CN103954723A - Detection method of common goldenrop particles - Google Patents
Detection method of common goldenrop particles Download PDFInfo
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- CN103954723A CN103954723A CN201410174576.XA CN201410174576A CN103954723A CN 103954723 A CN103954723 A CN 103954723A CN 201410174576 A CN201410174576 A CN 201410174576A CN 103954723 A CN103954723 A CN 103954723A
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Abstract
The invention relates to a detection method of common goldenrop particles. The detection method comprises inspection items of identifying characters and measuring content. The identification item comprises identification of scutellaria baicalensis, identification of liquorice, identification of radix bupleuri and identification of bupleurum longiradiatum; the item of measuring content is used for measuring the content of baicalin. In order to solve the problem of incapability of monitoring insufficiency or absence of corresponding raw materials of illegal manufacturers and monitoring the interfusion of bupleurum longiradiatum caused by the lack of one set of complete detection method for detecting active ingredients of common goldenrop particles at present, the method disclosed by the invention can be used for identifying components and measuring content scientifically, reasonably and feasibly; the method is capable of guaranteeing the clinical efficacy of common goldenrop particles, making clear quality indexes of several medicine materials and guaranteeing the use of all medicine materials in the common goldenrop particles, thereby guaranteeing the curative effect of the common goldenrop particles; in addition, bupleurum longiradiatum serving as a counterfeit drug of radix bupleuri can also be effectively detected, thereby avoiding the damage to the health of patients caused by two toxic ingredients comprising radix bupleuri toxin and acetyl radix bupleuri toxin in bupleurum longiradiatum.
Description
Technical field
The present invention relates to a kind of detection method of little bupleurum particles.
Background technology
Little bupleurum particles is kind of recording of < < Chinese Pharmacopoeia > > version in 2010, prescription is radix bupleuri, pinellia, the root of large-flowered skullcap, Radix Codonopsis, Radix Glycyrrhizae, ginger, date, it is the granule of pure Chinese medicine, there is the heat radiation of inducing sweat, the effect of soothing liver and harmonizing stomach, is used for the treatment of the common drug of fevers and chills alternate, fullness and discomfort in chest and hypochondrium, poor appetite, vexation and vomiting, bitter taste in the mouth and dry throat in the market.But up to the present, also do not have a set of perfect detection method to detect accordingly its effective ingredient, illegal manufacturer just may be in strict accordance with the dosage batching of writing out a prescription when producing medicine, wantonly reduce the raw material that price is high, cause the curative effect of medicine obviously to decline, affect the safe and effective of medicine, grievous injury patient's interests.On the other hand, the prescription of this medicine derives from the recipe Xiao Chaihu Tang in < < Treatise on Febrile and Miscellaneous Disease > >, in side, consumption maximum is radix bupleuri, account for 1/3rd of whole recipe quantity, its effect is mainly harmonizing Sharyang, harmonizing stomach and lowering adverse Qi, strengthening vital QI to eliminate pathogenic factors, but because radix bupleuri belongs to common medicine and use amount is larger, and the output of radix bupleuri is limited, so its adulterant bigleaf thorowax root is also had a mind to or is not intended to sneak into use, in bigleaf thorowax root, contain bupleurotoxin and acetylbupleurotoxin, these two kinds of compositions are noxious material, repeatedly occur because taking the poisoning Adverse drug events of medicine that is mixed with bigleaf thorowax root before this, so it is necessary to control bigleaf thorowax root.< < Shandong medical industry > > has delivered supplementary > > mono-literary composition of < < to the Chinese Pharmacopoeia version radix bupleuri method of inspection in 2000 in the 22 the 5th phase of volume in 2003, inquired into the discriminating control problem of bigleaf thorowax root medicinal material in pharmacopeia, but its method has obvious deficiency: place 24 hours, detection time is oversize, is unfavorable for drug production process control and drug quality supervision and check; Because of bupleurotoxin and acetylbupleurotoxin content relatively low, point sample is 2 μ l only, point sample amount is very few, the accuracy of impact check; Because of bupleurotoxin and acetylbupleurotoxin content relatively low, under uviol lamp, inspect, without chromogenic reagent, observe, observing effect is bad, the more important thing is many, the contained complicateds of little bupleurum particles prescription flavour of a drug, other composition can produce to be obscured and disturbs, so finding a kind of better way, to control bigleaf thorowax root be the task of top priority.
