CN103852555A - Quality detection method for Tibetan medicine composition rheumatism Theron preparation - Google Patents

Quality detection method for Tibetan medicine composition rheumatism Theron preparation Download PDF

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CN103852555A
CN103852555A CN201210523974.9A CN201210523974A CN103852555A CN 103852555 A CN103852555 A CN 103852555A CN 201210523974 A CN201210523974 A CN 201210523974A CN 103852555 A CN103852555 A CN 103852555A
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solution
medicinal material
rheumatism
take
sialon
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CN103852555B (en
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郑亭亭
江玉娟
任松鹏
宋洋
单玉刚
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JINHE TIBETAN MEDICINE CO., LTD.
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Shandong Arura Pharmaceutical Research & Development Co Ltd
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Abstract

The invention discloses a quality detection method for a Tibetan medicine composition rheumatism Theron preparation. With the adoption of the quality detection method, the quality standard of an existing rheumatism Theron preparation is correspondingly improved and an identification method for shellac and elecampane in a previous standard is optimized; the identification on juniperus formosana, syzygium aromaticum, meconopsis, rubia wallichiana decne and amomum kravanh is increased on the basis of the previous standard, and a content determination method of general flavone and swertia chirayita and a detection method for aristolochic acid A are increased, so as to further ensure the safe, efficient, uniform and stable product quality and the controllable quality.

Description

A kind of quality determining method of Tibetan medicinal composition rheumatism Sialon preparation
Technical field
The present invention relates to a kind of quality determining method of Tibetan medicinal composition, particularly a kind of quality determining method of Tibetan medicinal composition rheumatism Sialon preparation, belongs to medical technical field.
Background technology
Rheumatism one word originates from ancient Greek, and in B.C. 4th century, " Hippcrates complete or collected works " are about thinking in human dissection one literary composition: the body fluid of human body, because clammy bet causes disease in four limbs, internal organ, is rheumatism.China's Huangdi's Internal Classics three phase of cold-dampness heterozygosis of also keeping watch is called numbness.
Tibetan medicine for rheumatism research relatively early.Tibetan medicine thinks, the gout due to grand, red bar, blood, Baconic, pulse condition short and real compact as blood-head disease pulse condition slow, it is indefinite that urine resembles, and is heat symptom-complex, making a definite diagnosis errorless symptom is that urine has burnt angle taste, the dry pain in joint when morbidity.Arthritis with fixed pain caused by dampness is that the interior heat evil of articular cavity and yellow water gather, and infiltrates in articular cavity or spreads all over meat, bone, arteries and veins, muscle etc. and locate, and joint is as pulverized a kind of disease of sample pain.On Tibetanmedicine with bloodletting, rush down under, the three kinds of methods of taking medicine treat.Tibetan live in that in the cold environment on plateau, to suffer from rheumatism unavoidable for a long time, and Tibetan have found that in the process combating the disease for a long time Bailey Myospalax Born has good effect for rheumatism.Tibetan medicines add other medicinal materials with Bailey Myospalax Born, under the theoretical direction of Tibetan medicine's " dry yellow water regulates Baconic, the disorder such as grand ", have been mixed with rheumatism Sialon capsule.
Rheumatism Sialon capsule records in the national drug standards, standard number: WS-10788(ZD-0788)-2002.Arthritis, neck and shoulder ache, lumbago, talagia arthroncus finger-joint, carpal joint swelling and pain, gridle muscle pain that rheumatism Sialon capsule causes rheumatism, rheumatoid disease, ankylosing spondylitis all has definite curative effect, is one-level new drug prescription, rheumatism bone disease good medicine.
But, the quality inspection standard of existing rheumatism Sialon capsule only adopts TLC thin layer to differentiate that two taste medicinal material shellacs and the banksia rose, HPLC measure the content of an effective component gallic acid, and selected certified variety amount do not have representativeness very little, and can not effectively control the quality of other principal ingredients.Therefore existing quality determining method can not be controlled this product quality comprehensively and accurately, and quality standard has much room for improvement.
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide a kind of quality determining method of Tibetan medicinal composition rheumatism Sialon preparation.
Summary of the invention
The present invention has carried out corresponding raising to the quality standard of existing rheumatism Sialon preparation, discrimination method to shellac, the banksia rose in primary standard is optimized, and on the basis of primary standard, increase the discriminating of Chinese juniper, cloves, green suede wormwood artemisia, ZANGQIANCAO, cardamom, increase the content assaying method of general flavone, Indian Herba Swertiae bimaculatae, the inspection method of aristolochic acid A, has further guaranteed Product quality and safety, effective, homogeneous, stable, quality controllable.
Term explanation:
Rheumatism Sialon capsule is the nomenclature of drug that national standard for traditional Chinese medicines compilation (Chinese patent drug provincial standard rising national standard part) is recorded.
Other preparations that rheumatism Sialon preparation comprises rheumatism Sialon capsule and prepares with rheumatism Sialon capsule bulk drug formula.
