CN102671140A

CN102671140A - Anticancer traditional Chinese medicine combination oral liquid, preparation method and detection method thereof

Info

Publication number: CN102671140A
Application number: CN2012101726967A
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: 2012-09-19
Anticipated expiration: 2032-05-30
Also published as: CN102488863A; CN102657823B; CN102671140B; CN102657823A

Abstract

The invention discloses an anticancer traditional Chinese medicine combination oral liquid, a preparation method and a detection method thereof. The medicine combination is composed of thirty-four traditional Chinese medicines such as herba leonuri, ginseng and white paeony root. Active constituents are fully played through methods such as decoction, steam distillation and ethanol precipitation. Simultaneously, the invention further provides a determination method of content of the medicine combination. The method is high in specificity and good in precision, stability and repeatability.

Description

A kind of anticancer traditional Chinese medicine composition oral liquid 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 and preparation method thereof and quality determining method, belong to the Chinese medicine technical field with antitumaous effect.

Background technology

Cancer is the major disease that jeopardizes human health, and its fatality rate is the trend that rises year by year, and its generation and development are the complex lesions processes that multifactor participation, polygenes change and the multistage develops.Adopt mostly for treatment for cancer at present that operative treatment combines to put, embolic chemotherapy, but this method is big to patient's wound, dangerous high, and expensive, but curative effect is unsatisfactory.The weapon that Chinese medicine is prevented and cured diseases as China's tradition is accompanied by progressively deep in recent years research and discovery, in treatment of cancer, more and more comes into one's own.Research shows, in the treatment and prevention of tumour process, Chinese medicine mainly is to shift and the aspects such as attenuation synergistic of chemotherapy are played a role from human body immunity improving function, inducing apoptosis of tumour cell, inhibition tumor cell.

Chinese medicine composition compatibility according to the 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, and human body immunity improving power is very helpful for the auxiliary treatment of tumor.

Summary of the invention

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

Another object of the present invention is to provide the oral liquor of said composition, and the oral liquid quality of this method for preparing 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 oral liquid.

The objective of the invention is to realize through following technical scheme:

Chinese medicine composition according to the invention is processed (by weight) by following crude drug:

The crude drug of said Chinese medicine composition is preferably:

Said Pericarpium Zanthoxyli be charcoal Pericarpium Zanthoxyli, Hirudo be Hirudo (processed), triangular for triangular, the Rhizoma Cyperi of processed with vinegar be that Rhizoma Cyperi (processed with vinegar), Myrrha are that processed with vinegar, Semen Armeniacae Amarum are that Semen Armeniacae Amarum (parched), Fructus Foeniculi are that salt Fructus Foeniculi (parched), Oletum Trogopterori are that processed with vinegar Oletum Trogopterori, Rhizoma Corydalis are that Rhizoma Corydalis (processed with vinegar), Pollen Typhae are that cattail pollen charcoal, Olibanum are that processed with vinegar, Resina Toxicodendri are that to forge Resina Toxicodendri, Fructus Evodiae be that the liquorice beverage Fructus Evodiae of processing, Folium Artemisiae Argyi are for processing Folium Artemisiae Argyi.

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

Ginsenoside Rg1, Re total amount meter are not less than 100 μ g, the ginsenoside Rb1 is 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; Peoniflorin 50 μ g.

The invention described above traditional Chinese medicinal composition raw materials adds following adjuvant and is prepared into oral liquid after extracting; Said adjuvant comprises: correctives, sweeting agent, stabilization agent, thickening agent, dissolving adjuvant, pH regulator agent, coloring agent, essence; Said correctives is selected from one or more in following: polyvidone, Mentholum, glycyrrhizic acid dipotassium, malic acid, natrium malicum, tartaric acid, succinic acid, sodium succinate, acetic acid, glutamic acid, citric acid, sodium citrate, gluconic acid lactone, sodium chloride.Said sweeting agent is selected from one or more in following: sucrose, fructose, glucose, lactose, Sorbitol, maltose alcohol, erythrose, sucralose, Mel, glucide, stevioside extract, Aspartame, acesulfame potassium, sweet protein, Radix Glycyrrhizae, cyclamate.Stabilizing agent is optional to be descended a kind of or several kinds freely: polyvidone, glycerol, arabo-ascorbic acid and salt thereof, edetic acid, Metaphosphoric acid etc.Thickening agent is optional to be descended a kind of or several kinds freely: sodium carboxymethyl cellulose, agar, polyvidone, polyvinyl alcohol, xanthan gum.The dissolving adjuvant is optional to be descended a kind of or several kinds freely: polysorbate, Polyethylene Glycol, polyoxyethylene hardened castor oil, polyvidone, sodium hydroxide, polyoxyethylene sorbitan monoleate.The pH regulator agent be selected from following one or more: citric acid, sodium citrate, lactic acid, sodium hydroxide, hydrochloric acid, malic acid, natrium malicum, tartaric acid, succinic acid, acetic acid, gluconic acid, phosphoric acid etc.

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

Step 1: Carapax Trionycis decocte with water 2-4 time, each 2-4 hour, merge extractive liquid, filtered, filtrating simmer down to Carapax Trionycis decocting liquid;

Step 2: Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae vapor distillation extracted 5-7 hour, collected volatile oil and distillate,

Step 3: step 2 vapor distillation extraction residual medicine dreg and all the other crude drug decocte with water 2-4 time, each 1-3 hour, collecting decoction filtered, and filtrating is concentrated into clear paste 1,

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

Step 5: the Carapax Trionycis decocting liquid of clear paste 2 steps 1 in the step 4, boil, put coldly, add the volatile oil of step 2 and the adjuvant of distillate and oral liquid, add water and stir and make dissolving, leave standstill, filter, filtrating is processed oral liquid through conventional technology;

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

The adjuvant that adds oral liquid in the said step 5 is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion cyclamate.

Said method for preparing also comprises step 6: the assay of the total amount of emodin, chrysophanol; And/or following discriminating A, B, C is any or several kinds.

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

Carapax Trionycis decocte with water 2-4 time, each 2-4 hour, collecting decoction filtered, and filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae vapor distillation extracted 5-7 hour, and it is an amount of to collect volatile oil and distillate, medicinal residues and all the other 25-component decocte with water 2-4 time, each 1-3 hour; Collecting decoction filters, and filtrating is concentrated into the clear paste of relative density about 1.15 (50～60 ℃), adds ethanol and makes and contain the alcohol amount and reach 60-80%; Regulating pH value is 7.5～8.0, leaves standstill (0～5 ℃) 10-40 hour, filters filtrate recycling ethanol; Add water to the clear paste of relative density about 1.06 (50～60 ℃), add the Carapax Trionycis decocting liquid again, boiled 10-30 minute, put cold; Add volatile oil and distillate and 1-5 weight fraction polyoxyethylene sorbitan monoleate, 1-5 weight fraction cyclamate, add the water stirring and make dissolving, left standstill (0～5 ℃) 24～48 hours, filter; Filtrating adds water to 1000 parts by volume, embedding, and sterilization promptly gets; The relation of said parts by volume and weight portion is g/ml.

