CN102657823B

CN102657823B - Traditional Chinese medicine composition with anticancer effect and preparation method and detection method thereof

Info

Publication number: CN102657823B
Application number: CN2012101723278A
Authority: CN
Inventors: 汤美健; 钟强; 罗海龙; 陈勇; 杨胜玉
Original assignee: CHENGDU DIAO GROUP TIANFU MEDICINE Co Ltd
Current assignee: CHENGDU DIAO GROUP TIANFU MEDICINE Co Ltd
Priority date: 2011-12-01
Filing date: 2012-05-30
Publication date: 2013-03-06
Anticipated expiration: 2032-05-30
Also published as: CN102657823A; CN102671140B; CN102488863A; CN102671140A

Abstract

The invention discloses a medicine composition with anticancer effect and a preparation process and a quality detection method thereof. The medicine composition comprises 34 traditional Chinese medicines such as motherwort herb, ginseng, white peony root and the like. By the method of decoction, steam distillation, alcohol precipitation and the like, the active ingredients fully take effect. Meantime, the invention also provides a method for measuring the content of the medicine composition. The method has the advantages of strong specificity and good precision, stability and repeatability.

Description

A kind of Chinese medicine composition with antitumaous effect and preparation method thereof and detection method

Technical field

The present invention relates to a kind of Chinese medicine composition and preparation method thereof and detection method, be specifically related to a kind of Chinese medicine composition with antitumaous effect and preparation method thereof and quality determining method, belong to the traditional Chinese medicine technical field.

Background technology

Cancer is the major disease that jeopardizes human health, and its fatal rate is the trend that rises year by year, and its generation and development are the complex lesions processes of multifactor participation, polygenes change and Multi stage development.At present mostly adopt operative treatment in conjunction with the Radiotherapy chemotherapy method for the treatment of cancer, but the method is large to patient's wound, dangerous high, and somewhat expensive, but curative effect is unsatisfactory.The weapon that Chinese medicine is prevented and cured diseases as China's tradition is accompanied by in recent years progressively deep research and discovery, more and more comes into one's own in treatment of cancer.Studies show that, in the treatment and prevention of tumour process, Chinese medicine mainly be from improve body's immunity, inducing apoptosis of tumour cell, inhibition tumor cell shifts and the aspects such as attenuation synergistic of chemotherapy are played a role.

Chinese medicine composition compatibility of the present invention is reasonable, can regulate negative and positive of qi and blood and function, and the protection hemopoietic function of bone marrow can start the self-defense system again, improves immunity of organisms, and is very helpful for the auxiliary curing of tumour.

Summary of the invention

The object of the present invention is to provide a kind of Chinese medicine composition with antitumaous effect.

Another object of the present invention is to provide the preparation method of said composition.

Another object of the present invention is to provide the oral liquor of said composition, and the oral liquid quality of this preparation method preparation is high, clarify, contain amount temperature, good drug efficacy.

Another object of the present invention is to provide the quality determining method of said composition.

The objective of the invention is to be achieved through the following technical solutions:

Chinese medicine composition of the present invention is made (by weight) by following bulk drug:

The bulk drug of described Chinese medicine composition is preferably:

Described Chinese prickly ash is charcoal Chinese prickly ash, leech for leech processed, triangular are that vinegar prepared RHIZOMA SPARGANII with vine-gar, rhizoma cyperi are that vinegar prepared RHIZOMA CYPERI, myrrh are that vinegar is processed, semen armeniacae amarae is for stir-fry semen armeniacae amarae, fennel seeds are that salt fennel(parched), excrementum pteropi are that vinegar processs that excrementum pteropi, corydalis tuber are that vinegar processs that corydalis tuber, cattail pollen are that cattail pollen charcoal, frankincense are that vinegar is processed, dried lacquer is that to forge dried lacquer, evodia rutaecarpa be that the liquorice beverage evodia rutaecarpa of processing, tarragon are for processing tarragon.

Above-mentioned Chinese medicine composition routinely technique adds conventional auxiliary material and makes the clinical acceptable formulations such as tablet, capsule, oral liquid, pill, granule.

The invention described above traditional Chinese medicinal composition raw materials adds following auxiliary material and is prepared into oral liquid after extracting; Described auxiliary material comprises: flavouring, sweetener, stabilization agent, thickening agent, dissolving assistant, pH adjusting agent, colorant, essence; Described flavouring is selected from one or more in following: polyvidone, menthol, dipotassium glycyrrhizinate, malic acid, natrium malicum, tartrate, succinic acid, sodium succinate, acetic acid, glutamic acid, citric acid, sodium citrate, glucolactone, sodium chloride.Described sweetener is selected from one or more in following: sucrose, fructose, glucose, lactose, D-sorbite, maltitol, erythrose, Sucralose, honey, asccharin, steviol glycoside extract, Abbas are sweet, acesulfame potassium, sweet protein, Radix Glycyrrhizae, honey element.Stabilizing agent is optional to descend one or more freely: polyvidone, glycerine, arabo-ascorbic acid and salt thereof, edetic acid(EDTA), metaphosphoric acid etc.Thickening agent is optional to descend one or more freely: sodium carboxymethyl cellulose, agar, polyvidone, polyvinyl alcohol (PVA), xanthans.The dissolving assistant is optional to descend one or more freely: polysorbate, polyglycol, polyoxyethylene hardened castor oil, polyvidone, NaOH, polyoxyethylene sorbitan monoleate.PH adjusting agent be selected from following one or more: citric acid, sodium citrate, lactic acid, NaOH, hydrochloric acid, malic acid, natrium malicum, tartrate, succinic acid, acetic acid, gluconic acid, phosphoric acid etc.The auxiliary material of oral liquid most preferably is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion honey element.

Calculate with raw medicinal herbs (bulk drug) amount, the total amount that is equivalent to archen, Chrysophanol in the described composite preparation of 10g crude drug must not be less than 0.28mg.

The ginsenoside Rg ₁, Re total amount meter is not less than 100 μ g, ginsenoside Rb ₁Be not less than 50 μ g; Stachydrine is not less than 50 μ g; Rutaecarpin, Rutaecarpine total amount meter are not less than 100 μ g; Paeoniflorin 50 μ g.

The oral liquor of Chinese medicine composition is characterized in that the method comprises the steps:

Step 1: turtle shell boiling 2-4 time, each 2-4 hour, merge extract, filter filtrate simmer down to turtle shell decocting liquid;

Step 2: Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood steam distillation were extracted 5-7 hour, collected volatile oil and distillate,

Step 3: step 2 steam distillation is extracted the remaining dregs of a decoction and all the other crude drug boilings 2-4 time, and each 1-3 hour, collecting decoction filtered, and filtrate is concentrated into clearly cream 1,

Step 4: the clear cream 1 of step 3 adds ethanol and reaches 60-80% to containing the alcohol amount, and regulating the pH value is 7.5～8.0, leaves standstill, and filters, and filtrate recycling ethanol adds water and gets clearly cream 2,

Step 5: the turtle shell decocting liquid of clear cream 2 steps 1 in the step 4, boil, let cool, add the volatile oil of step 2 and the conventional auxiliary material of distillate and oral liquid, add the water stirring and make dissolving, leave standstill, filter, filtrate is made oral liquid through conventional technique;

Described step 2 and/or 4 leave standstill for: 0～5 ℃ leaves standstill;

The auxiliary material that adds oral liquid in the described step 5 is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion honey element.