Summary of the invention
Object of the present invention, is to provide a kind of detection method of little bupleurum particles, on the one hand, can effectively monitor illegal manufacturer and throws less or do not throw corresponding raw material, the problem that causes the curative effect of medicine to decline; On the other hand, control sneaking into of adulterant bigleaf thorowax root, prevented from taking the generation of rear poisoning Adverse drug events, guarantee that this drug safety is effective.
Technical scheme of the present invention is such:
The prescription of this little bupleurum particles is: radix bupleuri 150g, pinellia 56g, root of large-flowered skullcap 56g, Radix Codonopsis 56g, Radix Glycyrrhizae 56g, ginger 56g, date 56g.
The detection method of little bupleurum particles of the present invention, comprises proterties, discriminating, assay, inspection item; Wherein, differentiate and comprise the discriminating to the discriminating of the discriminating of the discriminating of the discriminating of radix bupleuri in particle, the root of large-flowered skullcap, Radix Codonopsis, Radix Glycyrrhizae, bigleaf thorowax root; Assay is the assay to scutelloside in particle.
Proterties: this product is yellow to tan particle; Taste is sweet.
Check: should meet every regulation relevant under granule item (appendix I C of < < Chinese Pharmacopoeia > > version in 2010).
Described detection method comprises following:
(1) discriminating of the root of large-flowered skullcap
Get this product 6g, porphyrize, adds ethanol 20ml, ultrasonic processing 20 minutes, filters filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get scutelloside reference substance appropriate, add methyl alcohol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, on the silica gel g thin-layer plate that to put respectively in same carboxymethylcellulose sodium solution of take containing 4% sodium acetate be binder, take ethyl acetate: butanone: formic acid: water=5:3:1:1 is developping agent, launches, take out, dry, spray is with 1% ferric trichloride ethanolic solution, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.
(2) discriminating of Radix Glycyrrhizae
Get this product 6g, porphyrize, adds ethanol 20ml, ultrasonic processing 20 minutes, filters filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Extracting Radix Glycyrrhizae control medicinal material 1g, adds water appropriate, decocts 30 minutes, let cool, filter, filtrate is concentrated into 20ml, with water saturated normal butyl alcohol jolting, extract 2 times, each 20ml, merges n-butanol extracting liquid, with the saturated water washing of normal butyl alcohol 2 times, each 10ml, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography, draw need testing solution 10 μ l and control medicinal material solution 5～10 μ l, put respectively on same silica gel g thin-layer plate, take methenyl choloride: methyl alcohol: water=40:10:1 is developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
(3) discriminating of radix bupleuri
Get this product 6g, add water 20ml, be stirred to dissolve, centrifugal, get supernatant, be added in polyamide column (100～200 orders, 8g, internal diameter is 2.5～3cm, wet method dress post) upper, difference water, 20% ethanol and each 100ml wash-out of 50% ethanol, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get radix bupleuri control medicinal material 1g, add water appropriate, decoct 1.5 hours, filter, filtrate is concentrated into about 10ml, is added in polyamide column (100～200 orders, 4g, internal diameter is 2cm, wet method dress post) upper, difference water 100ml and 50% ethanol 150ml wash-out, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography, test, draw need testing solution 2～10ul and control medicinal material solution 2ul, put respectively on same silica gel g thin-layer plate, take ethyl acetate: ethanol: water=12:2:1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid of 5% paradime thylaminobenzaldehyde, and it is clear that hot blast blows to spot colour developing, puts under ultraviolet lamp (365nm) and inspect.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
(4) discriminating of bigleaf thorowax root
Get this product 10g, add sherwood oil 20～40ml, soak 5～20 minutes, ultrasonic processing 10～30 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=7～9:0.7～1.3 are developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
(5) discriminating of Radix Codonopsis
Get this product 20g, porphyrize, add again normal butyl alcohol 30～70ml, ultrasonic processing 20～40 minutes, filter, filtrate is with the saturated solution washing of 10～30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 30～70ml, ultrasonic processing 20～40 minutes, filter, filtrate is with the saturated solution washing of 10～30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=13～17:3～5 are developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Wherein, discrimination method is as follows more specifically for bigleaf thorowax root:
Get this product 10g, add 60～90 ℃ of sherwood oil 30ml, soak 10 minutes, ultrasonic processing 15 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=8:1 as developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
The red sage root more specifically discrimination method is as follows:
Get this product 20g, porphyrize, add again normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=15: 4 is developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The assay of scutelloside:
According to high effective liquid chromatography for measuring
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase, and detection wavelength is 315nm, number of theoretical plate calculates and should be not less than 3000 by scutelloside peak.
