CN102998410A

CN102998410A - Blood nourishing and brain arousing particle quality detection method

Info

Publication number: CN102998410A
Application number: CN2011102690690A
Authority: CN
Inventors: 孙玉侠; 高展; 刘彦莉; 曹凤兰
Original assignee: Tasly Pharmaceutical Group Co Ltd
Current assignee: Tianshili Northeast Modern Chinese Medicine Resources Co Ltd
Priority date: 2011-09-13
Filing date: 2011-09-13
Publication date: 2013-03-27
Anticipated expiration: 2031-09-13
Also published as: CN102998410B

Abstract

The invention relates to the medicinal field, and concretely relates to a blood nourishing and brain arousing particle quality detection method. The method comprises identification of ursolic acid in Spica Prunellae, identification of Rhizoma Corydalis, identification of Semen Cassiae, identification of Chinese angelica and Ligusticum wallichii, identification of rosmarinic acid in Spica Prunellae, identification of alkaloids in Ligusticum wallichii, identification of Levistilide in Ligusticum wallichii, identification of Millettia dielsiana, identification of white peony root, identification of prepared rehmannia root, identification of Gambir Plant, and determination of the content of an active ingredient paeoniflorin in the blood nourishing and brain arousing particle.

Description

A kind of quality determining method of blood-nourishing and brain-refreshing granules

Technical field

The present invention relates to field of medicaments, be specifically related to a kind of quality determining method of blood-nourishing and brain-refreshing granules

Background technology

Blood-nourishing and brain-refreshing granules is the modern Chinese herbal medicine preparation of Tianjin Tasly Pharmaceutical Co., Ltd development, and obtains national New Drug Certificate in 1996, and is put into National essential drugs list in 1999, is put into national medical insurance medicine product catalogue in 2000.Blood-nourishing and brain-refreshing granules is comprised of Radix Angelicae Sinensis, Ligusticum wallichii, the root of herbaceous peony, reticulate millettia, cassia seed, corydalis tuber etc. 11 flavor Chinese medicines, and after modern high tech method extracts, adds suitable auxiliary material, a kind of granule of making through preparation processes such as mixing granulations.It is fast that said preparation has an effective constituent stripping, the bioavilability advantages of higher.Blood-nourishing and brain-refreshing granules has the flat liver of nourishing blood, and effect promoting blood circulation and removing obstruction in channels can be used for the headache of deficiency of blood liver due to high, staggers, the illness such as dysphoria, insomnia and dreamful sleep, has clinically significant curative effect.

The blood-nourishing and brain-refreshing granules quality determining method adopts thin-layered chromatography and high performance liquid chromatography more at present.Thin-layered chromatography because economical, easy, intuitive is strong, it is the highest to be applied to compound Chinese medicinal preparation discriminating frequency, remains so far " the discriminating means that Chinese pharmacopoeia, " British Pharmacopoeia ", India herbal medicine allusion quotation, European Pharmacopoeia etc. are generally used.High performance liquid chromatography is multiplex in the assay of effective constituent, and it has the advantages such as applied widely, that separating property good, analysis speed is fast, the mobile phase range of choice is wide, highly sensitive, chromatographic column can repeatedly be used.

But existing detection method complicated operation, precision is not high, goes wrong easily in the actual mechanical process.

Summary of the invention

Technical matters to be solved

The invention provides a kind of quality determining method of blood-nourishing and brain-refreshing granules, solved some important technological problems in the present detection.

Technical scheme

Its prescription of blood-nourishing and brain-refreshing granules of the present invention is as follows:

Main ingredient: Radix Angelicae Sinensis, Ligusticum wallichii, the root of herbaceous peony, prepared rhizome of rehmannia, yncaria stem with hooks, reticulate millettia, selfheal, cassia seed, mother-of-pearl, corydalis tuber, the root of Chinese wild ginger.

Auxiliary material is: dextrin, Steviosin.

Quality determining method of the present invention comprises discrimination method and content assaying method.

For this reason, the present invention at first provides a kind of discrimination method of blood-nourishing and brain-refreshing granules, and the method comprises the discriminating to following composition:

Ursolic acid is differentiated in the A selfheal

The B corydalis tuber is differentiated

The C cassia seed is differentiated

D Radix Angelicae Sinensis and Ligusticum wallichii are differentiated

Rosmarinic acid is differentiated in the E selfheal

Alkaloid is differentiated in the F Ligusticum wallichii

Levistilide A is differentiated in the G Ligusticum wallichii

The H reticulate millettia is differentiated

The I root of herbaceous peony is differentiated

The J prepared rhizome of rehmannia is differentiated

The K yncaria stem with hooks is differentiated

Discrimination method of the present invention adopts thin-layered chromatography.

Secondly, the present invention comprises also the effective constituent in the blood-nourishing and brain-refreshing granules is carried out assay that wherein said effective constituent is Paeoniflorin.Content assaying method of the present invention adopts high performance liquid chromatography.

Discrimination method of the present invention has 11, can use wherein a conduct to the discrimination method of blood-nourishing and brain-refreshing granules, also can multinomially be used in combination as to the discrimination method of blood-nourishing and brain-refreshing granules, multinomial being used in combination preferably, most preferably 11 are used in combination.

Content assaying method of the present invention, chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; Take isopropyl alcohol-methyl alcohol-5mmol/L citric acid soln (2-4: 18-22: 78-80) as mobile phase; The detection wavelength is 230-240nm.Number of theoretical plate calculates by the Paeoniflorin peak should be not less than 2000.

Discrimination method of the present invention, preferably adopt following methods:

The A selfheal is differentiated (ursolic acid)

Get this product 0.5-2.0g, add watery hydrochloric acid 0.2-1.0ml and ether 10-30ml, add hot reflux 20-40 minute, extract is waved most ether, and residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Other gets the ursolic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1-1.0mg, in contrast product solution.Test according to thin-layered chromatography, draw need testing solution 5-15 μ l, reference substance solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate-chloroform-glacial acetic acid (15-25: 2-4: 5-9: 0.1-0.5) as developping agent, launch, take out, dry, spray is with the 5-15% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 100-120 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

The B corydalis tuber is differentiated

Get this product 0.5-1.5g, put in the tool plug centrifuge tube, add 0.1-0.5mol/L sodium hydroxide solution 2-6ml, jolting makes dissolving, add ethyl acetate 5-10ml, shake well centrifugal 2-5 minute, is got upper solution, evaporate to dryness, residue add methyl alcohol 1-2ml makes dissolving, as need testing solution.Other gets corydalis tuber control medicinal material 0.5-2.0g, adds ammonia solution (get strong ammonia solution 1ml, add water 10ml, mixing) 0.5-2.0ml, grind, and the 5-20ml that adds methylene chloride, temperature was soaked 1-3 hour, filtered, and filtrate is concentrated into about 0.5-2.0ml, in contrast medicinal material solution.Get again the tetrahydropalmatine reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1-1.0mg, in contrast product solution.According to the thin-layered chromatography test, draw each 5-15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane-chloroform-methanol-strong ammonia solution (5-20: 6-10: 1-3: 0.2-0.5) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color; With the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

The C cassia seed is differentiated

Depend on pine torch control medicinal material 0.5-1.5g, add watery hydrochloric acid 0.5-1.0ml, ether 10-20ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate is reclaimed ether to doing, and residue adds respectively methyl alcohol 0.5-2.0ml makes dissolving, as cassia seed control medicinal material solution.Get again Physcion, Chrysophanol, archen and aurantio-obtusin reference substance, add absolute ethyl alcohol-ethyl acetate (1-4: 0.5-2) make the solution that every 1ml contains 0.5-1.0mg, as mixing reference substance solution.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 10-20 μ l in the item, control medicinal material solution 7-15 μ l and mixing reference substance solution 2-4 μ l, put respectively on same high-efficient silica gel G thin layer plate, take sherwood oil (30～60 ℃)-cyclohexane-ethyl acetate-formic acid (3-9: 10-14: 3-7: 0.2-0.8) as developping agent, launch, take out, dry, place more than the smoked 10min of ammonia steam, put under the daylight and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

D Radix Angelicae Sinensis-Ligusticum wallichii is differentiated

Get respectively Radix Angelicae Sinensis, each 0.5-2.0g of Ligusticum wallichii control medicinal material, the 10-30ml that adds diethyl ether, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate is reclaimed ether to doing, and residue adds respectively ethyl acetate 0.5-2.0ml, 1.0-2.0ml makes dissolving, as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 5-12 μ l in the item, Radix Angelicae Sinensis control medicinal material solution 5-15 μ l and Ligusticum wallichii control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, take normal hexane-ethyl acetate-formic acid (5-9: 0.5-1.5: 0.2-0.8) as developping agent, launch to take out more than the 8cm, dry, place under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.

