CN102426205A - Detection method of thrombus sumalin capsules - Google Patents
Detection method of thrombus sumalin capsules Download PDFInfo
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- CN102426205A CN102426205A CN2011103574729A CN201110357472A CN102426205A CN 102426205 A CN102426205 A CN 102426205A CN 2011103574729 A CN2011103574729 A CN 2011103574729A CN 201110357472 A CN201110357472 A CN 201110357472A CN 102426205 A CN102426205 A CN 102426205A
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Abstract
The invention relates to a detection method of thrombus sumalin capsules, belonging to the field of traditional Chinese medicine detection. The method comprises the following steps: carrying out developing identification on a same thin layer system of salvia miltiorrhiza, Ligusticum wallichii and borneol, carrying out gas chromatography identification on artificial musk, identifying the thin layer of artificial bezoar, and carrying out content determination of ginsenoside. The method has the advantages of strong specificity, rapidness, economy, simple operation and the like. According to the invention, by simultaneously adding the identification of artificial musk and artificial bezoar, the invention establishes a multi-index content determination; by concretely adding the content determination of Ligusticum wallichii and ginsenoside and cooperating with original content determination, the detection of components of the medicine is comprehensive, the content and stability are understood, and the method is helpful for explaining the medicinal material base of the medicine.
Description
Technical field
The invention belongs to modern Chinese traditional medicine field, be specifically related to a kind of detection method of XUESHUANXINMAINING capsule.
Background technology
Traditional Chinese medicine ingredients is complicated, and particularly Chinese medicine compound prescription often contains multiclass, multiple composition, has constituted a very complicated system.At present, the new active component Chinese medicine preparation and the research and development of modern Chinese traditional compound medicine are on the increase, and new technology and multiple different extracting mode also are used for the research and the production of medicine.
Along with the fast development of modern analysis measuring technology, equipment and instrument, the assay of Chinese medicine, chromatogram are differentiated the important means that becomes detection.But the assay or the discriminating of generally only setting up a certain index chemical constitution at present, to the simultaneously applied situation of above-mentioned multiple new technology, the assay or the discriminating of only setting up certain single component have bigger one-sidedness to a great extent.
The XUESHUANXINMAINING capsule has qi and activate blood circulation, and the pain-relieving functions of having one's ideas straightened out is used for apoplexy, the obstruction of qi in the chest due to the qi deficiency to blood stasis, and disease opinion is had a dizzy spell, hemiplegia, pained, palpitation uncomfortable in chest; Ishemic stroke convalescence, coronary disease and angina pectoris are seen above-mentioned disease person.
Its prescription is: Ligusticum wallichii, the red sage root, sophora flower, leech, ilex pubescens, muscone, calculus bovis factitius, ginseng stem and leave general saponin, borneol, the dried venom of toads;
Method for making: above ten flavors, get the red sage root, ilex pubescens, Ligusticum wallichii with 60% alcohol extract 3 times, add 8,5,5 times of amount ethanol successively, extracted respectively 3,2,1 hours, filter, merging filtrate, decompression recycling ethanol concentrates, drying, pulverizing, subsequent use; Other is ground into fine powder at the leech of fetching water, and is subsequent use; Get sophora flower again, add 5 times of water gagings, regulate pH value to 8～9 with saturated aqua calcis, be heated to little boiling, be incubated 30 minutes, filter while hot, the dregs of a decoction as above method extract 2 times again, merging filtrate, and concentrating under reduced pressure, 60 ℃ of dryings of low temperature are pulverized, and are subsequent use.The calculus bovis factitius of muscone, the dried venom of toads is ground into fine powder, presses facing-up method and borneol fine powder, ginseng stem and leave general saponin and above-mentioned two kinds of powder mixings, incapsulate, process 1000, promptly get.Every heavy 0.50g, oral, one time 4.