Technical scheme of the present invention is as follows:
A kind of bulk drug consists of Bailey Myospalax Born 3.2 weight portions, myrobalan's 36.3 weight portions, safflower 21.1 weight portions, cardamom 28.5 weight portions, ichor cream 10.6 weight portions, Indian Herba Swertiae bimaculatae 10.6 weight portions, sword bean 10.6 weight portions, caudate sweetleaf leaf 10.6 weight portions, ZANGQIANCAO 10.6 weight portions, shellac 10.6 weight portions, Chinese juniper 10.6 weight portions, borneol 3.2 weight portions, Tabasheer 10.6 weight portions, cloves 4.2 weight portions, nutmeg 6.3 weight portions, tsaoko 9.0 weight portions, agalloch eaglewood 5.3 weight portions, Santalum album 9.5 weight portions, santal 6.1 weight portions, green suede wormwood artemisia 6.1 weight portions, common bombax flower 11.3 weight portions, the banksia rose 9.5 weight portions, Cuminum celery 9.5 weight portions, banksia rose birthwort 5.3 weight portions, Chinese cassia tree 9.5 weight portions, spiral shell operculum 6.1 weight portions, the stem of noble dendrobium 6.1 weight portions, rhizoma nardostachyos 8.2 weight, parmelia saxatilis 14.7 weight portions, russian fenugreek herb 5.3 weight portions, terminaliae billericae,fructus 5.3 weight portions, the quality determining method of the rheumatism Sialon preparation of emblic 6.3 weight portions, is characterized in that, the method comprises one or more in following discriminating and/or assay:
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon solid pharmaceutical preparation 5～15g, add ethyl acetate 10～20mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into 1～2mL, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 5～10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take dimethylbenzene-methylene chloride-acetone-formic acid of volume ratio 4～6:4～6:0.5:1 as developping agent, launch, take out, dry, put respectively under daylight and inspect, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon solid pharmaceutical preparation 2～5g, the 10～20mL that adds methylene chloride, ultrasonic processing 20～40 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 5～10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the methylene chloride-cyclohexane of volume ratio 5～8:1～2 as developping agent, launch, take out, dry, spray is take volume ratio as 1% vanillic aldehyde sulfuric acid solution, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon solid pharmaceutical preparation 5～15g, the 50～150mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5～1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～5 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 7～9:3～1 as developping agent, launch, take out, dry, spray is take volume ratio as 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, add methyl alcohol 10～20mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into approximately 1～2mL, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the boiling range of volume ratio 2～6:1～2 as 60～90 ℃ of sherwood oil-acetone are as developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, add volume ratio 1% hydrochloric acid solution 20～30mL, ultrasonic processing 30～40 minutes, filter, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol dissolves about 2mL, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, drip again two ammoniacal liquor as developping agent take normal hexane-ethyl acetate-methyl alcohol of volume ratio 10～8:4～5:1, launch, take out, dry, spray, with rare bismuth potassium iodide solution, is inspected under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, adding boiling range is that 60-90 ℃ of sherwood oil 50～80mL is ultrasonic, and ultrasonic processing 30～40 minutes filters, and filtrate is concentrated into 1～2mL as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take sherwood oil (60～90 ℃)-ethyl acetate of volume ratio 8～9:2～1 as developping agent, launch, take out, dry, spray is take volume ratio as 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon solid pharmaceutical preparation 10～20g, the 80～100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8～9:2～1 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50mL measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, puts respectively in 25mL measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 8-10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15-20 minute, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon preparation fine powder 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50mL, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25mL puts in 100mL measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0mL, puts in 25mL measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0mL ", is measured absorbance in accordance with the law, reads the content (μ g/mL) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon preparation of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g;
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:70-75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1mL containing 45 μ g, to obtain final product;
The preparation of need testing solution: get the about 1g of Tibetan medicinal composition rheumatism Sialon preparation fine powder, accurately weighed, put in round-bottomed flask, precision adds ethanol 50mL, close plug, weighed weight, refluxing extraction 1 hour, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, get subsequent filtrate 25mL, be evaporated to dry, residue add water about 20mL make dissolve, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate liquid, is evaporated to dryly, and residue adds ethanol and dissolves and be settled in 5mL volumetric flask, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon preparation of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g;
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40-45:55-60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1mL containing 500ng, to obtain final product;
The preparation of need testing solution: get the about 10g of Tibetan medicinal composition rheumatism Sialon preparation fine powder, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25mL, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20mL to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20mL, after extraction, alkali lye uses volume parts to regulate pH2～3 than 5% hydrochloric acid, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1mL volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon preparation of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.
Preferably, a kind of bulk drug consists of Bailey Myospalax Born 3.2 weight portions, myrobalan's 36.3 weight portions, safflower 21.1 weight portions, cardamom 28.5 weight portions, ichor cream 10.6 weight portions, Indian Herba Swertiae bimaculatae 10.6 weight portions, sword bean 10.6 weight portions, caudate sweetleaf leaf 10.6 weight portions, ZANGQIANCAO 10.6 weight portions, shellac 10.6 weight portions, Chinese juniper 10.6 weight portions, borneol 3.2 weight portions, Tabasheer 10.6 weight portions, cloves 4.2 weight portions, nutmeg 6.3 weight portions, tsaoko 9.0 weight portions, agalloch eaglewood 5.3 weight portions, Santalum album 9.5 weight portions, santal 6.1 weight portions, green suede wormwood artemisia 6.1 weight portions, common bombax flower 11.3 weight portions, the banksia rose 9.5 weight portions, Cuminum celery 9.5 weight portions, banksia rose birthwort 5.3 weight portions, Chinese cassia tree 9.5 weight portions, spiral shell operculum 6.1 weight portions, the stem of noble dendrobium 6.1 weight portions, rhizoma nardostachyos 8.2 weight, parmelia saxatilis 14.7 weight portions, russian fenugreek herb 5.3 weight portions, terminaliae billericae,fructus 5.3 weight portions, the quality determining method of the rheumatism Sialon capsule of emblic 6.3 weight portions, is characterized in that, the method comprises one or more in following discriminating and/or assay:
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon capsule 's content 10g, add ethyl acetate 20mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2mL, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 5: 5: 0.5: dimethylbenzene-methylene chloride-acetone-formic acid of 0.1 is developping agent, launch, take out, dry, put respectively under daylight and inspect; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon capsule 's content 3g, the 15mL that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the volume ratio methylene chloride cyclohexane of 8: 1 as developping agent, launch, take out, dry, spray is with 1% vanillic aldehyde sulfuric acid solution, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon capsule 's content 10g, the 100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8.5:1.5 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon capsule 's content 5g, add methyl alcohol 10mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1mL, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, boiling range take volume ratio as 4:1 as 60～90 ℃ of sherwood oil acetone be developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon capsule 's content 10g, add volume ratio 1% hydrochloric acid solution 30mL, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol is dissolved to 2mL, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take volume ratio as 10:4:1 normal hexane-ethyl acetate-methyl alcohol, then to drip two ammoniacal liquor be developping agent, launches, take out, dry, spray, with rare bismuth potassium iodide solution, is inspected under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon capsule 's content 10g, the sherwood oil 50mL that adds boiling range and be 60-90 ℃ is ultrasonic, and ultrasonic processing 30 minutes filters, and filtrate is concentrated into 1mL as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the boiling range 60-90 ℃ of petroleum ether-ethyl acetate of volume ratio 8:2 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon capsule 's content 10g, the 100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8.5:1.5 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50mL measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, puts respectively in 25mL measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon capsule 's content 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50mL, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25mL puts in 100mL measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0mL, puts in 25mL measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0mL ", is measured absorbance in accordance with the law, reads the content (μ g/mL) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g.