Described preparation method also comprises step 6: the assay of the total amount of archen, Chrysophanol; And/or following discriminating A, B, C are any or several.

The present invention also provides the preparation method of said composition oral liquid, and the method comprises the steps:

Turtle shell boiling 2-4 time, each 2-4 hour, collecting decoction filtered, and filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, the dalbergia wood steam distillation was extracted 5-7 hour, it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings 2-4 time, each 1-3 hour, collecting decoction, filter, filtrate is concentrated into the about 1.15(50 of relative density～60 ℃) clear cream, add ethanol and make and contain alcohol and measure and reach 60-80%, regulating the pH value is 7.5～8.0, left standstill (0～5 ℃) 10-40 hour, and filtered filtrate recycling ethanol, add water to the about 1.06(50 of relative density～60 ℃) clear cream, add again the turtle shell decocting liquid, boiled 10-30 minute, let cool, add volatile oil and distillate and 1-5 weight fraction polyoxyethylene sorbitan monoleate, 1-5 weight fraction honey element, add the water stirring and make dissolving, left standstill (0～5 ℃) 24～48 hours, filter, filtrate adds water to 1000 parts by volume, embedding, sterilization, and get final product; The pass of described parts by volume and weight portion is g/ml.

The preparation method of described traditional Chinese medicine oral liquid is preferably:

More than 34 flavors, turtle shell boiling 3 times, each 3 hours, collecting decoction filtered, filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, the dalbergia wood steam distillation was extracted 6 hours, it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings 3 times, each 2 hours, collecting decoction, filter, filtrate is concentrated into the about 1.15(50 of relative density～60 ℃) clear cream, add ethanol and make and contain alcohol and measure and reach 60-80%, regulating the pH value is 7.5～8.0, left standstill 24 hours, and filtered filtrate recycling ethanol, add water to the about 1.06(50 of relative density～60 ℃) clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate and 2 weight fraction polyoxyethylene sorbitan monoleates, honey element 2 weight fraction, add the water stirring and make dissolving, left standstill 24～48 hours, filter, filtrate adds water to 1000 parts by volume, embedding, sterilization, and get final product.

The preparation method of composition oral liquid of the present invention, the method comprises the steps:

Choose following bulk drug:

Turtle shell adds 9 times of decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrate is concentrated into 160-200ml; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood adds 12 times of water, steam distillation was extracted 6 hours, collect volatile oil and distillate 40-60ml, the dregs of a decoction and all the other 25-component boilings three times add 12 times of amounts of water for the first time and decocted second 2 hours, add respectively 10 times in water for three times, 8 times, decocted each 1.5 hours, collecting decoction filters, and it is 1.15 clear cream that filtrate is concentrated into 50～60 ℃ of relative densities, adding ethanol makes and contains alcohol amount and reach 70%, adjusting pH value is 7.5～8.0,0～5 ℃ and left standstill 20-30 hour, filters, filtrate recycling ethanol, add water to 50～60 ℃ of relative densities and be 1.06 clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate and polyoxyethylene sorbitan monoleate, honey element adds the water stirring and makes dissolving, and 0～5 ℃ left standstill 24～48 hours, filter, filtrate adds water, embedding, 105 ℃ of sterilization 60min.

The detection method of drug composition oral liquid of the present invention comprises one or more in following discrimination method and/or the assay:

Differentiate

Differentiate A: the Chinese medicine composition oral liquid of getting suitable raw medicinal herbs amount 20-40g, add hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on strong acid cation exchange resin column, wash with water first, use 1-3mol/L ammonia solution wash-out again, collect the ammonia solution eluent, evaporate to dryness gets residue, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains the 0.5-2mg stachydrine hydrochloride; Draw above-mentioned two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate,, launch take the ratio of 3-12:1-5:0.5-2 as developping agent with n-butyl alcohol-hydrochloric acid-ethyl acetate, take out, dry, spray is with rare bismuth potassium iodide test solution;

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, extract 1-3 time with water saturated normal butyl alcohol, merge n-butanol extracting liquid, evaporate to dryness gets residue, residue adds ammonia solution makes dissolving, be added on the large pore resin absorption column, successively use respectively ammonia solution, water, 20-40% ethanol elution, discard respectively eluent; Continue and use the 60-80% ethanol elution, collect eluent, evaporate to dryness gets residue, and residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.2-1mg; Equivalent is drawn above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, lower floor's solution that chloroform-methanol-water take 8-20:2-12:1-5 as ratio spends the night in placement below 10 ℃ is developping agent, launch, take out, dry, spray is with ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing, puts under the uviol lamp and inspects;

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, extract 1-3 time the merging n-butanol extracting liquid with water saturated normal butyl alcohol, evaporate to dryness gets residue, and residue adds methyl alcohol makes dissolving, adds neutral alumina 1-3g, mix thoroughly, evaporate to dryness is added on the neutral alumina column, uses methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution; Draw each 10 μ l of two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate, take the chloroform-ethyl acetate of 5-10:0.5-2:1-8:0.5-2 ratio-methyl alcohol-strong ammonia solution as developping agent, launch, take out, dry, spray is with the vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing;

Content assaying method:

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take 60 ~ 80: 40 ~ 20 methyl alcohol-0.07% phosphoric acid solution is mobile phase; The detection wavelength is 250 ~ 260nm.Theoretical cam curve is calculated by the archen peak should be not less than 3000;

The preparation of reference substance solution: get the archen reference substance, the Chrysophanol reference substance adds methyl alcohol and makes mixed solution;

The preparation of need testing solution comprises the steps: step 1: described composition oral liquid water, hydrochloric acid, chloroform heating and refluxing extraction; Step 2: reflux extracting liquid gets chloroform solution and water liquid after cooling off, leaving standstill; Step 3: water liquid is used chloroform recovery again; Step 4: combining step 2,3 chloroform extracted solution, reclaim solvent to doing, get residue, residue adds methyl alcohol makes dissolving; The volumetric molar concentration of hydrochloric acid is in the described need testing solution preparation process 1: 2.5mol/L; Add hot reflux 0.5-2 hour in the described need testing solution preparation process 2; Chloroform recovery is 2-4 time in the described reference substance solution preparation process 3; Chloroform is: analyze pure.

The detection method of drug composition oral liquid of the present invention comprises following method:

Differentiate

Differentiate A: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, add watery hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on strong acid cation exchange resin column, wash with water to efflux closely colourlessly, discard water liquid, use again 1-3mol/L ammonia solution wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take n-butyl alcohol-hydrochloric acid-ethyl acetate (3-12:1-5:0.5-2) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, extract 1-3 time with water saturated normal butyl alcohol, each 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add ammonia solution 5ml makes dissolving, be added on the large pore resin absorption column, with ammonia solution 100ml washing, discard the ammonia eluent, wash with water again to neutrality, discard water elution liquid, use again 20-40% ethanol 50ml wash-out, discard ethanol eluate, continue with 60-80% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Other gets ginsenoside Re's reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, place lower floor's solution of spending the night below 10 ℃ as developping agent take chloroform-methanol-water (8-20:2-12:1-5), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at l05 ℃, puts that (365nm) inspects under the uviol lamp.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, extract 1-3 time with water saturated normal butyl alcohol, at every turn 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, add neutral alumina 1-3g, mix thoroughly, evaporate to dryness, be added on the neutral alumina column, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-ethyl acetate-methyl alcohol-strong ammonia solution (5-10:0.5-2:1-8:0.5-2) as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at l05 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

Content assaying method:

The preparation of need testing solution comprises the steps: step 1: described composition oral liquid water, hydrochloric acid, chloroform heating and refluxing extraction; Step 2: reflux extracting liquid gets chloroform solution and water liquid after cooling off, leaving standstill; Step 3: water liquid is used chloroform recovery again; Step 4: combining step 2,3 chloroform extracted solution, reclaim solvent to doing, get residue, residue adds methyl alcohol makes dissolving.The volumetric molar concentration of hydrochloric acid is in the described need testing solution preparation process 1: 2.5mol/L; Chloroform is: analyze pure.Add hot reflux 0.5-2 hour in the described need testing solution preparation process 2; Chloroform recovery is 2-4 time in the described reference substance solution preparation process 3.

