CN103197027A - Quality control method of astragalus-leech capsules capable of regulating collaterals - Google Patents
Quality control method of astragalus-leech capsules capable of regulating collaterals Download PDFInfo
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Abstract
The invention discloses a quality control method of astragalus-leech capsules capable of regulating collaterals. The quality control method comprises the steps of: respectively differentiating leech, red peony root, astragalus root, ginseng, chuanxiong rhizome, red sage root, ground beetle, cinnamon, borneol and polygonum multiflorum in medicaments by using a thin-layer chromatography; determining the content of astragaloside in the medicaments by using a thin-layer scanning method; and determining the content of tanshinol and paeoniflorin in the medicaments by using a liquid-phase chromatography. According to the quality control method disclosed by the invention, the medicinal material differentiation method is mature, feasible, negative and non-interfering and is strong in specificity, and the content determination method is easy to operate, high in precision and good in reproducibility, so that the quality control method disclosed by the invention can be used for controlling the quality of the medicaments accurately and stably, thereby being adaptable to the industrial stable production of the medicaments.
Description
Technical field
The present invention relates to a kind of Chinese medicine medicine that is used for the treatment of apoplexy sequelae, particularly relate to the method for quality control of this medicine.
Background technology
Astragalus leech network ressel freeing capsule is beneficial gas, invigorates blood circulation the medication of vein relaxing class.Be applicable to that the stroke in convalescent stage sequelae shows as hemiplegia, extremity numbness, facial paralysis, dysphonia, lassitude person's auxiliary curing.Its existing operative norm is National Drug Administration's standard (trying), has formulated following content in this quality standard.
1) get this product, put microscopically and observe: the body wall fragment is yellow or brown, circular trichopore is arranged, diameter 5-32 μ m, the bristle that the tool that has is different in size.How the elongated or long lanceolar of dorsoventral muscle fiber fractures, diameter 20-33 μ m, how single loose from, idol has several bunchys, bending or bird caging, and wall is thicker, and expand or inhomogeneous thickening the part sometimes, colourless or light brown, cell is obviously most, sees the sepia content sometimes.The body wall fragment is colourless, and there is superfine mycelium on the surface.Lithocyte similar round or rectangle, wall are simultaneously poor.
2) get this product content 5g, add zeyssatite 3g, fully mixing, add methyl alcohol 30ml, ultrasonic processing 20 minutes filters, filtrate water bath method, residue add water 30ml makes dissolving, extracts 3 times with the ether jolting, each 20ml, aqueous solution is standby, merges ether extracted liquid, flings to ether, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other leech control medicinal material 1.5g that fetches water adds methyl alcohol 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into about 1ml, in contrast medicinal material solution.Test according to thin-layered chromatography (appendix VI B), draw need testing solution 3 μ l, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with methenyl choloride-methyl alcohol (40:1), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 100 ℃.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
3) get differentiate 2) the following aqueous solution after the extracted by ether, extract 3 (20ml with water saturated normal butyl alcohol jolting, 10ml, 10ml), merge n-butanol extracting liquid, extract 3 times with ammonia solution, each 15ml, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue add water 3ml makes dissolving, is added on D101 type macroporous absorbent resin post (internal diameter 2cm, long 12cm) on, with water 50ml wash-out, discard water liquid, use 40% ethanol 30ml wash-out again, discard 40% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the Paeoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 2mg, in contrast product solution.Test according to thin-layered chromatography (appendix VI B), draw need testing solution 10 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with methenyl choloride-ethyl acetate-methyl alcohol-formic acid (40:5:10:0.2), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 100 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show identical bluish violet spot.
4) get Astragaloside IV, ginsenoside Rg1's reference substance, add methyl alcohol and make the solution that every 1ml contains 1mg, 2mg respectively, in contrast product solution.Test according to thin-layered chromatography (appendix VI B), draw each 2 μ l of above-mentioned reference substance solution, differentiate 3) under need testing solution 4 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with normal butyl alcohol-glacial acetic acid-water (8:1:1), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing in 100 ℃, puts respectively under daylight and the ultraviolet lamp (365nm) and inspects.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show spot and the fluorescence spot of same color respectively.
5) get this product content 10g, the 30ml that adds diethyl ether, ultrasonic processing 10 minutes filters, and filtrate volatilizes, and residue adds methenyl choloride 1ml makes dissolving, as need testing solution.Other gets Ligusticum wallichii control medicinal material 0.5g, shines medicinal material solution in pairs with legal system.According to thin-layered chromatography (appendix VI B) test, draw need testing solution 5 μ l, control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with normal hexane-ethyl acetate (9:1), launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
6) get this product content 5g, add water 20ml, ultrasonic processing 5 minutes, centrifugal, get supernatant, add watery hydrochloric acid and regulate pH to 2, add ethyl acetate 20ml, jolting is extracted, and extract is put evaporate to dryness in the water-bath, and residue adds methyl alcohol 2ml makes dissolving, as need testing solution.Other gets red sage root control medicinal material 0.5g, and it is an amount of to add water, decocts half an hour, filters, and filtrate is concentrated into about 10ml, shines medicinal material solution in pairs with legal system.Test according to thin-layered chromatography (appendix VI B), draw need testing solution 2 μ l, control medicinal material solution 1 μ l, so respectively on the silica gel g thin-layer plate, be developping agent with methenyl choloride-acetone-formic acid (8:1:1), launch, take out, dry, put in the ammonia steam smoked after, placement is spent the night, and puts under the ultraviolet lamp (365nm) and inspects.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show same color fluorescence spot.
7) check, should meet every regulation relevant under the capsule item.