In the side with hot temperature, go into the liver and gall warp, activating QI to alleviate the depression dispels pathogenic wind and remove dampness, the Ligusticum wallichii of promoting blood circulation and stopping pain and bitter tepor are gone into the conscience warp, promoting blood circulation, calming heart and tranquilizing mind be pain, the red sage root that nourishes blood is monarch drug in a prescription altogether.Minister is with ilex pubescens, leech, sophora flower, muscone, and is promoting blood circulation and removing blood stasis, the inducing resuscitation of having one's ideas straightened out, removes obstruction in channels to relieve pain.Assistant is promoting blood circulation and removing blood stasis with borneol, calculus bovis factitius, ginseng stem and leave general saponin three medicines assistant the monarch and his subjects, and the inducing resuscitation of having one's ideas straightened out is removed obstruction in channels to relieve pain, the hard kidney water of the liver that clears away heart-fire, and it is main taking stopgap measures, and takes into account the gesture of this void and Yu Huare.Make with the dried venom of toads, pain relieving is kept fit by the priming sick institute that goes directly, current channels and collaterals.
Discriminating in the original detection method of XUESHUANXINMAINING capsule comprises the discriminating of the red sage root, the discriminating of Ligusticum wallichii and the discriminating of borneol, and above-mentioned discriminating is separately carried out, and has shortcomings such as specificity is strong, need gradation operation.Aspect assay, the assay of tanshin polyphenolic acid B in pair red sage root is arranged at present, but the Ligusticum wallichii of monarch drug in a prescription does not have assay in the side of being all, only make that the assay to tanshin polyphenolic acid B in the monarch drug in a prescription has bigger one-sidedness to a great extent; The discriminating and the assay that originally in the detection method only there are pair sophora flower the discriminating and the assay aspect of ministerial drug; But the muscone of ministerial drug does not have discrimination method in the side of being all, and the medicine of the main sick and main symptom of ministerial drug to be auxiliary monarch drug in a prescription strengthen treatment, and costs an arm and a leg because of the muscone; As there are not the relevant detection means to cause illegal enterprise not fill out or fill out less the muscone easily; Only make discriminating and assay to sophora flower have bigger one-sidedness to a great extent, the discriminating to cholic acid in the calculus bovis factitius in the former capsule is to use the extracted by ether need testing solution, makes to contain ether in the development system; Spot spreads, hangover is serious, and identification result has much room for improvement; Also not to the assay of ginseng stem and leave general saponin, can not comprehensively reflect the content of the important composition of XUESHUANXINMAINING capsule simultaneously.
Summary of the invention
The present invention provides a kind of detection method of XUESHUANXINMAINING capsule, with solve the specificity that present detection method exists strong, need gradation operation, incomplete problem.
The technical scheme that the present invention takes is to comprise following discrimination method and content assaying method:
(1) discrimination method
(1) get the content of 20 of this capsules, porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; Get red sage root control medicinal material 1g, Ligusticum wallichii control medicinal material 1g respectively in addition, shine medicinal material solution in pairs with legal system; Get tanshinone IIA reference substance, borneol reference substance again, add ethyl acetate and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 1 μ l of above-mentioned five kinds of solution, put respectively on same silica gel g thin-layer plate, be at 11: 2 developping agent with 60～90 ℃-ethyl acetate of sherwood oil, launch, take out, dry; In the test sample chromatogram, with red sage root control medicinal material, the corresponding position of tanshinone IIA reference substance chromatogram on, the spot of apparent same color under the daylight; Put under the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the fluorescence spot of same color; Spray with 5% phosphomolybdic acid ethanol solution, it is clear to be heated to spot colour developing at 105 ℃ again; In the test sample chromatogram, with the corresponding position of borneol reference substance chromatogram on show the spot of same color;
