CN101028388B - Quality inspection of Chinese-medicinal preparation for treating shortsighness and asthenopia - Google Patents
Quality inspection of Chinese-medicinal preparation for treating shortsighness and asthenopia Download PDFInfo
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Abstract
A quality test method for the Chinese medicine used to treat ophthalmopathy, myopia and eye strain features that a qualitative discrimination is used for paeonol, chrysophanol, emodin, ararobinol, paeoniflorin, danshensu sodium and icariin, and a high-effect liquid-phase chlomatography is used to test the content of berberine hydrochloride.
Description
One, technical field:
The invention belongs to the Chinese traditional patent formulation formulation art, relate to a kind of quality determining method for the treatment of ophthalmic myopia and asthenopia Chinese medicine preparation specifically.
Two, background technology:
Treatment ophthalmic myopia and the bright medicine series of asthenopia Chinese medicine preparation Qi made with Fructus Lycii, Fructus Ligustri Lucidi, Radix Polygoni Multiflori etc. have the liver and the kidney tonifying, the effect of blood circulation-activating eyesight-improving.The eye that is used for due to the teenager hepatic and renal YIN deficiency is aching and tired, diseases such as eye socket pain.Yet the Chinese medicinal ingredients more complicated contains many unknown compositions.Can effectively control the product quality of treatment ophthalmic myopia and asthenopic Chinese medicine preparation at present, detection method that can be easy to operate is not also reported again.
Three, summary of the invention:
The object of the present invention is to provide a kind of can effectively control to treat ophthalmic myopia and asthenopic Chinese medicine preparation quality, the quality determining method that accuracy is high.
A kind of quality determining method for the treatment of ophthalmic myopia and asthenopia Chinese medicine preparation of the present invention is ground into fine powder with Radix Isatidis, Bombyx Batryticatus, and is standby; With Cortex Moutan, Radix Polygoni Multiflori, Cortex Phellodendri, Radix Paeoniae Rubra is ground into fine powder, all the other Fructus Lycii, Semen Cuscutae, Fructus Ligustri Lucidi, Fructus Leonuri, Fructus Corni, Herba Epimedii, Flos Eriocauli, the Herba Equiseti Hiemalis, Semen Cassiae, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, Radix Rehmanniae, Flos Carthami, Caulis Spatholobi 14 flavors, decoct with water three times, add 8 times of amounts of water for the first time, soaked 30 minutes, decocted 1 hour, second, respectively add 6 times of amounts of water for three times, decocted collecting decoction 1 hour, filter, relative density was 1.30 thick paste when filtrate decompression was concentrated into 60 ℃, added above-mentioned medical material fine powder, mixed, drying in the time of 60 ℃, be ground into fine powder, make granule, drying, the Borneolum Syntheticum porphyrize, add in the above-mentioned granule, mixing incapsulates, that is, its detection method may further comprise the steps:
(1) get this product content 5g, porphyrize, the 50ml that adds diethyl ether, low temperature reflux extracted 30 minutes, put coldly, filtered, and filtrate is concentrated into 10ml, as need testing solution; Other gets the Borneolum Syntheticum reference substance, adds diethyl ether to make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 2 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with 9: 1: 1 60-90 ℃ petroleum ether-toluene-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(2) get need testing solution under the discriminating (1), volatilize ether naturally, residue adds acetone 1ml makes dissolving, as need testing solution; Other gets the paeonol reference substance, adds acetone and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 3: 1 cyclohexane extraction-ethyl acetate, launch, take out, to dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get this product content 10g, porphyrize adds normal hexane 30ml, and supersound process 30 minutes filters, and filtrate is concentrated into 0.5ml, as need testing solution; Other depends on pine torch control medicinal material 1g, shines medical material solution in pairs with legal system; Get the chrysophanol reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(4) get this product content 10g, porphyrize adds methanol 40ml, put in the water-bath reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, adds hydrochloric acid 2ml again, puts in the water-bath and heats 30 minutes, cooling is extracted at twice with ether immediately, each 20ml, merge ether solution, volatilize, residue adds chloroform 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get emodin, physcione reference substance again, add methanol and make the mixed solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(5) get this product content 10g, porphyrize adds ethyl acetate 30ml, and reflux, extract, is 1 hour in the tepidarium, puts