CN1824245A - Quality control method of bone sinew medicinal pill preparation - Google Patents

Abstract

A quality control method for the Chinese medicine Bone-tandon pill features that the liquorice root, motherwort and paniculate swallowwort are discriminated by thin-layer chromatography method and the content of glycyrrhizic acid is measured by efficient liquid-phase chromatography method.

Description

The method of quality control of bone sinew medicinal pill preparation

Technical field: the present invention relates to a kind of method of quality control of bone sinew medicinal pill preparation, belong to the field of medicine technology.

Technical background: hypertrophic spondylitis, cervical spondylosis are a kind of commonly encountered diseases of the current people's health of harm.

Bone sinew medicinal pill preparation by Olibanum, Myrrha, the Radix Paeoniae Alba, Rhizoma Corydalis (vinegar system), Radix Notoginseng, the Radix Aucklandiae, Flos Carthami, Radix Curcumae, Radix Angelicae Pubescentis, Radix Achyranthis Bidentatae, Radix Gentianae Macrophyllae, Ramulus Cinnamomi, Sanguis Draxonis, Semen Strychni (system) totally ten four Chinese medicines form; Have blood circulation promoting and blood stasis dispelling, channels sootheing and network vessel quickening, the effect of wind-expelling pain-stopping is used for the treatment of hypertrophic spondylitis, cervical spondylosis, calcanean spur, proliferative arthritis, diseases such as Kaschin-Beck disease.Wherein " 'Gu Jin Wan ' capsule ", " iliacus ball sheet " are all on the books on the 13 in Chinese traditional patent formulation preparation promulgated by the ministries or commissions of the Central Government, this medicine is used for many years clinically, at the treatment hypertrophic spondylitis, cervical spondylosis, calcanean spur, proliferative arthritis, disease aspects such as Kaschin-Beck disease obtain satisfied therapeutic effect, but discover through us, existing bone sinew medicinal pill preparation exists quality control standard simply backward, the uppity shortcoming of product quality.Because the Radix Paeoniae Alba, Radix Notoginseng, Rhizoma Corydalis, Radix Angelicae Pubescentis, Radix Gentianae Macrophyllae, Semen Strychni are the medicine that plays a major role in the bone sinew medicinal pill preparation, wherein Semen Strychni still is the toxicity medical material, and only alkaloids medical material and Radix Angelicae Pubescentis medical material are carried out the chromogenic reaction discriminating in the existing preparation quality standard, other medical materials are not carried out assay or Study on Identification, so existing method of quality control can not effectively be controlled the quality of bone sinew medicinal pill 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 bone sinew medicinal pill preparation, and this method adopts according to thin layer chromatography Radix Glycyrrhizae, Herba Leonuri, Radix Cynanchi Paniculati are differentiated, adopt high performance liquid chromatography that the glycyrrhizic acid composition is 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 objective of the invention is to: a kind of method of quality control of bone sinew medicinal pill preparation is provided, and described iliacus ball Chinese medicine preparation is an oral formulations, preferably capsule, tablet or granule.

Its prescription of bone sinew medicinal pill preparation of the present invention and method for making are as follows:

Calculate according to components by weight percent, it mainly is prepared from by Olibanum 20-35 part, Myrrha 30-60 part, Radix Paeoniae Alba 70-130 part, Rhizoma Corydalis (vinegar system) 20-35 part, Radix Notoginseng 30-60 part, Radix Aucklandiae 20-35 part, Flos Carthami 30-60 part, Radix Curcumae 70-130 part, Radix Angelicae Pubescentis 160-220 part, Radix Achyranthis Bidentatae 30-60 part, Radix Gentianae Macrophyllae 160-220 part, Ramulus Cinnamomi 30-60 part, Sanguis Draxonis 20-35 part, Semen Strychni (system) 20-35 part.

Preferably, calculate according to components by weight percent, it mainly is prepared from for 27 parts by 27 parts of Olibanums, 46 parts of Myrrhas, 91 parts of the Radix Paeoniae Albas, 27 parts of Rhizoma Corydalis (vinegar system), Radix Notoginseng 46, part 27 parts of the Radix Aucklandiae, 46 parts on Flos Carthami, 91 parts of Radix Curcumaes, 183 parts of Radix Angelicae Pubescentiss, 46 parts of Radix Achyranthis Bidentataes, 183 parts of Radix Gentianae Macrophyllae, 46 parts of Ramulus Cinnamomi, 27 parts of Sanguis Draxonis, Semen Strychni (system)

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 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.

Preferred manufacturing procedure is as follows:

Preparation of the present invention all prepares capsule and pill according to the method about " 'Gu Jin Wan ' capsule ", " iliacus ball sheet " record on the Chinese traditional patent formulation preparation promulgated by the ministries or commissions of the Central Government, and tablet, granule, oral liquid etc. also can prepare according to routine techniques according to above method.

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 bone sinew medicinal pill preparation at above bone sinew medicinal pill preparation.At its characteristics and prescription of the present invention, we provide following method of quality control:

Character is observed, content is differentiated, official method is checked content, and gentiopicrin and the strychnine that contains carried out assay.

Wherein character is observed, step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet or granule respectively, add ethanol, warm macerating filters, and gets filtrate, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate, and evaporate to dryness adds boiling water and makes dissolving, filters while hot, puts coldly, with chloroform extraction 1-6 time, discards chloroform layer, the aqueous solution ethyl acetate extraction, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm observation down, apparent sapphirine fluorescence;

