (3) item; Calculate with the schisandrin area, obtain relative standard deviation RSD (%), the result sees table 25.

Table 25 schisandrin study on the stability experimental result table

Conclusion: get need testing solution, injecting chromatograph in the time of setting records average peak area and is: 304.98, and RSD (%) is 1.91% (n=5), shows that this need testing solution is stable in 12h.

(9) replica test

Get same these article of lot number,, draw need testing solution 10 μ L by the need testing solution method for preparing operation under (4) item; Inject high performance liquid chromatograph; Measure peak area by chromatographic condition under (3) item, calculate contained schisandrin content in each sample by one point external standard method, the result sees table 26.

Table 26 replica test is table as a result

The result shows: the schisandrin average content in this sample is 0.3362mg/g, and RSD is all less than 3%, so this method repeatability is good.

(10) average recovery test

Get this granule of same lot number, mixing is got about 0.3g, 9 parts; Accurate claim surely, put in the tool plug conical flask that (volume is respectively 2mL, 2.5mL, 3mL to per three parts of accurate respectively storing solutions that pipette the schisandrin reference substance that adds 40.345 μ g/mL concentration;), with sample mix, prepare need testing solution down by (4) item; Draw reference substance solution 5 μ L and need testing solution 10 μ L, inject high performance liquid chromatograph, measure peak area by chromatographic condition under (3) item; Be calculated as follows out the response rate, the result sees table 27, and computing formula is following:

The response rate=(C-A)/B * 100%;

A: the amount (μ g) that contains corresponding reference substance in the sample;

B: reference substance addition (μ g);

C: the amount of recording (μ g);

The average recovery result of table 27 schisandrin

Conclusion: it is 98.55% that this method is measured average recovery rate, and the RSD value is 2.86%.Explain that this method is feasible, the response rate is good.

(11) sample determination

The schisandrin assay prepares need testing solution down by (4) item in the three lot sample article; Draw respectively 10 μ L of reference substance solution and need testing solution, inject high performance liquid chromatograph, press (3) item chromatographic condition mensuration peak area down; Calculate the schisandrin content of three lot sample article, the result sees table 28.

Three batches in table 28 is spent schisandrin assay in the liver benefiting toxin expelling granule in vain

The result shows that three batches of average contents of spending every bag of (6g) middle schisandrin of the liver benefiting toxin expelling granule in vain are 1.9557 mg.Fluctuate according to total content and 20% to formulate this formulation content limit, limit every bag and spend the liver benefiting toxin expelling granule in vain and contain the schisandrin component content and must not be lower than 1.56mg.

Compared with prior art, the present invention improves spending the particulate preparation technology of the liver benefiting toxin expelling in vain, and it is more abundant to make in the medical material main active substances of enantiopathy virus hepatitis extract, and its clinical efficacy is better; And the liver benefiting toxin expelling granule of spending in vain that the present invention is directed to after the improvement has been set up system, complete, effective on-line quality testing method, and the specificity of said method is strong; Precision is high; Favorable reproducibility, the response rate is high, and measurement result is accurate; Reach the purpose of effective control drug quality, thereby guaranteed stablizing and safety of clinical administration, effective of product quality.

Description of drawings

Fig. 1 is the thin layer chromatography discriminating figure of Radix Et Rhizoma Rhei;

Fig. 2 is the thin layer chromatography discriminating figure of Fructus Schisandrae Chinensis;

Fig. 3 is the thin layer chromatography discriminating figure of Radix Glycyrrhizae;

Fig. 4 is the thin layer chromatography discriminating figure of Folium Ginkgo;

Fig. 5 is the HPLC figure of aloe-emodin, chrysophanic acid, emodin, chrysophanol reference substance;

Fig. 6 is the HPLC figure of the liver benefiting toxin expelling granule test sample in the total free anthraquinone constituents assay;

Fig. 7 is the HPLC figure of the negative sample that the liver benefiting toxin expelling granule lacks Radix Et Rhizoma Rhei in the total free anthraquinone constituents assay;

Fig. 8 is the HPLC figure of solvent blank sample in the total free anthraquinone constituents assay;

Fig. 9 is the HPLC figure of schisandrin reference substance;

Figure 10 is the HPLC figure of the liver benefiting toxin expelling granule test sample in the schisandrin assay;

Figure 11 is the HPLC figure of the negative sample that the liver benefiting toxin expelling granule lacks Fructus Schisandrae Chinensis in the schisandrin assay;

Figure 12 is the HPLC figure of solvent blank sample in the schisandrin assay.