8) assay is got content under the content uniformity item, porphyrize, mixing, get about 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds claims to decide weight, ultrasonic processing 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, discard filtrate just, precision is measured subsequent filtrate 25ml, reclaim methyl alcohol to doing, residue water 40ml dissolving while hot is transferred in the separating funnel, extracts 3 times with the ether jolting, be followed successively by 20ml, 15ml, 10ml, discard ether solution, water liquid continues and extracts four times with water saturated normal butyl alcohol, each 15ml.Merge normal butyl alcohol liquid, with 0.5% sodium hydroxide solution washing 3 times, each 20ml, discard alkali lye, with the water washing that normal butyl alcohol is saturated extremely neutral (each 20ml, 2-3 time), discard water lotion, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol makes dissolving, is transferred in the 5ml volumetric flask, adds methyl alcohol to scale, shake up, as need testing solution.It is an amount of that precision takes by weighing the Astragaloside IV reference substance in addition, adds methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (appendix VI B), accurate need testing solution 10 μ l, reference substance solution 2 μ l and the 4 μ l of drawing, the point of crossing is on same silica gel g thin-layer plate respectively, placing the lower floor's solution that spends the night below 10 ℃ with methenyl choloride-methanol-water (65:35:10) is developping agent, launch, take out, dry.Spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 100 ℃, take out, cover onesize glass plate at thin layer plate, use immobilization with adhesive tape on every side, scan according to thin-layered chromatography, wavelength: λ s=520nm, λ R=650nm measures test sample absorbance log integrated value and reference substance absorbance log integrated value, calculate, namely.Every of this product contains Astragaloside IV (C
41H
68O
14) must not be less than 0.060mg.
Above-mentioned quality standard exists more serious defective: at first, this standard is for the quantitative measurement imperfection of main effective ingredient content; Next, the composition medication amount of carrying out the chemistry discriminating is less.
Chinese medicine preparation steady quality, controlled and have that to produce repeatability be the prerequisite assurance that medicine has pharmacology and clinical effectiveness repeatability, can traditional Chinese medicine quality controlled and whether the traditional Chinese medicine quality standard is scientific, is the important restraining factors that influence industrialization of Chinese medicine.Quality controllability is the important indicator that medicine is estimated, and quality standard then is the imbody of drug quality controllability.
Summary of the invention
The method of quality control that the purpose of this invention is to provide a kind of above-mentioned astragalus leech network ressel freeing capsule is with the drug quality stable and controllable of guaranteeing to produce.
The method of quality control of astragalus leech network ressel freeing capsule provided by the invention is suitable for the medicine that is prepared from by following bulk drug: the Radix Astragali, leech, genseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine, Radix Angelicae Sinensis, Ligusticum wallichii, ilex pubescens, the radix paeoniae rubrathe, reticulate millettia, the red sage root, prepared fleece flower root, safflower, Herba Lycopi, ground bettle, earthworm, stiff silkworm, root tuber of aromatic turmeric, turmeric, scorpio, rhizoma Gastrodiae, Chinese cassia tree, notopterygium root, fructus gleditsiae, arisaema cum bile, borneol.Described method of quality control comprises discrimination method and content assaying method.
Wherein, described discrimination method comprises following discriminating:
1) gets described drug substance contents 5g, add zeyssatite 3g, fully mixing, add methyl alcohol 30ml, ultrasonic processing 20 minutes filters, filtrate water bath method, residue add water 30ml makes dissolving, extracts 3 times with the ether jolting, each 20ml, aqueous solution is standby, merges ether extracted liquid, flings to ether, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other leech control medicinal material 1.5g that fetches water adds methyl alcohol 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, in contrast medicinal material solution; Test according to thin-layered chromatography, drawing need testing solution 3 μ l, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with methenyl choloride: methyl alcohol=40:1, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to spot colour developing at 100 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
2) get 1) the following aqueous solution after the extracted by ether, extract 3 times with water saturated normal butyl alcohol jolting, be respectively 20ml 3 times, 10ml, 10ml merges n-butanol extracting liquid, extract 3 times with ammonia solution, each 15ml discards ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue adds water 3ml makes dissolving, be added on internal diameter 2cm, on the D101 type macroporous absorbent resin post of long 12cm, with water 50ml wash-out, discard water liquid, use 40% ethanol 30ml wash-out again, discard 40% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Paeoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=40:5:10:0.2 is developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to spot colour developing at 100 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
3) get Astragaloside IV, ginsenoside Rg1's reference substance, add methyl alcohol and make the solution that every 1ml contains 1mg, 2mg respectively, in contrast product solution; According to the thin-layered chromatography test, draw each 2 μ l, 2 of above-mentioned reference substance solution) following need testing solution 4 a μ l, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol: glacial acetic acid: water=8:1:1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 100 ℃, puts respectively under daylight and the 365nm ultraviolet lamp and inspects, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show spot and the fluorescence spot of same color respectively;
4) get described drug substance contents 10g, the 30ml that adds diethyl ether, ultrasonic processing 10 minutes filters, and filtrate volatilizes, and residue adds methenyl choloride 1ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; Test according to thin-layered chromatography, draw need testing solution 5 μ l, control medicinal material solution 1 μ l, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=9:1, launches, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;
5) get described drug substance contents 5g, add water 20ml, ultrasonic processing 5 minutes, centrifugal, get supernatant, add watery hydrochloric acid and regulate pH to 2, add ethyl acetate 20ml, jolting is extracted, and extract is put evaporate to dryness in the water-bath, and residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets red sage root control medicinal material 0.5g, and it is an amount of to add water, decocts half an hour, filters, and filtrate is concentrated into about 10ml, shines medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw need testing solution 2 μ l, control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride: acetone: formic acid=8:1:1 is developping agent, launch, take out, dry, put in the ammonia steam smoked after, placement is spent the night, and puts under the 365nm ultraviolet lamp and inspects, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;