The method for preparing of said Chinese medicine oral liquid is preferably:

More than 34 flavors, Carapax Trionycis decocte with water 3 times, each 3 hours, collecting decoction filtered, filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae vapor distillation extracted 6 hours, and it is an amount of to collect volatile oil and distillate, medicinal residues and all the other 25-component decocte with water 3 times, each 2 hours; Collecting decoction filters, and filtrating is concentrated into the clear paste of relative density about 1.15 (50～60 ℃), adds ethanol and makes and contain the alcohol amount and reach 60-80%; Regulating pH value is 7.5～8.0, leaves standstill 24 hours, filters filtrate recycling ethanol; Add water to the clear paste of relative density about 1.06 (50～60 ℃), add the Carapax Trionycis decocting liquid again, boiled 20 minutes, put cold; Add volatile oil and distillate and 2 weight fraction polyoxyethylene sorbitan monoleates, cyclamate 2 weight fraction, add the water stirring and make dissolving, left standstill 24～48 hours, filter; Filtrating adds water to 1000 parts by volume, embedding, and sterilization promptly gets.

The method for preparing of composition oral liquid according to the invention, this method comprises the steps:

Choose following crude drug:

Carapax Trionycis adds 9 times of decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrating is concentrated into 160-200ml; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae add 12 times of water, and vapor distillation extracted 6 hours, collect volatile oil and distillate 40-60ml, medicinal residues and all the other 25-component decocte with water three times; Add for the first time 12 times of amounts of water and decocted 2 hours, second and third time adds 10 times in water, 8 times respectively, decocts each 1.5 hours, collecting decoction; Filter, it is 1.15 clear paste that filtrating is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, and regulating pH value is 7.5～8.0; 0～5 ℃ left standstill 20-30 hour, filtered, and filtrate recycling ethanol adds water to 50～60 ℃ of relative densities and be 1.06 clear paste; Add the Carapax Trionycis decocting liquid again, boiled 20 minutes, put coldly, add volatile oil and distillate and polyoxyethylene sorbitan monoleate, cyclamate; Add the water stirring and make dissolving, 0～5 ℃ left standstill 24～48 hours, filtered; Filtrating 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: get the Chinese medicine composition oral liquid of 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, use earlier water elution; Reuse 1-3mol/L ammonia solution eluting is collected the ammonia solution eluent, and evaporate to dryness gets residue; Residue adds methanol makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methanol and processes the reference substance solution that every 1ml contains the 0.5-2mg stachydrine hydrochloride; Drawing above-mentioned two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate, is developing solvent with n-butyl alcohol-hydrochloric acid-ethyl acetate with the ratio of 3-12: 1-5: 0.5-2, launches, and takes out, and dries, and 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; With water saturated n-butanol extraction 1-3 time, merge n-butanol extracting liquid, evaporate to dryness gets residue; Residue adds ammonia solution makes dissolving; Be added on the macroporous adsorptive resins, successively use ammonia solution, water, 20-40% ethanol elution respectively, discard eluent respectively; Continue and use the 60-80% ethanol elution, collect eluent, evaporate to dryness gets residue, and residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methanol and processes the reference substance solution that every 1ml contains 0.2-1mg; Equivalent is drawn above-mentioned two kinds of solution, puts respectively on same silica gel g thin-layer plate, is that the chloroform-methanol-water of ratio is developing solvent at 10 ℃ of lower floor's solution that spend the night with held with 8-20: 2-12: 1-5; Launch; Take out, dry, spray is with ethanol solution of sulfuric acid; It is clear to be heated to the speckle 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, with water saturated n-butanol extraction 1-3 time, merging n-butanol extracting liquid, evaporate to dryness gets residue; Residue adds methanol makes dissolving, adds neutral alumina 1-3g, mixes evaporate to dryness thoroughly; Be added on the neutral alumina post, use methanol-eluted fractions, collect eluent; Evaporate to dryness, residue add methanol makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds methanol and processes the solution that every 1ml contains 0.2-1mg, as reference substance solution; Draw each 10 μ l of two kinds of solution of equivalent, put respectively on same silica gel g thin-layer plate, with 5-10: 0.5-2: 1-8: the chloroform-ethyl acetate of 0.5-2 ratio-methanol-strong ammonia solution is developing solvent; Launch, take out, dry; Spray is with the vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing;

Content assaying method:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With 60～80: 40～20 methanol-0.07% phosphoric acid solution is a mobile phase; The detection wavelength is 250～260nm.Theoretical cam curve is calculated by the emodin peak should be not less than 3000;

The preparation of reference substance solution: get the emodin reference substance, the chrysophanol reference substance adds methanol and processes mixed solution;

The preparation of need testing solution comprises the steps: step 1: said 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 reuse chloroform extraction; Step 4: the chloroform extracted solution of combining step 2,3, reclaim solvent to doing, get residue, residue adds methanol makes dissolving; The molar concentration of hydrochloric acid is in the said need testing solution preparation process 1: 2.5mol/L; In the said need testing solution preparation process 2 reflux 0.5-2 hour; Chloroform extraction is 2-4 time in the said reference substance solution preparation process 3; Chloroform is: analytical pure.

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

Differentiate

Differentiate A: take by weighing the Chinese medicine composition oral liquid of suitable raw medicinal herbs amount 20-40g, add dilute hydrochloric acid adjust pH to 1～2, centrifugal; Get supernatant, on strong acid cation exchange resin column, it is closely colourless that water is eluted to effluent; Discard water liquid, reuse 1-3mol/L ammonia solution eluting is collected eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.Other gets the stachydrine hydrochloride reference substance, adds methanol and processes the solution that every 1ml contains 0.5-2mg, as reference substance solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol-hydrochloric acid-ethyl acetate (3-12: 1-5: 0.5-2) be developing solvent, launch, take out that dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Differentiate B: take by weighing the Chinese medicine composition oral liquid of suitable raw medicinal herbs amount 20-40g, with water saturated n-butanol extraction 1-3 time, 30ml at every turn, merging n-butanol extracting liquid; Evaporate to dryness, residue add ammonia solution 5ml makes dissolving, is added on the macroporous adsorptive resins, washs with ammonia solution 100ml; Discard the ammonia eluent, the reuse water elution discards water elution liquid, reuse 20-40% ethanol 50ml eluting to neutral; Discard ethanol elution, continue, collect eluent with 60-80% ethanol 50ml eluting; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.Other gets ginsenoside Re's reference substance, adds methanol and processes the solution that every 1ml contains 0.2-1mg, as reference substance solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, (8-20: 2-12: 1-5) 10 ℃ of lower floor's solution that spend the night with held are developing solvent with chloroform-methanol-water; Launch; 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 that (365nm) inspects under the uviol lamp.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, with water saturated n-butanol extraction 1-3 time, 30ml at every turn, merging n-butanol extracting liquid; Evaporate to dryness, residue add methanol 2ml makes dissolving, adds neutral alumina 1-3g, mixes thoroughly; Evaporate to dryness is added on the neutral alumina post, uses methanol-eluted fractions, collects eluent; Evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets the peoniflorin reference substance, adds methanol and processes the solution that every 1ml contains 0.2-1mg, as reference substance solution.Draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-strong ammonia solution (5-10: 0.5-2: 1-8: 0.5-2) be developing solvent; Launch, take out, dry; Spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Content assaying method:

The preparation of need testing solution comprises the steps: step 1: said 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 reuse chloroform extraction; Step 4: the chloroform extracted solution of combining step 2,3, reclaim solvent to doing, get residue, residue adds methanol makes dissolving.The molar concentration of hydrochloric acid is in the said need testing solution preparation process 1: 2.5mol/L; Chloroform is: analytical pure.In the said need testing solution preparation process 2 reflux 0.5-2 hour; Chloroform extraction is 2-4 time in the said reference substance solution preparation process 3.

The detection method of Chinese medicine composition according to the 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 dilute 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), it is closely colourless that water is eluted to effluent; Discard water liquid, reuse 2mol/L ammonia solution 120ml eluting is collected eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution; Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developing solvent with n-butyl alcohol-hydrochloric acid-ethyl acetate (8: 3: 1), launches, and takes out, and dries, and spray is with rare bismuth potassium iodide test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Differentiate B: take by weighing the Chinese medicine composition oral liquid of suitable raw medicinal herbs amount 30g, with water saturated n-butanol extraction 2 times, each 30ml, merging n-butanol extracting liquid; Evaporate to dryness, residue add ammonia solution 5ml makes dissolving, is added on the D101 type macroporous adsorptive resins (internal diameter 1.5cm, the high 12cm of post); With ammonia solution 100ml washing, discard the ammonia eluent, the reuse water elution discards water elution liquid to neutral; Reuse 30% ethanol 50ml eluting discards 30% ethanol elution, continues with 70% ethanol 50ml eluting, collects eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methanol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With 10 ℃ of lower floor's solution that spend the night with held of chloroform-methanol-water (13: 7: 2) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts that (365nm) inspects under the uviol lamp; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Differentiate C: take by weighing and contain the Chinese medicine composition oral liquid that is equivalent to raw medicinal herbs amount 30g, with water saturated n-butanol extraction 2 times, each 30ml, merging n-butanol extracting liquid, evaporate to dryness; Residue adds methanol 2ml makes dissolving, adds neutral alumina 2g, mixes evaporate to dryness thoroughly; Be added on the neutral alumina post (100～200 orders, 3g, internal diameter 1cm), with methanol 50ml eluting; Collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds methanol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-ethyl acetate-methanol-strong ammonia solution (8: 1: 4: 1) be developing solvent; Launch, take out, dry; Spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

Content assaying method:

According to high effective liquid chromatography for measuring, chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With methanol-0.07% phosphoric acid solution (70: 30) is mobile phase; The detection wavelength is 254nm; Theoretical cam curve is calculated by the emodin peak should be not less than 3000; The preparation of reference substance solution: get the emodin reference substance, the chrysophanol reference substance is an amount of, accurately claim surely, add methanol and process the mixed solution that every 1ml contains 4 μ g, 8 μ g respectively, promptly get; The preparation of need testing solution: measure the said composition oral liquid that is equivalent to raw medicinal herbs 2g, add water 7ml and hydrochloric acid 1ml, add chloroform 10ml again, reflux 1 hour, cooling immediately; Shift in the separatory funnel, use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill; Obtain chloroform solution, water liquid reuse chloroform extraction 3 times, each 10ml, combined chloroform extracting solution; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get; Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get.

The present composition is used to treat primary hepatocarcinoma, pulmonary carcinoma, gastrointestinal cancer, breast carcinoma, gynecological 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 emodin, chrysophanol dissolving after the hydrolysis more complete; Cause extract incomplete shortcoming because tested composition is pasted the bottle wall with the deposition caking thereby overcome prior art, 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; Provide by self-sufficient and strategically located region Pharmaceutical joint stock company limited of Chengdu buchu group.

Emodin, chrysophanol reference substance: lot number emodin: 110756-200110, chrysophanol: 110796-200311, provide by Nat'l Pharmaceutical & Biological Products Control Institute, supply assay to use.

Reagent: except that methanol was chromatographically pure, all the other reagent were analytical pure.

2 test methods:

2.1 chromatographic condition and system suitability 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: methanol-0.07% phosphoric acid solution (70: 30); The detection wavelength is 254nm; Flow velocity: 1ml/min; Number of theoretical plate calculates by the emodin peak should be not less than 3000.

2.2 the preparation of reference substance solution

Precision takes by weighing emodin, chrysophanol reference substance 3.83mg, the 8.72mg that in silica gel drier, is dried to constant weight, puts in the 100ml measuring bottle, adds methanol to scale, shakes up, and precision is measured 5ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and promptly gets.(containing emodin 3.83 μ g, chrysophanol 8.72 μ g among every 1ml).

2.3 need testing solution preparation

Method 1: precision is measured these article 2ml, adds 2.5mol/L sulfuric acid solution 8ml, and reflux 2 hours is put coldly, extracts 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 liquid is incorporated in the filtrating, 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, adds methanol-acetone (1: 1) to scale; Shake up, filter, get subsequent filtrate, promptly get.

Method 2: precision is measured these article 2ml, places the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and filters, and precision is measured subsequent filtrate 5ml; Put in the 50ml round-bottomed flask, fling to methanol, add 2.5mol/I sulfuric acid solution 10ml, supersound process 5min adds chloroform 10ml again; Reflux 1h is put coldly, obtains chloroform layer, and acid solution is with chloroform extraction twice, each 10ml; Combined chloroform liquid, with anhydrous sodium sulfate dehydration, chloroform solution is put evaporate to dryness in the water-bath, and residue adds an amount of slight fever of methanol makes dissolving, moves in the 5ml measuring bottle; And add methanol constant volume, and shake up, filter, get subsequent filtrate, promptly get.

Method 3: precision is measured these article 2ml, adds water 7ml and hydrochloric acid 1ml, and water-bath backflow 1h is put cold; Extract (20,15,15ml) 3 times with the ether jolting, merge extractive liquid, use a small amount of anhydrous sodium sulfate dehydration, filtration; Filter washs with a small amount of ether, and washing liquid is incorporated in the filtrating, the low temperature evaporate to dryness, and residue is with dissolve with methanol and be transferred in the 5ml measuring bottle; Add methanol constant volume, shake up, centrifugal, promptly get need testing solution.

