CN102998410B - A kind of quality determining method of blood-nourishing and brain-refreshing granules - Google Patents
A kind of quality determining method of blood-nourishing and brain-refreshing granules Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000008187 granular material Substances 0.000 title claims abstract description 37
- 241000112528 Ligusticum striatum Species 0.000 claims abstract description 56
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 claims abstract description 29
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 claims abstract description 29
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 claims abstract description 26
- 244000179560 Prunella vulgaris Species 0.000 claims abstract description 25
- 235000008113 selfheal Nutrition 0.000 claims abstract description 25
- UBBRXVRQZJSDAK-ZJHGLIIDSA-N (Z)-6,6',7,3'a-Diligustilide Chemical compound C([C@H](C=C1C(=O)OC/2=C\CCC)[C@@H]3CC4)C[C@]1\2[C@H]3C1=C4C(=C/CCC)/OC1=O UBBRXVRQZJSDAK-ZJHGLIIDSA-N 0.000 claims abstract description 20
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 claims abstract description 18
- 229940096998 ursolic acid Drugs 0.000 claims abstract description 18
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 235000014489 Cinnamomum aromaticum Nutrition 0.000 claims abstract description 16
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- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 claims abstract description 13
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 claims abstract description 13
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 claims abstract description 13
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 24
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- ONBIUAZBGHXJDM-UHFFFAOYSA-J bismuth;potassium;tetraiodide Chemical compound [K+].[I-].[I-].[I-].[I-].[Bi+3] ONBIUAZBGHXJDM-UHFFFAOYSA-J 0.000 claims description 12
- LQGUBLBATBMXHT-UHFFFAOYSA-N chrysophanol Chemical compound C1=CC=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O LQGUBLBATBMXHT-UHFFFAOYSA-N 0.000 claims description 12
- 235000019441 ethanol Nutrition 0.000 claims description 12
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- 241000736199 Paeonia Species 0.000 claims description 11
- CNHOGQMRRDHYGI-NJCGARBWSA-N beta-onocerin Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CCC(C)=C2CCC1=C(C)CC[C@@H]2[C@]1(C)CC[C@H](O)C2(C)C CNHOGQMRRDHYGI-NJCGARBWSA-N 0.000 claims description 10
- FFWOKTFYGVYKIR-UHFFFAOYSA-N physcion Chemical compound C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1O FFWOKTFYGVYKIR-UHFFFAOYSA-N 0.000 claims description 10
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- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 8
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 8
- 229910052740 iodine Inorganic materials 0.000 claims description 8
- 239000011630 iodine Substances 0.000 claims description 8
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 8
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 8
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 8
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 claims description 6
- 229960000583 acetic acid Drugs 0.000 claims description 6
- NZPQWZZXRKZCDU-UHFFFAOYSA-N chrysophanol Natural products Cc1cc(O)c2C(=O)c3c(O)cccc3Oc2c1 NZPQWZZXRKZCDU-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims description 6
- OAMZXMDZZWGPMH-UHFFFAOYSA-N ethyl acetate;toluene Chemical compound CCOC(C)=O.CC1=CC=CC=C1 OAMZXMDZZWGPMH-UHFFFAOYSA-N 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 6
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- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000004811 liquid chromatography Methods 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
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- AEQDJSLRWYMAQI-UHFFFAOYSA-N Tetrahydropalmatine Natural products C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 claims description 5
- WLXGUTUUWXVZNM-UHFFFAOYSA-N anthraglycoside A Natural products C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1OC1OC(CO)C(O)C(O)C1O WLXGUTUUWXVZNM-UHFFFAOYSA-N 0.000 claims description 5
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- PKUBGLYEOAJPEG-UHFFFAOYSA-N physcion Natural products C1=C(C)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O PKUBGLYEOAJPEG-UHFFFAOYSA-N 0.000 claims description 5
- AEQDJSLRWYMAQI-KRWDZBQOSA-N tetrahydropalmatine Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3C[C@H]2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-KRWDZBQOSA-N 0.000 claims description 5
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
- 241000345998 Calamus manan Species 0.000 claims description 4
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims description 4
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- 229910021529 ammonia Inorganic materials 0.000 claims description 4
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to field of medicaments, be specifically related to a kind of quality determining method of blood-nourishing and brain-refreshing granules, method of the present invention, comprise the discriminating to following composition: in A selfheal, ursolic acid is differentiated, B corydalis tuber is differentiated, C cassia seed is differentiated, D Radix Angelicae Sinensis and Ligusticum wallichii are differentiated, in E selfheal, Rosmarinic acid is differentiated, in F Ligusticum wallichii, alkaloid is differentiated, in G Ligusticum wallichii, Levistilide A is differentiated, H reticulate millettia is differentiated, the I root of herbaceous peony is differentiated, J prepared rhizome of rehmannia is differentiated, K yncaria stem with hooks is differentiated, also comprises and carries out assay to the effective constituent Paeoniflorin in blood-nourishing and brain-refreshing granules.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of quality determining method of blood-nourishing and brain-refreshing granules
Background technology
Blood-nourishing and brain-refreshing granules is the modern Chinese herbal medicine preparation of Tianjin Tasly Pharmaceutical Co., Ltd's development, and obtains national New Drug Certificate in 1996, and is put into National essential drugs list in 1999, within 2000, is put into national medical insurance Drug catalogue.Blood-nourishing and brain-refreshing granules is made up of 11 taste Chinese medicines such as Radix Angelicae Sinensis, Ligusticum wallichii, the root of herbaceous peony, reticulate millettia, cassia seed, corydalis tubers, and after modern high tech method extracts, adds suitable auxiliary material, through a kind of granule that the production process such as mixing granulation are made.It is fast that said preparation has effective constituent stripping, bioavilability advantages of higher.Blood-nourishing and brain-refreshing granules has flat liver of nourishing blood, effect promoting blood circulation and removing obstruction in channels, can be used for the high caused headache of deficiency of blood liver, staggers, the illness such as dysphoria, insomnia and dreamful sleep, has significant curative effect clinically.
Current blood-nourishing and brain-refreshing granules quality determining method many employings thin-layered chromatography and high performance liquid chromatography.Thin-layered chromatography is strong due to economic, easy, intuitive, is applied to compound Chinese medicinal preparation and differentiates that frequency is the highest, remain the discriminating means that " Chinese Pharmacopoeia ", " British Pharmacopoeia ", India herbal medicine allusion quotation, European Pharmacopoeia etc. are generally applied so far.High performance liquid chromatography is used for the assay of effective constituent, and it has the advantages such as applied widely, separating property good, analysis speed is fast, mobile phase range of choice is wide, highly sensitive, chromatographic column can repeatedly use.
But existing detection method complicated operation, precision is not high, easily goes wrong in actual mechanical process.
Summary of the invention
technical matters to be solved
The invention provides a kind of quality determining method of blood-nourishing and brain-refreshing granules, solve some important technological problems in detecting at present.
technical scheme
Its prescription of blood-nourishing and brain-refreshing granules of the present invention is as follows:
Main ingredient: Radix Angelicae Sinensis, Ligusticum wallichii, the root of herbaceous peony, prepared rhizome of rehmannia, yncaria stem with hooks, reticulate millettia, selfheal, cassia seed, mother-of-pearl, corydalis tuber, the root of Chinese wild ginger.
Auxiliary material is: dextrin, Steviosin.
Quality determining method of the present invention, comprises discrimination method and content assaying method.
For this reason, first the present invention provides a kind of discrimination method of blood-nourishing and brain-refreshing granules, and the method comprises the discriminating to following composition:
In A selfheal, ursolic acid is differentiated
B corydalis tuber is differentiated
C cassia seed is differentiated
D Radix Angelicae Sinensis and Ligusticum wallichii are differentiated
In E selfheal, Rosmarinic acid is differentiated
In F Ligusticum wallichii, alkaloid is differentiated
In G Ligusticum wallichii, Levistilide A is differentiated
H reticulate millettia is differentiated
The I root of herbaceous peony is differentiated
J prepared rhizome of rehmannia is differentiated
K yncaria stem with hooks is differentiated
Discrimination method of the present invention adopts thin-layered chromatography.
Secondly, the present invention also comprises and carries out assay to the effective constituent in blood-nourishing and brain-refreshing granules, and wherein said effective constituent is Paeoniflorin.Content assaying method of the present invention adopts high performance liquid chromatography.
Discrimination method of the present invention has 11, can use wherein one as the discrimination method to blood-nourishing and brain-refreshing granules, also multinomially can combinationally use as the discrimination method to blood-nourishing and brain-refreshing granules, preferably multinomially to combinationally use, most preferably 11 combinationally use.
Content assaying method of the present invention, chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; With isopropanol-methanol-5mmol/L citric acid soln (2-4: 18-22: 78-80) for mobile phase; Determined wavelength is 230-240nm.Number of theoretical plate calculates should be not less than 2000 by Paeoniflorin peak.
Discrimination method of the present invention, preferably adopts following methods:
A selfheal differentiates (ursolic acid)
Get this product 0.5-2.0g, add watery hydrochloric acid 0.2-1.0ml and ether 10-30ml, add hot reflux 20-40 minute, most ether waved by extract, and residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Separately get ursolic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 0.1-1.0mg, product solution in contrast.Test according to thin-layered chromatography, draw need testing solution 5-15 μ l, reference substance solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-chloroform-glacial acetic acid (15-25: 2-4: 5-9: 0.1-0.5) for developping agent, launch, take out, dry, spray, with 5-15% ethanol solution of sulfuric acid, is heated to colour developing at 100-120 DEG C clear.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
B corydalis tuber is differentiated
Get this product 0.5-1.5g, put in tool plug centrifuge tube, add 0.1-0.5mol/L sodium hydroxide solution 2-6ml, jolting makes dissolving, add ethyl acetate 5-10ml, shake well, centrifugal 2-5 minute, gets upper solution, evaporate to dryness, residue adds methyl alcohol 1-2ml makes dissolving, as need testing solution.Separately get corydalis tuber control medicinal material 0.5-2.0g, add ammonia solution (get strong ammonia solution 1ml, add water 10ml, mixing) 0.5-2.0ml, grinding, add methylene chloride 5-20ml, temperature leaching 1-3 hour, filter, filtrate is concentrated into about 0.5-2.0ml, in contrast medicinal material solution.Get tetrahydropalmatine reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.1-1.0mg, product solution in contrast.According to thin-layered chromatography test, draw above-mentioned three kinds of solution each 5-15 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-chloroform-methanol-strong ammonia solution (5-20: 6-10: 1-3: 0.2-0.5) for developping agent, launch, take out, dry, spray with rare bismuth potassium iodide test solution.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color; On the position corresponding to reference substance chromatogram, the spot of aobvious same color.
C cassia seed is differentiated
Depend on pine torch control medicinal material 0.5-1.5g, add watery hydrochloric acid 0.5-1.0ml, ether 10-20ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate reclaims ether to dry, and residue adds methyl alcohol 0.5-2.0ml respectively makes dissolving, as cassia seed control medicinal material solution.Get Physcion, Chrysophanol, archen and aurantio-obtusin reference substance again, add absolute ethyl alcohol-ethyl acetate (1-4: 0.5-2) and make every 1ml respectively containing the solution of 0.5-1.0mg, as mixing reference substance solution.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 10-20 μ l in item, control medicinal material solution 7-15 μ l and mixing reference substance solution 2-4 μ l, put respectively on same high-efficient silica gel G thin layer plate, with sherwood oil (30 ~ 60 DEG C)-cyclohexane-ethyl acetate-formic acid (3-9: 10-14: 3-7: 0.2-0.8) for developping agent, launch, take out, dry, be placed in smoked more than the 10min of ammonia steam, inspect under putting daylight.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
D Radix Angelicae Sinensis-Ligusticum wallichii is differentiated
Get Radix Angelicae Sinensis, each 0.5-2.0g of Ligusticum wallichii control medicinal material respectively, add diethyl ether 10-30ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate reclaims ether to dry, and residue adds ethyl acetate 0.5-2.0ml, 1.0-2.0ml respectively makes dissolving, as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 5-12 μ l in item, Radix Angelicae Sinensis control medicinal material solution 5-15 μ l and Ligusticum wallichii control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate-formic acid (5-9: 0.5-1.5: 0.2-0.8) for developping agent, launch more than 8cm, take out, dry, inspect under being placed in ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color.
E selfheal differentiates (Rosmarinic acid)
Get Rosmarinic acid reference substance, add Diluted Alcohol and make the solution of every 1ml containing 0.5-1.0mg, product solution in contrast.Test according to thin-layered chromatography, draw " selfheal (ursolic acid) " and differentiate need testing solution 1-5 μ l, reference substance solution 1-4 μ l in item, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate-formic acid (1-3: 5-6: 0.2-0.5) for developping agent, launch, take out, dry, inspect under being placed in ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
F Ligusticum wallichii differentiates (alkaloids)
Get this product 5-20g, add ammoniacal liquor 3-8ml, and ether 30-50ml, close plug leaves standstill 30-90 minute, glass bar stirs evenly, and water-bath backflow 20-40 minute, takes out, let cool, extract reclaims ether to dry, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Get Ligusticum wallichii control medicinal material 0.5-2.0g, add ammoniacal liquor 3-8ml, and ether 10-30ml, close plug leaves standstill 50-70 minute, water-bath backflow 20-40 minute, takes out, lets cool, filter, filtrate reclaims ether to dry, and residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as Ligusticum wallichii control medicinal material solution.Test according to thin-layered chromatography, draw Ligusticum wallichii control medicinal material solution 7-10 μ l, need testing solution 7-12 μ l respectively, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate (7-11: 0.5-2) for developping agent, launch, take out, dry, spray, to improve bismuth potassium iodide solution-iodine liquor kalii iodide (1-4: 0.5-1) mixed solution, is inspected under being placed in daylight.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
G Ligusticum wallichii differentiates (Levistilide A)
Get this product 2-10g, add ether 20-60ml, water-bath backflow 20-60 minute, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Separately get Levistilide A reference substance, add ethyl acetate and make the solution of every 1ml containing 0.2-1.0mg, as Levistilide A reference substance solution.Test according to thin-layered chromatography, draw Levistilide A reference substance solution 2-6 μ l, need testing solution 6-10 μ l respectively, put respectively on same silica GF254 thin layer plate, with n-hexane-ethyl acetate (1-5: 0.5-2) for developping agent, launch, take out, dry, inspect under being placed in ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
H reticulate millettia is differentiated
Get this product 5-20g, add 0.1-0.5% sodium hydroxide solution 20-50ml, shake up, ultrasonic process 20-40min, take out, centrifugal (2000-6000 rev/min, 3-10 minute), get supernatant, with sherwood oil (60 ~ 90 DEG C) extraction 1-3 time, each 10-20ml, discard petroleum ether layer, water intaking layer adds 0.3-0.6ml watery hydrochloric acid, shake up, by extracted with diethyl ether 1-3 time, each 10-20ml, merge extracted with diethyl ether layer, reclaim ether to dry, residue adds methyl alcohol 0.5-2.0ml and dissolves, add silica gel 1-2g to mix thoroughly, volatilize solvent, put silicagel column (100 ~ 200 orders, 1-3g, internal diameter 1cm, dry column-packing) on, with methyl alcohol-methenyl choloride (0.5-1.5: 7-11) 30-50ml wash-out, collect methyl alcohol-methenyl choloride (0.5-1.5: 7-11) eluent, evaporate to dryness, residue adds methenyl choloride 0.5-1.5ml makes dissolving, as need testing solution.Separately get onocerin appropriate, add methyl alcohol and make the solution of every 1ml containing 0.1-0.5mg, product solution in contrast.Test according to thin-layered chromatography, draw onocerin reference substance solution 2-5 μ l, need testing solution 5-10 μ l respectively, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol (10-40: 1) for developping agent, launch more than 8cm, take out, dry, inspect under being placed in ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
The I root of herbaceous peony is differentiated
Test according to thin-layered chromatography, draw need testing solution 20-30 μ l and Paeoniflorin reference substance solution 10-20 μ l in assay item respectively, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetate-methanol-formic acid (20-50: 3-7: 5-15: 0.2) for developping agent, launch, take out, dry, spray, with 3-5% vanillin-sulfuric acid solution, is heated to colour developing at 100-120 DEG C clear.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
J prepared rhizome of rehmannia is differentiated
Get this product 15-20g, add water 50-100ml, in 50-60 DEG C of warm 20-50 minute, take out, ultrasonic process 20-40 minute, centrifugal, get supernatant, be extracted with ethyl acetate 1-3 time, each 20-40ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue adds ethyl acetate 3-10ml makes dissolving, adds neutral alumina 2-4g, mix thoroughly, load neutral alumina column (10-15g, internal diameter 15mm, 70 ~ 100 orders), with ethanol 20-50ml wash-out, collect eluent, evaporate to dryness, residue adds ethanol 0.5-2.0ml, make dissolving, as need testing solution.Separately get prepared rhizome of rehmannia control medicinal material 2-5g, the 30-80ml that adds water decocts 0.5-2.0 hour, and filter, filtrate is extracted with ethyl acetate 1-3 time, each 20-40ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml, makes dissolving, in contrast medicinal material solution.According to thin-layered chromatography test, draw need testing solution 20-25 μ l and control medicinal material solution 10-20 μ l respectively, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate (0.5-2: 1) for developping agent, launch, take out, dry, spray with DNPH ethanolic solution.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
K yncaria stem with hooks is differentiated
Get this product 5-20g, add strong ammonia solution 5-15ml and make infiltration, close plug 20-40 minute, add ether 50-100ml, add hot reflux 0.5-2.0 hour, let cool, divide and get ether layer, extract 1-3 time with the jolting of 3-8% hydrochloric acid solution, each 10-30ml, merge 3-8% hydrochloric acid solution, by ammoniacal liquor adjust ph to 9, extract 1-3 time with ether jolting, each 10-30ml, merges ether solution, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution.Another hook taking rattan control medicinal material 1-3g, adds strong ammonia solution 2-5ml and infiltrates, close plug 20-40 minute, and add diethyl ether 20-50ml, adds hot reflux 0.5-2.0 hour, let cool, and filter, filtrate evaporate to dryness, adds methyl alcohol 0.5-2.0ml and make dissolving, in contrast medicinal material solution.Test according to thin-layered chromatography, draw above-mentioned two kinds of solution each 5-20 μ l, put respectively on same silica gel g thin-layer plate, with toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor (6-10: 10-14: 1-3: 1-3: 0.5-0.8) for developping agent, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1-4: 0.5-1) mixed solution, in test sample chromatogram, with on control medicinal material chromatogram relevant position, show the principal spot of same color.
Content assaying method of the present invention, preferably adopts following methods:
With the content of high effective liquid chromatography for measuring Paeoniflorin
High-efficient liquid phase chromatogram condition: be filling agent with octadecylsilane chemically bonded silica; With isopropanol-methanol-5mmol/L citric acid soln (2-4: 18-22: 78-80) for mobile phase; Determined wavelength is 230-240nm.Number of theoretical plate calculates should be not less than 2000 by Paeoniflorin peak.
The preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 70-90% methyl alcohol and make the solution of every 1ml containing 15-25 μ g, to obtain final product.
The contents under this product content uniformity item is got in the preparation of need testing solution, and porphyrize, gets about 0.06-0.10g, accurately weighed, put in 20-50ml beaker, add sodium bicarbonate solution 5ml, ultrasonic process, is added on D101 type large pore resin absorption column, uses 10-20ml water elution, discard eluent, then use 6-10ml methanol-eluted fractions, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 4-6 minute, get supernatant, to obtain final product.
Determination method is accurate respectively draws reference substance solution and need testing solution each 5-15 μ l, injection liquid chromatography, measures, to obtain final product.
The most preferred content assaying method of the present invention is as follows:
High-efficient liquid phase chromatogram condition: be filling agent with octadecylsilane chemically bonded silica; With isopropanol-methanol-5mmol/L citric acid soln (2-4: 18-22: 78-80) for mobile phase; Determined wavelength is 230-240nm.Number of theoretical plate calculates should be not less than 2000 by Paeoniflorin peak.
The preparation of reference substance solution gets Paeoniflorin reference substance in right amount, accurately weighed, adds 80% methyl alcohol and makes the solution of every 1ml containing 20 μ g, to obtain final product.
The contents under this product content uniformity item is got in the preparation of need testing solution, porphyrize, gets about 0.08g, accurately weighed, put in 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic process (power 500W, frequency 30kHz) 5 minutes, be added in D101 type large pore resin absorption column (60 ~ 80 orders, internal diameter 6 ~ 8mm, height is 5cm about) on, with water 15ml wash-out, discard eluent, use methyl alcohol 8ml wash-out again, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
beneficial effect
In the present invention, TLC method qualitative detection can go out Radix Angelicae Sinensis in blood-nourishing and brain-refreshing granules, Ligusticum wallichii, reticulate millettia, cassia seed, selfheal, yncaria stem with hooks, the root of herbaceous peony, corydalis tuber and prepared rhizome of rehmannia, and the method is simple, stable, can be used as the qualitative checking method of blood-nourishing and brain-refreshing granules.HPLC method can measure the content of Paeoniflorin in blood-nourishing and brain-refreshing granules, and method is easy, and repeatability and good stability, result accurately and reliably, can be used as the detection method of content of blood-nourishing and brain-refreshing granules.
Flow phase system more different in test, find that isopropanol-methanol-aqueous solution of citric acid system can make Paeoniflorin in this product be well separated, and select through different proportion of mobile phase, therefore the present invention have finally chosen isopropanol-methanol-5mmol/L aqueous solution of citric acid (2-4: 20-22: 78-80) as mobile phase.
Accompanying drawing explanation
Fig. 1: Qingdao silica G plate, T=22 DEG C, RH=41%
From left to right: sample (lot number: 2011C10); Sample (lot number: 2011C11); ; Sample (lot number: 2011C12);
Ursolic acid reference substance
Fig. 2: Qingdao silica G plate, T=22 DEG C, RH=41%
From left to right: sample (lot number: 2010F27); Sample (lot number: 2010F28); Tetrahydropalmatine reference substance (middle inspection institute, 110726-201011); Corydalis tuber control medicinal material (middle inspection institute, 120928-200604); Sample (lot number: 2010F29);
Fig. 3: Yantai HSG, T=24 DEG C; RH=27%
From left to right: negative sample solution; Cassia seed control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03); Test sample (lot number: 2010B04);
Test sample (lot number: 2010B05); Test sample (lot number: 2010B06); Test sample (lot number: 2010B07);
Test sample (lot number: 2010B08); Test sample (lot number: 2010B09); Test sample (lot number: 2010B10);
Physcion reference substance; Chrysophanol reference substance; Archen reference substance; Aurantio-obtusin reference substance Fig. 4: Yantai silica G plate, T=21 DEG C; RH=23%
From left to right: blank sample solution; Radix Angelicae Sinensis control medicinal material; Need testing solution (lot number: 2009J23); Need testing solution (lot number: 2009K25); Need testing solution (lot number: 2009L03); Ligusticum wallichii control medicinal material Fig. 5: Yantai silica G plate, T=25 DEG C, RH=66%
From left to right: negative sample; Rosmarinic acid reference substance; Sample (lot number: 2010D01); Sample (lot number: 2010D02); Sample (lot number: 2010D03)
Fig. 6: Yantai silica G plate, T=26 DEG C, RH=50%
From left to right: negative (lacking Ligusticum wallichii); Radix Angelicae Sinensis control medicinal material; Test sample (lot number: 2009J23); Test sample (lot number: 2009L02); Test sample (lot number: 2009L03); Ligusticum wallichii control medicinal material 10 μ l;
Fig. 7: Yantai gel GF 254 plate, T=24 DEG C, RH=52%
From left to right: negative; Levistilide A; Test sample 2009J23; Test sample 2009L02; Test sample 2009L03 Fig. 8: Qingdao silica G plate, T=27 DEG C, RH=48%
From left to right: negative; Onocerin; Test sample 2009J23; Test sample 2009K25; Test sample 2010C30;
Fig. 9: Yantai silica G plate, T=25 DEG C, RH=36%
From left to right: negative sample solution; Paeoniflorin reference substance; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03); Test sample (lot number: 2010B04); Test sample (lot number: 2010B05); Test sample (lot number: 2010B06); Test sample (lot number: 2010B07);
Test sample (lot number: 2010B08); Test sample (lot number: 2010B09); Test sample (lot number: 2010B10);
Figure 10: Yantai silica G plate, T=25 DEG C, RH=47%
From left to right: negative sample solution; Prepared rhizome of rehmannia control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03);
Figure 11: Yantai silica G plate, T=22 DEG C; RH=28%
From left to right: negative sample solution; Yncaria stem with hooks control medicinal material; Test sample (lot number: 2010B01); Test sample (lot number: 2010B02); Test sample (lot number: 2010B03);
Figure 12: the HPLC collection of illustrative plates measuring paeoniflorin content
Embodiment
1 instrument and reagent
Waters 2695 high performance liquid chromatograph, Waters 2996PDA detecting device, Empower2 workstation; Milli-Q ultrapure water machine.
Paeoniflorin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 110736-200731), forulic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 0773-9910), Radix Angelicae Sinensis control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, 120927-200310), Ligusticum wallichii control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, 120918-200809), Rosmarinic acid reference substance (Tianjin one side Science and Technology Ltd.), selfheal control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, 120993-200604), cassia seed control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, 121544-200501), Chrysophanol reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 110796-200311), aurantio-obtusin (Tianjin one side Science and Technology Ltd.), onocerin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 111703-200501), prepared rhizome of rehmannia control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, 121196-200803)
Methyl alcohol, tetrahydrofuran are chromatographically pure, and it is pure that all the other reagent are analysis.
2 methods and result
Embodiment 1
Selfheal differentiates (ursolic acid)
The preparation of need testing solution: get this product 1g, adds watery hydrochloric acid 0.5ml and ether 20ml, and add hot reflux 30 minutes, most ether waved by extract, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution.
The preparation of reference substance solution: get ursolic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast.
The selection of developping agent: prepare 20: 3: 7: 0.2 ratio respectively, 15: 2: 5: 0.1 ratio, the cyclohexane-ethyl acetate-chloroform-glacial acetic acid of 25: 4: 9: 0.5 ratio is developping agent.
According to thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to colour developing at 105 DEG C clear.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.See figure (1-1).Be wherein that developping agent spot is the most obvious with the cyclohexane-ethyl acetate-chloroform-glacial acetic acid of 20: 3: 7: 0.2 ratio.
Embodiment 2
Corydalis tuber is differentiated
The preparation of need testing solution: get this product 1g, puts in tool plug centrifuge tube, and add 0.2mol/L sodium hydroxide solution 4ml, jolting makes dissolving, add ethyl acetate 5ml, shake well, centrifugal 2 minutes, get upper solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.
The preparation of control medicinal material solution: corydalis tuber control medicinal material 0.5g, adds ammonia solution (get strong ammonia solution 1ml, add water 10ml, mixing) 0.5ml, grinding, add methylene chloride 10ml, temperature leaching 2 hours, filter, filtrate is concentrated into about 1ml, in contrast medicinal material solution.
The preparation of reference substance solution: get tetrahydropalmatine reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast.
The selection of developping agent: prepare 15: 8: 2: 0.2 ratio respectively, 5: 6: 1: 0.2 ratio, the normal hexane-chloroform-methanol-strong ammonia solution of 20: 10: 3: 0.5 ratio is developping agent.
According to thin-layered chromatography test, draw each 10 μ l of need testing solution, control medicinal material solution and reference substance solution, put respectively on same silica gel g thin-layer plate, launch under above-mentioned developping agent condition, take out, dry, spray with rare bismuth potassium iodide test solution.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color; On the position corresponding to reference substance chromatogram, the spot of aobvious same color.See figure (2-1).Be wherein that developping agent spot is the most obvious with the normal hexane-chloroform-methanol-strong ammonia solution of 15: 8: 2: 0.2 ratio.
Embodiment 3
Cassia seed is differentiated
The preparation of need testing solution: get this product 2g, adds watery hydrochloric acid 1ml and ether 20ml, and add hot reflux 30 minutes, most ether waved by extract, and residue adds methyl alcohol 1ml makes dissolving, to obtain final product.
The preparation of blank solution: the blank pharmaceutical preparation getting scarce cassia seed, the preparation method according to need testing solution obtains blank solution.
The preparation of control medicinal material: depend on pine torch control medicinal material 1g, adds watery hydrochloric acid 0.5ml, ether 20ml, and water-bath refluxes 1 hour, takes out, lets cool, and filters, and filtrate reclaims ether to dry, and residue adds methyl alcohol 1ml respectively makes dissolving, to obtain final product.
The preparation of reference substance solution: get Chrysophanol reference substance, archen reference substance, Physcion reference substance and aurantio-obtusin, adds absolute ethyl alcohol-ethyl acetate (2: 1) and makes every 1ml respectively containing the solution of 0.5mg, product solution in contrast.
The selection of developping agent: prepare 6: 12: 5: 0.5 ratio respectively, the ratio of 3: 10: 3: 0.2, sherwood oil (30 ~ 60 DEG C)-cyclohexane-ethyl acetate-formic acid of 9: 14: 7: 0.8 ratio is that developping agent is according to thin-layered chromatography test, draw need testing solution 10 μ l, control medicinal material solution 7 and mixing reference substance solution 2 μ l, put respectively on same high-efficient silica gel G thin layer plate, launch under above-mentioned developping agent condition, take out, dry, be placed in smoked more than the 10min of ammonia steam, inspect under putting daylight.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.See figure (3-3).Be wherein that developping agent spot is the most obvious with the sherwood oil of 6: 12: 5: 0.5 ratio (30 ~ 60 DEG C)-cyclohexane-ethyl acetate-formic acid.
Embodiment 4
Radix Angelicae Sinensis-Ligusticum wallichii is differentiated
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, adds watery hydrochloric acid 1ml and ether 20ml, mixing, and 60 DEG C of heating water baths reflux 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 0.5ml and dissolves, and to obtain final product.
The preparation of control medicinal material solution: get Radix Angelicae Sinensis control medicinal material, each 1g of Ligusticum wallichii control medicinal material respectively, add diethyl ether 20ml, and water-bath refluxes 1 hour, take out, let cool, filter, filtrate reclaims ether to dry, and residue adds ethyl acetate 1ml respectively makes dissolving, respectively as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Radix Angelicae Sinensis, Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 9: 1: 0.5 ratios respectively, 5: 0.5: 0.2 ratios, the n-hexane-ethyl acetate-formic acid of 9: 1.5: 0.8 ratios is developping agent.
Test according to thin-layered chromatography, draw and differentiate need testing solution 10 μ l in (1) item, Radix Angelicae Sinensis control medicinal material solution 10 μ l and Ligusticum wallichii control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch more than 10cm, take out, dry, inspect under being placed in ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color.See figure (4-1).Be wherein that developping agent spot is the most obvious with the n-hexane-ethyl acetate-formic acid of 9: 1: 0.5 ratios.
Embodiment 5
Selfheal differentiates (Rosmarinic acid)
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, adds watery hydrochloric acid 1ml and ether 20ml, mixing, and 60 DEG C of heating water baths reflux 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 0.5ml and dissolves, and to obtain final product.
The preparation of reference substance solution: get Rosmarinic acid reference substance (Tianjin one side Science and Technology Ltd., content is 98%), adds Diluted Alcohol and makes the solution of every 1ml containing 0.5mg, product solution in contrast.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except selfheal and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 2: 4: 0.2 ratios respectively, 1: 5: 0.2 ratio, the n-hexane-ethyl acetate-formic acid of 3: 6: 0.5 ratios is developping agent.
According to thin-layered chromatography test, draw need testing solution 5 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, inspect under being placed in ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
See figure (5-1).Be wherein that developping agent spot is the most obvious with the n-hexane-ethyl acetate-formic acid of 2: 4: 0.2 ratios.
Embodiment 6
Ligusticum wallichii differentiates (alkaloids)
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, adds ammoniacal liquor 5ml, and ether 40ml, and close plug leaves standstill 60 minutes, and glass bar stirs evenly, and water-bath refluxes 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds absolute ethyl alcohol 1ml makes dissolving, to obtain final product.
The preparation of control medicinal material solution: get Ligusticum wallichii control medicinal material 1g, add ammoniacal liquor 5ml, and ether 20ml, close plug leaves standstill 60 minutes, water-bath refluxes 30 minutes, takes out, lets cool, and filters, filtrate reclaims ether to dry, and residue adds absolute ethyl alcohol 1ml makes dissolving, to obtain final product, as Ligusticum wallichii control medicinal material solution.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 9: 1 ratios respectively, 7: 0.5 ratios, the cyclohexane-ethyl acetate of 11: 2 ratios is developping agent.
Test according to thin-layered chromatography, draw Ligusticum wallichii control medicinal material solution 10 μ l, need testing solution 12 μ l respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray, to improve bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution, is inspected under being placed in daylight.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.See figure (6-1).Be wherein that developping agent spot is the most obvious with the cyclohexane-ethyl acetate of 9: 1 ratios.
Embodiment 7
Ligusticum wallichii differentiates (Levistilide A)
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 4g, adds ether 40ml, and water-bath refluxes 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 1ml makes dissolving, to obtain final product.
The preparation of reference substance solution: get Levistilide A reference substance 20mg, is placed in the brown volumetric flask of 50ml, adds acetic acid ethyl dissolution and be settled to scale, shaking up, to obtain final product, as Levistilide A reference substance solution.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 3: 1 ratios respectively, 1: 0.5 ratio, the n-hexane-ethyl acetate of 5: 2 ratios is developping agent.
According to thin-layered chromatography test, draw Levistilide A reference substance solution 6 μ l, need testing solution 10 μ l respectively, put respectively on same silica GF254 thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, inspect under being placed in ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.See figure (7-1).Be wherein that developping agent spot is the most obvious with the n-hexane-ethyl acetate of 3: 1 ratios.
Embodiment 8
Reticulate millettia is differentiated
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add 0.2% sodium hydroxide solution 30ml, shake up, ultrasonic process 30min, take out, centrifugal (2000 revs/min, 5 minutes), get supernatant, with sherwood oil (60 ~ 90 DEG C) extraction 2 times, each 15ml, discard petroleum ether layer, water intaking layer adds 0.4ml watery hydrochloric acid, shake up, by extracted with diethyl ether 3 times, each 15ml, merge extracted with diethyl ether layer, reclaim ether to dry, residue adds methyl alcohol 2ml and dissolves, add silica gel 1g to mix thoroughly, volatilize solvent, put silicagel column (100 ~ 200 orders, 2g, internal diameter 1cm, dry column-packing) on, with methyl alcohol-methenyl choloride (1: 9) 40ml wash-out, collect methyl alcohol-methenyl choloride (1: 9) eluent, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, obtain.
The preparation of reference substance solution: it is appropriate that precision takes onocerin, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except reticulate millettia and makes negative sample in accordance with the law, then obtain negative sample solution according to need testing solution preparation method.
The selection of developping agent: 30: 1 ratios, 10: 1 ratios, the methenyl choloride-methyl alcohol of 40: 1 ratios is developping agent.
According to thin-layered chromatography test, draw onocerin reference substance solution 2 ~ 5 μ l, need testing solution 5 ~ 10 μ l respectively, put respectively on same silica gel g thin-layer plate, in above-mentioned developping agent condition, launch 12cm, take out, dry, inspect under being placed in ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.See figure (8-1).Be wherein that developping agent spot is the most clear with the methenyl choloride-methyl alcohol of 30: 1 ratios.
Embodiment 9
The root of herbaceous peony is differentiated
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize, gets about 0.08g, accurately weighed, put in 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic process (power 500W, frequency 30kHz) 5 minutes, be added in D101 type large pore resin absorption column (60 ~ 80 orders, internal diameter 6 ~ 8mm, height is 5cm about) on, with water 15ml wash-out, discard eluent, use methyl alcohol 8ml wash-out again, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, to obtain final product.
The preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 80% methyl alcohol and make the solution of every 1ml containing 20 μ g, to obtain final product.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except the root of herbaceous peony and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 40: 5: 10: 0.2 ratio respectively, 20: 3: 5: 0.2 ratio, the methenyl choloride-acetate-methanol-formic acid of 50: 7: 15: 0.2 ratio is developping agent.
Test according to thin-layered chromatography, draw need testing solution 20 μ l and Paeoniflorin reference substance solution 10 μ l in assay item respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray, with 5% vanillin-sulfuric acid solution, is heated to colour developing at 105 DEG C clear.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.See figure (9-1).Be wherein that developping agent spot is the most obvious with the methenyl choloride-acetate-methanol-formic acid of 40: 5: 10: 0.2 ratio.
Embodiment 10
Prepared rhizome of rehmannia is differentiated
The preparation of need testing solution: get this product 20g, add water 100ml, warm 30 minutes in 60 DEG C, take out, ultrasonic process 30 minutes, centrifugal, get supernatant, be extracted with ethyl acetate 2 times, each 30ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue adds ethyl acetate 5ml makes dissolving, adds neutral alumina 3g, mix thoroughly, load neutral alumina column (10g, internal diameter 15mm, 70 ~ 100 orders), with ethanol 30ml wash-out, collect eluent, evaporate to dryness, residue adds ethanol 1ml, make dissolving, as need testing solution.
The preparation of control medicinal material solution: get prepared rhizome of rehmannia control medicinal material 4g, the 50ml that adds water decocts 1 hour, filter, filtrate is extracted with ethyl acetate 2 times, each 30ml, combined ethyl acetate extract, evaporate to dryness, and residue adds methyl alcohol 1ml, makes dissolving, in contrast medicinal material solution.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except prepared rhizome of rehmannia and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 1: 1 ratio respectively, 0.5: 1 ratio, the toluene-ethyl acetate of 2: 1 ratios is developping agent.
According to thin-layered chromatography test, draw need testing solution 20 μ l and control medicinal material solution 10 μ l respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray with DNPH ethanolic solution.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.See figure (10-1).Be wherein that developping agent spot is the most obvious with the toluene-ethyl acetate of 1: 1 ratio.
Embodiment 11
Yncaria stem with hooks is differentiated
The preparation of need testing solution: get this product 10g, adds strong ammonia solution 10ml and makes infiltration, close plug 30 minutes, add ether 100ml, add hot reflux 1 hour, let cool, divide and get ether layer, extract 2 times with 5% hydrochloric acid solution jolting, each 20ml, merge 5% hydrochloric acid solution, by ammoniacal liquor adjust ph to 9, extract 2 times with ether jolting, each 20ml, merges ether solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.
The preparation of control medicinal material solution: hook taking rattan control medicinal material 2g, add strong ammonia solution 3ml and infiltrate, close plug 30 minutes, add diethyl ether 40ml, adds hot reflux 1 hour, let cool, and filter, filtrate evaporate to dryness, adds methyl alcohol 1ml and make dissolving, in contrast medicinal material solution.
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except yncaria stem with hooks and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method.
The selection of developping agent: prepare 8: 12: 2: 2: 0.5 ratio respectively, 6: 10: 1: 1: 0.5 ratio, the toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor of 10: 14: 3: 3: 0.8 ratio is developping agent.Test according to thin-layered chromatography, draw need testing solution and each 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide (1: 1) mixed solution, in test sample chromatogram, with on control medicinal material chromatogram relevant position, show the principal spot of same color.See figure (11-1).Be wherein that developping agent spot is the most obvious with the toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor of 8: 12: 2: 2: 0.5 ratio.
Embodiment 12
Chromatographic condition and system suitability test take octadecylsilane chemically bonded silica as filling agent; With isopropanol-methanol-5mmol/L aqueous solution of citric acid (2: 22: 78) for mobile phase; Determined wavelength is 240nm; Column temperature 30 DEG C.Theoretical cam curve calculates should be not less than 2000 by Paeoniflorin peak.
The preparation of reference substance solution is got Paeoniflorin reference substance and is about 10mg, accurately weighed, puts in 50ml volumetric flask, adds 70% methyl alcohol and dissolves and be diluted to scale, shake up.Precision measures above-mentioned Paeoniflorin reference substance solution 5ml again, puts in 50ml volumetric flask, adds 70% methanol dilution to scale, shakes up, to obtain final product.
It is appropriate that this product is got in the preparation of need testing solution, porphyrize, get about 0.08g, accurately weighed, put in 10ml volumetric flask, add 0.2% sodium bicarbonate solution 5ml, ultrasonic process (power 500W, frequency 30kHz) 10 minutes, be added in D101 type large pore resin absorption column (60 ~ 80 orders, internal diameter 6 ~ 8mm, height is 5cm about) on, with water 15ml wash-out, discard eluent, then use methyl alcohol 8ml wash-out, collect eluent and add water to scale in 10ml measuring bottle, shake up, cross 0.45 μm of miillpore filter, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
Embodiment 13
Chromatographic condition and system suitability test take octadecylsilane chemically bonded silica as filling agent; With isopropanol-methanol-5mmol/L aqueous solution of citric acid (4: 20: 80) for mobile phase; Determined wavelength is 230nm; Column temperature 30 DEG C.Theoretical cam curve calculates should be not less than 2000 by Paeoniflorin peak.
The preparation of reference substance solution is got Paeoniflorin reference substance and is about 10mg, accurately weighed, puts in 50ml volumetric flask, adds 70% methyl alcohol and dissolves and be diluted to scale, shake up.Precision measures above-mentioned Paeoniflorin reference substance solution 5ml again, puts in 50ml volumetric flask, adds 70% methanol dilution to scale, shakes up, to obtain final product.
It is appropriate that this product is got in the preparation of need testing solution, porphyrize, get about 0.08g, accurately weighed, put in 10ml volumetric flask, add 0.2% sodium bicarbonate solution 5ml, ultrasonic process (power 500W, frequency 30kHz) 10 minutes, be added in D101 type large pore resin absorption column (60 ~ 80 orders, internal diameter 6 ~ 8mm, height is 5cm about) on, with water 15ml wash-out, discard eluent, then use methyl alcohol 8ml wash-out, collect eluent and add water to scale in 10ml measuring bottle, shake up, cross 0.45 μm of miillpore filter, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
Claims (3)
1. a detection method for blood-nourishing and brain-refreshing granules, is characterized in that, comprises the discriminating to following composition: in selfheal, ursolic acid is differentiated; Corydalis tuber is differentiated; Cassia seed is differentiated; Radix Angelicae Sinensis and Ligusticum wallichii are differentiated; In selfheal, Rosmarinic acid is differentiated; In Ligusticum wallichii, alkaloid is differentiated; In Ligusticum wallichii, Levistilide A is differentiated; Reticulate millettia is differentiated; The root of herbaceous peony is differentiated; Prepared rhizome of rehmannia is differentiated; Yncaria stem with hooks is differentiated;
In described selfheal, ursolic acid is differentiated, step is as follows:
Get this product 0.5-2.0g, add watery hydrochloric acid 0.2-1.0ml and ether 10-30ml, add hot reflux 20-40 minute, most ether waved by extract, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, separately get ursolic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 0.1-1.0mg, product solution in contrast, test according to thin-layered chromatography, draw need testing solution 5-15 μ l, reference substance solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-chloroform-glacial acetic acid=15-25: 2-4: 5-9: 0.1-0.5 for developping agent, launch, take out, dry, spray is with 5-15% ethanol solution of sulfuric acid, colour developing is heated to clear at 100-120 DEG C, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color,
Described corydalis tuber is differentiated, step is as follows:
Get this product 0.5-1.5g, put in tool plug centrifuge tube, add 0.1-0.5mol/L sodium hydroxide solution 2-6ml, jolting makes dissolving, add ethyl acetate 5-10ml, shake well, centrifugal 2-5 minute, get upper solution, evaporate to dryness, residue adds methyl alcohol 1-2ml makes dissolving, as need testing solution, separately get corydalis tuber control medicinal material 0.5-2.0g, add ammonia solution 0.5-2.0ml, grinding, add methylene chloride 5-20ml, temperature leaching 1-3 hour, filter, filtrate is concentrated into 0.5-2.0ml, medicinal material solution in contrast, get tetrahydropalmatine reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.1-1.0mg, product solution in contrast, test according to thin-layered chromatography, draw need testing solution, control medicinal material solution, reference substance solution each 5-15 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-chloroform-methanol-strong ammonia solution=5-20: 6-10: 1-3: 0.2-0.5 for developping agent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color, on the position corresponding to reference substance chromatogram, the spot of aobvious same color, described cassia seed is differentiated, step is as follows:
Depend on pine torch control medicinal material 0.5-1.5g, add watery hydrochloric acid 0.5-1.0ml, ether 10-20ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate reclaims ether to dry, residue adds methyl alcohol 0.5-2.0ml respectively makes dissolving, as cassia seed control medicinal material solution, get Physcion again, Chrysophanol, archen and aurantio-obtusin reference substance, add absolute ethyl alcohol-ethyl acetate=1-4: 0.5-2 and make every 1ml respectively containing the solution of 0.5-1.0mg, as mixing reference substance solution, test according to thin-layered chromatography, draw " in selfheal ursolic acid " and differentiate need testing solution 10-20 μ l under item, control medicinal material solution 7-15 μ l and mixing reference substance solution 2-4 μ l, put respectively on same high-efficient silica gel G thin layer plate, be that sherwood oil-cyclohexane-ethyl acetate-formic acid=3-9: 10-14: 3-7: the 0.2-0.8 of 30 ~ 60 DEG C is for developping agent with boiling range, launch, take out, dry, be placed in smoked more than the 10min of ammonia steam, inspect under putting daylight, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color,
Described Radix Angelicae Sinensis and Ligusticum wallichii are differentiated, step is as follows:
Get Radix Angelicae Sinensis respectively, the each 0.5-2.0g of Ligusticum wallichii control medicinal material, add diethyl ether 10-30ml, water-bath backflow 0.5-2.0 hour, take out, let cool, filter, filtrate reclaims ether to dry, residue adds ethyl acetate 0.5-2.0ml respectively, 1.0-2.0ml makes dissolving, as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution, test according to thin-layered chromatography, draw " in selfheal ursolic acid " and differentiate need testing solution 5-12 μ l under item, Radix Angelicae Sinensis control medicinal material solution 5-15 μ l and Ligusticum wallichii control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate-formic acid=5-9: 0.5-1.5: 0.2-0.8 for developping agent, launch more than 8cm, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color,
In described selfheal, Rosmarinic acid is differentiated, step is as follows:
Get Rosmarinic acid reference substance, add Diluted Alcohol and make the solution of every 1ml containing 0.5-1.0mg, product solution in contrast, test according to thin-layered chromatography, draw " in selfheal ursolic acid " and differentiate need testing solution 1-5 μ l, reference substance solution 1-4 μ l under item, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate-formic acid=1-3: 5-6: 0.2-0.5 for developping agent, launch, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color;
In described Ligusticum wallichii, alkaloid is differentiated, step is as follows:
Get this product 5-20g, add ammoniacal liquor 3-8ml, and ether 30-50ml, close plug leaves standstill 30-90 minute, glass bar stirs evenly, water-bath backflow 20-40 minute, take out, let cool, extract reclaims ether to dry, residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, get Ligusticum wallichii control medicinal material 0.5-2.0g, add ammoniacal liquor 3-8ml, and ether 10-30ml, close plug leaves standstill 50-70 minute, water-bath backflow 20-40 minute, take out, let cool, filter, filtrate reclaims ether to dry, residue adds absolute ethyl alcohol 0.5-2.0ml makes dissolving, as Ligusticum wallichii control medicinal material solution, test according to thin-layered chromatography, draw Ligusticum wallichii control medicinal material solution 7-10 μ l respectively, need testing solution 7-12 μ l, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate=7-11: 0.5-2 for developping agent, launch, take out, dry, spray is to improve bismuth potassium iodide solution-iodine liquor kalii iodide=1-4: 0.5-1 mixed solution, inspect under being placed in daylight, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color,
In described Ligusticum wallichii, Levistilide A is differentiated, step is as follows:
Get this product 2-10g, add ether 20-60ml, water-bath backflow 20-60 minute, take out, let cool, extract reclaims ether to dry, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, separately get Levistilide A reference substance, add ethyl acetate and make the solution of every 1ml containing 0.2-1.0mg, as Levistilide A reference substance solution, test according to thin-layered chromatography, draw Levistilide A reference substance solution 2-6 μ l respectively, need testing solution 6-10 μ l, put respectively on same silica GF254 thin layer plate, with normal hexane-ethyl acetate=1-5: 0.5-2 for developping agent, launch, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color,
Described reticulate millettia is differentiated, step is as follows:
Get this product 5-20g, add 0.1-0.5% sodium hydroxide solution 20-50ml, shake up, ultrasonic process 20-40min, take out, centrifugal, get supernatant, with petroleum ether extraction 1-3 time that boiling range is 60 ~ 90 DEG C, each 10-20ml, discard petroleum ether layer, water intaking layer adds 0.3-0.6ml watery hydrochloric acid, shake up, by extracted with diethyl ether 1-3 time, each 10-20ml, merge extracted with diethyl ether layer, reclaim ether to dry, residue adds methyl alcohol 0.5-2.0ml and dissolves, add silica gel 1-2g to mix thoroughly, volatilize solvent, put on silicagel column, with methyl alcohol-methenyl choloride=0.5-1.5: 7-11, 30-50ml wash-out, collect methyl alcohol-methenyl choloride=0.5-1.5: 7-11 eluent, evaporate to dryness, residue adds methenyl choloride 0.5-1.5ml makes dissolving, as need testing solution, separately get onocerin appropriate, add methyl alcohol and make the solution of every 1ml containing 0.1-0.5mg, product solution in contrast, test according to thin-layered chromatography, draw onocerin reference substance solution 2-5 μ l respectively, need testing solution 5-10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol=10-40: 1 for developping agent, launch more than 8cm, take out, dry, inspect under being placed in ultraviolet lamp 254nm, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color,
The described root of herbaceous peony is differentiated, step is as follows:
Test according to thin-layered chromatography, draw need testing solution 20-30 μ l and Paeoniflorin reference substance solution 10-20 μ l respectively, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol-formic acid=20-50: 3-7: 5-15: 0.2 for developping agent, launch, take out, dry, spray with 3-5% vanillin-sulfuric acid solution, colour developing is heated to clear at 100-120 DEG C, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
Wherein, the preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize, get 0.06-0.10g, accurately weighed, put in 20-50ml beaker, add sodium bicarbonate solution 5ml, ultrasonic process, be added on D101 type large pore resin absorption column, use 10-20ml water elution, discard eluent, then use 6-10ml methanol-eluted fractions, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 4-6 minute, gets supernatant and obtains need testing solution;
Described prepared rhizome of rehmannia is differentiated, step is as follows:
Get this product 15-20g, add water 50-100ml, in 50-60 DEG C of warm 20-50 minute, take out, ultrasonic process 20-40 minute, centrifugal, get supernatant, be extracted with ethyl acetate 1-3 time, each 20-40ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue adds ethyl acetate 3-10ml makes dissolving, add neutral alumina 2-4g, mix thoroughly, load neutral alumina column, with ethanol 20-50ml wash-out, collect eluent, evaporate to dryness, residue adds ethanol 0.5-2.0ml, make dissolving, as need testing solution, separately get prepared rhizome of rehmannia control medicinal material 2-5g, the 30-80ml that adds water decocts 0.5-2.0 hour, filter, filtrate is extracted with ethyl acetate 1-3 time, each 20-40ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml, make dissolving, medicinal material solution in contrast, test according to thin-layered chromatography, draw need testing solution 20-25 μ l and control medicinal material solution 10-20 μ l respectively, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate=0.5-2: 1 for developping agent, launch, take out, dry, spray is with 2, 4-dinitrophenylhydrazine ethanolic solution, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color,
Described yncaria stem with hooks is differentiated, step is as follows:
Get this product 5-20g, add strong ammonia solution 5-15ml and make infiltration, close plug 20-40 minute, add ether 50-100ml, add hot reflux 0.5-2.0 hour, let cool, divide and get ether layer, extract 1-3 time with the jolting of 3-8% hydrochloric acid solution, each 10-30ml, merge 3-8% hydrochloric acid solution, by ammoniacal liquor adjust ph to 9, extract 1-3 time with ether jolting, each 10-30ml, merge ether solution, evaporate to dryness, residue adds methyl alcohol 0.5-2.0ml makes dissolving, as need testing solution, another hook taking rattan control medicinal material 1-3g, add strong ammonia solution 2-5ml to infiltrate, close plug 20-40 minute, add diethyl ether 20-50ml, add hot reflux 0.5-2.0 hour, let cool, filter, filtrate evaporate to dryness, add methyl alcohol 0.5-2.0ml and make dissolving, medicinal material solution in contrast, test according to thin-layered chromatography, draw need testing solution, control medicinal material solution each 5-20 μ l, put respectively on same silica gel g thin-layer plate, with toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor=6-10: 10-14: 1-3: 1-3: 0.5-0.8 for developping agent, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide=1-4: 0.5-1 mixed solution, in test sample chromatogram, with on control medicinal material chromatogram relevant position, the principal spot of aobvious same color.
2. detection method according to claim 1, is characterized in that, in described selfheal, ursolic acid is differentiated, step is as follows:
The preparation of need testing solution: get this product 2g, adds watery hydrochloric acid 1.0ml and ether 20ml, and add hot reflux 30 minutes, most ether waved by extract, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution,
The preparation of reference substance solution: get ursolic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast,
The selection of developping agent: prepare 20: 3: 7: 0.2 ratio respectively, 15: 2: 5: 0.1 ratio, the cyclohexane-ethyl acetate-chloroform-glacial acetic acid of 25: 4: 9: 0.5 ratio is developping agent,
According to thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, colour developing is heated to clear, in test sample chromatogram, on the position corresponding to reference substance chromatogram at 105 DEG C, the spot of aobvious same color is wherein that developping agent spot is the most obvious with the cyclohexane-ethyl acetate-chloroform-glacial acetic acid of 20: 3: 7: 0.2 ratio;
Described corydalis tuber is differentiated, step is as follows:
The preparation of need testing solution: get this product 1g, puts in tool plug centrifuge tube, and add 0.2mol/L sodium hydroxide solution 4ml, jolting makes dissolving, add ethyl acetate 5ml, shake well, centrifugal 2 minutes, get upper solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution
The preparation of control medicinal material solution: corydalis tuber control medicinal material 0.5g, adds ammonia solution 0.5ml, grinding, add methylene chloride 10ml, temperature leaching 2 hours, and filter, filtrate is concentrated into 1ml, in contrast medicinal material solution,
The preparation of reference substance solution: get tetrahydropalmatine reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast,
The selection of developping agent: prepare 15: 8: 2: 0.2 ratio respectively, 5: 6: 1: 0.2 ratio, the normal hexane-chloroform-methanol-strong ammonia solution of 20: 10: 3: 0.5 ratio is developping agent,
Test according to thin-layered chromatography, draw each 10 μ l of need testing solution, control medicinal material solution and reference substance solution, put respectively on same silica gel g thin-layer plate, launch under above-mentioned developping agent condition, take out, dry, spray is with rare bismuth potassium iodide test solution, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color; On the position corresponding to reference substance chromatogram, the spot of aobvious same color is wherein that developping agent spot is the most obvious with the normal hexane-chloroform-methanol-strong ammonia solution of 15: 8: 2: 0.2 ratio;
Described cassia seed is differentiated, step is as follows:
The preparation of need testing solution: get this product 2g, adds watery hydrochloric acid 1.0ml and ether 20ml, and add hot reflux 30 minutes, most ether waved by extract, and residue adds methyl alcohol 1ml makes dissolving, to obtain final product,
The preparation of blank solution: the blank pharmaceutical preparation getting scarce cassia seed, the preparation method according to need testing solution obtains blank solution,
The preparation of control medicinal material: depend on pine torch control medicinal material 1g, adds watery hydrochloric acid 0.5ml, ether 20ml, and water-bath refluxes 1 hour, takes out, lets cool, and filters, and filtrate reclaims ether to dry, and residue adds methyl alcohol 1ml respectively makes dissolving, to obtain final product,
The preparation of reference substance solution: get Chrysophanol reference substance, archen reference substance, Physcion reference substance and aurantio-obtusin, add absolute ethyl alcohol-ethyl acetate=2: 1 makes every 1ml respectively containing the solution of 0.5mg, product solution in contrast,
The selection of developping agent: prepare 6: 12: 5: 0.5 ratio respectively, the ratio of 3: 10: 3: 0.2, the boiling range of 9: 14: 7: 0.8 ratio is the sherwood oil-cyclohexane-ethyl acetate-formic acid of 30 ~ 60 DEG C is developping agent
Test according to thin-layered chromatography, draw need testing solution 10 μ l, control medicinal material solution 7 and mixing reference substance solution 2 μ l, put respectively on same high-efficient silica gel G thin layer plate, launch under above-mentioned developping agent condition, take out, dry, be placed in smoked more than the 10min of ammonia steam, inspect under putting daylight, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, the sherwood oil-cyclohexane-ethyl acetate-formic acid being wherein 30 ~ 60 DEG C with the boiling range of 6: 12: 5: 0.5 ratio is that developping agent spot is the most obvious;
Described Radix Angelicae Sinensis and Ligusticum wallichii are differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, adds watery hydrochloric acid 1ml and ether 20ml, mixing, and 60 DEG C of heating water baths reflux 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 0.5ml and dissolves, and to obtain final product,
The preparation of control medicinal material solution: get Radix Angelicae Sinensis control medicinal material, each 1g of Ligusticum wallichii control medicinal material respectively, add diethyl ether 20ml, and water-bath refluxes 1 hour, takes out, let cool, filter, filtrate reclaims ether to dry, residue adds ethyl acetate 1ml respectively makes dissolving, respectively as Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material solution
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Radix Angelicae Sinensis, Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 9: 1: 0.5 ratios respectively, 5: 0.5: 0.2 ratios, the normal hexane-ethyl acetate-formic acid of 9: 1.5: 0.8 ratios is developping agent,
According to thin-layered chromatography test, draw need testing solution 10 μ l, Radix Angelicae Sinensis control medicinal material solution 10 μ l and Ligusticum wallichii control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch more than 10cm, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color is wherein that developping agent spot is the most obvious with the normal hexane-ethyl acetate-formic acid of 9: 1: 0.5 ratios;
In described selfheal, Rosmarinic acid is differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 2g, adds watery hydrochloric acid 1ml and ether 20ml, mixing, and 60 DEG C of heating water baths reflux 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 0.5ml and dissolves, and to obtain final product,
The preparation of reference substance solution: get Rosmarinic acid reference substance, adds Diluted Alcohol and makes the solution of every 1ml containing 0.5mg, product solution in contrast,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except selfheal and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 2: 4: 0.2 ratios respectively, 1: 5: 0.2 ratio, the n-hexane-ethyl acetate-formic acid of 3: 6: 0.5 ratios is developping agent,
Test according to thin-layered chromatography, draw need testing solution 5 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color is wherein that developping agent spot is the most obvious with the n-hexane-ethyl acetate-formic acid of 2: 4: 0.2 ratios;
In described Ligusticum wallichii, alkaloid is differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, adds ammoniacal liquor 5ml, and ether 40ml, and close plug leaves standstill 60 minutes, and glass bar stirs evenly, and water-bath refluxes 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds absolute ethyl alcohol 1ml makes dissolving, to obtain final product,
The preparation of control medicinal material solution: get Ligusticum wallichii control medicinal material 1g, add ammoniacal liquor 5ml, and ether 20ml, close plug leaves standstill 60 minutes, and water-bath refluxes 30 minutes, take out, let cool, filter, filtrate reclaims ether to dry, residue adds absolute ethyl alcohol 1ml makes dissolving, to obtain final product, as Ligusticum wallichii control medicinal material solution
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 9: 1 ratios respectively, 7: 0.5 ratios, the cyclohexane-ethyl acetate of 11: 2 ratios is developping agent,
According to thin-layered chromatography test, draw Ligusticum wallichii control medicinal material solution 10 μ l, need testing solution 12 μ l respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is to improve bismuth potassium iodide solution-iodine liquor kalii iodide=1: 1 mixed solution, inspect under being placed in daylight, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color is wherein that developping agent spot is the most obvious with the cyclohexane-ethyl acetate of 9: 1 ratios;
In described Ligusticum wallichii, Levistilide A is differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 4g, adds ether 40ml, and water-bath refluxes 30 minutes, takes out, lets cool, and extract reclaims ether to dry, and residue adds methyl alcohol 1ml makes dissolving, to obtain final product,
The preparation of reference substance solution: get Levistilide A reference substance 20mg, is placed in the brown volumetric flask of 50ml, adds acetic acid ethyl dissolution and be settled to scale, shaking up, to obtain final product, as Levistilide A reference substance solution,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except Ligusticum wallichii and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 3: 1 ratios respectively, 1: 0.5 ratio, the normal hexane-ethyl acetate of 5: 2 ratios is developping agent,
Test according to thin-layered chromatography, draw Levistilide A reference substance solution 6 μ l, need testing solution 10 μ l respectively, put respectively on same silica GF254 thin layer plate, under above-mentioned developping agent condition, launch, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color is wherein that developping agent spot is the most obvious with the normal hexane-ethyl acetate of 3: 1 ratios;
Described reticulate millettia is differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules 10g, add 0.2% sodium hydroxide solution 30ml, shake up, ultrasonic process 30min, take out, centrifugal, get supernatant, with the petroleum ether extraction 2 times that boiling range is 60 ~ 90 DEG C, each 15ml, discard petroleum ether layer, water intaking layer adds 0.4ml watery hydrochloric acid, shake up, by extracted with diethyl ether 3 times, each 15ml, merge extracted with diethyl ether layer, reclaim ether to dry, residue adds methyl alcohol 2ml and dissolves, add silica gel 1g to mix thoroughly, volatilize solvent, put on silicagel column, with methyl alcohol-methenyl choloride=1: the 40ml mixed solution wash-out of 9, collect methyl alcohol-methenyl choloride=1: the eluent of 9, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, obtain,
The preparation of reference substance solution: it is appropriate that precision takes onocerin, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, product solution in contrast,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except reticulate millettia and makes negative sample in accordance with the law, then obtain negative sample solution according to need testing solution preparation method,
The selection of developping agent: 30: 1 ratios, 10: 1 ratios, the methenyl choloride-methyl alcohol of 40: 1 ratios is developping agent,
Test according to thin-layered chromatography, draw onocerin reference substance solution 2 ~ 5 μ l, need testing solution 5 ~ 10 μ l respectively, put respectively on same silica gel g thin-layer plate, in above-mentioned developping agent condition, launch 12cm, take out, dry, inspect under being placed in ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color is wherein that developping agent spot is the most clear with the methenyl choloride-methyl alcohol of 30: 1 ratios;
The described root of herbaceous peony is differentiated, step is as follows:
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize, gets 0.08g, accurately weighed, put in 20ml beaker, add 0.2% sodium bicarbonate solution 5ml, ultrasonic process 5 minutes, is added on D101 type large pore resin absorption column, with water 15ml wash-out, discard eluent, then use methyl alcohol 8ml wash-out, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 5 minutes, get supernatant, to obtain final product
The preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 80% methyl alcohol and make the solution of every 1ml containing 20 μ g, to obtain final product,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except the root of herbaceous peony and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 40: 5: 10: 0.2 ratio respectively, 20: 3: 5: 0.2 ratio, the methenyl choloride-ethyl acetate-methyl alcohol-formic acid of 50: 7: 15: 0.2 ratio is developping agent,
Test according to thin-layered chromatography, draw need testing solution 20 μ l and Paeoniflorin reference substance solution 10 μ l respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 5% vanillin-sulfuric acid solution, colour developing is heated to clear, in test sample chromatogram, on the position corresponding to reference substance chromatogram at 105 DEG C, the spot of aobvious same color is wherein that developping agent spot is the most obvious with the methenyl choloride-ethyl acetate-methyl alcohol-formic acid of 40: 5: 10: 0.2 ratio;
Described prepared rhizome of rehmannia is differentiated, step is as follows:
The preparation of need testing solution: get this product 20g, add water 100ml, warm 30 minutes in 60 DEG C, takes out, ultrasonic process 30 minutes, centrifugal, get supernatant, be extracted with ethyl acetate 2 times, each 30ml, combined ethyl acetate, acetic acid ethyl fluid evaporate to dryness, residue adds ethyl acetate 5ml makes dissolving, add neutral alumina 3g, mix thoroughly, load neutral alumina column, with ethanol 30ml wash-out, collect eluent, evaporate to dryness, residue adds ethanol 1ml, make dissolving, as need testing solution
The preparation of control medicinal material solution: get prepared rhizome of rehmannia control medicinal material 4g, the 50ml that adds water decocts 1 hour, filter, filtrate is extracted with ethyl acetate 2 times, each 30ml, combined ethyl acetate extract, evaporate to dryness, and residue adds methyl alcohol 1ml, makes dissolving, in contrast medicinal material solution,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except prepared rhizome of rehmannia and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 1: 1 ratio respectively, 0.5: 1 ratio, the toluene-ethyl acetate of 2: 1 ratios is developping agent,
According to thin-layered chromatography test, draw need testing solution 20 μ l and control medicinal material solution 10 μ l respectively, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, spray is with 2,4-dinitrophenylhydrazine ethanolic solution, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color is wherein that developping agent spot is the most obvious with the toluene-ethyl acetate of 1: 1 ratio;
Described yncaria stem with hooks is differentiated, step is as follows:
The preparation of need testing solution: get this product 10g, adds strong ammonia solution 10ml and makes infiltration, close plug 30 minutes, add ether 100ml, add hot reflux 1 hour, let cool, divide and get ether layer, extract 2 times with 5% hydrochloric acid solution jolting, each 20ml, merge 5% hydrochloric acid solution, by ammoniacal liquor adjust ph to 9, extract 2 times with ether jolting, each 20ml, merges ether solution, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution
The preparation of control medicinal material solution: hook taking rattan control medicinal material 2g, add strong ammonia solution 3ml and infiltrate, close plug 30 minutes, add diethyl ether 40ml, adds hot reflux 1 hour, let cool, and filter, filtrate evaporate to dryness, adds methyl alcohol 1ml and make dissolving, in contrast medicinal material solution,
The preparation of blank solution: by prescription proportioning, gets other medicinal materials except yncaria stem with hooks and makes negative sample in accordance with the law, then obtain blank solution according to need testing solution preparation method,
The selection of developping agent: prepare 8: 12: 2: 2: 0.5 ratio respectively, 6: 10: 1: 1: 0.5 ratio, the toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor of 10: 14: 3: 3: 0.8 ratio is developping agent,
Test according to thin-layered chromatography, draw need testing solution and each 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, under above-mentioned developping agent condition, launch, take out, dry, improvement bismuth potassium iodide solution-iodine liquor kalii iodide=1: the mixed solution of 1, in test sample chromatogram, with on control medicinal material chromatogram relevant position, the principal spot of aobvious same color is wherein that developping agent spot is the most obvious with the toluene-dichloromethane-ethyl acetate-methyl alcohol-ammoniacal liquor of 8: 12: 2: 2: 0.5 ratio.
3. detection method according to claim 1, is characterized in that, also comprises and carries out assay to the effective constituent Paeoniflorin in blood-nourishing and brain-refreshing granules, and content assaying method adopts high performance liquid chromatography, and step is as follows:
Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; With isopropanol-methanol-5mmol/L citric acid soln=2-4: 18-22: 78-80 for mobile phase; Determined wavelength is 230-240nm, and number of theoretical plate calculates should be not less than 2000 by Paeoniflorin peak,
The preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 70-90% methyl alcohol and make the solution of every 1ml containing 15-25 μ g, obtain reference substance solution;
The preparation of need testing solution: get blood-nourishing and brain-refreshing granules, porphyrize, gets 0.06-0.10g, accurately weighed, put in 20-50ml beaker, add sodium bicarbonate solution 5ml, ultrasonic process, is added on D101 type large pore resin absorption column, uses 10-20ml water elution, discard eluent, then use 6-10ml methanol-eluted fractions, collect eluent in 10ml measuring bottle, add water to scale, shake up, centrifugal 4-6 minute, get supernatant and obtain need testing solution;
Determination method: accurate absorption reference substance solution and need testing solution each 5-15 μ l respectively, injection liquid chromatography, obtains chromatogram, calculates the content of Paeoniflorin in blood-nourishing and brain-refreshing granules.
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| CN103149320B (en) * | 2013-04-01 | 2014-08-13 | 山西振东开元制药有限公司 | Quality control method of loins-strengthening and kidney-invigorating medicine |
| CN103969394A (en) * | 2014-04-24 | 2014-08-06 | 四川蜀源博业医药科技有限公司 | Method for testing quality of medicine for treating alzheimer's disease |
| CN106645044A (en) * | 2015-11-03 | 2017-05-10 | 天士力制药集团股份有限公司 | Determination method of neuron protection bioactivity of Yangxue Qingnao granule |
| CN105699585B (en) * | 2016-02-05 | 2018-05-08 | 四川德成动物保健品有限公司 | Detection method for glutinous rehmannia in qingwen baidu powder |
| CN106290697A (en) * | 2016-08-29 | 2017-01-04 | 贵州信邦制药股份有限公司 | The discrimination method of Rhizoma Chuanxiong in SHIQUAN DABU JIU |
| CN107917966B (en) * | 2016-10-08 | 2022-02-18 | 天士力医药集团股份有限公司 | Detection method of blood-nourishing brain-nourishing preparation |
| CN107422063A (en) * | 2017-08-01 | 2017-12-01 | 安徽九洲方圆制药有限公司 | A kind of angelica sinensis and prepared RADIX ANGELICAE SINENSIS with yellow rice wine medicine materical crude slice and its thin-layer identification method of granule |
| CN109613166B (en) * | 2019-01-16 | 2020-01-07 | 鲁南制药集团股份有限公司 | Quality detection method of 'Jihui Tongbiang' capsule |
| CN110057960A (en) * | 2019-05-07 | 2019-07-26 | 哈尔滨快好药业有限公司 | The quality and safety control method of the fleece-flower root in a kind of neck recovering capsule |
| CN111122735B (en) * | 2019-12-30 | 2022-12-27 | 贵州景峰注射剂有限公司 | Ligusticum wallichii medicine quality detection method |
| CN116298053B (en) * | 2023-03-24 | 2023-08-22 | 遵义市中医院 | Quality control method of ginger stomach-invigorating granule |
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