The detection method of Chinese medicine composition of the present invention preferably includes following one or more methods:

Differentiate A: take by weighing the Chinese medicine composition of suitable raw medicinal herbs amount 30g, add watery hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on 001 * 7 type (732) Na-type strong acid cation exchange resin column (internal diameter 2cm, the high 15cm of post), wash with water to efflux closely colourless, discard water liquid, use again 2mol/L ammonia solution 120ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take n-butyl alcohol-hydrochloric acid-ethyl acetate (8:3:1) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 30g, extract 2 times with water saturated normal butyl alcohol, each 30ml merges n-butanol extracting liquid, evaporate to dryness, residue adds ammonia solution 5ml makes dissolving, be added on the D101 type large pore resin absorption column (internal diameter 1.5cm, the high 12cm of post), with ammonia solution 100ml washing, discard the ammonia eluent, wash with water again to neutrality, discard water elution liquid, use again 30% ethanol 50ml wash-out, discard 30% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Re's 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 (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, place lower floor's solution of spending the night below 10 ℃ as developping agent take chloroform-methanol-water (13:7:2), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at l05 ℃, puts that (365nm) inspects under the uviol lamp; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Differentiate C: take by weighing and contain the Chinese medicine composition oral liquid that is equivalent to raw medicinal herbs amount 30g, extract 2 times with water saturated normal butyl alcohol, at every turn 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, add neutral alumina 2g, mix thoroughly, evaporate to dryness, be added on the neutral alumina column (100～200 orders, 3g, internal diameter 1cm), with methyl alcohol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Paeoniflorin 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 (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-ethyl acetate-methyl alcohol-strong ammonia solution (8:1:4:1) as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at l05 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

Content assaying method:

According to high effective liquid chromatography for measuring, chromatographic condition and system suitability are take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-0.07% phosphoric acid solution (70: 30) as mobile phase; The detection wavelength is 254nm; Theoretical cam curve is calculated by the archen peak should be not less than 3000; The preparation of reference substance solution: get the archen reference substance, the Chrysophanol reference substance is an amount of, and is accurately weighed, add methyl alcohol and make the mixed solution that every 1ml contains respectively 4 μ g, 8 μ g, and get final product; The preparation of need testing solution: measure the described composition oral liquid that is equivalent to raw medicinal herbs 2g, add water 7ml and hydrochloric acid 1ml, add again chloroform 10ml, added hot reflux 1 hour, immediately cooling is shifted in the separating funnel, with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, and get final product; Determination method: precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.

The present composition is used for the treatment of primary carcinoma of liver, lung cancer, gastrointestinal cancer, breast cancer, gynecologic cancer; It is strong, quick, easy that present composition content assaying method has specificity.In the content assaying method of the present invention, when hydrolysis, added organic solvent, make archen, Chrysophanol dissolving after the hydrolysis more complete, thereby overcome prior art because tested composition is pasted the bottle wall and the precipitation caking causes extracting incomplete shortcoming, make the assay result more accurately, science, have a significant effect.

Experimental example 1: assay experiment

(1) investigation of sample method for hydrolysis

1 sample and reagent

The oral liquid of sample: embodiment 1 preparation, lot number 060502,060503,060504; Self-sufficient and strategically located region medicine company joint stock company limited of group difficult to understand provides by ground, Chengdu.

Archen, Chrysophanol reference substance: lot number archen: 110756-200110, Chrysophanol: 110796-200311 is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, for assay.

Reagent: except methyl alcohol was chromatographically pure, it is pure that all the other reagent are analysis.

2 test methods:

2.1 chromatographic condition and system flexibility experiment

High performance liquid chromatograph: Alltech426, the UVIS200 UV-detector; Chromatographic column: enlightening horse C18 post, 5 μ, 150*4.6mm; Column temperature: 35 ℃; Mobile phase: methyl alcohol-0.07% phosphoric acid solution (70:30); The detection wavelength is 254nm; Flow velocity: 1ml/min; Number of theoretical plate calculates by the archen peak should be not less than 3000.

2.2 the preparation of reference substance solution

Precision takes by weighing archen, Chrysophanol reference substance 3.83mg, the 8.72mg that is dried to constant weight in silica gel drier, puts in the 100ml measuring bottle, adds methyl alcohol to scale, shakes up, and precision is measured 5ml, puts in the 50ml measuring bottle, adds methyl alcohol to scale, shakes up, and get final product.(containing archen 3.83 μ g, Chrysophanol 8.72 μ g among every 1ml).

2.3 need testing solution preparation

Method 1: precision is measured this product 2ml, adds 2.5mol/L sulfuric acid solution 8ml, adds hot reflux 2 hours, let cool, extract 4 times (30ml, 15ml, 15ml, 15ml) with the ether jolting, merge ether solution, use anhydrous sodium sulfate dehydration, filter, filter washs with a small amount of ether, washing lotion is incorporated in the filtrate, the slow evaporate to dryness ether of low temperature, and residue dissolves with methanol-acetone (1:1) and is transferred in the 5ml measuring bottle, add methanol-acetone (1:1) to scale, shake up, filter, get subsequent filtrate, and get final product.

Method 2: precision is measured this product 2ml, places the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shake up, filter, precision is measured subsequent filtrate 5ml, put in the 50ml round-bottomed flask, fling to methyl alcohol, add 2.5mol/L sulfuric acid solution 10ml, ultrasonic processing 5min adds chloroform 10ml again, adds hot reflux 1h, let cool, divide and get chloroform layer, twice of chloroform recovery of acid solution, each 10ml, combined chloroform liquid is with anhydrous sodium sulfate dehydration, chloroform solution is put evaporate to dryness in the water-bath, residue adds an amount of low-grade fever of methyl alcohol makes dissolving, moves in the 5ml measuring bottle, and adds methanol constant volume, shake up, filter, get subsequent filtrate, and get final product.

Method 3: precision is measured this product 2ml, adds water 7ml and hydrochloric acid 1ml, water-bath backflow 1h, let cool, extract (20,15,15ml) 3 times with the ether jolting, merge extract, use a small amount of anhydrous sodium sulfate dehydration, filter, filter washs with a small amount of ether, washing lotion is incorporated in the filtrate, the low temperature evaporate to dryness, and residue dissolves with methyl alcohol and is transferred in the 5ml measuring bottle, add methanol constant volume, shake up, centrifugal, namely get need testing solution.

Method 4: precision is measured this product 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, adds hot reflux 1 hour, immediately cooling is shifted in the separating funnel, and with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, and get final product.

