CN100418563C - Quality control method of Chinese medicinal preparation - Google Patents

Abstract

The present invention discloses a quality control method of a Chinese medicine preparation, which aims to overcome the defects that the quality control method is underdeveloped, and the quality of products is difficult to control, which exist in the existing antithrombus preparation. In the present invention, tangkuei tail, pberetima, nuxvomica poisonnut, musk, yanbusuo, toad, liquorice, etc. are identified by using the thin-layer chromatography method; the content of the components of Tanshinone IIA and the content of the components of Strychnine are mearsured by using the high-efficiency liquid chromatography method. The quality control method of a Chinese medicine preparation has the advantages of high precision, high sensibility and high stability; the finished product quantity of the raw dose taken every day is used as the unit quantity of the preparation, and every unit quantity contains at least 52 mu g of Tanshinone IIA (C19H18O3) of danshen root; every unit quantity contains 47 to 86 mu g of nuxvomica poisonnut of Strychnine (C21H22N2O2).

Description

A kind of method of quality control of Chinese medicine preparation

Technical field:

The present invention relates to a kind of method of quality control of Chinese medicine preparation, particularly thromboembolism preventing quality of the pharmaceutical preparations control method.

Background technology:

Thromboembolism preventing preparation raw material medicine is made up of 19 flavor Chinese medicines such as Radix Angelicae Sinensis, Radix Salviae Miltiorrhizae, Bombyx Batryticatus, Gekko Swinhonis, Eupolyphaga Seu Steleophaga, Scolopendra, Hirudo, Nidus Vespae, Pheretima, Semen Strychni, Moschus, Venenum Bufonis, Radix Glycyrrhizae, Rhizoma Smilacis Glabrae, Rhizoma Corydalis, Rhizoma Drynariae, Zaocys, Tabanus, Squama Manis, and domestic have multiple dosage form to go on the market.But discover that through us existing thromboembolism preventing preparation exists method of quality control to fall behind the uppity shortcoming of product quality.Radix Angelicae Sinensis, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae are not differentiated in the existing method of quality control, or tanshinone and strychnine composition are carried out assay.So existing method of quality control can not effectively be controlled the quality of thromboembolism preventing preparation, thereby will influence the production of product and ensure the quality of products.

For effectively controlling product quality, we have set up the new method of quality control of thromboembolism preventing preparation, this method adopts according to thin layer chromatography Radix Angelicae Sinensis, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae etc. is differentiated, adopts high performance liquid chromatography that tanshinone and strychnine composition are carried out assay.This method of quality control precision, sensitivity, stability are all good, guarantee " safety, the homogeneous, stable, effective, controlled " of product quality.

Summary of the invention:

The object of the invention is to provide the method for quality control of thromboembolism preventing preparation.

The present invention be directed to the thromboembolism preventing preparation and propose method of quality control, this thromboembolism preventing preparation comprises oral formulations such as capsule, tablet, oral liquid, syrup, granule.

Described thromboembolism preventing preparation of the present invention is made by following parts by weight of Chinese traditional medicine raw material:

Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

More than composition can be made into 1000 doses of pharmaceutical preparatioies, described 1000 doses of fingers, and the final drug preparation of making, as make 1000 of capsule preparations, and 1000 in tablet, granule 1000g, liquid preparation 1000ml etc.,

More than form being by weight as proportioning, can increasing or reduce according to corresponding proportion when producing, can be unit with the kilogram as large-scale production, or is unit with the ton.

The preparation method of thromboembolism preventing preparation of the present invention can adopt following method:

By the raw material of Chinese medicine of above-mentioned prescription is processed through extraction or other modes, making pharmaceutically active substance, subsequently, is raw material with this material, add the medicine acceptable carrier when needing, make capsule, tablet, oral liquid, syrup, granule according to the routine techniques of galenic pharmacy.Described active substance can obtain by the method that is selected from following mode, as: by pulverize, squeeze, calcine, grind, sieve, percolation, extraction, water are carried, alcohol extraction, ester are carried, methods such as ketone is carried, chromatography obtain, these active substances can be the materials of extractum form, can be that dry extract also can be a fluid extract, can also be the high-purity extract, make different concentration according to the different needs decision of preparation.

Thromboembolism preventing preparation of the present invention, when making preparation, can add the medicine acceptable carrier as required, these carriers can be any carriers that is fit to make the thromboembolism preventing preparation, as: benzoic acid or potassium salt, mannitol, sorbitol, sorbic acid or potassium salt, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, Nepal's methyl ester, Nepal's ethyl ester, Nepal's propyl ester, Nepal's butyl ester, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin A, vitamin C, vitamin E, vitamin D, azone, the EDTA disodium, EDTA calcium sodium, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, propylene glycol, ethanol, soil temperature 60-80, Arlacel-80, Cera Flava, lanoline, liquid paraffin, hexadecanol, gallate ester, agar, triethanolamine, basic amino acid, carbamide, allantoin, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material etc.

The present invention is to provide method of quality control, be the quality of control thromboembolism preventing preparation at above thromboembolism preventing preparation.At its characteristics and prescription of the present invention, we provide following method of quality control:

Method of quality control of the present invention may further comprise the steps:

Character is observed, and official method is checked content, and content Radix Angelicae Sinensis, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae are differentiated, tanshinone and the strychnine that contains carried out assay.

Therefore, the main step of method of quality control of the present invention is:

A. the Radix Angelicae Sinensis in the pharmaceutical preparation of the present invention, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae are differentiated;

B. measure the content of tanshinone and strychnine in the pharmaceutical preparation of the present invention;

Wherein the concrete steps of A discriminating are as follows:

(1) get thromboembolism preventing preparation content, put microscopically and observe: mycelium is closely colourless, and elongated curling becomes entangled in the body wall.Body wall fragment dark-brown or yellow, on it give birth to short and thick or elongated bristle, the circular trichopore after the Chang Kejian bristle comes off.Parenchyma cell contains prism of calcium oxalate around the fibre bundle, forms crystalline cellulose; Bordered pit vessel is bigger.The needle-like calcium oxalate crystal bundle is present in the mucilage cell or is dispersed in.Gelatinized starch grain agglomerate is faint yellow or closely colourless.Scutellum is near colourless or faint yellow; Rhabdium as seen, and is faint yellow or closely colourless, how cataclasm, and the side is seen and is strip and block more.