(2) muscone's gas chromatography is differentiated: get the content of 20 of this capsules, and porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; It is an amount of to get the muskone reference substance, adds absolute ethyl alcohol and processes the solution that every 1ml contains 0.1mg, as reference substance solution; According to vapor-phase chromatography test, the DB-FFAP capillary column: column length is 30m, and internal diameter is 0.32mm, and film thickness is 0.5 μ m, and column temperature is temperature programme: initial temperature is 200 ℃, keeps 10 minutes, is warming up to 250 ℃ with the speed of 20 ℃ of per minutes, keeps 10 minutes; Accurate respectively above-mentioned reference substance solution and each 1 μ l of need testing solution of drawing, inject gas chromatograph is measured; In the record test sample chromatogram with the identical chromatographic peak of reference substance chromatographic peak retention time;
(3) get the content of 6 of this capsules, porphyrize adds methyl alcohol 50ml, and reflux 1 hour filters, and filtrating evaporate to dryness, residue add water 30ml dissolving, with extracted by ether 2 times, and each 20ml, the merging ether solution, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets calculus bovis factitius control medicinal material 0.5g, adds methyl alcohol 5ml, and cold soaking spends the night, and filters, and filtrating is as control medicinal material solution; Get the cholic acid reference substance again, add methyl alcohol and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 3 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate; With normal hexane-ethyl acetate-acetic acid-methyl alcohol 20: 25: 2: 3 upper solution was a developping agent; Launch, take out, dry; Spray is with 5% phosphomolybdic acid ethanol solution, and it is clear to be heated to the spot colour developing at 105 ℃;
(2) content assaying method:
(1) Ligusticum wallichii assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With methyl alcohol-1% acetum is moving phase at 25: 75; The detection wavelength is 321nm, and number of theoretical plate calculates by the forulic acid peak should be not less than 4000;
The preparation of reference substance solution: it is an amount of to get the forulic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds 70% methyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.3g～0.5g, and accurate title is fixed; Put in the tool plug conical flask, the accurate 70% methyl alcohol 20ml that adds claims to decide weight, sonicated 30 minutes, power 250W; Frequency 50kHz is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 70% methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method: accurate respectively reference substance solution 5 μ l and the need testing solution 10 μ l of drawing, inject liquid chromatograph, measure, promptly get.
(2) ginseng stem and leave general saponin assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water is moving phase at 20: 80; The detection wavelength is 203nm, and number of theoretical plate calculates by the ginsenoside Re peak should be not less than 6000;
The preparation of reference substance solution: get ginsenoside Rg1's reference substance and ginsenoside Re's reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains ginsenoside Rg1 0.15mg, ginsenoside Re 0.50mg, promptly get;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.4～0.6g, and accurate title is fixed, puts in the tool plug conical flask, adds methenyl choloride 50ml, and ultrasonic Extraction 30 minutes filters, and discards filtrating; The dregs of a decoction volatilize solvent together with filter paper, and the accurate methyl alcohol 50ml that adds claims decide weight, and refluxing extraction 1 hour is put coldly, and weight decided in title again; Supply the weight that subtracts mistake with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, evaporate to dryness; Residue adds water-saturated n-butanol solution 20ml dissolving, with the washing of 50ml ammonia solution once, discards the ammonia solution layer, and normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol makes dissolving in right amount; Be transferred in the 10ml measuring bottle, add methyl alcohol to scale and make dissolving, shake up, filter, promptly get.
Advantage of the present invention is: through the extraction of easy steps, same thin layer system launches, and reaches the purpose of differentiating three kinds of compositions, has characteristics such as specificity is strong, quick, economic, easy and simple to handle; Through setting up discriminating, and cooperate the discriminating and the assay of original sophora flower, can more comprehensively detect the composition and its compatibility of ministerial drug the muscone; The present invention sets up many indexs assay; Concrete passing through increases the assay to Ligusticum wallichii, cooperates existing assay to the red sage root, sophora flower, can more comprehensively detect the composition of monarch drug in a prescription; Understand its content and stability, also help to explain the medical substance basis of medicine.
Embodiment
The XUESHUANXINMAINING capsule is provided by prescription and explained hereafter in the background technology by Jilin Huakang Pharmaceutical Co., Ltd.