2.4 determination method is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, the injection liquid chromatography is measured, and be get final product.

3 results

The archen that the need testing solution that makes according to method 4 is measured and the content of Chrysophanol are apparently higher than other three groups, so adopt the method as the method for assay need testing solution preparation.See Table 1

The comparison of table 1 method for hydrolysis

(2) methodological study

1 linear the investigation

Precision is measured archen (38.3 μ g/ml), Chrysophanol (87.2 μ g/ml) reference substance solution 1.0,2.0,3.0,4.0,5.0,6.0 μ l respectively, sample introduction, and the record chromatogram is measured its peak area, the results are shown in Table 4.And with peak area value (Y) sample size (X) is returned, get archen typical curve equation and be: Y=2865509.1X+7066.3, r=0.9998; Chrysophanol typical curve equation is: Y=3891218.2X+265424.9, r=0.9998.And with peak area value (A) to sample size (C) mapping, get a straight line, the result shows that archen is good in 0.0383 ~ 0.2298 μ g scope internal linear, Chrysophanol is good in 0.0872 ~ 0.5232 μ g scope internal linear.

The linearity of table 2 archen, Chrysophanol is investigated

2 precision are investigated

Get archen, the Chrysophanol reference substance solution 20 μ l of same concentration, continuous sample introduction is 5 times under identical chromatographic conditions, and the record peak area is investigated instrument precision.Archen, Chrysophanol RSD are respectively 1.93%, 0.96%, and precision is investigated the result and met the requirements.The results are shown in Table 3.

The precision of table 3 archen, Chrysophanol is investigated the result

	Sequence number	Peak area	RSD(%)
					1	227015.60
	2	218583.54
				Archen	3	223751.87	1.93%
	4	230185.39
					5	226489.35
	1	702079.68
					2	713654.34
Chrysophanol	3	709375.34	0.96%
					4	720274.14
	5	715385.83

The study on the stability of 3 archens, Chrysophanol

Archen in archen, Chrysophanol standard items and the test sample, Chrysophanol all were stable in 48 hours.

4 reappearances are investigated

Get same lot number embodiment 1 oral liquid (060401), press 5 parts of the parallel preparations of preparation method of need testing solution, sample introduction 20 μ l get simultaneously reference substance solution 20 μ l sample introductions and measure respectively, by external standard method content and calculate RSD, the results are shown in Table 4.Its RSD is 0.94%, and reappearance is investigated the result and met the requirements.

Table 4 archen, Chrysophanol repeatability are investigated the result

Sequence number	Total content (mg/ props up)	RSD（%）
			1	0.5795
2	0.5730
			3	0.5835	0.94%
4	0.5814
			5	0.5878

5 application of sample recovery tests

Sample (060503) 1ml that precision is measured known content gets 6 parts altogether, adds respectively a certain amount of reference substance, presses preparation method's preparation of need testing solution.Sample introduction is measured respectively, calculates average recovery.The results are shown in Table 5.Archen, Chrysophanol average recovery are 97.91%.

Table 5 archen, Chrysophanol application of sample recovery test result

6 sample sizes are measured

The content assaying method that adopts text to draft is measured 6 batches of embodiment 1 oral liquids, the results are shown in Table 6.

Table 6 sample size measurement result (n=2)

Lot number	Archen and Chrysophanol total content (mg/ props up)	RSD(%)
			060301	0.5155	0.52
060401	0.4568	0.80
			060402	0.4051	1.13
060501	0.2977	1.61
			060502	0.3422	1.78
060503	0.5816	125
			060504	0.5127	0.79

7 specificities are investigated

According to the method described above, the embodiment 1 oral liquid negative control product of embodiment 1 oral liquid and scarce rheum officinale are measured, the negative control product occur without absorption peak in corresponding retention time as a result.Illustrate that other compositions are noiseless to the mensuration of archen, Chrysophanol, the method for drafting has specificity and feasibility.

The need testing solution preparation method investigated during experimental example 2 root of herbaceous peony thin layers were differentiated

The preparation of need testing solution 1: get Chinese medicine composition oral liquid 30ml of the present invention, extract 2 times with water saturated normal butyl alcohol, each 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, add neutral alumina 2g, mix thoroughly, evaporate to dryness, be added on the neutral alumina column (100～200 orders, 3g, internal diameter 1cm), with methyl alcohol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution 1;

The preparation of need testing solution 2: the 20ml jolting that adds diethyl ether is extracted, discard ether solution, water liquid extracts 3 times with water saturated normal butyl alcohol jolting again, and each 15ml merges normal butyl alcohol liquid, water 30ml washing, discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue add ethanol 2ml makes dissolving, adding neutral alumina 1g low temperature mixes thoroughly, drying, neutral alumina pillar (200～300 orders, a 2g who fills in advance packs into, on the internal diameter 10～15mm), with ethanol 60ml wash-out, collect eluent, evaporate to dryness, residue adds ethanol 1ml makes dissolving, as need testing solution 2;

The preparation of reference substance solution 1: get the Paeoniflorin reference substance, add methyl alcohol and make the solution that every 1ml contains 0.5mg, product solution 1 in contrast.

Thin-layer chromatography: according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-ethyl acetate-methyl alcohol-strong ammonia solution (8:1:4:1) as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at l05 ℃.

Conclusion: in the test sample chromatogram, method 1 with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, method 2 with the corresponding position of reference substance chromatogram on the spot of aobvious same color, interference component is arranged.

Experimental example 3: the investigation of the thin-layer identification method specificity of motherwort

The preparation of need testing solution: get three crowdes of each 30ml of embodiment 1 oral liquid, add watery hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on 001 * 7 type (732) Na-type strong acid cation exchange resin column (internal diameter 2cm, the high 15cm of post), wash with water to efflux closely colourless, discard water liquid, use again 2mol/L ammonia solution 120ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution.

The preparation of reference substance solution: get the stachydrine hydrochloride reference substance, add methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution.

Lack the preparation of motherwort negative control solution: in prescription ratio and method for making, make the negative sample that lacks motherwort, get 30ml, according to the need testing solution preparation method, make negative control solution.

Thin-layer chromatography: according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of mentioned solution, put on same silica gel g thin-layer plate respectively and (be followed successively by from left to right reference substance solution, three batch sample solution, negative control solution), take n-butyl alcohol-hydrochloric acid-ethyl acetate (8:3:1) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution.

Conclusion: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, negative control is noiseless in the relevant position.

The thin-layer identification method specificity of experimental example 4 ginsengs is investigated

Need testing solution preparation: get three crowdes of each 30ml of Chinese medicine composition oral liquid of the present invention, extract 2 times with water saturated normal butyl alcohol, each 30ml merges n-butanol extracting liquid, evaporate to dryness, residue adds ammonia solution 5ml makes dissolving, be added on the D101 type large pore resin absorption column (internal diameter 1.5cm, the high 12cm of post), with ammonia solution 100ml washing, discard the ammonia eluent, wash with water again to neutrality, discard water elution liquid, use again 30% ethanol 50ml wash-out, discard 30% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution.

The preparation of reference substance solution: get ginsenoside Re's reference substance, add methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.

The shortage of staff joins the preparation of negative control solution: in prescription ratio and method for making, make the negative sample of shortage of staff's ginseng, get 30ml, according to the need testing solution preparation method, make negative control solution.

