CN101897942B

CN101897942B - Method for detecting medicine composition for warming spleen and tonifying kidney and releasing turbidity and eliminating stasis

Info

Publication number: CN101897942B
Application number: CN2009102112689A
Authority: CN
Inventors: 张建立; 曹相林; 于文占; 褚清宗; 熊胜辉; 高万峰
Original assignee: BEIJING SIHUANKEBAO PHARMACEUTICAL Co Ltd
Current assignee: BEIJING SIHUANKEBAO PHARMACEUTICAL Co Ltd
Priority date: 2009-05-31
Filing date: 2009-11-05
Publication date: 2012-02-08
Anticipated expiration: 2029-11-05
Also published as: CN101897942A

Abstract

The invention relates to a method for detecting medicine composition for warming the spleen and tonifying the kidney and releasing turbidity and eliminating stasis. The method includes content determination and identification methods or detection of limited quantity of aconitine. In the content determination method, the high performance liquid chromatography is adopted. The detection method of the invention also includes identification of ginseng, liquorice and rhizoma zingiberis and detection of aconitine. The detection method of the invention features good specificity, high stability, good repeatability and high precision, thus being more suitable for industrialized production and ensuring safe, effective and reliable clinical medication. In addition, the preparation prepared by the alcohol extraction method has better effect.

Description

Warming spleen and tonifying kidney, the detection method of letting out the pharmaceutical composition of turbid blood stasis dispelling

Technical field

The present invention relates to a kind of detection method of Chinese medicine composition, particularly relate to warming spleen and tonifying kidney, let out the quality determining method of the pharmaceutical composition of turbid blood stasis dispelling.

Background technology

Chronic renal failure (be called for short CRF) is because various chronic renal diseases develop into a kind of serious syndrome due to late period.The category that belongs to diseases such as Chinese medicine " poison of drowning ", " chronic consumptive disease ", " obstruction and rejection ".According to foreign statistic, annual sickness rate 100-150 people/1,000,000 infer that according to China's 2,000,000 urban populations investigation end-stage renal failure takes place in 568 people/1,000,000.In the nephrotic, more be prone to renal insufficiency old age.Along with the aggravation of China's population aging degree, this sick incidence rate increases gradually, and the breach of the treatment new drug of nephropathy will increase gradually.This shows the demand of such medicine in market.CRF is clinical common difficult disease, lacks the efficacious therapy method at present.The clinical no specific medicament of doctor trained in Western medicine.Have only Coated Aldehyde Oxystarch, Thebe woods on the market.Be used for this sick Chinese patent medicine and also have only several kinds of (the accurate word of medicine is defended in 96 Yunnan) SHENSHUAINING capsule and (97 defend the accurate word of medicine) NIAODUQING KELIs etc.Particularly these two kinds of Western medicine can only adsorb the excessive blood urea nitrogen and the serum creatinine of retention in the blood.And can not fundamentally solve such problem.Severe patient has only the dialysis of employing to delay the state of an illness or carry out kidney transfer operation.This method costs an arm and a leg, and general patient can't afford.So, how to treat and delay the progress of CRF, become current important subject.Along with China joined WTO, Chinese medicine will progressively go to the world, because China's tradition ancient prescription treatment disease is occupied suitable advantage, especially not have under the situation of specific drug in difficult disease, and Chinese medicine more shows its strong point.Special Chinese medicine has played huge effect in the process of carrying out non-dialysis therapy for treating primary disease, and it not only can make patient's clinical symptoms improve, and has delayed the progress of the CRF state of an illness.

Find warming spleen and tonifying kidney after deliberation, (crude drug consists of: Radix Et Rhizoma Rhei 300-1000 weight portion, Radix Aconiti Lateralis Preparata (system) 100-600 weight portion, Rhizoma Zingiberis 100-600 weight portion, Radix Ginseng 100-600 weight portion, Radix Glycyrrhizae 60-300 weight portion to let out the pharmaceutical composition of turbid blood stasis dispelling.) be the most effectively one of prescription of present clinical treatment CRF, said preparation adopts water decoction at present, and decoction is inconvenient; The volatile ingredient loss is many, and dose is big, and does not have corresponding quality determining method; The present invention is directed to warming spleen and tonifying kidney; Let out the modern granular preparation of the pharmaceutical composition of turbid blood stasis dispelling, quality controllable detection method is provided, guarantee the curative effect of medicine.

Summary of the invention

The object of the invention is to provide warming spleen and tonifying kidney, lets out the detection method of the pharmaceutical composition of turbid blood stasis dispelling.

The present invention provides above-mentioned warming spleen and tonifying kidney, lets out the drug regimen object detecting method of turbid blood stasis dispelling, comprises in the detection of following assay and/or discrimination method and/or aconitine limit one or more:

Content assaying method comprises the steps:

According to HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; The methanol of 80-90: 10-20-0.1% phosphoric acid solution is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates and should be less than 3000 for a short time by the emodin peak;

The preparation of reference substance solution: precision takes by weighing emodin and the chrysophanol reference substance is an amount of respectively, and add 1-4: 1 dehydrated alcohol-ethyl acetate is processed the mixed solution that every 1ml contains emodin 8 μ g, chrysophanol 20 μ g, promptly gets;

The preparation of need testing solution: get warming spleen and tonifying kidney, let out the pharmaceutical composition of turbid blood stasis dispelling and put in the volumetric flask, add the 1.5-3mol/L sulfuric acid solution, add chloroform again, 80-95 ℃ reflux 1-3 hour; Cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container, incorporates in the separatory funnel; Obtain chloroform layer, acid solution is with chloroform extraction 2-4 time, each 15ml, combined chloroform liquid; Naturally volatilize, residue is used 4-1: 1 dehydrated alcohol-ethyl acetate dissolving in the dislocation 25ml measuring bottle, and is diluted to scale; Shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45 μ m);

Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, promptly get; The warming spleen and tonifying kidney that is equivalent to crude drug 6-9g, the pharmaceutical composition of letting out turbid blood stasis dispelling contains emodin (C ₁₅H ₁₀O ₅) and chrysophanol (C ₁₅H ₁₀O ₄) total, must not be less than 20mg.