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1mL containing 45 μ g, to obtain final product;
The preparation of need testing solution: get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in round-bottomed flask, precision adds ethanol 50mL, close plug, weighed weight, refluxing extraction 1 hour, let cool, weighed weight again, supply the weight of less loss with ethanol, shake up, filter, get subsequent filtrate 25mL, be evaporated to dry, residue add water about 20mL make dissolve, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate liquid, be evaporated to dry, residue adds ethanol and dissolves and be settled in 5mL volumetric flask, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g.
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40:60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1mL containing 500ng, to obtain final product;
The preparation of need testing solution: get the about 10g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25mL, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20mL to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20mL, after extraction, alkali lye uses volume parts to regulate pH2～3 than 5% hydrochloric acid, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1mL volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon capsule of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.
The pass of weight portion of the present invention and parts by volume is the relation of g/mL or kg/L.
Beneficial effect of the present invention:
Under rheumatism Sialon capsule proper mass normal term, only have the thin layer of shellac, the banksia rose to differentiate the assay item of item and gallic acid, can not effectively control the quality of other principal ingredients.The present invention has carried out corresponding raising to primary standard, discrimination method to shellac, the banksia rose in primary standard is optimized, and on the basis of primary standard, increase the discriminating of Chinese juniper, cloves, green suede wormwood artemisia, ZANGQIANCAO, cardamom, increase the content assaying method of general flavone, Indian Herba Swertiae bimaculatae, the inspection method of aristolochic acid A, security, homogeneity, the stability of product are further guaranteed, make product quality more guaranteed, and then guarantee the healthy of the clinical efficacy of product and extensive patients.This law also can be used for other preparations of rheumatism Sialon simultaneously, as rheumatism Sialon particle, rheumatism Sialon ball, rheumatism Sialon sheet etc.
Experimental example below and embodiment are used for further illustrating but are not limited to the present invention.
The rheumatism Sialon capsule detecting is that Qinghai gold is scolded the production of Tibetan medicine medicine company incorporated company.
Experimental example 1: identification experiment
1, the discriminating of shellac
(1) need testing solution preparation method research:
The ultrasonic extraction of methyl alcohol: get rheumatism Sialon capsule 's content 10g, add methyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil extracts: get rheumatism Sialon capsule 's content 10g, add the sherwood oil 30mL of boiling range 30-60 ℃, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
(2) research of thin layer condition:
Adopt silica G plate, toluene-chloroform for developping agent-acetone-formic acid system, has compared the expansion effect of different volumes ratio (4:4:0.6:0.1), (5:5:0.5:0.1), (6:6:0.4:0.1), and inspection method is to inspect under daylight.
Adopt silica G plate, dimethylbenzene-methylene chloride for developping agent-acetone-formic acid system, has compared the expansion effect of different proportion (4:4:0.6:0.1), (5:5:0.5:0.1), (6:6:0.4:0.1), and inspection method is to inspect under daylight.
Adopt silica G plate, cyclohexane-ethyl acetate for developping agent-glacial acetic acid system, has compared the expansion effect of different proportion (4:1:1), (4:2:1), (4:0.5:1), and inspection method is inspected for putting under ultraviolet lamp (365nm).
Adopt silica G plate, toluene-methylene chloride for developping agent-acetone system, compare different proportion (4:5:0.5), (5:5:0.5) expansion effect (6:5:1), it is 10% ethanol solution of sulfuric acid that developer is selected volume fraction, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Dimethylbenzene-methylene chloride-formic acid of volume ratio 6:5:0.5, the development system such as petroleum ether-ethyl acetate, dimethylbenzene-acetone of volume ratio 1:1 that volume ratio 1:1 boiling range is 60-90 ℃ have also been selected in this experiment; Mass volume ratio is that 1% vanillic aldehyde ethanol solution of sulfuric acid, mass volume ratio are the developers such as 5% ethanol solution of sulfuric acid.
Test by said system, select test condition and be:
Need testing solution preparation method: ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 20mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2mL, as need testing solution.
Development system: volume ratio is 5: 5: 0.5: dimethylbenzene-methylene chloride-acetone-formic acid of 0.1
Inspection method: inspect under daylight.
Concrete test method is:
Get rheumatism Sialon capsule 's content 5～15g, add ethyl acetate 10～20mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into 1～2mL, as need testing solution.Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution.Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 5～10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take dimethylbenzene-methylene chloride-acetone-formic acid of volume ratio 4～6:4～6:0.5:1 as developping agent, launch, take out, dry, put respectively under daylight and inspect.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color, negative noiseless.