Embodiment 1
Comprise following discrimination method and content assaying method:
(1) discrimination method
Get the content of 20 of this capsules, porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; Get red sage root control medicinal material 1g, Ligusticum wallichii control medicinal material 1g respectively in addition, shine medicinal material solution in pairs with legal system; Get tanshinone IIA reference substance, borneol reference substance again, add ethyl acetate and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 1 μ l of above-mentioned five kinds of solution, put respectively on same silica gel g thin-layer plate, be at 11: 2 developping agent with 60～90 ℃-ethyl acetate of sherwood oil, launch, take out, dry; In the test sample chromatogram, with red sage root control medicinal material, the corresponding position of Tanshinone I I A reference substance chromatogram on, the spot of apparent same color under the daylight; Put under the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the fluorescence spot of same color; Spray with 5% phosphomolybdic acid ethanol solution, it is clear to be heated to spot colour developing at 105 ℃ again; In the test sample chromatogram, with the corresponding position of borneol reference substance chromatogram on show the spot of same color;
(2) muscone's gas chromatography is differentiated: get the content of 20 of this capsules, and porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; It is an amount of to get the muskone reference substance, adds absolute ethyl alcohol and processes the solution that every 1ml contains 0.1mg, as reference substance solution; According to vapor-phase chromatography test, the DB-FFAP capillary column: column length is 30m, and internal diameter is 0.32mm, and film thickness is 0.5 μ m, and column temperature is temperature programme: initial temperature is 200 ℃, keeps 10 minutes, is warming up to 250 ℃ with the speed of 20 ℃ of per minutes, keeps 10 minutes; Accurate respectively above-mentioned reference substance solution and each 1 μ l of need testing solution of drawing, inject gas chromatograph is measured; In the record test sample chromatogram with the identical chromatographic peak of reference substance chromatographic peak retention time;
(3) get the content of 6 of this capsules, porphyrize adds methyl alcohol 50ml, and reflux 1 hour filters, and filtrating evaporate to dryness, residue add water 30ml dissolving, with extracted by ether 2 times, and each 20ml, the merging ether solution, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets calculus bovis factitius control medicinal material 0.5g, adds methyl alcohol 5ml, and cold soaking spends the night, and filters, and filtrating is as control medicinal material solution; Get the cholic acid reference substance again, add methyl alcohol and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 3 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate; With normal hexane-ethyl acetate-acetic acid-methyl alcohol 20: 25: 2: 3 upper solution was a developping agent; Launch, take out, dry; Spray is with 5% phosphomolybdic acid ethanol solution, and it is clear to be heated to the spot colour developing at 105 ℃;
(2) content assaying method:
The Ligusticum wallichii assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With methyl alcohol-1% acetum is moving phase at 25: 75; The detection wavelength is 321nm, and number of theoretical plate calculates by the forulic acid peak should be not less than 4000;
The preparation of reference substance solution: it is an amount of to get the forulic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds 70% methyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.5g, and accurate title is fixed; Put in the tool plug conical flask, the accurate 70% methyl alcohol 20ml that adds claims to decide weight, sonicated 30 minutes, power 250W; Frequency 50kHz is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 70% methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Accurate respectively reference substance solution 5 μ l and the need testing solution 10 μ l of drawing of determination method inject liquid chromatograph, measure, and promptly get.
The ginseng stem and leave general saponin assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water is moving phase at 20: 80; The detection wavelength is 203nm, and number of theoretical plate calculates by the ginsenoside Re peak should be not less than 6000;
The preparation of reference substance solution: get ginsenoside Rg1's reference substance and ginsenoside Re's reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains ginsenoside Rg1 0.15mg, ginsenoside Re 0.50mg, promptly get;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.5g, and accurate title is fixed, puts in the tool plug conical flask, adds methenyl choloride 50ml, and ultrasonic Extraction 30 minutes filters, and discards filtrating; The dregs of a decoction volatilize solvent together with filter paper, and the accurate methyl alcohol 50ml that adds claims decide weight, and refluxing extraction 1 hour is put coldly, and weight decided in title again; Supply the weight that subtracts mistake with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, evaporate to dryness; Residue adds water-saturated n-butanol solution 20ml dissolving, with the washing of 50ml ammonia solution once, discards the ammonia solution layer, and normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol makes dissolving in right amount; Be transferred in the 10ml measuring bottle, add methyl alcohol to scale and make dissolving, shake up, filter, promptly get.
Embodiment 2
Discrimination method is with embodiment 1.