The E selfheal is differentiated (Rosmarinic acid)

Get the Rosmarinic acid reference substance, add Diluted Alcohol and make the solution that every 1ml contains 0.5-1.0mg, in contrast product solution.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 1-5 μ l, reference substance solution 1-4 μ l in the item, put respectively on same silica gel g thin-layer plate, take normal hexane-ethyl acetate-formic acid (1-3: 5-6: 0.2-0.5) as developping agent, launch, take out, dry, place under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

The F Ligusticum wallichii is differentiated (alkaloids)

Get this product 5-20g, add ammoniacal liquor 3-8ml, and ether 30-50ml, close plug left standstill 30-90 minute, glass bar stirs evenly, and water-bath backflow 20-40 minute is taken out, and lets cool, extract reclaims ether to doing, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Get Ligusticum wallichii control medicinal material 0.5-2.0g, add ammoniacal liquor 3-8ml, and ether 10-30ml, close plug left standstill 50-70 minute, water-bath backflow 20-40 minute is taken out, and lets cool, and filters, filtrate is reclaimed ether to doing, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as Ligusticum wallichii control medicinal material solution.Test according to thin-layered chromatography, draw respectively Ligusticum wallichii control medicinal material solution 7-10 μ l, need testing solution 7-12 μ l, put respectively on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate (7-11: 0.5-2) as developping agent, launch, take out, dry, solution-(1-4: 0.5-1) mixed solution places under the daylight and inspects the iodine liquor kalii iodide with the improvement bismuth potassium iodide in spray.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

The G Ligusticum wallichii is differentiated (Levistilide A)

Get this product 2-10g, add ether 20-60ml, water-bath backflow 20-60 minute is taken out, and lets cool, and extract reclaims ether to doing, and residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Other gets the Levistilide A reference substance, adds ethyl acetate and makes the solution that every 1ml contains 0.2-1.0mg, as the Levistilide A reference substance solution.Test according to thin-layered chromatography, draw respectively Levistilide A reference substance solution 2-6 μ l, need testing solution 6-10 μ l, put respectively on same silica GF254 thin layer plate, take normal hexane-ethyl acetate (1-5: 0.5-2) as developping agent, launch, take out, dry, place under the ultraviolet lamp (254nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

The H reticulate millettia is differentiated

Get this product 5-20g, add 0.1-0.5% sodium hydroxide solution 20-50ml, shake up, ultrasonic processing 20-40min, take out centrifugal (2000-6000 rev/min, 3-10 minute), get supernatant, with sherwood oil (60～90 ℃) extraction 1-3 time, each 10-20ml discards petroleum ether layer, the water intaking layer adds 0.3-0.6ml watery hydrochloric acid, shake up, use extracted with diethyl ether 1-3 time, each 10-20ml, merge the extracted with diethyl ether layer, reclaim ether to doing, residue adds methyl alcohol 0.5-2.0ml dissolving, adds silica gel 1-2g and mixes thoroughly, volatilize solvent, put silicagel column (100～200 orders, 1-3g, internal diameter 1cm, dry column-packing) on, with methyl alcohol-methenyl choloride (0.5-1.5: 7-11) 30-50ml wash-out, collection methyl alcohol-methenyl choloride (0.5-1.5: 7-11) eluent, evaporate to dryness, residue adds methenyl choloride 0.5-1.5ml makes dissolving, as need testing solution.It is an amount of that other gets onocerin, adds methyl alcohol and make the solution that every 1ml contains 0.1-0.5mg, in contrast product solution.Test according to thin-layered chromatography, draw respectively onocerin reference substance solution 2-5 μ l, need testing solution 5-10 μ l, put respectively on same silica gel g thin-layer plate, take methenyl choloride-methyl alcohol (10-40: 1) as developping agent, launch more than the 8cm, take out, dry, place under the ultraviolet lamp (254nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

The I root of herbaceous peony is differentiated

Test according to thin-layered chromatography, draw respectively need testing solution 20-30 μ l and Paeoniflorin reference substance solution 10-20 μ l in the assay item, put respectively on same silica gel g thin-layer plate, take methenyl choloride-ethyl acetate-methyl alcohol-formic acid (20-50: 3-7: 5-15: 0.2) as developping agent, launch, take out, dry, spray is with 3-5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to colour developing at 100-120 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

The J prepared rhizome of rehmannia is differentiated

Get this product 15-20g, add water 50-100ml, in 50-60 ℃ of warm 20-50 minute, take out, ultrasonic processing 20-40 minute, centrifugal, get supernatant, use ethyl acetate extraction 1-3 time, each 20-40ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue add ethyl acetate 3-10ml makes dissolving, adds neutral alumina 2-4g, mix thoroughly, the neutral alumina column (10-15g, internal diameter 15mm, 70～100 orders) of packing into, with ethanol 20-50ml wash-out, collect eluent, evaporate to dryness, residue add ethanol 0.5-2.0ml, make dissolving, as need testing solution.Other gets prepared rhizome of rehmannia control medicinal material 2-5g, adds water 30-80ml and decocts 0.5-2.0 hour, filters, and filtrate is used ethyl acetate extraction 1-3 time, each 20-40ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml, makes dissolving, in contrast medicinal material solution.According to the thin-layered chromatography test, draw respectively need testing solution 20-25 μ l and control medicinal material solution 10-20 μ l, put respectively on same silica gel g thin-layer plate, take toluene-ethyl acetate (0.5-2: 1) as developping agent, launch, take out, dry, spray is with the DNPH ethanolic solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

The K yncaria stem with hooks is differentiated

Get this product 5-20g, add strong ammonia solution 5-15ml and make infiltration, close plug 20-40 minute, add ether 50-100ml, add hot reflux 0.5-2.0 hour, let cool, divide and get ether layer, extract 1-3 time with the jolting of 3-8% hydrochloric acid solution, each 10-30ml, merge the 3-8% hydrochloric acid solution, regulate pH value to 9 with ammoniacal liquor, extract 1-3 time with the ether jolting, each 10-30ml merges ether solution, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Hook taking rattan control medicinal material 1-3g adds strong ammonia solution 2-5ml and infiltrates in addition, close plug 20-40 minute, and the 20-50ml that adds diethyl ether adds hot reflux 0.5-2.0 hour, lets cool, and filters, and the filtrate evaporate to dryness adds methyl alcohol 0.5-2.0ml and makes dissolving, in contrast medicinal material solution.Test according to thin-layered chromatography, draw each 5-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene-methylene chloride-ethyl acetate-methyl alcohol-ammoniacal liquor (6-10: 10-14: 1-3: 1-3: 0.5-0.8) as developping agent, launch, take out, dry, and improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1-4: 0.5-1) mixed solution, in the test sample chromatogram, with control medicinal material chromatogram relevant position on, the principal spot of aobvious same color.

Content assaying method of the present invention, preferably adopt following methods:

Content with the high effective liquid chromatography for measuring Paeoniflorin

High-efficient liquid phase chromatogram condition: be filling agent with octadecylsilane chemically bonded silica; Take isopropyl alcohol-methyl alcohol-5mmol/L citric acid soln (2-4: 18-22: 78-80) as mobile phase; The detection wavelength is 230-240nm.Number of theoretical plate calculates by the Paeoniflorin peak should be not less than 2000.

The preparation of reference substance solution: it is an amount of to get the Paeoniflorin reference substance, accurately weighed, adds 70-90% methyl alcohol and makes the solution that every 1ml contains 15-25 μ g, and get final product.

The internal memory thing under this product content uniformity item is got in the preparation of need testing solution, and porphyrize is got about 0.06-0.10g, accurately weighed, put in the 20-50ml beaker, add sodium bicarbonate solution 5ml, ultrasonic processing is added on the D101 type large pore resin absorption column, uses the 10-20ml water elution, discard eluent, use again the 6-10ml methanol-eluted fractions, collect eluent in the 10ml measuring bottle, add water to scale, shake up, centrifugal 4-6 minute, get supernatant, and get final product.