Interpretation of result: shellac TLC result shows, under dimethylbenzene-methylene chloride-acetone-formic acid condition of developping agent take volume ratio as 4～6:4～6:0.5:1, there is good expansion effect, Volume fraction 5: 5: 0.5: launch effect under dimethylbenzene-methylene chloride-acetone-formic acid condition of 0.1 best, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 1.The method can be used as the discrimination method of shellac in rheumatism Sialon capsule.
2, the discriminating of the banksia rose
(1) need testing solution preparation method research:
The ultrasonic extraction of methyl alcohol: get rheumatism Sialon capsule 's content 3g, add methyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 3g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil extracts: get rheumatism Sialon capsule 's content 3g, add the sherwood oil 30mL of boiling range 30-60 ℃, ultrasonic processing 30min, filters, and filtrate volatilizes, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Methylene chloride extract: get rheumatism Sialon capsule 's content 3g, the 30mL that adds methylene chloride, ultrasonic processing 30min, filter, filtrate evaporate to dryness, residue add methylene chloride 2mL dissolve, as need testing solution.
Extract volatile oil: get rheumatism Sialon capsule 's content 3g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
(2) research of thin layer condition:
Adopt silica G plate, normal hexane for developping agent-ethyl acetate system, compare the expansion effect of different volumes ratio (5:1), (5:2), (5:3), it is 1% vanillic aldehyde ethanol solution of sulfuric acid that developer is selected mass volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, sherwood oil for developping agent-ethyl acetate system, has compared the expansion effect of different volumes ratio (6:2), (6:1), (6:3), and developer is selected mass volume ratio is 1% vanillic aldehyde sulfuric acid solution, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, ethyl acetate for developping agent-cyclohexane system, the relatively expansion effect of different volumes ratio (5:1), (6:1), (4:1) lower floor, developer is selected mass volume ratio 1% vanillic aldehyde sulfuric acid solution, and inspection method is to inspect under daylight.
Adopt silica G plate, methylene chloride for developping agent-cyclohexane system, has compared the expansion effect of different volumes ratio (8:1), (5:1), (4:1), and developer is selected volume fraction is 10% ethanol solution of sulfuric acid, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Chloroform-normal hexane of volume ratio 5:1, the development system such as cyclohexane-ethyl acetate, the diformazan benzol-cyclohexane of volume ratio 5:2 of volume ratio 5:1 have also been selected in this experiment; Mass volume ratio is that sulfuric acid solution, the mass volume ratio that 1% ethanol solution of sulfuric acid, mass volume ratio are 1% is the developers such as 1% vanillic aldehyde sulfuric acid solution.
Test by said system, select experiment condition and be:
Need testing solution preparation method: the ultrasonic extraction of methylene chloride: get rheumatism Sialon capsule 's content 5g, the 15mL that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution.
Development system: methylene chloride-cyclohexane.
Developer: mass volume ratio is 1% vanillic aldehyde ethanol solution of sulfuric acid.
Inspection method: 105 ℃ to be heated to spot colour developing clear, inspects under daylight.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 2～5g, the 10～50mL that adds methylene chloride, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into 1～5mL, as need testing solution; Separately get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Separately get in the negative control sample of the scarce banksia rose of prescription ratio preparation, be made in the same way of negative control solution.Test according to appendix VI B thin-layered chromatography of Pharmacopoeia of the People's Republic of China version in 2010, draw banksia rose control medicinal material solution 1～5 μ L, need testing solution 3～8 μ L, negative control solution 3～8 μ L, put respectively on same silica gel g thin-layer plate, methylene chloride-cyclohexane take volume ratio as 5～8:1～3 is developping agent, launches, and takes out, dry, spray is take mass volume ratio as 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, negative noiseless.
Interpretation of result: santal TLC result shows, under methylene chloride-cyclohexane condition of developping agent take volume ratio as 5～8:1～3, there is good expansion effect, under the methylene chloride-cyclohexane condition that is 8:1 in volume ratio, launch effect best, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 2.The method can be used as the discrimination method of banksia rose medicinal material in rheumatism Sialon capsule.
3, the discriminating of ZANGQIANCAO
(1) need testing solution preparation method research:
The ultrasonic extraction of methyl alcohol: get rheumatism Sialon capsule 's content 10g, add methyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Normal butyl alcohol extracts: get rheumatism Sialon capsule 's content 10g, add normal butyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Methyl alcohol is ultrasonic, adjust pH, and methylene chloride extracts: get this product content 10g, add methyl alcohol 30mL, ultrasonic processing 30 minutes, filters, filtrate evaporate to dryness, it is that 1% hydrochloric acid 20mL makes to dissolve that residue adds volume ratio, filters, filtrate adds ammoniacal liquor and is adjusted to alkalescence, with methylene chloride jolting extraction 2 times, and each 20mL, combined dichloromethane liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as need testing solution.
Sherwood oil (60-90 ℃) refluxing extraction: get rheumatism Sialon capsule 's content 10g, add sherwood oil (60-90 ℃) 30mL, refluxing extraction 30min, filters, and filtrate is concentrated into 2mL, as need testing solution.
(2) research of thin layer condition:
Adopt silica G plate, sherwood oil for developping agent (60-90 ℃)-ethyl formate-formic acid system, compare the expansion effect of different proportion (15:5:1), (4:1:1), (15:10:1), it is 5% vanillic aldehyde sulfuric acid solution that developer is selected mass volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, sherwood oil for developping agent (60-90 ℃)-acetone system, has compared the expansion effect of different volumes ratio (4:1), (5:1), (5:2), and inspection method is to inspect under ultraviolet lamp (365nm).
Adopt gel GF 254 plate, dimethylbenzene for developping agent-acetone system, compare the expansion effect of different proportion (4:1), (6:1), (2:1), inspection method is to inspect under ultraviolet lamp (254nm), and then spray is with 5% vanillic aldehyde sulfuric acid solution, 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.