It is appropriate that scutelloside reference substance is got in the preparation of reference substance solution, accurately weighed, adds 70% ethanol and make every 1ml containing 60ug solution, obtains.
This product under content uniformity item is got in the preparation of need testing solution, mixes, and gets in right amount porphyrize, get about 3g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 50KHZ) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, obtain.
Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
Every bag of this product in scutelloside (C21H18O11), must not be less than 20.0mg containing the root of large-flowered skullcap.
The ratio of developping agent of the present invention and the ratio of mobile phase are all in volume ratio.
Technique effect of the present invention:
The present invention by little bupleurum particles the discriminating of the root of large-flowered skullcap, the discriminating of the discriminating of Radix Glycyrrhizae, radix bupleuri, the discriminating of the discriminating of bigleaf thorowax root, Radix Codonopsis, the assay of scutelloside, allow several medicinal materials have clear and definite quality index, guaranteed the use of ingredients in little bupleurum particles, thereby guaranteed the curative effect of medicine, on the other hand, the adulterant bigleaf thorowax root of radix bupleuri also can effectively detect, and has avoided these two kinds of toxic substances harm patients' of the bupleurotoxin that contains in bigleaf thorowax root and acetylbupleurotoxin health.The present invention can reflect the quality of little bupleurum particles better and more comprehensively, and can effectively control illegal manufacturers produce little bupleurum particles of poor quality, thereby has guaranteed curative effect and the safety of medicine, has guaranteed patient's interests, and the present invention is scientific and reasonable, practical.
Embodiment
Embodiment 1:
[prescription] radix bupleuri 150g pinellia 56g root of large-flowered skullcap 56g Radix Codonopsis 56g Radix Glycyrrhizae 56g ginger 56g date 56g
[method for making] above seven tastes, radix bupleuri, the root of large-flowered skullcap, Radix Codonopsis, Radix Glycyrrhizae and date boiling secondary, each 1.5 hours, collecting decoction, filtered, and filtrate is concentrated in right amount.Pinellia, ginger are made solvent with 70% ethanol, flood and carry out diacolation after 24 hours, collect percolate 600ml, reclaim ethanol, merge with above-mentioned concentrate, are concentrated in right amount, add appropriate sucrose, and granulation is dry, makes 1000g, obtains.
[proterties] this product is yellow to tan particle; Taste is sweet.
The discriminating of [discriminating] (1) root of large-flowered skullcap
Get this product 6g, porphyrize, adds ethanol 20ml, ultrasonic processing 20 minutes, filters filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get scutelloside reference substance appropriate, add methyl alcohol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, on the silica gel g thin-layer plate that to put respectively in same carboxymethylcellulose sodium solution of take containing 4% sodium acetate be binder, take ethyl acetate: butanone: formic acid: water=5:3:1:1 is developping agent, launches, take out, dry, spray is with 1% ferric trichloride ethanolic solution, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.
(2) discriminating of Radix Glycyrrhizae
Get this product 6g, porphyrize, adds ethanol 20ml, ultrasonic processing 20 minutes, filters filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Extracting Radix Glycyrrhizae control medicinal material 1g, adds water appropriate, decocts 30 minutes, let cool, filter, filtrate is concentrated into 20ml, with water saturated normal butyl alcohol jolting, extract 2 times, each 20ml, merges n-butanol extracting liquid, with the saturated water washing of normal butyl alcohol 2 times, each 10ml, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography, draw need testing solution 10 μ l and control medicinal material solution 5～10 μ l, put respectively on same silica gel g thin-layer plate, take methenyl choloride: methyl alcohol: water=40:10:1 is developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
(3) discriminating of radix bupleuri
Get this product 6g, add water 20ml, be stirred to dissolve, centrifugal, get supernatant, be added in polyamide column (100～200 orders, 8g, internal diameter is 2.5～3cm, wet method dress post) upper, difference water, 20% ethanol and each 100ml wash-out of 50% ethanol, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get radix bupleuri control medicinal material 1g, add water appropriate, decoct 1.5 hours, filter, filtrate is concentrated into about 10ml, is added in polyamide column (100～200 orders, 4g, internal diameter is 2cm, wet method dress post) upper, difference water 100ml and 50% ethanol 150ml wash-out, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography, test, draw need testing solution 2～10 μ l and control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, take ethyl acetate: ethanol: water=12:2:1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid of 5% paradime thylaminobenzaldehyde, and it is clear that hot blast blows to spot colour developing, puts under ultraviolet lamp (365nm) and inspect.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
(4) discriminating of bigleaf thorowax root
Get this product 10g, add 60～90 ℃ of sherwood oil 20ml, soak 5 minutes, ultrasonic processing 10 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=7:0.7 as developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
(5) discriminating of Radix Codonopsis
Get this product 20g, porphyrize, add again normal butyl alcohol 30ml, ultrasonic processing 20 minutes, filter, filtrate is used the saturated solution washing of 10ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 30ml, ultrasonic processing 20 minutes, filter, filtrate is used the saturated solution washing of 10ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=13:3 as developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
[inspection] should meet every regulation relevant under granule item (appendix I C of < < Chinese Pharmacopoeia > > version in 2010).
The assay of [assay] scutelloside:
According to high effective liquid chromatography for measuring
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase, and detection wavelength is 315nm, number of theoretical plate calculates and should be not less than 3000 by scutelloside peak.
It is appropriate that scutelloside reference substance is got in the preparation of reference substance solution, accurately weighed, adds 70% ethanol and make every 1ml containing 60ug solution, obtains.
This product under content uniformity item is got in the preparation of need testing solution, mixes, and gets in right amount porphyrize, get about 3g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic processing (power 250W, frequency 50KHZ) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, obtain.
Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
Every bag of this product in scutelloside (C21H18O11) 21.9mg, is no less than 20.0mg containing the root of large-flowered skullcap.
Embodiment 2:
In the present embodiment, except the discriminating of bigleaf thorowax root is different with the discrimination method of Radix Codonopsis, all the other detection methods are all identical with embodiment 1, and concrete discrimination method is as follows:
The discriminating of bigleaf thorowax root:
Get this product 10g, add 60～90 ℃ of sherwood oil 30ml, soak 10 minutes, ultrasonic processing 15 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=8:1 as developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
The discrimination method of the red sage root:
Get this product 20g, porphyrize, add again normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=15: 4 is developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Embodiment 3:
In the present embodiment, except the discriminating of bigleaf thorowax root is different with the discrimination method of Radix Codonopsis, all the other detection methods are all identical with embodiment 1, and concrete discrimination method is as follows:
The discriminating of bigleaf thorowax root
Get this product 10g, add sherwood oil 40ml, soak 20 minutes, ultrasonic processing 30 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=9:1.3 as developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
(5) discriminating of Radix Codonopsis
