CN101274036B - Method for testing conghuang preparation for tonifying kidney - Google Patents
Method for testing conghuang preparation for tonifying kidney Download PDFInfo
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Abstract
The invention relates to a method for inspecting a Conghuang kidney-reinforcing preparation, which used for controlling the quality of the Conghuang kidney reinforcing preparation, wherein, the preparation refers to granules, tablets and hard gelatin capsules. When disclosing the method for inspecting a new Conghuang kidney reinforcing preparation, relatively great improvement is made to the existing methods for inspecting the quality standards of medical pills by the invention, which is particularly characterized in that: thin layers qualitative differential diagnosis of prepared rhizome of rehmannia, seed of Chinese dodder and fruit of Chinese magnolia vine (steaming with wine) in a prescription is added, the preparation method of desertliving cistache differential diagnosis sample solution is changed and determination of echinacoside with little interference but high content by high performance liquid chromatography is modified. The quality controllability of the new Conghuang kidney-reinforcing preparation can be improved by using the inspecting method of the invention, which is favorable to the quality stability of products.
Description
Technical field
The invention belongs to the Chinese patent drug field of quality control.
Background technology
The yellow kidney tonifying prescription of desert is formed the effect that have nourishing kidney yin, strengthens muscles and bones by saline cistanche, prepared rhizome of rehmannia, the seed of Chinese dodder, the fruit of Chinese magnoliavine (wine steaming) four traditional Chinese medicine material.Can process formulations such as ball, particle, sheet, hard shell capsules here.At present, only recorded pill standard (" country of National Drug Administration standard for traditional Chinese medicines compilation " internal medicine kidney is a fascicle) in the disclosed drug standards, yellow other formulation standards of kidney tonifying of desert are not appeared in the newspapers.Have only microscopical identification in the pill standard, saline cistanche TLC differentiates and acteoside HPLC assay.This medicine prescription is made up of four traditional Chinese medicine, and obvious pill standard and imperfection can not effectively guarantee the quality of product.
Simultaneously; We also find in test: the yellow kidney tonifying novel form of these three kinds of deserts of the particle that relates to, sheet and hard shell capsules saline cistanche in the pill standard is differentiated the test liquid that the test liquid preparation method obtains under the item, and there is a certain distance in the spot of spot on its thin-layer chromatography and betaine reference substance chromatogram on the position.
In addition, the yellow kidney tonifying novel forms of these three kinds of deserts of particle, sheet and hard shell capsules are when doing the HPLC assay of acteoside, and the negative test liquid peak that disturbs exists.Testing different chromatographic columns and moving phase composition all can not well separate the peak of Interference Peaks with acteoside.
Summary of the invention
The object of the invention provides the detection method of the yellow kidney-tonifying preparation of a kind of desert, thereby improves the yellow kidney tonifying novel form of desert quality controllability, guarantees the stable of product quality.Wherein preparation is meant particle, sheet and hard shell capsules.
The present invention has carried out bigger improvement to the pill standard detecting method; Be specially: increased the thin-layer qualitative discriminating of prepared rhizome of rehmannia, the seed of Chinese dodder, the fruit of Chinese magnoliavine (wine steaming) in the prescription; Changed the preparation method of saline cistanche discriminating test liquid, changed high performance liquid chromatography assay composition for disturbing the echinacoside little, that content is high.
Why change that to measure echinacoside be because discovery has bigger interference during with containing the survey method and detect the acteoside content of the yellow kidney tonifying novel form of desert in the former pill standard into.Tested methyl alcohol-acetonitrile-1% acetate (13: 10: 77; 11: 9: 80,15: 10: 75), methyl alcohol-0.1% formic acid (28: 72,35: 65) moving phase; And kromasil-C18 (250mm * 4.6mm; 5 μ m) and Ultimate-C18 (250mm * 4.6mm, 5 μ m) chromatographic column, the result all can not make the peak of Interference Peaks and acteoside separate.And do not find obvious Interference Peaks when detecting echinacoside.Because the complicacy of Chinese medicine; And can not obtain former pill standard and declare the research of comparing of the pill sample of producer; Can't confirm now than the reason of larger interference problem so exist when producing this detection acteoside, possibly be that used medicinal material place of production difference or the equal reason of the concrete operations parameter in the manufacturing process causes.