Dimethylbenzene-ethyl acetate of volume ratio 4:1, the development system such as cyclohexane ethyl acetate, dimethylbenzene-methyl alcohol of volume ratio 9:1 of volume ratio 4:1 have also been selected in this experiment.
Test by said system, select experiment condition and be:
Need testing solution preparation method: get rheumatism Sialon capsule 's content 5g, add methyl alcohol 30mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1mL, as need testing solution.
Sherwood oil (60～90 ℃)-acetone of development system: volume ratio 4:1
Inspection method: put under 365nm ultraviolet lamp and inspect.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 5～15g, add methyl alcohol 10～50mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into about 1mL, as need testing solution.Get ZANGQIANCAO control medicinal material 0.5g, be made in the same way of control medicinal material solution.Separately get in the negative control sample of the scarce ZANGQIANCAO of prescription ratio preparation, with need testing solution, preparation method makes negative control solution.Test according to appendix VI B thin-layered chromatography of Pharmacopoeia of the People's Republic of China version in 2010, draw control medicinal material solution 2～8 μ L, each 5～15 μ L of test sample and negative control solution, put respectively on same silica gel g thin-layer plate, take sherwood oil (60～90 ℃)-acetone of volume ratio 2～6:1～2 as developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless.
Interpretation of result: ZANGQIANCAO TLC result shows, under dimethylbenzene-ethanol condition of developping agent with volume ratio 2～6:1～2, there is good expansion effect, under the sherwood oil that is 4:1 in volume ratio (60～90 ℃)-acetone condition, launch effect best, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 3.The method can be used as the discrimination method of ZANGQIANCAO medicinal material in rheumatism Sialon capsule.
4, the discriminating of Chinese juniper
(1) need testing solution preparation method research:
Volume fraction 95% alcohol extract: get rheumatism Sialon capsule 's content 5g, add volume fraction 95% ethanol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, it is that 95% alcohol 2mL dissolves that residue adds volume fraction, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 5g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil (60～90 ℃) extracts: get rheumatism Sialon capsule 's content 5g, add sherwood oil (60～90 ℃) 30mL, ultrasonic processing 30min, filters, and filtrate is concentrated into 1mL, as need testing solution.
Extract volatile oil: get rheumatism Sialon capsule 's content 5g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
(2) research of thin layer condition:
Adopt silica G plate, methenyl choloride-ethyl acetate for developping agent-formic acid system, compare the expansion effect of different volumes ratio (6:4:1), (6:6:1), (6:8:1), it is 1% ferric trichloride ethanolic solution that developer is selected mass volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, the upper solution system of dimethylbenzene-ethyl acetate for developping agent-formic acid-water, compare the expansion effect of different volumes ratio (7:10:3:4), (10:10:3:4), (4:10:3:4), it is 1% ferric trichloride ethanolic solution that developer is selected mass volume ratio, under daylight, inspects.
Adopt gel GF 254 plate, dimethylbenzene for developping agent-acetone system, compare the expansion effect of different volumes ratio (4:1), (6:1), (2:1), inspection method is to inspect under ultraviolet lamp (254nm), and then spray is with mass volume ratio 5% vanillic aldehyde sulfuric acid solution, 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, sherwood oil for developping agent-ethyl acetate system, compare the expansion effect of different volumes ratio (8:2), (9:1), (8.5:1.5), it is 10% ethanol solution of sulfuric acid that developer is selected volume fraction, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
The development systems such as the dimethylbenzene-ethyl acetate-formic acid, 4:1 cyclohexane-ethyl acetate, 1:1 dimethylbenzene-ethyl acetate of volume ratio 7:3:1 have also been selected in this experiment; Mass volume ratio is that 1% aluminium choride ethanolic solution, mass volume ratio are the developers such as 5% phosphomolybdic acid ethanol solution.
Test by said system, select test condition and be:
Need testing solution preparation method: get rheumatism Sialon capsule 's content 5g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
Developping agent: petroleum ether-ethyl acetate system.
Developping agent: volume ratio is 8.5:1.5.
Inspection method: spray is with volume ratio 1% ethanol solution of sulfuric acid, 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 5～10g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after having decocted, collect ethyl acetate part, as need testing solution; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution.Separately get in the negative control sample of the Chinese juniper of prescription ratio preparation, with need testing solution, preparation method makes negative control solution.Test and draw the each 3 μ L of above-mentioned two kinds of solution according to appendix VI B thin-layered chromatography of Pharmacopoeia of the People's Republic of China version in 2010, put respectively on same silica gel g thin-layer plate, take volume ratio 8～9:2～1 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and 105 ℃ to be dried to spot colour developing clear.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless.
Interpretation of result: Chinese juniper TLC result shows, petroleum ether-ethyl acetate take volume ratio as 8～9:2～1 has good expansion effect under developping agent condition, under the petroleum ether-ethyl acetate condition that is 8.5:1.5 in volume ratio, effect is best, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 4.The method can be used as the discrimination method of Chinese juniper in rheumatism Sialon capsule.
5, the discriminating of green suede wormwood artemisia
(1) need testing solution preparation method research:
The ultrasonic extraction of methyl alcohol: get rheumatism Sialon capsule 's content 10g, add methyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil extracts: get rheumatism Sialon capsule 's content 10g, add sherwood oil 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Acid solution decocts, adjust pH, methylene chloride extracts: get rheumatism Sialon capsule 's content 10g, add volume ratio 1% hydrochloric acid solution 30mL, ultrasonic processing 30min, filter, filtrate with 1% NaOH adjust alkali to pH10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol dissolves about 2mL, as need testing solution.