(3) get capsule, tablet or granule respectively, add ethanol, supersound process, filter, filtrate evaporate to dryness, residue add water makes dissolving, filter, filtrate is colourless to ether layer with ether extraction, discards ether solution, the water saturated n-butanol extraction of reuse 1-6 time, merge n-butanol extracting liquid, water washing 1-6 time discards water liquid, evaporate to dryness, residue adds ethanol makes dissolving, admixes neutral alumina, stirs dry in the water-bath, the little column top of neutral alumina of packing into and filling in advance, with methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds dissolve with ethanol, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.2-1g, adds ethanol, and jolting filters, and filtrate evaporate to dryness, residue add ethanol makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, it is clear to be heated 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 capsule, tablet or granule respectively, put in the tool plug triangular flask, after adding ammonia and stirring moistening, add diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 1-6 time, transfer pH9～13 with ammonia, again with chloroform extraction 1-6 time, combined chloroform liquid, wash with water 1-5 time, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol and makes dissolving, as need testing solution; Other gets the Rhizoma Corydalis control medicinal material, is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add dissolve with methanol, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet or granule respectively, add petroleum ether (60～90 ℃), close plug, supersound process filters, and filtrate is as need testing solution; Other gets the Radix Angelicae Pubescentis control medicinal material, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol or acetonitrile: water=3: 5-8 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, uses dissolve with methanol, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item respectively, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol that adds, close plug claims to decide weight, supersound process, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, get supernatant and use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In the said preparation, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item respectively, tablet or granule, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform and strong ammonia solution, close plug, jolting gently, claim to decide weight, place, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate, put in the separatory funnel, extract 2-10 time, merge sulphuric acid liquid with sulfuric acid solution (3 → 100), add strong ammonia solution and regulate pH value to 8～12, with chloroform extraction 2-10 time, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, shake up, use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine;

Preferable methods is:

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet 1-5g respectively; Or get granule 5-15g, and add ethanol 10-50ml, warm macerating 10-60 minute, filter, get filtrate 1-5ml, evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 1～5 of bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate 5-15ml, and evaporate to dryness adds boiling water 10-20ml and makes dissolving, filters while hot, put coldly, with chloroform extraction 1-6 time, discard chloroform layer, aqueous solution is with ethyl acetate 5-20ml extraction, extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule respectively, tablet 1-5g, or get granule 5-15g, add ethanol 20-100ml, supersound process 10-60 minute, filter the filtrate evaporate to dryness, residue adds water 10-30ml makes dissolving, filters, and filtrate is colourless to ether layer with ether extraction, discard ether solution, the water saturated n-butanol extraction of reuse 1-6 time merges n-butanol extracting liquid, water washing 1-6 time, discard water liquid, evaporate to dryness, residue add ethanol 0.5-3ml makes dissolving, admix neutral alumina, stir drying in the water-bath, the little column top of neutral alumina of packing into and filling in advance is with methanol 20-100ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.2-1ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.2-1g, adds ethanol 5-30ml, and jolting 3-15 minute, filter, filtrate evaporate to dryness, residue add ethanol 0.5-2ml makes dissolving, in contrast medical material solution; According to the thin layer chromatography test, draw each 5-25 μ l of above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

(4) get capsule, tablet 1-5g respectively; Or get granule 5-15g, and put in the tool plug triangular flask, after adding ammonia and stirring moistening, 30-100ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 1-6 time, transfer pH9～13 with ammonia, again with chloroform extraction 1-6 time, combined chloroform liquid, wash with water 1-5 time, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 0.5-2ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 0.3-1g and is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 0.3-1mg, in contrast product solution; Test according to thin layer chromatography, draw each 5-15 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet 0.5-3g respectively; Or get granule 3-10g, and add petroleum ether (60～90 ℃) 2-20ml, close plug, supersound process 2-20 minute, filter, filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 0.3-1g, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw each 5-25 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol or acetonitrile: water=3: 5-8 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.03-0.1mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 0.1-1g under the content uniformity item respectively; Or get granule 1-2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 30-100ml that adds, close plug claims to decide weight, supersound process 5-50 minute, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, get supernatant and use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and each 3-15 μ l of need testing solution of drawing injects hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 0.05-2mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 2-5g respectively; Or get granule 5-15g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 20-100ml and strong ammonia solution 2-10ml, close plug, jolting gently, claim to decide weight, placed 10-50 hour, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 5-15ml, puts in the separatory funnel, extracts 2-10 time with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 8～12, use chloroform extraction 2-10 time, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and each 3-25 μ l of need testing solution of drawing injects hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine;

Method of the present invention obtains through research, it is considered herein that, Radix Gentianae Macrophyllae is the medicine of the main effect of performance in the bone sinew medicinal pill preparation, and wherein gentiopicrin is a main effective ingredient in the Radix Gentianae Macrophyllae, so contained Determination of gentiopicroside is measured in this preparation reply Radix Gentianae Macrophyllae.But how to measure Determination of gentiopicroside is difficult point of the present invention place in the preparation, and through experimentation, we select high performance liquid chromatography, and with methanol or acetonitrile: water=3: 5-8 is that mobile phase is measured Determination of gentiopicroside in the preparation.Semen Strychni also is the medicine of the main effect of performance in this preparation in addition, and Semen Strychni is the toxicity medical material, if the control of Semen Strychni content is bad in the preparation, also can produce harmful effect to the patient.Because strychnine is a main effective ingredient in the Semen Strychni, so among the present invention, should the content of Semen Strychni in the preparation be controlled with strychnine as detecting index, through research, the present invention adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is the content that mobile phase is measured strychnine in the preparation, the result, the negative sample chromatogram is at non-false positive peak, detection material position, and detection material is separated fully with close impurity peaks, separating degree＞1.5.

In addition, because Olibanum, Myrrha, Rhizoma Corydalis, Semen Strychni, Radix Notoginseng, Sanguis Draxonis are used as medicine with powder in this preparation, wherein Olibanum, Myrrha, Sanguis Draxonis are resin secretions, do not have obvious microscopic features, differentiate so the present invention carries out microscopic character of powder to Rhizoma Corydalis, Semen Strychni, Radix Notoginseng in the preparation.In addition, the present invention also carries out the chromogenic reaction discriminating to Radix Angelicae Pubescentis medical material in the preparation and alkaloids medical material.Simultaneously, the present invention also carries out the thin layer Study on Identification to the Radix Paeoniae Alba, Rhizoma Corydalis and Radix Angelicae Pubescentis, and to select with Radix Paeoniae Alba control medicinal material be contrast, and with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is that white Peony Root in the preparation is differentiated in developing solvent; With the Rhizoma Corydalis control medicinal material is contrast, and with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is that Rhizoma Corydalis radix scutellariae medicinal materials in the preparation is differentiated in developing solvent; With the Radix Angelicae Pubescentis control medicinal material is contrast, is that Radix Angelicae Pubescentis medical material in the preparation is differentiated in developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1.Its separating degree is good as a result, and the speckle colour developing is clear, and negative control is noiseless, the method favorable reproducibility.So adopt method of quality control of the present invention can effectively control the quality of this bone sinew medicinal pill preparation, thereby guarantee the clinical efficacy of said preparation.