Determination method is accurate reference substance solution and each 5-15 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product.

The most preferred content assaying method of the present invention is as follows:

It is an amount of that the Paeoniflorin reference substance is got in the preparation of reference substance solution, accurately weighed, adds 80% methyl alcohol and make the solution that every 1ml contains 20 μ g, and get final product.

The internal memory thing under this product content uniformity item is got in the preparation of need testing solution, porphyrize is got about 0.08g, and is accurately weighed, put in the 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic processing (power 500W, frequency 30kHz) 5 minutes, be added in D101 type large pore resin absorption column (60～80 orders, internal diameter 6～8mm, high about 5cm) on, water 15ml wash-out, discard eluent, use again methyl alcohol 8ml wash-out, collect eluent in the 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, and get final product.

Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product.

Beneficial effect

Among the present invention, but TLC method qualitative detection goes out Radix Angelicae Sinensis in the blood-nourishing and brain-refreshing granules, Ligusticum wallichii, reticulate millettia, cassia seed, selfheal, yncaria stem with hooks, the root of herbaceous peony, corydalis tuber and prepared rhizome of rehmannia, and the method is simple, stable, can be used as the qualitative checking method of blood-nourishing and brain-refreshing granules.The HPLC method can be measured the content of Paeoniflorin in the blood-nourishing and brain-refreshing granules, and method is easy, repeatability and good stability, and the result can be used as the detection method of content of blood-nourishing and brain-refreshing granules accurately and reliably.

More different flow phase system in the test, find that isopropyl alcohol-methyl alcohol-aqueous solution of citric acid system can make that Paeoniflorin is well separated in this product, and select through different proportion of mobile phase, so the present invention has finally selected isopropyl alcohol-methyl alcohol-5mmol/L aqueous solution of citric acid (2-4: 20-22: 78-80) as mobile phase.

Description of drawings

Fig. 1: Qingdao silica G plate, T=22 ℃, RH=41%

From left to right: sample (lot number: 2011C10); Sample (lot number: 2011C11); Sample (lot number: 2011C12);

The ursolic acid reference substance

Fig. 2: Qingdao silica G plate, T=22 ℃, RH=41%

From left to right: sample (lot number: 2010F27); Sample (lot number: 2010F28); The tetrahydropalmatine reference substance (middle inspection institute, 110726-201011); The corydalis tuber control medicinal material (middle inspection institute, 120928-200604); Sample (lot number: 2010F29);

Fig. 3: Yantai HSG, T=24 ℃; RH=27%

From left to right: negative sample solution; The cassia seed control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03); Test sample (lot number: 2010B04);

Test sample (lot number: 2010B05); Test sample (lot number: 2010B06); Test sample (lot number: 2010B07);

Test sample (lot number: 2010B08); Test sample (lot number: 2010B09); Test sample (lot number: 2010B10);

The Physcion reference substance; The Chrysophanol reference substance; The archen reference substance; Aurantio-obtusin reference substance Fig. 4: Yantai silica G plate, T=21 ℃; RH=23%

From left to right: blank sample solution; The Radix Angelicae Sinensis control medicinal material; Need testing solution (lot number: 2009J23); Need testing solution (lot number: 2009K25); Need testing solution (lot number: 2009L03); Ligusticum wallichii control medicinal material Fig. 5: Yantai silica G plate, T=25 ℃, RH=66%

From left to right: negative sample; The Rosmarinic acid reference substance; Sample (lot number: 2010D01); Sample (lot number: 2010D02); Sample (lot number: 2010D03)

Fig. 6: Yantai silica G plate, T=26 ℃, RH=50%

From left to right: negative (lacking Ligusticum wallichii); The Radix Angelicae Sinensis control medicinal material; Test sample (lot number: 2009J23); Test sample (lot number: 2009L02); Test sample (lot number: 2009L03); Ligusticum wallichii control medicinal material 10 μ l;

Fig. 7: Yantai gel GF 254 plate, T=24 ℃, RH=52%

From left to right: feminine gender; Levistilide A; Test sample 2009J23; Test sample 2009L02; Test sample 2009L03 Fig. 8: Qingdao silica G plate, T=27 ℃, RH=48%

From left to right: feminine gender; Onocerin; Test sample 2009J23; Test sample 2009K25; Test sample 2010C30;

Fig. 9: Yantai silica G plate, T=25 ℃, RH=36%

From left to right: negative sample solution; The Paeoniflorin reference substance; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03); Test sample (lot number: 2010B04); Test sample (lot number: 2010B05); Test sample (lot number: 2010B06); Test sample (lot number: 2010B07);

Figure 10: Yantai silica G plate, T=25 ℃, RH=47%

From left to right: negative sample solution; The prepared rhizome of rehmannia control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03);

Figure 11: Yantai silica G plate, T=22 ℃; RH=28%

From left to right: negative sample solution; The yncaria stem with hooks control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03);

Figure 12: the HPLC collection of illustrative plates of measuring paeoniflorin content

Embodiment

1 instrument and reagent

Waters 2695 high performance liquid chromatographs, Waters 2996PDA detecting device, Empower2 workstation; The Milli-Q ultrapure water machine.

Paeoniflorin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 110736-200731), forulic acid reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 0773-9910), Radix Angelicae Sinensis control medicinal material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 120927-200310), Ligusticum wallichii control medicinal material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 120918-200809), Rosmarinic acid reference substance (Tianjin one side Science and Technology Ltd.), the selfheal control medicinal material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 120993-200604), cassia seed control medicinal material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 121544-200501), the Chrysophanol reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 110796-200311), aurantio-obtusin (Tianjin one side Science and Technology Ltd.), onocerin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 111703-200501), and the prepared rhizome of rehmannia control medicinal material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 121196-200803)

Methyl alcohol, tetrahydrofuran are chromatographically pure, and it is pure that all the other reagent are analysis.

2 methods and result

Embodiment 1

Selfheal is differentiated (ursolic acid)

The preparation of need testing solution: get this product 1g, add watery hydrochloric acid 0.5ml and ether 20ml, added hot reflux 30 minutes, extract is waved most ether, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution.

The preparation of reference substance solution: get the ursolic acid reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1mg, in contrast product solution.

The selection of developping agent: prepared respectively 20: 3: 7: 0.2 ratio, 15: 2: 5: 0.1 ratio, 25: 4: 9: the cyclohexane-ethyl acetate of 0.5 ratio-chloroform-glacial acetic acid was developping agent.

According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, to dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.See figure (1-1).Wherein take 20: 3: 7: the cyclohexane-ethyl acetate of 0.2 ratio-chloroform-glacial acetic acid as the developping agent spot the most obvious.

Embodiment 2

Corydalis tuber is differentiated

The preparation of need testing solution: get this product 1g, put in the tool plug centrifuge tube, add 0.2mol/L sodium hydroxide solution 4ml, jolting makes dissolving, add ethyl acetate 5ml, shake well centrifugal 2 minutes, is got upper solution, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution.

The preparation of control medicinal material solution: corydalis tuber control medicinal material 0.5g, add ammonia solution (get strong ammonia solution 1ml, add water 10ml, mixing) 0.5ml, grind, the 10ml that adds methylene chloride, temperature was soaked 2 hours, filtered, and filtrate is concentrated into about 1ml, in contrast medicinal material solution.

The preparation of reference substance solution: get the tetrahydropalmatine reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1mg, in contrast product solution.

The selection of developping agent: prepared respectively 15: 8: 2: 0.2 ratio, 5: 6: 1: 0.2 ratio, 20: 10: 3: the normal hexane-chloroform-methanol of 0.5 ratio-strong ammonia solution was developping agent.

According to the thin-layered chromatography test, draw each 10 μ l of need testing solution, control medicinal material solution and reference substance solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, to dry, spray is with rare bismuth potassium iodide test solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color; With the corresponding position of reference substance chromatogram on, the spot of aobvious same color.See figure (2-1).Wherein take 15: 8: 2: the normal hexane-chloroform-methanol of 0.2 ratio-strong ammonia solution as the developping agent spot the most obvious.

Embodiment 3

Cassia seed is differentiated

The preparation of need testing solution: get this product 2g, add watery hydrochloric acid 1ml and ether 20ml, added hot reflux 30 minutes, extract is waved most ether, and residue adds methyl alcohol 1ml makes dissolving, and get final product.