(2) get thromboembolism preventing preparation content 3g, add normal hexane 20ml supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution.Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate (6-13: 0.7-1.4) be developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

(3) get thromboembolism preventing preparation content 10g, add little the boiling backflow 0.5-1.5 hour of water 250ml heating, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution.Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate (6-12: 0.7-1.3) be developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

(4) get thromboembolism preventing preparation content 8g, put in the 500ml round-bottomed flask, add water 200ml, benzene 1.0ml, mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and kept little 1.5-2.5 of boiling hour, puts coldly, divides and gets the benzene layer as need testing solution.Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution.According to gas chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 E), be immobile phase with 100% methyl silicone (OV-1); Fid detector; Elastic quartz capillary tube chromatographic column (30m * 0.25mm * 0.32 μ m); Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min.Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(5) get thromboembolism preventing preparation content 20g, add petroleum ether (30～60 ℃) 50ml merceration 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina (100～200 order) 4g, packing into, (1.5 * 20cm), usefulness chloroform 50ml eluting is collected eluent to the neutral alumina post, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution.Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast.According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D), be filler with octadecylsilane chemically bonded silica; Water-acetonitrile (48-70: 26-52) be mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 296 ± 3nm; Column temperature: 20-60 ℃.Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak.Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(6) get thromboembolism preventing preparation content 10g, add water 40ml, hydrochloric acid 3ml heats little boiling backflow 1-2 hour, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 0.5-1.5 hour discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution.Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-benzene-ethyl acetate-glacial acetic acid (7-14: 11-30: 3-11: 0.2-1.1) be developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

(7) get thromboembolism preventing preparation content 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divide three extractions (20,20,10ml) with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions (20,20,10ml) with chloroform 50ml again, combined chloroform liquid washes with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-ethyl acetate (1.2-2.9: 0.9-2.8) be developing solvent, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp (365nm) and is inspected after the smoked several seconds.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Wherein the concrete steps of B mensuration content are as follows:

A. tanshinone assay:

Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water (64-110: 9.5-22); Flow velocity: 1.0ml/min; Column temperature: 25-55 ℃; Detect wavelength 270 ± 3nm.Number of theoretical plate is with Tanshinone I I A (C ₁₉H ₁₈O ₃) the peak meter, should be not less than 2000.

The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets (containing tanshinone 16 μ g among every 1ml).

Thromboembolism preventing preparation content 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, placement is spent the night, supersound process 10-60 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane (0.45 μ m).

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

B. strychnine assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine (52-94: 16-33: 0.9-3.4) be mobile phase; Flow velocity: 1.0ml/min; Column temperature: 20-60 ℃; The detection wavelength is 254 ± 2nm.Number of theoretical plate calculates by the strychnine peak should be not less than 2000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly.

Thromboembolism preventing preparation content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 2-8ml, close plug, jolting gently, claim to decide weight, placed supersound process 0-40 minute 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 5-9 time with sulfuric acid solution (3 → 100), each 25ml merges sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, with chloroform extraction 5-9 time, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Thromboembolism preventing preparation described in the above method is meant that final drug of the present invention is as capsule, tablet, oral liquid, syrup, granule.

Method of quality control of the present invention is more effective to the quality control of product, and method precision, sensitivity, stability are all higher.Can control product quality preferably, guarantee " safety, the homogeneous, stable, effective, controlled " of product quality.

The used preparation of each experimental example test sample of the present invention all can adopt embodiment 1 described thrombus-resisting capsules preparation, and also available have other preparations that same materials is formed with embodiment 1 described thrombus-resisting capsules agent.

The content assaying method research of experimental example 1 Tanshinone I I

1. instrument and reagent

1.1 instrument day island proper Tianjin LC-10ATVP high performance liquid chromatograph, the LC-10ATvp infusion pump; SPD-M10Avp secondary array detector; The SCL-10Avp system controller; CTO-10ASvp thermocolumn case; The CLASS-VP6.1 workstation data processing system; Microsyringe (25 μ l), HAMILTON company; Ultrasonic washing unit (250W, Beijing's armarium two factories).

1.1.2 reagent methanol (analytical pure), water are the secondary double distilled water, (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides the tanshinone reference substance, for assay usefulness, lot number: 0766-9908);

1.2 chromatographic condition chromatographic column: Hypersil C ₁₈(5um, 250 * 5.0i.d.mm) spies of Dalian Erie; Mobile phase: methanol-water (85: 15); Detect wavelength: 270nm; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Sample size: 5 μ l.

1.3 system suitability test

The negative need testing solution of getting Tanshinone I I A reference substance reference substance solution, need testing solution and scarce red rooted salvia respectively injects chromatograph of liquid, the record chromatograph.Retention time (the t of tanshinone as can be known _R) being about 9.2 minutes, the negative sample chromatogram is at non-false positive peak, place, tanshinone peak position, and theoretical cam curve is calculated as 5200 with the tanshinone peak.With other component peaks separating degree＞1.5.

1.4 linear relationship is investigated

1.4.1 the preparation of standard solution

It is an amount of that precision takes by weighing Tanshinone I I A reference substance, adds dissolve with methanol and make the solution that every 1ml contains Tanshinone I I A0.0245mg.

1.4.2 the drafting of standard curve

Precision is measured the solution 2.0ml of 0.0245mg, 4.0ml, 6.0ml, 8.0ml, 10.0ml, put respectively in the 10ml brown bottle, add methanol constant volume to scale, shake up, accurate respectively amount is drawn above-mentioned five kinds of solution, 5 μ l and is injected chromatograph of liquid, the record chromatograph, see Table 1, with peak area A (μ Vs) concentration C (μ g/ml) is carried out linear regression and calculate, must equation of linear regression be: A=328359.82C+6495.9, r=0.9999, the standard solution 6.5ml that precision is measured 0.0245mg/ml puts in the 10ml brown bottle, adds methanol constant volume to scale, shake up, get reference substance solution (0.0245mg/ml).Accurate amount is drawn this reference substance solution 5 μ l and is injected chromatograph of liquid, the gained peak area is 454684, one need testing solution sample introduction is analyzed the gained peak area calculate content with regression equation and one point method respectively, result's relative deviation 0.74% is so quality standard of the present invention adopts one point method to calculate content.The range of linearity: 4.9～245g/ml.

Table 1 tanshinone linear relationship is investigated

1.4.3 the selective extraction method of need testing solution extraction time: merceration spends the night, supersound extraction, and measurement result sees Table 2.

The investigation (n=3) of the different ultrasonic times of table 2 methanol

As known from Table 2, merceration spends the night, and supersound extraction 30min can extract safety with the tanshinone in the preparation, so select supersound process 30min.

1.4.4 precision test

Get this product (lot number 20011105) content, prepare test liquid, repeat sample introduction 5 times by the preparation method of test liquid under the assay item in the quality standard of the present invention, measure peak area, result of calculation is listed table 3 in, average content 0.02108, RSD is 0.46%, and precision is good.