Method of quality control of the present invention is the preferred plan that obtains through a large amount of screenings, and following experimentation is a preferred process of the present invention.

One, gentiopicroside in different morphological study on determination method

1, need testing solution preparation method research:

The thing of getting it filled, porphyrize extracts with methods such as immersion, water-bath reflux, extract,, ultrasonic extraction, Soxhlet reflux, extract, respectively and measures its content after 1 hour, the results are shown in following table:

Ultrasonic time	Soak	The water-bath reflux, extract,	Supersound extraction	The Soxhlet reflux, extract,
					Content (%) RSD (%)	0.6572 2.65	1.0415 2.18	1.1217 2.64	1.0480 2.07

Result of the test shows, soak not extract gentiopicrin in its preparation is extracted fully, supersound extraction, water-bath reflux, extract,, Soxhlet reflux, extract, all can be extracted gentiopicrin in the preparation fully, and the three differs minimum, for easy and simple to handle, select ultrasonic extracting method for use.

2, the selection of mobile phase:

Mobile phase 1: the mixed solution with methanol, water different proportion is a mobile phase.

Mobile phase 2: the mixed solution with acetonitrile, water different proportion is a mobile phase.

Mobile phase 3: the mixed solution with acetonitrile, phosphoric acid, triethylamine, water different proportion is a mobile phase.

Mobile phase 4: the mixed solution with methanol, water, acetonitrile different proportion is a mobile phase.

The result: with methanol or acetonitrile: water=1-9: 9-1 is mobile phase; The negative sample chromatogram is at non-false positive peak, place, gentiopicrin peak position, and the gentiopicrin peak separates fully (separating degree＞1.5) with close impurity peaks, and promptly the gentiopicrin peak separates with other components fully under this condition.Optimal flow is mutually: methanol: water=3: 7.

3, repeatability test

The product of getting it filled, the preparation method by test liquid under the assay item among the present invention prepares 5 parts of test liquids respectively, and sample introduction is measured peak area, the results are shown in following table.

The sample introduction number of times	1	2	3	4	5	Meansigma methods	RSD(％)
								Content (%)	1.038	1.059	1.093	1.062	1.045	1.059	2.00

The result shows that this method repeatability is good.

4, recovery test

Adopt the application of sample absorption method, it is an amount of that precision takes by weighing 5 parts of samples of having measured content respectively, puts in the tool plug conical flask, (with methanol is solvent to accurate adding gentiopicrin reference substance solution, 0.0256mg/ml) 50ml, close plug claims to decide weight, supersound process 15 minutes, put coldly, supply the weight that subtracts mistake, shake up with methanol, get supernatant and filter, make test liquid with filter membrane (0.45 μ m).The accurate respectively 5 μ l of absorption inject chromatograph of liquid, and the record chromatograph is measured content, and calculate recovery rate, average recovery rate are 99.43%, and RSD is 1.61%.Prove that this method is feasible.

Test number (TN)	Sample size (g)	Contain gentiopicrin (mg)	Add gentiopicrin (mg)	The amount of recording (mg)	The response rate (%)	Average recovery rate (%)	RSD (％)
								1 2 3 4 5	0.0901 0.1051 0.1075 0.1200 0.1089	0.9542 1.1130 1.1384 1.2708 1.1533	1.28	2.1979 2.3752 2.4224 2.5672 2.4308	97.16 98.61 100.3 101.3 99.80	99.43	1.61

Two, strychnine content assaying method research

1, need testing solution preparation method research:

Be easy under alkali condition by the characteristic of organic solvent extraction according to strychnine, we have selected in test to be usually used in extracting alkaloidal chloroform-dense ammonia or methanol-dense ammonia is extracting solution, the results are shown in following table:

Extracting method	Methanol-dense ammonia (20: 1)	Chloroform-dense ammonia (50: 1)	Chloroform-dense ammonia (50: 6)
				Content (%) RSD (%)	0.0941 3.53	0.1114 2.21	0.1519 1.21

Result of the test shows, is that extractant can extract strychnine fully with chloroform-dense ammonia.

2, the selection of mobile phase:

Mobile phase 1: the mixed solution with normal hexane, dichloromethane, methanol, strong ammonia solution different proportion is a mobile phase.

Mobile phase 2: the mixed solution with ether, acetonitrile, diethylamine different proportion is a mobile phase.

Mobile phase 3: the mixed solution with ether, methanol, diethylamine different proportion is a mobile phase.

Mobile phase 4: the mixed solution with methanol, acetonitrile, water, glacial acetic acid different proportion is a mobile phase.

Result: with ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is a mobile phase; The negative sample chromatogram is at non-false positive peak, place, strychnine peak position, and the strychnine peak separates fully (separating degree＞1.5) with close impurity peaks, and promptly the strychnine peak separates with other components fully under this condition.Optimal flow is mutually: ether: methanol: diethylamine=80: 20: 1.

3, repeatability test

The product of getting it filled, the preparation method by test liquid under the assay item among the present invention prepares 5 parts of test liquids respectively, and sample introduction is measured peak area, and average content is 0.1556%, and RSD is 2.41%.The results are shown in following table:

The sample introduction number of times	1	2	3	4	5	Meansigma methods	RSD(％)
								Content (%)	0.1599	0.1546	0.1557	0.1577	0.1499	0.1556	2.41

The result shows that this method repeatability is good.