coldly, filters, and discards acetic acid ethyl fluid, and residue is flung to solvent, adds ethanol 30ml reflux, extract, 1 hour, filters, and filtrate is concentrated into 5ml, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 35: 1 chloromethanes-methanol is developing solvent, launches, and takes out, and dries, and spray is with 5% vanillin sulfuric acid solution, and 100 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(6) get this product content 5g, porphyrize adds water 15ml and makes dissolving, adds dilute hydrochloric acid and regulates pH value to 2, adds ethyl acetate 30ml, and jolting is extracted, and extracting solution is put evaporate to dryness in the water-bath, and residue adds methanol 2ml makes dissolving, as need testing solution; Other gets the danshensu sodium reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 25: 10: 4 chloroform-acetone-formic acid was developing solvent, launched, and took out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to speckle colour developing at 100 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(7) get this product content 10g, porphyrize is put in the extractor of the rope third constellations, adds 70% alcohol reflux to colourless, the extracting solution water-bath boils off ethanol, add hot water 15ml, mixing filters while hot, filtrate moves in the separatory funnel, add the chloroform jolting and extract 3 times, for the first time 15ml, 10ml for the second time, 10ml for the third time, chloroform liquid discards, and water liquid extracts 5 times with water saturated n-butyl alcohol jolting, is respectively 15,15,10,10,10ml, merge n-butyl alcohol liquid, with ammonia solution washing 5 times, each 15ml continues with the saturated water 25ml washing of n-butyl alcohol; Divide and get n-butyl alcohol liquid, water bath method, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution; Other gets the icariin reference substance, adds dehydrated alcohol and makes 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 two kinds of solution, put respectively on same silica gel g thin-layer plate, the upper strata liquid of placing below 10 ℃ with 1.3: 1: 1 butyl acetate-formic acid-water is developing solvent, and presaturation 15 minutes launches, take out, dry, spray is with 5% aluminum chloride alcoholic solution, and 105 ℃ to be heated to speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color; Under the 365nm ultra-violet lamp, inspect, show the fluorescence speckle of same color;
(8) berberine hydrochloride content:
The preparation of reference substance solution: get berberine hydrochloride reference substance 10mg, the accurate title, decide, put in the 100ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, and precision is measured 5ml, put in the 50ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, promptly gets hydrochloric berberine 0.01mg reference substance solution among every 1ml;
The preparation of need testing solution: get the content under this product content uniformity item, mixing is got about 1.0g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol-hydrochloric acid solution the 50ml that adds 100: 1, close plug claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate, promptly;
According to high effective liquid chromatography for measuring, be filler with the octadecylsilane chemically bonded silica; Dodecyl sodium sulfate with sodium dihydrogen phosphate-0.025mol/L of acetonitrile-0.1mol/L of 50: 25: 25 is a mobile phase; The detection wavelength is 265nm; Number of theoretical plate calculates by the berberine hydrochloride peak should be not less than 3000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure the content of berberine hydrochloride in the calculation sample.
The present invention draws by test, and every contains Cortex Phellodendri with berberine hydrochloride (C
20H
18ClNO
4) meter, must not be less than 0.20mg.
Treatment ophthalmic myopia of the present invention and asthenopia Chinese medicine preparation comprise pill, granule, capsule, tablet regular dosage form.
The meaning that quality standard of the present invention improves is: the invention provides the qualitative identification to paeonol, chrysophanol, emodin, physcione, peoniflorin, danshensu sodium, icariin, made best test sample preparation method, found suitable developing solvent, can carry out effective qualitative identification said preparation.The invention provides the content assaying method of berberine hydrochloride in addition, because berberine hydrochloride and other component separating degree, repeatability, precision, the response rate are good, so adopt the content of berberine hydrochloride in high effective liquid chromatography for measuring this product, make best test sample preparation method and mobile phase compound method, improved the content detection accuracy of berberine hydrochloride greatly.The foundation of this method helps on the market true and false quality of treatment ophthalmic myopia and asthenopia Chinese medicine preparation being differentiated.