Thin-layer chromatography: according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of mentioned solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder and (be followed successively by from left to right reference substance solution, three batch sample solution, negative control solution), place lower floor's solution of spending the night below 10 ℃ as developping agent take chloroform-methanol-water (13:7:2), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at l05 ℃, puts that (365nm) inspects under the uviol lamp.

The screening of experimental example 5 oral liquid additive types of the present invention

The present invention corrects oral liquid smell, taste by adding respective additive, reduces this oral liquid to gastral pungency, improves the content of effective constituent in the preparation.

Press embodiment 1 prescription proportioning, turtle shell adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood adds 12 times of water, steam distillation was extracted 6 hours, and it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings three times, add for the first time 12 times in water, decocted 2 hours, add for the second time 10 times in water, decocted 1.5 hours, and added for the third time 8 times in water, decocted collecting decoction 1.5 hours, filter, it is 1.15 clear cream that filtrate is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, and regulating the pH value is 7.5～8.0,0～5 ℃ left standstill 24 hours, filter, filtrate recycling ethanol adds water to 50～60 ℃ of relative densities and is 1.06 clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, and let cool, add volatile oil and distillate, according to table 8, add and respectively to organize adjuvant, add water stir make the dissolving mixing after, left standstill 1 hour.

5 health volunteers contain in mouth into the about 5mL of oral liquid, do not swallow, and make it spread all over tongue, spue after about 15 seconds.Estimate with the degree of the unhappy tastes such as the bitter taste of 5 grade oral disposition liquid shown in the table 7, pungency and the degree of unhappy smell, calculating mean value the results are shown in Table 8.

The unhappy taste of table 7 oral liquid and the scoring of smell degree

Table 8 different formulations oral liquid taste and smell appraisal result (n=5)

Adjuvant in the table 8 " ‰ " is weight ratio, when solution adds water to 1000ml, adds the weight ratio of adjuvant, and the present invention amounts to the raw material of about 1000g, after extracting, when extract adds water to 1000ml, adds 2g honey element, 2g Tween-80.

By above experiment as seen: experiment can effectively reduce its disagreeable taste and smell with adding (honey element, Tween-80) and (honey element, lecithin) in the oral liquid, and is used in combination effect and is much better than independent result of use.

The impact of active constituent content in the experimental example 6 adjuvant oral disposition liquid

The preparation of oral liquid: press embodiment 1 prescription proportioning, turtle shell adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood adds 12 times of water, steam distillation was extracted 6 hours, it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings three times add 12 times in water for the first time, decocted 2 hours, and added 10 times in water for the second time, decocted 1.5 hours, add for the third time 8 times in water, decocted 1.5 hours, collecting decoction filters, and it is 1.15 clear cream that filtrate is concentrated into 50～60 ℃ of relative densities, adding ethanol makes and contains alcohol amount and reach 70%, adjusting pH value is 7.5～8.0,0～5 ℃ and left standstill 24 hours, filters, filtrate recycling ethanol, add water to 50～60 ℃ of relative densities and be 1.06 clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate, respectively according to table 9 prescription 1,2,3 add adjuvant, add the water stirring and make dissolving, 0～5 ℃ left standstill 24～48 hours, filter, filtrate adds water to 1000ml, gets the oral liquid I, the oral liquid II, the oral liquid III.

Table 9 oral liquid additive formulations

Experimental technique:

Chromatographic condition: Agilent1100 high performance liquid chromatograph; Agilent C18 chromatographic column; Mobile phase: methyl alcohol-0.07% phosphoric acid solution (70:30); Detect wavelength: 254nm.

The preparation of reference substance solution: precision takes by weighing the Chrysophanol reference substance, adds methyl alcohol and makes the solution that every 1ml contains 13 μ g, and get final product.

The preparation of need testing solution:

Precision is measured oral liquid I, oral liquid II, each 2ml of oral liquid III respectively, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, added hot reflux 1 hour, immediately cooling is shifted in the separating funnel, with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, namely get need testing solution 1,2,3.

Assay method: precision is drawn reference substance solution and 3 kinds of each 20 μ L of need testing solution respectively, and the injection liquid chromatography is measured, and one point external standard method is measured the content of Chrysophanol in the oral liquid.

Liquid chromatogram is seen Fig. 1-4; Assay the results are shown in Table 10.

Table 10 assay result

	Peak area	Content mg/10ml
			Reference substance	512.00366
Test sample 1	612.33069	0.389
			Test sample 2	598.91439	0.380
Test sample 3	585.02478	0.371

Experimental result shows: 2 ‰ Tween-80s: 2 ‰ honey elements can increase active constituent content in the oral liquid.

Confirm that oral liquid optimum addn of the present invention consists of: Tween 80, honey element.

The screening of experimental example 7 oral liquid adjuvant use amounts of the present invention

The preparation of oral liquid: press embodiment 1 prescription proportioning, turtle shell adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood adds 12 times of water, steam distillation was extracted 6 hours, it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings three times, add for the first time 12 times in water, decocted 2 hours, and added 10 times in water for the second time, decocted 1.5 hours, and added for the third time 8 times in water, decocted 1.5 hours, collecting decoction, filter, it is 1.15 clear cream that filtrate is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, regulating the pH value is 7.5～8.0,0～5 ℃ left standstill 24 hours, filtered filtrate recycling ethanol, add water to 50～60 ℃ of relative densities and be 1.06 clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate, add adjuvant according to table 12 prescription respectively, add the water stirring and make dissolving, 0～5 ℃ left standstill 24～48 hours, filter, filtrate adds water to 1000ml.The selection result sees Table 11.

The different amount of table 11 additive combination evaluation result

Prescription	Tween-80 (‰)	Honey element (‰)	Taste	Smell
						1	1	1	4.5	4.6
2	1	2	4.8	4.7
					3	1	3	4.5	4.6
4	2	1	4.5	4.6
					5	2	2	4.7	4.7
6	2	3	4.5	4.6
					7	3	1	4.5	4.6
8	3	2	4.5	4.6
					9	3	3	4.4	4.5

Can find out that by above-mentioned evaluation result Tween-80 (‰) and honey element (‰) all have preferably taste and smell in the 1-3:1-3 scope.

Experimental example 8 adjuvants are on the impact of drug effect

1, different oral liquids of the present invention and endoxan share the synergistic effect to lotus S 180 mouse

Select Huaxi Medical Univ's oncology to inoculate the mouse (S180, ascitic type) of lotus S180 sarcoma, be divided at random five groups, respectively conduct in rear 24 hours in inoculation: ⑴ blank group (distilled water 0.4ml/ props up, gavage); ⑵ endoxan group (10mg/Kg, hypodermic injection); ⑶ endoxan group (10mg/Kg, hypodermic injection) is share the oral liquid I and (is experimental example 6 preparation oral liquid I, 12.10g crude drug/Kg, gavage) ⑷ endoxan group (10mg/Kg, hypodermic injection) share oral liquid II (for experimental example 6 preparation oral liquid II, 12.10g crude drug/Kg, gavage).Each organizes administration every day 1 time, continuous 10 days.After the last administration 24 hours, claim that taking off cervical vertebra behind the weight of animals puts to death, strip knurl soma, thymus gland, spleen and weigh.Calculate as follows the heavy and tumour inhibiting rate of knurl, carry out statistics (t check) and process, and calculate the organ coefficient of Thymus and spleen according to body weight.Test findings sees Table 12:

The different oral liquids of the present invention of table 12 are on the impact of Transplantable Murine 180 knurl bulk-growths

From above-mentioned test findings as can be known endoxan murine sarcoma S180 is truly had therapeutic action, when dosage is 10mg/Kg hypodermic injection administration, continuous 10 days, can make the heavy inhibiting rate of knurl reach 45.52%, P＜0.01.After endoxan share with two kinds of oral liquids respectively, then tumor-inhibiting action strengthens, tumor-inhibiting action increase to 53.73% and the 58.09%(P value all＜0.01), increase by 8.21% and 12.57% than simple application endoxan, and share oral liquid II ratio, to share oral liquid I cancer suppressing ratio high by 4.36%.Therefore, can think that oral liquid of the present invention strengthens the chemotherapeutics endoxan to the tumor-inhibiting action of mouse S180, and a certain amount of tween of adding helps to strengthen this synergistic effect in oral liquid of the present invention.