Bibliographical information is arranged, and working the major component of improving the sexual function effect in the saline cistanche is the benzyl carbinol glycosides compounds.Echinacoside, acteoside all belong to the higher benzyl carbinol glycosides compounds of content.Through our detection to commercially available 5 different places of production saline cistanche medicinal materials (wherein 2 Cistanche tubulosas for the Xinjiang product), the content of finding 4 place of production echinacosides is all greater than acteoside content, and the content of two kinds of compositions is suitable in the medicinal material in another place of production.So changing into detecting disturbs the content of the echinacoside little, that content is high also can reflect product quality preferably.
The method for making of the yellow kidney tonifying novel form of desert involved in the present invention, except that auxiliary material kind and consumption, process total amount, drying process, finally formulation different, other is all according to prescription in the pill standard and method for making manufacturing.Whole manufacturing approach and pill relatively do not have change big, essence.So according to the classification of medicine registration management way, the yellow kidney tonifying particle of desert involved in the present invention, sheet or hard capsule preparation should belong to 8 kind new medicines (being the imitation medicine dosage changing form).
The invention provides the detection method of the yellow kidney-tonifying preparation of desert---particle, sheet, hard shell capsules---, comprise with thin-layer chromatography (TLC) saline cistanche, prepared rhizome of rehmannia, the seed of Chinese dodder, the fruit of Chinese magnoliavine (wine steaming) are carried out qualitative identification and measure active component---the content of echinacoside (echinacoside) in the saline cistanche with high performance liquid chromatography (HPLC).
1, the thin layer of saline cistanche is differentiated
It is an amount of to get the yellow kidney-tonifying preparation of desert (particle, sheet or hard shell capsules), and porphyrize takes by weighing powder 0.5～1.0g, adds water 20ml; Ultrasonic 10min makes to dissolve and looses, and centrifugal (4500r/min) 5min gets supernatant vacuum filtration again, the strong acid cation exchange resin column of filtrating; Wash post with 20ml, use 0.5～4mol/l ammonia spirit, 20～60ml wash-out again, collect eluent, evaporate to dryness; With ethanol 2ml dissolving, leave standstill, get supernatant as need testing solution.Other gets the negative sample of misrun desert cistanche, processes negative test liquid with method.The betaine reference substance adds ethanol and processes the reference substance solution that every 1ml contains 5mg.Draw each 10 μ l of need testing solution and negative test liquid, reference substance solution 5 μ l, point sample is on same silica gel g thin-layer plate, with methyl alcohol-glacial acetic acid-water (10～20 respectively; 0.5～2: 2～8) be developping agent, launch, take out, dry that spray is with the improvement bismuth potassium iodide test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.The negative sample chromatogram does not have corresponding spot.
2, the thin layer of prepared rhizome of rehmannia is differentiated
It is an amount of to get the yellow kidney-tonifying preparation of desert (particle, sheet or hard shell capsules), and porphyrize takes by weighing powder 1.0～3.0g; Add water 30ml, sonicated 10min, centrifugal (4500r/min) 5min; Get supernatant and extract 2 times with normal butyl alcohol (chloroform, ethyl acetate), each 15ml merges extract; Extract is concentrated into dried, and residue adds ethanol 1ml dissolving, as need testing solution.Other gets the negative sample that lacks prepared rhizome of rehmannia, processes negative test liquid with method.The 5 hydroxymethyl furfural reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.5mg.Draw each 10 μ l of need testing solution and negative test liquid, reference substance solution 5 μ l, point sample is in same silica G F respectively
254On the thin layer plate, (1～8: 2～10) upper strata liquid is developping agent, launches, and takes out, and dries, and places under the 254nm uviol lamp and inspects with sherwood oil II-ethyl acetate.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color.The negative sample chromatogram does not have corresponding blackening point.