The specific embodiment

Below in conjunction with embodiment the present invention is further described.

Embodiment 1: take by weighing Herba Hedyotidis Diffusae 20kg, Ganoderma 10kg, Rhizoma Polygonati 8kg, Radix Astragali 10kg, Rhizoma Alismatis 8kg, Radix Et Rhizoma Rhei 8kg, Herba Cistanches 4kg, Carapax Trionycis 10kg, Fructus Schisandrae Chinensis 8kg, Folium Ginkgo 8kg and Radix Glycyrrhizae 6kg; Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis medical material are added 6 times of amount ethanol extractions of 70% 3 times, and each 2h filters; Alcohol extract is evaporated to density 1.2 (density bottle survey) for 70 ℃, and thick extractum gets dry extract in the dry 36h of 80 ℃ of vacuum decompressions; Pulverize sieve No. 3, got the alcohol extraction powder.All the other 8 flavor crude drug mix with the alcohol extraction medicinal residues and add 6 times of water gagings immersion 0.5h, add 6 times of water gagings 0.5h that is decocted first again, add 12 times of amounts of water altogether; Decoct three times, decocting time is respectively: 3h, 3h and 1h, and 85 ℃ are evaporated to density 1.2 (density bottle survey); 80 ℃ of drying under reduced pressure 48h; Get dry extract, pulverized sieve No. 3, get water and carry powder.Alcohol extraction powder and water are put forward powder mixes, do sweeting agent, do wetting agent (every 100g mixture adds 17.5mL 80% ethanol) with 80% ethanol in ratio adding adjuvant soluble starch and 0.2% aspartame of 1:1; Wet granulation, 75 ℃ of dry 4-6h of granule, granulate; Cross No. 1 and screen out bulky grain; Cross No. 5 and screen out powder (cross No. 5 sieve gained fine powders and add granulation next time),, promptly get by every bag 6g packing.

Embodiment 2: take by weighing Herba Hedyotidis Diffusae 5kg, Ganoderma 2kg, Rhizoma Polygonati 1kg, Radix Astragali 2kg, Rhizoma Alismatis 1kg, Radix Et Rhizoma Rhei 1kg, Herba Cistanches 1kg, Carapax Trionycis 2kg, Fructus Schisandrae Chinensis 1kg, Folium Ginkgo 1kg and Radix Glycyrrhizae 1kg; Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis medical material are added 6 times of amount 70% ethanol extractions 3 times, and each 2h filters, and alcohol extract is evaporated to density 1.2 for 70 ℃, and thick extractum gets dry extract in the dry 36h of 80 ℃ of vacuum decompressions, and crushing screening gets the alcohol extraction powder; All the other 8 flavor crude drug mix with the alcohol extraction medicinal residues and add 6 times of water gagings immersion 0.5h, add 6 times of water gagings 0.5h that is decocted first again, add 12 times of amounts of water altogether; Decoct three times, decocting time is respectively: 3h, 3h and 1h, and 85 ℃ are evaporated to density 1.2; 80 ℃ of drying under reduced pressure 48h; Get dry extract, crushing screening gets water and carries powder; Alcohol extraction powder and water are put forward powder mixes, do sweeting agent, do wetting agent with 80% ethanol by 1: 1 part by weight adding adjuvant soluble starch and 0.2% aspartame, wet granulation, 75 ℃ of dry 4-6h of granule, granulate sieves, and packing promptly gets.