The precision of tanshinone test in the table 3 preparation test sample

1.4.5 repeatability test

Get this product (lot number 20011105) content, by 5 parts of test liquids of preparation method preparation of test liquid under the assay item in the quality standard of the present invention, sample introduction respectively, measure peak area, result of calculation is listed table 4 in, and average content is 0.02209%, RSD is 1.12%, and repeatability is good.

The repeatability of tanshinone test in the table 4 preparation test sample

1.4.6 tanshinone stability test in the need testing solution

Get this product (lot number 20011105) content, preparation method by test liquid under the assay item in the quality standard of the present invention prepares test liquid, respectively at 0,1,2,4,8 hour mensuration tanshinone content (seeing Table 5), the result shows that tanshinone is stable relatively in 8 hours in the need testing solution.

The stability test of tanshinone in the table 5 preparation test sample

1.4.7 recovery test

Adopt the application of sample absorption method, it is an amount of that precision takes by weighing 5 parts of samples (lot number 20011105, average content are 0.02209%) of having measured content respectively, put in the tool plug conical flask, accurate adding tanshinone reference substance solution (wherein is solvent, 0.00808mg/ml) 50ml with methanol, close plug claims to decide weight, and placement is spent the night, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, make test liquid with filter membrane (0.45um).The accurate respectively 5ul of absorption injects chromatograph of liquid, and the record chromatograph is measured content, calculate recovery rate (the results are shown in Table 6), and average recovery rate is 99.21%, RSD is 1.34%.

Tanshinone I I A measures recovery test in table 6 need testing solution

1.4.8 sample determination

Prepare test liquid and reference substance solution by quality standard, sample introduction 5 μ l write down chromatograph respectively, measure peak area, are calculated as follows content:

In the formula: Ai: the need testing solution peak area

As: reference substance solution peak area

Cs: reference substance solution concentration (μ g/ml)

W: test sample sample weighting amount (mg)

W ₁: average particle heavy (mg)

According to 3 batch samples in strict accordance with the production technology preparation, Tanshinone I I content in the working sample, measurement result sees Table 7.

Tanshinone I I assay result in table 73 batch samples

Tanshinone I I content limit is calculated as follows in the preparation:

Tanshinone I I content lower bound=preparation contains red rooted salvia (g/g) * medical material and contains Tanshinone I I amount lower bound (pharmacopeia regulation) * yield=(200/2184) * 0.2% * 95%=1.740 * 10 ^-4Every content lower bound=1.740 * 10 ^-4* 0.3g/ grain * 10 ⁶=52ug/ grain.

So deciding every of this product contains Radix Salviae Miltiorrhizae with Tanshinone I I (C ₁₉H ₁₈O ₃) must not be less than 52ug.Measurement result per sample, the Tanshinone I I (C in 3 batch samples ₁₉H ₁₈O ₃) content all greater than the 52ug/ grain.

The content assaying method research of experimental example 2 strychnine

2. instrument and reagent

2.1 instrument day island proper Tianjin LC-10ATVP high performance liquid chromatograph, the LC-10ATvp infusion pump; SPD-M10Avp secondary array detector; The SCL-10Avp system controller; CTO-10ASvp thermocolumn case; The CLASS-VP6.1 workstation data processing system; Microsyringe (25 μ l), HAMILTON company; Ultrasonic washing unit (250W, Beijing's armarium two factories).

2.1.2 reagent chloroform (analytical pure), cyclohexane extraction (analytical pure), ethylenediamine (analytical pure), ammonia (analytical pure), concentrated sulphuric acid (analytical pure), ethyl acetate (analytical pure), (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides the strychnine reference substance, for assay usefulness, lot number: 0705-200005);

2.1.3 chromatographic condition chromatographic column: Hypersil C ₁₈(5um, 250 * 5.0i.d.mm) spies of Dalian Erie; Mobile phase: methanol-water (85: 15); Detect wavelength: 270nm; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Sample size: 5 μ l.

2.2 system suitability test

The negative need testing solution of getting strychnine reference substance reference substance solution, need testing solution and scarce Semen Strychni medical material respectively injects chromatograph of liquid, the record chromatograph.Retention time (the t of strychnine as can be known _R) being about 12.3 minutes, the negative sample chromatogram is at non-false positive peak, place, strychnine peak position, and strychnine separates fully (separating degree＞1.5) with close impurity peaks, promptly separate fully with other component peaks under this experimental condition.Theoretical cam curve is calculated as 4500 with the strychnine peak.

2.3 linear relationship is investigated

2.4 the preparation of standard solution

It is an amount of that precision takes by weighing the strychnine reference substance, adds the ethyl acetate dissolving and make the reference substance solution that every 1ml contains strychnine 0.206mg.

2.5 the drafting of standard curve

Precision is measured reference substance solution 2,4,6,8,10ul, inject chromatograph of liquid respectively, the record chromatograph, see Table 8, with peak area A (μ Vs) prime number x (μ g) being carried out linear regression calculates, getting equation of linear regression is: A=1466315.7767 * X+19673.9000, r=0.9999, accurate amount is drawn this reference substance solution 5 μ l and is injected chromatograph of liquid, the gained peak area is 1528781, one need testing solution sample introduction is analyzed the gained peak area calculate content with regression equation and one point method respectively, result's relative deviation 0.045% is so quality standard of the present invention adopts one point method to calculate content.The range of linearity: 0.412～2.06 μ g.

Table 8 strychnine linear relationship is investigated

Select under alkali condition, to be easy to by the feature of organic solvent extraction 2.6 extract according to strychnine, we have selected in test to be usually used in extracting alkaloidal chloroform-dense ammonia is extracting solution, because the preparation composition is more, contained strychnine is difficult for extracting fully, extract the back with chloroform-dense ammonia (50: 2), chloroform-dense ammonia (50: 5), chloroform-dense ammonia (50: 8) respectively and survey content, the results are shown in Table 9.Result of the test shows, strychnine can be extracted fully for extractant with chloroform-dense ammonia (50: 8), so this quality standard employing chloroform-dense ammonia (50: 8) is extractant.

The different influences (n=2) of extracting solvent of table 9 to the strychnine assay

2.7 the selective extraction method of supersound extraction time: placed 24 hours, supersound extraction, measurement result sees Table 10.

The investigation of the different ultrasonic times of table 10 (n=3)

As known from Table 10, placed 24 hours, supersound extraction 20min can extract safety with the strychnine in the preparation, so select to place supersound extraction 20min 24 hours.

2.8 extracting the number of times of sample solution and the result that influences of strychnine assay investigates with sulfuric acid solution (3 → 100) extraction merging sulphuric acid liquid, add strong ammonia solution and reconcile pH value to 9-10, the reuse chloroform extraction, its extraction time and strychnine assay the results are shown in Table 11.

