CN101672834A - Method for detecting quality of Chinese medicinal preparation for treating diabetic retinopathy - Google Patents
Method for detecting quality of Chinese medicinal preparation for treating diabetic retinopathy Download PDFInfo
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- CN101672834A CN101672834A CN 200810183368 CN200810183368A CN101672834A CN 101672834 A CN101672834 A CN 101672834A CN 200810183368 CN200810183368 CN 200810183368 CN 200810183368 A CN200810183368 A CN 200810183368A CN 101672834 A CN101672834 A CN 101672834A
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- methyl alcohol
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- ethanol
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- 238000002360 preparation method Methods 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 39
- 206010012689 Diabetic retinopathy Diseases 0.000 title claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 134
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 125
- 239000000463 material Substances 0.000 claims abstract description 47
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 39
- RXUWDKBZZLIASQ-UHFFFAOYSA-N Puerarin Natural products OCC1OC(Oc2c(O)cc(O)c3C(=O)C(=COc23)c4ccc(O)cc4)C(O)C(O)C1O RXUWDKBZZLIASQ-UHFFFAOYSA-N 0.000 claims abstract description 25
- HKEAFJYKMMKDOR-VPRICQMDSA-N puerarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 HKEAFJYKMMKDOR-VPRICQMDSA-N 0.000 claims abstract description 25
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 241000405414 Rehmannia Species 0.000 claims abstract description 13
- 235000015468 Lycium chinense Nutrition 0.000 claims abstract description 12
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 12
- 244000037364 Cinnamomum aromaticum Species 0.000 claims abstract description 11
- 235000014489 Cinnamomum aromaticum Nutrition 0.000 claims abstract description 11
- 238000005251 capillar electrophoresis Methods 0.000 claims abstract description 11
- 239000009636 Huang Qi Substances 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 172
- 238000012360 testing method Methods 0.000 claims description 112
- 239000013558 reference substance Substances 0.000 claims description 59
- 239000000284 extract Substances 0.000 claims description 38
- 239000000047 product Substances 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000000706 filtrate Substances 0.000 claims description 24
- 239000003795 chemical substances by application Substances 0.000 claims description 20
- 238000004587 chromatography analysis Methods 0.000 claims description 20
- 239000000741 silica gel Substances 0.000 claims description 20
- 229910002027 silica gel Inorganic materials 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 16
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 16
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 14
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 244000241872 Lycium chinense Species 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 10
- 239000007921 spray Substances 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- 241000545744 Hirudinea Species 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000010992 reflux Methods 0.000 claims description 8
- 229910021529 ammonia Inorganic materials 0.000 claims description 7
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000012085 test solution Substances 0.000 claims description 7
- 238000003556 assay Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000009472 formulation Methods 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 claims description 6
- 238000002137 ultrasound extraction Methods 0.000 claims description 6
- YEZWWUMWIFKEQM-UHFFFAOYSA-N O.OC.ClC(Cl)Cl.CCOC(C)=O Chemical compound O.OC.ClC(Cl)Cl.CCOC(C)=O YEZWWUMWIFKEQM-UHFFFAOYSA-N 0.000 claims description 5
- DLUOMMXONAIKQD-UHFFFAOYSA-N OC=O.ClC(Cl)Cl.CCOC(C)=O Chemical compound OC=O.ClC(Cl)Cl.CCOC(C)=O DLUOMMXONAIKQD-UHFFFAOYSA-N 0.000 claims description 5
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims description 5
- 235000011613 Pinus brutia Nutrition 0.000 claims description 5
- 241000018646 Pinus brutia Species 0.000 claims description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 5
- CZCSLHYZEQSUNV-UHFFFAOYSA-N [Na].OB(O)O Chemical compound [Na].OB(O)O CZCSLHYZEQSUNV-UHFFFAOYSA-N 0.000 claims description 5
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 claims description 5
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 claims description 5
- 229910021538 borax Inorganic materials 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Substances OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 239000012982 microporous membrane Substances 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 239000011591 potassium Substances 0.000 claims description 5
- 239000013643 reference control Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 239000004328 sodium tetraborate Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 244000046146 Pueraria lobata Species 0.000 claims description 4
- 235000010575 Pueraria lobata Nutrition 0.000 claims description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 4
- 239000000469 ethanolic extract Substances 0.000 claims description 4
- 238000011003 system suitability test Methods 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 101800000263 Acidic protein Proteins 0.000 claims description 2
- QIIDATRCGITYRZ-UHFFFAOYSA-N Catalpol Natural products OCC1OC(OC2OC=CC3C(O)C(=C(CO)C23)O)C(O)C(O)C1O QIIDATRCGITYRZ-UHFFFAOYSA-N 0.000 claims description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims description 2
- 240000001398 Typha domingensis Species 0.000 claims description 2
- 239000003957 anion exchange resin Substances 0.000 claims description 2
- LHDWRKICQLTVDL-PZYDOOQISA-N catalpol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@@H]2[C@@]3(CO)O[C@H]3[C@@H](O)[C@@H]2C=CO1 LHDWRKICQLTVDL-PZYDOOQISA-N 0.000 claims description 2
- UXSACQOOWZMGSE-UHFFFAOYSA-N catalposide Natural products OC1C(O)C(O)C(CO)OC1OC1C2C3(CO)OC3C(OC(=O)C=3C=CC(O)=CC=3)C2C=CO1 UXSACQOOWZMGSE-UHFFFAOYSA-N 0.000 claims description 2
- HXOLFXRMWWHLMH-UHFFFAOYSA-L disodium boric acid carbonate Chemical compound [Na+].[Na+].OB(O)O.[O-]C([O-])=O HXOLFXRMWWHLMH-UHFFFAOYSA-L 0.000 claims description 2
- 239000000796 flavoring agent Substances 0.000 claims description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 claims description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hcl hcl Chemical compound Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 2
- JWHHANVGNNWIRI-UHFFFAOYSA-N methanol phosphoric acid hydrate Chemical compound O.OC.OP(O)(O)=O JWHHANVGNNWIRI-UHFFFAOYSA-N 0.000 claims description 2
- LHDWRKICQLTVDL-UHFFFAOYSA-N methyl iridoid glycoside Natural products OC1C(O)C(O)C(CO)OC1OC1C2C3(CO)OC3C(O)C2C=CO1 LHDWRKICQLTVDL-UHFFFAOYSA-N 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- ZEFCBWQRHWLUTC-UHFFFAOYSA-M sodium;2,3-dihydroxybutanedioic acid;2,3,4-trihydroxy-4-oxobutanoate Chemical compound [Na+].OC(=O)C(O)C(O)C(O)=O.OC(=O)C(O)C(O)C([O-])=O ZEFCBWQRHWLUTC-UHFFFAOYSA-M 0.000 claims description 2
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 claims description 2
- 239000001117 sulphuric acid Substances 0.000 claims description 2
- 235000011149 sulphuric acid Nutrition 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims 2
- 239000012141 concentrate Substances 0.000 claims 1
- 238000001035 drying Methods 0.000 claims 1
- 235000019634 flavors Nutrition 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 244000241838 Lycium barbarum Species 0.000 abstract 1
- 235000015459 Lycium barbarum Nutrition 0.000 abstract 1
- 239000004480 active ingredient Substances 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 238000002203 pretreatment Methods 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 235000019441 ethanol Nutrition 0.000 description 31
- 239000000523 sample Substances 0.000 description 29
- 210000004185 liver Anatomy 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 5
- 241000201295 Euphrasia Species 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000035922 thirst Effects 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
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- 230000002040 relaxant effect Effects 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
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- 206010013954 Dysphoria Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000001431 Psychomotor Agitation Diseases 0.000 description 1
- 241000219780 Pueraria Species 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 description 1
- 208000009205 Tinnitus Diseases 0.000 description 1
- 206010047513 Vision blurred Diseases 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 206010029410 night sweats Diseases 0.000 description 1
- 230000036565 night sweats Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical compound C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 1
- 235000021286 stilbenes Nutrition 0.000 description 1
- 231100000886 tinnitus Toxicity 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for detecting the quality of Chinese medicinal preparation for treating diabetic retinopathy. The method comprises the following steps: carrying out pretreatments ofa sample including extraction, concentration, filtration, defatting, and the like by using reagents such as methanol, ethanol, aether and ethyl acetate; then, carrying out thin-layer identification ofa plurality of medicinal materials such as radix astragali, rehmannia root, barbary wolfberry fruit and cassia seed in the sample and particularly carrying out identification of bloodsuckers by meansof an efficient capillary electrophoresis fingerprint chromatogram; and measuring the content of an active ingredient, puerarin in the preparation by means of an HPLC method,. The method provides reliable guarantee for stable and controllable quality and stable and exact therapeutic effect of products.
Description
Technical field
The present invention relates to a kind of quality determining method, particularly relate to a kind of quality determining method of Chinese medicine six class compound preparations, belong to the field of Chinese medicines.
Background technology
The bright particle of stilbene is to have the modern preparation of Chinese medicine that nourishing generate fluid, nourishing the liver and kidney, vein relaxing make eye bright, be used for the treatment of DRP, belong to deficiency of qi and yin, kidney deficiency and liver, obstruction of collaterals by blood stasis person, card is seen blurring of vision, dryness with foreign body sensation in the eyes, spiritlessness and weakness, soreness and weakness of waist and knees, dysphoria in chestpalms-soles, night sweat, thirst and liking drink, dizziness and tinnitus, constipation etc.Radix Astragali function tonifying middle-Jiao and Qi in this Chinese medicine is the key medicine for the treatment of diabete at all times; Pueraria root functional is analgesic to promote the production of body fluid, relieving restlessness is quenched the thirst, but elevate a turnable ladder yang-energy also promotes to reach on the body fluid and makes eye bright; Fruit of Chinese wolfberry replenishing the vital essence and the blood, benefiting shrewd head, the moon of auxiliary root of kudzu vine nourishing the liver and kidney; The glutinous rehmannia nourishing yin and nourishing blood is successive dynasties treatment deficiency of Yin blood-head and hemorrhage, the key medicine of quenching one's thirst etc.; Cassia seed clears liver and improve vision, and auxiliary root of kudzu vine benefit the moon purges heat, the energy nourishing the liver and kidney of holding concurrently; Semen Leonuri makes eye bright beneficial smart, can assist a ruler in governing a country root of kudzu vine moistening lung and promote the production of body fluid to quench thirst, and helps Radix Astragali tonifying middle-Jiao and Qi: cattail pollen energy promoting blood circulation and hemostasis, leech cures mainly the disease of all extravasated blood.All medicines share, the interpretation of the cause, onset and process of an illness of link closely DRP caused by liver and kidney deficiency, eyes losing nutrition, and the merit that concurrence nourishing generate fluid, nourishing the liver and kidney, vein relaxing make eye bright is the active drug of DRP.For promoting the Chinese medicine preparation modernization, it is necessary that Chinese medicine preparation is constantly studied quality determining method.
Summary of the invention
The object of the invention is to provide a kind of Chinese medicine six class compound preparation quality determining methods.The present invention seeks to be achieved through the following technical solutions.
The quality determining method of compound preparation of the present invention comprises one or more of following discriminating and/or quality determination:
Differentiate: a, get solid pharmaceutical preparation 1.5-3g, add ethanol 20-60ml, sonicated 10-40 minute, filter, filtrate hydro-oxidation potassium 0.5-1.5g makes dissolving, water-bath backflow 1-3 hour, evaporate to dryness, residue adds water 10-40ml makes dissolving, extracts the extract evaporate to dryness with normal butyl alcohol 10-30ml, add the D101 macroporous resin column of having handled well on the water 1-5ml dissolving back, water 50-120ml successively, 10%7 pure 30-80ml, 30% ethanol 30-80ml wash-out, eluent discards, and uses 70% ethanol 30-100ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol 0.5-3ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, product solution in contrast, test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 10 μ l of above-mentioned two solution, putting respectively on same silica gel g thin-layer plate, is developping agent with chloroform-methanol-water=45-65: 20-40: 5-15 in lower floor's solution of placing 12 hours below 10 ℃, launches, take out, dry.Spray is with the 5%-10% ethanol solution of sulfuric acid, and 105 ℃ of bakings are clear to developing the color, and inspect under the daylight.In the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color;
B, get solid pharmaceutical preparation 3-5g, add diethyl ether 30-60ml or methyl alcohol 10-40ml, sonicated 10-40 minute, filter, filtrate evaporate to dryness, residue add methyl alcohol 0.5-1.5ml and make need testing solution A; Or add water 5-15ml dissolving, on macroreticular resin and the anion exchange resins handled well, use Different concentrations of alcohol and water elution respectively, collect eluent, evaporated under reduced pressure adds ethanol 5-15ml dissolving as need testing solution B; Other gets the glutinous rehmannia control medicinal material, adds methyl alcohol and is prepared into the solution that every 1ml contains 0.5mg, in contrast the product solution A; Or get the Catalpol reference substance, add the solution that methyl alcohol is prepared into 0.5mg/ml, in contrast the product solution B.Test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 10 μ l of reference substance solution A and need testing solution A, put respectively on same silica gel g thin-layer plate, make into strips, with chloroform-ethyl acetate-methanol-water=10-20: 30-50: 18-25: 5-15 or lower floor's solution of 12 hours of placement below chloroform-methanol-dense ammonia=12-20: 3-6: 0.5-1.510 ℃ is developping agent, launch, take out, dry, spray is with Goden test solution or anisaldehyde-concentrated sulphuric acid test solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance chromatogram relevant position on show the spot of same color;
C, get solid pharmaceutical preparation 0.5-1.5g, add water 20-40ml, heated and boiled 15 minutes is put coldly, filters, and filtrate is extracted with ethyl acetate 10-30ml jolting, and extract is concentrated into about 0.5-1.5ml, as need testing solution; Other gets fruit of Chinese wolfberry control medicinal material 0.5g, shine medicinal material solution in pairs with legal system, test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), drawing each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, is developping agent with ethyl acetate-chloroform-formic acid=1-5: 1-5: 0.5-1.5 or ether-glacial acetic acid-methyl alcohol=3-8: 0.5-1.5: 0.5-1.5, launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material chromatogram relevant position on show the fluorescence principal spot of same color;
D, get solid pharmaceutical preparation 1-3g, add methyl alcohol 10-30ml, sonicated 20-50 minute, filter, filtrate evaporate to dryness, residue added water 5-15ml and hydrochloric acid 0.5-1.5ml reflux 30 minutes, immediately cooling, with twice of extracted by ether, each 10-30ml merges ether solution, volatilizes, residue adds methyl alcohol 0.5-1.5ml makes dissolving, as need testing solution; Other depends on pine torch control medicinal material 1g, with method preparation, as cassia seed control medicinal material solution, gets the archen reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution; According to thin-layered chromatography, draw each 1-2 μ l of above-mentioned three solution, put respectively on same silica gel H-0.5%CMC thin layer plate, upper solution with sherwood oil (30-60 ℃)-ethyl formate-formic acid=10-20: 3-8: 0.5-1.5 is a developping agent, launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with reference substance and control medicinal material chromatogram relevant position on show identical fluorescent orange spot; Put in the ammonia steam smoked after, spot becomes redness;
E, take by weighing solid pharmaceutical preparation 2-5g, add the 10-50ml extract, extract comprises one or more in acidic protein extract, basic protein extract, neutral protein extract, the 0.9%NaCl extract, extract 20-60min, extracting method be ultrasonic or grind in one or both, leave standstill, supernatant is through 2000-5000r/min centrifuging 20-50min, behind 0.45 μ m filtering with microporous membrane, as need testing solution.Other leech control medicinal material 0.5g that fetches water makes reference substance solution with method.Measure according to capillary electrophoresis (2005 editions appendix VIF of Chinese Pharmacopoeia), electrophoretic buffer is one or more in boric acid-sodium chloride-borax system, sodium dihydrogen phosphate-citric acid system, boric acid-sodium carbonate system, glycocoll+sodium chloride-hydrochloric acid system, the tartrate-sodium tartrate system, the pH value of buffer system is 8.0-9.5, and the electrophoretic separation time is 10-40 minute.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, identical HPCE peak appears, the relative effective time of each major component is identical;
Assay: measure according to high performance liquid chromatography (2005 editions appendix VID of Chinese Pharmacopoeia); Chromatographic condition and system suitability test, with the octadecylsilane chemically bonded silica is filling agent, and methyl alcohol-0.1% citric acid soln=20-30: 70-80 or methanol-water-chloroform=20-30: 70-80: 0.5-1.5 or methanol-water-phosphoric acid=270-290: 710-730: 0.10-0.20 is a moving phase; The detection wavelength is 240-250nm, and number of theoretical plate calculates by Puerarin should be not less than 4000; The preparation of reference substance solution: it is an amount of that precision takes by weighing the Puerarin reference substance, and add 30% ethanol and make the solution that every 1ml contains 80 μ g, both; The preparation of need testing solution: get solid pharmaceutical preparation 0.1-0.3, porphyrize, the methyl alcohol or the ethanol extract that add 10%-50%, ultrasonic 20-50 minute, the neutral alumina post of having handled well on the extract, the 30-70% ethanol elution is collected the eluent evaporate to dryness, be dissolved in 30% ethanolic solution of 10-80ml, promptly; Determination method: accurate respectively reference substance and the need testing solution drawn, inject liquid chromatograph, measure, promptly.
The quality determining method of herbal mixture of the present invention be preferably as follows differentiate and or quality determination in one or more.
Differentiate: a, get this product 3g, add ethanol 40ml, sonicated 30 minutes, filter, filtrate hydro-oxidation potassium 1g makes dissolving, and water-bath refluxed 2 hours, evaporate to dryness, residue adds water 20ml makes dissolving, extracts the extract evaporate to dryness with normal butyl alcohol 20ml, add the D101 macroporous resin column of having handled well on the water 2ml dissolving back, water 100ml, 10% ethanol 60ml, 30% ethanol 60ml wash-out successively, eluent discards, and uses 70% ethanol 80ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, product solution in contrast, test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 10 μ l of above-mentioned two solution, putting respectively on same silica gel g thin-layer plate, is developping agent with chloroform-methanol-water=65: 30: 10 in lower floor's solution of placing 12 hours below 10 ℃, launches, take out, dry.Spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ of bakings are clear to developing the color, and inspect under the daylight.In the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color.B, get this product 4.5g, the 50ml that adds diethyl ether, sonicated 30 minutes filters, and filtrate evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the glutinous rehmannia control medicinal material, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution, test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 10 μ l of above-mentioned two solution, put on same silica gel g thin-layer plate respectively, make into strips, with chloroform-ethyl acetate-methanol-water=15: 40: 22: lower floor's solution of placing 12 hours below 10 10 ℃ was developping agent, launch, take out, dry, spray is with the anisaldehyde test solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance chromatogram relevant position on show the spot of same color.
C, get this product 0.5g, add water 35ml, heated and boiled 15 minutes is put coldly, filters, and filtrate is extracted with ethyl acetate 15ml jolting, and extract is concentrated into about 1ml, as need testing solution; Other gets fruit of Chinese wolfberry control medicinal material 0.5g, shine medicinal material solution in pairs with legal system, test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), drawing each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, is developping agent with ethyl acetate-chloroform-formic acid (3: 2: 1), launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material chromatogram relevant position on show the fluorescence principal spot of same color.D, get this product 2g, add methyl alcohol 20ml, sonicated 30 minutes filters the filtrate evaporate to dryness, residue added water 10ml and hydrochloric acid 1ml reflux 30 minutes, cooling immediately, and with extracted by ether twice, each 20ml, merge ether solution, volatilize, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other depends on pine torch control medicinal material 1g, with method preparation, as cassia seed control medicinal material solution, gets the archen reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin-layered chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 1-2 μ l of above-mentioned three solution, put respectively on same silica gel H-0.5%CMC thin layer plate, upper solution with sherwood oil (30-60 ℃)-ethyl formate-formic acid (15: 5: 1) is a developping agent, launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with reference substance and control medicinal material chromatogram relevant position on show identical fluorescent orange spot; Put in the ammonia steam smoked after, spot becomes redness.
E, get this product 3g, add in the 30ml0.9%NaCl extract, ultrasonic Extraction 30min leaves standstill, and supernatant is through 2000-5000r/min centrifuging 25min, behind 0.45 μ m filtering with microporous membrane, as need testing solution.Other leech control medicinal material 0.5g that fetches water makes reference substance solution with method.Measuring according to capillary electrophoresis (2005 editions appendix VIF of Chinese Pharmacopoeia), is electrophoretic buffer with boric acid-sodium chloride-borax system, and the pH regulator of buffer system is 8.98, and the electrophoretic separation time is 30 minutes.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, identical HPCE peak appears, the relative effective time of each major component is identical;
Assay: measure according to high performance liquid chromatography (2005 editions appendix VID of Chinese Pharmacopoeia), chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica, and methyl alcohol-0.1% citric acid soln=25-75 is a moving phase; The detection wavelength is 250nm, and number of theoretical plate calculates by Puerarin should be not less than 4000; The preparation of reference substance solution: it is an amount of that precision takes by weighing the Puerarin reference substance, adds 30% ethanol and make the solution that every 1ml contains 80 μ g, promptly; The preparation of need testing solution: get solid pharmaceutical preparation 0.1g, porphyrize, add 10ml 30% ethanol extract, ultrasonic 30 minutes, the neutral alumina post of having handled well on the extract, 50% ethanol elution, collect the eluent evaporate to dryness, be dissolved in 30% ethanolic solution of 20ml, shake up, filter, get subsequent filtrate, promptly; Determination method: accurate respectively reference substance and the need testing solution 10 μ l of drawing, inject liquid chromatograph, measure, promptly.Unit formulation contains puerarin content and must not be less than 0.64% in the solid pharmaceutical preparation.
Beneficial effect of the present invention: the present invention measures project by test of many times has been worked out the multi-flavor medicinal material in the quality standard of this kind discriminating project and content of puerarin.By setting up strong discriminating of clear and definite specificity and the good content assaying method of reappearance, stability and precision, can effectively control the quality of product, make said preparation reach stable, controlled, efficiently reach safety, be strictness control to the feed intake requirement and the quality of product, be to guarantee the product curative effect, satisfy the effective ways of needs of patients better.
Embodiment:
The discriminating of 1 Radix Astragali
This product 3g is got in the preparation of need testing solution, porphyrize adds ethanol 40ml, sonicated 30 minutes, filter, filtrate hydro-oxidation potassium 1g makes dissolving, and water-bath refluxed 2 hours, evaporate to dryness, residue adds water 20ml makes dissolving, extract with normal butyl alcohol 20ml, the extract evaporate to dryness adds D101 macroporous resin column (the internal diameter 1.5cm that has handled well on the water 2ml dissolving back, long 12cm), water 100ml, 10% ethanol 60ml, 30% ethanol 60ml wash-out successively, eluent discards, and uses 70% ethanol 80ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution.
The preparation of negative sample solution: do not contain the sample of the Radix Astragali by preparation process preparation, prepare equally with the preparation method of need testing solution, as negative sample solution.
The preparation of reference substance solution: get the Astragaloside IV reference substance and add methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution.
According to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, be developping agent with chloroform-methanol-water (65: 30: 10) in lower floor's solution of placing 12 hours below 10 ℃, launch, take out, dry.Spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ of bakings are clear to developing the color, and inspect under the daylight.In the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color.
The discriminating of 2 glutinous rehmannia
The need testing solution preparation: get this product 4.5g, the 50ml that adds diethyl ether, sonicated 30 minutes filters, and filtrate evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution.
Glutinous rehmannia control medicinal material formulations prepared from solutions: get glutinous rehmannia control medicinal material 2g, the 50ml that adds diethyl ether, sonicated 30 minutes filters, and filtrate evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as glutinous rehmannia control medicinal material solution.
The preparation of glutinous rehmannia negative sample: get the preparation 4.5g that lacks glutinous rehmannia, the preparation method prepares equally by need testing solution, promptly gets negative sample solution.
Test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, make into strips, (15: 40: 22: 10) lower floor's solution of placing 12 hours below 10 ℃ was developping agent, launched with chloroform-ethyl acetate-methanol-water, take out, dry, spray is with the anisaldehyde test solution, and it is clear to be heated to spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance chromatogram relevant position on show the spot of same color.
The discriminating of 3 fruits of Chinese wolfberry
Need testing solution preparation: get this product 0.5g, add water 35ml, heated and boiled 15 minutes is put coldly, filters, and filtrate is extracted with ethyl acetate 15ml jolting, and extract is concentrated into about 1ml, as need testing solution
Fruit of Chinese wolfberry control medicinal material formulations prepared from solutions: get fruit of Chinese wolfberry control medicinal material 0.5g, make control medicinal material solution by the need testing solution preparation method
Fruit of Chinese wolfberry negative sample formulations prepared from solutions: get the preparation 2g that lacks the fruit of Chinese wolfberry, the preparation method prepares with method by need testing solution, promptly gets negative sample solution.
According to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, be developping agent with ethyl acetate-chloroform-formic acid (3: 2: 1), launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material chromatogram relevant position on show the fluorescence principal spot of same color
The thin layer of 4 cassia seeds is differentiated
Need testing solution preparation: get this product 2g, add methyl alcohol 20ml, sonicated 30 minutes, filter, filtrate evaporate to dryness, residue added water 10ml and hydrochloric acid 1ml reflux 30 minutes, immediately cooling, with twice of extracted by ether, each 20ml merges ether solution, volatilizes, residue adds methyl alcohol 1ml makes dissolving, as need testing solution
Reference substance solution preparation: get the archen reference substance and add methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution
Cassia seed control medicinal material formulations prepared from solutions: depend on that the pine torch control medicinal material is an amount of, porphyrize takes by weighing 1g, presses need testing solution preparation method preparation.
Cassia seed negative controls: get the preparation 2g that lacks cassia seed, be equipped with method by need testing solution preparation method system, promptly.
According to the thin-layered chromatography test, draw each 1-2 μ l of above-mentioned three solution, put respectively on same silica gel H-0.5%CMC thin layer plate, upper solution with sherwood oil (30-60 ℃)-ethyl formate-formic acid (15: 5: 1) is a developping agent, launches, and takes out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with reference substance and control medicinal material chromatogram relevant position on show identical fluorescent orange spot; Put in the ammonia steam smoked after, spot becomes redness.
The discriminating of 5 leech
The preparation of need testing solution: get this product 3g, add in the 30ml0.9%NaCl extract, ultrasonic Extraction 30min leaves standstill, and supernatant is through 2000-5000r/min centrifuging 25min, behind 0.45 μ m filtering with microporous membrane, as need testing solution.
The preparation of leech control medicinal material solution: take by weighing leech control medicinal material 0.5g, the preparation method prepares with method by need testing solution, as need testing solution.
Measuring according to capillary electrophoresis (2005 editions appendix VIF of Chinese Pharmacopoeia), is electrophoretic buffer with boric acid-sodium chloride-borax system, and the pH regulator of buffer system is 8.98, and the electrophoretic separation time is 30 minutes.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, identical HPCE peak appears, the relative effective time of each major component is identical.
6HPLC measures content of puerarin
(1) test of chromatographic condition and system suitability is a filling agent with octadecylsilane chemically bonded silica; Methyl alcohol-0.1% citric acid soln (25: 75) is a moving phase; The detection wavelength is 250nm.Number of theoretical plate calculates by puerarin peak should be not less than 4000.
(2) the preparation precision of reference substance solution takes by weighing Puerarin reference substance 100mg, puts in the 25ml measuring bottle, adds 30% ethanol to scale, shakes up, and precision is measured 2ml, puts in the 10ml measuring bottle, adds 30% ethanol to scale, shakes up, and promptly gets (containing Puerarin 80ug among every 1ml).
(3) selection of need testing solution extraction conditions
According to the literature, the Puerarin extracting method mainly contains ultrasonic and refluxes two kinds.Heating reflux method length consuming time, it is more to extract impurity; Ultrasonic extraction is to utilize hyperacoustic wave effect, mechanical crushing and cavitation, and it is less to extract impurity, and weak point consuming time, easy and simple to handle, and the extraction ratio no significant difference of heating reflux method and ultrasonic extraction.Thereby we determine to adopt ultrasonic extraction to handle sample.
The A test sample extracts choice of Solvent
This compound preparation selects methyl alcohol, 50% methyl alcohol, 30% ethanol, 95% ethanol, absolute ethyl alcohol to compare as extracting solvent according to the character of Puerarin, the results are shown in Table 1.
Table 1 test sample extracts choice of Solvent table as a result
Hence one can see that, adopts 30% ethanol as extracting the solvent content of puerarin for the highest, so the extraction solvent of 30% ethanol as sample adopted in this test
Determining of B extraction time
After the extraction solvent is determined, further investigate extraction time, the results are shown in Table 2.
Table 2 test sample extraction time selection result table
This shows that the extraction effect of 30 minutes and 40 minutes is suitable, so selected get final product in 30 minutes for extraction time.
According to " extraction choice of Solvent " and " determining of extraction time " test, we determine that optimum extraction condition is for adding 30% ethanol, Extraction by Ultrasound 30 minutes.
The preparation of C need testing solution
Get this product 4.5g, porphyrize is got 0.3g, accurate claims surely, adds 30% ethanol 30ml, puts in the tool plug conical flask, claims decide weight, and sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with 30% ethanol, shakes up.Precision is measured 5ml, puts in the 10ml measuring bottle,, add 30% ethanol to scale, shake up, filter, get subsequent filtrate, promptly.
(4) methodological study
The accurate absorption of A linear dependence investigation reference substance solution 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, 6.0ml, 7.0ml put in the 10ml measuring bottle, add 30% ethanol dilution to scale, shake up, mass concentration be respectively 8.0,16.0,24.0,32.0,40.0,48.0, the reference substance solution of 56.0ug/ml, each accurate 20ul of absorption injects liquid chromatograph, the record chromatogram.With reference substance concentration is horizontal ordinate, and the peak area integrated value is an ordinate, the drawing standard curve, and calculating regression equation is A=71327C-11860 r=0.9997, the results are shown in Table 3.
Table 3 linear dependence is investigated
The result shows that the concentration of Puerarin in 8.0～56.0ug/ml scope and peak area are good dominance relation.
The accurate need testing solution 20 μ l that draw of B precision test repeat sample introduction and measure puerarin content 6 times, and the calculating relative standard deviation is RSD=0.80%, the results are shown in Table 4.
Table 4 Precision test result
The C stability test is got respectively with a need testing solution 20 μ l, measures its content at regular intervals by above-mentioned chromatographic condition, investigates the stability of sample solution, calculates relative standard deviation, the results are shown in Table 5.
Table 5 sample stability test findings
More than experiment shows that content of puerarin is basicly stable in 8 hours in the need testing solution.
D reappearance test precision takes by weighing 6 parts in sample, makes need testing solution as stated above, and by measuring peak area with the chromatographic condition of drafting, calculating relative standard deviation is 0.91%, sees Table 6
Table 6 reproducible test results
The E recovery test
Adopt the application of sample absorption method, get five parts of the Chinese medicine preparations of known content, it is an amount of to add the Puerarin reference substance respectively, makes need testing solution, measures by above-mentioned chromatographic condition, with the following formula calculate recovery rate, the results are shown in Table 7, and the result shows that this law has the good recovery.
Table 7 determination of recovery rates result
The accurate respectively reference substance solution 20 μ l that draw of the assay result of (5) five batches of compound Chinese medicinal preparation samples measure according to above-mentioned chromatographic condition, and the record peak area calculates content.Sample determination sees Table 8.
Table 8 sample determination result
According to puerarin content in five batch samples, and, promptly contain puerarin content in the Chinese medicine preparation and must not be lower than 0.64% with 75% content lower limit of measurement result mean value as this compound preparation.
Claims (2)
1, a kind of quality determining method of Chinese patent drug of treatment of diabetic retinopathy change, it is characterized in that described preparation is to carry thing through the concentrate drying gained by the pure water of eight flavor medicinal materials such as the root of kudzu vine, the Radix Astragali, glutinous rehmannia, cassia seed, Semen Leonuri, cattail pollen, the fruit of Chinese wolfberry and leech is two, this quality determining method comprises one or more of discriminating and/or quality determination.
Differentiate: a, get solid pharmaceutical preparation 1.5-3g, add ethanol 20-60ml, sonicated 10-40 minute, filter, filtrate hydro-oxidation potassium 0.5-1.5g makes dissolving, water-bath backflow 1-3 hour, evaporate to dryness, residue adds water 10-40ml makes dissolving, extracts the extract evaporate to dryness with normal butyl alcohol 10-30ml, add the D101 macroporous resin column of having handled well on the water 1-5ml dissolving back, water 50-120ml successively, 10% ethanol 30-80ml, 30% ethanol 30-80ml wash-out, eluent discards, and uses 70% ethanol 30-100ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol 0.5-3ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two solution, putting respectively on same silica gel g thin-layer plate, is developping agent with chloroform-methanol-water=45-65: 20-40: 5-15 in lower floor's solution of placing 12 hours below 10 ℃, launches, take out, dry.Spray is with the 5%-10% ethanol solution of sulfuric acid, and 105 ℃ of bakings are clear to developing the color, and inspect under the daylight.In the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color;
B, get solid pharmaceutical preparation 3-5g, add diethyl ether 30-60ml or methyl alcohol 10-40ml, sonicated 10-40 minute, filter, filtrate evaporate to dryness, residue add methyl alcohol 0.5-1.5ml and make need testing solution A; Or add water 5-15ml dissolving, on macroreticular resin and the anion exchange resins handled well, use Different concentrations of alcohol and water elution respectively, collect eluent, evaporated under reduced pressure adds ethanol 5-15ml dissolving as need testing solution B; Other gets the glutinous rehmannia control medicinal material, adds methyl alcohol and is prepared into the solution that every 1ml contains 0.5mg, in contrast the product solution A; Or get the Catalpol reference substance, add the solution that methyl alcohol is prepared into 0.5mg/ml, in contrast the product solution B.Test according to thin-layered chromatography, draw each 10 μ l of reference substance solution A and need testing solution A, put respectively on same silica gel g thin-layer plate, making into strips, is developping agent with chloroform-ethyl acetate-methanol-water=10-20: 30-50: 18-25: 5-15 or lower floor's solution of 12 hours of placement below chloroform-methanol-dense ammonia=12-20: 3-6: 0.5-1.510 ℃, launches, take out, dry, spray is with Goden test solution or anisaldehyde-concentrated sulphuric acid test solution, and it is clear to be heated to spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance chromatogram relevant position on show the spot of same color;
C, get solid pharmaceutical preparation 0.5-1.5g, add water 20-40ml, heated and boiled 15 minutes is put coldly, filters, and filtrate is extracted with ethyl acetate 10-30ml jolting, and extract is concentrated into about 0.5-1.5ml, as need testing solution; Other gets fruit of Chinese wolfberry control medicinal material 0.5g, shine medicinal material solution in pairs with legal system, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two solution, putting respectively on same silica gel g thin-layer plate, is that developping agent launches with ethyl acetate-chloroform-formic acid=1-5: 1-5: 0.5-1.5 or ether-glacial acetic acid-methyl alcohol=3-8: 0.5-1.5: 0.5-1.5, takes out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material chromatogram relevant position on show the fluorescence principal spot of same color;
D, get solid pharmaceutical preparation 1-3g, add methyl alcohol 10-30ml, sonicated 20-50 minute, filter, filtrate evaporate to dryness, residue added water 5-15ml and hydrochloric acid 0.5-1.5ml reflux 30 minutes, immediately cooling, with twice of extracted by ether, each 10-30ml merges ether solution, volatilizes, residue adds methyl alcohol 0.5-1.5ml makes dissolving, as need testing solution; Other depends on pine torch control medicinal material 1g, with method preparation, as cassia seed control medicinal material solution, gets the archen reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution; According to thin-layered chromatography, draw each 1-2 μ l of above-mentioned three solution, put respectively on same silica gel H-0.5%CMC thin layer plate, upper solution with sherwood oil (30-60 ℃)-ethyl formate-formic acid=10-20: 3-8: 0.5-1.5 is a developping agent, launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with reference substance and control medicinal material chromatogram relevant position on show identical fluorescent orange spot; Put in the ammonia steam smoked after, spot becomes redness;
E, take by weighing solid pharmaceutical preparation 2-5g, add the 10-50ml extract, extract comprises one or more in acidic protein extract, basic protein extract, neutral protein extract, the 0.9%NaCl extract, extract 20-60min, extracting method be ultrasonic or grind in one or both, leave standstill, supernatant is through 2000-5000r/min centrifuging 20-50min, behind 0.45 μ m filtering with microporous membrane, as need testing solution.Other leech control medicinal material 0.5g that fetches water makes reference substance solution with method.Measure according to capillary electrophoresis, electrophoretic buffer is one or more in boric acid-sodium chloride-borax system, sodium dihydrogen phosphate-citric acid system, boric acid-sodium carbonate system, glycocoll+sodium chloride-hydrochloric acid system, the tartrate-sodium tartrate system, the pH value of buffer system is 8.0-9.5, and the electrophoretic separation time is 10-40 minute.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, identical HPCE peak appears, the relative effective time of each major component is identical;
Assay: shine high effective liquid chromatography for measuring: chromatographic condition and system suitability test, with the octadecylsilane chemically bonded silica is filling agent, and methyl alcohol-0.1% citric acid soln=20-30: 70-80 or methanol-water-chloroform=20-30: 70-80: 0.5-1.5 or methanol-water-phosphoric acid=270-290: 710-730: 0.10-0.20 is a moving phase; The detection wavelength is 240-250nm, and theoretical cam curve is calculated by Puerarin should be not less than 4000; The preparation of reference substance solution: it is an amount of that precision takes by weighing the Puerarin reference substance, and add 30% ethanol and make the solution that every 1ml contains 80 μ g, both; The preparation of need testing solution: get solid pharmaceutical preparation 0.1-0.3g, porphyrize, the methyl alcohol or the ethanol extract that add 10%-50%, ultrasonic 20-50 minute, the neutral alumina post of having handled well on the extract, the 30-70% ethanol elution is collected the eluent evaporate to dryness, be dissolved in 30% ethanolic solution of 10-80ml, promptly; Determination method: accurate respectively reference substance and the need testing solution 20 μ l of drawing, inject liquid chromatograph, measure, promptly.
2, quality determining method as claimed in claim 1 is characterized in that comprising in this method in following discriminating and/or the assay one or more:
Differentiate: a, get this product 3g, add ethanol 40ml, sonicated 30 minutes, filter, filtrate hydro-oxidation potassium 1g makes dissolving, and water-bath refluxed 2 hours, evaporate to dryness, residue adds water 20ml makes dissolving, extracts the extract evaporate to dryness with normal butyl alcohol 20ml, add the D101 macroporous resin column of having handled well on the water 2ml dissolving back, water 100ml, 10% ethanol 60ml, 30% ethanol 60ml wash-out successively, eluent discards, and uses 70% ethanol 80ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two solution, putting respectively on same silica gel g thin-layer plate, is developping agent with chloroform-methanol-water=65: 30: 10 in lower floor's solution of placing 12 hours below 10 ℃, launches, take out, dry.Spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ of bakings are clear to developing the color, and inspect under the daylight.In the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color.
B, get this product 4.5g, the 50ml that adds diethyl ether, sonicated 30 minutes filters, and filtrate evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the glutinous rehmannia control medicinal material, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two solution, put on same silica gel g thin-layer plate respectively, make into strips, with chloroform-ethyl acetate-methanol-water=15: 40: 22: lower floor's solution of placing 12 hours below 1010 ℃ was developping agent, launch, take out, dry, spray is with the anisaldehyde test solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with reference substance chromatogram relevant position on show the spot of same color.
C, get this product 0.5g, add water 35ml, heated and boiled 15 minutes is put coldly, filters, and filtrate is extracted with ethyl acetate 15ml jolting, and extract is concentrated into about 1ml, as need testing solution; Other gets fruit of Chinese wolfberry control medicinal material 0.5g, shine medicinal material solution in pairs with legal system, test according to thin-layered chromatography, drawing each 10 μ l of above-mentioned two solution, put respectively on same silica gel g thin-layer plate, is developping agent with ethyl acetate-chloroform-formic acid (3: 2: 1), launch, take out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with control medicinal material chromatogram relevant position on show the fluorescence principal spot of same color.
D, get this product 2g, add methyl alcohol 20ml, sonicated 30 minutes filters the filtrate evaporate to dryness, residue added water 10ml and hydrochloric acid 1ml reflux 30 minutes, cooling immediately, and with extracted by ether twice, each 20ml, merge ether solution, volatilize, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other depends on pine torch control medicinal material 1g, with method preparation, as cassia seed control medicinal material solution, gets the archen reference substance and adds methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution; According to the thin-layered chromatography test, draw each 1-2 μ l of above-mentioned three solution, put respectively on same silica gel H-0.5%CMC thin layer plate, upper solution with sherwood oil (30-60 ℃)-ethyl formate-formic acid (15: 5: 1) is a developping agent, launches, and takes out, dry, put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with reference substance and control medicinal material chromatogram relevant position on show identical fluorescent orange spot; Put in the ammonia steam smoked after, spot becomes redness;
E, get this product 3g, add in the 30ml0.9%NaCl extract, ultrasonic Extraction 30min leaves standstill, and supernatant is through 2000-5000r/min centrifuging 25min, behind 0.45 μ m filtering with microporous membrane, as need testing solution.Other leech control medicinal material 0.5g that fetches water makes reference substance solution with method.Measuring according to capillary electrophoresis, is electrophoretic buffer with boric acid-sodium chloride-borax system, and the pH regulator of buffer system is 8.98, and the electrophoretic separation time is 30 minutes.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, identical HPCE peak appears, the relative effective time of each major component is identical;
Assay: according to high effective liquid chromatography for measuring, chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica, and methyl alcohol-0.1% citric acid soln=25-75 is a moving phase; The detection wavelength is 250nm, and number of theoretical plate calculates by Puerarin should be not less than 4000; The preparation of reference substance solution: it is an amount of that precision takes by weighing the Puerarin reference substance, adds 30% ethanol and make the solution that every 1ml contains 80 μ g, promptly; The preparation of need testing solution: get solid pharmaceutical preparation 0.1g, porphyrize, add 10ml 30% ethanol extract, ultrasonic 30 minutes, the neutral alumina post of having handled well on the extract, 50% ethanol elution, collect the eluent evaporate to dryness, be dissolved in 30% ethanolic solution of 20ml, shake up, filter, get subsequent filtrate, promptly; Determination method: accurate respectively reference substance and the need testing solution 10 μ l of drawing, inject liquid chromatograph, measure, promptly.Unit formulation contains puerarin content and must not be less than 0.64% in the solid pharmaceutical preparation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807948A (en) * | 2015-05-11 | 2015-07-29 | 中国人民解放军第三七一医院 | Quality control method for anti-vertigo granules |
| CN104940219A (en) * | 2015-06-01 | 2015-09-30 | 哈尔滨医科大学 | Chinese medicinal composition for diabetic retinopathy simple stage treatment and preparation method thereof |
| CN105675791A (en) * | 2016-02-05 | 2016-06-15 | 四川德成动物保健品有限公司 | Antipyretic-and-antitoxic-powder pretreatment method for detecting rehmannia in thin-layer chromatography mode |
| CN105699585A (en) * | 2016-02-05 | 2016-06-22 | 四川德成动物保健品有限公司 | Test method for radix rehmanniae in scourge-clearing toxin-vanquishing powder |
| CN112020738A (en) * | 2018-04-23 | 2020-12-01 | 陈浩能 | Method and apparatus for product monitoring |
| CN112255324A (en) * | 2020-08-31 | 2021-01-22 | 广州泽力医药科技有限公司 | Method for establishing fingerprint of wolfberry fruit and kudzu vine root solid beverage and fingerprint thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345569C (en) * | 2005-03-17 | 2007-10-31 | 河北以岭医药研究院有限公司 | Medicine for treating glycuresis retinal disease and its prepn. method |
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- 2008-12-01 CN CN200810183368.0A patent/CN101672834B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807948A (en) * | 2015-05-11 | 2015-07-29 | 中国人民解放军第三七一医院 | Quality control method for anti-vertigo granules |
| CN104940219A (en) * | 2015-06-01 | 2015-09-30 | 哈尔滨医科大学 | Chinese medicinal composition for diabetic retinopathy simple stage treatment and preparation method thereof |
| CN105675791A (en) * | 2016-02-05 | 2016-06-15 | 四川德成动物保健品有限公司 | Antipyretic-and-antitoxic-powder pretreatment method for detecting rehmannia in thin-layer chromatography mode |
| CN105699585A (en) * | 2016-02-05 | 2016-06-22 | 四川德成动物保健品有限公司 | Test method for radix rehmanniae in scourge-clearing toxin-vanquishing powder |
| CN112020738A (en) * | 2018-04-23 | 2020-12-01 | 陈浩能 | Method and apparatus for product monitoring |
| CN112255324A (en) * | 2020-08-31 | 2021-01-22 | 广州泽力医药科技有限公司 | Method for establishing fingerprint of wolfberry fruit and kudzu vine root solid beverage and fingerprint thereof |
| CN112255324B (en) * | 2020-08-31 | 2022-11-29 | 广州泽力医药科技有限公司 | Method for establishing fingerprint of wolfberry fruit and kudzuvine root solid beverage and fingerprint thereof |
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|---|---|
| CN101672834B (en) | 2014-08-13 |
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