Discrimination method comprises one or more in the following method:

A, get the warming spleen and tonifying kidney that is equivalent to crude drug 10-25g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the vitriolic 40-50% alcoholic solution of 6-8% 40-60ml, supersound process 15-30 minute; Filter with filter cloth, filtrating is put in the conical flask, and water-bath backflow 1-3 hour is filtered with filter cloth; Filtrating is with 60-90 ℃ of petroleum ether extraction 2-4 time, 20ml at every turn, combining extraction liquid; Add the 0.1mol/L sodium hydroxide solution and be washed till the nothing pink, the reuse washing is to neutrality, ether layer evaporate to dryness; Residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, adds the vitriolic 40-50% alcoholic solution of 6-8% 40-60ml, supersound process 15-30 minute, filters with filter cloth; Filtrating is put in the conical flask, and water-bath backflow 1-3 hour is filtered with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 2-4 time; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as control medicinal material solution; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; Benzene-acetone with 4-6: 1-3 is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the 365nm uviol lamp and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the speckle or the fluorescence speckle of same color respectively;

B, get the warming spleen and tonifying kidney that is equivalent to crude drug 5-15, let out the pharmaceutical composition of turbid blood stasis dispelling, add 3ml hydrochloric acid and 20ml chloroform, reflux 0.5-2 hour, put coldly, filter, evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; In addition extracting liquorice control medicinal material 0.5g adds 3ml hydrochloric acid and 20ml chloroform, reflux 0.5-2 hour, put cold, filtration, evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as control medicinal material solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography; Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, be developing solvent with petroleum ether-benzene-ethyl acetate-glacial acetic acid of 3-7: 8-15: 2-6: 0.4-0.8; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively;

C, get the warming spleen and tonifying kidney that is equivalent to crude drug 35-45g, let out the pharmaceutical composition of turbid blood stasis dispelling, extract volatile oil,, obtain the ether layer, volatilize to 0.5ml, as need testing solution with a small amount of ether flushing volatile oil extractor with volatile oil extractor; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with volatile oil extractor, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With 7-11: benzene-ether of 1 is developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

The aconitine limit detection method is:

Get the warming spleen and tonifying kidney that is equivalent to crude drug 10-20g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 15-30 minute 1-3 hour; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 1-3 time; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 2-4 time; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata (system) control medicinal material coarse powder 7.2g, adds the ammonia solution moistening, soaks into the 60ml supersound process that adds diethyl ether 15-30 minute 1-3 hour; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 1-3 time; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 2-4 time; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as control medicinal material solution; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Test according to thin layer chromatography; Accurate need testing solution and each 10 μ l of reference substance solution of drawing; Put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 1: 1-3: E-C-methanol of 1 is developing solvent, launch to take out to dry; Put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

The present invention provides above-mentioned warming spleen and tonifying kidney, lets out the drug regimen object detecting method of turbid blood stasis dispelling, preferably includes in the detection of following assay and/or discrimination method and/or aconitine limit one or more:

Content assaying method comprises the steps:

According to HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; With 85: 15 methanol-0.1% phosphoric acid solution is mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

The preparation of reference substance solution: precision takes by weighing emodin and the chrysophanol reference substance is an amount of respectively, and the dehydrated alcohol-ethyl acetate that adds 2: 1 is processed the mixed solution that every 1ml contains emodin 8 μ g, chrysophanol 20 μ g, promptly gets;

The preparation of need testing solution: get the warming spleen and tonifying kidney that is equivalent to crude drug 0.2g-0.3g, let out the pharmaceutical composition of turbid blood stasis dispelling, porphyrize is put in the 150ml ground conical flask, adds 2mol/L sulfuric acid solution 20ml; Add chloroform 20ml again, 90 ℃ of reflux 2 hours, cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container; Incorporate in the separatory funnel, obtain chloroform layer, acid solution is with chloroform extraction 3 times, each 15ml, combined chloroform liquid; Naturally volatilize, residue in the dislocation 25ml measuring bottle, and is diluted to scale with dehydrated alcohol-ethyl acetate dissolving in 2: 1; Shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45 μ m);

Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, promptly get; The warming spleen and tonifying kidney that is equivalent to crude drug 7.8g, the pharmaceutical composition of letting out turbid blood stasis dispelling contains emodin (C ₁₅H ₁₀O ₅) and chrysophanol (C ₁₅H ₁₀O ₄) total, must not be less than 20mg.

Discrimination method comprises one or more in the following method

A, get the warming spleen and tonifying kidney that is equivalent to crude drug 15.6g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 7% vitriolic 45% alcoholic solution 50ml, supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, adds 7% vitriolic 45% alcoholic solution 50ml, and supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as control medicinal material solution; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-acetone of 5: 2 was developing solvent, launched, and took out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the 365nm uviol lamp and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the speckle or the fluorescence speckle of same color respectively;

B, get the warming spleen and tonifying kidney that is equivalent to crude drug 7.8g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; In addition extracting liquorice control medicinal material 0.5g adds 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour, put cold, filtration, evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as control medicinal material solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography; Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 5: 10: 4: petroleum ether-benzene of 0.6-ethyl acetate-glacial acetic acid is developing solvent; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively;

C, get the warming spleen and tonifying kidney that is equivalent to crude drug 39g, let out the pharmaceutical composition of turbid blood stasis dispelling, extract volatile oil,, obtain the ether layer, volatilize to 0.5ml, as need testing solution with a small amount of ether flushing volatile oil extractor with volatile oil extractor; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with volatile oil extractor, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-ether of 9: 1 was developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

The aconitine limit detection method is:

Get the warming spleen and tonifying kidney that is equivalent to crude drug 15.6g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata (system) control medicinal material coarse powder 7.2g, adds the ammonia solution moistening, soaks into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as control medicinal material solution; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Test according to thin layer chromatography; Accurate need testing solution and each 10 μ l of reference substance solution of drawing; Put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, be developing solvent with E-C-methanol of 1: 2: 1, launch to take out and to dry; Put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

Said warming spleen and tonifying kidney, the crude drug of letting out the pharmaceutical composition of turbid blood stasis dispelling consists of (weight ratio):

Radix Et Rhizoma Rhei 300-1000 weight portion Radix Aconiti Lateralis Preparata (system) 100-600 weight portion Rhizoma Zingiberis 100-600 weight portion

Radix Ginseng 100-600 weight portion Radix Glycyrrhizae 60-300 weight portion.