6) get described drug substance contents 5g, add methyl alcohol 25ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets ground bettle control medicinal material 1g, shines medicinal material solution in pairs with legal system; Test according to thin-layered chromatography, draw need testing solution 2 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with toluene: methylene chloride: acetone=5:5:0.5 is developping agent, launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, 105 ℃ to be heated to spot colour developing clear, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
7) get described drug substance contents 20g, add ethanol 40ml, cold soaking 20 minutes, jolting constantly filters, and filtrate volatilizes, and adds ethanol 1ml and makes dissolving, as need testing solution; Other gets Chinese cassia tree control medicinal material 2g, shines medicinal material solution in pairs with legal system; Get the cinnaldehydrum reference substance again, add ethanol and make the reference substance solution that every 1ml contains 1 μ l; Test according to thin-layered chromatography, draw need testing solution 15 μ l, control medicinal material solution 2 μ l, reference substance solution 2 μ l, putting respectively on same silica gel g thin-layer plate, is developping agent with 60-90 ℃ of sherwood oil: ethyl acetate=17:3, launches, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
8) get described drug substance contents 5g, add methenyl choloride 30ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and adds chloroform 1ml and makes dissolving, as need testing solution; It is an amount of that other gets the borneol control medicinal material, adds methenyl choloride and make the control medicinal material solution that every 1ml contains 10mg; Test according to thin-layered chromatography, drawing need testing solution 10 μ l, control medicinal material solution 6 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with cyclohexane: ethyl acetate=17:3, launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, 105 ℃ to be heated to spot colour developing clear, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
9) get described drug substance contents 5g, add ethyl acetate 20ml, concentrated hydrochloric acid 0.5ml, ultrasonic processing 20 minutes filters, and filtrate volatilizes, and adds ethyl acetate 1ml and makes dissolving, as need testing solution; Other gets fleece-flower root control medicinal material 0.1g, shines medicinal material solution in pairs with legal system; Get the archen reference substance again, add methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin-layered chromatography, draw need testing solution 15 μ l, control medicinal material solution 5 μ l, reference substance solution 2 μ l, put respectively on same silica gel g thin-layer plate, with toluene: the upper solution of ethyl acetate: formic acid=20:2:1 is developping agent, launches, take out, dry, it is smoked clear to the spot colour developing to put in the ammonia steam, in the test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, show the spot of same color.
Described content assaying method comprises the mensuration of Astragaloside IV, danshensu and paeoniflorin content.
The present invention is specifically with the content of the described Astragaloside IV of following tlc scanning determination:
1) preparation of reference substance solution: precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.5mg;
2) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds claims to decide weight, ultrasonic processing 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, discard filtrate just, precision is measured subsequent filtrate 25ml, reclaims methyl alcohol to doing residue water 40ml dissolving while hot, be transferred in the separating funnel, extract 3 times with the ether jolting, each consumption is respectively 20,15,10ml, discard ether solution, water liquid continues and extracts 4 times with water saturated normal butyl alcohol, each 15ml, merge normal butyl alcohol liquid, with 0.5% sodium hydroxide solution washing 3 times, each 20ml discards alkali lye, extremely neutral with the water washing that normal butyl alcohol is saturated, discard water lotion, normal butyl alcohol evaporate to dryness, residue add methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution;
3) the accurate need testing solution 10 μ l that draw, reference substance solution 2 μ l and 4 μ l, the point of crossing is on same silica gel g thin-layer plate respectively, with methenyl choloride: methyl alcohol: water=65:35:10 mixed solution is placed the lower floor's solution that spends the night below 10 ℃ be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 100 ℃, takes out, cover onesize glass plate at thin layer plate, use immobilization with adhesive tape on every side, with wavelength X S=520nm, λ R=650nm scans, and measures the integrated value of test sample absorbance log and the integrated value of reference substance absorbance log, calculate, namely.
The present invention is specifically with the content of the described danshensu of following high effective liquid chromatography for measuring:
1) chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Be the phase that flows with 1% glacial acetic acid: methyl alcohol=94:6; Detect wavelength 280nm; Number of theoretical plate calculates by the Sodium Danshensu peak and is not less than 3000;
2) preparation of reference substance solution: precision takes by weighing the Sodium Danshensu reference substance, adds 50% methyl alcohol and makes the reference substance solution that every 1ml contains the 0.135mg danshensu;
3) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 4g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 2% hydrochloric acid solution 50ml that adds, close plug claims to decide weight, power 150W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with 2% hydrochloric acid solution, shake up, add sodium chloride 5g, shake up, centrifugal, precision is measured supernatant 25ml, put in the separating funnel, extract 4 times with the ethyl acetate jolting, each consumption is respectively 50,30,20,20ml, combined ethyl acetate liquid, reclaim ethyl acetate to doing, residue dissolves with 50% methyl alcohol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate, namely;
4) precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, namely.
The present invention is specifically with the described content of paeoniflorin of following high effective liquid chromatography for measuring:
1) chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; With acetonitrile: 0.1% phosphoric acid=16:84 is the phase that flows; Detect wavelength 230nm, number of theoretical plate calculates by the Paeoniflorin peak and is not less than 3000;
2) preparation of reference substance solution: precision takes by weighing the Paeoniflorin reference substance, adds 50% methyl alcohol and makes the reference substance solution that every 1ml contains 0.1mg;
3) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 4g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims to decide weight, the ultrasonic processing of power 150W, frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methyl alcohol, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate, namely;
4) precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, namely.
The present invention has set up the method for quality control of astragalus leech network ressel freeing capsule, in the method for setting up, the discrimination method mature and feasible of medicinal material, specificity is strong, and is negative noiseless, the content assaying method processing ease, the precision height, favorable reproducibility uses method of quality control of the present invention can accurately, stably control the drug quality of astragalus leech network ressel freeing capsule, to adapt to the industrialization steady production of medicine.
Embodiment
Embodiment 1: the preparation of astragalus leech network ressel freeing capsule.