In the content assaying method, the preparation of need testing solution in the Ligusticum wallichii assay: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.3g, and accurate title is fixed, and all the other are with embodiment 1.
The preparation of need testing solution in the ginseng stem and leave general saponin assay: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.4g, and accurate title is fixed, and all the other are with embodiment 1.
Embodiment 3
Discrimination method is with embodiment 1.
In the content assaying method, the preparation of need testing solution in the Ligusticum wallichii assay: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.4g, and accurate title is fixed, and all the other are with embodiment 1.
The preparation of need testing solution in the ginseng stem and leave general saponin assay: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.6g, and accurate title is fixed, and all the other are with embodiment 1.
Every of these article contain Ligusticum wallichii with forulic acid (C
10H
10O
4) meter, be no less than 0.10mg.
Every of these article contain ginseng stem and leave general saponin with ginsenoside Rg1 (C
42H
72O
14) and ginsenoside Re (C
48H
82O
18) the total amount meter, be no less than 4.0mg.
Test Example 1 further specifies Ligusticum wallichii content assaying method of the present invention through Test Example below.
Instrument, reagent and sample
Agilent 1200 high performance liquid chromatographs; Forulic acid reference substance (lot number: 110773-200611), provide, supply assay to use by Chinese pharmaceutical biological product check; Reagent: methyl alcohol is chromatographically pure, and water is purified water, and it is pure that other reagent is analysis.Sample is provided by Jilin Huakang Pharmaceutical Co., Ltd.
The test of 1 precision
The instrument precision test is pressed the chromatographic condition of the embodiment of the invention 1 to a need testing solution, and METHOD FOR CONTINUOUS DETERMINATION 6 times, results peaks area average are 359.1244, and RSD is 0.12% (n=6), and the result sees table 1.
Table 1 forulic acid instrument precision test findings
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | Average | RSD ％ |
| Peak area | 359.7123 | 358.7959 | 359.0851 | 359.1065 | 358.5632 | 359.4838 | 359.1244 | 0.12 |
The result shows that the precision of instrument is good.
2 replica tests are to same lot number sample, measure 6 times by method parallel extraction of the present invention, and ferulaic acid content average out to 0.140mg/ sheet as a result, RSD=0.31% (n=6), the result sees table 2.
Table 2 forulic acid reappearance experimental result
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | Mean value | RSD(％) |
| Content (mg/ sheet) | 0.140 | 0.140 | 0.140 | 0.140 | 0.141 | 0.141 | 0.140 | 0.37 |
The result shows that the repeatability of this law is good.
The test of 3 specificities
For further investigating the rationality of test, make the negative control solution that does not contain the Ligusticum wallichii medicinal material according to recipe quantity, measure in accordance with the law, negative controls is not having chromatographic peak with the corresponding retention time in reference substance peak place as a result, shows noiselessly, and specificity is good.
4 linear relationships and scope
Accurate respectively forulic acid reference substance solution (lot number: 110773-200611, concentration 0.02046mg/ml) 1 μ l, the 3 μ l of drawing; 5 μ l; 7 μ l, 9 μ l inject liquid chromatograph, and chromatographic condition of the present invention is measured; Sample size (μ g) with reference substance is a horizontal ordinate, and chromatographic peak area is an ordinate drawing standard curve.The result sees table 3.
Table 3 forulic acid linear relationship is investigated the result
| Sample size (μ g) | 0.02046 | 0.06138 | 0.1023 | 0.14322 | 0.18414 |
| The forulic acid peak area | 82.37376 | 245.863 | 412.1724 | 576.1788 | 738.6967 |
Regression equation is: y=4015.1x+0.3165, and R=0.99999, the result shows that forulic acid is good log-linear relation in 0.02046 μ g～0.18414 μ g scope.
5 accuracy tests
Precision is measured test sample (Jilin Huakang Pharmaceutical Co., Ltd's lot number: 110201) 6 parts of measuring known content with method; Every part of 0.25g; Accurate reference substance solution (0.0043mg/ml) 20ml that adds extracts, measures calculate recovery rate by method of the present invention; Average recovery rate=99.07%, RSD=0.20% (n=6).Meet the requirement of quantitative test.The result sees table 4.