Method 4: precision is measured these article 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, reflux 1 hour, and cooling is immediately shifted in the separatory funnel; Use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill, and obtains chloroform solution, water liquid reuse chloroform extraction 3 times; Each 10ml, the combined chloroform extracting solution, decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle; Add methanol to scale, shake up, filter, get subsequent filtrate, promptly get.

2.4 accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and promptly get.

3 results

The emodin 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 this method of employing is 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 emodin (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, and the result sees table 4.And with peak area value (Y) sample size (X) is returned, emodin standard curve equation be: Y=2865509.1X+7066.3, r=0.9998; Chrysophanol standard curve equation is: Y=3891218.2X+265424.9, r=0.9998.And with peak area value (A) to sample size (C) mapping, a straight line, the result shows that emodin 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 emodin, chrysophanol is investigated

2 precision are investigated

Get emodin, 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.Emodin, chrysophanol RSD are respectively 1.93%, 0.96%, and precision is investigated the result and met the requirements.The result sees table 3.

The precision of table 3 emodin, chrysophanol is investigated the result

	Sequence number	Peak area	RSD(％)
					1	227015.60
	2	218583.54
				Emodin	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 emodin, chrysophanol

Emodin in emodin, chrysophanol standard substance and the test sample, chrysophanol all were stable in 48 hours.

4 repeatability are investigated

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

Table 4 emodin, 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 a certain amount of reference substance respectively, presses the method for preparing preparation of need testing solution.Sample introduction is measured respectively, calculates average recovery.The result sees table 5.Emodin, chrysophanol average recovery are 97.91%.

Table 5 emodin, 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, and the result sees table 6.

Table 6 sample size is measured result (n=2)

Lot number	Emodin 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	1.25
			060504	0.5127	0.79

7 specificities are investigated

According to the method described above, the embodiment 1 oral liquid negative control article of embodiment 1 oral liquid and scarce Radix Et Rhizoma Rhei are measured, the negative control article do not have the absworption peak appearance in corresponding retention time as a result.Explain that other compositions are noiseless to the mensuration of emodin, chrysophanol, institute's method suggested has specificity and feasibility.

The need testing solution method for preparing was investigated during experimental example 2 Radix Paeoniae Alba thin layers were differentiated

The preparation of need testing solution 1: get Chinese medicine composition oral liquid 30ml of the present invention, with water saturated n-butanol extraction 2 times, each 30ml merges n-butanol extracting liquid, evaporate to dryness; Residue adds methanol 2ml makes dissolving, adds neutral alumina 2g, mixes evaporate to dryness thoroughly; Be added on the neutral alumina post (100～200 orders, 3g, internal diameter 1cm), with methanol 50ml eluting; Collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution 1;

The preparation of need testing solution 2: the 20ml jolting that adds diethyl ether is extracted, and discards ether solution, and the water saturated n-butyl alcohol jolting of water liquid reuse is extracted 3 times, each 15ml; Merge n-butyl alcohol liquid, water 30ml washing discards water liquid, n-butyl alcohol liquid evaporate to dryness; Residue adds ethanol 2ml makes dissolving, adds neutral alumina 1g low temperature and mixes thoroughly, drying, neutral alumina pillar (200～300 orders that fill in advance of packing into; 2g on the internal diameter 10～15mm), with ethanol 60ml eluting, collects eluent; Evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution 2;

The preparation of reference substance solution 1: get the peoniflorin reference substance, add methanol and process the solution that every 1ml contains 0.5mg, as reference substance solution 1.

Thin layer chromatography: according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-ethyl acetate-methanol-strong ammonia solution (8: 1: 4: 1) be developing solvent; Launch, take out, dry; Spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃.

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

Experimental example 3: the investigation of the thin layer discrimination method specificity of Herba Leonuri

The preparation of need testing solution: get three crowdes of each 30ml of embodiment 1 oral liquid, add dilute 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), it is closely colourless that water is eluted to effluent; Discard water liquid, reuse 2mol/L ammonia solution 120ml eluting is collected eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.

The preparation of reference substance solution: get the stachydrine hydrochloride reference substance, add methanol and process the solution that every 1ml contains 1mg, as reference substance solution.

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

Thin layer chromatography: according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned solution, put on same silica gel g thin-layer plate respectively and (be followed successively by reference substance solution from left to right; Three lot sample article solution, negative control solution), be developing solvent with n-butyl alcohol-hydrochloric acid-ethyl acetate (8: 3: 1); Launch; Take out, dry, spray is with rare bismuth potassium iodide test solution.

Conclusion: in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color, negative control is noiseless in the relevant position.

The thin layer discrimination method specificity of experimental example 4 Radix Ginsengs is investigated

Need testing solution preparation: get three crowdes of each 30ml of Chinese medicine composition oral liquid of the present invention, with water saturated n-butanol extraction 2 times, each 30ml, merging n-butanol extracting liquid; Evaporate to dryness, residue add ammonia solution 5ml makes dissolving, is added on the D101 type macroporous adsorptive resins (internal diameter 1.5cm, the high 12cm of post); With ammonia solution 100ml washing, discard the ammonia eluent, the reuse water elution discards water elution liquid to neutral; Reuse 30% ethanol 50ml eluting discards 30% ethanol elution, continues with 70% ethanol 50ml eluting, collects eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.

The preparation of reference substance solution: get ginsenoside Re's reference substance, add methanol and process the solution that every 1ml contains 0.5mg, as reference substance solution.

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

Thin layer chromatography: according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned solution, put respectively in same be (to be followed successively by reference substance solution from left to right on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; Three lot sample article solution, negative control solution), be developing solvent with 10 ℃ of lower floor's solution that spend the night with held of chloroform-methanol-water (13: 7: 2); Launch; 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 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 abnormal smells from the patient, taste through adding respective additive, reduces this oral liquid to gastral zest, improves content of effective in the preparation.

Press embodiment 1 prescription, Carapax Trionycis adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae add 12 times of water, and vapor distillation extracted 6 hours, and it is an amount of to collect volatile oil and distillate, and medicinal residues and all the other 25-component decocte with water three times add 12 times in water for the first time, decocted 2 hours; Add for the second time 10 times in water, decocted 1.5 hours, add 8 times in water for the third time, decocted 1.5 hours, collecting decoction filters; It is 1.15 clear paste that filtrating is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, regulates pH value and is 7.5～8.0,0～5 ℃ and left standstill 24 hours; Filter, filtrate recycling ethanol adds water to 50～60 ℃ of relative densities and is 1.06 clear paste, adds the Carapax Trionycis decocting liquid again; Boiled 20 minutes, and put coldly, add volatile oil and distillate, according to table 8; Add and respectively organize additive, add water to 1000ml, stirring was left standstill 1 hour after making the dissolving mixing.