The preparation of blank solution: get the blank pharmaceutical preparation that lacks cassia seed, make blank solution according to the preparation method of need testing solution.

The preparation of control medicinal material: depend on pine torch control medicinal material 1g, add watery hydrochloric acid 0.5ml, ether 20ml, water-bath refluxed 1 hour, took out, and let cool, and filtered, and filtrate is reclaimed ether to doing, and residue adds respectively methyl alcohol 1ml makes dissolving, and get final product.

The preparation of reference substance solution: get Chrysophanol reference substance, archen reference substance, Physcion reference substance and aurantio-obtusin, add absolute ethyl alcohol-ethyl acetate (2: 1) and make the solution that every 1ml contains 0.5mg, in contrast product solution.

The selection of developping agent: prepared respectively 6: 12: 5: 0.5 ratio, 3: 10: 3: 0.2 ratio, 9: 14: 7: the sherwood oil of 0.8 ratio (30～60 ℃)-cyclohexane-ethyl acetate-formic acid was that developping agent is according to the thin-layered chromatography test, draw need testing solution 10 μ l, control medicinal material solution 7 and mixing reference substance solution 2 μ l, put respectively on same high-efficient silica gel G thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, place more than the smoked 10min of ammonia steam, put under the daylight and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.See figure (3-3).Wherein take 6: 12: 5: the sherwood oil of 0.5 ratio (30～60 ℃)-cyclohexane-ethyl acetate-formic acid as the developping agent spot the most obvious.

Embodiment 4

Radix Angelicae Sinensis-Ligusticum wallichii is differentiated

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, add watery hydrochloric acid 1ml and ether 20ml, mixing, 60 ℃ of heating water baths refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds methyl alcohol 0.5ml dissolving, and get final product.

The preparation of control medicinal material solution: get respectively Radix Angelicae Sinensis control medicinal material, each 1g of Ligusticum wallichii control medicinal material, the 20ml that adds diethyl ether, water-bath refluxed 1 hour, take out, let cool, filter, filtrate is reclaimed ether to doing, and residue adds respectively ethyl acetate 1ml makes dissolving, respectively as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution.

The preparation of blank solution: by the prescription proportioning, get except Radix Angelicae Sinensis, Ligusticum wallichii other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepare respectively 9: 1: 0.5 ratios, 5: 0.5: 0.2 ratios, the normal hexane-ethyl acetate of 9: 1.5: 0.8 ratios-formic acid is developping agent.

Test according to thin-layered chromatography, draw and differentiate need testing solution 10 μ l in (1) item, Radix Angelicae Sinensis control medicinal material solution 10 μ l and Ligusticum wallichii control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch to take out more than the 10cm, dry, place under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.See figure (4-1).Wherein the most obvious take the normal hexane-ethyl acetate of 9: 1: 0.5 ratios-formic acid as the developping agent spot.

Embodiment 5

Selfheal is differentiated (Rosmarinic acid)

The preparation of reference substance solution: get Rosmarinic acid reference substance (Tianjin one side Science and Technology Ltd., content is 98%), add Diluted Alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.

The preparation of blank solution: by the prescription proportioning, get except selfheal other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepare respectively 2: 4: 0.2 ratios, 1: 5: 0.2 ratio, the normal hexane-ethyl acetate of 3: 6: 0.5 ratios-formic acid is developping agent.

According to the thin-layered chromatography test, draw need testing solution 5 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, place under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

See figure (5-1).Wherein the most obvious take the normal hexane-ethyl acetate of 2: 4: 0.2 ratios-formic acid as the developping agent spot.

Embodiment 6

Ligusticum wallichii is differentiated (alkaloids)

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add ammoniacal liquor 5ml, and ether 40ml, close plug left standstill 60 minutes, and glass bar stirs evenly, and water-bath refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds absolute ethyl alcohol 1ml makes dissolving, and get final product.

The preparation of control medicinal material solution: get Ligusticum wallichii control medicinal material 1g, add ammoniacal liquor 5ml, and ether 20ml, close plug left standstill 60 minutes, water-bath refluxed 30 minutes, took out, and let cool, and filtered, filtrate is reclaimed ether to doing, and residue adds absolute ethyl alcohol 1ml makes dissolving, and get final product, as Ligusticum wallichii control medicinal material solution.

The preparation of blank solution: by the prescription proportioning, get except Ligusticum wallichii other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepare respectively 9: 1 ratios, 7: 0.5 ratios, the cyclohexane-ethyl acetate of 11: 2 ratios is developping agent.

Test according to thin-layered chromatography, draw respectively Ligusticum wallichii control medicinal material solution 10 μ l, need testing solution 12 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray places under the daylight and inspects with improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.See figure (6-1).Wherein the most obvious take the cyclohexane-ethyl acetate of 9: 1 ratios as the developping agent spot.

Embodiment 7

Ligusticum wallichii is differentiated (Levistilide A)

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 4g, add ether 40ml, water-bath refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds methyl alcohol 1ml makes dissolving, and get final product.

The preparation of reference substance solution: get Levistilide A reference substance 20mg, place the brown volumetric flask of 50ml, add acetic acid ethyl dissolution and be settled to scale, shake up, and get final product, as the Levistilide A reference substance solution.

The selection of developping agent: prepare respectively 3: 1 ratios, 1: 0.5 ratio, the normal hexane-ethyl acetate of 5: 2 ratios is developping agent.

According to the thin-layered chromatography test, draw respectively Levistilide A reference substance solution 6 μ l, need testing solution 10 μ l, put respectively on same silica GF254 thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, place under the ultraviolet lamp (254nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.See figure (7-1).Wherein the most obvious take the normal hexane-ethyl acetate of 3: 1 ratios as the developping agent spot.

Embodiment 8

Reticulate millettia is differentiated

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add 0.2% sodium hydroxide solution 30ml, shake up, ultrasonic processing 30min takes out centrifugal (2000 rev/mins, 5 minutes), get supernatant, with sherwood oil (60～90 ℃) extraction 2 times, each 15ml discards petroleum ether layer, and the water intaking layer adds 0.4ml watery hydrochloric acid, shake up, use extracted with diethyl ether 3 times, each 15ml, merge the extracted with diethyl ether layer, reclaim ether to doing, residue adds methyl alcohol 2ml dissolving, adds silica gel 1g and mixes thoroughly, volatilize solvent, put silicagel column (100～200 orders, 2g, internal diameter 1cm, dry column-packing) on, with methyl alcohol-methenyl choloride (1: 9) 40ml wash-out, collect methyl alcohol-methenyl choloride (1: 9) eluent, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, and get final product.

The preparation of reference substance solution: it is an amount of that precision takes by weighing onocerin, adds methyl alcohol and make the solution that every 1ml contains 0.1mg, in contrast product solution.

The preparation of blank solution: by the prescription proportioning, get except reticulate millettia other medicinal materials and make negative sample in accordance with the law, make negative sample solution according to the need testing solution preparation method again.

The selection of developping agent: 30: 1 ratios, 10: 1 ratios, the methenyl choloride-methyl alcohol of 40: 1 ratios is developping agent.

According to the thin-layered chromatography test, draw respectively onocerin reference substance solution 2～5 μ l, need testing solution 5～10 μ l, put respectively on same silica gel g thin-layer plate, in above-mentioned developping agent condition, launch 12cm, take out, dry, place under the ultraviolet lamp (254nm) and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.See figure (8-1).Wherein the most clear take the methenyl choloride-methyl alcohol of 30: 1 ratios as the developping agent spot.

Embodiment 9

The root of herbaceous peony is differentiated

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize is got about 0.08g, and is accurately weighed, put in the 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic processing (power 500W, frequency 30kHz) 5 minutes, be added on the D101 type large pore resin absorption column (60～80 orders, internal diameter 6～8mm, high about 5cm), water 15ml wash-out discards eluent, uses methyl alcohol 8ml wash-out again, collect eluent in the 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, and get final product.

The preparation of reference substance solution: it is an amount of to get the Paeoniflorin reference substance, accurately weighed, adds 80% methyl alcohol and makes the solution that every 1ml contains 20 μ g, and get final product.

The preparation of blank solution: by the prescription proportioning, get except the root of herbaceous peony other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepared respectively 40: 5: 10: 0.2 ratio, 20: 3: 5: 0.2 ratio, 50: 7: 15: the methenyl choloride-ethyl acetate of 0.2 ratio-methyl alcohol-formic acid was developping agent.