Four, the specific embodiment:
Experimental example:
Radix Isatidis, Bombyx Batryticatus are ground into fine powder, standby; With Cortex Moutan, Radix Polygoni Multiflori, Cortex Phellodendri, Radix Paeoniae Rubra is ground into fine powder, all the other Fructus Lycii, Semen Cuscutae, Fructus Ligustri Lucidi, Fructus Leonuri, Fructus Corni, Herba Epimedii, Flos Eriocauli, the Herba Equiseti Hiemalis, Semen Cassiae, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, Radix Rehmanniae, Flos Carthami, Caulis Spatholobi 14 flavors, decoct with water three times, add 8 times of amounts of water for the first time, soaked 30 minutes, decocted 1 hour, second, respectively add 6 times of amounts of water for three times, decocted collecting decoction 1 hour, filter, relative density was 1.30 thick paste when filtrate decompression was concentrated into 60 ℃, added above-mentioned medical material fine powder, mixed, drying in the time of 60 ℃, be ground into fine powder, make granule, drying, the Borneolum Syntheticum porphyrize, add in the above-mentioned granule, mixing incapsulates, that is, its detection method may further comprise the steps:
(1) get this product content 5g, porphyrize, the 50ml that adds diethyl ether, low temperature reflux extracted 30 minutes, put coldly, filtered, and filtrate is concentrated into 10ml, as need testing solution; Other gets the Borneolum Syntheticum reference substance, adds diethyl ether to make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 2 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with 9: 1: 1 60-90 ℃ petroleum ether-toluene-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(2) get need testing solution under the discriminating (1), volatilize ether naturally, residue adds acetone 1ml makes dissolving, as need testing solution; Other gets the paeonol reference substance, adds acetone and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 3: 1 cyclohexane extraction-ethyl acetate, launch, take out, to dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get this product content 10g, porphyrize adds normal hexane 30ml, and supersound process 30 minutes filters, and filtrate is concentrated into 0.5ml, as need testing solution; Other depends on pine torch control medicinal material 1g, shines medical material solution in pairs with legal system; Get the chrysophanol reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(4) get this product content 10g, porphyrize adds methanol 40ml, put in the water-bath reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, adds hydrochloric acid 2ml again, puts in the water-bath and heats 30 minutes, cooling is extracted at twice with ether immediately, each 20ml, merge ether solution, volatilize, residue adds chloroform 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get emodin, physcione reference substance again, add methanol and make the mixed solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(5) get this product content 10g, porphyrize adds ethyl acetate 30ml, and reflux, extract, is 1 hour in the tepidarium, puts coldly, filters, and discards acetic acid ethyl fluid, and residue is flung to solvent, adds ethanol 30ml reflux, extract, 1 hour, filters, and filtrate is concentrated into 5ml, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 35: 1 chloromethanes-methanol is developing solvent, launches, and takes out, and dries, and spray is with 5% vanillin sulfuric acid solution, and 100 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(6) get this product content 5g, porphyrize adds water 15ml and makes dissolving, adds dilute hydrochloric acid and regulates pH value to 2, adds ethyl acetate 30ml, and jolting is extracted, and extracting solution is put evaporate to dryness in the water-bath, and residue adds methanol 2ml makes dissolving, as need testing solution; Other gets the danshensu sodium reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 25: 10: 4 chloroform-acetone-formic acid was developing solvent, launched, and took out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to speckle colour developing at 100 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(7) get this product content 10g, porphyrize is put in the extractor of the rope third constellations, adds 70% alcohol reflux to colourless, the extracting solution water-bath boils off ethanol, add hot water 15ml, mixing filters while hot, filtrate moves in the separatory funnel, add the chloroform jolting and extract 3 times, for the first time 15ml, 10ml for the second time, 10ml for the third time, chloroform liquid discards, and water liquid extracts 5 times with water saturated n-butyl alcohol jolting, is respectively 15,15,10,10,10ml, merge n-butyl alcohol liquid, with ammonia solution washing 5 times, each 15ml continues with the saturated water 25ml washing of n-butyl alcohol; Divide and get n-butyl alcohol liquid, water bath method, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution; Other gets the icariin reference substance, adds dehydrated alcohol and makes 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 two kinds of solution, put respectively on same silica gel g thin-layer plate, the upper strata liquid of placing below 10 ℃ with 1.3: 1: 1 butyl acetate-formic acid-water is developing solvent, and presaturation 15 minutes launches, take out, dry, spray is with 5% aluminum chloride alcoholic solution, and 105 ℃ to be heated to speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color; Under the 365nm ultra-violet lamp, inspect, show the fluorescence speckle of same color;
(8) berberine hydrochloride content:
The preparation of reference substance solution: get berberine hydrochloride reference substance 10mg, the accurate title, decide, put in the 100ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, and precision is measured 5ml, put in the 50ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, promptly gets hydrochloric berberine 0.01mg reference substance solution among every 1ml;
The preparation of need testing solution: get the content under this product content uniformity item, mixing is got about 1.0g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol-hydrochloric acid solution the 50ml that adds 100: 1, close plug claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate, promptly;
According to high effective liquid chromatography for measuring, be filler with the octadecylsilane chemically bonded silica; Dodecyl sodium sulfate with sodium dihydrogen phosphate-0.025mol/L of acetonitrile-0.1mol/L of 50: 25: 25 is a mobile phase; The detection wavelength is 265nm; Number of theoretical plate calculates by the berberine hydrochloride peak should be not less than 3000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure the content of berberine hydrochloride in the calculation sample.