4, recovery test

Adopt the application of sample absorption method, it is an amount of that precision takes by weighing 5 parts of samples (average content is 0.1556%) of having measured content respectively, puts in the tool plug conical flask, accurate strychnine reference substance solution (0.055mg/ml is a solvent with the chloroform) 50ml and strong ammonia solution 6ml, the close plug of adding, jolting gently claims to decide weight, places 24 hours, claim again to decide weight, supply the weight that subtracts mistake, shake well with chloroform, filter, precision is measured subsequent filtrate 10ml, puts in the separatory funnel, extract 5 times with sulfuric acid solution (3 → 100), each 15ml merges sulphuric acid liquid, adds strong ammonia solution and regulates pH value to 9～10, with chloroform extraction 5 times, each 20ml, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, be transferred in the 10ml measuring bottle, and be diluted to scale, shake up, filter with filter membrane (0.45 μ m), make test liquid.The accurate respectively 5 μ l of absorption inject chromatograph of liquid, and the record chromatograph is measured content, and calculate recovery rate, average recovery rate are 99.66%, and RSD is 2.67%.Prove that this method is feasible.

Test number (TN)	Sample size (g)	Contain strychnine (mg)	Add strychnine (mg)	The amount of recording (mg)	The response rate (%)	Average recovery rate (%)	RSD (％)
								1 2 3 4 5	1.3241 1.3254 1.9032 1.8518 1.9586	2.0603 2.0623 2.9614 2.8814 3.0476	2.75	4.7198 4.7381 5.7025 5.7014 5.8552	96.71 97.30 99.68 102.5 102.1	99.66	2.67

Three, Radix Paeoniae Alba thin layer Study on Identification

With white Peony Root in the Radix Paeoniae Alba control medicinal material discriminating side.

Need testing solution preparation method one: get this product, add the ethanol jolting and extract, filter, an amount of dissolve with ethanol of filtrate evaporate to dryness, residue is as need testing solution.The negative need testing solution that lacks white Peony Root with the method preparation.

Need testing solution preparation method two: get this product content and add the ethanol supersound process, filter the filtrate evaporate to dryness, residue adds water makes dissolving, colourless to ether layer with ether extraction, discards ether solution, the water saturated n-butanol extraction of reuse, merge n-butanol extracting liquid, washing discards water liquid, evaporate to dryness, residue adds ethanol 1ml makes dissolving, admixes a little neutral alumina, stirs dry in the water-bath, a little neutral alumina pillar (200～300 order of neutral alumina pillar of packing into and filling in advance, 1g, internal diameter 10～15mm) tops are with methanol-eluted fractions, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.5ml, as need testing solution.The negative need testing solution that lacks white Peony Root with the method preparation.

Developing solvent is selected: respectively with the mixed solution of chloroform, acetone, methanol and glacial acetic acid different proportion; Mixed solution with chloroform, acetone and glacial acetic acid different proportion; Mixed solution with normal hexane, ethyl acetate different proportion is developing solvent.

The result: adopt method two to prepare need testing solution, with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is developing solvent, good separating effect, and clear spot, negative control is noiseless, the method favorable reproducibility.Best developing solvent is a chloroform: acetone: methanol: glacial acetic acid=5: 2: 1: 0.2.

Four, Rhizoma Corydalis thin layer Study on Identification

With corydalis tuber medicinal material in the Rhizoma Corydalis control medicinal material discriminating side

Need testing solution preparation method one: get this product, add petroleum ether (60～90 ℃) supersound process, filter, filtrate evaporate to dryness, residue add methanol makes dissolving, as need testing solution.The negative need testing solution that lacks corydalis tuber medicinal material with the method preparation.

Need testing solution preparation method two: get this product, add chloroform (with an amount of moistening of ammonia) supersound process, filtrate is washed with acetum, merges acetate solution, transfer pH9～10 with ammonia, use chloroform extraction, combined chloroform liquid, evaporate to dryness, residue add methanol makes dissolving, as need testing solution.The negative need testing solution that lacks corydalis tuber medicinal material with the method preparation.

Need testing solution preparation method three: get this product, after adding ammonia and stirring moistening in right amount, add diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant is used 10% acetic acid extraction, merges acetate layer, transfer pH10～11 with ammonia, again with chloroform extraction, combined chloroform liquid, wash with water to neutrality, add an amount of anhydrous sodium sulfate, filter, filter evaporate to dryness, add methanol 2ml and make dissolving, as need testing solution.The negative need testing solution that lacks corydalis tuber medicinal material with the method preparation.

Developing solvent is selected: respectively with the mixed solution of chloroform, ethyl acetate, methanol different proportion; Mixed solution with petroleum ether, ether, ethyl acetate different proportion; Mixed solution with cyclohexane extraction, ether, ethyl acetate different proportion is developing solvent.

The result: employing method three preparation need testing solutions, with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is developing solvent, good separating effect, clear spot, negative control is noiseless, the method favorable reproducibility.Best developing solvent is a cyclohexane extraction: ether: ethyl acetate=9: 2: 4.

Five, Radix Angelicae Pubescentis thin layer Study on Identification

With Radix Angelicae Pubescentis medical material in the Radix Angelicae Pubescentis control medicinal material discriminating side

Need testing solution preparation method one: get this product, the dipping that adds diethyl ether spends the night, and filters, and filtrate evaporate to dryness, residue add chloroform 1ml makes dissolving, as need testing solution.The negative need testing solution that lacks the Radix Angelicae Pubescentis medical material with the method preparation.

Need testing solution preparation method two: get this product, add the methanol supersound process, filter, extractions that add diethyl ether of filtrate evaporate to dryness, residue, the merging ether solution, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.The negative need testing solution that lacks the Radix Angelicae Pubescentis medical material with the method preparation.

Need testing solution preparation method three: get this product, add petroleum ether (60～90 ℃) supersound process, filter, filtrate is as need testing solution.The negative need testing solution that lacks the Radix Angelicae Pubescentis medical material with the method preparation.

Developing solvent is selected: respectively with the mixed solution of cyclohexane extraction, ether different proportion; Mixed solution with toluene, ethyl acetate different proportion; Mixed solution with cyclohexane extraction, ethyl acetate different proportion is developing solvent.