3, the thin layer of the seed of Chinese dodder is differentiated
It is an amount of to get the yellow kidney-tonifying preparation of desert (particle, sheet or hard shell capsules), and porphyrize takes by weighing powder 1.0～3.0g; Add chloroform (absolute ethyl alcohol, methyl alcohol, ethyl acetate, normal butyl alcohol) 10ml, sonicated 15min filters; Filtrating is concentrated into dried, adds ethanol 1ml dissolved residue, as need testing solution.Other gets the negative sample that lacks the seed of Chinese dodder, processes negative test liquid with method.Naphthalene plain reference substance in mountain adds ethanol and processes the reference substance solution that every 1ml contains 0.2mg.Draw each 10 μ l of need testing solution and negative test liquid, reference substance solution 5 μ l, point sample is on same silica gel g thin-layer plate respectively; With toluene-ethyl acetate-methyl alcohol (4～10: 4～8: 1～6) be developping agent; Launch, take out, dry; Spray places under the 365nm uviol lamp and inspects with 3% aluminium choride ethanolic solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.The negative sample chromatogram does not have corresponding spot.
4, the thin layer of the fruit of Chinese magnoliavine (wine steaming) is differentiated
It is an amount of to get the yellow kidney-tonifying preparation of desert (particle, sheet or hard shell capsules), and porphyrize takes by weighing powder 8.0～15.0g; Add methyl alcohol (absolute ethyl alcohol, chloroform, ethyl acetate, normal butyl alcohol) 20ml, sonicated or reflux 30min filter; Filtrating is concentrated into about 1ml, as need testing solution.Other gets the negative sample that lacks the fruit of Chinese magnoliavine, processes negative test liquid with method.The deoxyschizandrin reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.4mg.Draw each 10 μ l of need testing solution, negative test liquid and reference substance solution, point sample is on same silica GF254 thin layer plate respectively, and (8～20: 2～8: 0.5～2) upper strata liquid is developping agent with sherwood oil I-ethyl formate-formic acid; Launch; Take out, dry, place under the 254nm uviol lamp and inspect.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color.The negative sample chromatogram does not have corresponding blackening point.
5, echinacoside assay in the saline cistanche
Chromatographic condition and system suitability test chromatographic column adopting octadecylsilane chemically bonded silica are filler; Moving phase is methyl alcohol-acetonitrile-1% acetum (15: 10: 75); The detection wavelength is 334nm.Theoretical cam curve is calculated by the echinacoside peak should be not less than 4000.
Reference substance solution prepares precision, and to take by weighing the echinacoside reference substance an amount of, puts in the brown bottle, adds moving phase and be made into the reference substance solution that every 1ml contains 0.16mg
It is an amount of that the yellow kidney-tonifying preparation of desert (particle, sheet or hard shell capsules) is got in the need testing solution preparation, and porphyrize is got 0.5g, and accurate title is fixed, puts in the brown measuring bottle of 50ml, and precision adds moving phase 20ml; Close plug shakes up, and claims to decide weight, soaks sonicated 40min 0.5 hour; Take out, put coldly, claim again to decide weight, add moving phase and supply the weight that subtracts mistake, shake up; Centrifugal, get supernatant, with the micro-filtration membrane filtration of 0.45 μ m, filtrating is put in the brown bottle, promptly gets.
Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure.
Calculating is calculated as follows:
In the formula:
W: echinacoside content, %;
Cs: the concentration of echinacoside in the reference substance, mg/ml;
M: the sample weighting amount of test sample, mg;
As: the chromatographic peak area of echinacoside in the reference substance;
Ai: the chromatographic peak area of echinacoside in the test sample.
The content assaying method of echinacoside has passed through the methodological study test (because tablet and hard shell capsules involved in the present invention all is to form through machine pressing or filling from granule in the yellow kidney-tonifying preparation of desert according to the invention; The just variation of profile; There is not composition to change, so if the subjects that following summary of the invention adopted does not have explanation to be granule.)：
(1) instrument and reagent
SPD-10Avp Ultraviolet Detector day island proper Tianjin company; P4000 quaternary pump U.S. power & light company; AT-130 chromatographic column constant temperature oven Tianjin Hengao Technology Development Co., Ltd.; HN1006 ultrasonic cleaning machine China south China ultrasonic device factory; HM202 electronic analytical balance Japan AND company
Echinacoside (111670-200502) Nat'l Pharmaceutical & Biological Products Control Institute; The yellow kidney-tonifying preparation particle of desert (lot number 070801,070802,070803, specification are the 2.0g/ bag) provides for Guangzhou Meichen Pharmaceutical Co., Ltd.; The second eyeball is a chromatographically pure Di Ma company.All the other reagent are pure for analyzing.