Prescription: Radix Astragali 325g, leech 130g, genseng 130g, the tuber of dwarf lilyturf 32.5g, fruit of Chinese magnoliavine 65g, Radix Angelicae Sinensis 65g, Ligusticum wallichii 65g, ilex pubescens 325g, radix paeoniae rubrathe 65g, reticulate millettia 65g, red sage root 65g, prepared fleece flower root 162.5g, safflower 32.5g, Herba Lycopi 32.5g, ground bettle 32.5g, earthworm 32.5g, stiff silkworm 6.5g, root tuber of aromatic turmeric 65g, turmeric 32.5g, scorpio 13g, rhizoma Gastrodiae 13g, Chinese cassia tree 13g, notopterygium root 32.5g, fructus gleditsiae 6.5g, arisaema cum bile 13g, borneol 13g.
Method for making: above 20 Six-elements, except borneol, leech, ground bettle, Chinese cassia tree, scorpio, stiff silkworm, arisaema cum bile are ground into fine powder, sieve mixing; All the other Radixs Astragali etc. 19 flavor, the boiling secondary adds 10 times of amounts of water for the first time, adds 5 times of amounts of water for the second time, decocts 2 hours at every turn, collects volatile oil in the decoction process simultaneously, and volatile oil is standby after with beta-cyclodextrin inclusion compound; Collecting decoction filters, and it is 1.20-1.25(80 ℃ that filtrate is concentrated into relative density) clear cream, add fine powders such as leech, mixing, after the low temperature drying, be ground into fine powder, with borneol and volatile oil beta cyclodextrin inclusion complex facing-up, sieve again, mixing incapsulates, and makes 1000, namely.
Proterties: this product is capsule, and content is the powder of yellowish-brown; Gas perfume (or spice), salty, hot, cool, the little hardship of distinguishing the flavor of.
Function cures mainly: beneficial gas, invigorate blood circulation vein relaxing.Be applicable to that the stroke in convalescent stage sequelae shows as hemiplegia, extremity numbness, facial paralysis, dysphonia, lassitude person's auxiliary curing.
Specification: every dress 0.5g.
Usage and dosage: oral, one time 4,2 times on the one, breakfast serviced once after meal or followed the doctor's advice the evening before yesterday.
Storage: sealing, put the dry place of shady and cool ventilation.
Embodiment 2: the thin layer of leech is differentiated.
Get embodiment 1 drug substance contents 5g, add zeyssatite 3g, fully mixing, add methyl alcohol 30ml, ultrasonic processing 20 minutes filters, filtrate water bath method, residue add water 30ml makes dissolving, extracts 3 times with the ether jolting, each 20ml, aqueous solution is standby, merges ether extracted liquid, flings to ether, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other leech control medicinal material 1.5g that fetches water adds methyl alcohol 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into about 1ml, in contrast medicinal material solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 3 μ l, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with methenyl choloride-methyl alcohol (40: 1), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 100 ℃.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Embodiment 3: the thin layer of the radix paeoniae rubrathe is differentiated.
Get the aqueous solution after the extracted by ether among the embodiment 2, extract 3 (20ml, 10ml with water saturated normal butyl alcohol jolting, 10ml), merge n-butanol extracting liquid, extract 3 times with ammonia solution, each 15ml, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue add water 3ml makes dissolving, is added on D101 type macroporous absorbent resin post (internal diameter 2cm, long 12cm) on, with water 50ml wash-out, discard water liquid, use 40% ethanol 30ml wash-out again, discard 40% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the Paeoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 2mg, in contrast product solution.According to thin-layered chromatography (" 2010 editions one appendix VI B of Chinese pharmacopoeia) test, draw need testing solution 10 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol-formic acid (40: 5: 10: 0.2) be developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing at 100 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
Embodiment 4: the thin layer of the Radix Astragali and genseng is differentiated.
Get Astragaloside IV, ginsenoside Rg1's reference substance, add methyl alcohol and make the solution that every 1ml contains 1mg, 2mg respectively, in contrast product solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw the need testing solution 4 μ l of each 2 μ l, embodiment 3 of above-mentioned reference substance solution, put respectively on same silica gel g thin-layer plate, be developping agent with normal butyl alcohol-glacial acetic acid-water (8: 1: 1), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 100 ℃, puts respectively under daylight and the ultraviolet lamp (365nm) and inspects.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show spot and the fluorescence spot of same color respectively.
Embodiment 5: the thin layer of Ligusticum wallichii is differentiated.
Get embodiment 1 drug substance contents 10g, the 30ml that adds diethyl ether, ultrasonic processing 10 minutes filters, and filtrate volatilizes, and residue adds methenyl choloride 1ml makes dissolving, as need testing solution.Other gets Ligusticum wallichii control medicinal material 0.5g, shines medicinal material solution in pairs with legal system.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 5 μ l, control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with normal hexane-ethyl acetate (9: 1), launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Embodiment 6: the thin layer of the red sage root is differentiated.
Get embodiment 1 drug substance contents 5g, add water 20ml, ultrasonic processing 5 minutes, centrifugal, get supernatant, add watery hydrochloric acid and regulate pH to 2, add ethyl acetate 20ml, jolting is extracted, and extract is put evaporate to dryness in the water-bath, and residue adds methyl alcohol 2ml makes dissolving, as need testing solution.Other gets red sage root control medicinal material 0.5g, and it is an amount of to add water, decocts half an hour, filters, and filtrate is concentrated into about 10ml, shines medicinal material solution in pairs with legal system.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 2 μ l, control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with methenyl choloride-acetone-formic acid (8: 1: 1), launch, take out, dry, put in the ammonia steam smoked after, placement is spent the night, and puts under the ultraviolet lamp (365nm) and inspects.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Embodiment 7: the thin layer of ground bettle is differentiated.
Get embodiment 1 drug substance contents 5g, add methyl alcohol 25ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets ground bettle control medicinal material 1g, shines medicinal material solution in pairs with legal system.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 2 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with toluene-methylene chloride-acetone (5: 5: 0.5), launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, and 105 ℃ to be heated to the spot colour developing clear.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Embodiment 8: the thin layer of Chinese cassia tree is differentiated.