Table 4 forulic acid accuracy test result
Test Example 2 adopts vapor-phase chromatography to differentiate the muskone among the muscone.
Agilent 7890A gas chromatograph, (column length is 30m to the DB-FFAP capillary column, and internal diameter is 0.32mm; Film thickness is 0.5 μ m), column temperature is temperature programme: initial temperature is 200 ℃, keeps 10 minutes; Speed with 20 ℃ of per minutes is warming up to 250 ℃, keeps 10 minutes.Fid detector.Prepare need testing solution and muskone reference substance solution by the inventive method, prepare the negative control solution that does not contain the muscone, in 5 batches of sample test sample chromatograms, all appear and the identical chromatographic peak of reference substance chromatographic peak retention time with method, negative noiseless.
Test Example 3:
Embodiment 1 is seen in the preparation of test sample, control medicinal material and reference substance, and other gets the negative control that does not contain calculus bovis factitius, processes negative control solution with method.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned four kinds of solution, put respectively in same with silica gel g thin-layer plate on; With normal hexane-ethyl acetate-acetic acid-methyl alcohol 20: 25: 2: 3 was developping agent; Launch, take out, dry; Spray is with 5% phosphomolybdic acid ethanol solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance, the corresponding position of control medicinal material chromatogram on, show the spot of same color.Negative control is noiseless.Tested 5 lot sample article, all can reappear.
Test Example 4: followingly further specify genseng stem-leaf total saponin content assaying method of the present invention through Test Example.
1, stability test
To with a need testing solution,, measure once at regular intervals by chromatographic condition of the present invention; Investigate 25 hours altogether, measured 6 times, measure ginsenoside Rg1 and ginsenoside Re's content and average out to 4.3mg/ sheet 6 times as a result; RSD=0.63% (n=6) sees table 5 for details.
Table 5 ginsenoside stability test result
The result shows that need testing solution is stable in 25 hours.
2, precision test
The instrument precision test is pressed chromatographic condition of the present invention to a need testing solution, continuous sample introduction 6 times, and ginsenoside Rg1 and ginsenoside Re's peak area in the working sample, the RSD value is respectively 1.00% and 0.47%, and the result sees table 6.
Table 6 instrument precision test findings
The result shows that the precision of instrument is good.
3, replica test is to same sample, by 6 parts of method replicate determinations of the present invention, calculate these article contain ginsenoside Rg1 and ginsenoside Re and, mean value is the 4.3mg/ sheet, the RSD value is 1.39%, the result sees table 6.
Table 6 ginsenoside reappearance experimental result
The result shows that the repeatability of this law is good.
4, specificity test
Be further to investigate the specificity of test, do not contain the negative control solution of ginsenoside according to the recipe quantity preparation, measure in accordance with the law, negative control solution is not having chromatographic peak with the corresponding retention time of reference substance chromatographic peak place as a result, shows noiselessly, and specificity is good.
5, linear relationship and scope
Accurate respectively ginsenoside Rg1's reference substance solution (lot number: 110703-200425 of drawing; Concentration 0.159mg/ml) and ginsenoside Re's reference substance solution (lot number: 110754-200421; Concentration 0.2406mg/ml) inject liquid chromatograph, measure by chromatographic condition of the present invention, the result sees the following form.Sample size (μ g) with reference substance is a horizontal ordinate, and chromatographic peak area is an ordinate drawing standard curve.The result sees table 7, table 8.
Table 7 ginsenoside Rg1 linear relationship is investigated the result
| Sample size (μ g) | 0.159 | 0.477 | 0.795 | 1.113 | 1.431 |
| The Rg1 peak area | 66605 | 213226 | 349093 | 496380 | 633770 |
Regression equation is: Y=-2556.2+445749.7X, and R=0.9999, the result shows that the ginsenoside Rg1 is good log-linear relation (ginsenoside Rg1's linear relationship is seen assay Fig. 4 .12) in 0.159 μ g～1.431 μ g scopes
Table 8 ginsenoside Re linear relationship is investigated the result
| Sample size (μ g) | 0.7218 | 1.203 | 2.406 | 3.609 | 4.812 |
| The Re peak area | 250029 | 421690 | 816128 | 1220516 | 1638418 |
Regression equation is: Y=8238.5+337645.5X, and R=0.9999, the result shows that the ginsenoside Re is good linear relation (ginsenoside Re's linear relationship is seen assay Fig. 4 .13) in 0.7218 μ g～4.812 μ g scopes.