The result: employing method three preparation need testing solutions are developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1, good separating effect, and clear spot, negative control is noiseless, the method favorable reproducibility.Best developing solvent is a cyclohexane extraction: cyclohexane extraction: ethyl acetate=5: 1.5.

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:

Further specify the present invention by the following examples, but not as limitation of the present invention.

Embodiment 1

The method of quality control of 'Gu Jin Wan ' capsule agent, tablet or granule.

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, each 3g of tablet respectively, or get granule 10g; Add ethanol 20ml, warm macerating 30 minutes filters, and gets filtrate 2ml, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 1～2 of bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate 10ml, and evaporate to dryness adds boiling water 15ml and makes dissolving, filters while hot, put coldly, use the chloroform extraction secondary, each 10ml discards chloroform layer, aqueous solution extracts with ethyl acetate 10ml, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule, each 2g of tablet respectively, or get granule 10g; Add ethanol 40ml, supersound process 20 minutes filters, filtrate evaporate to dryness, residue add water 20ml makes dissolving, filters, filtrate is colourless to ether layer with ether extraction, discards ether solution, the water saturated n-butanol extraction of reuse three times, each 20ml merges n-butanol extracting liquid, water washing 3 times, each 20ml discards water liquid, evaporate to dryness, residue adds ethanol 1ml makes dissolving, admixes neutral alumina, stirs dry in the water-bath, neutral alumina pillar (200～300 orders of packing into and filling in advance, 1g, internal diameter 10～15mm) tops are with methanol 40ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.5ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.5g, adds ethanol 10ml, and jolting 5 minutes filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medical material solution; According to the thin layer chromatography test, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 8 μ l, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=5: 2: 1: 0.2 is developing solvent, launches, and takes out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated 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 capsule, each 2g of tablet respectively, or get granule 10g; Put in the tool plug triangular flask, after adding ammonia and stirring moistening, 60ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel, divide three extractions, be respectively 20ml, 20ml, 10ml with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions with chloroform 50ml again, be respectively 20ml, 20ml, 10ml, combined chloroform liquid, wash with water 3 times, each 30ml adds anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 1ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 0.5g, is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=9: 2: 4 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, each 1g of tablet respectively, or get granule 5g; Add petroleum ether (60～90 ℃) 5ml, close plug, supersound process 5 minutes filters, and filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 0.5g, is equipped with control medicinal material solution with legal system; According to thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ethyl acetate=5: 1.5 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; Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol: water=3: 7 is mobile phase; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 254nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.05mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 0.2g under the content uniformity item respectively; Or get granule 1g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 50ml, the close plug of adding, claim decide weight, supersound process 15 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 get need testing solution with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol: diethylamine=80: 20: 1 is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 254nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 0.1mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 3.5g respectively; Or get granule 10g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 6ml, close plug, jolting gently, claim to decide weight, placed 24 hours, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 10ml, puts in the separatory funnel, extracts 5 times with sulfuric acid solution (3 → 100), each 15ml merges sulphuric acid liquid, adds strong ammonia solution and regulates pH value to 9～10, with chloroform extraction 5 times, each 20ml, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, filter, promptly get need testing solution with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine.

Embodiment 2:

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet 5g respectively; Or get granule 15g, and adding ethanol 50ml, warm macerating 60 minutes filters, and gets filtrate 5ml, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 5 of bismuth potassium iodide test solutions, promptly generates salmon precipitation; Other gets filtrate 15ml, and evaporate to dryness adds boiling water 20ml and makes dissolving, filters while hot, puts coldly, with chloroform extraction 6 times, discards chloroform layer, and aqueous solution extracts with ethyl acetate 20ml, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule respectively, tablet 5g, or get granule 15g, add ethanol 100ml, supersound process 60 minutes filters the filtrate evaporate to dryness, residue adds water 30ml makes dissolving, filters, and filtrate is colourless to ether layer with ether extraction, discard ether solution, the water saturated n-butanol extraction of reuse 6 times merges n-butanol extracting liquid, water washing 6 times, discard water liquid, evaporate to dryness, residue add ethanol 3ml makes dissolving, admix neutral alumina, stir drying in the water-bath, the little column top of neutral alumina of packing into and filling in advance is with methanol 100ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 1ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 1g, adds ethanol 30ml, and jolting 15 minutes filters, and filtrate evaporate to dryness, residue add ethanol 2ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=10: 0.5: 5: 1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, it is clear to be heated 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 capsule, tablet 5g respectively; Or get granule 15g, and put in the tool plug triangular flask, after adding ammonia and stirring moistening, 100ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 6 times, transfer ph13 with ammonia, again with chloroform extraction 6 times, combined chloroform liquid, wash with water 5 times, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 2ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 1g and is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=5: 5: 1 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet 3g respectively; Or get granule 10g, and add petroleum ether (60～90 ℃) 20ml, close plug, supersound process 20 minutes filters, and filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 1g, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ethyl acetate=1: 9 is developing solvent, launches, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With acetonitrile: water=3: 8 is mobile phase; Flow velocity: 1.5ml/min; Column temperature: 60 ℃; Detect wavelength 500nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.1mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 1g under the content uniformity item respectively; Or get granule 2g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 100ml, the close plug of adding, claim decide weight, supersound process 50 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, get the filter membrane filtration of supernatant, promptly get need testing solution with 0.65 μ m; Accurate respectively reference substance solution and each 3 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: acetonitrile: diethylamine=90: 90: 0.5 is a mobile phase; Flow velocity: 1.5ml/min; Column temperature: 60 ℃; Detect wavelength 500nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 2mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 5g respectively; Or get granule 15g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 100ml and strong ammonia solution 10ml, close plug, jolting gently, claim to decide weight, placed 50 hours, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 15ml, puts in the separatory funnel, extracts 10 times with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 12, use chloroform extraction 10 times, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, the filter membrane filtration with 0.65 μ m promptly gets need testing solution; Accurate respectively reference substance solution and each 3 μ l of need testing solution of drawing inject hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine;