Warming spleen and tonifying kidney; Let out the preparation of drug combination method of turbid blood stasis dispelling: Radix Et Rhizoma Rhei, Radix Aconiti Lateralis Preparata (system), Rhizoma Zingiberis, Radix Ginseng, Radix Glycyrrhizae add the pharmacy acceptable auxiliary in proportion and process the acceptable any conventional dosage form of pharmaceutics after conventional process is extracted, comprise capsule, tablet, granule, gel, slow releasing agent, oral liquid or drop pill; Said common process method comprises water extraction or alcohol extraction or first water extraction water extraction again after alcohol extraction or the first alcohol extraction again, and it is further active constituent-enriched after above-mentioned conventional the extraction, can also to cross macroporous resin column;

Above-mentioned common process water extracting method can for: each 10-30 doubly measures, and extracts 1-3 hour at every turn 1-4 time;

Above-mentioned common process alcohol extraction method can for: 30-80% ethanol, 4-15 doubly measured, extract respectively 1-3 hour;

The said macroporous resin column method of crossing is: extracting solution filters, and filtrating is through macroporous resin, and elder generation is with the ethanol elution of 4-6 times of water gaging or 20% following concentration; Eluent discards, and reuse adds 1-10 times of eluting of 50-80% ethanol, collects eluent; Decompression recycling ethanol gets the purification extracting solution to there not being the alcohol flavor.

Said pharmacy acceptable auxiliary is: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, fixed, the Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods of acetic acid chloroethene; Substrate comprises: PEG6000, PEG4000, insect wax etc.

Above-mentioned warming spleen and tonifying kidney is let out alcohols extraction method in the preparation of drug combination method of turbid blood stasis dispelling.

Warming spleen and tonifying kidney, the quality determining method specificity of pharmaceutical composition of letting out turbid blood stasis dispelling is good, stability height, favorable reproducibility, precision are high, is fit to industrialized production more, has really guaranteed safety of clinical administration, effectively, reliably.

The inventor Different Extraction Method to the influence of rat adenine induced CRF contrast effect experiment in, the unexpected discovery has marked difference with the compare method of traditional water decoction of the crude drug of ethanol extraction.Therefore, the present invention also provides warming spleen and tonifying kidney, and the pharmaceutical composition alcohol extraction method of letting out turbid blood stasis dispelling prepares the application of preparation in preparation treatment chronic kidney hypofunction medicine.Said alcohol extraction method is: crude drug: Radix Et Rhizoma Rhei 300-1000 weight portion, Radix Aconiti Lateralis Preparata (system) 100-600 weight portion, Rhizoma Zingiberis 100-600 weight portion, Radix Ginseng 100-600 weight portion, Radix Glycyrrhizae 60-300 weight portion; The medicinal 30-80% ethanol of above-mentioned raw materials 4-15 doubly measures and extracts 1-3 hour respectively.

Said alcohol extraction method is preferably: Rhizoma Zingiberis 240 weight portions add 25 times of water gagings, soak after 16 hours and distill 6 hours, extract volatile oil, the centrifugal filtration of distillate, and other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600 weight portions, Radix Aconiti Lateralis Preparata 360 weight portions, Radix Ginseng 240 weight portions, Radix Glycyrrhizae 120 weight portions are with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation; When being concentrated into relative density 1.22-1.25 (50 ℃), drying, it is subsequent use to be ground into fine powder; Volatile oil and subsequent use fine powder are processed conventional formulation.

Following experimental example and embodiment do further explanation to the present invention, but do not constitute restriction of the present invention.

(these article are the product of embodiment 1 preparation in the following experimental example)

The research of experimental example 1 discrimination method

1, Radix Ginseng is differentiated

The ginsenoside is under acid condition, and hydrolyzable is panoxadiol, panaxatriol, so be the index of Radix Ginseng qualitative identification with panoxadiol, panaxatriol.

1.1 development system: proof development system has: benzene-acetone (5: 2); Chloroform-ether (1: 1).The former development system chromatography effect is better, so the developing solvent of selecting for use benzene-acetone (5: 2) to differentiate as Radix Ginseng.

1.2 developer: the developer of testing has: 10% ethanol solution of sulfuric acid and 30% ethanol solution of sulfuric acid.The former colour developing is more clear, thus with 10% ethanol solution of sulfuric acid as developer.

1.3 lamellae: proof lamellae has the G lamellae of silica gel; Contain the CMC-Na silica gel g thin-layer plate, activation 30 minutes (105 ℃) respectively.It is better that the result contains CMC-Na silica gel g thin-layer plate chromatography effect.

1.4 thin layer chromatography result: it is clear that the test sample speckle that makes separates, and the chromatography effect is better, and negative sample is noiseless, can reach the discriminating purpose.

2, Radix Glycyrrhizae is differentiated

(A) method for preparing of need testing solution

(1) get these article 5g, porphyrize adds hydrochloric acid 3ml and chloroform 20ml, and reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution.

(2) get these article 5g, porphyrize adds hydrochloric acid 3ml and chloroform 20ml, and supersound process 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution.

(B) selection of developing solvent

(1) petroleum ether-benzene-ethyl acetate-glacial acetic acid is (10: 20: 7: 0.5)

(2) petroleum ether-benzene-ethyl acetate-glacial acetic acid is (5: 10: 4: 0.6)

Extracting liquorice control medicinal material 0.5g shines medical material solution in pairs with legal system in addition.

Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again.According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in being on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; Launch with developing solvent, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively.

The discrimination test result of table 1 Radix Glycyrrhizae

According to above experimental result, draw the method for preparing of selecting test sample for use and be: get these article 5g, porphyrize adds hydrochloric acid 3ml and chloroform 20ml, and reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution.The developing solvent system is petroleum ether-benzene-ethyl acetate-glacial acetic acid (5: 10: 4: 0.6).

3, the discriminating of Rhizoma Zingiberis

Mainly contain volatile oil in the Rhizoma Zingiberis, but volatile oil contents is very low, and the Radix Et Rhizoma Rhei composition disturbs bigger.

3.1 development system: the development system of testing has: a. petroleum ether-ethyl acetate (85: 15); B. benzene; C. normal hexane-ethyl acetate (85: 15); D. chloroform-methanol (9: 1); E. petroleum ether-ethyl acetate (19: 1); F. cyclohexane extraction-ether (1: 1); G. cyclohexane extraction-ether-ethyl acetate (6: 2: 2); H. benzene-ether (9: 1).Through overtesting, better with h item development system chromatography effect, so with the developing solvent of benzene-ether (9: 1) as the Rhizoma Zingiberis discriminating.

3.2 developer: the developer of testing has: 1% vanillin sulfuric acid solution, 1% vanillin test solution, 2% vanillin test solution, and clear with 1% vanillin sulfuric acid solution colour developing, so be developer with 1% vanillin sulfuric acid solution.