Repeat above-mentioned steps once, the meansigma methods of content of berberine hydrochloride is the 0.28mg/ grain in the calculation sample.
With the described quality determining method repetition test of embodiment 9 times, draw content of berberine hydrochloride (mg/ grain) meansigma methods, the results are shown in following table:
Laboratory report:
1, instrument and reagent
Instrument: Agilentl 100 type high performance liquid chromatographs, Anjelen Sci. ﹠ Tech. Inc.G1311A type quaternary gradient pump, G1314A type UV-detector able to programme, G2170AA chem workstation.
Reagent: acetonitrile is a chromatographically pure, and water is ultra-pure water, and other chemical reagent is analytical pure.
Reference substance: berberine hydrochloride, lot number: 0714-9903 is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute for assay usefulness.
Test sample: Qi ' Yanming ' capsules for clearing, specification: every dress 0.4g; Produce by our company.
2, chromatographic condition
Chromatographic column: Kromasil 5u C
18100A, 250 * 4.6mm;
The dodecyl sodium sulfate of sodium dihydrogen phosphate-0.025mol/L of mobile phase: acetonitrile-0.1mol/L (50: 25: 25).
Detect wavelength: get berberine hydrochloride reference substance solution (C=0.036mg/ml), put in the ultraviolet spectrophotometer, carry out continuous wavelength scanning from 200～300nm, the result shows, the berberine hydrochloride reference substance has independently absworption peak at the 265nm place, so select the detection wavelength of 265nm as this product for use.
Under above-mentioned analysis condition, the berberine hydrochloride peak reaches baseline separation fully, and the separating degree of its peak and adjacent impurity peaks is greater than more than 1.7, and number of theoretical plate is pressed the berberine hydrochloride peak and calculated greater than 3000.
3, extraction conditions is selected and is extracted the completeness investigation
Extract our biliographic data of choice of Solvent, selected methanol and methanol-hydrochloric acid (100: 1) supersound process for use 30 minutes, carry out the comparison of extraction conditions.Result of the test shows that the impurity that adopts methanol-hydrochloric acid (100: 1) extracting method to extract disturbs few, the extraction efficiency height.The efficient of methanol extraction is low, and impurity disturbs more, and separating degree is undesirable, so select the text method as extraction conditions.
Extract completeness and investigate according to the extraction conditions analysis, the preparation process of text sample solution mainly is subjected to the influence of supersound process, and therefore, we investigate it and compare.
The supersound process time gets same batch sample (lot number: 20030303), about 1.5g accurate claims surely, carries out supersound process by 20,30,40 minutes different times respectively, measures, and result of the test is asked for an interview table 1 in accordance with the law extracting complete influence.
Test of table 1 supersound process and result
Result of the test shows that the sample size of supersound process after 30 minutes is tending towards constant substantially, is the supersound process time so select 30 minutes.
4, methodological study
The accurate absorption of the investigation of linear relationship and scope concentration is berberine hydrochloride reference substance solution 2,4,8,12, the 16 μ l of 0.036mg/ml, injects chromatograph of liquid respectively, measures in accordance with the law, and the result asks for an interview table 2.