(2) chromatographic condition adopts kromasil-C18 post (250mm * 4.6mm, 5 μ m) chromatographic column, 30 ℃ of column temperatures; Moving phase is methyl alcohol-acetonitrile-1% acetum (15: 10: 75); Flow velocity is 1ml/min; The detection wavelength is 334nm.
(3) reference substance solution prepares precision to take by weighing the echinacoside reference substance an amount of, puts in the brown bottle, adds moving phase and is made into the reference substance solution that every 1ml contains 0.16mg
(4) the need testing solution preparation is got these article in right amount, and porphyrize is got 0.5g, and accurate the title decides, and puts in the brown measuring bottle of 50ml, and precision adds moving phase 20ml; Close plug shakes up, and claims to decide weight, soaks sonicated 40min 0.5 hour; Take out, put coldly, claim again to decide weight, add moving phase and supply the weight that subtracts mistake, shake up; Centrifugal, get supernatant, with the micro-filtration membrane filtration of 0.45 μ m, filtrating is put in the brown bottle, promptly gets.Get the negative sample 0.3g of misrun desert cistanche, process negative test liquid with method.
(5) linear relationship is investigated accurate absorption above-mentioned reference substance solution 3 μ l, 5 μ l, 7 μ l, 10 μ l, 12 μ l inject liquid chromatograph respectively, are horizontal ordinate with peak area A, are ordinate with sample size W (mg), calculate regression equation: W=-3.3143 * 10
-5+ 7.97149 * 10
-10A, R=0.99996.Echinacoside is at 0.48 μ g---between the 1.92 μ g good linear relationship is arranged.
(6) accurate each the 5 μ l of above-mentioned reference substance solution, test liquid and negative test liquid that draw of negative control test inject liquid chromatograph, and result's negative test liquid chromatogram on echinacoside retention time position is not obviously disturbed.
(7) average recovery test precision suitably takes by weighing 9 parts of these article, and per 3 parts is 1 group, and every group all adds above-mentioned reference substance solution 3ml, 2ml, 1ml respectively by high, medium and low three concentration.According to above-mentioned need testing solution preparation method preparation and mensuration.Average recovery rate is 99.1% as a result, RSD%=1.52%.
(8) the accurate contrast liquid 5 μ l that draw of precision test inject liquid chromatograph, repeat the record peak area 6 times.Its RSD=1.03% shows that this method has good precision.
(9) stability test is injected liquid chromatograph, the record peak area respectively at 0,2,4,6,8 hour accurate each 5 μ l of test liquid that draw.Its RSD=1.41% shows that test liquid is stable in 0-8 hour.
(10) replica test is to same lot sample article, according to the said determination method, and replication 6 times.Its RSD=1.01% shows that this method has good repeatability.
(11) sample determination is measured three lot sample article according to the method described above, result's see the following form (by formula calculate gained mass percentage content multiply by the preparation specification be listed unit content)
The content (n=3) of echinacoside in the yellow kidney tonifying particle of table 1 desert
The present invention has the following advantages:
1, the present invention carries out the thin-layer chromatography qualitative identification respectively to the middle four traditional Chinese medicine material of the yellow kidney-tonifying preparation---particle, sheet, hard shell capsules---of desert, and contained a kind of active component is carried out assay.Compare with the pill standard detecting method, the present invention is more perfect, more scientific and reasonable.。
2, the present invention propose first in the yellow kidney-tonifying preparation of desert except that saline cistanche other three distinguish the flavor of medicinal materials the thin layer discrimination method; And the part that is not suitable for detecting the yellow kidney tonifying novel form of desert (particle, sheet, hard shell capsules) in the pill standard detecting method carried out bigger modification (changed the preparation method that saline cistanche is differentiated test liquid, changed high performance liquid chromatography assay composition and be the echinacoside that disturbs less, content is high).Thereby improved the quality control level of the yellow kidney tonifying novel form of desert, ensured the stable of product quality
3, strong, the good reproducibility of specificity of the present invention can be used for the daily quality control of this medicine.
Description of drawings
Provide accompanying drawing below and be used to further specify the present invention, but the present invention is not constituted restriction.
Fig. 1 is the TLC figure of saline cistanche of the present invention;
Fig. 2 is the TLC figure of prepared rhizome of rehmannia of the present invention;
Fig. 3 is the TLC figure of the seed of Chinese dodder of the present invention;
Fig. 4 is the TLC figure of the fruit of Chinese magnoliavine of the present invention;
Fig. 5 is the yellow capsule for tonifying kidney HPLC of a desert of the present invention chromatogram.