3.3 lamellae: proof lamellae has the G lamellae of silica gel; Contain the CMC-Na silica gel g thin-layer plate, activation 30 minutes (105 ℃) respectively.It is better that the result contains CMC-Na silica gel g thin-layer plate chromatography effect.

3.4 thin layer chromatography result: it is clear that the test sample speckle that makes separates, and chromatography is effective, and the retinue negative sample is noiseless, can be used for the discriminating of Rhizoma Zingiberis composition.

4, aconitine detects

Development system: the development system of testing has: a. benzene-ethyl acetate-diethylamine (14: 4: 1); B. E-C-methanol (1: 2: 1).Latter's development system chromatography effect is better, so with E-C-methanol (1: 2: 1).

The thin layer chromatography result: it is clear that the test sample speckle that makes separates, and chromatography is effective, and the retinue negative sample is noiseless, can be used for the aconitine limit inspection.

Detection sensitivity: the dilution of aconitine reference substance solution is 0.18mg/ml, and visible through thin-layer chromatogram, point sample 2ul can debate spotting out, so detection sensitivity is 0.36ug.

The research of experimental example 2 content assaying methods

1, instrument, reagent, reagent: HP1100 high performance liquid chromatograph; Tianjin, island LC-10ATP chromatograph of liquid; Emodin, chrysophanol reference substance, purchase in Nat'l Pharmaceutical & Biological Products Control Institute classification: supply assay usefulness, lot number is respectively 0756-200110,0796-200006; Agents useful for same is analytical pure; The product of embodiment 1 preparation is by Jilin Province Medicine Research Institute development and production, lot number 20010611,20011222,20020105.

2, chromatographic condition

2.1 detection wavelength determination

Get emodin and chrysophanol reference substance solution, carry out spectral scan at 190-600nm respectively, maximum absorption band is respectively at 254nm and 436nm.Selecting octadecylsilane chemically bonded silica for use is filler; With methanol-0.1% phosphoric acid solution (85: 15) is mobile phase, gets emodin, chrysophanol reference substance solution, need testing solution, negative control solution, injects chromatograph of liquid respectively, under two wavelength, measures.Test is found, test sample chromatographic peak hangover when measuring wavelength 254nm.When measuring wavelength 436nm, emodin, chrysophanol and other component are separated better in the test sample, and negative control solution is noiseless, therefore, measure wavelength and are chosen to be 436nm.

2.2 the mobile phase ratio is selected

Selecting octadecylsilane chemically bonded silica for use is filler; The detection wavelength is 436nm, and the ratio of adjustment methanol and 0.1% phosphoric acid solution selects for use 3 mobile phase ratios to be respectively: (1) methanol-0.1% phosphoric acid solution (80: 20); (2) methanol-0.1% phosphoric acid solution (85: 15); (3) methanol-0.1% phosphoric acid solution (90: 10) is investigated the influence of mobile phase ratio adjustment to main peak and impurity peaks separation case and appearance time.Accurate respectively each the 20 μ l of need testing solution that draw inject liquid chromatograph, and each peak-to-peak minimum separation degree and appearance time data see Table 2.

Table 2 separating degree and appearance time data

Can be known by above result: (2), (3) all can make main peak and assorted peak separating degree reach requirement, but appearance time (2) is smaller than (3), and the separating degree of (1) does not reach requirement.Take all factors into consideration, select the mobile phase ratio of (2), i.e. methanol-0.1% phosphoric acid solution (85: 15).

3, the screening of need testing solution method for preparing: adopt sulphuric acid hydrolysis, the method for chloroform reflux, extract,, condition such as chloroform consumption is optimized selection during respectively to vitriolic concentration, consumption, hydrolysis time, temperature, reflux, extract.

3.1 hydrolysis sees Table 3 with the concentration and the time screening of sulfuric acid.

Table 3 hydrolysis is with sulfuric acid concentration and time result of the test

This shows that select sulfuric acid concentration 2.0mol/L for use, hydrolysis time 2h is advisable.

3.2 table 4 is seen in the screening of sulphuric acid consumption

Table 4 sulphuric acid different amounts is measured the result

It is thus clear that, selecting 2.0mol/L sulphuric acid 20ml for use, hydrolysis 2h is advisable.

3.3 chloroform reflux, extract, consumption is seen table 5 during hydrolysis

Table 5 chloroform reflux, extract, is used the quantitative determination result

So chloroform consumption 20ml.

3.4 the different hydrolysis temperature measuring of hydrolysis temperature result sees table 6

The different hydrolysis temperature measuring of table 6 result

It is thus clear that hydrolysis temperature is advisable for selected 90 ℃.

Conclusion: through the screening of above pre-treating method, visible optimum condition is 2.0mol/L sulphuric acid consumption 20ml, adds chloroform 20ml, hydrolysis time 2h, 90 ℃ of hydrolysis temperatures.

4, linear relationship is investigated:

Precision takes by weighing emodin reference substance 5.39mg and chrysophanol reference substance 4.95mg puts respectively in the 25ml measuring bottle; Add dissolve with methanol and be diluted to scale; Get emodin reference substance solution 4ml and chrysophanol reference substance solution 10ml respectively in the 100ml measuring bottle, add mobile phase and be diluted to scale.Process respectively every 1ml contain emodin and chrysophanol be respectively 8.624 with the reference substance solution of mixing of 19.8 μ g.Precision is measured above-mentioned mixing reference substance solution 5 μ l respectively, 10 μ l, and 20 μ l, 40 μ l, 50 μ l, sample introduction is measured peak area successively, and the result sees table 7,8.

Table 7 emodin linear relationship is investigated

It is thus clear that emodin is good in 0.04312～0.43120 μ g scope internal linear.

Table 8 chrysophanol linear relationship

It is thus clear that chrysophanol is good in 0.099～0.990 μ g scope internal linear.

5, precision: draw " 4, linear relationship investigate " mixing reference substance solution 20 μ l down, repetition sample introduction 5 times, the result sees table 9.

Table 9 Precision test result

This law precision is good.

6, repeatability test: get lot number and be 20010611 sample, press content assaying method, process 5 parts of need testing solutions respectively, carry out quantitative assay, the result sees table 10.