Table 11 extracts the number of times and the strychnine assay result (n=2) of sample solution

From table as can be known, extract 7 times and do not have significant difference, so this quality standard application extraction time is 7 times with 8 times, 9 times result.

2.9 precision test

Get this product (lot number 20011105) content, prepare test liquid, repeat sample introduction 5 times by the preparation method of test liquid under the assay item in the quality standard of the present invention, measure peak area, result of calculation is listed table 12 in, average content 0.02067, RSD is 0.27%, and precision is good.

The precision of strychnine test in the table 12 preparation test sample

2.10 repeatability test

Get this product (lot number 20011105) content, by 5 parts of test liquids of preparation method preparation of test liquid under the assay item in the quality standard of the present invention, sample introduction respectively, measure peak area, result of calculation is listed table 13 in, and average content is 0.02078%, RSD is 1.72%, and repeatability is good.

The repeatability of strychnine test in the table 13 preparation test sample

2.11 strychnine stability test in the need testing solution

Get this product (lot number 20011105) content, preparation method by test liquid under the assay item in the quality standard of the present invention prepares test liquid, respectively at 0,2,4,6,8 hour mensuration strychnine content (seeing Table 14), average content is 0.02066%, RSD is 0.36%, the result shows that strychnine is stable relatively in 8 hours in the need testing solution.

The stability test of strychnine in the table 14 preparation test sample

2.12 recovery test

Adopt the application of sample absorption method, it is an amount of that precision takes by weighing 5 parts of samples (lot number 20011105, average content are 0.02078%) of having measured content respectively, put in the tool plug conical flask, accurate adding strychnine reference substance solution (wherein is solvent, 0.024mg/ml) 50ml and strong ammonia solution 8ml with the chloroform, close plug, jolting gently claims to decide weight, placed 24 hours, supersound process 20 minutes is put cold, claim again to decide weight, supply the weight that subtracts mistake, fully shake up with extracting solution, filter, make test liquid.Precision is measured subsequent filtrate 25m, puts in the separatory funnel, extracts 7 times with sulfuric acid solution (3 → 100), each 30ml merges sulphuric acid liquid, adds strong ammonia solution and reconciles pH value to 9-10, reuse chloroform extraction 7 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, make test liquid.The accurate respectively 5ul of absorption injects chromatograph of liquid, and the record chromatograph is measured content, calculate recovery rate (the results are shown in Table 15), and average recovery rate is 98.34%, RSD is 1.60%.

Strychnine is measured recovery test in table 15 need testing solution

2.13 sample determination

Prepare test liquid and reference substance solution by quality standard, sample introduction 5 μ l write down chromatograph respectively, measure peak area, are calculated as follows content:

In the formula: Ai: the need testing solution peak area

As: reference substance solution peak area

Cs: reference substance solution concentration (μ g/ml)

W: test sample sample weighting amount (g)

W ₁: average particle heavy (g)

According to 3 batch samples in strict accordance with the production technology preparation, strychnine content in the working sample, measurement result sees Table 16.

Strychnine assay result in table 163 batch sample

The strychnine content limit is calculated as follows in the preparation:

Strychnine content lower bound=preparation contains Semen Strychni medical material (g/g) * medical material and contains strychnine content lower bound (pharmacopeia regulation) * yield=(30/2184) * 1.2% * 95%=1.5659 * 10 ^-4Every content lower bound=1.5659 * 10 ^-4* 0.3g/ grain * 10 ⁶=46.98ug/ grain; High limit=the preparation of strychnine content contains Semen Strychni medical material (g/g) * medical material and contains strychnine content lower bound (pharmacopeia regulation) * yield=(30/2184) * 2.2% * 95%=2.8709 * 10 ^-4Every content lower bound=2.8709 * 10 ^-4* 0.3g/ grain * 10 ⁶=86.13ug/ grain;

So deciding every of this product contains Semen Strychni with strychnine (C ₂₁H ₂₂N ₂O ₂) count 47-86ug.Measurement result per sample, the strychnine (C in 3 batch samples ₂₁H ₂₂N ₂O ₂) content all in this limits.

The invention has the advantages that: method of quality control of the present invention has guaranteed that the quality inspection standard of preparation of the present invention can be than the qualitative character of effectively controlling preparation comprehensively, have accuracy and advance, can be used as the effective technology means of the stability of quality control and investigation technology.Be of great importance to improving the quality of products.

The specific embodiment:

The present invention is further illustrated below in conjunction with fact Example, and following each fact Example only is used to illustrate the present invention, to the present invention and unrestricted.

Following inventive embodiments all can realize above-mentioned effect.

Embodiment 1 thrombus-resisting capsules

[prescription] Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

[method for making] above raw material, except that Moschus, Venenum Bufonis, 17 flavors such as all the other Radix Angelicae Sinensis are ground into fine powder, with Moschus, Venenum Bufonis difference porphyrize, with above-mentioned powder facing-up, sieve, and mixing incapsulates, promptly.

[discriminating]

(1) get thrombus-resisting capsules content 2g, put microscopically and observe: mycelium is closely colourless, and elongated curling becomes entangled in the body wall.Body wall fragment dark-brown or yellow, on it give birth to short and thick or elongated bristle, the circular trichopore after the Chang Kejian bristle comes off.Parenchyma cell contains prism of calcium oxalate around the fibre bundle, forms crystalline cellulose; Bordered pit vessel is bigger.The needle-like calcium oxalate crystal bundle is present in the mucilage cell or is dispersed in.Gelatinized starch grain agglomerate is faint yellow or closely colourless.Scutellum is near colourless or faint yellow; Rhabdium as seen, and is faint yellow or closely colourless, how cataclasm, and the side is seen and is strip and block more.