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.With 5 grades shown in the table 7 degree of unhappy tastes such as the bitter taste of oral liquid, zest and the degree of unhappy abnormal smells from the patient are estimated, calculating mean value, the result sees table 8.

Unhappy taste of table 7 oral liquid and the scoring of abnormal smells from the patient degree

Table 8

Additive in the table 8 " ‰ " is a weight ratio, when solution adds water to 1000ml, adds the weight ratio of additive, and the present invention amounts to the raw material of about 1000g, after extracting, when extracting solution adds water to 1000ml, adds 2g cyclamate, 2g tween 80.

Visible through above experiment: experiment can effectively reduce its disagreeable taste and abnormal smells from the patient with adding (cyclamate, tween 80) and (cyclamate, lecithin) in the oral liquid, and the combination result of use is much better than independent result of use.

Experimental example 6 additives are to the influence of active constituent content in the oral liquid

The preparation of oral liquid: press embodiment 1 prescription proportioning, Carapax Trionycis adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae add 12 times of water, and vapor distillation extracted 6 hours, and it is an amount of to collect volatile oil and distillate, medicinal residues and all the other 25-component decocte with water three times; Add for the first time 12 times in water, decocted 2 hours, add 10 times in water for the second time, decocted 1.5 hours, add 8 times in water for the third time, decocted collecting decoction 1.5 hours; Filter, it is 1.15 clear paste that filtrating is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, and regulating pH value is 7.5～8.0; 0～5 ℃ left standstill 24 hours, filtered, and filtrate recycling ethanol adds water to 50～60 ℃ of relative densities and be 1.06 clear paste; Add the Carapax Trionycis decocting liquid again, boiled 20 minutes, put coldly, add volatile oil and distillate; Add additive according to table 9 prescription 1,2,3 respectively, add the water stirring and make dissolving, 0～5 ℃ left standstill 24～48 hours; Filter, filtrating adds water to 1000ml, gets oral liquid I, oral liquid II, oral liquid III.

Table 9 oral liquid additive formulations

Experimental technique:

Chromatographic condition: Agilentl 100 high performance liquid chromatographs; Agilent C18 chromatographic column; Mobile phase: methanol-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 methanol and processes the solution that every 1ml contains 13 μ g, promptly gets.

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, reflux 1 hour, cooling immediately; Shift in the separatory funnel, use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill; Obtain chloroform solution, water liquid reuse chloroform extraction 3 times, each 10ml, combined chloroform extracting solution; Decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, adds methanol to scale; Shake up, filter, get subsequent filtrate, promptly get test sample and dissolve

night

1,2,3.

Assay method: accurate respectively reference substance solution and 3 kinds of each 20 μ L of need testing solution of drawing, inject chromatograph of liquid, to measure, one point external standard method is measured the content of chrysophanol in the oral liquid.

Liquid chromatogram is seen Fig. 1-4; The assay result sees 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 ‰ cyclamates can increase active constituent content in the oral liquid.

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

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

The preparation of oral liquid: press embodiment 1 prescription proportioning, Carapax Trionycis adds 9 times of water decoctings and boils 3 times, and each 3 hours, collecting decoction filtered, and filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae add 12 times of water, and vapor distillation extracted 6 hours, and it is an amount of to collect volatile oil and distillate, medicinal residues and all the other 25-component decocte with water three times; Add for the first time 12 times in water, decocted 2 hours, add 10 times in water for the second time, decocted 1.5 hours, add 8 times in water for the third time, decocted collecting decoction 1.5 hours; Filter, it is 1.15 clear paste that filtrating is concentrated into 50～60 ℃ of relative densities, adds ethanol and makes and contain the alcohol amount and reach 70%, and regulating pH value is 7.5～8.0; 0～5 ℃ left standstill 24 hours, filtered, and filtrate recycling ethanol adds water to 50～60 ℃ of relative densities and be 1.06 clear paste; Add the Carapax Trionycis decocting liquid again, boiled 20 minutes, put cold; Add volatile oil and distillate, add additive according to table 12 prescription respectively, add the water stirring and make dissolving; 0～5 ℃ left standstill 24～48 hours, filtered, and filtrating adds water to 1000ml.The selection result is seen table 11.

The not commensurability additive combination evaluation result of table 11

Can find out that through above-mentioned evaluation result tween 80 (‰) and cyclamate (‰) are at 1-3: in the 1-3 scope, all have taste and abnormal smells from the patient preferably.

Experimental example 8 additives are to the influence of drug effect

1, different oral liquids of the present invention and cyclophosphamide share the potentiation to lotus S180 mice

Select for use Huaxi Medical Univ's oncology to inoculate the mice (S180, ascitic type) of lotus S180 sarcoma, be divided into five groups at random, conduct respectively in back 24 hours: (1) blank group (distilled water 0.4ml/ props up, and irritates stomach) in inoculation; (2) cyclophosphamide group (10mg/Kg, subcutaneous injection); (3) cyclophosphamide group (10mg/Kg, subcutaneous injection) is share oral liquid I (for experimental example 6 prepares oral liquid I, 12.10g crude drug/Kg; Irritate stomach) (4) cyclophosphamide group (10mg/Kg; Subcutaneous injection) share oral liquid II (be experimental example 6 preparation oral liquid II, 12.10g crude drug/Kg irritates stomach).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 tumor soma, thymus, spleen and weigh.Calculate the heavy and tumour inhibiting rate of tumor by following formula, carry out statistics (t check) and handle, and calculate the organ coefficient of thymus and spleen according to body weight.Result of the test is seen table 12:

The different oral liquids of the present invention of table 12 are to the influence of transplantability mice 180 tumor bulk-growths

Can know that from above-mentioned result of the test cyclophosphamide truly has therapeutical effect to murine sarcoma S 180,, continuous 10 days, can make the heavy suppression ratio of tumor reach 45.52%, P＜0.01 when dosage is the administration of 10mg/Kg subcutaneous injection.After cyclophosphamide share with two kinds of oral liquids respectively; Then tumor-inhibiting action strengthens; Tumor-inhibiting action increases to 53.73% and 58.09% (the P value all＜0.01), increases by 8.21% and 12.57% than simple application cyclophosphamide, 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 tumor-inhibiting action of chemotherapeutics cyclophosphamide to mice S 180, and a certain amount of tween of adding helps to strengthen this potentiation in oral liquid of the present invention.