Embodiment 3:

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet 1g respectively; Or get granule 5g, and adding ethanol 10ml, warm macerating 10 minutes filters, and gets filtrate 1ml, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 1 of bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate 5ml, and evaporate to dryness adds boiling water 10ml and makes dissolving, filters while hot, puts coldly, with chloroform extraction 1 time, discards chloroform layer, and aqueous solution extracts with ethyl acetate 5ml, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule respectively, tablet 1g, or get granule 5g, add ethanol 20ml, supersound process 10 minutes filters the filtrate evaporate to dryness, residue adds water 10ml makes dissolving, filters, and filtrate is colourless to ether layer with ether extraction, discard ether solution, the water saturated n-butanol extraction of reuse 1 time merges n-butanol extracting liquid, water washing 1 time, discard water liquid, evaporate to dryness, residue add ethanol 0.5ml makes dissolving, admix neutral alumina, stir drying in the water-bath, the little column top of neutral alumina of packing into and filling in advance is with methanol 20ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.2ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.2g, adds ethanol 5ml, and jolting 3 minutes filters, and filtrate evaporate to dryness, residue add ethanol 0.5ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw each 25 μ l of above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=1: 5: 0.5: 0.1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, it is clear to be heated 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 capsule, tablet 1g respectively; Or get granule 5g, and put in the tool plug triangular flask, after adding ammonia and stirring moistening, 30ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 1 time, transfer pH9 with ammonia, again with chloroform extraction 1 time, combined chloroform liquid, wash with water time, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 0.5ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 0.3g and is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 0.3mg, in contrast product solution; Test according to thin layer chromatography, draw each 15 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=20: 0.5: 10 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet 0.5g respectively; Or get granule 10g, and add petroleum ether (60～90 ℃) 2ml, close plug, supersound process 2 minutes filters, and filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 1g, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ethyl acetate=9: 1 is developing solvent, launches, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol: water=3: 5 is mobile phase; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Detect wavelength 200nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.03mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 0.1g under the content uniformity item respectively; Or get granule 1g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 30ml, the close plug of adding, claim decide weight, supersound process 5 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, get the filter membrane filtration of supernatant, promptly get need testing solution with 0.25 μ m; Accurate respectively reference substance solution and each 15 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol: diethylamine=10: 10: 3 is a mobile phase; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Detect wavelength 200nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 0.05mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 2g respectively; Or get granule 5g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 20ml and strong ammonia solution 2ml, close plug, jolting gently, claim to decide weight, placed 10 hours, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 5ml, puts in the separatory funnel, extracts 2 times with sulfuric acid solution (3 → 100), merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 8, use chloroform extraction 2-10 time, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, the filter membrane filtration with 0.2 μ m promptly gets need testing solution; Accurate respectively reference substance solution and each 25 μ l of need testing solution of drawing inject hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine,

Claims (8)

1, a kind of method of quality control of bone sinew medicinal pill preparation is characterized in that, character is observed, and content is differentiated, official method is checked content, and gentiopicrin and the strychnine that contains carried out assay.

2, the method for claim 1 is characterized in that, described bone sinew medicinal pill preparation is an oral formulations.

3, the method for claim 2 is characterized in that, described oral formulations is pill, capsule, tablet, oral liquid, syrup or granule.

4, the method for claim 1, it is characterized in that described bone sinew medicinal pill preparation is made by following parts by weight of Chinese traditional medicine raw material: Olibanum 20-35 part, Myrrha 30-60 part, Radix Paeoniae Alba 70-130 part, Rhizoma Corydalis vinegar system 20-35 part, Radix Notoginseng 30-60 part, Radix Aucklandiae 20-35 part, Flos Carthami 30-60 part, Radix Curcumae 70-130 part, Radix Angelicae Pubescentis 160-220 part, Radix Achyranthis Bidentatae 30-60 part, Radix Gentianae Macrophyllae 160-220 part, Ramulus Cinnamomi 30-60 part, Sanguis Draxonis 20-35 part, Semen Strychni system 20-35 part.

5, the method for claim 1, it is characterized in that described bone sinew medicinal pill preparation is made by following parts by weight of Chinese traditional medicine raw material: 27 parts of Olibanums, 46 parts of Myrrhas, 91 parts of the Radix Paeoniae Albas, 27 parts of Rhizoma Corydalis vinegar systems, Radix Notoginseng 46, part 27 parts of the Radix Aucklandiae, 46 parts on Flos Carthami, 91 parts of Radix Curcumaes, 183 parts of Radix Angelicae Pubescentiss, 46 parts of Radix Achyranthis Bidentataes, 183 parts of Radix Gentianae Macrophyllae, 46 parts of Ramulus Cinnamomi, 27 parts of Sanguis Draxonis, 27 parts of Semen Strychni systems.

6, the method for claim 1 is characterized in that, wherein character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet or granule respectively, add ethanol, warm macerating filters, and gets filtrate, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate, and evaporate to dryness adds boiling water and makes dissolving, filters while hot, puts coldly, with chloroform extraction 1-6 time, discards chloroform layer, the aqueous solution ethyl acetate extraction, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm observation down, apparent sapphirine fluorescence;