Get embodiment 1 drug substance contents 20g, add ethanol 40ml, cold soaking 20 minutes, jolting constantly filters, and filtrate volatilizes, and adds ethanol 1ml and makes dissolving, as need testing solution.Other gets Chinese cassia tree control medicinal material 2g, shines medicinal material solution in pairs with legal system.Get the cinnaldehydrum reference substance again, add ethanol and make the reference substance solution that every 1ml contains 1 μ l.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 15 μ l, control medicinal material solution 2 μ l, reference substance solution 2 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with sherwood oil (60-90 ℃)-ethyl acetate (17: 3), launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution.In the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color.
Embodiment 9: the thin layer of borneol is differentiated.
Get embodiment 1 drug substance contents 5g, add methenyl choloride 30ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and adds chloroform 1ml and makes dissolving, as need testing solution.It is an amount of that other gets the borneol control medicinal material, adds methenyl choloride and make the control medicinal material solution that every 1ml contains 10mg.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 10 μ l, control medicinal material solution 6 μ l, put respectively on same silica gel g thin-layer plate, be developping agent with cyclohexane-ethyl acetate (17: 3), launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, and 105 ℃ to be heated to the spot colour developing clear.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Embodiment 10: the thin layer of the fleece-flower root is differentiated.
Get embodiment 1 drug substance contents 5g, add ethyl acetate 20ml, concentrated hydrochloric acid 0.5ml, ultrasonic processing 20 minutes filters, and filtrate volatilizes, and adds ethyl acetate 1ml and makes dissolving, as need testing solution.Other gets fleece-flower root control medicinal material 0.1g, shines medicinal material solution in pairs with legal system.Get the archen reference substance again, add methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw need testing solution 15 μ l, control medicinal material solution 5 μ l, reference substance solution 2 μ l, put respectively on same silica gel g thin-layer plate, upper solution with toluene-ethyl acetate-formic acid (20: 2: 1) is developping agent, launch, take out, dry, it is smoked clear to the spot colour developing to put in the ammonia steam.In the test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, show the spot of same color.
Embodiment 11: the mensuration of Astragaloside content.
With reference to primary standard, with the Astragaloside content in the tlc scanning determination Radix Astragali.
Get embodiment 1 drug substance contents, porphyrize, mixing, get about 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds claims to decide weight, ultrasonic processing 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, discard filtrate just, precision is measured subsequent filtrate 25ml, reclaims methyl alcohol to doing residue water 40ml dissolving while hot, be transferred in the separating funnel, extract 3 times with the ether jolting, be followed successively by 20,15,10ml, discard ether solution, water liquid continues and extracts 4 times with water saturated normal butyl alcohol, each 15ml, merge normal butyl alcohol liquid, with 0.5% sodium hydroxide solution washing 3 times, each 20ml, discard alkali lye, with the water washing that normal butyl alcohol is saturated extremely neutral (each 20ml, 2-3 time), discard water lotion, the normal butyl alcohol evaporate to dryness, residue adds methyl alcohol makes dissolving, is transferred in the 5ml measuring bottle, adds methyl alcohol to scale, shake up, as need testing solution.Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.5mg.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, the accurate need testing solution 10 μ l that draw, reference substance solution 2 μ l and 4 μ l, the point of crossing is on same silica gel g thin-layer plate respectively, placing the lower floor's solution that spends the night below 10 ℃ with methenyl choloride-methanol-water (65: 35: 10) is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 100 ℃, takes out, cover onesize glass plate at thin layer plate, use immobilization with adhesive tape on every side, the photograph thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B thin layer chromatography scanning) scan wavelength: λ S=520nm, λ R=650nm, measure test sample absorbance log integrated value and reference substance absorbance log integrated value, calculate, namely.Every contains the Radix Astragali with Astragaloside IV (C
41H
68O
14) meter, be no less than 0.06mg.
Embodiment 11: the mensuration of content of Danshensu.
Only adopt the content of Astragaloside IV in the tlc scanning determination Radix Astragali in the proper mass standard, for further accurately controlling the astragalus leech network ressel freeing capsule quality, cure mainly and the medicinal material dosage according to the astragalus leech network ressel freeing capsule function, select the red sage root and the radix paeoniae rubrathe two flavor medicinal materials, adopt the HPLC method that its effective constituent is carried out assay, wherein red sage root selection danshensu is index, and it is index that the radix paeoniae rubrathe is selected Paeoniflorin.
1, instrument and reagent.
The Agilent high performance liquid chromatograph, the Agilent1100 chem workstation; KQ3200DB type numerical control supersonic washer (Kunshan Ultrasonic Instruments Co., Ltd.); Electronic balance (German Sai Duolisi BP211D); Methyl alcohol is chromatographically pure (Tianjin Siyou Fine Chemicals Co., Ltd.); Water is distilled water; It is pure that other chemical reagent is analysis; The Sodium Danshensu reference substance is (for survey usefulness, lot number: 110855-200809) provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute are provided.
2, chromatographic condition.
Chromatographic column: octadecylsilane chemically bonded silica is filling agent, 4.6mm * 250mm; Column temperature: room temperature; Phase flows: 1% glacial acetic acid-methyl alcohol (94: 6); Flow velocity: 1ml/min; Detect wavelength 280nm.Number of theoretical plate calculates by the Sodium Danshensu peak should be not less than 3000.
3, the preparation of reference substance solution.
Precision takes by weighing the Sodium Danshensu reference substance, adds 50% methyl alcohol and makes the solution (be equivalent to every 1ml and contain danshensu 0.135mg) that every 1ml contains 0.15mg, namely.
4, the preparation of need testing solution.