2, different oral liquids of the present invention and fluorouracil share the potentiation to lotus H22 mice

Behind mouse inoculation H22, be divided into 4 groups at random, conduct respectively in 24 hours: (1) blank group (distilled water 0.4ml/ props up, and irritates stomach) (2) fluorouracil group (5-Fu) (10mg/Kg, subcutaneous injection); (3) fluorouracil (10mg/Kg, subcutaneous injection) share oral liquid I (for experimental example 6 prepares oral liquid I, 12.10g crude drug/Kg; Irritate stomach) (4) fluorouracil (10mg/Kg; Subcutaneous injection) share oral liquid II (be experimental example 6 preparation oral liquid II, 12.10g crude drug/Kg irritates stomach).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 tumor soma, thymus, spleen and weigh.Calculate the heavy and tumour inhibiting rate of tumor by following formula, carry out statistics (t check) and handle, and calculate the organ coefficient of thymus and spleen according to body weight.The result sees table 13.

The different oral liquids of the present invention of table 13 are to the influence of transplantability rat liver cancer (H22) tumor bulk-growth

Experimental result shows that fluorouracil has certain inhibitory action to the tumor bulk-growth of transplantability rat liver cancer (H22).Its suppression ratio can reach 34.85% (p＜0.05).After fluorouracil and two kinds of oral liquids (12.10g crude drug/Kg irritates stomach) share, increase by 11.36% and 15.15% respectively than simple cancer suppressing ratio, and share oral liquid II ratio, to share oral liquid I cancer suppressing ratio high by 3.79% with fluorouracil.Therefore, can think that oral liquid of the present invention has certain potentiation to fluorouracil in treatment transplantability rat liver cancer (H22), and the adding tween helps to strengthen this potentiation 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

The specific embodiment

Embodiment 1:

More than 30 four Chinese medicine materials, Carapax Trionycis decocte with water 3 times (adding 9 times of water), each 3 hours, collecting decoction filtered, filtrating is concentrated into an amount of (about 180ml); Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae vapor distillation extract 6 hours (adding 12 times of water), collect volatile oil and distillate an amount of (about 50ml), medicinal residues and all the other 25-component decocte with water three times, 2 hours (adding 12 times in water) for the first time; Second and third time each 1.5 hours (adds respectively 10 times in water, 8 times),, collecting decoction filters, and filtrating is concentrated into the clear paste that relative density is 1.15 (50～60 ℃); Add ethanol and make and contain alcohol amount and reach 70%, regulating pH value is 7.5～8.0, leaves standstill (0～5 ℃) 24 hours, filters; Filtrate recycling ethanol, adding water to relative density is the clear paste of 1.06 (50～60 ℃), adds the Carapax Trionycis decocting liquid again, boils 20 minutes; Put coldly, add volatile oil and distillate and polyoxyethylene sorbitan monoleate, cyclamate, add water and stir and make dissolving, left standstill (0～5 ℃) 24～48 hours; Filter, filtrating adds water to ormal weight (1000ml), embedding; Sterilization (105 ℃ 60min), promptly get.Every 10ml.

Embodiment 2:

More than 34 flavors, Carapax Trionycis decocts 3 times with 9 times of water gagings, each 3 hours, collecting decoction filtered, filtrating is concentrated into 180ml; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae add 12 times of water extraction 6 hours, collect volatile oil and the about 50ml of Aromatic water, and medicinal residues and all the other 25-component decocte with water 3 times add 12 times of amounts of water for the first time, decoct 2 hours; Second and third time respectively adds 10 times of amounts of water, decocts 1.5 hours, and collecting decoction is concentrated into relative density about 1.15; Add ethanol and make and contain alcohol amount and reach 70%, regulating pH value is 7.5～8.0, leaves standstill 24 hours, filters; Filtrate recycling ethanol, it is about 1.06 to add water to relative density, adds the Carapax Trionycis decocting liquid again, boils 20 minutes; Put coldly, add volatile oil, Aromatic water, tween 80 2 ‰, cyclamate 2 ‰.Add water to 1000ml, left standstill 24～48 hours, get supernatant, embedding, sterilization, packing promptly gets.

Embodiment 3:

Method for preparing: above 34 flavors, Carapax Trionycis decocte with water 3 times, each 3 hours, collecting decoction filtered, and filtrating is concentrated into an amount of; Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Curcumae Longae, Rhizoma Alpiniae Officinarum, Cortex Cinnamomi, Flos Caryophylli, Resina Ferulae, Lignum Dalbergiae Odoriferae vapor distillation extracted 6 hours, and it is an amount of to collect volatile oil and distillate, medicinal residues and all the other 25-component decocte with water three times, 2 hours for the first time; Second and third time each 1.5 hours, collecting decoction filters, and filtrating is concentrated into the clear paste of relative density about 1.15 (50～60 ℃); Add ethanol and make and contain alcohol amount and reach 70%, regulating pH value is 7.5～8.0, leaves standstill 24 hours, filters; Filtrate recycling ethanol adds water to the clear paste of relative density about 1.06 (50～60 ℃), adds the Carapax Trionycis decocting liquid again, boils 20 minutes; Put coldly, add volatile oil and distillate, process granule through conventional technology.

Embodiment 4: embodiment 1 oral liquid detection method

Differentiate:

(1) get oral liquid 30ml, add dilute 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), it is closely colourless that water is eluted to effluent; Discard water liquid, reuse 2mol/L ammonia solution 120ml eluting is collected eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.Other gets the stachydrine hydrochloride reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With n-butyl alcohol-hydrochloric acid-ethyl acetate (8: 3: 1) is developing solvent, launches, and takes out; Dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

(2) get oral liquid 30ml, with water saturated n-butanol extraction 2 times, each 30ml merges n-butanol extracting liquid; Evaporate to dryness, residue add ammonia solution 5ml makes dissolving, is added on the D101 type macroporous adsorptive resins (internal diameter 1.5cm, the high 12cm of post); With ammonia solution 100ml washing, discard the ammonia eluent, the reuse water elution discards water elution liquid to neutral; Reuse 30% ethanol 50ml eluting discards 30% ethanol elution, continues with 70% ethanol 50ml eluting, collects eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.Other gets ginsenoside Re's reference substance, adds methanol and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With 10 ℃ of lower floor's solution that spend the night with held of chloroform-methanol-water (13: 7: 2) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts that (365nm) inspects under the uviol lamp.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

(3) get oral liquid 30ml, with water saturated n-butanol extraction 2 times, each 30ml merges n-butanol extracting liquid, evaporate to dryness; Residue adds methanol 2ml makes dissolving, adds neutral alumina 2g, mixes evaporate to dryness thoroughly; Be added on the neutral alumina post (100～200 orders, 3g, internal diameter 1cm), with methanol 50ml eluting; Collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets the peoniflorin reference substance, adds methanol and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-ethyl acetate-methanol-strong ammonia solution (8: 1: 4: 1) be developing solvent; Launch, take out, dry; Spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Assay: measure according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2010).

Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With methanol-0.07% phosphoric acid solution (70: 30) is mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the emodin peak should be not less than 3000.The emodin reference substance is got in the preparation of reference substance solution, the chrysophanol reference substance is an amount of, and accurate the title decides, and adds methanol and processes the mixed solution that every 1ml contains 4 μ g, 8 μ g respectively, promptly gets.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, reflux 1 hour, and cooling is immediately shifted in the separatory funnel; Use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill, and obtains chloroform solution, water liquid reuse chloroform extraction 3 times; Each 10ml, the combined chloroform extracting solution, decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle; Add methanol to scale, shake up, filter, get subsequent filtrate, promptly get.

Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get.Every of these article contain Radix Et Rhizoma Rhei with emodin (C ₁₅H ₁₀O ₅), chrysophanol (C ₁₅H ₁₀O ₄) total amount meter 0.58mg.

Embodiment 5: embodiment 1 oral 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 methanol 1ml makes dissolving, as need testing solution.Other gets Fructus Evodiae control medicinal material 0.5g, adds ethanol 10ml, and reflux 30 minutes filters, and filtrating is as control medicinal material solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 5 μ l of above-mentioned two kinds of solution respectively, put respectively on same silica gel g thin-layer plate; With n-butyl alcohol-glacial acetic acid-water (7: 1: 2) is developing solvent, launches, and takes out; Dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

Assay: measure according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2010).Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-0.07% phosphoric acid solution (70: 30) is mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the emodin peak should be not less than 3000.The emodin reference substance is got in the preparation of reference substance solution, the chrysophanol reference substance is an amount of, and accurate the title decides, and adds methanol and processes the mixed solution that every 1ml contains 4 μ g, 8 μ g respectively, promptly gets.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, reflux 1 hour, and cooling is immediately shifted in the separatory funnel; Use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill, and obtains chloroform solution, water liquid reuse chloroform extraction 3 times; Each 10ml, the combined chloroform extracting solution, decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle; Add methanol to scale, shake up, filter, get subsequent filtrate, promptly get.Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get.Every of these article contain Radix Et Rhizoma Rhei with emodin (C ₁₅H ₁₀O ₅), chrysophanol (C ₁₅H ₁₀O ₄) total amount meter 0.58mg.

Embodiment 6: embodiment 1 oral liquid detection method

Differentiate:

(5) get oral liquid 30ml, add hydrochloric acid 3ml, put in the water-bath reflux 1 hour, put coldly, 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 emodin reference substance, chrysophanol reference substance, and chlorination is copied into the mixed solution that every 1ml contains 1mg, as reference substance solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw above-mentioned two kinds of each 8ul of solution, put respectively on same silica gel g thin-layer plate; Upper strata liquid with petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent; Launch, take out, dry; Put in the ammonia steam smoked after, inspect under the daylight.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of 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 process the solution that every 1ml contains 16 μ l, as reference substance solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developing solvent with cyclohexane extraction-ethyl acetate (10: 1), launches; Take out; Dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Assay: measure according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2010).Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-0.07% phosphoric acid solution (70: 30) is mobile phase; The detection wavelength is 254nm.Theoretical cam curve is calculated by the emodin peak should be not less than 3000.The emodin reference substance is got in the preparation of reference substance solution, the chrysophanol reference substance is an amount of, and accurate the title decides, and adds methanol and processes the mixed solution that every 1ml contains 4 μ g, 8 μ g respectively, promptly gets.The preparation of need testing solution: precision is measured oral liquid 2ml, adds water 7ml and hydrochloric acid 1ml, adds chloroform 10ml again, reflux 1 hour, and cooling is immediately shifted in the separatory funnel; Use the minimum of chloroform washing container, washing liquid is incorporated in the separatory funnel, leaves standstill, and obtains chloroform solution, water liquid reuse chloroform extraction 3 times; Each 10ml, the combined chloroform extracting solution, decompression and solvent recovery is to doing, and residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle; Add methanol to scale, shake up, filter, get subsequent filtrate, promptly get.

Embodiment 7 dwell temperature are investigated test

The method for preparing of this experimental basis enforcement 1 is processed sample 1, and wherein twice dwell temperature is (0～5 ℃); Press embodiment 1 technology, all change twice dwell temperature into (20 ℃), process sample 2, carry out accelerated test; The main situation of investigating character deposition, content's index.Experimental condition: under 40 ± 2 ℃ of conditions, made an experiment and see the following form 14 in 0,1,2,3,6 month.

Table 14 accelerated test result

On the theory, along with the reduction of temperature, sedimentation effect is obvious more, and materials such as emodin can go down along with precipitate sedimentation together.Get oral liquid of the present invention in the preparation process, comprise and leave standstill process twice, once be precipitate with ethanol after; Once for after adding volatile oil and distillate and polyoxyethylene sorbitan monoleate, cyclamate; When under low temperature, leaving standstill, main component can appear by sedimentation, influence the content of active component.

Prove that through accelerated test different with the understanding of routine, medicinal liquid after oral liquid cold preservation of the present invention is left standstill deposition is not only few, and emodin, chrysophanol content stable content, the quality standard requirement met.

Claims

1. Chinese medicine composition oral liquid with antitumaous effect is characterized in that this oral liquid processed by following raw material of Chinese medicine medicine:

2. oral liquid as claimed in claim 1 is characterized in that this oral liquid is to be processed by following crude drug:

Calculate with the raw material dose, the total amount that is equivalent to emodin, chrysophanol in the said compositions of 10g crude drug is no less than 0.28mg.

3. according to claim 1 or claim 2 oral liquid is characterized in that, traditional Chinese medicinal composition raw materials adds following adjuvant and is prepared into oral liquid after extracting; Said adjuvant comprises: one or more in correctives, sweeting agent, stabilization agent, thickening agent, dissolving adjuvant, pH regulator agent, coloring agent, the essence; Said correctives be selected from following one or more: polyvidone, Mentholum, glycyrrhizic acid dipotassium, malic acid, natrium malicum, tartaric acid, succinic acid, sodium succinate, acetic acid, glutamic acid, citric acid, sodium citrate, gluconic acid lactone, sodium chloride; Said sweeting agent is selected from one or more in following: sucrose, fructose, glucose, lactose, Sorbitol, maltose alcohol, erythrose, sucralose, Mel, glucide, stevioside extract, Aspartame, acesulfame potassium, sweet protein, Radix Glycyrrhizae, cyclamate; Stabilizing agent is optional to be descended a kind of or several kinds freely: polyvidone, glycerol, arabo-ascorbic acid and salt thereof, edetic acid, Metaphosphoric acid; Thickening agent be selected from following one or more: sodium carboxymethyl cellulose, agar, polyvidone, polyvinyl alcohol, xanthan gum; The dissolving adjuvant be selected from following one or more: polysorbate, Polyethylene Glycol, polyoxyethylene hardened castor oil, polyvidone, sodium hydroxide, polyoxyethylene sorbitan monoleate; The pH regulator agent be selected from following one or more: citric acid, sodium citrate, lactic acid, sodium hydroxide, hydrochloric acid, malic acid, natrium malicum, tartaric acid, succinic acid, acetic acid, gluconic acid, phosphoric acid.

