The method of quality control of ZHANGYANMING PIAN
Technical field
The present invention relates to a kind of method of quality control of ZHANGYANMING PIAN.
Background technology
ZHANGYANMING PIAN be a kind of be used for the treatment of the initial stage and year in mid-term cataract Chinese medicine, this product records in the 18 in ministerial standard Chinese traditional patent formulation preparation by grass-leaved sweetflag, cassia seed, 22 flavor medicinal materials such as golden cypress are made, because the part medicinal material does not have reference substance, or owing to big some composition difficulty of prescription is separated, simultaneously, do not have content assaying method in the proper mass control method, be not enough to control the quality of finished product.
Summary of the invention
The objective of the invention is for a kind of root of kudzu vine in original ZHANGYANMING PIAN that improved is provided, the qualitative authentication method of golden cypress, increased the medicine assay step, can measure the ZHANGYANMING PIAN content of effective, with the specificity and the validity of the control that improves the quality, help improving the method for quality control of the quality of ZHANGYANMING PIAN finished product.
Main technical schemes of the present invention is: the method for quality control of this ZHANGYANMING PIAN comprises following steps;
(1) gets 15 of ZHANGYANMING PIAN finished products, remove sugar-coat, porphyrize, add methyl alcohol 30ML, soaked 3 hours, filter, filter residue washs with methyl alcohol 10ml, and washing lotion is incorporated in the filtrate, adds activated charcoal 4g, put in the water-bath warm 5 minutes, and filtered, filtrate is concentrated into about 1ml, add 2 of freshly prepared aniline solutions (get aniline 15ml, add concentrated hydrochloric acid 1ml, mixing), promptly show pale brown look, crossfade into brownish red.
(2) get 30 of ZHANGYANMING PIAN finished products, remove sugar-coat, porphyrize is put in the conical flask, add ethanol 50ml, reflux 30 minutes, inclining ethanol extract, filter, add 70% ethanol 50ml again, reflux 30 minutes, filter, filtrate merges, and is concentrated into about 15ml, move in the separating funnel, add ethyl acetate and extract 2 times, each 20ml, merge ethyl acetate extract (aqueous solution is standby), with reclaiming ethyl acetate behind the anhydrous sodium sulfate dehydration to about 0.5ml, as need testing solution.Other gets root of kudzu vine control medicinal material 1g, shines medicinal material solution in pairs with legal system; Get the Puerarin reference substance again, add absolute ethyl alcohol and make every 1ml and contain 0.35mg solution, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-water (7: 3: 0.3) is developping agent, launch, take out, dry, for a moment smoked with the ammonia steam, put under the ultraviolet light (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence principal spot of same color.With the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
(3) get the aqueous solution after ethyl acetate extracts under (2) item, regulate pH value greater than 10 with ammonia solution, add chloroform 40ml and extract, chloroform solution anhydrous sodium sulfate dehydration, evaporate to dryness, residue add absolute ethyl alcohol 1ml makes dissolving, as need testing solution.Other gets golden cypress control medicinal material 0.2g, adds acidic ethanol 20ml, and reflux 30 minutes is filtered, and evaporate to dryness is regulated pH value greater than 10 with ammonia solution, shines medicinal material solution in pairs with legal system.Get the Berberine hydrochloride reference substance again, add methyl alcohol and make the solution that every 1ml contains 0.03mg, in contrast product solution.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 2 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-glacial acetic acid-water (14: 2: 5) is developping agent, launches, and takes out, dry, put ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
The method of quality control of ZHANGYANMING PIAN of the present invention, the photograph thin-layered chromatography is carried out assay to the effective constituent Berberine hydrochloride of golden cypress in the ZHANGYANMING PIAN finished product.