Embodiment
Enumerate embodiment further explain the present invention of detection below, but to not restriction of the present invention.
Embodiment one
1. instrument
Booth Electronic Instruments Plant in Hai'an on the ultraviolet point sample instrument; SPD-10Avp Ultraviolet Detector day island proper Tianjin company; P4000 quaternary pump U.S. power & light company; AT-130 chromatographic column constant temperature oven Tianjin Hengao Technology Development Co., Ltd.; HN1006 ultrasonic cleaning machine China south China ultrasonic device factory; HM202 electronic analytical balance Japan AND company; The 4K15C high speed freezing centrifuge; Germany SIGMA company.
2. reagent
Silica G Qingdao Marine Chemical Co., Ltd.; Betaine (894-200202), 5 hydroxymethyl furfural (111626-200402), deoxyschizandrin (765-200104), mountain naphthalene plain (0861-200002), echinacoside (111670-200502) Nat'l Pharmaceutical & Biological Products Control Institute; The yellow kidney tonifying particle (lot number 070801, specification 2.0g/ bag) of desert, the yellow kidney invigorating tablet (lot number 070811, specification 0.5g/ sheet) of desert, the yellow kidney tonifying hard shell capsules of desert (lot number 070821, specification 0.4g/ grain) provides for Guangzhou Meichen Pharmaceutical Co., Ltd.; The second eyeball is a chromatographically pure Di Ma company.All the other reagent are pure for analyzing.
3. thin layer is differentiated (with the granule is example, and tablet is identical with it with hard capsule)
3.1. saline cistanche
It is an amount of to get the yellow kidney tonifying particle of desert, and porphyrize takes by weighing powder 0.5～1.0g, adds water 20ml; Ultrasonic 10min makes to dissolve and looses, and centrifugal (4500r/min) 5min gets supernatant vacuum filtration again, the strong acid cation exchange resin column of filtrating; Wash post with 20ml, use 0.5～4mol/l ammonia spirit, 20～60ml wash-out again, collect eluent, evaporate to dryness; With ethanol 2ml dissolving, leave standstill, get supernatant as need testing solution.The betaine reference substance adds ethanol and processes the reference substance solution that every 1ml contains 5mg.Draw need testing solution 10 μ l, reference substance solution 5 μ l, point sample is on same silica gel g thin-layer plate, with methyl alcohol-glacial acetic acid-water (10～20: 0.5～2: 2～8) be developping agent, launch, take out, dry that spray is with the improvement bismuth potassium iodide test solution respectively.As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
3.2. prepared rhizome of rehmannia
It is an amount of to get the yellow kidney tonifying particle of desert, and porphyrize takes by weighing powder 1.0～3.0g; Add water 30ml, sonicated 10min, centrifugal (4500r/min) 5min; Get supernatant and extract 2 times with normal butyl alcohol (chloroform, ethyl acetate), each 15ml merges extract; Extract is concentrated into dried, and residue adds ethanol 1ml dissolving, as need testing solution.The 5 hydroxymethyl furfural reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.5mg.Draw need testing solution 10 μ l, reference substance solution 5 μ l, point sample is in same silica G F respectively
254On the thin layer plate, (1～8: 2～10) upper strata liquid is developping agent, launches, and takes out, and dries, and places under the 254nm uviol lamp and inspects with sherwood oil II-ethyl acetate.As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color.
3.3. the seed of Chinese dodder
It is an amount of to get the yellow kidney tonifying particle of desert, and porphyrize takes by weighing powder 1.0～3.0g, adds chloroform (absolute ethyl alcohol, methyl alcohol, ethyl acetate, normal butyl alcohol) 10ml, and sonicated 15min filters, and filtrating is concentrated into dried, adds ethanol 1ml dissolved residue, as need testing solution.Naphthalene plain reference substance in mountain adds ethanol and processes the reference substance solution that every 1ml contains 0.2mg.Draw need testing solution 10 μ l, reference substance solution 5 μ l, point sample is on same silica gel g thin-layer plate respectively; With toluene-ethyl acetate-methyl alcohol (4～10: 4～8: 1～6) be developping agent, launch, take out; Dry, spray places under the 365nm uviol lamp and inspects with 3% aluminium choride ethanolic solution.As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
3.4. the fruit of Chinese magnoliavine (wine steaming)
It is an amount of to get the yellow kidney tonifying particle of desert, and porphyrize takes by weighing powder 8.0～15.0g, adds methyl alcohol (absolute ethyl alcohol, chloroform, ethyl acetate, normal butyl alcohol) 20ml, and sonicated or reflux 30min filter, and filtrating is concentrated into about 1ml, as need testing solution.The deoxyschizandrin reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.4mg.Draw each 10 μ l of need testing solution and reference substance solution, point sample is in same silica G F respectively
254On the thin layer plate, (8～20: 2～8: 0.5～2) upper strata liquid is developping agent, launches, and takes out, and dries, and places under the 254nm uviol lamp and inspects with sherwood oil I-ethyl formate-formic acid.As a result in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color.
" 1 " of indication is a test liquid among Fig. 1, and " 2 " are contrast liquid, and " 3 " are negative test liquids.
" 1 " of indication is a test liquid among Fig. 2, and " 2 " are contrast liquid, and " 3 " are negative test liquids.
To reach " 3 " be reference substance in " 1 " of indication among Fig. 3, and " 2 " reach " 5 " is test liquid, and " 4 " are negative test liquids.
Among Fig. 4 " 1 " of indication reach " 4 " be the contrast liquid, " 2 " reach " 5 " is test liquid, " 3 " are negative test liquids.
4. assay (with the granule is example, and tablet and hard capsule division result are outer, and other are identical with it).
4.1. chromatographic condition chromatographic column adopting kromasil-C18 post (250mm * 4.6mm, 5 μ m); 30 ℃ of column temperatures; Moving phase is methyl alcohol-acetonitrile-1% acetum (15: 10: 75); Flow velocity is 1ml/min; The detection wavelength is 334nm.
To take by weighing the echinacoside reference substance an amount of 4.2. reference substance solution prepares precision, puts in the brown bottle, adds moving phase and be made into the reference substance solution that every 1ml contains 0.16mg
4.3. the need testing solution preparation is got, and the yellow kidney tonifying particle of desert is an amount of, porphyrize is got 0.5g, and accurate the title decides, and puts in the brown measuring bottle of 50ml, and precision adds moving phase 20ml; Close plug shakes up, and claims to decide weight, soaks sonicated 40min 0.5 hour; Take out, put coldly, claim again to decide weight, add moving phase and supply the weight that subtracts mistake, shake up; Centrifugal, get supernatant, with the micro-filtration membrane filtration of 0.45 μ m, filtrating is put in the brown bottle, promptly gets.
4.4. accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure.(by formula calculating the gained mass percentage content multiply by the preparation specification and is listed unit content.)
4.5. granule contains echinacoside 18.1mg for every bag as a result
Every in tablet contains echinacoside 4.5mg
Every of hard capsule contains echinacoside 3.9mg
" I " of indication is test liquid among Fig. 5, and " II " is contrast liquid, and " III " is negative test liquid, and " a " is echinacoside.
Fig. 1,2,3,4, the result of negative test shown in 5 are in order to verify and explain the present invention.Detection method is being carried out to do negative test when specificity is investigated.After detection method is confirmed, in daily detection, generally need not do negative test.