Table 2 linear relationship is investigated test and result
With the sample size is abscissa, and the peak area integrated value is a vertical coordinate, the drawing standard curve, and carry out rectilinear regression, regression equation is Y=3421.7X-28.54, r=9991.Result of the test shows that this method sample size is good linear relationship in 0.072～0.576mg scope.
Accurate same reference substance solution (C=0.036mg/ml) the 10 μ l that draw of precision test repeat sample introduction 5 times continuously, measure the peak area integrated value, and result of the test is asked for an interview table 3.
Test of table 3 precision and result
Result of the test shows that the berberine hydrochloride reference substance repeats sample introduction and records relative standard deviation RSD＜2.0% of peak area integrated value for 5 times, so think that this method has good precision.
The simulation prescription of removing Cortex Phellodendri is got in negative blank test, makes the blank sample that does not contain Cortex Phellodendri, makes blank solution by the need testing solution method for making.20030303) and reference substance solution (C=0.014mg/ml) other gets need testing solution (lot number:, press the text method and inject hplc determination.Result of the test shows, in the test sample chromatograph, with the corresponding retention time of reference substance chromatograph position on, the single chromatographic peak of a correspondence is arranged, and in the blank liquid chromatography, does not have corresponding peak in this retention time corresponding position and occur.Illustrate that negative blank is noiseless to its mensuration, the specificity of its method is better.
Stability test is accurate draw with a collection of (lot number: 20030303) need testing solution and reference substance solution (C=0.014mg/ml), by 0,0.5,1,2,4 hour interval, inject chromatograph of liquid respectively and measure, result of the test please sees Table 4 respectively.
Test of table 4 study on the stability and result
Result of the test shows that the berberine hydrochloride of sample and reference substance is measured through this method, and comparatively stable in 4 hours, its relative standard deviation is respectively RSD=2.13% and RSD=2.39%.
Replica test gets with a collection of that (lot number: 20030303) sample is 5 parts, repeats handle to measure by the text method, and the result asks for an interview table 5.
Table 5 sample replica test and result
Result of the test shows that the relative standard deviation RSD=2.23% of 5 replications of this method illustrates that its repeatability is better.
Recovery test adopts application of sample recovery test method, precision takes by weighing with a collection of (lot number: 20030303) known content (0.183mg/ grain, amount to 0.4575mg/g) each about 1.0g of sample, the accurate title, decide, accurate respectively each the about 0.28～0.36mg of berberine hydrochloride reference substance that adds, measure, result of the test is asked for an interview table 6 in accordance with the law.
Table 6 application of sample recovery test and result
Result of the test shows that this method has the higher response rate, and its average recovery rate is 99.03%, and relative standard deviation is RSD=1.25%.
5, berberine hydrochloride content in the sample
Get version in 2005 and carry out 8 batch samples that the back produces, carry out berberine hydrochloride content by the text method, the result asks for an interview table 7.
Table 7 eight batch sample assay results (n=2)
6, the assay of berberine hydrochloride in the Cortex Phellodendri medical material
Get the Cortex Phellodendri medical material of separate sources, carry out assay by the text method, the result asks for an interview table 8
Table 8 Cortex Phellodendri medical material assay result (n=2)
7, content limit determines
In eight batch samples, the measured result of berberine hydrochloride, high-load is the 0.483mg/ grain, minimum is the 0.201mg/ grain, and prescribed limit of Cortex Phellodendri medical material Chinese Pharmacopoeia version in 2005 must not be and is less than 0.60%, and the high-load of three batches of separate sources medical materials of actual measurement is 1.08%, minimum content is 0.63%, consider the prescription consumption, factor affecting such as process loss and content uniformity are so contain Cortex Phellodendri with berberine hydrochloride (C in every of this product
20H
18ClNO
4) meter, must not be less than 0.20mg.