6, accuracy test
6.1 ginsenoside Rg1's accuracy test
Precision takes by weighing 6 parts of the test samples of known content, every part of 0.25g, accurate ginsenoside Rg1's reference substance solution (0.0324mg/ml that adds; Purity 96.3%) 20ml extracts, measures calculate recovery rate by method of the present invention; Average recovery rate=96.5%, RSD=2.32% (n=6).Meet the requirement of quantitative test.The result sees table 9.
Table 9 ginsenoside Rg1 accuracy test result
6.2 ginsenoside Re's accuracy test
Precision takes by weighing 6 parts of test samples measuring known content with method, every part of 0.25g, accurate ginsenoside Re's reference substance solution (0.2256mg/ml that adds; Purity 88.8%) 20ml extracts, measures calculate recovery rate by the method for working out standard; Average recovery rate=96.4%, RSD=1.34% (n=6).Meet the requirement of quantitative test.The result sees table 10.
Table 10 ginsenoside Re accuracy test result
Claims (1)
1. the detection method of an XUESHUANXINMAINING capsule is characterized in that: comprise following discrimination method and content assaying method:
(1) discrimination method
(1) get the content of 20 of this capsules, porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; Get red sage root control medicinal material 1g, Ligusticum wallichii control medicinal material 1g respectively in addition, shine medicinal material solution in pairs with legal system; Get tanshinone IIA reference substance, borneol reference substance again, add ethyl acetate and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 1 μ l of above-mentioned five kinds of solution, put respectively on same silica gel g thin-layer plate, 11:2 is a developping agent with 60～90 ℃-ethyl acetate of sherwood oil, launches, and takes out, and dries; In the test sample chromatogram, with red sage root control medicinal material, the corresponding position of tanshinone IIA reference substance chromatogram on, the spot of apparent same color under the daylight; Put under the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the fluorescence spot of same color; Spray with 5% phosphomolybdic acid ethanol solution, it is clear to be heated to spot colour developing at 105 ℃ again; In the test sample chromatogram, with the corresponding position of borneol reference substance chromatogram on show the spot of same color;
(2) muscone's gas chromatography is differentiated: get the content of 20 of this capsules, and porphyrize, the 40ml cold soaking 1 hour of adding diethyl ether filters, and filtrating volatilizes, and residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; It is an amount of to get the muskone reference substance, adds absolute ethyl alcohol and processes the solution that every 1ml contains 0.1mg, as reference substance solution; According to vapor-phase chromatography test, the DB-FFAP capillary column: column length is 30m, and internal diameter is 0.32mm, and film thickness is 0.5 μ m, and column temperature is temperature programme: initial temperature is 200 ℃, keeps 10 minutes, is warming up to 250 ℃ with the speed of 20 ℃ of per minutes, keeps 10 minutes; Accurate respectively above-mentioned reference substance solution and each 1 μ l of need testing solution of drawing, inject gas chromatograph is measured; In the record test sample chromatogram with the identical chromatographic peak of reference substance chromatographic peak retention time;
(3) get the content of 6 of this capsules, porphyrize adds methyl alcohol 50ml, and reflux 1 hour filters; The filtrating evaporate to dryness, residue adds water 30ml dissolving, with extracted by ether 2 times, 20ml at every turn; Merge ether solution, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets calculus bovis factitius control medicinal material 0.5g, adds methyl alcohol 5ml, and cold soaking spends the night, and filters, and filtrating is as control medicinal material solution; Get the cholic acid reference substance again, add methyl alcohol and process the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 3 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate; Upper solution with normal hexane-ethyl acetate-acetic acid-methyl alcohol 20:25:2:3 is a developping agent; Launch, take out, dry; Spray is with 5% phosphomolybdic acid ethanol solution, and it is clear to be heated to the spot colour developing at 105 ℃;