(2) get thrombus-resisting capsules content 3g, add normal hexane 20ml supersound process 15 minutes, filter, filtrate is concentrated into 1ml, as need testing solution.Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, idea is same respectively is on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate (9: 1) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.(3) get this product content 10g, add little the boiling of water 250ml heating and refluxed 1 hour, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution.Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate (9: 1) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

(4) get thrombus-resisting capsules content 8g, put in the 500ml round-bottomed flask, add water 200ml, benzene 1.0ml, mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and keep little and boiled 2 hours, puts coldly, divides and gets the benzene layer as need testing solution.Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution.According to gas chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 E), be immobile phase with 100% methyl silicone (OV-1); Fid detector; Elastic quartz capillary tube chromatographic column (30m * 0.25mm * 0.32 μ m); Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min.Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(5) get thrombus-resisting capsules content 20g, add petroleum ether (30～60 ℃) 50ml merceration 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina (100～200 order) 4g, packing into, (1.5 * 20cm), usefulness chloroform 50ml eluting is collected eluent to the neutral alumina post, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution.Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast.According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D), be filler with octadecylsilane chemically bonded silica; Water-acetonitrile (60: 40) is a mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 296nm; Column temperature: 40 ℃.Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak.Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(6) get thrombus-resisting capsules content 10g, add water 40ml, little the boiling of hydrochloric acid 3ml heating refluxed 1.5 hours, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 1 hour discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution.Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-benzene-ethyl acetate-glacial acetic acid (10: 20: 7: 0.5) be developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

(7) get thrombus-resisting capsules content 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divide three extractions (20,20,10ml) with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions (20,20,10ml) with chloroform 50ml again, combined chloroform liquid washes with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with petroleum ether (30～60 ℃)-ethyl acetate (2: 1.7), put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp (365nm) and is inspected after the smoked several seconds.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

[assay]

A. Tanshinone I I A assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water (85: 15); Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 270nm.Number of theoretical plate is with tanshinone (C ₁₉H ₁₈O ₃) the peak meter, should be not less than 2000.

The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets (containing tanshinone 16 μ g among every 1ml).

This product content 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, placement is spent the night, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane (0.45 μ m).

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Every of present embodiment this product contains Radix Salviae Miltiorrhizae with Tanshinone I I A (C ₁₉H ₁₈O ₃) must not count and be less than 52 μ g.

B. the assay of strychnine is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine (75: 25: 2) is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 254nm.Number of theoretical plate calculates by the strychnine peak should be not less than 2000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly.

This product content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 8ml, close plug, jolting gently, claim to decide weight, placed supersound process 20 minutes 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 7 times, each 25ml with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 7 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Every contains Semen Strychni with strychnine (C ₂₁H ₂₂N ₂O ₂) count 47～86 μ g.

Embodiment 2 antithrombotic tablets

[prescription] Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

[method for making] above raw material, except that Moschus, Venenum Bufonis, 17 flavors such as all the other Radix Angelicae Sinensis are ground into fine powder, with Moschus, Venenum Bufonis difference porphyrize,, sieve with above-mentioned powder facing-up, mixing, granulate, often regulation becomes tablet, the 0.4g/ sheet, promptly.

[method of quality control] discriminating, content assaying method are with embodiment 1, and wherein every of content contains Radix Salviae Miltiorrhizae with Tanshinone I I A (C ₁₉H ₁₈O ₃) must not count and be less than 52 μ g; Every contains strychnine (C ₂₁H ₂₂N ₂O ₂) count 47～86 μ g).

Embodiment 3 thromboembolism preventing granules

[prescription] Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

[method for making] above raw material, except that Moschus, Venenum Bufonis, 17 flavors such as all the other Radix Angelicae Sinensis are ground into fine powder, with Moschus, Venenum Bufonis difference porphyrize,, sieve with above-mentioned powder facing-up, mixing, often regulation becomes granule, the 5g/ bag.Promptly.

[method of quality control] discriminating, content assaying method are with embodiment 1, and wherein content contains Radix Salviae Miltiorrhizae with tanshinone (C for every bag ₁₉H ₁₈O ₃) must not count and be less than 52 μ g; Every bag contains strychnine (C ₂₁H ₂₂N ₂O ₂) count 47～86 μ g.

Embodiment 4 thrombus-resisting capsules

[prescription] Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

[method for making] above raw material, except that Moschus, Venenum Bufonis, 17 flavors such as all the other Radix Angelicae Sinensis are ground into fine powder, with Moschus, Venenum Bufonis difference porphyrize, with above-mentioned powder facing-up, sieve, and mixing incapsulates, promptly.

[discriminating]

(1) gets Thrombus-resisting capsulesContent, put microscopically and observe: mycelium is closely colourless, and elongated curling becomes entangled in the body wall.Body wall fragment dark-brown or yellow, on it give birth to short and thick or elongated bristle, the circular trichopore after the Chang Kejian bristle comes off.Parenchyma cell contains prism of calcium oxalate around the fibre bundle, forms crystalline cellulose; Bordered pit vessel is bigger.The needle-like calcium oxalate crystal bundle is present in the mucilage cell or is dispersed in.Gelatinized starch grain agglomerate is faint yellow or closely colourless.Scutellum is near colourless or faint yellow; Rhabdium as seen, and is faint yellow or closely colourless, how cataclasm, and the side is seen and is strip and block more.

(2) get Thrombus-resisting capsulesContent 3g adds normal hexane 20ml supersound process 10 minutes, filters, and filtrate is concentrated into 1ml, as need testing solution.Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate (6: 0.7) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

(3) get thromboembolism preventing preparation content 10g, add little the boiling of water 250ml heating and refluxed 0.5 hour, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution.Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate (6: 0.7) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

(4) get Thrombus-resisting capsulesContent 8g puts in the 500ml round-bottomed flask, adds water 200ml, benzene 1.0ml, and mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and keep little and boiled 1.5 hours, puts coldly, divides and gets the benzene layer as need testing solution.Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution.According to gas chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 E), be immobile phase with 100% methyl silicone (OV-1); Fid detector; Elastic quartz capillary tube chromatographic column (30m * 0.25mm * 0.32 μ m); Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min.Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(5) get Thrombus-resisting capsulesContent 20g adds petroleum ether (30～60 ℃) 50ml merceration 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina (100～200 order) 4g, packing into, (1.5 * 20cm), usefulness chloroform 50ml eluting is collected eluent to the neutral alumina post, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution.Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast.According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D), be filler with octadecylsilane chemically bonded silica; Water-acetonitrile (48: 26) is a mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 293nm; Column temperature: 20 ℃.Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak.Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(6) get Thrombus-resisting capsulesContent 10g adds water 40ml, and little the boiling of hydrochloric acid 3ml heating refluxed 1 hour, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 0.5 hour discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution.Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-benzene-ethyl acetate-glacial acetic acid (7: 11: 3: 0.2) be developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

(7) get Thrombus-resisting capsulesContent 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divide three extractions (20,20,10ml) with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions (20,20,10ml) with chloroform 50ml again, combined chloroform liquid washes with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with petroleum ether (30～60 ℃)-ethyl acetate (1.2: 0.9), put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp (365nm) and is inspected after the smoked several seconds.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

[assay]

A. tanshinone assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water (64: 9.5); Flow velocity: 1.0ml/min; Column temperature: 25 ℃; Detect wavelength 273nm.Number of theoretical plate is with Tanshinone I I A (C ₁₉H ₁₈O ₃) the peak meter, should be not less than 2000.

The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets (containing tanshinone 16 μ g among every 1ml).