Test according to thin-layered chromatography, draw respectively need testing solution 20 μ l and Paeoniflorin reference substance solution 10 μ l in the assay item, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.See figure (9-1).Wherein take 40: 5: 10: the methenyl choloride-ethyl acetate of 0.2 ratio-methyl alcohol-formic acid as the developping agent spot the most obvious.

Embodiment 10

Prepared rhizome of rehmannia is differentiated

The preparation of need testing solution: get this product 20g, add water 100ml, warm 30 minutes in 60 ℃, take out, ultrasonic processing 30 minutes, centrifugal, get supernatant, use ethyl acetate extraction 2 times, each 30ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue add ethyl acetate 5ml makes dissolving, adds neutral alumina 3g, mix thoroughly, the neutral alumina column (10g, internal diameter 15mm, 70～100 orders) of packing into, with ethanol 30ml wash-out, collect eluent, evaporate to dryness, residue add ethanol 1ml, make dissolving, as need testing solution.

The preparation of control medicinal material solution: get prepared rhizome of rehmannia control medicinal material 4g, add water 50ml and decocted 1 hour, filter, filtrate is used ethyl acetate extraction 2 times, each 30ml, and the combined ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 1ml, make dissolving, in contrast medicinal material solution.

The preparation of blank solution: by the prescription proportioning, get except prepared rhizome of rehmannia other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepare respectively 1: 1 ratio, 0.5: 1 ratio, the toluene-ethyl acetate of 2: 1 ratios is developping agent.

According to the thin-layered chromatography test, draw respectively need testing solution 20 μ l and control medicinal material solution 10 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, to dry, spray is with the DNPH ethanolic solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.See figure (10-1).Wherein the most obvious take the toluene-ethyl acetate of 1: 1 ratio as the developping agent spot.

Embodiment 11

Yncaria stem with hooks is differentiated

The preparation of need testing solution: get this product 10g, add strong ammonia solution 10ml and make infiltration, close plug 30 minutes, add ether 100ml, added hot reflux 1 hour, let cool, divide and get ether layer, extract 2 times with 5% hydrochloric acid solution jolting, each 20ml, merge 5% hydrochloric acid solution, regulate pH value to 9 with ammoniacal liquor, extract 2 times with the ether jolting, each 20ml merges ether solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.

The preparation of control medicinal material solution: hook taking rattan control medicinal material 2g, add strong ammonia solution 3ml and infiltrate, close plug 30 minutes, the 40ml that adds diethyl ether added hot reflux 1 hour, let cool, and filtered, and the filtrate evaporate to dryness adds methyl alcohol 1ml and makes dissolving, in contrast medicinal material solution.

The preparation of blank solution: by the prescription proportioning, get except yncaria stem with hooks other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again.

The selection of developping agent: prepared respectively 8: 12: 2: 2: 0.5 ratios, 6: 10: 1: 1: 0.5 ratio, 10: 14: 3: the toluene-methylene chloride of 3: 0.8 ratios-ethyl acetate-methyl alcohol-ammoniacal liquor was developping agent.Test according to thin-layered chromatography, draw each 10 μ l of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution is in the test sample chromatogram, with control medicinal material chromatogram relevant position on, the principal spot of aobvious same color.See figure (11-1).Wherein take 8: 12: 2: the toluene-methylene chloride of 2: 0.5 ratios-ethyl acetate-methyl alcohol-ammoniacal liquor as the developping agent spot the most obvious.

Embodiment 12

Chromatographic condition and system suitability test are take octadecylsilane chemically bonded silica as filling agent; Take isopropyl alcohol-methyl alcohol-5mmol/L aqueous solution of citric acid (2: 22: 78) as mobile phase; The detection wavelength is 240nm; 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Paeoniflorin peak should be not less than 2000.

The about 10mg of Paeoniflorin reference substance is got in the preparation of reference substance solution, and is accurately weighed, puts in the 50ml volumetric flask, adds the dissolving of 70% methyl alcohol and is diluted to scale, shakes up.Precision is measured above-mentioned Paeoniflorin reference substance solution 5ml again, puts in the 50ml volumetric flask, adds 70% methyl alcohol and is diluted to scale, shakes up, and get final product.

It is an amount of that this product is got in the preparation of need testing solution, and porphyrize is got about 0.08g, accurately weighed, put in the 10ml volumetric flask, add 0.2% sodium bicarbonate solution 5ml, ultrasonic processing (power 500W, frequency 30kHz) 10 minutes is added in D101 type large pore resin absorption column (60～80 orders, internal diameter 6～8mm, high about 5cm) on, water 15ml wash-out, discard eluent, use again methyl alcohol 8ml wash-out, collect eluent and in the 10ml measuring bottle, add water to scale, shake up, cross 0.45 μ m miillpore filter, and get final product.

Embodiment 13

Chromatographic condition and system suitability test are take octadecylsilane chemically bonded silica as filling agent; Take isopropyl alcohol-methyl alcohol-5mmol/L aqueous solution of citric acid (4: 20: 80) as mobile phase; The detection wavelength is 230nm; 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Paeoniflorin peak should be not less than 2000.

Claims

1. the quality determining method of a blood-nourishing and brain-refreshing granules is characterized in that, comprises the discriminating to following composition:

Ursolic acid is differentiated in the A selfheal

The B corydalis tuber is differentiated

The C cassia seed is differentiated

D Radix Angelicae Sinensis and Ligusticum wallichii are differentiated

Rosmarinic acid is differentiated in the E selfheal

Alkaloid is differentiated in the F Ligusticum wallichii

Levistilide A is differentiated in the G Ligusticum wallichii

The H reticulate millettia is differentiated

The I root of herbaceous peony is differentiated

The J prepared rhizome of rehmannia is differentiated

The K yncaria stem with hooks is differentiated.

2. according to claim 1 quality determining method is characterized in that, discrimination method adopts thin-layered chromatography.

3. according to claim 1 quality determining method is characterized in that, also comprises the effective constituent Paeoniflorin in the blood-nourishing and brain-refreshing granules is carried out assay, and content assaying method adopts high performance liquid chromatography.

4. according to claim 1 quality determining method is characterized in that, uses wherein a conduct to the discrimination method of blood-nourishing and brain-refreshing granules, or multinomial being used in combination as the discrimination method to blood-nourishing and brain-refreshing granules.

5. according to claim 4 quality determining method is characterized in that, is that 11 discrimination methods are used in combination.

6. according to claim 5 quality determining method is characterized in that, is that 11 discrimination methods are used in combination, and simultaneously the effective constituent Paeoniflorin in the blood-nourishing and brain-refreshing granules is carried out assay.

7. according to claim 1 quality determining method is characterized in that, ursolic acid is differentiated in the described selfheal, and step is as follows:

Get this product 0.5-2.0g, add watery hydrochloric acid 0.2-1.0ml and ether 10-30ml, add hot reflux 20-40 minute, extract is waved most ether, residue adds methyl alcohol 0.5-2.0ml makes dissolving, and as need testing solution, other gets the ursolic acid reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1-1.0mg, product solution according to the thin-layered chromatography test, is drawn need testing solution 5-15 μ l in contrast, reference substance solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate-chloroform-glacial acetic acid (15-25: 2-4: 5-9: 0.1-0.5) as developping agent, launch, take out, dry, spray is with the 5-15% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 100-120 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

Described corydalis tuber differentiates that step is as follows:

Get this product 0.5-1.5g, put in the tool plug centrifuge tube, add 0.1-0.5mol/L sodium hydroxide solution 2-6ml, jolting makes dissolving, adds ethyl acetate 5-10ml, shake well, centrifugal 2-5 minute, get upper solution, evaporate to dryness, residue adds methyl alcohol 1-2ml makes dissolving, and as need testing solution, other gets corydalis tuber control medicinal material 0.5-2.0g, add ammonia solution 0.5-2.0ml, grind, 5-20ml adds methylene chloride, temperature was soaked 1-3 hour, filter, filtrate is concentrated into about 0.5-2.0ml, in contrast medicinal material solution, get again the tetrahydropalmatine reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1-1.0mg, product solution is tested according to thin-layered chromatography in contrast, draw each 5-15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane-chloroform-methanol-strong ammonia solution (5-20: 6-10: 1-3: 0.2-0.5) as developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color; With the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