2, different oral liquids of the present invention and fluorouracil share the synergistic effect to mice bearing H_ 22

Behind mouse inoculation H22, be divided at random 4 groups, respectively conduct in 24 hours: ⑴ blank group (distilled water 0.4ml/ props up, gavage) ⑵ fluorouracil group (5-Fu) (10mg/Kg, hypodermic injection); ⑶ fluorouracil (10mg/Kg, hypodermic injection) share the oral liquid I and (is experimental example 6 preparation oral liquid I, 12.10g crude drug/Kg, gavage) ⑷ fluorouracil (10mg/Kg, hypodermic injection) share oral liquid II (for experimental example 6 preparation oral liquid II, 12.10g crude drug/Kg, gavage).Each organizes administration every day 1 time, continuous 10 days.After the last administration 24 hours, claim that taking off cervical vertebra behind the weight of animals puts to death, strip knurl soma, thymus gland, spleen and weigh.Calculate as follows the heavy and tumour inhibiting rate of knurl, carry out statistics (t check) and process, and calculate the organ coefficient of Thymus and spleen according to body weight.The results are shown in Table 13.

The different oral liquids of the present invention of table 13 are on the impact of Transplantable Murine liver cancer (H22) knurl bulk-growth

Experimental result shows that fluorouracil has certain inhibiting effect to the knurl bulk-growth of Transplantable Murine liver cancer (H22).Its inhibiting rate can reach 34.85%(p＜0.05).After fluorouracil and two kinds of oral liquids (12.10g crude drug/Kg, gavage) share, increase respectively 11.36% and 15.15% than simple cancer suppressing ratio with fluorouracil, and share oral liquid II ratio, to share oral liquid I cancer suppressing ratio high by 3.79%.Therefore, can think that oral liquid of the present invention has certain synergistic effect to fluorouracil in treatment Transplantable Murine liver cancer (H22), and the adding tween helps to strengthen this synergistic effect in oral liquid of the present invention.

Description of drawings

Fig. 1 Chrysophanol reference substance chromatogram

Fig. 2 test sample 1 chromatogram

Fig. 3 test sample 2 chromatograms

Fig. 4 test sample 3 chromatograms

Following embodiment all can be realized the effect of above-mentioned experimental example

Embodiment

Embodiment 1:

More than 30 four traditional Chinese medicine materials, turtle shell boiling 3 times (adding 9 times of water), each 3 hours, collecting decoction filtered, filtrate is concentrated in right amount (about 180ml); Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, the dalbergia wood steam distillation is extracted 6 hours (adding 12 times of water), collect volatile oil and distillate an amount of (about 50ml), the dregs of a decoction and all the other 25-component boilings three times, 2 hours (adding 12 times in water) of the first time, second, three times each (added respectively 10 times in water in 1.5 hours, 8 times), collecting decoction, filter, it is 1.15(50～60 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol and make and contain the alcohol amount and reach 70%, regulating the pH value is 7.5～8.0, left standstill (0～5 ℃) 24 hours, and filtered filtrate recycling ethanol, add water to relative density and be 1.06(50～60 ℃) clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate and Tween-80 2 ‰, honey element 2 ‰ adds the water stirring and makes dissolving, leaves standstill (0～5 ℃) 24～48 hours, filter, filtrate adds water to ormal weight (1000ml), and embedding is sterilized (105 ℃, 60min), and get final product.Every 10ml.

Embodiment 2:

More than 34 flavors, turtle shell decocts 3 times with 9 times of water gagings, each 3 hours, collecting decoction filtered, filtrate is concentrated into 180ml; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood adds 12 times of water extractions to be got 6 hours, collected volatile oil and the about 50ml of aqua aromatica, the dregs of a decoction and all the other 25-component boilings 3 times, for the first time add 12 times of amounts of water, decocted second 2 hours, respectively add 10 times of amounts of water for three times, decocted 1.5 hours, collecting decoction is concentrated into relative density about 1.15, add ethanol and make and contain alcohol amount and reach 70%, regulating the pH value is 7.5～8.0, leaves standstill 24 hours, filter, filtrate recycling ethanol, it is about 1.06 to add water to relative density, adds the turtle shell decocting liquid again, boiled 20 minutes, let cool, add volatile oil, aqua aromatica, Tween-80 2 ‰, honey element 2 ‰, add water to 1000ml, left standstill 24～48 hours, get supernatant, embedding, sterilization, packing, and get final product.

Embodiment 3:

The preparation method: above 34 flavors, turtle shell boiling 3 times, each 3 hours, collecting decoction filtered, and filtrate is concentrated in right amount; Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, the dalbergia wood steam distillation was extracted 6 hours, it is an amount of to collect volatile oil and distillate, the dregs of a decoction and all the other 25-component boilings three times, 2 hours for the first time, second, three times each 1.5 hours, collecting decoction, filter, filtrate is concentrated into the about 1.15(50 of relative density～60 ℃) clear cream, add ethanol and make and contain alcohol and measure and reach 70%, regulating the pH value is 7.5～8.0, left standstill 24 hours, and filtered filtrate recycling ethanol, add water to the about 1.06(50 of relative density～60 ℃) clear cream, add again the turtle shell decocting liquid, boiled 20 minutes, let cool, add volatile oil and distillate, through the agent of conventional technique granulation.

Embodiment 4: embodiment 1 Liquid detection method

Differentiate:

(1) get oral liquid 30ml, add watery hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on 001 * 7 type (732) Na-type strong acid cation exchange resin column (internal diameter 2cm, the high 15cm of post), wash with water to efflux closely colourless, discard water liquid, use again 2mol/L ammonia solution 120ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution.Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take n-butyl alcohol-hydrochloric acid-ethyl acetate (8:3:1) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

(2) get oral liquid 30ml, extract 2 times with water saturated normal butyl alcohol, each 30ml merges n-butanol extracting liquid, evaporate to dryness, residue adds ammonia solution 5ml makes dissolving, be added on the D101 type large pore resin absorption column (internal diameter 1.5cm, the high 12cm of post), with ammonia solution 100ml washing, discard the ammonia eluent, wash with water again to neutrality, discard water elution liquid, use again 30% ethanol 50ml wash-out, discard 30% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution.Other gets ginsenoside Re's 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 (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, place lower floor's solution of spending the night below 10 ℃ as developping agent take chloroform-methanol-water (13:7:2), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at l05 ℃, puts that (365nm) inspects under the uviol lamp.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

(3) get oral liquid 30ml, extract 2 times with water saturated normal butyl alcohol, each 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, add neutral alumina 2g, mix thoroughly, evaporate to dryness, be added on the neutral alumina column (100～200 orders, 3g, internal diameter 1cm), with methyl alcohol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the Paeoniflorin 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 (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-ethyl acetate-methyl alcohol-strong ammonia solution (8:1:4:1) as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at l05 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

Assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 D).