The preparation of need testing solution is got Thrombus-resisting capsulesContent 3g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 50ml, the close plug of adding, claim decide weight, placement is spent the night, and supersound process 10 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane (0.45 μ m).

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Every of this product contains Radix Salviae Miltiorrhizae with Tanshinone I I A (C ₁₉H ₁₈O ₃) must not count and be less than 52 μ g.

B. the assay of strychnine is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine (52: 16: 0.9) is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 20 ℃; The detection wavelength is 252nm.Number of theoretical plate calculates by the strychnine peak should be not less than 2000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly.

The preparation of need testing solution is got Thrombus-resisting capsulesContent 10g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 2ml, close plug, jolting gently, claim to decide weight, placed supersound process 20 minutes 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 5 times, each 25ml with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 5 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.Every of this product contains Semen Strychni with strychnine (C ₂₁H ₂₂N ₂O ₂) count 47～86 μ g.

Embodiment 5 thrombus-resisting capsules

[prescription] Radix Angelicae Sinensis 200g, Radix Salviae Miltiorrhizae 200g, Bombyx Batryticatus (parched with bran) 100g, Gekko Swinhonis 100g, Eupolyphaga Seu Steleophaga 200g, Scolopendra 50g, Hirudo 200g, Nidus Vespae 100g, Pheretima 100g, Semen Strychni (system) 30g, Moschus 3g, Venenum Bufonis (processed with wine) 1g, Radix Glycyrrhizae 100g, Rhizoma Smilacis Glabrae 200g, Rhizoma Corydalis (vinegar system) 100g, Rhizoma Drynariae (system) 200g, Zaocys (processed with wine) 200g, Tabanus (removing wing) 50g, Squama Manis (husky scalding) 50g

[method for making] above raw material, except that Moschus, Venenum Bufonis, 17 flavors such as all the other Radix Angelicae Sinensis are ground into fine powder, with Moschus, Venenum Bufonis difference porphyrize, with above-mentioned powder facing-up, sieve, and mixing incapsulates, promptly.

[discriminating]

(1) gets Thrombus-resisting capsulesContent, put microscopically and observe: mycelium is closely colourless, and elongated curling becomes entangled in the body wall.Body wall fragment dark-brown or yellow, on it give birth to short and thick or elongated bristle, the circular trichopore after the Chang Kejian bristle comes off.Parenchyma cell contains prism of calcium oxalate around the fibre bundle, forms crystalline cellulose; Bordered pit vessel is bigger.The needle-like calcium oxalate crystal bundle is present in the mucilage cell or is dispersed in.Gelatinized starch grain agglomerate is faint yellow or closely colourless.Scutellum is near colourless or faint yellow; Rhabdium as seen, and is faint yellow or closely colourless, how cataclasm, and the side is seen and is strip and block more.

(2) get Thrombus-resisting capsulesContent 3g adds normal hexane 20ml supersound process 20 minutes, filters, and filtrate is concentrated into 1ml, as need testing solution.Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate (13: 1.4) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

(3) get Thrombus-resisting capsulesContent 10g adds little the boiling of water 250ml heating and refluxed 1.5 hours, filters, and filtrate is put and is concentrated into extractum in the water-bath, adds methanol 8ml, stirs, and places 1 hour, filters, and filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution.Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate is developing solvent at 12: 1.3, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

(4) get Thrombus-resisting capsulesContent 8g puts in the 500ml round-bottomed flask, adds water 200ml, benzene 1.0ml, and mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and keep little and boiled 2.5 hours, puts coldly, divides and gets the benzene layer as need testing solution.Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution.According to gas chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 E), be immobile phase with 100% methyl silicone (OV-1); Fid detector; Elastic quartz capillary tube chromatographic column (30m * 0.25mm * 0.32 μ m); Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min.Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(5) get Thrombus-resisting capsulesContent 20g adds petroleum ether (30～60 ℃) 50ml merceration 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina (100～200 order) 4g, packing into, (1.5 * 20cm), usefulness chloroform 50ml eluting is collected eluent to the neutral alumina post, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution.Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast.According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D), be filler with octadecylsilane chemically bonded silica; Water-acetonitrile (70: 52) is a mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 299nm; Column temperature: 60 ℃.Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak.Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram.In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged.

(6) get Thrombus-resisting capsulesContent 10g adds water 40ml, and little the boiling of hydrochloric acid 3ml heating refluxed 2 hours, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 1.5 hours discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution.Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-benzene-ethyl acetate-glacial acetic acid (14: 30: 11: 1.1) be developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

(7) get Thrombus-resisting capsulesContent 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divide three extractions (20,20,10ml) with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions (20,20,10ml) with chloroform 50ml again, combined chloroform liquid washes with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with petroleum ether (30～60 ℃)-ethyl acetate (2.9: 2.8), put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp (365nm) and is inspected after the smoked several seconds.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

[assay]

A. Tanshinone I I A assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water (110: 22); Flow velocity: 1.0ml/min; Column temperature: 55 ℃; Detect wavelength 273nm.Number of theoretical plate is with tanshinone (C ₁₉H ₁₈O ₃) the peak meter, should be not less than 2000.

The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets (containing Tanshinone I I A16 μ g among every 1ml).

The preparation of need testing solution is got Thrombus-resisting capsulesContent 3g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 50ml, the close plug of adding, claim decide weight, placement is spent the night, and supersound process 60 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane (0.45 μ m).

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Every of this product contains Radix Salviae Miltiorrhizae with Tanshinone I I A (C ₁₉H ₁₈O ₃) must not count and be less than 52 μ g.

B. the assay of strychnine is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine (94: 33: 3.4) is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 60 ℃; The detection wavelength is 256nm.Number of theoretical plate calculates by the strychnine peak should be not less than 2000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly.

The preparation of need testing solution is got Thrombus-resisting capsulesContent 10g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 8ml, close plug, jolting gently, claim to decide weight, placed supersound process 40 minutes 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 9 times, each 25ml with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 9 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Every of this product contains Semen Strychni with strychnine (C ₂₁H ₂₂N ₂O ₂) count 47～86 μ g.