Claims (1)
1. the detection method of the yellow kidney-tonifying preparation of desert is characterized in that with thin-layer chromatography saline cistanche, prepared rhizome of rehmannia, the seed of Chinese dodder, wine being steamed and concocts that the fruit of Chinese magnoliavine carries out qualitative identification and with the content of active component echinacoside in the high-performance liquid chromatogram determination saline cistanche;
(1) it is an amount of that saline cistanche is got the yellow kidney tonifying particle of desert, sheet or hard shell capsules formulation preparation, and porphyrize takes by weighing powder 0.5～1.0g, adds water 20ml; Ultrasonic 10min makes to dissolve and looses, and centrifugal commentaries on classics 5min gets supernatant vacuum filtration again; The strong acid cation exchange resin column of filtrating is washed post with 20ml, uses 0.5～4mol/l ammonia spirit, 20～60ml wash-out again; Collect eluent, evaporate to dryness dissolves with ethanol 2ml; Leave standstill, get supernatant as need testing solution, the betaine reference substance adds ethanol and processes the reference substance solution that every 1ml contains 5mg; Draw need testing solution 10 μ l, reference substance solution 5 μ l, point sample is a developping agent with methyl alcohol-glacial acetic acid-water on same silica gel g thin-layer plate respectively, ratio is 10～20: 0.5～2: 2～8, launch, take out, to dry, spray is with the improvement bismuth potassium iodide test solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
(2) it is an amount of that prepared rhizome of rehmannia is got the yellow kidney tonifying particle of desert, sheet or hard shell capsules formulation preparation, and porphyrize takes by weighing powder 1.0～3.0g; Add water 30ml, sonicated 10min, centrifugal commentaries on classics 5min; Get supernatant with normal butyl alcohol, chloroform or ethyl acetate extraction 2 times, each 15ml merges extract; Extract is concentrated into dried, and residue adds ethanol 1ml dissolving, as need testing solution; The 5 hydroxymethyl furfural reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.5mg, draws need testing solution 10 μ l, reference substance solution 5 μ l, and point sample is in same silica G F respectively
254On the thin layer plate, be that 1～8: 2～10 sherwood oil II-ethyl acetate upper strata liquid are developping agent, launch, take out, dry, place under the 254nm uviol lamp and inspect with ratio; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color;
(3) it is an amount of that the seed of Chinese dodder is got the yellow kidney tonifying particle of desert, sheet or hard shell capsules formulation preparation, and porphyrize takes by weighing powder 1.0～3.0g, adds chloroform, absolute ethyl alcohol, methyl alcohol, ethyl acetate or normal butyl alcohol 10ml; Sonicated 15min filters, and filtrating is concentrated into dried, adds ethanol 1ml dissolved residue; As need testing solution, naphthalene plain reference substance in mountain adds ethanol and processes the reference substance solution that every 1ml contains 0.2mg, draws need testing solution 10 μ l, reference substance solution 5 μ l; Point sample is on same silica gel g thin-layer plate respectively, and be 4～10: 4～8 with ratio: 1～6 toluene-ethyl acetate-methyl alcohol is developping agent, launches, and takes out; Dry, spray places under the 365nm uviol lamp and inspects with 3% aluminium choride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;
(4) it is an amount of that the wine steaming process of preparing Chinese medicine fruit of Chinese magnoliavine is got the yellow kidney tonifying particle of desert, sheet or hard shell capsules formulation preparation, and porphyrize takes by weighing powder 8.0～15.0g; Add methyl alcohol, absolute ethyl alcohol, chloroform, ethyl acetate or normal butyl alcohol 20ml; Sonicated or reflux 30min filter, and filtrating is concentrated into about 1ml; As need testing solution, the deoxyschizandrin reference substance adds ethanol and processes the reference substance solution that every 1ml contains 0.4mg; Draw each 10 μ l of need testing solution reference substance solution, point sample is in same silica G F respectively
254On the thin layer plate, be 8～20: 2～8 with ratio: 0.5～2 sherwood oil I-ethyl formate-formic acid upper strata liquid is developping agent, launches, and takes out, and dries, and places under the 254nm uviol lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the blackening point of same color,
(5) assay of active component echinacoside in the saline cistanche:
Chromatographic condition and system suitability test chromatographic column adopting octadecylsilane chemically bonded silica are filler; Moving phase is that ratio is 15: 10: 75 methyl alcohol-acetonitrile-1% acetums; The detection wavelength is 334nm, and theoretical cam curve is calculated by the echinacoside peak should be not less than 4000;
Reference substance solution prepares precision, and to take by weighing the echinacoside reference substance an amount of, puts in the brown bottle, adds moving phase and be made into the reference substance solution that every 1ml contains 0.16mg;
It is an amount of that these article are got in the need testing solution preparation, and porphyrize is got 0.5g, and accurate the title decides, and puts in the brown measuring bottle of 50ml, and precision adds moving phase 20ml; Close plug shakes up, and claims to decide weight, soaks sonicated 40min 0.5 hour; Take out, put coldly, claim again to decide weight, add moving phase and supply the weight that subtracts mistake, shake up; Centrifugal, get supernatant, with the micro-filtration membrane filtration of 0.45 μ m, filtrating is put in the brown bottle, promptly gets;
Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure echinacoside content.
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