Embodiment 3: take by weighing Herba Hedyotidis Diffusae 25kg, Ganoderma 15kg, Rhizoma Polygonati 10kg, Radix Astragali 15kg, Rhizoma Alismatis 10kg, Radix Et Rhizoma Rhei 10kg, Herba Cistanches 5kg, Carapax Trionycis 15kg, Fructus Schisandrae Chinensis 10kg, Folium Ginkgo 10kg and Radix Glycyrrhizae 6kg; Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis medical material are added 6 times of amount 70% ethanol extractions 3 times, and each 2h filters, and alcohol extract is evaporated to density 1.2 for 70 ℃, and thick extractum gets dry extract in the dry 36h of 80 ℃ of vacuum decompressions, and crushing screening gets the alcohol extraction powder; All the other 8 flavor crude drug mix with the alcohol extraction medicinal residues and add 6 times of water gagings immersion 0.5h, add 6 times of water gagings 0.5h that is decocted first again, add 12 times of amounts of water altogether; Decoct three times, decocting time is respectively: 3h, 3h and 1h, and 85 ℃ are evaporated to density 1.2; 80 ℃ of drying under reduced pressure 48h; Get dry extract, crushing screening gets water and carries powder; Alcohol extraction powder and water are put forward powder mixes, do sweeting agent, do wetting agent with 80% ethanol by 1: 1 part by weight adding adjuvant soluble starch and 0.2% aspartame, wet granulation, 75 ℃ of dry 4-6h of granule, granulate sieves, and packing promptly gets.

Embodiment 4: the particulate complete detection method of the liver benefiting toxin expelling of spending in vain according to the invention is:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; Acetonitrile: water=45%:55% is a mobile phase; Flow velocity is 1.0 mLmin ^-125 ℃ of column temperatures; Detecting wavelength is 250 nm; Theoretical cam curve is calculated with the schisandrin peak and is not less than 2000;

Claims

1. spend the liver benefiting toxin expelling granule in vain for one kind; It is characterized in that: it is to be crude drug with Herba Hedyotidis Diffusae 5 ~ 25kg, Ganoderma 2 ~ 15kg, Rhizoma Polygonati 1 ~ 10kg, the Radix Astragali 2 ~ 15kg, Rhizoma Alismatis 1 ~ 10kg, Radix Et Rhizoma Rhei 1 ~ 10kg, Herba Cistanches 1 ~ 5kg, Carapax Trionycis 2 ~ 15kg, Fructus Schisandrae Chinensis 1 ~ 10kg, Folium Ginkgo 1 ~ 10kg and Radix Glycyrrhizae 1 ~ 6kg; Extract the back and add an amount of soluble starch and aspartame, and be that wetting agent is processed with 80% ethanol.

2. the liver benefiting toxin expelling granule of spending in vain according to claim 1; It is characterized in that: it is to be crude drug with Herba Hedyotidis Diffusae 20kg, Ganoderma 10kg, Rhizoma Polygonati 8kg, Radix Astragali 10kg, Rhizoma Alismatis 8kg, Radix Et Rhizoma Rhei 8kg, Herba Cistanches 4kg, Carapax Trionycis 10kg, Fructus Schisandrae Chinensis 8kg, Folium Ginkgo 8kg and Radix Glycyrrhizae 6kg; Extract the back and add an amount of soluble starch and aspartame, and be that wetting agent is processed with 80% ethanol.

3. like each described particulate method for preparing of the liver benefiting toxin expelling of spending in vain among the claim 1-2, it is characterized in that: Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis, Rhizoma Alismatis medical material are added 6 times of amount 70% ethanol extractions 3 times, each 2h; Filter; Alcohol extract is evaporated to density 1.2 for 70 ℃, and thick extractum gets dry extract in the dry 36h of 80 ℃ of vacuum decompressions; Crushing screening gets the alcohol extraction powder; All the other 8 flavor crude drug mix with the alcohol extraction medicinal residues and add 6 times of water gagings immersion 0.5h, add 6 times of water gagings 0.5h that is decocted first again, add 12 times of amounts of water altogether; Decoct three times, decocting time is respectively: 3h, 3h and 1h, and 85 ℃ are evaporated to density 1.2; 80 ℃ of drying under reduced pressure 48h; Get dry extract, crushing screening gets water and carries powder; Alcohol extraction powder and water are put forward powder mixes, do sweeting agent, do wetting agent with 80% ethanol by 1: 1 part by weight adding adjuvant soluble starch and 0.2% aspartame, wet granulation, 75 ℃ of dry 4-6h of granule, granulate sieves, and packing promptly gets.