Chromatographic condition and system suitability are take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-0.07% phosphoric acid solution (70: 30) as mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the archen peak should be not less than 3000.The archen reference substance is got in the preparation of reference substance solution, the Chrysophanol reference substance is an amount of, and is accurately weighed, adds methyl alcohol and makes the mixed solution that every 1ml contains respectively 4 μ g, 8 μ g, and get final product.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, adds hot reflux 1 hour, immediately cooling is shifted in the separating funnel, and with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, and get final product.

Determination method: precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.Every of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅), Chrysophanol (C ₁₅H ₁₀O ₄) total amount count 0.58mg.

Embodiment 5: embodiment 1 Liquid detection method

Differentiate:

(4) get oral liquid 40ml, regulate pH value to alkalescence with strong ammonia solution, add diethyl ether and extract twice, each 30ml merges ether solution, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution.Other gets evodia rutaecarpa control medicinal material 0.5g, adds ethanol 10ml, adds hot reflux 30 minutes, filters, and filtrate is medicinal material solution in contrast.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw respectively each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal butyl alcohol-glacial acetic acid-water (7:1:2) as developping agent, launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.

Assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 D).Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-0.07% phosphoric acid solution (70: 30) as mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the archen peak should be not less than 3000.The archen reference substance is got in the preparation of reference substance solution, the Chrysophanol reference substance is an amount of, and is accurately weighed, adds methyl alcohol and makes the mixed solution that every 1ml contains respectively 4 μ g, 8 μ g, and get final product.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, adds hot reflux 1 hour, immediately cooling is shifted in the separating funnel, and with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, and get final product.Determination method: precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.Every of this product contains rheum officinale with archen (C ₁₅H ₁₀O ₅), Chrysophanol (C ₁₅H ₁₀O ₄) total amount count 0.58mg.

Embodiment 6: embodiment 1 Liquid detection method

Differentiate:

(5) get oral liquid 30ml, add hydrochloric acid 3ml, put and added hot reflux in the water-bath 1 hour, let cool, extract 2 times with the ether jolting, each 30ml merges ether solution, and evaporate to dryness, residue add chloroform 2ml makes dissolving, as need testing solution.Other gets archen reference substance, Chrysophanol reference substance, and chlorination is copied into the mixed solution that every 1ml contains 1mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw above-mentioned two kinds of each 8ul of solution, put respectively on same silica gel g thin-layer plate, take the upper strata liquid of sherwood oil (30～60 ℃)-ethyl formate-formic acid (15:5:1) as developping agent, launch, take out, dry, put in the ammonia steam smoked after, inspect under the daylight.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(6) get oral liquid 30ml, add diethyl ether and extract 2 times, each 30ml merges ether solution, volatilizes, and the residue 1ml that adds diethyl ether makes dissolving, as need testing solution.Other gets the eugenol reference substance, adds diethyl ether to make the solution that every 1ml contains 16 μ l, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B), draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate (10:1) as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

Assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 D).Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-0.07% phosphoric acid solution (70: 30) as mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the archen peak should be not less than 3000.The archen reference substance is got in the preparation of reference substance solution, the Chrysophanol reference substance is an amount of, and is accurately weighed, adds methyl alcohol and makes the mixed solution that every 1ml contains respectively 4 μ g, 8 μ g, and get final product.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, adds hot reflux 1 hour, immediately cooling is shifted in the separating funnel, and with a small amount of chloroform washing container, washing lotion is incorporated in the separating funnel, leave standstill, divide and get chloroform solution, water liquid is used chloroform recovery 3 times again, each 10ml, the combined chloroform extract, decompression and solvent recovery is to doing, and residue adds methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, and get final product.

Embodiment 7 dwell temperature are investigated test

The preparation method of this experimental basis enforcement 1 makes sample 1, and wherein twice dwell temperature is (0～5 ℃); Press embodiment 1 technique, all change twice dwell temperature into (20 ℃), make sample 2, carry out accelerated test; The main situation of investigating proterties precipitation, content's index.Test condition: under 40 ± 2 ℃ of conditions, tested in 0,1,2,3,6 month and see the following form 14.

Table 14 accelerated test result

On the theory, along with the reduction of temperature, sedimentation effect is more obvious, and the materials such as archen can go down along with sediment together sedimentation.Get oral liquid of the present invention in preparation process, comprise and leave standstill process twice, once be alcohol precipitation after, once for after adding volatile oil and distillate and polyoxyethylene sorbitan monoleate, honey element, when under low temperature, leaving standstill, principal ingredient can appear by sedimentation, affect the content of active component.

Prove that by accelerated test different from the understanding of routine, the liquid precipitation after oral liquid of the present invention refrigeration is left standstill is not only few, and archen, Determination of chrysophanol stable content, the quality standard requirement met.

Claims

1. the oral liquor with Chinese medicine composition of antitumaous effect is characterized in that the method comprises the steps:

Described Chinese medicine composition oral liquid is to be made by following bulk drug:

The preparation method of described Chinese medicine composition oral liquid comprises the steps:

Step 5: the turtle shell decocting liquid of clear cream 2 steps 1 in the step 4, boil, let cool, add the volatile oil of step 2 and the conventional auxiliary material of distillate and oral liquid, add the water stirring and make dissolving, leave standstill, filter, filtrate is made oral liquid through conventional technique.

2. the method for claim 1 is characterized in that preparation method's step 4 of Chinese medicine composition oral liquid and/or the dwell temperature of step 5 are: 0～5 ℃.

3. the method for claim 1 is characterized in that this Chinese medicine composition oral liquid is to be made by following bulk drug:

Motherwort 140 weight portion safflowers 17.5 weight portion charcoals Chinese prickly ash 17.5 weight portions processed

Leech 17.5 weight portion Radix Angelicae Sinensis 35 weight portion bushes 17.5 weight portions processed

Vinegar prepared RHIZOMA SPARGANII with vine-gar 17.5 weight portions 17.5 weight portion Ligusticum wallichiis pointed at both ends 17.5 weight portions

Dalbergia wood 17.5 weight portion vinegar prepared RHIZOMA CYPERIs 17.5 weight portion ginsengs 52.5 weight portions

Galangal 17.5 weight portion turmerics 10.5 weight portion vinegar stir-baked MYRRHA with vinegar 17.5 weight portions

Fry semen armeniacae amarae 26.25 weight portion rheum officinales 70 weight portion perilla seeds 17.5 weight portions

Salt fennel(parched) 26.25 weight portion peach kernels 26.25 weight portions

Vinegar is processed excrementum pteropi 17.5 weight portions

Gadfly 17.5 weight portion turtle shells 140 weight portion cloves 21.25 weight portions

Vinegar is processed the corydalis tuber 17.5 weight portion root of herbaceous peonys 35 weight portion cattail pollen charcoals 17.5 weight portions

Vinegar stir-baked OLIBANUM 17.5 weight portions are forged dried lacquer 17.5 weight portions

Liquorice beverage is processed evodia rutaecarpa 17.5 weight portions

Asafoetide 17.5 weight portion Chinese cassia trees 17.5 weight portions are processed tarragon 17.5 weight portions

Prepared rhizome of rehmannia 35 weight portions;

Calculate with the raw material dose, the total amount that is equivalent to archen, Chrysophanol in the described composition of 10g bulk drug is no less than 0.28mg.