(3) get capsule, tablet or granule respectively, add ethanol, supersound process, filter, filtrate evaporate to dryness, residue add water makes dissolving, filter, filtrate is colourless to ether layer with ether extraction, discards ether solution, the water saturated n-butanol extraction of reuse 1-6 time, merge n-butanol extracting liquid, water washing 1-6 time discards water liquid, evaporate to dryness, residue adds ethanol makes dissolving, admixes neutral alumina, stirs dry in the water-bath, the little column top of neutral alumina of packing into and filling in advance, with methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds dissolve with ethanol, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.2-1g, adds ethanol, and jolting filters, and filtrate evaporate to dryness, residue add ethanol makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, it is clear to be heated 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 capsule, tablet or granule respectively, put in the tool plug triangular flask, after adding ammonia and stirring moistening, add diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 1-6 time, transfer pH9～13 with ammonia, again with chloroform extraction 1-6 time, combined chloroform liquid, wash with water 1-5 time, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol and makes dissolving, as need testing solution; Other gets the Rhizoma Corydalis control medicinal material, is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add dissolve with methanol, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet or granule respectively, add 60～90 ℃ of petroleum ether, close plug, supersound process filters, and filtrate is as need testing solution; Other gets the Radix Angelicae Pubescentis control medicinal material, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol or acetonitrile: water=3: 5-8 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, uses dissolve with methanol, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item respectively, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol that adds, close plug claims to decide weight, supersound process, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, get supernatant and use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In the said preparation, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item respectively, tablet or granule, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform and strong ammonia solution, close plug, jolting gently, claim to decide weight, place, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate, put in the separatory funnel, extract 2-10 time, merge sulphuric acid liquid with sulfuric acid solution 3 → 100, add strong ammonia solution and regulate pH value to 8～12, with chloroform extraction 2-10 time, combined chloroform liquid, evaporate to dryness, residue adds the ethyl acetate dissolving, shake up, use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and the need testing solution drawn injects hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine.

7, the method for claim 1 is characterized in that,

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, tablet 1-5g respectively; Or get granule 5-15g, and add ethanol 10-50ml, warm macerating 10-60 minute, filter, get filtrate 1-5ml, evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 1～5 of bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate 5-15ml, and evaporate to dryness adds boiling water 10-20ml and makes dissolving, filters while hot, put coldly, with chloroform extraction 1-6 time, discard chloroform layer, aqueous solution is with ethyl acetate 5-20ml extraction, extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule respectively, tablet 1-5g, or get granule 5-15g, add ethanol 20-100ml, supersound process 10-60 minute, filter the filtrate evaporate to dryness, residue adds water 10-30ml makes dissolving, filters, and filtrate is colourless to ether layer with ether extraction, discard ether solution, the water saturated n-butanol extraction of reuse 1-6 time merges n-butanol extracting liquid, water washing 1-6 time, discard water liquid, evaporate to dryness, residue add ethanol 0.5-3ml makes dissolving, admix neutral alumina, stir drying in the water-bath, the little column top of neutral alumina of packing into and filling in advance is with methanol 20-100ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.2-1ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.2-1g, adds ethanol 5-30ml, and jolting 3-15 minute, filter, filtrate evaporate to dryness, residue add ethanol 0.5-2ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw each 5-25 μ l of above-mentioned need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=1-10: 0.5-5: 0.5-5: 0.1-1 is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, it is clear to be heated 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 capsule, tablet 1-5g respectively; Or get granule 5-15g, and put in the tool plug triangular flask, after adding ammonia and stirring moistening, 30-100ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel,, merge acetate layer with 10% acetic acid extraction 1-6 time, transfer pH9～13 with ammonia, again with chloroform extraction 1-6 time, combined chloroform liquid, wash with water 1-5 time, add anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 0.5-2ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 0.3-1g and is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 0.3-1mg, in contrast product solution; Test according to thin layer chromatography, draw each 5-15 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=5-20: 0.5-5: 1-10 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, tablet 0.5-3g respectively; Or get granule 3-10g, and add 60～90 ℃ of 2-20ml of petroleum ether, close plug, supersound process 2-20 minute, filter, filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 0.3-1g, is equipped with control medicinal material solution with legal system; Test according to thin layer chromatography, draw each 5-25 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with cyclohexane extraction: ethyl acetate=1-9: 9-1, 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol or acetonitrile: water=3: 5-8 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.03-0.1mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 0.1-1g under the content uniformity item respectively; Or get granule 1-2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 30-100ml that adds, close plug claims to decide weight, supersound process 5-50 minute, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, get supernatant and use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and each 3-15 μ l of need testing solution of drawing injects hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol or acetonitrile: diethylamine=10-90: 90-10: 0.5-3 is a mobile phase; Flow velocity: 0.8-1.5ml/min; Column temperature: 30-60 ℃; Detect wavelength 200-500nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 0.05-2mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 2-5g respectively; Or get granule 5-15g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 20-100ml and strong ammonia solution 2-10ml, close plug, jolting gently, claim to decide weight, placed 10-50 hour, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 5-15ml, puts in the separatory funnel, extracts 2-10 time with sulfuric acid solution 3 → 100, merge sulphuric acid liquid, add strong ammonia solution and regulate pH value to 8～12, use chloroform extraction 2-10 time, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, use filter membrane to filter, promptly get need testing solution less than 0.65 μ m; Accurate respectively reference substance solution and each 3-25 μ l of need testing solution of drawing injects hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine.

8, the method for claim 1 is characterized in that, step is as follows:

Character is observed, and step is:

For capsule: content shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For tablet: medicine shows brown; Gas perfume (or spice), bitter in the mouth, puckery;

For granule: product is brown granule;

Content is differentiated step is:

(1) get capsule, tablet or granule respectively, put microscopically and observe: gelatinized starch grain agglomerate is faint yellow or closely colourless; Hypodermis prothenchyma (of wood) green-yellow, the cell polygon, class is square or strip, and wall is crooked slightly, lignify, the one-tenth beaded that has thickens, and pit is fine and closely woven; Stone cell is faint yellow, and to 60 μ m, wall is thicker approximately for similar round or Long Circle, diameter, and pit is fine and closely woven; Spiral duct diameter 16～32 μ m; Starch grain is a lot of, simple grain circle, semicircle or circle polygon, diameter 4～30 μ m; Composite grain is made up of gradation surplus 2～10; The resin canal fragment contains yellow secretions; Scalariform, reticulate pattern and spiral duct diameter 15～55 μ m; Calcium oxalate cluster crystal is rare, diameter 50～80 μ m; Nonglandular hair is unicellular, and base portion expands like stone cell, and how cataclasm wall is extremely thick,, lignify; The albuminous cell polygon, wall thickness includes fatty oil and aleurone grain;