(2) research of thin layer condition::
Adopt silica G plate, developping agent petroleum ether-ethyl acetate system, compare the expansion effect of different volumes volume ratio (8:2), (8:1), (8.5:1.5), it is 10% ethanol solution of sulfuric acid that developer is selected volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt gel GF 254 plate, ethyl acetate for developping agent-methanol-water system, has compared the expansion effect of different volumes ratio (5:1:0.5), (5:0.5:0.5), (5:1.5:0.5), and inspection method inspects for putting under 254nm ultraviolet lamp.
Adopt silica G plate, normal hexane-ethyl acetate for developping agent-methyl alcohol adds two ammonia spirit systems again, the expansion effect that has compared different volumes ratio (10:4:0.1), (10:2:0.5), (10:8:1), developer is selected rare bismuth potassium iodide solution, under inspection method daylight, inspect.
The development systems such as dimethylbenzene-ethyl formate-methylene chloride of volume ratio 10:1:0.5 methenyl choloride-methanol-water, volume ratio 10:1.5:0.5 have also been selected in this experiment; Mass volume ratio is that vanillic aldehyde ethanol solution of sulfuric acid, the mass volume ratio that 1% aluminium choride ethanolic solution, mass volume ratio are 1% is the developers such as % ethanol solution of sulfuric acid.
Test by said system, select test condition and be:
Need testing solution preparation method: get rheumatism Sialon capsule 's content 5g, add volume ratio 1% hydrochloric acid solution 30mL, ultrasonic processing 30 minutes, filter, filtrate with 1% NaOH adjust alkali to pH10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol dissolves about 2mL, as need testing solution.
Normal hexane-ethyl acetate-methyl alcohol of development system: volume ratio 10:4:1, then drip two ammoniacal liquor systems.
Developer: rare bismuth potassium iodide solution.
Inspection method: inspect under daylight.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 5～10g, add volume ratio 1% hydrochloric acid solution 30～40mL, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol dissolves about 2mL, as need testing solution.Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution.Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, with volume ratio 8～10:4～6:1 normal hexane-ethyl acetate-methyl alcohol, then to drip two ammoniacal liquor be developping agent, launches, take out, dry, spray is with rare bismuth potassium iodide solution, experience under daylight.In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, negative noiseless.
Interpretation of result: green suede wormwood artemisia TLC result shows, normal hexane-ethyl acetate-the methyl alcohol of developping agent take volume ratio as 8～10:4～6:1 drips again good expansion effect under two ammoniacal liquor conditions, normal hexane-ethyl acetate-the methyl alcohol that is 10:4:1 in volume ratio adds that under two ammoniacal liquor conditions, to launch effect best again, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 5.The method can be used as the discrimination method of rheumatism Sialon capsule medium green suede wormwood artemisia medicinal material.
6, the discriminating of cloves
(1) need testing solution preparation method research:
The ultrasonic extraction of methyl alcohol: get rheumatism Sialon capsule 's content 10g, add methyl alcohol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil extracts: get rheumatism Sialon capsule 's content 5g, add sherwood oil 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds methyl alcohol 2mL and dissolves, as need testing solution.
Extract volatile oil: get rheumatism Sialon capsule 's content 10g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
(2) research of thin layer condition:
Adopt silica G plate, sherwood oil for developping agent (60～90 ℃)-ethyl acetate system, compare the expansion effect of different volumes ratio (7:1.5), (8.5:1.5), (9:1), it is 1% ethanol solution of sulfuric acid that developer is selected mass volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, methylene chloride-methanol for developping agent-formic acid system, compare the expansion effect of different volumes ratio (7:0.7:0.2), (8:1:0.2), (8:0.5:0.2), it is 10% ethanol solution of sulfuric acid that developer is selected volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, ethyl acetate-glacial acetic acid for developping agent-methyl alcohol system, compare the expansion effect of different volumes ratio (7:3:1), (8:2:1), (7:1:1), developer is selected volume ratio mark 10% ethanol solution of sulfuric acid, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
The development system such as dimethylbenzene-ethyl formate, ethyl acetate-formic acid-water of volume ratio 9:1:1 of volume ratio 5:5 has also been selected in this experiment; Mass volume ratio is the developers such as 1% aluminium choride ethanolic solution, the mass volume ratio vanillic aldehyde sulfuric acid solution that is 1%.
Test by said system, select test condition and be:
Need testing solution preparation method: get rheumatism Sialon capsule 's content 10g, add sherwood oil (60-90 ℃) 50mL ultrasonic, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 1mL as need testing solution.Development system: lower floor's solution of methenyl choloride-methanol-water-formic acid
Developer: volume fraction 10% ethanol solution of sulfuric acid.
Inspection method: 105 ℃ to be heated to spot colour developing clear, inspects under daylight.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 5～10g, add sherwood oil (60-90 ℃) 10～50mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into 2mL, as need testing solution.Separately get cloves control medicinal material 1g, be made in the same way of control medicinal material solution.Separately get in the negative control sample of the scarce cloves of prescription ratio preparation, with need testing solution, preparation method makes negative control solution.Test according to thin-layered chromatography (appendix VI B of Pharmacopoeia of the People's Republic of China version in 2010), draw each 2～8 μ L of above-mentioned 3 kinds of solution, put respectively on same silica gel g thin-layer plate, boiling range take volume ratio as 9～1:1～2 as lower floor's solution of the petroleum ether-ethyl acetate of 60～90 ℃ be developping agent, launch, take out, dry, spray is with volume ratio 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, negative noiseless.
Interpretation of result: cloves TLC result shows, lower floor's solution of sherwood oil (60～90 ℃)-ethyl acetate take volume ratio as 9～1:1～2 is developping agent, there is good expansion effect, under lower floor's solution condition of the sherwood oil that is 8.5:1.5 in volume ratio (60～90 ℃)-ethyl acetate, launch effect best.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 6.The method can be used as the discrimination method of cloves medicinal material in rheumatism Sialon capsule.