4. according to claim 1 or claim 2 oral liquor is characterized in that this method comprises the steps:

Step 5: the Carapax Trionycis decocting liquid of clear paste 2 steps 1 in the step 4, boil, put coldly, add the volatile oil of step 2 and the conventional adjuvant of distillate and oral liquid, add water and stir and make dissolving, leave standstill, filter, filtrating is processed oral liquid through conventional technology;

The dwell temperature of said step 4 and/or step 5 is: 0～5 ℃.

5. method as claimed in claim 4 is characterized in that the conventional adjuvant that adds oral liquid in the said step 5 is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion cyclamate.

6. method as claimed in claim 4 is characterized in that this method also comprises step 6: the assay of the total amount of emodin, chrysophanol;

The content assaying method of the total amount of said emodin, chrysophanol is:

The preparation of need testing solution comprises the steps: step a: said 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 reuse chloroform extraction; Steps d: the chloroform extracted solution of combining step b, c, reclaim solvent to doing, get residue, residue adds methanol makes dissolving.

7. method as claimed in claim 6 is characterized in that in the content assaying method of total amount of said emodin, chrysophanol, in the said need testing solution preparation among the step a molar concentration of hydrochloric acid be: 2-3mol/L; Among the said need testing solution preparation process b reflux 0.5-2 hour; Chloroform extraction is 2-4 time among the said reference substance solution preparation process c.

8. like the detection method of the Chinese medicine composition oral liquid of the said method of claim 4 preparation, it is characterized in that this method mainly comprises one or more in following discrimination method and/or the assay:

Discrimination method:

Differentiate B: the Chinese medicine composition oral liquid that takes by weighing suitable raw medicinal herbs amount 20-40g; With water saturated n-butanol extraction 1-3 time, merge n-butanol extracting liquid, evaporate to dryness gets residue; Residue adds ammonia solution makes dissolving; Be added on the macroporous adsorptive resins, successively use ammonia solution, water, 20-40% ethanol elution respectively, discard eluent respectively; Continue and use the 60-80% ethanol elution, collect this eluent, evaporate to dryness gets residue, and residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methanol and processes the reference substance solution that every 1ml contains 0.2-1mg; Equivalent is drawn above-mentioned two kinds of solution, puts respectively on same silica gel g thin-layer plate, is that the chloroform-methanol-water of ratio is developing solvent at 10 ℃ of lower floor's solution that spend the night with held with 8-20: 2-12: 1-5; Launch; Take out, dry, spray is with ethanol solution of sulfuric acid; It is clear to be heated to the speckle colour developing, puts under the uviol lamp and inspects;

Content assaying method:

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With 60～80: 40～20 methanol-0.07% phosphoric acid solution is a mobile phase; The detection wavelength is 250～260nm; Theoretical cam curve is calculated by the emodin peak should be not less than 3000;

The preparation of need testing solution comprises the steps: step 1: said 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 reuse chloroform extraction; Step 4: the chloroform extracted solution of combining step 2,3, reclaim solvent to doing, get residue, residue adds methanol makes dissolving; The molar concentration of hydrochloric acid is in the said need testing solution preparation process 1: 2.5mol/L; In the said need testing solution preparation process 2 reflux 0.5-2 hour; Chloroform extraction is 2-4 time in the said reference substance solution preparation process 3.

9. detection method as claimed in claim 8 is characterized in that this method mainly comprises the steps:

Differentiate

Differentiate A: take by weighing the Chinese medicine composition oral liquid of suitable raw medicinal herbs amount 20-40g, add dilute hydrochloric acid and transfer p H-number to 1～2, centrifugal; Get supernatant, on strong acid cation exchange resin column, it is closely colourless that water is eluted to effluent; Discard water liquid, reuse 1-3mol/L ammonia solution eluting is collected eluent; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution; Other gets the stachydrine hydrochloride reference substance, adds methanol and processes the solution that every 1ml contains 0.5-2mg, as reference substance solution; Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developing solvent with the n-butyl alcohol-hydrochloric acid-ethyl acetate of the ratio of 3-12: 1-5: 0.5-2, launches, and takes out, and dries, and spray is with rare bismuth potassium iodide test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Differentiate B: take by weighing the Chinese medicine composition oral liquid of suitable raw medicinal herbs amount 20-40g, with water saturated n-butanol extraction 1-3 time, 30ml at every turn, merging n-butanol extracting liquid; Evaporate to dryness, residue add ammonia solution 5ml makes dissolving, is added on the macroporous adsorptive resins, washs with ammonia solution 100ml; Discard the ammonia eluent, the reuse water elution discards water elution liquid, reuse 20-40% ethanol 50ml eluting to neutral; Discard ethanol elution, continue, collect eluent with 60-80% ethanol 50ml eluting; Evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Re's reference substance, adds methanol and processes the solution that every 1ml contains 0.2-1mg, as reference substance solution; Draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8-20: 2-12: the chloroform-methanol-water of 1-5 ratio is developing solvent at 10 ℃ of lower floor's solution that spend the night with held; Launch; 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 uviol lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Differentiate C: take by weighing to contain and be equivalent to raw medicinal herbs amount 20-40g oral liquid, with water saturated n-butanol extraction 1-3 time, 30ml at every turn, merging n-butanol extracting liquid; Evaporate to dryness, residue add methanol 2ml makes dissolving, adds neutral alumina 1-3g, mixes thoroughly; Evaporate to dryness is added on the neutral alumina post, uses methanol-eluted fractions, collects eluent; Evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds methanol and processes the solution that every 1ml contains 0.2-1mg, as reference substance solution; Draw each 10 μ l of above-mentioned two kinds of solution; Put respectively on same silica gel g thin-layer plate, with 5-10: 0.5-2: 1-8: the chloroform-ethyl acetate of 0.5-2 ratio-methanol-strong ammonia solution is developing solvent, launches; Take out; Dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

Content assaying method:

The preparation of need testing solution comprises the steps: step 1: said 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 reuse chloroform extraction; Step 4: the chloroform extracted solution of combining step 2,3, reclaim solvent to doing, get residue, residue adds methanol makes dissolving; The molar concentration of hydrochloric acid is in the said need testing solution preparation process 1: 2.5mol/L; Chloroform is: analytical pure; In the said need testing solution preparation process 2 reflux 0.5-2 hour; Chloroform extraction is 2-4 time in the said reference substance solution preparation process 3;

Algoscopy: equivalent is drawn reference substance solution and need testing solution 1 respectively, injects chromatograph of liquid, measures, and promptly gets.

10. oral liquid as claimed in claim 3 is characterized in that the adjuvant of oral liquid is: 1-5 weight portion polyoxyethylene sorbitan monoleate, 1-5 weight portion cyclamate.