Get the astragalus leech network ressel freeing capsule content, porphyrize, mixing, get about 4g, the accurate title, decide, and puts in the tool plug conical flask, accurate hydrochloric acid solution (1 → 50) 50ml that adds, close plug claims to decide weight, ultrasonic processing (power 150W, frequency 40kHz) 30 minutes is put cold, claim again to decide weight, supply the weight that subtracts mistake with hydrochloric acid solution (1 → 50), shake up, add sodium chloride 5g, shake up, centrifugal, precision is measured supernatant 25ml, puts in the separating funnel, extracts 4 times (50 with the ethyl acetate jolting, 30,20,20ml), combined ethyl acetate liquid reclaims ethyl acetate to doing, and residue dissolves with 50% methyl alcohol, be transferred in the 10ml measuring bottle, and be diluted to scale, and shake up, filter with miillpore filter (0.45 μ m), get subsequent filtrate, namely.
5, the mensuration of content of Danshensu.
Accurate reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph respectively, measure, namely.Every in preparation contains the red sage root with danshensu (C
9H
10O
5) meter, must not be less than 0.15mg.
6, negative sample test.
Prepare the negative preparation that does not contain the red sage root by the astragalus leech network ressel freeing capsule prescription, extract, measure by the need testing solution preparation method.Under the chromatographic condition of determining, record the chromatogram of danshensu reference substance, test sample and negative control product respectively, collection of illustrative plates is the result show: with the corresponding section of Sodium Danshensu retention time, no chromatographic peak occurs in the negative control product chromatogram, namely under the condition determination of determining, other flavour of a drug are noiseless to the mensuration of the red sage root in the preparation prescription.
7, the investigation of linear relationship.
Precision takes by weighing the Sodium Danshensu reference substance, adds 50% methyl alcohol and makes the solution that every 1ml contains 0.1633mg.Accurate 1,2.5,5,10,15, the 20 μ l sample introductions of drawing are measured (seeing Table 1) in accordance with the law respectively, and (μ g) is horizontal ordinate with Sodium Danshensu, and peak area integrated value (A) calculates regression equation for ordinate: y=648.67x-3.5208, r=1.Show that Sodium Danshensu content has good linear relationship in 0.1633 μ g-3.2666 μ g scope.
8, precision test.
The accurate Sodium Danshensu reference substance solution 10 μ l that draw repeat sample introduction 6 times, the results are shown in Table 2, and the RSD that tries to achieve the Sodium Danshensu peak area value is 0.15%, shows that the precision of method is good.
9, stability test.
The accurate absorption with a need testing solution 10 μ l, every 2 hours sample introductions once, sample introduction 6 times the results are shown in Table 3, and the RSD that tries to achieve the Sodium Danshensu peak area value is 1.35%, shows that test sample is good at 10 hours internal stabilities.
10, replica test.
Get same lot number sample (lot number: 090708), porphyrize, mixing is got about 4g, accurate claims surely, the preparation method extracts by need testing solution, 6 parts of parallel preparations, assay is surveyed 2 times, is averaged for every part.As calculated, average content is 0.5307mg/g, RSD is 1.07%.The results are shown in Table 4.
11, average recovery test.
Get 6 parts in the sample of known content respectively, accurately claim surely, accurately respectively add a certain amount of reference substance, the preparation method extracts by need testing solution, assay.The results are shown in Table 5.
The average average recovery of Sodium Danshensu is that 102.53%, RSD is 1.48% as a result, shows that between 95%-105% the method recovery is good.
Embodiment 12: the mensuration of paeoniflorin content.
1, instrument and reagent.
The Agilent high performance liquid chromatograph, the Agilent1100 chem workstation; KQ3200DB type numerical control supersonic washer (Kunshan Ultrasonic Instruments Co., Ltd.); Electronic balance (German Sai Duolisi BP211D); Acetonitrile is chromatographically pure (Tianjin fine chemicals company limited); Water is distilled water; It is pure that other chemical reagent is analysis; The Paeoniflorin reference substance is (for survey usefulness, lot number: 110736-200934) provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute are provided.
2, chromatographic condition.
Chromatographic column: octadecylsilane chemically bonded silica is filling agent, 4.6mm * 250mm; Column temperature: room temperature; Phase flows: acetonitrile-0.1% phosphoric acid (16: 84); Flow velocity: 1ml/min; Detect wavelength 230nm, number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000.
3, the preparation of reference substance solution.
Precision takes by weighing the Paeoniflorin reference substance, adds 50% methyl alcohol and makes the solution that every 1ml contains 0.1mg, namely.
4, the preparation of need testing solution.
Get the astragalus leech network ressel freeing capsule content, porphyrize, mixing, get about 4g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims to decide weight, ultrasonic processing (power 150W, frequency 40kHz) 30 minutes is put cold, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol, shake up, filter with miillpore filter (0.45 μ m), get subsequent filtrate, namely.
5, the mensuration of content of Danshensu.
Accurate reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph respectively, measure, namely.Every in preparation contains the radix paeoniae rubrathe with Paeoniflorin (C
23H
28O
11) meter, must not be less than 0.15mg.
6, negative sample test.
Prepare the negative preparation that does not contain the radix paeoniae rubrathe by the astragalus leech network ressel freeing capsule prescription, extract, measure by the need testing solution preparation method.Under the chromatographic condition of determining, record the chromatogram of Paeoniflorin reference substance, test sample and negative control product respectively, collection of illustrative plates is the result show: with the corresponding section of Paeoniflorin retention time, no chromatographic peak occurs in the negative control product chromatogram, namely under the condition determination of determining, other flavour of a drug are noiseless to the mensuration of the radix paeoniae rubrathe in the preparation prescription.
7, the investigation of linear relationship.
Precision takes by weighing the Paeoniflorin reference substance, adds 50% methyl alcohol and makes the solution that every 1ml contains 0.1012mg.Accurate 1,2.5,5,10,15, the 20 μ l sample introductions of drawing are measured (seeing Table 6) in accordance with the law respectively, and (μ g) is horizontal ordinate with Paeoniflorin, and peak area (A) calculates regression equation for ordinate: y=1148.2x-6.1517, r=1.Show that paeoniflorin content has good linear relationship in 0.1012 μ g-2.0240 μ g scope.