Get this product 20g, porphyrize, add again normal butyl alcohol 70ml, ultrasonic processing 40 minutes, filter, filtrate is used the saturated solution washing of 30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 70ml, ultrasonic processing 40 minutes, filter, filtrate is used the saturated solution washing of 30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=17:5 as developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Claims (4)
1. the detection method of a little bupleurum particles, comprise proterties, discriminating, assay, inspection item, it is characterized in that: described discrimination method comprise the discriminating of the root of large-flowered skullcap, the discriminating of the discriminating of Radix Glycyrrhizae, radix bupleuri, the discriminating of the discriminating of bigleaf thorowax root, Radix Codonopsis, described assay is the assay of scutelloside, and detection method is as follows:
(1) discriminating of the root of large-flowered skullcap
Get this product 6g, porphyrize, add ethanol 20ml, ultrasonic processing 20 minutes, filter, filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get scutelloside reference substance, add methyl alcohol and make every 1ml containing the solution of 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, on the silica gel g thin-layer plate that to put respectively in same carboxymethylcellulose sodium solution of take containing 4% sodium acetate be binder, take ethyl acetate: butanone: formic acid: water=5:3:1:1 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,
(2) discriminating of Radix Glycyrrhizae
Get this product 6g, porphyrize, add ethanol 20ml, ultrasonic processing 20 minutes, filter, filtrate evaporate to dryness, residue adds water 20ml and dissolves, with hydrochloric acid, adjust pH value to 2～3, with ethyl acetate jolting, extract 2 times, each 20ml, combined ethyl acetate extract, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, extracting Radix Glycyrrhizae control medicinal material 1g, add water appropriate, decoct 30 minutes, let cool, filter, filtrate is concentrated into 20ml, with water saturated normal butyl alcohol jolting, extract 2 times, each 20ml, merge n-butanol extracting liquid, with the saturated water washing of normal butyl alcohol 2 times, each 10ml, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw need testing solution 10 μ l and control medicinal material solution 5～10 μ l, put respectively on same silica gel g thin-layer plate, take methenyl choloride: methyl alcohol: water=40:10:1 is developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color,
(3) discriminating of radix bupleuri
Get this product 6g, add water 20ml, be stirred to dissolve, centrifugal, get supernatant, be added on polyamide column a, difference water, each 100ml wash-out of 20% ethanol and 50% ethanol, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get radix bupleuri control medicinal material 1g, add water appropriate, decoct 1.5 hours, filter, filtrate is concentrated into 10ml, be added on polyamide column b, difference water 100ml and 50% ethanol 150ml wash-out, collect 50% ethanol eluate, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw need testing solution 2～10ul and control medicinal material solution 2ul, put respectively on same silica gel g thin-layer plate, take ethyl acetate: ethanol: water=12:2:1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid of 5% paradime thylaminobenzaldehyde, it is clear that hot blast blows to spot colour developing, putting wavelength is to inspect under 365nm ultraviolet lamp, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color,
(4) discriminating of bigleaf thorowax root
Get this product 10g, add sherwood oil 20～40ml, soak 5～20 minutes, ultrasonic processing 10～30 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=7～9:0.7～1.3 are developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
(5) discriminating of Radix Codonopsis
Get this product 20g, porphyrize, add again normal butyl alcohol 30～70ml, ultrasonic processing 20～40 minutes, filter, filtrate is with the saturated solution washing of 10～30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 30～70ml, ultrasonic processing 20～40 minutes, filter, filtrate is with the saturated solution washing of 10～30ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=13～17:3～5 are developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color,
(6) assay of scutelloside:
Take octadecylsilane chemically bonded silica as filling agent, take methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase, detection wavelength is 315nm, number of theoretical plate calculates and should be not less than 3000 by scutelloside peak, get scutelloside reference substance appropriate, add 70% ethanol and make every 1ml containing 60ug solution, obtain reference substance solution, get this product under content uniformity item, mix, porphyrize, get 3g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic processing 30 minutes, let cool, weighed weight again, with 70% ethanol, supply the weight of less loss, shake up, filter, get subsequent filtrate, obtain need testing solution, precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measure, obtain.