Described cassia seed differentiates that step is as follows:

Depend on pine torch control medicinal material 0.5-1.5g, add watery hydrochloric acid 0.5-1.0ml, ether 10-20ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate is reclaimed ether to doing, residue adds respectively methyl alcohol 0.5-2.0ml makes dissolving, as cassia seed control medicinal material solution, get again Physcion, Chrysophanol, archen and aurantio-obtusin reference substance, add absolute ethyl alcohol-ethyl acetate (1-4: 0.5-2) make the solution that every 1ml contains 0.5-1.0mg, as mixing reference substance solution, test according to thin-layered chromatography, draw " ursolic acid in the selfheal " and differentiate a lower need testing solution 10-20 μ l, control medicinal material solution 7-15 μ l and mixing reference substance solution 2-4 μ l, put respectively on same high-efficient silica gel G thin layer plate, take sherwood oil (30～60 ℃)-cyclohexane-ethyl acetate-formic acid (3-9: 10-14: 3-7: 0.2-0.8) as developping agent, launch, take out, dry, place more than the smoked 10min of ammonia steam, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

Described Radix Angelicae Sinensis and Ligusticum wallichii differentiate that step is as follows:

Get respectively Radix Angelicae Sinensis, each 0.5-2.0g of Ligusticum wallichii control medicinal material, 10-30ml adds diethyl ether, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate is reclaimed ether to doing, residue adds respectively ethyl acetate 0.5-2.0ml, 1.0-2.0ml make dissolving, as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution according to the thin-layered chromatography test, is drawn " ursolic acid in the selfheal " and is differentiated a lower need testing solution 5-12 μ l, Radix Angelicae Sinensis control medicinal material solution 5-15 μ l and Ligusticum wallichii control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, take normal hexane-ethyl acetate-formic acid (5-9: 0.5-1.5: 0.2-0.8) as developping agent, launch more than the 8cm, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color;

Rosmarinic acid is differentiated in the described selfheal, and step is as follows:

Get the Rosmarinic acid reference substance, add Diluted Alcohol and make the solution that every 1ml contains 0.5-1.0mg, product solution in contrast, test according to thin-layered chromatography, draw " ursolic acid in the selfheal " and differentiate lower need testing solution 1-5 μ l, a reference substance solution 1-4 μ l, put respectively on same silica gel g thin-layer plate, take normal hexane-ethyl acetate-formic acid (1-3: 5-6: 0.2-0.5) as developping agent, launch, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Alkaloid is differentiated in the described Ligusticum wallichii, and step is as follows:

Get this product 5-20g, add ammoniacal liquor 3-8ml, and ether 30-50ml, close plug left standstill 30-90 minute, glass bar stirs evenly, water-bath backflow 20-40 minute, take out, let cool, extract reclaims ether to doing, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, get Ligusticum wallichii control medicinal material 0.5-2.0g, add ammoniacal liquor 3-8ml, and ether 10-30ml, close plug left standstill 50-70 minute, water-bath backflow 20-40 minute is taken out, and lets cool, filter, filtrate is reclaimed ether to doing, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as Ligusticum wallichii control medicinal material solution, test according to thin-layered chromatography, draw respectively Ligusticum wallichii control medicinal material solution 7-10 μ l, need testing solution 7-12 μ l puts respectively on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate (7-11: 0.5-2) as developping agent, launch, take out, dry, spray is with improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1-4: 0.5-1) mixed solution, place under the daylight and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

Levistilide A is differentiated in the described Ligusticum wallichii, and step is as follows:

Get this product 2-10g, add ether 20-60ml, water-bath backflow 20-60 minute, take out, let cool, extract reclaims ether to doing, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, other gets the Levistilide A reference substance, adds ethyl acetate and makes the solution that every 1ml contains 0.2-1.0mg, as the Levistilide A reference substance solution, according to the thin-layered chromatography test, draw respectively Levistilide A reference substance solution 2-6 μ l, need testing solution 6-10 μ l puts respectively on same silica GF254 thin layer plate, take normal hexane-ethyl acetate (1-5: 0.5-2) as developping agent, launch, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Described reticulate millettia differentiates that step is as follows:

Get this product 5-20g, add 0.1-0.5% sodium hydroxide solution 20-50ml, shake up, ultrasonic processing 20-40min, take out, centrifugal, get supernatant, with sherwood oil (60～90 ℃) extraction 1-3 time, each 10-20ml, discard petroleum ether layer, the water intaking layer adds 0.3-0.6ml watery hydrochloric acid, shakes up, and uses extracted with diethyl ether 1-3 time, each 10-20ml, merge the extracted with diethyl ether layer, reclaim ether to doing, residue adds methyl alcohol 0.5-2.0ml dissolving, adding silica gel 1-2g mixes thoroughly, volatilize solvent, put on the silicagel column, with methyl alcohol-methenyl choloride (0.5-1.5: 7-11) 30-50ml wash-out, collect methyl alcohol-methenyl choloride (0.5-1.5: 7-11) eluent, evaporate to dryness, residue add methenyl choloride 0.5-1.5ml makes dissolving, as need testing solution, it is an amount of that other gets onocerin, add methyl alcohol and make the solution that every 1ml contains 0.1-0.5mg, product solution is tested according to thin-layered chromatography in contrast, draw respectively onocerin reference substance solution 2-5 μ l, need testing solution 5-10 μ l, put respectively on same silica gel g thin-layer plate, take methenyl choloride-methyl alcohol (10-40: 1) as developping agent, launch more than the 8cm, take out, dry, place under the ultraviolet lamp (254nm) and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

The described root of herbaceous peony differentiates that step is as follows:

Test according to thin-layered chromatography, draw respectively need testing solution 20-30 μ l and Paeoniflorin reference substance solution 10-20 μ l in the assay item, put respectively on same silica gel g thin-layer plate, take methenyl choloride-ethyl acetate-methyl alcohol-formic acid (20-50: 3-7: 5-15: 0.2) as developping agent, launch, take out, dry, spray is with 3-5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to colour developing at 100-120 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

Described prepared rhizome of rehmannia differentiates that step is as follows:

Get this product 15-20g, add water 50-100ml, in 50-60 ℃ of warm 20-50 minute, take out, ultrasonic processing 20-40 minute, centrifugal, get supernatant, use ethyl acetate extraction 1-3 time, each 20-40ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue add ethyl acetate 3-10ml makes dissolving, adds neutral alumina 2-4g, mix thoroughly, the neutral alumina column of packing into ethanol 20-50ml wash-out, is collected eluent, evaporate to dryness, residue adds ethanol 0.5-2.0ml, makes dissolving, as need testing solution, other gets prepared rhizome of rehmannia control medicinal material 2-5g, add water 30-80ml and decocted 0.5-2.0 hour, filter, filtrate is used ethyl acetate extraction 1-3 time, each 20-40ml, the combined ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 0.5-2.0ml, make dissolving, medicinal material solution according to the thin-layered chromatography test, is drawn respectively need testing solution 20-25 μ l and control medicinal material solution 10-20 μ l in contrast, put respectively on same silica gel g thin-layer plate, take toluene-ethyl acetate (0.5-2: 1) as developping agent, launch, take out, dry, spray is with the DNPH ethanolic solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

Described yncaria stem with hooks differentiates that step is as follows:

Get this product 5-20g, add strong ammonia solution 5-15ml and make infiltration, close plug 20-40 minute, add ether 50-100ml, add hot reflux 0.5-2.0 hour, let cool, divide and get ether layer, extract 1-3 time with the jolting of 3-8% hydrochloric acid solution, each 10-30ml merges the 3-8% hydrochloric acid solution, regulate pH value to 9 with ammoniacal liquor, extract 1-3 time with the ether jolting, each 10-30ml merges ether solution, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml makes dissolving, and as need testing solution, other is hook taking rattan control medicinal material 1-3g, adding strong ammonia solution 2-5ml infiltrates, close plug 20-40 minute, the 20-50ml that adds diethyl ether adds hot reflux 0.5-2.0 hour, let cool, filter, the filtrate evaporate to dryness adds methyl alcohol 0.5-2.0ml and makes dissolving, medicinal material solution in contrast, according to the thin-layered chromatography test, draw each 5-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene-methylene chloride-ethyl acetate-methyl alcohol-ammoniacal liquor (6-10: 10-14: 1-3: 1-3: 0.5-0.8) as developping agent, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1-4: 0.5-1) mixed solution, in the test sample chromatogram, with control medicinal material chromatogram relevant position on, the principal spot of aobvious same color.