(2) content assaying method:
(1) Ligusticum wallichii assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; 25:75 is a moving phase with methyl alcohol-1% acetum; The detection wavelength is 321nm, and number of theoretical plate calculates by the forulic acid peak should be not less than 4000;
The preparation of reference substance solution: it is an amount of to get the forulic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds 70% methyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.3g ~ 0.5g, and accurate title is fixed; Put in the tool plug conical flask, the accurate 70% methyl alcohol 20ml that adds claims to decide weight, sonicated 30 minutes, power 250W; Frequency 50kHz is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 70% methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method: accurate respectively reference substance solution 5 μ l and the need testing solution 10 μ l of drawing, inject liquid chromatograph, measure, promptly get;
(2) ginseng stem and leave general saponin assay
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water 20:80 is moving phase; The detection wavelength is 203nm, and number of theoretical plate calculates by the ginsenoside Re peak should be not less than 6000;
The preparation of reference substance solution: get ginsenoside Rg1's reference substance and ginsenoside Re's reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains ginsenoside Rg1 0.15mg, ginsenoside Re 0.50mg, promptly get;
The preparation of need testing solution: get the content of 20 of this capsules, the accurate title, decide, and porphyrize is got 0.4 ~ 0.6g, and accurate title is fixed, puts in the tool plug conical flask, adds methenyl choloride 50ml, and ultrasonic Extraction 30 minutes filters, and discards filtrating; The dregs of a decoction volatilize solvent together with filter paper, and the accurate methyl alcohol 50ml that adds claims decide weight, and refluxing extraction 1 hour is put coldly, and weight decided in title again; Supply the weight that subtracts mistake with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, evaporate to dryness; Residue adds water-saturated n-butanol solution 20ml dissolving, with the washing of 50ml ammonia solution once, discards the ammonia solution layer, and normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol makes dissolving in right amount; Be transferred in the 10ml measuring bottle, add methyl alcohol to scale and make dissolving, shake up, filter, promptly get.
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| CN106483260A (en) * | 2015-10-09 | 2017-03-08 | 厦门中药厂有限公司 | A kind of pill of Eight Treasures capsule quality standard control method |
| CN109085284A (en) * | 2018-08-28 | 2018-12-25 | 王智森 | A kind of quick thin-layer identification method of multiple medicine taste of Ershiwuwei zhenzhu wan |
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| CN102507844A (en) * | 2011-11-13 | 2012-06-20 | 吉林华康药业股份有限公司 | Testing method for Xueshuan xinmaining tablet |
| CN102507844B (en) * | 2011-11-13 | 2013-10-16 | 吉林华康药业股份有限公司 | Testing method for Xueshuan xinmaining tablet |
| CN103884676A (en) * | 2014-02-27 | 2014-06-25 | 浙江大学 | Rapid detection method for contents of multi-index ingredients in traditional Chinese medicinal materials |
| CN103884676B (en) * | 2014-02-27 | 2016-06-22 | 浙江大学 | A kind of rapid assay methods of Chinese crude drug multi-target ingredient content |
| CN106483260A (en) * | 2015-10-09 | 2017-03-08 | 厦门中药厂有限公司 | A kind of pill of Eight Treasures capsule quality standard control method |
| CN109085284A (en) * | 2018-08-28 | 2018-12-25 | 王智森 | A kind of quick thin-layer identification method of multiple medicine taste of Ershiwuwei zhenzhu wan |
| CN110632240A (en) * | 2019-11-08 | 2019-12-31 | 亳州职业技术学院 | Identification kit and identification method for traditional Chinese medicine ground beetles |
| CN110632240B (en) * | 2019-11-08 | 2021-11-02 | 亳州职业技术学院 | Identification kit and identification method for traditional Chinese medicine ground beetles |
| CN113720958A (en) * | 2021-08-25 | 2021-11-30 | 国药集团精方（安徽）药业股份有限公司 | Quality detection method of Shenyanshu capsule |
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