Table 10 reproducible test results

Annotate: A is peak area average content: 0.66%RSD:2.14%

7, stability test

Through test, test sample is stable in 48 hours.The result sees table 11

Table 11 sample stability result of the test

Experimental example 3 effect experiments---to the influence of rat adenine induced CRF

1, sample preparation

Water is put forward sample sets: Rhizoma Zingiberis 240g adds 25 times of water gagings, soaks after 16 hours and distills 6 hours, extracts volatile oil, the centrifugal filtration of distillate, and other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600g, Radix Aconiti Lateralis Preparata 360g, Radix Ginseng 240g, Radix Glycyrrhizae 120g soaked 16 hours with 6～20 times of water gagings, and heating and refluxing extraction 3 times respectively adds 6～20 times of water gagings the 2nd, 3 time; The each extraction 2 hours is when the aqueous solution merging filtrate after filtration while hot and the distillation is concentrated into relative density 1.20-1.25 (50 ℃); Add microcrystalline Cellulose 200g; Mixing is dried to dry extract, and it is subsequent use to be ground into fine powder; Get 8 times of betacyclodextrins to volatile oil, add 15 times of water gagings, heating makes dissolving about 80 ℃, when water temperature is reduced to 40 ℃; Add volatile oil, 900 rev/mins were stirred 2 hours, and cold preservation 48 hours filters; The low amounts of water washing, 40 ℃ of drying under reduced pressure get betacyclodextrin clathrate dry powder, and are subsequent use; Get dry extract, betacyclodextrin clathrate, add microcrystalline Cellulose 54-37g, starch 381-356g, mixing, with the moistening granulation of 20% ethanol, 55-60 ℃ of drying processed 1000g, promptly gets.

The alcohol extraction sample sets: Rhizoma Zingiberis 240g adds 25 times of water gagings, soaks after 16 hours and distills 6 hours, extracts volatile oil, the centrifugal filtration of distillate, and other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600g, Radix Aconiti Lateralis Preparata 360g, Radix Ginseng 240g, Radix Glycyrrhizae 120g be with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation, when being concentrated into relative density 1.22-1.25 (50 ℃), add microcrystalline Cellulose 200g; Mixing is dried to dry extract, and it is subsequent use to be ground into fine powder; Get 8 times of betacyclodextrins to volatile oil, add 15 times of water gagings, heating makes dissolving about 80 ℃, when water temperature is reduced to 40 ℃; Add volatile oil, 900 rev/mins were stirred 2 hours, and cold preservation 48 hours filters; The low amounts of water washing, 40 ℃ of drying under reduced pressure get betacyclodextrin clathrate dry powder, and are subsequent use; Get dry extract, betacyclodextrin clathrate, add microcrystalline Cellulose 54-37g, starch 381-356g, mixing, with the moistening granulation of 20% ethanol, 55-60 ℃ of drying processed 1000g, promptly gets.

The positive drug group: NIAODUQING KELI, commercially available, the suspension of desired concn is all processed in above-mentioned test medication with distilled water, supplies gastric infusion.

Model control group: give distilled water with volume.

2, method and result

2.1 Preparation of model: get 60 of the male rats of body weight 150 ± 5.73g, be divided into 6 groups: the normal control group, raise with normal diet; All the other 5 groups of feedstuffs that give 0.75% adenine.Gave for 4 weeks continuously, duplicate rat adenine chronic kidney hypofunction model.

2.2 grouping administration: give the adenine feedstuff after 4 weeks; Model control group is irritated stomach and is given distilled water 10ml/kg, positive drug group (5g/kg), water and put forward sample sets (1.95 (crude drug) g/kg), alcohol extraction sample sets (1.95 (crude drug) g/kg); Irritate stomach every day once, gave for 4 weeks continuously.

2.3 result of the test: renal function is measured: visible from the result of table 12, and water is put forward sample sets, alcohol extraction sample sets and positive drug group and model control group more all has reduction effect in various degree, wherein alcohol extraction sample sets obviously is superior to water and puies forward sample sets.

Table 12 distinct methods extract is to the influence of pharmacology index

Group	BUN(mmol/L)	Scr(mmol/L)
			Model control group	39.23±42.561	281.15±113.650
The positive drug group	35.28±7.886	245.98±65.879
			Water is put forward sample sets	32.81±10.409	236.50±89.182
The alcohol extraction sample sets	17.10±11.307	118.40±92.969
			BUN/Scr reduces percentage rate (%)	47.88 ^＊ 51.53 ^#	49.94 ^＊ 51.87 ^#

Annotate: sample extraction 2 times, 2 hours/inferior.

" * ": alcohol appearance group and water sample group are than " # ": alcohol appearance group and positive drug group ratio

The specific embodiment

Embodiment 1: granule and detection method

Radix Et Rhizoma Rhei 600g Radix Aconiti Lateralis Preparata (system) 360g Rhizoma Zingiberis 240g Radix Ginseng 240g Radix Glycyrrhizae 120g

Method for making: the above five tastes, Rhizoma Zingiberis add 25 times of water gagings, soak after 16 hours and distill 6 hours, extract volatile oil, the centrifugal filtration of distillate, and other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei, Radix Aconiti Lateralis Preparata, Radix Ginseng, Radix Glycyrrhizae be with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation, when being concentrated into relative density 1.22-1.25 (50 ℃), add microcrystalline Cellulose 200g; Mixing is dried to dry extract, and it is subsequent use to be ground into fine powder; Get 8 times of betacyclodextrins to volatile oil, add 15 times of water gagings, heating makes dissolving about 80 ℃, when water temperature is reduced to 40 ℃; Add volatile oil, 900 rev/mins were stirred 2 hours, and cold preservation 48 hours filters; The low amounts of water washing, 40 ℃ of drying under reduced pressure get betacyclodextrin clathrate dry powder, and are subsequent use; Get dry extract, betacyclodextrin clathrate, add microcrystalline Cellulose 54-37g, starch 381-356g, mixing, with the moistening granulation of 20% ethanol, 55-60 ℃ of drying processed 1000g, promptly gets.

Function with cure mainly: warming spleen and tonifying kidney, let out turbid blood stasis dispelling.It is early stage to be used for chronic renal failure azotemia phase and uremia, and Chinese medical discrimination belongs to deficiency of spleen-YANG and kidneyYANG.This product can reduce blood urea nitrogen, creatinine, stablizes renal function, suppresses urine albumen, regulates electrolyte disturbance, and renal anemia is had some improvement.

Usage and consumption: boiled water is taken after mixing it with water, a 5g, 3 times on the one.

Specification: every bag of 5g

Differentiate: A, get these article 10g, add 7% vitriolic 45% alcoholic solution 50ml, supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with petroleum ether (60-90 ℃) extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, shines medical material solution in pairs with legal system; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography (appendix VI of Chinese Pharmacopoeia version in 2000) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-acetone (5: 2) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the uviol lamp (365nm) and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the speckle or the fluorescence speckle of same color respectively.