Claims (3)
1. a quality determining method for the treatment of ophthalmic myopia and asthenopia Chinese medicine preparation is ground into fine powder with Radix Isatidis, Bombyx Batryticatus, and is standby; With Cortex Moutan, Radix Polygoni Multiflori, Cortex Phellodendri, Radix Paeoniae Rubra is ground into fine powder, all the other Fructus Lycii, Semen Cuscutae, Fructus Ligustri Lucidi, Fructus Leonuri, Fructus Corni, Herba Epimedii, Flos Eriocauli, the Herba Equiseti Hiemalis, Semen Cassiae, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, Radix Rehmanniae, Flos Carthami, Caulis Spatholobi 14 flavors, decoct with water three times, for the first time add 8 times of amounts of water, soaked 30 minutes, decocted 1 hour, second, respectively add 6 times of amounts of water for three times, decocted 1 hour, collecting decoction filters, relative density was 1.30 thick paste when filtrate decompression was concentrated into 60 ℃, add above-mentioned medical material fine powder, mix drying in the time of 60 ℃, be ground into fine powder, make granule, drying, Borneolum Syntheticum porphyrize, add in the above-mentioned granule, mixing promptly, is characterized in that may further comprise the steps:
(1) get this product 5g, porphyrize, the 50ml that adds diethyl ether, low temperature reflux extracted 30 minutes, put coldly, filtered, and filtrate is concentrated into 10ml, as need testing solution; Other gets the Borneolum Syntheticum reference substance, adds diethyl ether to make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 2 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with 9: 1: 1 60-90 ℃ petroleum ether-toluene-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(2) get need testing solution under the discriminating (1), volatilize ether naturally, residue adds acetone 1ml makes dissolving, as need testing solution; Other gets the paeonol reference substance, adds acetone and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 3: 1 cyclohexane extraction-ethyl acetate, launch, take out, to dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get this product 10g, porphyrize adds normal hexane 30ml, and supersound process 30 minutes filters, and filtrate is concentrated into 0.5ml, as need testing solution; Other depends on pine torch control medicinal material 1g, shines medical material solution in pairs with legal system; Get the chrysophanol reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(4) get this product 10g, porphyrize adds methanol 40ml, put in the water-bath reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, adds hydrochloric acid 2ml again, puts in the water-bath and heats 30 minutes, cooling is extracted at twice with ether immediately, each 20ml, merge ether solution, volatilize, residue adds chloroform 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get emodin, physcione reference substance again, add methanol and make the mixed solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, launch with 15: 5: 1 30-60 ℃ petroleum ether-Ethyl formate-formic acid, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color; Put in the ammonia steam smoked after, speckle becomes redness;
(5) get this product 10g, porphyrize adds ethyl acetate 30ml, and reflux, extract, is 1 hour in the tepidarium, puts coldly, filters, and discards acetic acid ethyl fluid, and residue is flung to solvent, adds ethanol 30ml reflux, extract, 1 hour, filters, and filtrate is concentrated into 5ml, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 5: 1 chloroform-methanol, launch, take out, to dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(6) get this product 5g, porphyrize adds water 15ml and makes dissolving, adds dilute hydrochloric acid and regulates pH value to 2, adds ethyl acetate 30ml, and jolting is extracted, and extracting solution is put evaporate to dryness in the water-bath, and residue adds methanol 2ml makes dissolving, as need testing solution; Other gets the danshensu sodium reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 25: 10: 4 chloroform-acetone-formic acid was developing solvent, launched, and took out, dry, spray is with 10% phosphomolybdic acid ethanol solution, and it is clear to be heated to speckle colour developing at 100 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(7) get this product 10g, porphyrize is put in the apparatus,Soxhlet's, adds 70% alcohol reflux to colourless, the extracting solution water-bath boils off ethanol, add hot water 15ml, mixing filters while hot, filtrate moves in the separatory funnel, add the chloroform jolting and extract 3 times, for the first time 15ml, 10ml for the second time, 10ml for the third time, chloroform liquid discards, and water liquid extracts 5 times with water saturated n-butyl alcohol jolting, is respectively 15,15,10,10,10ml, merge n-butyl alcohol liquid, with ammonia solution washing 5 times, each 15ml continues with the saturated water 25ml washing of n-butyl alcohol; Divide and get n-butyl alcohol liquid, water bath method, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution; Other gets the icariin reference substance, adds dehydrated alcohol and makes 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 two kinds of solution, put respectively on same silica gel g thin-layer plate, the upper strata liquid of placing below 10 ℃ with 1.3: 1: 1 butyl acetate-formic acid-water is developing solvent, and presaturation 15 minutes launches, take out, dry, spray is with 5% aluminum chloride alcoholic solution, and 105 ℃ to be heated to speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color; Under the 365nm ultra-violet lamp, inspect, show the fluorescence speckle of same color;
(8) berberine hydrochloride content:
The preparation of reference substance solution: get berberine hydrochloride reference substance 10mg, the accurate title, decide, put in the 100ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, and precision is measured 5ml, put in the 50ml measuring bottle, methanol-the hydrochloric acid solution that adds 100: 1 shakes up to scale, promptly gets hydrochloric berberine 0.01mg reference substance solution among every 1ml;
The preparation of need testing solution: get the content under this product content uniformity item, mixing is got about 1.0g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, accurate methanol-hydrochloric acid solution the 50ml that adds 100: 1, close plug claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate, promptly;
According to high effective liquid chromatography for measuring, be filler with the octadecylsilane chemically bonded silica; Dodecyl sodium sulfate with sodium dihydrogen phosphate-0.025mol/L of acetonitrile-0.1mol/L of 50: 25: 25 is a mobile phase; The detection wavelength is 265nm; Number of theoretical plate falls calculating by berberine hydrochloride should be not less than 3000; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure the content of berberine hydrochloride in the calculation sample.