4. the method for claim 1 is characterized in that the auxiliary material that adds oral liquid in the Chinese medicine composition oral liquor step 5 is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion honey element.

5. the method for claim 1 is characterized in that the method also comprises step 6: the assay of the total amount of archen, Chrysophanol;

The content assaying method of the total amount of described archen, Chrysophanol is:

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take 60～80: 40～20 methyl alcohol-0.07% phosphoric acid solution is mobile phase; The detection wavelength is 250～260nm.Theoretical cam curve is calculated by the archen peak should be not less than 3000;

The preparation of need testing solution comprises the steps: step a: described composite preparation water, hydrochloric acid, chloroform heating and refluxing extraction; Step b: reflux extracting liquid gets chloroform solution and water liquid after cooling off, leaving standstill; Step c: water liquid is used chloroform recovery again; Steps d: the chloroform extracted solution of combining step b, c, reclaim solvent to doing, get residue, residue adds methyl alcohol makes dissolving.

6. method as claimed in claim 5 is characterized in that in the content assaying method of total amount of described archen, Chrysophanol, in the described need testing solution preparation among the step a volumetric molar concentration of hydrochloric acid be: 2-3mol/L; Add hot reflux 0.5-2 hour among the described need testing solution preparation process b; Chloroform recovery is 2-4 time among the described reference substance solution preparation process c.

7. the detection method of a Chinese medicine composition oral liquid is characterized in that the method mainly comprises one or more in following discrimination method and/or the assay:

Discrimination method:

Differentiate A: the Chinese medicine composition oral liquid of getting suitable raw medicinal herbs amount 20-40g, add hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on strong acid cation exchange resin column, wash with water first, use 1-3mol/L ammonia solution wash-out again, collect the ammonia solution eluent, evaporate to dryness gets residue, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains the 0.5-2mg stachydrine hydrochloride; Draw above-mentioned two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate,, launch take the ratio of 3-12: 1-5: 0.5-2 as developping agent with n-butyl alcohol-hydrochloric acid-ethyl acetate, take out, dry, spray is with rare bismuth potassium iodide test solution;

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, extract 1-3 time with water saturated normal butyl alcohol, merge n-butanol extracting liquid, evaporate to dryness gets residue, residue adds ammonia solution makes dissolving, be added on the large pore resin absorption column, successively use respectively ammonia solution, water, 20-40% ethanol elution, discard respectively eluent; Continue and use the 60-80% ethanol elution, collect this eluent, evaporate to dryness gets residue, and residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.2-1mg; Equivalent is drawn above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, lower floor's solution that chloroform-methanol-water take 8-20: 2-12: 1-5 as ratio spends the night in placement below 10 ℃ is developping agent, launch, take out, dry, spray is with ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing, puts under the uviol lamp and inspects;

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, extract 1-3 time the merging n-butanol extracting liquid with water saturated normal butyl alcohol, evaporate to dryness gets residue, and residue adds methyl alcohol makes dissolving, adds neutral alumina 1-3g, mix thoroughly, evaporate to dryness is added on the neutral alumina column, uses methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution; Draw each 10 μ l of two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate, take 5-10: 0.5-2: 1-8: the chloroform-ethyl acetate of 0.5-2 ratio-methyl alcohol-strong ammonia solution is as developping agent, launch, take out, dry, spray is with the vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing;

Content assaying method:

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take 60～80: 40～20 methyl alcohol-0.07% phosphoric acid solution is mobile phase; The detection wavelength is 250～260nm; Theoretical cam curve is calculated by the archen peak should be not less than 3000;

The preparation of need testing solution comprises the steps: step 1: described composition oral liquid water, hydrochloric acid, chloroform heating and refluxing extraction; Step 2: reflux extracting liquid gets chloroform solution and water liquid after cooling off, leaving standstill; Step 3: water liquid is used chloroform recovery again; Step 4: combining step 2,3 chloroform extracted solution, reclaim solvent to doing, get residue, residue adds methyl alcohol makes dissolving; The volumetric molar concentration of hydrochloric acid is in the described need testing solution preparation process 1: 2.5mol/L; Add hot reflux 0.5-2 hour in the described need testing solution preparation process 2; Chloroform recovery is 2-4 time in the described reference substance solution preparation process 3;

Described Chinese medicine composition is to be made by following bulk drug:

The oral liquor of described Chinese medicine composition comprises the steps:

8. detection method as claimed in claim 7 is characterized in that described Chinese medicine composition is to be made by following bulk drug:

Salt fennel(parched) 26.25 weight portion peach kernels 26.25 weight portions

Vinegar is processed excrementum pteropi 17.5 weight portions

Liquorice beverage is processed evodia rutaecarpa 17.5 weight portions

Prepared rhizome of rehmannia 35 weight portions;

9. detection method as claimed in claim 7 is characterized in that the method mainly comprises following method:

Differentiate

Differentiate A: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, add watery hydrochloric acid adjust pH to 1～2, centrifugal, get supernatant, on strong acid cation exchange resin column, wash with water to efflux closely colourlessly, discard water liquid, use again 1-3mol/L ammonia solution wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the n-butyl alcohol-hydrochloric acid-ethyl acetate of the ratio of 3-12: 1-5: 0.5-2 as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g, extract 1-3 time with water saturated normal butyl alcohol, each 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add ammonia solution 5ml makes dissolving, be added on the large pore resin absorption column, with ammonia solution 100ml washing, discard the ammonia eluent, wash with water again to neutrality, discard water elution liquid, use again 20-40% ethanol 50ml wash-out, discard ethanol eluate, continue with 60-80% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution; Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 8-20: 2-12: the chloroform-methanol-water of 1-5 ratio is being placed lower floor's solution of spending the night as developping agent below 10 ℃, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃, puts under the 365nm uviol lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, extract 1-3 time with water saturated normal butyl alcohol, at every turn 30ml, merge n-butanol extracting liquid, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, add neutral alumina 1-3g, mix thoroughly, evaporate to dryness, be added on the neutral alumina column, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2-1mg, in contrast product solution; Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 5-10: 0.5-2: 1-8: the chloroform-ethyl acetate of 0.5-2 ratio-methyl alcohol-strong ammonia solution is as developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

Content assaying method:

The preparation of need testing solution comprises the steps: step 1: described composition oral liquid water, hydrochloric acid, chloroform heating and refluxing extraction; Step 2: reflux extracting liquid gets chloroform solution and water liquid after cooling off, leaving standstill; Step 3: water liquid is used chloroform recovery again; Step 4: combining step 2,3 chloroform extracted solution, reclaim solvent to doing, get residue, residue adds methyl alcohol makes dissolving; The volumetric molar concentration of hydrochloric acid is in the described need testing solution preparation process 1: 2.5mol/L; Chloroform is: analyze pure; Add hot reflux 0.5-2 hour in the described need testing solution preparation process 2; Chloroform recovery is 2-4 time in the described reference substance solution preparation process 3;

Determination method: equivalent is drawn reference substance solution and need testing solution l respectively, and the injection liquid chromatography is measured, and be get final product.