B, get these article 5g, add 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; Extracting liquorice control medicinal material 0.5g shines medical material solution in pairs with legal system in addition; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B); Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with petroleum ether-benzene-ethyl acetate-glacial acetic acid (5: 10: 4: 0.6) be developing solvent; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively.

C, get these article 25g, extract volatile oil,, obtain the ether layer, volatilize to 0.5ml, as need testing solution with a small amount of ether flushing volatile oil extractor with volatile oil extractor; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with method, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-ether (9: 1) is developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Aconitine limit inspection: get these article 10g, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata (system) control medicinal material coarse powder 7.2g, shines medical material solution in pairs with legal system; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000); Accurate need testing solution and each 10 μ l of reference substance solution of drawing; Put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, be developing solvent with E-C-methanol (1: 2: 1), launch to take out and to dry; Put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

Assay: according to HPLC, chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Methanol-0.1% phosphoric acid solution (85: 15) is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000; The preparation of reference substance solution: precision takes by weighing emodin and the chrysophanol reference substance is an amount of respectively, adds dehydrated alcohol-ethyl acetate (2: 1) and processes the mixed solution that every 1ml contains emodin 8 μ g, chrysophanol 20 μ g, promptly gets; The preparation of need testing solution: get the content under these article content uniformity item, porphyrize, precision takes by weighing 0.17g, puts in the 150ml ground conical flask, adds 2mol/L sulfuric acid solution 20ml; Add chloroform 20ml again, 90 ℃ of reflux 2 hours, cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container; Incorporate in the separatory funnel, obtain chloroform layer, acid solution is with chloroform extraction 3 times, each 15ml, combined chloroform liquid; Naturally volatilize, residue in the dislocation 25ml measuring bottle, and is diluted to scale with dehydrated alcohol-ethyl acetate (2: 1) dissolving; Shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45 μ m); Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, promptly get; These article contain emodin (C for every bag ₁₅H ₁₀O ₅) and chrysophanol (C ₁₅H ₁₀O ₄) total, must not be less than 20mg.

Embodiment 2: granule and detection method

Content assaying method:

According to HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; 81: 19 methanol-0.1% phosphoric acid solution is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

The preparation of reference substance solution: precision takes by weighing emodin and the chrysophanol reference substance is an amount of respectively, adds 2: 1 dehydrated alcohol-ethyl acetates and processes the mixed solution that every 1ml contains emodin 8 μ g, chrysophanol 20 μ g, promptly gets;

The preparation of need testing solution: get the content under these article content uniformity item, porphyrize, precision takes by weighing 0.17g, puts in the 150ml ground conical flask, adds 2mol/L sulfuric acid solution 20ml; Add chloroform 20ml again, 90 ℃ of reflux 2 hours, cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container; Incorporate in the separatory funnel, obtain chloroform layer, acid solution is with chloroform extraction 3 times, each 15ml, combined chloroform liquid; Naturally volatilize, residue in the dislocation 25ml measuring bottle, and is diluted to scale with dehydrated alcohol-ethyl acetate dissolving in 2: 1; Shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45 μ m);

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, promptly get; The warming spleen and tonifying kidney that is equivalent to crude drug 7.8g, the pharmaceutical composition of letting out turbid blood stasis dispelling contains emodin (C ₁₅H ₁₀O ₅) and chrysophanol (C ₁₅H ₁₀O ₄) total, must not be less than 20mg.

Embodiment 3: detection method

Warming spleen and tonifying kidney, the pharmaceutical composition of letting out turbid blood stasis dispelling is the preparation of embodiment 2 methods.

A, get the warming spleen and tonifying kidney that is equivalent to crude drug 15.6g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 7% vitriolic 45% alcoholic solution 50ml, supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, shines medical material solution in pairs with legal system; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With 5: 2 benzene-acetone was developing solvent, launched, and took out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the uviol lamp (365nm) and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the speckle or the fluorescence speckle of same color respectively;

B, get the warming spleen and tonifying kidney that is equivalent to crude drug 7.8g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; Extracting liquorice control medicinal material 0.5g shines medical material solution in pairs with legal system in addition; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography; Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 5: 10: 4: 0.6 petroleum ether-benzene-ethyl acetate-glacial acetic acid is developing solvent; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively;

Embodiment 4: detection method

Content assaying method:

According to HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; 85: 15 methanol-0.1% phosphoric acid solution is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

The aconitine limit detection method is:

Get the warming spleen and tonifying kidney that is equivalent to crude drug 15.6g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata (system) control medicinal material coarse powder 7.2g, shines medical material solution in pairs with legal system; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, the accurate respectively 10 μ l of need testing solution and reference substance solution that draw, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With 1: 2: 1 E-C-methanol was developing solvent; Launch to take out and to dry, put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

Embodiment 5: capsule and detection method

Method for making: the above five tastes, Rhizoma Zingiberis add 25 times of water gagings, soak after 16 hours and distill 6 hours, extract volatile oil, the centrifugal filtration of distillate, and other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei, Radix Aconiti Lateralis Preparata, Radix Ginseng, Radix Glycyrrhizae be with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation, when being concentrated into relative density 1.22-1.25 (50 ℃), add microcrystalline Cellulose 200g; Mixing is dried to dry extract, and it is subsequent use to be ground into fine powder; Get 8 times of betacyclodextrins to volatile oil, add 15 times of water gagings, heating makes dissolving about 80 ℃, when water temperature is reduced to 40 ℃; Add volatile oil, 900 rev/mins were stirred 2 hours, and cold preservation 48 hours filters; The low amounts of water washing, 40 ℃ of drying under reduced pressure get betacyclodextrin clathrate dry powder, and are subsequent use; Get dry extract, betacyclodextrin clathrate, add adjuvant and process capsule.

Content assaying method: according to HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; 88: 12 methanol-0.1% phosphoric acid solution is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

Discrimination method:

C, get the warming spleen and tonifying kidney that is equivalent to crude drug 39g, let out the pharmaceutical composition of turbid blood stasis dispelling, extract volatile oil,, obtain the ether layer, volatilize to 0.5ml, as need testing solution with a small amount of ether flushing volatile oil extractor with volatile oil extractor; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with method, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With 9: 1 benzene-ether was developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Embodiment 6: detection method

Get the warming spleen and tonifying kidney that is equivalent to crude drug 15.6g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 7% vitriolic 45% alcoholic solution 50ml, supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, shines medical material solution in pairs with legal system; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With 5: 2 benzene-acetone was developing solvent, launched, and took out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the uviol lamp (365nm) and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the speckle or the fluorescence speckle of same color respectively;

Embodiment 7: detection method

Get the warming spleen and tonifying kidney that is equivalent to crude drug 7.8g, let out the pharmaceutical composition of turbid blood stasis dispelling, add 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; Extracting liquorice control medicinal material 0.5g shines medical material solution in pairs with legal system in addition; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography; Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 5: 10: 4: 0.6 petroleum ether-benzene-ethyl acetate-glacial acetic acid is developing solvent; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively;

Embodiment 8: detection method

These article of getting 25g extracts volatile oil with volatile oil extractor, with a small amount of ether flushing volatile oil extractor, obtains the ether layer, volatilizes to 0.5ml, as need testing solution; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with method, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-ether (9: 1) is developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Embodiment 9: detection method

Claims

1. warming spleen and tonifying kidney, the detection method of letting out the pharmaceutical composition of turbid blood stasis dispelling is characterized in that the content assaying method of emodin and chrysophanol is in this method:

HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; The methanol of 80-90: 10-20-0.1% phosphoric acid solution is a mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

The preparation of need testing solution: get this warming spleen and tonifying kidney, let out the pharmaceutical composition of turbid blood stasis dispelling and put in the volumetric flask, add the 1.5-3mol/L sulfuric acid solution, add chloroform again, 80-95 ℃ reflux 1-3 hour; Cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container, incorporates in the separatory funnel; Obtain chloroform layer, acid solution is with chloroform extraction 2-4 time, each 15ml, combined chloroform liquid; Naturally volatilize, residue is used 4-1: 1 dehydrated alcohol-ethyl acetate dissolving in the dislocation 25ml measuring bottle, and is diluted to scale; Shake up, filter, get subsequent filtrate, promptly get with 0.45 μ m microporous filter membrane;

Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, promptly get; It is C that the kidney tonifying detoxicating preparation that is equivalent to crude drug 6-9g contains molecular formula ₁₅H ₁₀O ₅Emodin and molecular formula be C ₁₅H ₁₀O ₄The chrysophanol total, must not be less than 20mg;

Said warming spleen and tonifying kidney, the pharmaceutical composition of letting out turbid blood stasis dispelling are to be processed by following crude drug:

Radix Et Rhizoma Rhei 300-1000 weight portion Radix Aconiti Lateralis Preparata 100-600 weight portion Rhizoma Zingiberis 100-600 weight portion

Radix Ginseng 100-600 weight portion Radix Glycyrrhizae 60-300 weight portion.

2. detection method as claimed in claim 1 is characterized in that the content assaying method of emodin and chrysophanol is preferably in this method:

HPLC, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; With 85: 15 methanol-0.1% phosphoric acid solution is mobile phase; Detect wavelength 436nm; Number of theoretical plate calculates by the emodin peak should be no less than 3000;

The preparation of need testing solution: get the warming spleen and tonifying kidney that is equivalent to crude drug 0.2g-0.3g, let out the pharmaceutical composition of turbid blood stasis dispelling, porphyrize is put in the 150ml ground conical flask, adds 2mol/L sulfuric acid solution 20ml; Add chloroform 20ml again, 90 ℃ of reflux 2 hours, cooling in the dislocation separatory funnel, is used the minimum of chloroform washing container; Incorporate in the separatory funnel, obtain chloroform layer, acid solution is with chloroform extraction 3 times, each 15ml, combined chloroform liquid; Naturally volatilize, residue in the dislocation 25ml measuring bottle, and is diluted to scale with dehydrated alcohol-ethyl acetate dissolving in 2: 1; Shake up, filter, get subsequent filtrate, promptly get with 0.45 μ m microporous filter membrane;

Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, promptly get; It is C that the kidney tonifying detoxicating preparation that is equivalent to crude drug 7.8g contains molecular formula ₁₅H ₁₀O ₅Emodin and molecular formula be C ₁₅H ₁₀O ₄The chrysophanol total, must not be less than 20mg;

Radix Et Rhizoma Rhei 600 weight portion Radix Aconiti Lateralis Preparata 360 weight portion Rhizoma Zingiberiss 240 weight portion Radix Ginsengs 240 weight portion Radix Glycyrrhizaes 120 weight portions.

3. according to claim 1 or claim 2 detection method is characterized in that this method comprises that following Radix Ginseng is differentiated and/or Radix Glycyrrhizae is differentiated and/or Rhizoma Zingiberis is differentiated and/or aconitine limit one or more in detecting:

A, get the warming spleen and tonifying kidney that is equivalent to crude drug 10-25g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the vitriolic 40-50% alcoholic solution of 6-8% 40-60ml, supersound process 15-30 minute; Filter with filter cloth, filtrating is put in the conical flask, and water-bath backflow 1-3 hour is filtered with filter cloth; Filtrating is with 60-90 ℃ of petroleum ether extraction 2-4 time, 20ml at every turn, combining extraction liquid; Add the 0.1mol/L sodium hydroxide solution and be washed till the nothing pink, the reuse washing is to neutrality, ether layer evaporate to dryness; Residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, adds the vitriolic 40-50% alcoholic solution of 6-8% 40-60ml, supersound process 15-30 minute, filters with filter cloth; Filtrating is put in the conical flask, and water-bath backflow 1-3 hour is filtered with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 2-4 time; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as control medicinal material solution; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; Benzene-acetone with 4-6: 1-3 is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the 365nm uviol lamp and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the fluorescence speckle of same color respectively;

D, aconitine limit detect: get the warming spleen and tonifying kidney that is equivalent to crude drug 10-20g, let out the pharmaceutical composition of turbid blood stasis dispelling, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 15-30 minute 1-3 hour; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 1-3 time; Each 20ml, combining extraction liquid is transferred pH11-12 with strong caustic, with chloroform extraction 2-4 time; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata control medicinal material coarse powder 7.2g, adds the ammonia solution moistening, soaks into the 60ml supersound process that adds diethyl ether 15-30 minute 1-3 hour; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 1-3 time; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 2-4 time; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as control medicinal material solution; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Test according to thin layer chromatography; Accurate need testing solution and each 10 μ l of reference substance solution of drawing; Put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 1: 1-3: E-C-methanol of 1 is developing solvent, launch to take out to dry; Put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

4. detection method as claimed in claim 3 is characterized in that this method comprises one or more in following Radix Ginseng discriminating and/or Radix Glycyrrhizae discriminating and/or Rhizoma Zingiberis discriminating and/or the aconitine limit detection:

A, get this pharmaceutical composition that is equivalent to crude drug 15.6g, add 7% vitriolic 45% alcoholic solution 50ml, supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as need testing solution; Other gets Radix Ginseng control medicinal material 2.4g, adds 7% vitriolic 45% alcoholic solution 50ml, and supersound process 20 minutes is filtered with filter cloth; Filtrating is put in the conical flask, and water-bath refluxed 2 hours, filters with filter cloth, and filtrating is with 60-90 ℃ of petroleum ether extraction 3 times; Each 20ml, combining extraction liquid adds the 0.1mol/L sodium hydroxide solution and is washed till the nothing pink, and the reuse washing is to neutral; Ether layer evaporate to dryness, residue is used dissolve with methanol, makes into 0.5ml, as control medicinal material solution; Other gets the panoxadiol, and panaxatriol's reference substance adds methanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-acetone of 5: 2 was developing solvent, launched, and took out; Dry; Spray is with 10% ethanol solution of sulfuric acid, after 105 ℃ of heating several minutes are clear to the speckle colour developing, put under daylight and the 365nm uviol lamp and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the fluorescence speckle of same color respectively;

B, get this pharmaceutical composition that is equivalent to crude drug 7.8g, add 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour is put coldly, filters, and evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as need testing solution; In addition extracting liquorice control medicinal material 0.5g adds 3ml hydrochloric acid and 20ml chloroform, reflux 1 hour, put cold, filtration, evaporate to dryness, residue add anhydrous alcohol solution makes into 1ml, as control medicinal material solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution again; Test according to thin layer chromatography; Draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent, with the sodium carboxymethyl cellulose with 5: 10: 4: petroleum ether-benzene of 0.6-ethyl acetate-glacial acetic acid is developing solvent; Launch, take out, dry; Spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to the speckle colour developing at 110 ℃, puts under the daylight and inspects; In the test sample chromatograph, on control medicinal material and the corresponding position of reference substance chromatograph, show the same color speckle respectively;

C, get this pharmaceutical composition that is equivalent to crude drug 39g, extract volatile oil,, obtain the ether layer, volatilize to 0.5ml, as need testing solution with a small amount of ether flushing volatile oil extractor with volatile oil extractor; Other gets Rhizoma Zingiberis control medicinal material 50g, is ground into the beans size, soaks 16 hours, extracts volatile oil with volatile oil extractor, draws 1 and drips 15 times of ether dilutions, as control medicinal material solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose; With benzene-ether of 9: 1 was developing solvent; Launch, take out, dry; Spray is heated several minutes to clear spot with 1% vanillin sulfuric acid solution at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

D, aconitine limit detect: get this pharmaceutical composition that is equivalent to crude drug 15.6g, add the ammonia solution moistening, soaked into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as need testing solution; Other takes by weighing Radix Aconiti Lateralis Preparata control medicinal material coarse powder 7.2g, adds the ammonia solution moistening, soaks into the 60ml supersound process that adds diethyl ether 20 minutes 2 hours; Close plug, placement is spent the night, and filters, with 0.5mol/L hydrochloric acid solution extraction 2 times; Each 20ml, combining extraction liquid is transferred pH 11-12 with strong caustic, with chloroform extraction 3 times; Combining extraction liquid, evaporate to dryness, residue add dehydrated alcohol 0.5ml dissolving, as control medicinal material solution; Precision takes by weighing the aconitine reference substance in addition, adds dehydrated alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Test according to thin layer chromatography; Accurate need testing solution and each 10 μ l of reference substance solution of drawing; Put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, be developing solvent with E-C-methanol of 1: 2: 1, launch to take out and to dry; Put in the iodine vapor smoked after, inspect under the daylight; In the test sample chromatograph, the speckle that on control medicinal material and the corresponding position of reference substance chromatograph, occurs respectively should or speckle not occur less than the speckle of reference substance.

5. according to claim 1 or claim 2 detection method is characterized in that said warming spleen and tonifying kidney, lets out the pharmaceutical composition of turbid blood stasis dispelling and is processed by following method: Rhizoma Zingiberis 240 weight portions are added 25 times of water gagings; Soak after 16 hours and distilled 6 hours; Extract volatile oil, the centrifugal filtration of distillate, other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600 weight portions, Radix Aconiti Lateralis Preparata 360 weight portions, Radix Ginseng 240 weight portions, Radix Glycyrrhizae 120 weight portions are with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation; When being concentrated into 50 ℃ of following relative densities and being 1.22-1.25, drying, it is subsequent use to be ground into fine powder; Volatile oil and subsequent use fine powder are processed conventional formulation.

6. detection method as claimed in claim 3 is characterized in that said warming spleen and tonifying kidney, lets out the pharmaceutical composition of turbid blood stasis dispelling and is processed by following method: Rhizoma Zingiberis 240 weight portions are added 25 times of water gagings; Soak after 16 hours and distilled 6 hours; Extract volatile oil, the centrifugal filtration of distillate, other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600 weight portions, Radix Aconiti Lateralis Preparata 360 weight portions, Radix Ginseng 240 weight portions, Radix Glycyrrhizae 120 weight portions are with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70% ethanol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation; When being concentrated into 50 ℃ of following relative densities and being 1.22-1.25, drying, it is subsequent use to be ground into fine powder; Volatile oil and subsequent use fine powder are processed conventional formulation.

7. detection method as claimed in claim 4 is characterized in that said warming spleen and tonifying kidney, lets out the pharmaceutical composition of turbid blood stasis dispelling and is processed by following method: Rhizoma Zingiberis 240 weight portions are added 25 times of water gagings; Soak after 16 hours and distilled 6 hours; Extract volatile oil, the centrifugal filtration of distillate, other device of filtrating is collected; Medicinal residues and Radix Et Rhizoma Rhei 600 weight portions, Radix Aconiti Lateralis Preparata 360 weight portions, Radix Ginseng 240 weight portions, Radix Glycyrrhizae 120 weight portions are with 7 times of amount 70% soak with ethanol 16 hours, and heating and refluxing extraction 3 times respectively adds 6 times of amount 70%Z alcohol the 2nd, 3 time; The each extraction 2 hours filters while hot, and filtrate recycling ethanol is to there not being the alcohol flavor; Merge with the aqueous solution after the distillation; When being concentrated into 50 ℃ of following relative densities and being 1.22-1.25, drying, it is subsequent use to be ground into fine powder; Volatile oil and subsequent use fine powder are processed conventional formulation.