According to claim 1 or claim 2 spend the particulate detection method of the liver benefiting toxin expelling in vain, it is characterized in that: described detection method comprises character, discriminating, inspection and assay project; Wherein differentiate it is thin layer chromatography discriminating to the Folium Ginkgo in the preparation, Radix Glycyrrhizae, Radix Et Rhizoma Rhei, Fructus Schisandrae Chinensis; Assay is a content of measuring the contained total free anthraquinone constituents of Radix Et Rhizoma Rhei and the contained schisandrin of Fructus Schisandrae Chinensis in the preparation with HPLC respectively.

5. the particulate detection method of the liver benefiting toxin expelling of spending in vain according to claim 4 is characterized in that: the discrimination method of Folium Ginkgo is to be contrast with the Folium Ginkgo control medicinal material, is the thin layer chromatography of developing solvent with ethyl acetate-butanone-formic acid-water=5:3:1:1; The discrimination method of Radix Glycyrrhizae is to be contrast with Radix Glycyrrhizae control medicinal material and ammonium glycyrrhizinate reference substance, is the thin layer chromatography of developing solvent with ethyl acetate-formic acid-glacial acetic acid-water=15:1:1:2; The discrimination method of Radix Et Rhizoma Rhei is to be contrast with the Radix Et Rhizoma Rhei control medicinal material, is the thin layer chromatography of developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1; The discrimination method of Fructus Schisandrae Chinensis is to be contrast with Fructus Schisandrae Chinensis control medicinal material and deoxyschizandrin reference substance, is the thin layer chromatography of developing solvent with the upper solution of 30～60 ℃ of petroleum ether-Ethyl formate-formic acid=15:5:1.

6. according to claim 4 or the 5 described particulate detection methods of the liver benefiting toxin expelling of spending in vain, it is characterized in that: concrete discrimination method is:

(1) thin layer chromatography of Folium Ginkgo is differentiated: get this granule 4g, add 40% methanol 20mL, reflux 30min is put coldly, filters, and gets filtrating 10mL and is concentrated into 2mL, as need testing solution; Other gets Folium Ginkgo control medicinal material 1g, shines medical material solution in pairs with legal system; According to " above-mentioned need testing solution 10 μ L and control medicinal material solution 6 μ L are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin layer chromatography, and putting in the same carboxymethylcellulose sodium solution that contains 4% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive preparation; With ethyl acetate-butanone-formic acid-water=5:3:1:1 is developing solvent, launches, and takes out; Dry; Spray is with 3% aluminum chloride alcoholic solution, and hot blast drying is put under the 365nm ultra-violet lamp and inspected; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence principal spot of same color;

7. the particulate detection method of the liver benefiting toxin expelling of spending in vain according to claim 4; It is characterized in that: the content assaying method of total free anthraquinone constituents is to be contrast with aloe-emodin reference substance, chrysophanic acid reference substance, emodin reference substance and chrysophanol reference substance in the Radix Et Rhizoma Rhei, and with acetonitrile: 0.1% phosphoric acid=58%-85%:42%-15% is the HPLC of mobile phase; The content assaying method of schisandrin is to be contrast with the schisandrin reference substance in the Fructus Schisandrae Chinensis, is the HPLC of mobile phase with acetonitrile: water=45%:55%.

8. according to claim 4 or the 7 described particulate detection methods of the liver benefiting toxin expelling of spending in vain, it is characterized in that: concrete content assaying method is:

The preparation of need testing solution: get this granule 0.6g under the content uniformity item, the accurate title, decide, and puts in the 25mL volumetric flask, adds methanol 20mL, takes out behind the supersound process 45min, adds methanol to scale, shakes up, and gets subsequent filtrate, promptly gets need testing solution;

9. according to claim 4, the 5 or 7 described particulate detection methods of the liver benefiting toxin expelling of spending in vain, it is characterized in that: described detection method comprises: