CN101732607B - Method for detecting quality of huaqi Chinese medicinal preparation - Google Patents
Method for detecting quality of huaqi Chinese medicinal preparation Download PDFInfo
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- CN101732607B CN101732607B CN201010041802.9A CN201010041802A CN101732607B CN 101732607 B CN101732607 B CN 101732607B CN 201010041802 A CN201010041802 A CN 201010041802A CN 101732607 B CN101732607 B CN 101732607B
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for detecting quality of a huaqi Chinese medicinal preparation, and belongs to the technical field of medicaments. The huaqi Chinese medicinal preparation comprises astragalus, rehmannia root, Siberian solomonseal rhizoma, shizandra berry, common macrocarpium fruit and the like, has the effects of supplementing qi, nourishing yin, moistening dryness and clearing heat, and is used for treating wasting-thirst deficiency of qi and yin and dryness-heat and other symptoms. The method for detecting the quality of the huaqi Chinese medicinal preparation directly provides a detection index, a technical method and the like of the huaqi preparation for relevant production and detection institutions and the like. Therefore, the method brings convenience to control the quality of the Chinese medicinal preparation in a better way, ensures the safety and curative effect of the medicament, can guide production better and provides a product with high quality for customers.
Description
Technical field:
The present invention relates to a kind of quality determining method of spending stilbene Chinese medicine preparation, belong to the technical field of medicine.
Background technology:
The present invention is a kind of quality determining method of spending stilbene Chinese medicine preparation, belongs to the technical field of medicine.Flower stilbene Chinese medicine preparation is to form with taste medicines such as the Radix Astragali, Radix Rehmanniae, Rhizoma Polygonati, Fructus Schisandrae Chinensis, Fructus Corni, has the effect of supplementing QI and nourishing YIN, moistening for dryness and clearing away heat, belongs to the deficiency of both QI and YIN scorching card etc. of holding concurrently for quenching one's thirst.The invention provides a kind of quality determining method of spending stilbene Chinese medicine preparation, providing directly of method provides detection index, technical method of colored stilbene preparation etc. to relevant production, testing agency etc.; To better control the quality of this Chinese medicine preparation, guarantee safety and the curative effect of medication, can better instruct production, for consumer provides a kind of colory product.
Summary of the invention
The object of the invention is to: a kind of quality determining method of spending stilbene Chinese medicine preparation is provided, and this method provides the index that detects, means of detection, technical method etc. to relevant production, testing agency; To better control the quality of this Chinese medicine preparation, guarantee the safety of medication, can better instruct production, make controlling of production process more rationally strict, make consumer's energy full appreciation product quality.
The present invention relates to Fructus Corni or its extract, rhubarb medicinal material or its extract, schisandra chinensis medicinal material or its extract, Milkvetch Root or its extract, red rooted salvia or its extract, the method of quality control of the Chinese medicine preparation that rhizoma ane marrhenae or its extract etc. are made according to certain ratio prescription, mainly comprises discriminating, the projects such as assay, it is by ursolic acid, loganin, Fructus Corni control medicinal material, Rhizoma Anemarrhenae control medicinal material, timosaponin, Sarsasapogenin, Cortex Phellodendri control medicinal material, in berberine hydrochloride, Radix Et Rhizoma Rhei control medicinal material, emodin, chrysophanic acid, physcione, ponticin, Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, and schisantherin A, schisantherin B, Radix Astragali control medicinal material, astragaloside, kaempferol, rutin, Quercetin, Quercitroside, isorhamnetin, glucose, Radix Salviae Miltiorrhizae control medicinal material, Tanshinone I, Tanshinone I I A, Tanshinone I I B, salvianolic acid A, salvianolic acid B, salvianolic acid C, danshensu, danshensuan B, in all or part of kind as the detection index of the quality control of this Chinese medicine preparation.
Above-mentioned a kind of quality determining method of spending stilbene Chinese medicine preparation, in employing following methods, all or part of method as quality testing is as differentiating item:
Authentication technique for Pollen Pini: get this product and pulverize, adopt microscopical identification;
Authentication technique for Pollen Pini: adopt thin layer chromatography, use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Pollen Pini control medicinal material product in contrast, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Authentication technique for Fructus Corni: adopt thin layer chromatography, use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with ursolic acid, loganin, all or part of kind product in contrast in Fructus Corni control medicinal material, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Authentication technique for the Rhizoma Anemarrhenae: adopt thin layer chromatography, use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Rhizoma Anemarrhenae control medicinal material, timosaponin, all or part of kind product in contrast in Sarsasapogenin, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Authentication technique for Cortex Phellodendri: adopt thin layer chromatography, use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Cortex Phellodendri control medicinal material, all or part of kind product in contrast in berberine hydrochloride, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Authentication technique for Radix Et Rhizoma Rhei adopts thin layer chromatography, uses silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Radix Et Rhizoma Rhei control medicinal material, emodin, chrysophanic acid, physcione, all or part of kind product in contrast in ponticin, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Discrimination method for Fructus Schisandrae Chinensis: adopt thin layer chromatography, generally use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, and schisantherin A, all or part of kind product in contrast in schisantherin B, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Discrimination method for the Radix Astragali: adopt thin layer chromatography, generally use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Radix Astragali control medicinal material, astragaloside, kaempferol, rutin, Quercetin, Quercitroside, isorhamnetin, all or part of kind product in contrast in glucose, sample pre-treatments comprises direct sample or with the method for reconcentration after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be ethyl acetate, methanol, water, normal hexane, chloroform, acetone, n-butyl alcohol, diethylamine, pyridine, toluene, formic acid, cyclohexane extraction, benzene, glacial acetic acid, one or more reagent in petroleum ether are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with ethanol solution of sulfuric acid, concentrated sulfuric acid solution, anisaldehyde sulfuric acid solution, 10% sulfuric acid solution, the method of the solution colour developings such as 5% ferric chloride alcoholic solution,
Discrimination method for Radix Salviae Miltiorrhizae: adopt thin layer chromatography, generally use silica gel G or silica gel G F
254or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30 μ l, with Radix Salviae Miltiorrhizae control medicinal material, Tanshinone I, Tanshinone I I A, Tanshinone I I B, salvianolic acid A, salvianolic acid B, salvianolic acid C, danshensu, all or part of kind product in contrast in danshensuan B, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be normal hexane, ethyl acetate, petroleum ether, Ethyl formate, formic acid, dimethylbenzene, toluene, one or more reagent of cyclohexane extraction are formulated according to a certain percentage, the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with chromotropic acid sulfuric acid solution, anisaldehyde sulfuric acid solution, iodine vapor, the method of 10% phosphomolybdic acid ethanol solution solution colour developing,
Above-mentioned a kind of quality determining method of spending stilbene Chinese medicine preparation, adopts following methods to differentiate:
Discriminating for Pollen Pini: get this product content, put micro-Microscopic observation: pollen grain is oval, long 45～55 μ m, diameter 29～40 μ m, smooth surface, both sides respectively have 1 to expand air bag, and airbag wall has obvious reticular texture, mesh polygon.
Discriminating for Fructus Corni: get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.Separately get ursolic acid reference substance, add dehydrated alcohol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, cyclohexane extraction-ethyl acetate-the glacial acetic acid (9: 3: 0.3) of take is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
Discriminating for Fructus Schisandrae Chinensis: get deoxyschizandrin reference substance, add chloroform and make every 1ml containing the solution of 1mg, in contrast product solution.Get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005) test, draw need testing solution 10 μ l and above-mentioned reference substance solution 2 μ l, put in same silica gel G F respectively
254on lamellae, the cyclohexane extraction-ethyl acetate (7: 3) of take is developing solvent, launches, and takes out, and dries, and puts under ultra-violet lamp (254mm) and inspects.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
Discriminating for Pollen Pini: get Pollen Pini control medicinal material 3g, the 60ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds 2ml chloroform to be made to dissolve, in contrast medical material solution.Get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 5 μ l of need testing solution and above-mentioned reference substance solution, put respectively on same silica gel g thin-layer plate, petroleum ether-the ethyl acetate (9: 1) of take is developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle of aobvious same color.
Discriminating for the Rhizoma Anemarrhenae: get this product content 3g, add ethanol 50ml, reflux 1 hour, filters, filtrate adds hydrochloric acid 2ml, continue backflow 1 hour, with 40% sodium hydroxide solution, adjust pH to neutral, reclaim ethanol extremely without alcohol taste, add water 20ml, add benzene 30ml, jolting is extracted, and water liquid discards, reclaim benzene to about 2ml, put on processed good neutral alumina column (internal diameter 15mm, 5g, wet method dress post), with benzene-chloroform (4: 1) 50ml eluting, collect eluent, reclaim eluent to about 0.5ml, as need testing solution.Separately get Sarsasapogenin reference substance, add benzene and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, (upper solution of 30～60 ℃-Ethyl formate-formic acid (15: 5: 1) is developing solvent to take petroleum ether, launch, take out, dry, spray is with vanillin-dehydrated alcohol-concentrated sulphuric acid (0.4: 8: 1.5) solution, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
Discriminating for Cortex Phellodendri: get this product content 5g, add kieselguhr 5g, mix, add dehydrated alcohol 80ml, reflux 1 hour, filters while hot, and filtrate is concentrated into about 10ml, put processed good neutral alumina column (internal diameter 15mm, 10g, wet method dress post) upper, with dehydrated alcohol 30ml eluting, collect eluent, be concentrated into about 5ml; As need testing solution.Separately get berberine hydrochloride reference substance, add methanol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 5 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, take ethyl acetate-butanone-formic acid-water (10: 7: 1: 1) be developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical fluorescence speckle.
Discriminating for Radix Et Rhizoma Rhei: get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.Get emodin reference substance, add methanol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 5 μ l of need testing solution 10 μ l, above-mentioned control medicinal material and reference substance solution, put respectively on same silica gel g thin-layer plate, the upper solution of petroleum ether (30～60 ℃)-ethyl acetate-glacial acetic acid (15: 1.5: 1) of take is developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, aobvious five identical orange-yellow fluorescence principal spots, with aobvious identical orange-yellow fluorescence speckle on the corresponding position of reference substance chromatograph.Put in ammonia smoked after, under daylight, inspect, speckle becomes redness.
Above-mentioned a kind of quality determining method of spending stilbene Chinese medicine preparation, adopts all or part of content assaying method as quality testing in following methods:
Method 1: adopt schisandrin in high effective liquid chromatography for measuring this product, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, and the cruel first of Fructus Schisandrae Chinensis, all or part of kind in the cruel second of Fructus Schisandrae Chinensis, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, use the chromatographic column of C8 or C18 type filler, with methanol, water, second eyeball, glacial acetic acid, oxolane, one or more kind solvents in phosphoric acid solution are mobile phase under conventional proper ratio condition, detect wavelength within the scope of 200～600nm,
Method 2: adopt evaporative light scattering detector and high performance liquid chromatography coupling method to measure astragaloside in this product, kaempferol, Quercetin, all or part of kind in isorhamnetin, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, use the chromatographic column of C8 or C18 type filler, with methanol, water, second eyeball, glacial acetic acid, oxolane, one or more kind solvents in phosphoric acid solution are mobile phase under conventional proper ratio condition, detect wavelength within the scope of 200～600nm,
Method 3: adopt astragaloside in tlc determination this product, kaempferol, Quercetin, all or part of kind in isorhamnetin, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, using silica gel G or silica GF254 or silica gel H is lamellae, point sample amount is arbitrary volume between 0.5～30ul, with astragaloside, kaempferol, Quercetin, all or part of kind product in contrast in isorhamnetin, sample pre-treatments comprises direct sample, with the method for reconcentration after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, developing solvent can be ethyl acetate, methanol, water, normal hexane, chloroform, acetone, n-butyl alcohol, diethylamine, pyridine, toluene, formic acid, cyclohexane extraction, benzene, glacial acetic acid, one or more reagent in petroleum ether are formulated according to a certain percentage, and the condition of inspecting comprises under ultra-violet lamp inspects, after ammonia fumigating, put again under ultra-violet lamp and inspect, or spray is with ethanol solution of sulfuric acid, concentrated sulfuric acid aqueous solution, anisaldehyde sulfuric acid solution, 10% sulfuric acid solution, the method scanning with thin-layer chromatogram scanner after the solution colour developings such as 5% ferric chloride alcoholic solution,
Method 4: adopt Tanshinone I in high effective liquid chromatography for measuring this product, tanshinone ⅡA, Tanshinone II B, salvianolic acid A, salvianolic acid B, salvianolic acid C, danshensu, all or part of kind in danshensuan B, sample pre-treatments comprises direct sample or with reconcentration method after ethyl acetate or chloroform or dichloromethane or n-butanol extraction, use the chromatographic column of C8 or C18 type filler, with methanol, water, second eyeball, glacial acetic acid, oxolane, one or more kind solvents in phosphoric acid solution are mobile phase under conventional proper ratio condition, detect wavelength within the scope of 200～600nm,
Above-mentioned a kind of quality determining method of spending stilbene Chinese medicine preparation of stating, adopts following technology as content assaying method:
Assay for the Radix Astragali:
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; The acetonitrile-water (32: 68) of take is mobile phase; Evaporative light scattering detector.Number of theoretical plate calculates and should be not less than 4000 by astragaloside peak.
It is appropriate that the preparation precision of reference substance solution takes astragaloside reference substance, adds methanol and make every 1ml containing the solution of 0.5mg, obtains.
20 of this product are got in the preparation of need testing solution, accurately weighed, incline and content, mix, get about 6g, accurately weighed, add magnesium oxide 6g, mix, put in apparatus,Soxhlet's, add chloroform heating and refluxing extraction 4 hours, discard chloroform liquid, residue is waved most chloroform, add methanol 100ml, heating and refluxing extraction 6 hours, extracting solution reclaim solvent as for, residue adds water 40ml, slight fever makes to dissolve, with water saturated n-butyl alcohol jolting, extract 4 (30ml, 30ml, 20ml, 20ml), merge n-butyl alcohol liquid, with ammonia solution jolting washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds dehydrated alcohol 5～7ml to be made to dissolve, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 16cm), with water 80ml eluting, discard water liquid.Use 40% ethanol 50ml eluting again, discard eluent, continue with 70% ethanol 150ml eluting, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in 5ml measuring bottle, adds methanol to scale, shakes up, and obtains.
Accurate reference substance solution 10 μ l, the 20 μ l of drawing of algoscopy, need testing solution 20 μ l, injection liquid chromatography, measures, and with external standard two-point method logarithmic equation, calculates, and obtains.
Assay for Radix Salviae Miltiorrhizae:
Adopt the content of hplc simultaneous determination salvianolic acid B.
Chromatographic condition chromatographic column is Lichrosorb C18.Mobile phase: A pump methanol: acetonitrile (3: 1) B pump is containing the water of 10ml/L formic acid.Two pumps carry out gradient elution simultaneously, and elution time is 60min.Flow velocity is 1ml/min; Detecting wavelength Tanshinone I I A is 270nm, and salvianolic acid B is 286nm.
The preparation precision of reference substance solution takes salvianolic acid B reference substance 1.52mg, puts in the brown volumetric flask of 5ml simultaneously, adds 850ml/L methanol to scale, shakes up and obtain high concentration reference substance solution, according to the reference substance solution that need to be diluted to variable concentrations of following experiment.
The about 0.3g of Danshen Root (crossing sieve No. 3) is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, adds 850ml/L methanol supersound extraction 15min, and supersonic frequency is 30kHz, and power is 30W, gets subsequent filtrate, obtains.
Algoscopy is accurate above-mentioned contrast liquid and the need testing solution 10ul of drawing respectively, injects high performance liquid chromatograph, measures, and obtains.
Compared with prior art, method of quality control provided by the invention can better be controlled a kind of product quality of spending stilbene Chinese medicine preparation, guarantee the safety of medication, so more be conducive to instruct and produce, make technology controlling and process more strict rationally, allow consumer's full appreciation product quality, this quasi drugs of relieved use.The assay index components that the present invention chooses is this product according to theory of Chinese medical science monarch, minister, the monarch drug of helping, make sequence and the effective active composition of ministerial drug, to controlling drug quality, is very necessary, and the index of controlling as content is very desirable.Find under study for action the Rhizoma Polygonati in we, the Radix Astragali, the Rhizoma Anemarrhenae contains a large amount of glucides, and Fructus Schisandrae Chinensis, the Radix Astragali all contains polysaccharide, because glycoside material is that a class polarity is larger, baroque compound, it is without uv absorption, without single-minded developer, add saponin mensuration and be often subject to sugared interference, while adopting routine techniques condition to measure, during due to ultraviolet detection end absorption, sensitivity is low, noise effect is large, and there is Interference Peaks, be difficult to use, therefore, the applicant equates a series of investigations of condition determination through flowing, the applicant adopts high performance liquid chromatography-evaporative light scattering detector method to carry out the assay of Astragaloside IV in Radix Astragali, result separating degree is good, favorable reproducibility, the response rate is high, the collection of illustrative plates obtaining can be good at testing product quality.So the present invention selected a series of composition and method as controlling, the technological means of testing product quality, make the production technology of flower stilbene Chinese medicine preparation have guarantee, the preparation obtaining is more reliable, has reached the object of invention.
For proof, utilize method of quality control provided by the invention can better control a kind of product quality of spending stilbene Chinese medicine preparation, the medicine obtaining has effective effect, and applicant has carried out series of experiments;
Experimental example 1 content of danshinolic acid B method for measuring is investigated
1.1 instrument conditions: Shimadzu high performance liquid chromatograph, LC-2010A HT.
1.2 reagents: methanol (chromatographically pure, analytical pure), acetonitrile (chromatographically pure), formic acid (analytical pure), water is high purity water; Salvianolic acid B reference substance (Nat'l Pharmaceutical & Biological Products Control Institute provides, for assay), sample YIXINSHU JIAONANG (Guizhou Xin Bang pharmaceutical Co. Ltd).
1.3 chromatographic conditions: chromatographic column: VP-ODS; Mobile phase: methanol-acetonitrile-0.5% aqueous formic acid (28: 8: 64) column temperature: 30 ℃; Detect wavelength: 286nm; Flow velocity: 1ml/min; Sample size: 10ul.
1.4 system suitability test
Get respectively salvianolic acid B reference substance, flower astragalus capsules test sample (lot number: 20081201) solution lacks the negative blank solution injecting chromatograph of red rooted salvia, record chromatogram, from figure, the retention time of salvianolic acid B is 16.1 minutes, negative blank chromatogram is at salvianolic acid B position Wu Feng, and salvianolic acid B other peak separating degrees close with it are (separating degree > 1.5) completely, and under this experiment condition, salvianolic acid B is separated with other components complete.In theoretical cam curve reference substance: be 2952; In theoretical cam curve sample: salvianolic acid B is 2965; The separating degree of salvianolic acid B and other component peaks is greater than 1.5.Therefore theoretical cam curve is calculated with salvianolic acid B peak, should be not less than 2000.
1.5 linear relationships are investigated
1.5.1 the preparation of standard solution
It is appropriate that precision is got salvianolic acid B reference substance, adds dissolve with methanol, makes every 1ml containing the reference substance solution of salvianolic acid B 0.1392mg.
1.5.2 the drafting of standard curve
Accurate above-mentioned reference substance solution 1ul, 2ul, 4ul, 8ul, 12ul, 16ul, the 20ul of drawing.Injection liquid chromatography, records chromatograph and the results are shown in Table 1 respectively, with peak area A, mass number X (μ g) is carried out to linear regression calculating, obtains equation of linear regression and is:
Salvianolic acid B: A=1015012X-28940, r=0.9995 (salvianolic acid B is good in 0.1392 μ g～2.784 μ g scope internal linear relation).
Table 1 salvianolic acid B linear relationship is investigated
The selective extraction method of 1.6 need testing solution extraction times: reflux, extract, (75% methanol), measurement result table 2.
Table 2 different time extracts investigates (sample: flower astragalus capsules 20081201)
Result: as seen from Table 2, extract and the salvianolic acid B in preparation can be extracted completely for 60 minutes, therefore select heating and refluxing extraction 1 hour.
1.7 precision test
Get salvianolic acid B reference substance 10ul, repeat sample introduction 6 times, measure peak area, be averaging peak area and relative mean standard deviation (RSD).The results are shown in Table 3:
The precision test that table 3 flower stilbene is measured
1.8 stability test
Get this product (lot number: 20081201) content, put in tool plug conical flask, precision adds 75% methanol 50ml, weighed weight, reflux 1 hour, taking-up lets cool, and supplies the weight of less loss with 75% methanol, shakes up, and filters, and obtains need testing solution.Measure respectively at 0,1,2,4,8,12 hour, result is listed table 4 in, and salvianolic acid B average peak area is that 528853, RSD is 1.69%, illustrates that need testing solution is good at 12 hours internal stabilities.
In table 4 flower astragalus capsules, salvianolic acid B is measured stability test
1.9 replica test
Get this product (lot number: 20051201) content, preparation method is the same, 5 parts of test liquids of parallel preparation, respectively sample introduction 10 μ l, measure peak area, result of calculation is in Table 5, the average content of salvianolic acid B is 2.35mg/g, RSD is 1.56%.
In table 5 flower astragalus capsules, salvianolic acid B is measured replica test
1.10 recovery test
Adopt application of sample absorption method, precision takes 6 parts of about 0.5g of sample (content of salvianolic acid is 2.35mg/g) that measured content respectively, and precision adds salvianolic acid B (0.2304mg/ml) contrast liquid 5ml, puts in tool plug conical flask, add 75% methanol to 50ml, weighed weight, reflux 1 hour, taking-up lets cool, with 75% methanol, supply the weight of less loss, shake up, filter, obtain need testing solution.Precision is drawn each 10 μ l of above-mentioned test liquid respectively, and injecting chromatograph is measured, and obtains.The results are shown in Table 6.
Table 6 flower astragalus capsules (lot number: 20081201), salvianolic acid B is measured recovery test
1.11 sample determination
By preceding method, prepare test liquid and contrast solution, sample introduction 10 μ l, record chromatogram respectively, measure peak area, by external standard method, calculate content, and in 10 batch samples, content of danshinolic acid B measurement result is in Table 7.
Content of danshinolic acid B measurement result in table 7 10 batch samples
The methodological study of experimental example 2 Determination of Astragalosides
2.1 instrument conditions: Shimadzu high performance liquid chromatograph, LC-2010A HT; Evaporative light scattering detection: ELSD2000
2.2 reagents: acetonitrile is chromatographically pure, and methanol, ethyl acetate, n-butyl alcohol, ammonia are analytical pure, potassium hydroxide is chemical pure, water is redistilled water; Astragaloside (Nat'l Pharmaceutical & Biological Products Control Institute provides, for assay), sample flower astragalus capsules (Guizhou Xin Bang pharmaceutical Co. Ltd).
2.3 chromatographic conditions: chromatographic column: the U.S. C18 of Phenomenex company post; Mobile phase: acetonitrile-water (37: 63) column temperature: 25 ℃; Flow velocity: 0.5ml/min; ELSD drift tube temperature: 100 ℃; Flow rate of carrier gas: 2.7L/min; Sample size: 10ul.
2.4 system suitability test
Get respectively astragaloside reference substance, flower astragalus capsules test sample (lot number: 20081201) solution lacks the negative blank solution injecting chromatograph of Milkvetch Root, record chromatogram, from figure, the retention time of astragaloside is 16.1 minutes, negative blank chromatogram is at astragaloside position Wu Feng, and astragaloside other peak separating degrees close with it are (separating degree > 1.5) completely, and under this experiment condition, red astragaloside is separated with other components complete.
2.5 standard curves and the range of linearity
Precision takes astragaloside reference substance 19.8mg, adds the reference substance solution that 50ml methanol is made 0.396mg/ml, in contrast product stock solution.Precision is drawn above-mentioned reference substance solution 1ml, 3ml, 5ml, 7ml, 9ml respectively, put in 10ml measuring bottle, add methanol and be diluted to scale, shake up, respectively sample introduction 10 (ul), the common logarithm value of sample size (ug) of take is abscissa, the common logarithm value of peak area of take is vertical coordinate, makes standard curve, and obtaining regression equation is Y=4.9358+1.4956X, r=0.9995, sample size is good in 0.396 ~ 3.564ug scope internal linear relation.Because of this standard curve initial point only, sample determination adopts external standard two-point method to calculate.
2.6 Precision Experiment
Get with a need testing solution, repeat continuously sample introduction 6 times, each sample introduction 10ul, measures peak area, and RSD is 0.48%.
2.7 stability test
Get need testing solution, respectively at 0h, 2h, 4h, 6h, 8h, 24h, sample introduction, carry out stability test, measurement result shows, within 24h, astragaloside chromatographic peak area is without obvious change.RSD is 1.1%.
2.8 repeatability tests
Get same sample and prepare 6 parts of test liquids by the preparation method of need testing solution, difference sample introduction, the peak area of mensuration astragaloside, calculates content, and RSD is 1.15%.
2.9 recovery test
Precision takes 18.8mg astragaloside reference substance and puts in 200ml measuring bottle, adds 2%KOH-methanol and makes to dissolve and be diluted to scale, shakes up, in contrast product stock solution.Precision takes sample 0.15g, and precision adds reference substance stock solution 5ml, by the method under " 1.8 " item, makes need testing solution, and each sample size is 10ul.Average recovery rate is that 97.94%, RSD is 0.68%.
The assay of 2.10 samples is got 10 batch samples, by assay item below legal system available test solution, carries out measurement result in Table 8:
Determination of Astragaloside result in table 8 10 batch samples
Specific embodiment
1 one kinds of quality determining methods of spending stilbene Chinese medicine capsules of embodiment, mainly comprise the projects such as discriminating, assay.
1, prescription:
Radix Astragali 270g Radix Rehmanniae 200g Radix Pseudostellariae 60g Rhizoma Dioscoreae 45g Rhizoma Polygonati 90g Fructus Corni 60g
Fructus Schisandrae Chinensis 60g Pollen Pini 70g Rhizoma Anemarrhenae 40g Cortex Phellodendri 60g Cortex Mori 150g Radix Salviae Miltiorrhizae 45g
Radix Et Rhizoma Rhei 30g Semen Litchi 360g Endothelium Corneum Gigeriae Galli 30g Stigma Maydis 1200g
2, method for making: above ten Six-elements, Endothelium Corneum Gigeriae Galli powder is broken into fine powder; The Radix Astragali, Radix Pseudostellariae, the Rhizoma Anemarrhenae, Cortex Phellodendri, Stigma Maydis decoct with water 2 times, and 2 hours for the first time, 1.5 hours for the second time, collecting decoction, filtered, and filtrate decompression is concentrated into the thick paste of relative density 1.30～1.35 (50 ℃); Radix Rehmanniae, Rhizoma Dioscoreae, Rhizoma Polygonati, Cortex Mori, Semen Litchi decoct with water 2 times, each 2 hours, collecting decoction, filter, filtrate decompression is concentrated into relative density 1.08～1.12 (50 ℃), lets cool to room temperature, add 1.5 times of amount ethanol and make precipitation, standing, get supernatant and reclaim after ethanol, be evaporated to the thick paste of relative density 1.30～1.35 (50 ℃); 80% alcohol reflux three times for Fructus Corni, Fructus Schisandrae Chinensis, Radix Salviae Miltiorrhizae, Radix Et Rhizoma Rhei, 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, filter, merging filtrate, reclaims ethanol, and being evaporated to relative density is the thick paste of 1.30～1.35 (50 ℃).Merge above thick paste, mix with Endothelium Corneum Gigeriae Galli fine powder, Pollen Pini, dry, pulverize, make 1000 capsules, obtain.
3, differentiate:
(1) get this product content, put micro-Microscopic observation: pollen grain is oval, long 45～55 μ m, diameter 29～40um, smooth surface, both sides respectively have 1 to expand air bag, and airbag wall has obvious reticular texture, mesh polygon.
(2) get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.Separately get ursolic acid reference substance, add dehydrated alcohol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, cyclohexane extraction-ethyl acetate-the glacial acetic acid (9: 3: 0.3) of take is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(3) get deoxyschizandrin reference substance, add chloroform and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005) test, draw need testing solution 10 μ l and above-mentioned reference substance solution 2 μ l under the item of [discriminating] (2), put in same silica gel G F respectively
254on lamellae, the cyclohexane extraction-ethyl acetate (7: 3) of take is developing solvent, launches, and takes out, and dries, and puts under ultra-violet lamp (254mm) and inspects.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(4) get Pollen Pini control medicinal material 3g, the 60ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds 2ml chloroform to be made to dissolve, in contrast medical material solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution and each 5 μ l of above-mentioned reference substance solution under the item of [discriminating] (2), put respectively on same silica gel g thin-layer plate, petroleum ether-the ethyl acetate (9: 1) of take is developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle of aobvious same color.
(5) get this product content 3g, add ethanol 50ml, reflux 1 hour, filters, filtrate adds hydrochloric acid 2ml, continue backflow 1 hour, with 40% sodium hydroxide solution, adjust pH to neutral, reclaim ethanol extremely without alcohol taste, add water 20ml, add benzene 30ml, jolting is extracted, and water liquid discards, reclaim benzene to about 2ml, put on processed good neutral alumina column (internal diameter 15mm, 5g, wet method dress post), with benzene-chloroform (4: 1) 50ml eluting, collect eluent, reclaim eluent to about 0.5ml, as need testing solution.Separately get Sarsasapogenin reference substance, add benzene and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, the upper solution of petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) of take is developing solvent, launch, take out, dry, spray is with vanillin-dehydrated alcohol-concentrated sulphuric acid (0.4: 8: 1.5) solution, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(6) get this product content 5g, add kieselguhr 5g, mix, add dehydrated alcohol 80ml, reflux 1 hour, filters while hot, and filtrate is concentrated into about 10ml, put processed good neutral alumina column (internal diameter 15mm, 10g, wet method dress post) upper, with dehydrated alcohol 30ml eluting, collect eluent, be concentrated into about 5ml; As need testing solution.Separately get berberine hydrochloride reference substance, add methanol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 5 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, take ethyl acetate-butanone-formic acid-water (10: 7: 1: 1) be developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical fluorescence speckle.
(7) get Radix Et Rhizoma Rhei control medicinal material 0.2g, according to the lower need testing solution preparation method of [discriminating] (2) item, make control medicinal material solution.Get again emodin reference substance, add methanol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each the 5 μ l of need testing solution 10 μ l, above-mentioned control medicinal material and reference substance solution under the item of [discriminating] (2), put respectively on same silica gel g thin-layer plate, the upper solution of petroleum ether (30～60 ℃)-ethyl acetate-glacial acetic acid (15: 1.5: 1) of take is developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, aobvious five identical orange-yellow fluorescence principal spots, with aobvious identical orange-yellow fluorescence speckle on the corresponding position of reference substance chromatograph.Put in ammonia smoked after, under daylight, inspect, speckle becomes redness.
(8) get this product content 3g, add methanol 30ml, reflux 1 hour, let cool, filter, reclaim methanol, residue adds water 25ml to be made to dissolve, with water-saturated n-butanol, extract (25,20ml) 2 times, merge n-butyl alcohol liquid, with 5% sodium bicarbonate solution 25ml, wash, discard soda solution, n-butyl alcohol liquid is put evaporate to dryness in water-bath, and residue adds methanol 5ml to be made to dissolve, as need testing solution.Separately get Stigma Maydis control medicinal material 3g, add methanol 30ml, be made in the same way of control medicinal material solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 5 μ l of above-mentioned two kinds of solution, put on same silica gel g thin-layer plate respectively, the chloroform-methanol-water (10: 2: 0.15) of take is developing solvent, launches, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is dried approximately 5～7 minutes at 110 ℃, take out, be engraved under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle of aobvious same color.
4, assay:
The assay of astragaloside
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; The acetonitrile-water (32: 68) of take is mobile phase; Evaporative light scattering detector.Number of theoretical plate calculates and should be not less than 4000 by astragaloside peak.
It is appropriate that the preparation precision of reference substance solution takes astragaloside reference substance, adds methanol and make every 1ml containing the solution of 0.5mg, obtains.
20 of this product are got in the preparation of need testing solution, accurately weighed, incline and content, mix, get about 6g, accurately weighed, add magnesium oxide 6g, mix, put in apparatus,Soxhlet's, add chloroform heating and refluxing extraction 4 hours, discard chloroform liquid, residue is waved most chloroform, add methanol 100ml, heating and refluxing extraction 6 hours, extracting solution reclaims solvent to dry, residue adds water 40ml, slight fever makes to dissolve, with water saturated n-butyl alcohol jolting, extract 4 (30ml, 30ml, 20ml, 20ml), merge n-butyl alcohol liquid, with ammonia solution jolting washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds dehydrated alcohol 5～7ml to be made to dissolve, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 16cm), with water 80ml eluting, discard water liquid.Use 40% ethanol 50ml eluting again, discard eluent, continue with 70% ethanol 150ml eluting, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in 5ml measuring bottle, adds methanol to scale, shakes up, and obtains.
Accurate reference substance solution 10 μ l, the 20 μ l of drawing of algoscopy, need testing solution 20 μ l, injection liquid chromatography, measures, and with external standard two-point method logarithmic equation, calculates, and obtains.
This product contains the Radix Astragali with astragaloside (C
41h
65o
4) meter, every must not be less than 0.10mg.
The assay of Radix Salviae Miltiorrhizae:
Chromatographic condition chromatographic column is Lichrosorb C18.Mobile phase: A pump methanol: acetonitrile (3: 1); B pump is containing the water of 10ml/L formic acid.Two pumps carry out gradient elution simultaneously, and elution time is 60min.Flow velocity is 1ml/min; Detecting wavelength salvianolic acid B is 286nm.
The preparation precision of reference substance solution takes salvianolic acid B reference substance 1.52mg, puts in the brown volumetric flask of 5ml simultaneously, adds 850ml/L methanol to scale, shakes up and obtain high concentration reference substance solution, according to the reference substance solution that need to be diluted to variable concentrations of following experiment.
The about 0.3g of Danshen Root (crossing sieve No. 3) is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, adds 850ml/L methanol supersound extraction 15min, and supersonic frequency is 30kHz, and power is 30W, gets subsequent filtrate, obtains.
Algoscopy is accurate above-mentioned contrast liquid and the need testing solution 10ul of drawing respectively, injects high performance liquid chromatograph, measures, and obtains.
This product is containing Radix Salviae Miltiorrhizae in salvianolic acid B, and every must not be less than 1.10mg.
2 one kinds of quality determining methods of spending stilbene Chinese medicine capsules of embodiment, mainly comprise the projects such as discriminating, assay.
1, prescription:
Radix Astragali 270g Radix Rehmanniae 200g Radix Pseudostellariae 60g Rhizoma Dioscoreae 45g Rhizoma Polygonati 90g Fructus Corni 60g
Fructus Schisandrae Chinensis 60g Pollen Pini 70g Rhizoma Anemarrhenae 40g Cortex Phellodendri 60g Cortex Mori 150g Radix Salviae Miltiorrhizae 45g
Radix Et Rhizoma Rhei 30g Semen Litchi 360g Endothelium Corneum Gigeriae Galli 30g Stigma Maydis 1200g
2, method for making: above ten Six-elements, Endothelium Corneum Gigeriae Galli powder is broken into fine powder; The Radix Astragali, Radix Pseudostellariae, the Rhizoma Anemarrhenae, Cortex Phellodendri, Stigma Maydis decoct with water 2 times, and 2 hours for the first time, 1.5 hours for the second time, collecting decoction, filtered, and filtrate decompression is concentrated into the thick paste of relative density 1.30～1.35 (50 ℃); Radix Rehmanniae, Rhizoma Dioscoreae, Rhizoma Polygonati, Cortex Mori, Semen Litchi decoct with water 2 times, each 2 hours, collecting decoction, filter, filtrate decompression is concentrated into relative density 1.08～1.12 (50 ℃), lets cool to room temperature, add 1.5 times of amount ethanol and make precipitation, standing, get supernatant and reclaim after ethanol, be evaporated to the thick paste of relative density 1.30～1.35 (50 ℃); 80% alcohol reflux three times for Fructus Corni, Fructus Schisandrae Chinensis, Radix Salviae Miltiorrhizae, Radix Et Rhizoma Rhei, 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, filter, merging filtrate, reclaims ethanol, and being evaporated to relative density is the thick paste of 1.30～1.35 (50 ℃).Merge above thick paste, mix with Endothelium Corneum Gigeriae Galli fine powder, Pollen Pini, dry, pulverize, make 1000 capsules, obtain.
3, differentiate:
(1) get this product content, put micro-Microscopic observation: pollen grain is oval, long 45～55 μ m, diameter 29～40um, smooth surface, both sides respectively have 1 to expand air bag, and airbag wall has obvious reticular texture, mesh polygon.
(2) get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution.Separately get ursolic acid reference substance, add dehydrated alcohol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, cyclohexane extraction-ethyl acetate-the glacial acetic acid (9: 3: 0.3) of take is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(3) get deoxyschizandrin reference substance, add chloroform and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005) test, draw need testing solution 10 μ l and above-mentioned reference substance solution 2 μ l under the item of [discriminating] (2), put in same silica gel G F respectively
254on lamellae, the cyclohexane extraction-ethyl acetate (7: 3) of take is developing solvent, launches, and takes out, and dries, and puts under ultra-violet lamp (254mm) and inspects.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(4) get this product content 3g, add ethanol 50ml, reflux 1 hour, filters, filtrate adds hydrochloric acid 2ml, continue backflow 1 hour, with 40% sodium hydroxide solution, adjust pH to neutral, reclaim ethanol extremely without alcohol taste, add water 20ml, add benzene 30ml, jolting is extracted, and water liquid discards, reclaim benzene to about 2ml, put on processed good neutral alumina column (internal diameter 15mm, 5g, wet method dress post), with benzene-chloroform (4: 1) 50ml eluting, collect eluent, reclaim eluent to about 0.5ml, as need testing solution.Separately get Sarsasapogenin reference substance, add benzene and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, the upper solution of petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) of take is developing solvent, launch, take out, dry, spray is with vanillin-dehydrated alcohol-concentrated sulphuric acid (0.4: 8: 1.5) solution, and hot blast blows to clear spot.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
(5) get this product content 5g, add kieselguhr 5g, mix, add dehydrated alcohol 80ml, reflux 1 hour, filters while hot, and filtrate is concentrated into about 10ml, put processed good neutral alumina column (internal diameter 15mm, 10g, wet method dress post) upper, with dehydrated alcohol 30ml eluting, collect eluent, be concentrated into about 5ml; As need testing solution.Separately get berberine hydrochloride reference substance, add methanol and make every 1ml containing the solution of 0.5mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw need testing solution 5 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, take ethyl acetate-butanone-formic acid-water (10: 7: 1: 1) be developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical fluorescence speckle.
(6) get Radix Et Rhizoma Rhei control medicinal material 0.2g, according to the lower need testing solution preparation method of [discriminating] (2) item, make control medicinal material solution.Get again emodin reference substance, add methanol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each the 5 μ l of need testing solution 10 μ l, above-mentioned control medicinal material and reference substance solution under the item of [discriminating] (2), put respectively on same silica gel g thin-layer plate, the upper solution of petroleum ether (30～60 ℃)-ethyl acetate-glacial acetic acid (15: 1.5: 1) of take is developing solvent, launch, take out, dry, put under ultra-violet lamp (365nm) and inspect.In test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, aobvious five identical orange-yellow fluorescence principal spots, with aobvious identical orange-yellow fluorescence speckle on the corresponding position of reference substance chromatograph.Put in ammonia smoked after, under daylight, inspect, speckle becomes redness.
4, assay:
The assay of astragaloside
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; The acetonitrile-water (32: 68) of take is mobile phase; Evaporative light scattering detector.Number of theoretical plate calculates and should be not less than 4000 by astragaloside peak.
It is appropriate that the preparation precision of reference substance solution takes astragaloside reference substance, adds methanol and make every 1ml containing the solution of 0.5mg, obtains.
20 of this product are got in the preparation of need testing solution, accurately weighed, incline and content, mix, get about 6g, accurately weighed, add magnesium oxide 6g, mix, put in apparatus,Soxhlet's, add chloroform heating and refluxing extraction 4 hours, discard chloroform liquid, residue is waved most chloroform, add methanol 100ml, heating and refluxing extraction 6 hours, extracting solution reclaims solvent to dry, residue adds water 40ml, slight fever makes to dissolve, with water saturated n-butyl alcohol jolting, extract 4 (30ml, 30ml, 20ml, 20ml), merge n-butyl alcohol liquid, with ammonia solution jolting washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds dehydrated alcohol 5～7ml to be made to dissolve, pass through D
101type macroporous adsorptive resins (internal diameter 1.5cm, long 16cm), with water 80ml eluting, discards water liquid.Use 40% ethanol 50ml eluting again, discard eluent, continue with 70% ethanol 150ml eluting, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in 5ml measuring bottle, adds methanol to scale, shakes up, and obtains.
Accurate reference substance solution 10 μ l, the 20 μ l of drawing of algoscopy, need testing solution 20 μ l, injection liquid chromatography, measures, and with external standard two-point method logarithmic equation, calculates, and obtains.
This product contains the Radix Astragali with astragaloside (C
41h
65o
4) meter, every must not be less than 0.10mg.
Claims (2)
1. spend a detection method for stilbene Chinese medicine capsules, it is characterized in that assay take astragaloside or/and the index that salvianolic acid is assay:
(1) assay of astragaloside
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Take acetonitrile-water ratio as 32:68 be mobile phase; Evaporative light scattering detector; Number of theoretical plate calculates and should be not less than 4000 by astragaloside peak;
It is appropriate that the preparation precision of reference substance solution takes astragaloside reference substance, adds methanol and make every 1ml containing the solution of 0.5mg, obtains;
20 of this product are got in the preparation of need testing solution, accurately weighed, incline and content, mix, get 6g, accurately weighed, add magnesium oxide 6g, mix, put in apparatus,Soxhlet's, add chloroform heating and refluxing extraction 4 hours, discard chloroform liquid, residue is waved most chloroform, add methanol 100ml, heating and refluxing extraction 6 hours, extracting solution reclaims solvent to dry, residue adds water 40ml, slight fever makes to dissolve, with water saturated n-butyl alcohol jolting, extracting 4 each n-butyl alcohol consumptions is: 30ml, 30ml, 20ml, 20ml, merge n-butyl alcohol liquid, with ammonia solution jolting washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds dehydrated alcohol 5～7ml to be made to dissolve, pass through D
101type macroporous adsorptive resins, internal diameter is 1.5cm, long 16cm, with water 80ml eluting, discards water liquid, use 40% ethanol 50ml eluting again, discard eluent, continue with 70% ethanol 150ml eluting, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in 5ml measuring bottle, adds methanol to scale, shakes up, and obtains,
Accurate reference substance solution 10 μ l, the 20 μ l of drawing of algoscopy, need testing solution 20 μ l, injection liquid chromatography, measures, and with external standard two-point method logarithmic equation, calculates, and obtains;
(2) assay of salvianolic acid B:
Chromatographic condition chromatographic column is Lichrosorb C18, mobile phase: A pump methanol: acetonitrile ratio is 3:1, and B pump is containing the water of 10ml/L formic acid; Two pumps carry out gradient elution simultaneously, and elution time is 60min, and flow velocity is 1ml/min; Detecting wavelength salvianolic acid B is 286nm;
The preparation precision of reference substance solution takes salvianolic acid B reference substance 1.52mg, puts in the brown volumetric flask of 5ml simultaneously, adds 850ml/L methanol to scale, shakes up and obtain high concentration reference substance solution, according to the reference substance solution that need to be diluted to variable concentrations of following experiment;
The preparation of need testing solution is got Danshen Root and is crossed sieve 0.3g No. 3, accurately weighed, puts in tool plug conical flask, adds 850ml/L methanol supersound extraction 15min, and supersonic frequency is 30kHz, and power is 30W, gets subsequent filtrate, obtains;
Algoscopy is accurate above-mentioned contrast liquid and the need testing solution 10ul of drawing respectively, injects high performance liquid chromatograph, measures, and obtains.
2. the detection method of colored stilbene Chinese medicine capsules according to claim 1, it is characterized in that its authentication technique adopt following one or more:
(1) get this product content, put micro-Microscopic observation: pollen grain is oval, long 45～55 μ m, diameter 29～40um, smooth surface, both sides respectively have 1 to expand air bag, and airbag wall has obvious reticular texture, mesh polygon;
(2) get this product content 10g, add kieselguhr 5g, mix, the 100ml that adds diethyl ether, reflux 1 hour, filters, and reclaims ether to dry, and residue adds chloroform 2ml to be made to dissolve, as need testing solution; Separately get ursolic acid reference substance, add dehydrated alcohol and make every 1ml containing the solution of 0.5mg, in contrast product solution; According to thin layer chromatography, according to appendix VI B test of Chinese Pharmacopoeia version in 2005, draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, cyclohexane extraction-ethyl acetate-the glacial acetic acid of ratio 9:3:0.3 of take is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and hot blast blows to clear spot, in test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color;
(3) get deoxyschizandrin reference substance, add chloroform and make every 1ml containing the solution of 1mg, product solution in contrast, according to thin layer chromatography, appendix VI B test of Chinese Pharmacopoeia version in 2005, draw need testing solution 10 μ l and above-mentioned reference substance solution 2 μ l under differentiating 2, put respectively on same silica GF254 lamellae, the cyclohexane extraction-ethyl acetate of ratio 7:3 of take is developing solvent, launches, take out, dry, put under ultra-violet lamp 254mm and inspect, in test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color;
(4) get this product content 3g, add ethanol 50ml, reflux 1 hour, filter, filtrate adds hydrochloric acid 2ml, continues to reflux 1 hour, with 40% sodium hydroxide solution, adjusts pH to neutral, reclaim ethanol extremely without alcohol taste, add water 20ml, add benzene 30ml, jolting is extracted, water liquid discards, reclaim benzene to 2ml, put processed good neutral alumina column, neutral alumina column adopts internal diameter 15mm, 5g, wet method dress post, with benzene-chloroform 50ml eluting of ratio 4:1, collects eluent, reclaim eluent to 0.5ml, as need testing solution; Separately get Sarsasapogenin reference substance, add benzene and make every 1ml containing the solution of 0.5mg, in contrast product solution; According to thin layer chromatography, press appendix VI B test of Chinese Pharmacopoeia version in 2005, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, take the petroleum ether of 30～60 ℃ of boiling ranges: Ethyl formate: the upper solution that formic acid ratio is 15:5:1 is developing solvent, launch, take out, dry, spray is with vanillin-dehydrated alcohol-concentrated sulfuric acid solution of ratio 0.4:8:1.5, and hot blast blows to clear spot, in test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color;
(5) get this product content 5g, add kieselguhr 5g, mix, add dehydrated alcohol 80ml, reflux 1 hour, filters while hot, filtrate is concentrated into 10ml, put processed good neutral alumina column, neutral alumina column is with internal diameter 15mm, 10g, wet method dress post, with dehydrated alcohol 30ml eluting, collect eluent, be concentrated into 5ml; As need testing solution; Separately get berberine hydrochloride reference substance, add methanol and make every 1ml containing the solution of 0.5mg, in contrast product solution; According to thin layer chromatography, appendix VIB test of Chinese Pharmacopoeia version in 2005, draws need testing solution 5 μ l, reference substance solution 1 μ l, puts respectively on same silica gel g thin-layer plate, ethyl acetate-butanone-formic acid-the water of ratio 10:7:1:1 of take is developing solvent, launch, take out, dry, put under ultra-violet lamp 365nm and inspect, in test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical fluorescence speckle;
(6) get Radix Et Rhizoma Rhei control medicinal material 0.2g, according to 2 lower need testing solution preparation methoies of discriminating, make control medicinal material solution, get again emodin reference substance, add methanol and make every 1ml containing the solution of 1mg, in contrast product solution, according to thin layer chromatography, appendix VIB test of Chinese Pharmacopoeia version in 2005, draw the need testing solution 10 μ l under differentiating 2, each 5 μ l of above-mentioned control medicinal material and reference substance solution, put respectively on same silica gel g thin-layer plate, petroleum ether with 30～60 ℃ of boiling ranges: ethyl acetate: glacial acetic acid, ratio is that the upper solution of 15:1.5:1 is developing solvent, launch, take out, dry, put under ultra-violet lamp 365nm and inspect, in test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, aobvious five identical orange-yellow fluorescence principal spots, with aobvious identical orange-yellow fluorescence speckle on the corresponding position of reference substance chromatograph, put in ammonia smoked after, under daylight, inspect, speckle becomes redness.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260204A (en) * | 1999-12-15 | 2000-07-19 | 邵玉君 | Medicament for treating diabetes and its preparation method |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260204A (en) * | 1999-12-15 | 2000-07-19 | 邵玉君 | Medicament for treating diabetes and its preparation method |
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| CN101732607A (en) | 2010-06-16 |
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Address after: 550014 Luodian County, Qiannan Buyei and Miao Autonomous Prefecture, Jiefang Road Province Ping Town, No., No. 96 Patentee after: GUIZHOU XINBANG PHARMACEUTICAL Co.,Ltd. Address before: 227 No. 550014 Guizhou Guiyang Xinbang Baiyun Road Economic Development Zone Patentee before: GUIZHOU XINBANG PHARMACEUTICAL Co.,Ltd. |
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