(2) get capsule, each 3g of tablet respectively, or get granule 10g; Add ethanol 20ml, warm macerating 30 minutes filters, and gets filtrate 2ml, and evaporate to dryness adds dilute hydrochloric acid and makes acidity, filters, and filtrate adds 1～2 of bismuth potassium iodide test solution, promptly generates salmon precipitation; Other gets filtrate 10ml, and evaporate to dryness adds boiling water 15ml and makes dissolving, filters while hot, put coldly, use the chloroform extraction secondary, each 10ml discards chloroform layer, aqueous solution extracts with ethyl acetate 10ml, and extracting solution is put on chromatography filter paper, puts ultra-violet lamp 365nm and observes down, shows sapphirine fluorescence;

(3) get capsule, each 2g of tablet respectively, or get granule 10g; Add ethanol 40ml, supersound process 20 minutes filters, filtrate evaporate to dryness, residue add water 20ml makes dissolving, filters, filtrate is colourless to ether layer with ether extraction, discards ether solution, the water saturated n-butanol extraction of reuse three times, each 20ml merges n-butanol extracting liquid, water washing 3 times, each 20ml discards water liquid, evaporate to dryness, residue adds ethanol 1ml makes dissolving, admixes neutral alumina, stirs dry in the water-bath, neutral alumina pillar 200～300 orders of packing into and filling in advance, 1g, internal diameter 10～15mm top is with methanol 40ml eluting, collect eluent, evaporate to dryness, residue add dissolve with ethanol makes into 0.5ml, as need testing solution; Other gets Radix Paeoniae Alba control medicinal material 0.5g, adds ethanol 10ml, and jolting 5 minutes filters, and filtrate evaporate to dryness, residue add ethanol 1ml makes dissolving, in contrast medical material solution; According to the thin layer chromatography test, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 8 μ l, put respectively on same silica gel g thin-layer plate, with chloroform: acetone: methanol: glacial acetic acid=5: 2: 1: 0.2 is developing solvent, launches, and takes out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated 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 capsule, each 2g of tablet respectively, or get granule 10g; Put in the tool plug triangular flask, after adding ammonia and stirring moistening, 60ml adds diethyl ether, close plug, jolting constantly, placement is spent the night, the leaching supernatant, put in the separatory funnel, divide three extractions, be respectively 20ml, 20ml, 10ml with 10% acetic acid 50ml, merge acetate layer, transfer pH10～11 with ammonia, divide three extractions with chloroform 50ml again, be respectively 20ml, 20ml, 10ml, combined chloroform liquid, wash with water 3 times, each 30ml adds anhydrous sodium sulfate, filter, the filtrate evaporate to dryness adds methanol 1ml and makes dissolving, as need testing solution; Other gets Rhizoma Corydalis control medicinal material 0.5g, is equipped with control medicinal material solution with legal system; Get the tetrahydropalmatine reference substance again, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ether: ethyl acetate=9: 2: 4 is developing solvent, launch, take out, dry, iodine is after the smoked several seconds, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

(5) get capsule, each 1g of tablet respectively, or get granule 5g; Add 60～90 ℃ of 5ml of petroleum ether, close plug, supersound process 5 minutes filters, and filtrate is as need testing solution; Other gets Radix Angelicae Pubescentis control medicinal material 0.5g, is equipped with control medicinal material solution with legal system; According to thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with cyclohexane extraction: ethyl acetate=5: 1.5 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;

Official method checks that to content step is:

Should meet every regulation relevant under Chinese Pharmacopoeia capsule, tablet or the granule item;

Gentiopicrin and the strychnine that contains carried out assay, and step is:

Gentiopicrin adopts high performance liquid chromatography, is filler with octadecylsilane chemically bonded silica; With methanol: water=3: 7 is mobile phase; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 254nm; Theoretical cam curve is pressed the gentiopicrin peak and is calculated, and should be not less than 3000; Precision takes by weighing the gentiopicrin reference substance, makes with dissolve with methanol to contain gentiopicrin 0.05mg among every 1ml, promptly gets reference substance solution; Get capsule, tablet 0.2g under the content uniformity item respectively; Or get granule 1g, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol 50ml, the close plug of adding, claim decide weight, supersound process 15 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 get need testing solution with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject hplc determination content; In this oral formulations, contain Radix Gentianae Macrophyllae in the capsule, must not be less than in 5mg/g, the tablet and contain Radix Gentianae Macrophyllae, must not be less than in 5mg/g, the granule and contain Radix Gentianae Macrophyllae, must not be less than 0.5mg/g in gentiopicrin in gentiopicrin in gentiopicrin;

Strychnine adopts high performance liquid chromatography, is filler with silica gel; With ether: methanol: diethylamine=80: 20: 1 is a mobile phase; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; Detect wavelength 254nm; Theoretical cam curve should be not less than 2000 in the strychnine peak; Precision takes by weighing the strychnine reference substance, makes the solution that every 1ml contains 0.1mg with the ethyl acetate dissolving, promptly gets reference substance solution; Get capsule under the content uniformity item, tablet 3.5g respectively; Or get granule 10g, the accurate title, decide, and puts in the tool plug conical flask, precision adds chloroform 50ml and strong ammonia solution 6ml, close plug, jolting gently, claim to decide weight, placed 24 hours, claim to decide weight again, supply the weight that subtracts mistake with chloroform, shake well filters, precision is measured subsequent filtrate 10ml, puts in the separatory funnel, extracts 5 times with sulfuric acid solution 3 → 100, each 15ml merges sulphuric acid liquid, adds strong ammonia solution and regulates pH value to 9～10, with chloroform extraction 5 times, each 20ml, combined chloroform liquid, evaporate to dryness, residue add the ethyl acetate dissolving, are transferred in the 10ml measuring bottle, and be diluted to scale, shake up, filter, promptly get need testing solution with filter membrane 0.45 μ m; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject hplc determination content; In the said preparation, contain Semen Strychni in the capsule, should be in 0.8～2.5mg/g, the tablet and contain Semen Strychni, should be in 0.8～2.5mg/g, the granule and contain Semen Strychni, should be 0.08～0.25mg/g in strychnine in strychnine in strychnine.