2. a kind of quality determining method for the treatment of ophthalmic myopia and asthenopia Chinese medicine preparation as claimed in claim 1, it is characterized in that: the every 0.4g of described Chinese medicine preparation contains Cortex Phellodendri in berberine hydrochloride, must not be less than 0.20mg.
3. a kind of quality determining method for the treatment of ophthalmic myopia and asthenopia Chinese medicine preparation as claimed in claim 1 or 2, it is characterized in that: the dosage form of described Chinese medicine preparation comprises pill, granule, capsule, tablet regular dosage form.
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| WO2021252717A1 (en) * | 2020-06-11 | 2021-12-16 | The Trustees Of Columbia University In The City Of New York | Methods and compositions for preventing and treating myopia with berberine, a berberidaceaen alkaloid, and derivatives thereof |
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| CN102520110B (en) * | 2011-12-16 | 2014-04-23 | 西藏奇正藏药股份有限公司 | Method for detecting ten-flavour rankincense preparation |
| CN107561206A (en) * | 2017-08-28 | 2018-01-09 | 广西壮族自治区药用植物园 | A kind of thin-layer chromatography detection method of ＂ FUYANLINGs ＂ lotions |
| CN110857938B (en) * | 2018-08-23 | 2022-03-11 | 完美（中国）有限公司 | Method for simultaneously identifying lucid ganoderma and vine of multiflower knotweed in traditional Chinese medicine composition |
| CN110857939B (en) * | 2018-08-23 | 2022-03-18 | 完美（中国）有限公司 | Detection method of traditional Chinese medicine composition with sleep improvement function |
| CN110464825B (en) * | 2019-08-28 | 2021-10-08 | 贵州景诚制药有限公司 | Rehmannia root potion pharmaceutical composition, preparation method and detection method |
| CN114569661B (en) * | 2020-11-30 | 2023-10-03 | 西安碑林药业股份有限公司 | Preparation method of traditional Chinese medicine composition and traditional Chinese medicine composition |
| CN115541799B (en) * | 2022-10-10 | 2024-07-19 | 湖南春光九汇现代中药有限公司 | Thin layer identification method of pipewort formula granule and standard decoction |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1453019A (en) * | 2003-05-21 | 2003-11-05 | 西安碑林药业股份有限公司 | Chinese medicine for treating eye diseases of middle-aged and old people and its prepn |
| CN1544075A (en) * | 2003-11-13 | 2004-11-10 | 西安碑林药业股份有限公司 | Hypometropia treating and asthenopia relieving Chinese traditional medicine and its preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1453019A (en) * | 2003-05-21 | 2003-11-05 | 西安碑林药业股份有限公司 | Chinese medicine for treating eye diseases of middle-aged and old people and its prepn |
| CN1544075A (en) * | 2003-11-13 | 2004-11-10 | 西安碑林药业股份有限公司 | Hypometropia treating and asthenopia relieving Chinese traditional medicine and its preparation |
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| WO2021252717A1 (en) * | 2020-06-11 | 2021-12-16 | The Trustees Of Columbia University In The City Of New York | Methods and compositions for preventing and treating myopia with berberine, a berberidaceaen alkaloid, and derivatives thereof |
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