8, precision test.
The above-mentioned Paeoniflorin reference substance solution 10 μ l of accurate absorption repeat sample introduction 6 times, the results are shown in Table 7, and the RSD that tries to achieve the Paeoniflorin peak area value is 0.31%, shows that the precision of this method is good.
9, stability test.
The accurate absorption with a need testing solution 10 μ l, every 2 hours sample introductions once, sample introduction 6 times the results are shown in Table 8, and the RSD that tries to achieve the Paeoniflorin peak area value is 0.99%, shows that test sample is good at 10 hours internal stabilities.
10, replica test.
Get the sample of same lot number, porphyrize, mixing is got about 4g, and accurate the title, decide, and the preparation method extracts by need testing solution, 6 parts of parallel preparations, assay is surveyed 2 times, is averaged for every part.As calculated, average content is 0.3527mg/g, RSD is 1.09%.The results are shown in Table 9.
11, average recovery test.
Get 6 parts in the sample of known content respectively, accurately claim surely, accurately respectively add a certain amount of reference substance, the preparation method extracts by need testing solution, assay.The results are shown in Table 10.
The average average recovery of Paeoniflorin is that 99.55%, RSD is 1.75% as a result, shows that between 95%-105% the method recovery is good.
Claims (5)
1. the method for quality control of astragalus leech network ressel freeing capsule, described astragalus leech network ressel freeing capsule is the medicine that is prepared from by following bulk drug: the Radix Astragali, leech, genseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine, Radix Angelicae Sinensis, Ligusticum wallichii, ilex pubescens, the radix paeoniae rubrathe, reticulate millettia, the red sage root, prepared fleece flower root, safflower, Herba Lycopi, ground bettle, earthworm, stiff silkworm, root tuber of aromatic turmeric, turmeric, scorpio, rhizoma Gastrodiae, Chinese cassia tree, notopterygium root, fructus gleditsiae, arisaema cum bile, borneol, and the discrimination method in the described method of quality control comprises following discriminating:
1) gets described drug substance contents 5g, add zeyssatite 3g, fully mixing, add methyl alcohol 30ml, ultrasonic processing 20 minutes filters, filtrate water bath method, residue add water 30ml makes dissolving, extracts 3 times with the ether jolting, each 20ml, aqueous solution is standby, merges ether extracted liquid, flings to ether, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other leech control medicinal material 1.5g that fetches water adds methyl alcohol 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, in contrast medicinal material solution; Test according to thin-layered chromatography, drawing need testing solution 3 μ l, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with methenyl choloride: methyl alcohol=40:1, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to spot colour developing at 100 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
2) get 1) the following aqueous solution after the extracted by ether, extract 3 times with water saturated normal butyl alcohol jolting, be respectively 20ml 3 times, 10ml, 10ml merges n-butanol extracting liquid, extract 3 times with ammonia solution, each 15ml discards ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue adds water 3ml makes dissolving, be added on internal diameter 2cm, on the D101 type macroporous absorbent resin post of long 12cm, with water 50ml wash-out, discard water liquid, use 40% ethanol 30ml wash-out again, discard 40% ethanol eluate, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Paeoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=40:5:10:0.2 is developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to spot colour developing at 100 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
3) get Astragaloside IV, ginsenoside Rg1's reference substance, add methyl alcohol and make the solution that every 1ml contains 1mg, 2mg respectively, in contrast product solution; According to the thin-layered chromatography test, draw each 2 μ l, 2 of above-mentioned reference substance solution) following need testing solution 4 a μ l, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol: glacial acetic acid: water=8:1:1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 100 ℃, puts respectively under daylight and the 365nm ultraviolet lamp and inspects, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show spot and the fluorescence spot of same color respectively;
4) get described drug substance contents 10g, the 30ml that adds diethyl ether, ultrasonic processing 10 minutes filters, and filtrate volatilizes, and residue adds methenyl choloride 1ml makes dissolving, as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; Test according to thin-layered chromatography, draw need testing solution 5 μ l, control medicinal material solution 1 μ l, putting respectively on same silica gel g thin-layer plate, is developping agent with normal hexane: ethyl acetate=9:1, launches, take out, dry, put under the 365nm ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;
5) get described drug substance contents 5g, add water 20ml, ultrasonic processing 5 minutes, centrifugal, get supernatant, add watery hydrochloric acid and regulate pH to 2, add ethyl acetate 20ml, jolting is extracted, and extract is put evaporate to dryness in the water-bath, and residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets red sage root control medicinal material 0.5g, and it is an amount of to add water, decocts half an hour, filters, and filtrate is concentrated into about 10ml, shines medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw need testing solution 2 μ l, control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride: acetone: formic acid=8:1:1 is developping agent, launch, take out, dry, put in the ammonia steam smoked after, placement is spent the night, and puts under the 365nm ultraviolet lamp and inspects, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;
It is characterized in that the discrimination method in the described method of quality control also comprises following discriminating:
6) get described drug substance contents 5g, add methyl alcohol 25ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets ground bettle control medicinal material 1g, shines medicinal material solution in pairs with legal system; Test according to thin-layered chromatography, draw need testing solution 2 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with toluene: methylene chloride: acetone=5:5:0.5 is developping agent, launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, 105 ℃ to be heated to spot colour developing clear, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
7) get described drug substance contents 20g, add ethanol 40ml, cold soaking 20 minutes, jolting constantly filters, and filtrate volatilizes, and adds ethanol 1ml and makes dissolving, as need testing solution; Other gets Chinese cassia tree control medicinal material 2g, shines medicinal material solution in pairs with legal system; Get the cinnaldehydrum reference substance again, add ethanol and make the reference substance solution that every 1ml contains 1 μ l; Test according to thin-layered chromatography, draw need testing solution 15 μ l, control medicinal material solution 2 μ l, reference substance solution 2 μ l, putting respectively on same silica gel g thin-layer plate, is developping agent with 60-90 ℃ of sherwood oil: ethyl acetate=17:3, launches, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
8) get described drug substance contents 5g, add methenyl choloride 30ml, ultrasonic processing 30 minutes filters, and filtrate volatilizes, and adds chloroform 1ml and makes dissolving, as need testing solution; It is an amount of that other gets the borneol control medicinal material, adds methenyl choloride and make the control medicinal material solution that every 1ml contains 10mg; Test according to thin-layered chromatography, drawing need testing solution 10 μ l, control medicinal material solution 6 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with cyclohexane: ethyl acetate=17:3, launch, take out, dry, spray is with 2% vanillic aldehyde sulfuric acid test solution, 105 ℃ to be heated to spot colour developing clear, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
9) get described drug substance contents 5g, add ethyl acetate 20ml, concentrated hydrochloric acid 0.5ml, ultrasonic processing 20 minutes filters, and filtrate volatilizes, and adds ethyl acetate 1ml and makes dissolving, as need testing solution; Other gets fleece-flower root control medicinal material 0.1g, shines medicinal material solution in pairs with legal system; Get the archen reference substance again, add methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin-layered chromatography, draw need testing solution 15 μ l, control medicinal material solution 5 μ l, reference substance solution 2 μ l, put respectively on same silica gel g thin-layer plate, with toluene: the upper solution of ethyl acetate: formic acid=20:2:1 is developping agent, launches, take out, dry, it is smoked clear to the spot colour developing to put in the ammonia steam, in the test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, show the spot of same color.