The method of quality control of ZHANGYANMING PIAN of the present invention in the assay that the effective constituent Berberine hydrochloride of golden cypress carries out in the ZHANGYANMING PIAN finished product, is got 20 of ZHANGYANMING PIAN finished products, remove sugar-coat, the accurate title, decided porphyrize, precision takes by weighing about 0.5g, puts in the 100ml volumetric flask, adds 0.1% (V/V) hydrochloric acid solution 1ml, placed 10 minutes abundant wetting back, it is an amount of to add methyl alcohol, and sonicated 30 minutes is put cold, add methyl alcohol and be diluted to scale, shake up, placed 12 hours, accurate supernatant (the filtering in case of necessity) 50ml that draws, evaporate to dryness, it is abundant wetting that residue adds 3 in water, adds absolute ethyl alcohol 10ml and make dissolving, and (105 ℃ activate 1 hour to be added on the neutral alumina post, 100~200 orders, 4g, internal diameter 2cm) on, container washs with the gradation of 10ml absolute ethyl alcohol, washing lotion is added on the neutral alumina post, add absolute ethyl alcohol 80ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol to be made dissolving and is settled to 2ml, as need testing solution.Other precision takes by weighing through 12 hours Berberine hydrochloride reference substance of phosphorus pentoxide vacuum drying an amount of, adds methyl alcohol and makes the solution that every 1ml contains 0.03mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), the accurate need testing solution 5 μ l that draw, reference substance solution 1 μ l and 3 μ l, the point of crossing is on same silica gel g thin-layer plate respectively, and (12: 6: 3: 3: 1`5) be developping agent, groove added isopyknic strong ammonia solution in addition with benzene-ethyl acetate-methyl alcohol-isopropyl alcohol-strong ammonia solution, pre-equilibration 15 minutes, launch, take out, dry.Carry out fluorescent scanning according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B thin layer chromatography scanning), excitation wavelength is 366nm, measures the integrated value of test sample and reference substance fluorescence intensity, calculates, promptly.Calculate according to ZHANGYANMING PIAN finished product dry product, every contains golden cypress in Berberine hydrochloride, and it is qualified being no less than setting value.
Above-mentioned steps need not carried out according to sequencing, and simultaneously can also carry out conventional sense, and as the observation of proterties, this ZHANGYANMING PIAN finished product is a sugar coated tablet, shows sepia after removing sugar-coat, and it is sweet to distinguish the flavor of, little acid.This finished product also should meet relevant every regulation under an appendix ID of Chinese Pharmacopoeia version in 2000 the tablet item.
The ZHANGYANMING PIAN finished product that meets above condition is qualified.
The present invention is the root of kudzu vine in having improved original ZHANGYANMING PIAN, the qualitative authentication method of golden cypress, increased the medicine assay step, can measure the ZHANGYANMING PIAN content of effective, with the specificity and the validity of the control that improves the quality, help the quality that improves the ZHANGYANMING PIAN finished product is effectively controlled.
Embodiment
The method of quality control of a kind of ZHANGYANMING PIAN of present embodiment comprises following steps:
(1) gets 15 of ZHANGYANMING PIAN finished products, remove sugar-coat, porphyrize, add methyl alcohol 30ml, soaked 3 hours, filter, filter residue washs with methyl alcohol 10ml, and washing lotion is incorporated in the filtrate, adds activated charcoal 4g, put in the water-bath warm 5 minutes, and filtered, filtrate is concentrated into about 1ml, add 2 of freshly prepared aniline solutions (get aniline 15ml, add concentrated hydrochloric acid 1ml, mixing), promptly show pale brown look, crossfade into brownish red.