Claims (7)

1. the method for quality control of a thromboembolism preventing preparation, comprise character is observed, official method is checked content, content Radix Angelicae Sinensis, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae are differentiated, Tanshinone I I A and the strychnine that contains carried out assay; It is characterized in that, described content Radix Angelicae Sinensis, Pheretima, Semen Strychni, Moschus, Rhizoma Corydalis, Bufo siccus and Radix Glycyrrhizae are differentiated, through following method:

A. get thromboembolism preventing preparation content 3g, add normal hexane 20ml supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution; Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2000 B thin layer chromatography, 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate 6-13: 0.7-1.4 is developing solvent, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

B. get thromboembolism preventing preparation content 10g, add little the boiling backflow 0.5-1.5 hour of water 250ml heating, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution; Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2000 B thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate 6-12: 0.7-1.3 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

C. get thromboembolism preventing preparation content 20g, add 30～60 ℃ of petroleum ether 50ml mercerations 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with 100～200 order neutral alumina 4g, 1.5 * 20cm neutral alumina post of packing into chloroform 50ml eluting, is collected eluent, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution; Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast; According to an appendix VI of Chinese Pharmacopoeia version in 2000 D high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; Water-acetonitrile 48-70: 26-52 is a mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 296 ± 3nm; Column temperature: 20-60 ℃; Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak; Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram; In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged;

D. get thromboembolism preventing preparation content 10g, add water 40ml, hydrochloric acid 3ml heats little boiling backflow 1-2 hour, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 0.5-1.5 hour discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition; Test according to thin layer chromatography, 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 adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of petroleum ether-benzene-ethyl acetate-glacial acetic acid 7-14: 11-30: 3-11: 0.2-1.1 is developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

E. get thromboembolism preventing preparation content 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divides three extractions with 10% acetic acid 50ml, each 20,20,10ml merge acetate layer, transfer pH10～11 with ammonia, divide three extractions with chloroform 50ml again, each 20,20,10ml, combined chloroform liquid, wash with water to neutrality, add an amount of anhydrous sodium sulfate, filter, filtrate evaporate to dryness, residue add methanol 0.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with 30～60 ℃ of petroleum ether-ethyl acetate 1.2-2.9: 0.9-2.8, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the 365nm ultra-violet lamp and is inspected after the smoked several seconds; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Describedly the tanshinone that contains and strychnine are carried out assay may further comprise the steps:

According to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water 64-110: 9.5-22; Flow velocity: 1.0ml/min; Column temperature: 25-55 ℃; Detect wavelength 270 ± 3nm; Number of theoretical plate is with Tanshinone I I AC ₁₉H ₁₈O ₃The peak meter should be not less than 2000;

The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets to contain tanshinone 16 μ g among every 1ml;

Thromboembolism preventing preparation content 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, placement is spent the night, supersound process 10-60 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

2. the method for claim 1 is characterized in that, the content assaying method in this method is:

According to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine 52-94: 16-33: 0.9-3.4 is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 20-60 ℃; The detection wavelength is 254 ± 2nm; Number of theoretical plate calculates by the strychnine peak should be not less than 2000;

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly;

Thromboembolism preventing preparation content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 2-8ml, close plug, jolting gently, claim to decide weight, placed supersound process 0-40 minute 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, press 3 → 100 usefulness sulphuric acid solution extraction 5-9 time, each 25ml merges sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, with chloroform extraction 5-9 time, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly;

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

3. the described method of claim 1 is characterized in that, content assaying method is in this method:

According to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine is a mobile phase at 75: 25: 2; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 254nm; Number of theoretical plate calculates by the strychnine peak should be not less than 2000;

It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly;

This product content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 8ml, close plug, jolting gently, claim to decide weight, placed supersound process 20 minutes 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 7 times, each 25ml with sulfuric acid solution 3 → 100, merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 7 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly;

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly; Every contains Semen Strychni with strychnine C ₂₁H ₂₂N ₂O ₂Count 47～86 μ g.

4. the method for claim 1 is characterized in that, the following method of process:

Differentiate:

A. get thromboembolism preventing preparation content 3g, add normal hexane 20ml supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution; Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system; 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate 6-13: 0.7-1.4 is developing solvent, launches, and takes out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

B. get thromboembolism preventing preparation content 10g, add little the boiling backflow 0.5-1.5 hour of water 250ml heating, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution; Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system; Test according to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate 6-12: 0.7-1.3 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

C. get thrombus-resisting capsules preparation content 8g, put in the 500ml round-bottomed flask, add water 200ml, benzene 1.0ml, mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and kept little 1.5-2.5 of boiling hour, puts coldly, divides and gets the benzene layer as need testing solution; Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution; According to gas chromatography, be immobile phase with 100% methyl silicone OV-1; Fid detector; Elastic quartz capillary tube chromatographic column 30m * 0.25mm * 0.32 μ m; Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min; Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram; In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged;

D. get thrombus-resisting capsules preparation content 20g, add 30～60 ℃ of petroleum ether 50ml mercerations 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina 100～200 order 4g, the neutral alumina post 1.5 * 20cm that packs into chloroform 50ml eluting, collects eluent, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution; Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast; According to high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; Water-acetonitrile 48-70: 26-52 is a mobile phase; Flow velocity: 1.0ml/min; Detect wavelength 296 ± 3nm; Column temperature: 20-60 ℃; Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak; Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram; In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged;

E. get thrombus-resisting capsules preparation content 10g, add water 40ml, hydrochloric acid 3ml heats little boiling backflow 1-2 hour, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 0.5-1.5 hour discards ether solution, medicinal residues wave in, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition; Test according to thin layer chromatography, 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 adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of petroleum ether-benzene-ethyl acetate-glacial acetic acid 7-14: 11-30: 3-11: 0.2-1.1 is developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

F. get thrombus-resisting capsules preparation content 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, the leaching supernatant is put in the separatory funnel, divides three extractions with 10% acetic acid 50ml, be 20ml for the first time, being 20ml for the second time, is 10ml for the third time, merges acetate layer, transfer pH10～11 with ammonia, divide three extractions with chloroform 50ml again, each 20,20,10ml, combined chloroform liquid, wash with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with 30～60 ℃ of petroleum ether-ethyl acetate 1.2-2.9: 0.9-2.8, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp 365nm and is inspected after the smoked several seconds; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Assay:

A. tanshinone assay: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water 64-110: 9.5-22; Flow velocity: 1.0ml/min; Column temperature: 25-55 ℃; Detect wavelength 270 ± 3nm; Number of theoretical plate is with tanshinone C ₁₉H ₁₈O ₃The peak meter should be not less than 2000; The preparation precision of reference substance solution takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets to contain tanshinone 16 μ g among every 1ml; Thromboembolism preventing preparation content 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, placement is spent the night, supersound process 10-60 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly;

B. strychnine assay: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine 52-94: 16-33: 0.9-3.4 is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 20-60 ℃; The detection wavelength is 254 ± 2nm; Number of theoretical plate calculates by the strychnine peak should be not less than 2000; It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly; Thromboembolism preventing preparation content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 2-8ml, close plug, jolting gently, claim to decide weight, placed supersound process 0-40 minute 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 5-9 time, each 25ml with sulfuric acid solution 3 → 100, merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 5-9 time, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly; Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