2. the method for quality control of astragalus leech network ressel freeing capsule according to claim 1 is characterized in that content assaying method in the described method of quality control comprises the mensuration of Astragaloside content, content of Danshensu and paeoniflorin content.
3. the method for quality control of astragalus leech network ressel freeing capsule according to claim 2 is characterized in that the content with the described Astragaloside IV of following tlc scanning determination:
1) preparation of reference substance solution: precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.5mg;
2) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds claims to decide weight, ultrasonic processing 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, discard filtrate just, precision is measured subsequent filtrate 25ml, reclaims methyl alcohol to doing residue water 40ml dissolving while hot, be transferred in the separating funnel, extract 3 times with the ether jolting, each consumption is respectively 20,15,10ml, discard ether solution, water liquid continues and extracts 4 times with water saturated normal butyl alcohol, each 15ml, merge normal butyl alcohol liquid, with 0.5% sodium hydroxide solution washing 3 times, each 20ml discards alkali lye, extremely neutral with the water washing that normal butyl alcohol is saturated, discard water lotion, normal butyl alcohol evaporate to dryness, residue add methyl alcohol makes dissolving, be transferred in the 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution;
3) the accurate need testing solution 10 μ l that draw, reference substance solution 2 μ l and 4 μ l, the point of crossing is on same silica gel g thin-layer plate respectively, with methenyl choloride: methyl alcohol: water=65:35:10 mixed solution is placed the lower floor's solution that spends the night below 10 ℃ be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 100 ℃, takes out, cover onesize glass plate at thin layer plate, use immobilization with adhesive tape on every side, with wavelength X S=520nm, λ R=650nm scans, and measures the integrated value of test sample absorbance log and the integrated value of reference substance absorbance log, calculate, namely.
4. the method for quality control of astragalus leech network ressel freeing capsule according to claim 2 is characterized in that the content with the described danshensu of following high effective liquid chromatography for measuring:
1) chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; Be the phase that flows with 1% glacial acetic acid: methyl alcohol=94:6; Detect wavelength 280nm; Number of theoretical plate calculates by the Sodium Danshensu peak and is not less than 3000;
2) preparation of reference substance solution: precision takes by weighing the Sodium Danshensu reference substance, adds 50% methyl alcohol and makes the reference substance solution that every 1ml contains the 0.135mg danshensu;
3) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 4g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 2% hydrochloric acid solution 50ml that adds, close plug claims to decide weight, power 150W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with 2% hydrochloric acid solution, shake up, add sodium chloride 5g, shake up, centrifugal, precision is measured supernatant 25ml, put in the separating funnel, extract 4 times with the ethyl acetate jolting, each consumption is respectively 50,30,20,20ml, combined ethyl acetate liquid, reclaim ethyl acetate to doing, residue dissolves with 50% methyl alcohol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate, namely;
4) precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, namely.
5. the method for quality control of astragalus leech network ressel freeing capsule according to claim 2 is characterized in that with the described content of paeoniflorin of following high effective liquid chromatography for measuring:
1) chromatographic condition and system suitability test: be filling agent with the octadecylsilane chemically bonded silica; With acetonitrile: 0.1% phosphoric acid=16:84 is the phase that flows; Detect wavelength 230nm, number of theoretical plate calculates by the Paeoniflorin peak and is not less than 3000;
2) preparation of reference substance solution: precision takes by weighing the Paeoniflorin reference substance, adds 50% methyl alcohol and makes the reference substance solution that every 1ml contains 0.1mg;
3) preparation of need testing solution: get described drug substance contents, porphyrize, mixing, get 4g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 50ml that adds, close plug claims to decide weight, the ultrasonic processing of power 150W, frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methyl alcohol, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate, namely;
4) precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, namely.
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| CN109932470A (en) * | 2019-04-12 | 2019-06-25 | 安徽中医药大学第一附属医院 | The alkalization method of silica G plate in a kind of traditional Chinese medicine of licorice root preparation indentification by TLC |
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| CN104161847A (en) * | 2014-07-25 | 2014-11-26 | 北京汉典制药有限公司 | Quality detection method of traditional Chinese medicinal composition for curing diabetic retinopathy |
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| CN114839301B (en) * | 2022-04-19 | 2024-11-01 | 王喜军 | Qihe capsule UPLC-UV contrast fingerprint and establishing method and application thereof |
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