7, the discriminating of cardamom
(1) need testing solution preparation method research:
Volume fraction 95% alcohol extract: get rheumatism Sialon capsule 's content 10g, add volume fraction 95% ethanol 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, it is that 95% alcohol 2mL dissolves that residue adds volume fraction, as need testing solution.
Ethyl acetate extracts: get rheumatism Sialon capsule 's content 10g, add ethyl acetate 30mL, ultrasonic processing 30min, filters, filtrate evaporate to dryness, and residue adds ethyl acetate 2mL and dissolves, as need testing solution.
Sherwood oil (60～90 ℃) extracts: get rheumatism Sialon capsule 's content 10g, add sherwood oil (60～90 ℃) 30mL, ultrasonic processing 30min, filters, and filtrate is concentrated into 1mL, as need testing solution.
Extract volatile oil: get rheumatism Sialon capsule 's content 10g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
(2) research of thin layer condition:
Adopt silica G plate, methenyl choloride-ethyl acetate for developping agent-formic acid system, compare the expansion effect of different volumes ratio (6:4:1), (6:6:1), (6:8:1), it is 1% ferric trichloride ethanolic solution that developer is selected mass volume ratio, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, the upper solution system of dimethylbenzene-ethyl acetate for developping agent-formic acid-water, compare the expansion effect of different volumes ratio (7:10:3:4), (10:10:3:4), (4:10:3:4), it is 1% ferric trichloride ethanolic solution that developer is selected mass volume ratio, under daylight, inspects.
Adopt gel GF 254 plate, dimethylbenzene for developping agent-acetone system, compare the expansion effect of different volumes ratio (4:1), (6:1), (2:1), inspection method is to inspect under ultraviolet lamp (254nm), and then spray is with 5% vanillic aldehyde sulfuric acid solution, 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.
Adopt silica G plate, sherwood oil for developping agent-ethyl acetate system, compare the expansion effect of different volumes ratio (8:2), (9:1), (8.5:1.5), it is 10% ethanol solution of sulfuric acid that developer is selected volume fraction, inspection method is 105 ℃, and to be heated to spot colour developing clear, under daylight, inspects.
Dimethylbenzene-ethyl acetate-formic acid of volume ratio 7:3:1, the development system such as cyclohexane-ethyl acetate, dimethylbenzene-ethyl acetate of volume ratio 1:1 of volume ratio 4:1 have also been selected in this experiment; Mass volume ratio is that 1% aluminium choride ethanolic solution, mass volume ratio are the developers such as 5% phosphomolybdic acid ethanol solution.
Test by said system, select test condition and be:
Need testing solution preparation method: get rheumatism Sialon capsule 's content 5g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after decoction completes, collect ethyl acetate part, as need testing solution;
Developping agent: petroleum ether-ethyl acetate system.
Developping agent: volume ratio is 8.5:1.5.
Inspection method: spray is with volume ratio 1% ethanol solution of sulfuric acid, 105 ℃ to be heated to spot colour developing clear, under daylight, inspects.
Concrete testing program is:
Get rheumatism Sialon capsule 's content 10～20g, put in 100mL round-bottomed flask, 50mL adds water, decoct 1h, collect volatile oil (starting to add a small amount of water and 1mL ethyl acetate in volatile oil determination apparatus) with volatile oil determination apparatus, after having decocted, collect ethyl acetate part, as need testing solution; Get cardamom control medicinal material 0.5g, be made in the same way of control medicinal material solution.Separately get in the negative control sample of the cardamom of prescription ratio preparation, with need testing solution, preparation method makes negative control solution.Test according to appendix VI B thin-layered chromatography of Pharmacopoeia of the People's Republic of China version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8～9:2～1 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and 105 ℃ to be dried to spot colour developing clear.。In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless.
Interpretation of result: cardamom TLC result shows, petroleum ether-ethyl acetate take volume ratio as 8～9:2～1 has good expansion effect under developping agent condition, under the petroleum ether-ethyl acetate condition that is 8.5:1.5 in volume ratio, effect is best, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color, negative noiseless, the results are shown in Figure 7.The method can be used as the discrimination method of cardamom in rheumatism Sialon capsule.
Experimental example 2: the checking experiment of aristolochic acid A
1. instrument, reagent and test sample
Instrument: Agilent 1200 type high performance liquid chromatographs; Shimadzu AuW220D electronic balance.
Reference substance: aristolochic acid A reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 110746-200806.
Sample: rheumatism Sialon capsule, 0.3g/ grain (Qinghai gold is scolded Tibetan medicine medicine company incorporated company), lot number: 20110601,20110602,20110603.
2. detect the selection of wavelength
Get aristolochic acid A reference substance solution, in 190 ~ 400nm wavelength coverage, scan, aristolochic acid A has absorption maximum at 315nm wavelength place, therefore be detection wavelength according to the selected 315nm of ultraviolet absorpting spectrum.
3. the selection of mobile phase
Methanol-water system, methyl alcohol-volume parts are studied respectively than under 1% glacial acetic acid aqueous solution system mobile phase condition, the separating effect of aristolochic acid A chromatographic peak, found that take methyl alcohol-volume parts than 1% glacial acetic acid aqueous solution system as mobile phase, the peak shape of aristolochic acid A chromatographic peak is better, methyl alcohol-volume parts is than 1% glacial acetic acid aqueous solution in the time that volume parts ratio is 40:60, and aristolochic acid A can reach good chromatographic resolution effect.
4. the preparation of reference substance solution
Get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1mL containing 500ng, to obtain final product.
5. the preparation of need testing solution
By method under assay item, need testing solution is detected.Press the method under the preparation of need testing solution, investigated respectively under Different Extraction Method, different solvents, different extraction time condition the extraction effect of aristolochic acid A.Determine extracting method take the content of aristolochic acid A in every gram of medicine as index, extract solvent, extraction time.The results are shown in Table 1, table 2 and table 3.