5. the method for claim 1 is characterized in that, the following method of process:

Differentiate:

A. get thrombus-resisting capsules preparation content 3g, add normal hexane 20ml supersound process 15 minutes, filter, filtrate is concentrated into 1ml, as need testing solution; Other gets Radix Angelicae Sinensis control medicinal material 0.1g, shines medical material solution in pairs with legal system; 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 adhesive with the sodium carboxymethyl cellulose, with normal hexane-ethyl acetate is developing solvent at 9: 1, launches, and takes out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color; Get this product content 10g, add little the boiling of water 250ml heating and refluxed 1 hour, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution; Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system; According to thin layer chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate is developing solvent at 9: 1, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

B. get thrombus-resisting capsules preparation content 10g, add little the boiling of water 250ml heating and refluxed 1 hour, filter, filtrate is put and is concentrated into extractum in the water-bath, add methanol 8ml, stir, placed 1 hour, filter, filtrate is concentrated into 0.6～0.8ml, adds benzene 0.6ml, and jolting divides and gets the benzene layer as need testing solution; Other gets Pheretima control medicinal material 1g, shines medical material solution in pairs with legal system; According to thin layer chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate is developing solvent at 9: 1, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

C. get thrombus-resisting capsules preparation content 8g, put in the 500ml round-bottomed flask, add water 200ml, benzene 1.0ml, mixing connects volatile oil determination apparatus and reflux condensing tube, is heated to and boils, and keep little and boiled 2 hours, puts coldly, divides and gets the benzene layer as need testing solution; Other gets the muscone reference substance, adds benzene and makes the solution that every 1ml contains 0.2mg, in contrast product solution; According to gas chromatography, be immobile phase with 100% methyl silicone OV-1; Fid detector; Elastic quartz capillary tube chromatographic column 30m * 0.25mm * 0.32 μ m; Temperature programming: 200 ℃-1min-3 ℃/min-240 ℃-0min-20 ℃/min-260 ℃-5min; Draw each 4 μ l of above-mentioned two kinds of solution respectively, inject gas chromatograph, record chromatogram; In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged;

D. get thrombus-resisting capsules preparation content 20g, add 30～60 ℃ of petroleum ether 50ml mercerations 30 minutes, jolting constantly, discard petroleum ether liquid, medicinal residues volatilize, and add chloroform 80ml supersound process 30 minutes, filter, the filtrate evaporate to dryness, residue adds methanol 40ml dissolving, filters, and filtrate is concentrated into about 2ml, in water-bath, stir dry with neutral alumina 100～200 order 4g, the neutral alumina post 1.5 * 20cm that packs into chloroform 50ml eluting, collects eluent, evaporate to dryness, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, as need testing solution; Other gets cinobufagin, each is an amount of for the resibufogenin reference substance, adds methanol and makes the mixed solution that every 1ml contains cinobufagin 7 μ g, resibufogenin 18 μ g, product solution in contrast; According to high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; Water-acetonitrile is a mobile phase at 60: 40; Flow velocity: 1.0ml/min; Detect wavelength 296nm; Column temperature: 40 ℃; Number of theoretical plate should be not less than 4000 respectively in cinobufagin, resibufogenin peak; Draw each 20 μ l of above-mentioned two kinds of solution respectively, inject chromatograph of liquid, the record chromatogram; In the test sample chromatograph, the chromatographic peak with the identical retention time of reference substance chromatograph should be arranged;

E. get thrombus-resisting capsules preparation content 10g, add water 40ml, little the boiling of hydrochloric acid 3ml heating refluxed 1.5 hours, filter, discard filtrate, medicinal residues volatilize, the 30ml reflux, extract, that adds diethyl ether 1 hour discards ether solution, and medicinal residues volatilize, add chloroform 30ml reflux, extract, 1 hour, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, filter, filtrate is as need testing solution; Extracting liquorice subacid reference substance adds dehydrated alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution in addition; Test according to thin layer chromatography, 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 adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of petroleum ether-benzene-ethyl acetate-glacial acetic acid 10: 20: 7: 0.5 was developing solvent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

F. get thrombus-resisting capsules preparation content 5g, add ammonia an amount of stir moistening after, the 40ml that adds diethyl ether placed 24 hours, jolting constantly, leaching supernatant, put in the separatory funnel, divide three extractions with 10% acetic acid 50ml, each 20,20,10ml merges acetate layer, transfer pH10～11 with ammonia, divide three extractions with chloroform 50ml again, each 20,20,10ml, combined chloroform liquid, wash with water to neutrality, add an amount of anhydrous sodium sulfate, filter the filtrate evaporate to dryness, residue adds methanol 0.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds methanol and makes the solution that every 1ml contains 0.5ml, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 5 μ l, reference substance solution 1 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be at 2: 1.7 developing solvent with 30～60 ℃ of petroleum ether-ethyl acetates, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, iodine was put under the ultra-violet lamp 365nm and is inspected after the smoked several seconds; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Assay:

A. Tanshinone I I A assay: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Mobile phase: methanol-water 85: 15; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 270nm; Number of theoretical plate is with tanshinone C ₁₉H ₁₈O ₃The peak meter should be not less than 2000; The preparation precision of reference substance solution takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets to contain tanshinone 16 μ g among every 1ml; This product content 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, placement is spent the night, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, get supernatant and filter, promptly with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly; Every of this product contains Radix Salviae Miltiorrhizae with Tanshinone I I AC ₁₉H ₁₈O ₃Meter must not be less than 52 μ g;

B. the assay of strychnine is according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with silica gel; Chloroform-cyclohexane extraction-diethylamine is a mobile phase at 75: 25: 2; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 254nm; Number of theoretical plate calculates by the strychnine peak should be not less than 2000; It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds ethyl acetate and make the solution that every 1ml contains 0.2mg, promptly; This product content 10g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 8ml, close plug, jolting gently, claim to decide weight, placed supersound process 20 minutes 24 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with extracting solution, shake well filters, and precision is measured subsequent filtrate 25ml, put in the separatory funnel, extract 7 times, each 25ml with sulfuric acid solution 3 → 100, merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 9～10, use chloroform extraction 7 times, each 30ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shake up, promptly; Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly; Every contains Semen Strychni with strychnine C ₂₁H ₂₂N ₂O ₂Count 47～86 μ g.

6. any one method of claim 1-5 is characterized in that, described thromboembolism preventing preparation is an oral formulations.

7. the method for claim 6 is characterized in that, described oral formulations is capsule, tablet, oral liquid, syrup or granule.