8. according to claim 2 quality determining method is characterized in that, ursolic acid is differentiated in the described selfheal, and step is as follows:

The preparation of need testing solution: get this product 2g, add watery hydrochloric acid 1.0ml and ether 20ml, added hot reflux 30 minutes, extract is waved most ether, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution,

The preparation of reference substance solution: get the ursolic acid reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1mg, product solution in contrast,

The selection of developping agent: prepared respectively 20: 3: 7: 0.2 ratio, 15: 2: 5: 0.1 ratio, 25: 4: 9: the cyclohexane-ethyl acetate of 0.5 ratio-chloroform-glacial acetic acid was developping agent,

According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ be heated to the colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, wherein take 20: 3: 7: the cyclohexane-ethyl acetate of 0.2 ratio-chloroform-glacial acetic acid as the developping agent spot the most obvious;

Described corydalis tuber differentiates that step is as follows:

The preparation of need testing solution: get this product 1g, put in the tool plug centrifuge tube, add 0.2mol/L sodium hydroxide solution 4ml, jolting makes dissolving, add ethyl acetate 5ml, shake well centrifugal 2 minutes, is got upper solution, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution

The preparation of control medicinal material solution: corydalis tuber control medicinal material 0.5g, add ammonia solution 0.5ml, grind, the 10ml that adds methylene chloride, temperature was soaked 2 hours, filtered, and filtrate is concentrated into about 1ml, medicinal material solution in contrast,

The preparation of reference substance solution: get the tetrahydropalmatine reference substance, add methyl alcohol and make the solution that every 1ml contains 0.1mg, product solution in contrast,

The selection of developping agent: prepared respectively 15: 8: 2: 0.2 ratio, 5: 6: 1: 0.2 ratio, 20: 10: 3: the normal hexane-chloroform-methanol of 0.5 ratio-strong ammonia solution was developping agent,

Test according to thin-layered chromatography, draw each 10 μ l of need testing solution, control medicinal material solution and reference substance solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with rare bismuth potassium iodide test solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color; With the corresponding position of reference substance chromatogram on, the spot of aobvious same color, wherein take 15: 8: 2: the normal hexane-chloroform-methanol of 0.2 ratio-strong ammonia solution as the developping agent spot the most obvious;

Described cassia seed differentiates that step is as follows:

The preparation of need testing solution: get this product 2g, add watery hydrochloric acid 1.0ml and ether 20ml, added hot reflux 30 minutes, extract is waved most ether, and residue adds methyl alcohol 1ml makes dissolving, and get final product,

The preparation of blank solution: get the blank pharmaceutical preparation that lacks cassia seed, make blank solution according to the preparation method of need testing solution,

The preparation of control medicinal material: depend on pine torch control medicinal material 1g, add watery hydrochloric acid 0.5ml, ether 20ml, water-bath refluxed 1 hour, took out, and let cool, and filtered, and filtrate is reclaimed ether to doing, and residue adds respectively methyl alcohol 1ml makes dissolving, and get final product,

The preparation of reference substance solution: get Chrysophanol reference substance, archen reference substance, Physcion reference substance and aurantio-obtusin, add absolute ethyl alcohol-ethyl acetate (2: 1) and make the solution that every 1ml contains 0.5mg, product solution in contrast,

The selection of developping agent: prepared respectively 6: 12: 5: 0.5 ratio, 3: 10: 3: 0.2 ratio, 9: 14: 7: the sherwood oil of 0.8 ratio (30～60 ℃)-cyclohexane-ethyl acetate-formic acid was that developping agent is according to the thin-layered chromatography test, draw need testing solution 10 μ l, control medicinal material solution 7 and mixing reference substance solution 2 μ l, put respectively on same high-efficient silica gel G thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, place more than the smoked 10min of ammonia steam, put under the daylight and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, wherein take 6: 12: 5: the sherwood oil of 0.5 ratio (30～60 ℃)-cyclohexane-ethyl acetate-formic acid as the developping agent spot the most obvious;

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, add watery hydrochloric acid 1ml and ether 20ml, mixing, 60 ℃ of heating water baths refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds methyl alcohol 0.5ml dissolving, and get final product,

The preparation of control medicinal material solution: get respectively Radix Angelicae Sinensis control medicinal material, each 1g of Ligusticum wallichii control medicinal material, the 20ml that adds diethyl ether, water-bath refluxed 1 hour, took out, let cool, filter, filtrate is reclaimed ether to doing, residue adds respectively ethyl acetate 1ml makes dissolving, respectively as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution

The preparation of blank solution: by the prescription proportioning, get except Radix Angelicae Sinensis, Ligusticum wallichii other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepare respectively 9: 1: 0.5 ratios, 5: 0.5: 0.2 ratios, the normal hexane-ethyl acetate of 9: 1.5: 0.8 ratios-formic acid is developping agent,

According to the thin-layered chromatography test, draw need testing solution 10 μ l, Radix Angelicae Sinensis control medicinal material solution 10 μ l and Ligusticum wallichii control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch to take out more than the 10cm, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color, wherein the most obvious take the normal hexane-ethyl acetate of 9: 1: 0.5 ratios-formic acid as the developping agent spot;

The preparation of reference substance solution: get the Rosmarinic acid reference substance, add Diluted Alcohol and make the solution that every 1ml contains 0.5mg, product solution in contrast,

The preparation of blank solution: by the prescription proportioning, get except selfheal other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepare respectively 2: 4: 0.2 ratios, 1: 5: 0.2 ratio, the normal hexane-ethyl acetate of 3: 6: 0.5 ratios-formic acid is developping agent,

Test according to thin-layered chromatography, draw need testing solution 5 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, wherein the most obvious take the normal hexane-ethyl acetate of 2: 4: 0.2 ratios-formic acid as the developping agent spot;

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add ammoniacal liquor 5ml, and ether 40ml, close plug left standstill 60 minutes, and glass bar stirs evenly, and water-bath refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds absolute ethyl alcohol 1ml makes dissolving, and get final product,

The preparation of control medicinal material solution: get Ligusticum wallichii control medicinal material 1g, add ammoniacal liquor 5ml, and ether 20ml, close plug left standstill 60 minutes, and water-bath refluxed 30 minutes, take out, let cool, filter, filtrate is reclaimed ether to doing, residue adds absolute ethyl alcohol 1ml makes dissolving, and get final product, as Ligusticum wallichii control medicinal material solution

The preparation of blank solution: by the prescription proportioning, get except Ligusticum wallichii other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepare respectively 9: 1 ratios, 7: 0.5 ratios, the cyclohexane-ethyl acetate of 11: 2 ratios is developping agent,

Test according to thin-layered chromatography, draw respectively Ligusticum wallichii control medicinal material solution 10 μ l, need testing solution 12 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution, place under the daylight and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, wherein the most obvious take the cyclohexane-ethyl acetate of 9: 1 ratios as the developping agent spot;

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 4g, add ether 40ml, water-bath refluxed 30 minutes, took out, and let cool, and extract reclaims ether to doing, and residue adds methyl alcohol 1ml makes dissolving, and get final product,

The preparation of reference substance solution: get Levistilide A reference substance 20mg, place the brown volumetric flask of 50ml, add acetic acid ethyl dissolution and be settled to scale, shake up, and get final product, as the Levistilide A reference substance solution,

The selection of developping agent: prepare respectively 3: 1 ratios, 1: 0.5 ratio, the normal hexane-ethyl acetate of 5: 2 ratios is developping agent,

Test according to thin-layered chromatography, draw respectively Levistilide A reference substance solution 6 μ l, need testing solution 10 μ l, put respectively on same silica GF254 thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, wherein the most obvious take the normal hexane-ethyl acetate of 3: 1 ratios as the developping agent spot;

Described reticulate millettia differentiates that step is as follows:

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add 0.2% sodium hydroxide solution 30ml, shake up, ultrasonic processing 30min takes out, and is centrifugal, get supernatant, with sherwood oil (60～90 ℃) extraction 2 times, each 15ml, discard petroleum ether layer, the water intaking layer adds 0.4ml watery hydrochloric acid, shakes up, with extracted with diethyl ether 3 times, each 15ml merges the extracted with diethyl ether layer, reclaim ether to doing, residue adds methyl alcohol 2ml dissolving, adds silica gel 1g and mixes thoroughly, volatilize solvent, put on the silicagel column, with methyl alcohol-methenyl choloride (1: 9) 40ml wash-out, collect methyl alcohol-methenyl choloride (1: 9) eluent, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, and get final product

The preparation of reference substance solution: it is an amount of that precision takes by weighing onocerin, and add methyl alcohol and make the solution that every 1ml contains 0.1mg, product solution in contrast,

The preparation of blank solution: by the prescription proportioning, get except reticulate millettia other medicinal materials and make negative sample in accordance with the law, make negative sample solution according to the need testing solution preparation method again,

The selection of developping agent: 30: 1 ratios, 10: 1 ratios, the methenyl choloride-methyl alcohol of 40: 1 ratios is developping agent,

Test according to thin-layered chromatography, draw respectively onocerin reference substance solution 2～5 μ l, need testing solution 5～10 μ l, put respectively on same silica gel g thin-layer plate, in above-mentioned developping agent condition, launch 12cm, take out, dry, place under the ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, wherein the most clear take the methenyl choloride-methyl alcohol of 30: 1 ratios as the developping agent spot;

The described root of herbaceous peony differentiates that step is as follows:

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize is got about 0.08g, accurately weighed, put in the 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic processing 5 minutes is added on the D101 type large pore resin absorption column water 15ml wash-out, discard eluent, use again methyl alcohol 8ml wash-out, collect eluent in the 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, and get final product

The preparation of reference substance solution: it is an amount of to get the Paeoniflorin reference substance, accurately weighed, and add 80% methyl alcohol and make the solution that every 1ml contains 20 μ g, and get final product,

The preparation of blank solution: by the prescription proportioning, get except the root of herbaceous peony other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepared respectively 40: 5: 10: 0.2 ratio, 20: 3: 5: 0.2 ratio, 50: 7: 15: the methenyl choloride-ethyl acetate of 0.2 ratio-methyl alcohol-formic acid was developping agent,

Test according to thin-layered chromatography, draw respectively need testing solution 20 μ l and Paeoniflorin reference substance solution 10 μ l in the assay item, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, 105 ℃ be heated to the colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, wherein take 40: 5: 10: the methenyl choloride-ethyl acetate of 0.2 ratio-methyl alcohol-formic acid as the developping agent spot the most obvious;

Described prepared rhizome of rehmannia differentiates that step is as follows:

The preparation of need testing solution: get this product 20g, add water 100ml, in 60 ℃ warm 30 minutes, take out, ultrasonic processing 30 minutes, centrifugal, get supernatant, use ethyl acetate extraction 2 times, each 30ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue add ethyl acetate 5ml makes dissolving, add neutral alumina 3g, mix thoroughly, the neutral alumina column of packing into is with ethanol 30ml wash-out, collect eluent, evaporate to dryness, residue add ethanol 1ml, make dissolving, as need testing solution

The preparation of control medicinal material solution: get prepared rhizome of rehmannia control medicinal material 4g, add water 50ml and decocted 1 hour, filter, filtrate is with ethyl acetate extraction 2 times, each 30ml, and the combined ethyl acetate extract, evaporate to dryness, residue add methyl alcohol 1ml, make dissolving, medicinal material solution in contrast,

The preparation of blank solution: by the prescription proportioning, get except prepared rhizome of rehmannia other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepare respectively 1: 1 ratio, 0.5: 1 ratio, the toluene-ethyl acetate of 2: 1 ratios is developping agent,

According to the thin-layered chromatography test, draw respectively need testing solution 20 μ l and control medicinal material solution 10 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 2,4-dinitrophenylhydrazine ethanolic solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, wherein the most obvious take the toluene-ethyl acetate of 1: 1 ratio as the developping agent spot; Described yncaria stem with hooks differentiates that step is as follows:

The preparation of need testing solution: get this product 10g, add strong ammonia solution 10ml and make infiltration, close plug 30 minutes, add ether 100ml, added hot reflux 1 hour, let cool, divide and get ether layer, extract 2 times with 5% hydrochloric acid solution jolting, each 20ml, merge 5% hydrochloric acid solution, regulate pH value to 9 with ammoniacal liquor, extract 2 times with the ether jolting, each 20ml merges ether solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution

The preparation of control medicinal material solution: hook taking rattan control medicinal material 2g, add strong ammonia solution 3ml and infiltrate, close plug 30 minutes, the 40ml that adds diethyl ether added hot reflux 1 hour, let cool, and filtered, and the filtrate evaporate to dryness adds methyl alcohol 1ml and makes dissolving, medicinal material solution in contrast,

The preparation of blank solution: by the prescription proportioning, get except yncaria stem with hooks other medicinal materials and make negative sample in accordance with the law, make blank solution according to the need testing solution preparation method again,

The selection of developping agent: prepared respectively 8: 12: 2: 2: 0.5 ratios, 6: 10: 1: 1: 0.5 ratio, 10: 14: 3: the toluene-methylene chloride of 3: 0.8 ratios-ethyl acetate-methyl alcohol-ammoniacal liquor was developping agent,

Test according to thin-layered chromatography, draw each 10 μ l of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution, in the test sample chromatogram, with control medicinal material chromatogram relevant position on, the principal spot of aobvious same color, wherein take 8: 12: 2: the toluene-methylene chloride of 2: 0.5 ratios-ethyl acetate-methyl alcohol-ammoniacal liquor as the developping agent spot the most obvious.

9. according to claim 3 quality determining method is characterized in that, described content assaying method, and step is as follows:

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; Take isopropyl alcohol-methyl alcohol-5mmo1/L citric acid soln (2-4: 18-22: 78-80) as mobile phase; The detection wavelength is 230-240nm, and number of theoretical plate calculates by the Paeoniflorin peak should be not less than 2000,

The preparation of reference substance solution: it is an amount of to get the Paeoniflorin reference substance, accurately weighed, adds 70-90% methyl alcohol and makes the solution that every 1ml contains 15-25 μ g, obtains reference substance solution;

The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize is got 0.06-0.10g, accurately weighed, put in the 20-50ml beaker, add sodium bicarbonate solution 5ml, ultrasonic processing is added on the D101 type large pore resin absorption column, uses the 10-20ml water elution, discard eluent, use again the 6-10ml methanol-eluted fractions, collect eluent in the 10ml measuring bottle, add water to scale, shake up, centrifugal 4-6 minute, get supernatant and obtain need testing solution;

Determination method: precision is drawn reference substance solution and each 5-15 μ l of need testing solution respectively, and the injection liquid chromatography obtains chromatogram, calculates the content of Paeoniflorin in the blood-nourishing and brain-refreshing granules.

10. according to claim 9 quality determining method is characterized in that step is as follows:

Chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Take isopropyl alcohol-methyl alcohol-5mmol/L aqueous solution of citric acid (2: 18: 80) as mobile phase; The detection wavelength is 230nm; 30 ℃ of column temperatures, theoretical cam curve calculate by the Paeoniflorin peak should be not less than 2000,

The preparation of reference substance solution: get the about 10mg of Paeoniflorin reference substance, accurately weighed, put in the 50ml volumetric flask, add the dissolving of 70% methyl alcohol and be diluted to scale, shake up, precision is measured above-mentioned Paeoniflorin reference substance solution 5ml again, puts in the 50ml volumetric flask, adds 70% methyl alcohol and is diluted to scale, shake up, and get final product

It is an amount of that blood-nourishing and brain-refreshing granules is got in the preparation of need testing solution, and porphyrize is got about 0.08g, and is accurately weighed, put in the 10ml volumetric flask, add 0.2% sodium bicarbonate solution 5ml, power 500W, the ultrasonic processing of frequency 30kHz 10 minutes is added in 60～80 orders, internal diameter 6～8mm, on the D101 type large pore resin absorption column of high about 5cm, water 15ml wash-out discards eluent, use again methyl alcohol 8ml wash-out, collect eluent and in the 10ml measuring bottle, add water to scale, shake up, cross 0.45 μ m miillpore filter, and get final product

Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and obtains chromatogram, calculates the content of Paeoniflorin in the blood-nourishing and brain-refreshing granules.