2. the detection method of little bupleurum particles according to claim 1, is characterized in that:
(1) bigleaf thorowax root is differentiated more specifically
Get this product 10g, add 60～90 ℃ of sherwood oil 30ml, soak 10 minutes, ultrasonic processing 15 minutes, filter, filtrate is waved to 1ml, as need testing solution, separately get bupleurotoxin, acetylbupleurotoxin reference substance, add methyl alcohol and make every 1ml containing the mixed solution of bupleurotoxin reference substance 1mg and acetylbupleurotoxin reference substance 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take normal hexane: ethyl acetate=8:1 as developping agent, launch, take out, dry, spray is with 10% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot that must not show same color,
(2) Radix Codonopsis is differentiated more specifically
Get this product 20g, porphyrize, add again normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get Radix Codonopsis control medicinal material 1g, boiling 1 hour, let cool, filter, filtrate is put in separating funnel, add normal butyl alcohol 50ml, ultrasonic processing 30 minutes, filter, filtrate is used the saturated solution washing of 20ml normal butyl alcohol, discard water liquid, normal butyl alcohol liquid is concentrated into dry, residue adds methyl alcohol 1ml to be made to dissolve, medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take toluene: ethyl acetate=15: 4 is developping agent, launch, take out, dry, spray is with 20% phosphomolybdic acid ethanol solution, at 105 ℃, be heated to spot colour developing clear, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
3. the detection method of little bupleurum particles according to claim 1, it is characterized in that: the described polyamide column a of step (3) is that polyamide is crossed 100～200 mesh sieves, take the polyamide 8g after sieving, select the chromatographic column that internal diameter is 2.5～3cm, wet method dress post.
4. the detection method of little bupleurum particles according to claim 1, is characterized in that: the described polyamide column b of step (3) is that polyamide is crossed 100～200 orders, takes the polyamide 4g after sieving, and selects the chromatographic column that internal diameter is 2cm, wet method dress post.
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| CN107014945A (en) * | 2017-05-23 | 2017-08-04 | 四川逢春制药有限公司 | A kind of detection method for the Chinese medicine preparation for treating acute infectious hepatitis |
| CN107102093A (en) * | 2017-05-23 | 2017-08-29 | 四川逢春制药有限公司 | The detection method of compound pellet mattress cream |
| CN107202856A (en) * | 2017-05-23 | 2017-09-26 | 四川逢春制药有限公司 | A kind of detection method for the Chinese medicine preparation for treating flu |
| CN108414668A (en) * | 2018-06-15 | 2018-08-17 | 四川逢春制药有限公司 | A kind of detection method of Chinese medicine detoxification particles |
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| CN105929102A (en) * | 2016-05-18 | 2016-09-07 | 四川逢春制药有限公司 | Detection method of bear gall pills |
| CN105974045A (en) * | 2016-05-18 | 2016-09-28 | 四川逢春制药有限公司 | Detection method of traditional Chinese medicine pills |
| CN105974045B (en) * | 2016-05-18 | 2017-10-17 | 四川逢春制药有限公司 | A kind of detection method of medicine pill |
| CN107014945A (en) * | 2017-05-23 | 2017-08-04 | 四川逢春制药有限公司 | A kind of detection method for the Chinese medicine preparation for treating acute infectious hepatitis |
| CN107102093A (en) * | 2017-05-23 | 2017-08-29 | 四川逢春制药有限公司 | The detection method of compound pellet mattress cream |
| CN107202856A (en) * | 2017-05-23 | 2017-09-26 | 四川逢春制药有限公司 | A kind of detection method for the Chinese medicine preparation for treating flu |
| CN107102093B (en) * | 2017-05-23 | 2018-05-22 | 四川逢春制药有限公司 | The detection method of compound pellet mattress cream |
| CN107014945B (en) * | 2017-05-23 | 2018-07-31 | 四川逢春制药有限公司 | A kind of detection method of Chinese medicine preparation that treating acute infectious hepatitis |
| CN109030639A (en) * | 2018-05-31 | 2018-12-18 | 上海交通大学医学院附属新华医院 | The measuring method of content of baicalin in a kind of Gandhi's capsule |
| CN108414668A (en) * | 2018-06-15 | 2018-08-17 | 四川逢春制药有限公司 | A kind of detection method of Chinese medicine detoxification particles |
| WO2021179616A1 (en) * | 2020-03-12 | 2021-09-16 | 广州白云山光华制药股份有限公司 | Novel use of xiaochaihu granules in combination with chloroquine phosphate |
| CN111830187A (en) * | 2020-06-02 | 2020-10-27 | 鉴甄检测技术（上海）有限公司 | Rapid thin-layer identification method for multiple medicines in small radix bupleuri granule finished product |
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Application publication date: 20140730 |