(2) get 30 of ZHANGYANMING PIAN finished products, remove sugar-coat, porphyrize is put in the conical flask, add ethanol 50ml, reflux 30 minutes, inclining ethanol extract, filter, add 70% ethanol 50ml again, reflux 30 minutes, filter, filtrate merges, and is concentrated into about 15ml, move in the separating funnel, add ethyl acetate and extract 2 times, each 20ml, merge ethyl acetate extract (aqueous solution is standby), with reclaiming ethyl acetate behind the anhydrous sodium sulfate dehydration to about 0.5ml, as need testing solution.Other gets root of kudzu vine control medicinal material 1g, shines medicinal material solution in pairs with legal system; Get the Puerarin reference substance again, add absolute ethyl alcohol and make every 1ml and contain 0.35mg solution, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-water (7: 3: 0.3) is developping agent, launch, take out, dry, for a moment smoked with the ammonia steam, put under the ultraviolet light (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence principal spot of same color.With the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
(3) get the aqueous solution after ethyl acetate extracts under (2) item, regulate pH value greater than 10 with ammonia solution, add chloroform 40ml and extract, chloroform solution anhydrous sodium sulfate dehydration, evaporate to dryness, residue add absolute ethyl alcohol 1ml makes dissolving, as need testing solution.Other gets golden cypress control medicinal material 0.2g, adds acidic ethanol 20ml, and reflux 30 minutes is filtered, and evaporate to dryness is regulated pH value greater than 10 with ammonia solution, shines medicinal material solution in pairs with legal system.Get the Berberine hydrochloride reference substance again, add methyl alcohol and make the solution that every 1ml contains 0.03mg, in contrast product solution.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 2 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-glacial acetic acid-water (14: 2: 5) is developping agent, launches, and takes out, dry, put ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
The method of quality control of the ZHANGYANMING PIAN of present embodiment, photograph thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) is carried out assay to the effective constituent Berberine hydrochloride of golden cypress in the ZHANGYANMING PIAN finished product.
The method of quality control of the ZHANGYANMING PIAN of present embodiment in the assay that the effective constituent Berberine hydrochloride of golden cypress carries out in the ZHANGYANMING PIAN finished product, is got 20 of ZHANGYANMING PIAN finished products, remove sugar-coat, the accurate title, decided porphyrize, precision takes by weighing about 0.5g, puts in the 100ml volumetric flask, adds 0.1% (V/V) hydrochloric acid solution 1ml, placed 10 minutes abundant wetting back, it is an amount of to add methyl alcohol, and sonicated 30 minutes is put cold, add methyl alcohol and be diluted to scale, shake up, placed 12 hours, accurate supernatant (the filtering in case of necessity) 50ml that draws, evaporate to dryness, it is abundant wetting that residue adds 3 in water, adds absolute ethyl alcohol 10ml and make dissolving, and (105 ℃ activate 1 hour to be added on the neutral alumina post, 100~200 orders, 4g, internal diameter 2cm) on, container washs with the gradation of 10ml absolute ethyl alcohol, washing lotion is added on the neutral alumina post, add absolute ethyl alcohol 80ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol to be made dissolving and is settled to 2ml, as need testing solution.Other precision takes by weighing through 12 hours Berberine hydrochloride reference substance of phosphorus pentoxide vacuum drying an amount of, adds methyl alcohol and makes the solution that every 1ml contains 0.03mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), the accurate need testing solution 5 μ l that draw, reference substance solution 1 μ l and 3 μ l, the point of crossing is a developping agent with benzene-ethyl acetate-methyl alcohol-isopropyl alcohol-strong ammonia solution (12: 6: 3: 3: 1.5) on same silica gel g thin-layer plate respectively, and groove adds isopyknic strong ammonia solution in addition, pre-equilibration 15 minutes, launch, take out, dry.Carry out fluorescent scanning according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B thin layer chromatography scanning), excitation wavelength lambda=366nm, the integrated value of measurement test sample and reference substance fluorescence intensity is calculated, promptly.Calculate according to ZHANGYANMING PIAN finished product dry product, every contains golden cypress in Berberine hydrochloride, and it is qualified being no less than setting value.
In the present embodiment, contain golden cypress in Berberine hydrochloride in the every ZHANGYANMING PIAN finished product, setting value is 3mg.
Above-mentioned steps need not carried out according to sequencing, and simultaneously can also carry out conventional sense, and as the observation of proterties, this ZHANGYANMING PIAN finished product is a sugar coated tablet, shows sepia after removing sugar-coat, and it is sweet to distinguish the flavor of, little acid.This finished product also should meet relevant every regulation under an appendix ID of Chinese Pharmacopoeia version in 2000 the tablet item.
The ZHANGYANMING PIAN finished product that meets above condition is qualified.