Table 1 extracting method is investigated test findings
Figure BDA00002541818100161
Table 2 extracts solvent and investigates test findings
Table 3 extraction time is investigated test findings
Figure BDA00002541818100172
Result shows: ultrasonic basically identical with the content measured aristolochic acid A of refluxing extraction, consider that ultrasonic extraction is easy and simple to handle, therefore selective extraction method is ultrasonic extraction; Methyl alcohol extracts the content of measured aristolochic acid A apparently higher than ethanol, therefore selective extraction solvent is methyl alcohol; Extract 40 minutes and the content of 50 minutes measured aristolochic acid As basically identical, therefore the selective extraction time is 40 minutes.Preferably concrete grammar is as follows:
Get the about 10g of Tibetan medicinal composition rheumatism Sialon capsule 's content fine powder, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25mL, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20mL to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20mL, after extraction, alkali lye uses volume parts to regulate pH2 ~ 3 than 5% hydrochloric acid, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1mL volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain.
6. system suitability
Press method under assay item, accurate aristolochic acid A reference substance solution, the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography, records chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, aristolochic acid A is adjacent chromatographic peak is all greater than 1.5.The results are shown in accompanying drawing 8, Fig. 9.
7. the preparation of typical curve and the investigation of linear relationship
Precision measures aristolochic acid A reference substance stock solution solution (938.71ng/mL) 1mL, 3mL, 5mL, 8mL, 10mL, put respectively in 10mL volumetric flask, methyl alcohol is diluted to scale, shake up, each accurate sample introduction 20 μ L, take peak area A as ordinate, reference substance concentration C is horizontal ordinate, carry out linear regression, obtain aristolochic acid A regression equation: A=0.4263C+0.2744, related coefficient: R=0.9997.Result shows, aristolochic acid A is within the scope of 93.871ng/mL ~ 938.71ng/mL, and peak area A and reference substance concentration C linear relationship are good.The results are shown in Table 4.
Table 4 linear relationship is investigated result
8. precision test
The accurate aristolochic acid A reference substance solution 20 μ L that draw respectively, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, this instrument precision is good.The results are shown in Table 5.
Table 5 Precision test result
Figure BDA00002541818100181
9. quantitative limit
Accurate absorption in aristolochic acid A contrast solution (469.35ng/mL) 1mL to 25mL volumetric flask, dilute and is settled to scale with methyl alcohol, shakes up accurate absorption 20 μ L, injection liquid chromatography.The signal to noise ratio (S/N ratio) of result aristolochic acid A chromatographic peak is about 10.Result shows: when aristolochic acid A concentration is 18.77ng/mL, signal to noise ratio (S/N ratio) is about 10, and aristolochic acid A is quantitatively limited to 0.375ng.
10. stability test
After prepared by need testing solution, the accurate 20 μ L that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 8 hours, records peak area and calculates relative standard deviation.Result shows, in need testing solution, aristolochic acid A measurement result in 8 hours is stable.The results are shown in Table 6.
Table 6 stability test result
Figure BDA00002541818100182
11. replica tests
Precision takes with a collection of Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 10g of content fine powder, accurately weighed, put in the conical flask of tool plug, totally 6 parts, prepare need testing solution by the method under the preparation of need testing solution, accurate 20 μ L, injection liquid chromatography, the content of aristolochic acid A in calculation sample drawn respectively.Result shows, this analytical approach repeatability is good.The results are shown in Table 7.
Table 7 replica test result
Figure BDA00002541818100183
Figure BDA00002541818100191
12. recovery tests
Precision takes with a collection of Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 5g of content fine powder, 6 parts, each precision adds aristolochic acid A reference substance, measures its content, calculate recovery rate totally.Result shows, this assay method measurement result is accurate.The results are shown in Table 8.
Table 8 recovery test result
Figure BDA00002541818100192
13. sample inspections
Get three batches of Tibetan medicinal composition rheumatism Sialon capsules, measure and calculate Determination of Aristolochic Acid A.The results are shown in Table 9.
Table 9 sample check result
Figure BDA00002541818100193
Experimental example 3: the assay experiment of general flavone
1. instrument, reagent and test sample
Instrument: Shimadzu UV-2450 type ultraviolet-visible spectrophotometer; Shimadzu AuW220D type electronic balance.
Reference substance: control substance of Rutin (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 100080-200306.
Sample: rheumatism Sialon capsule, 0.3g/ grain (Qinghai gold is scolded Tibetan medicine medicine company incorporated company), lot number: 20110601,20110602,20110603.
2. the preparation of reference substance solution
Precision takes control substance of Rutin 10.06mg, puts in 50mL measuring bottle, adds appropriate amount of ethanol, puts in water-bath low-grade fever and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and obtains that (every 1mL containing rutin 201.2 μ g).
3. detect the selection of wavelength
The accurate reference substance solution 0.0 of label taking, 3.0mL, put respectively in 25mL measuring bottle, respectively adds water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15 minutes, make full wavelength scanner with ultraviolet-visible spectrophotometer at 400 ~ 600nm, determine maximum absorption wavelength.Known according to ultraviolet absorpting spectrum, reference substance solution has absorption maximum at 500nm wavelength place, therefore determine that 500nm is for detecting wavelength.
4. the preparation of typical curve and the investigation of linear relationship
Precision measures control substance of Rutin liquid (201.2 μ g/mL) 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, puts respectively in 25mL measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; According to UV-VIS spectrophotometry (appendix V A of " Chinese Pharmacopoeia " version in 2010), measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve, obtains regression equation A=0.0101C+0.0021, coefficient R=0.9995.Result shows, rutin-within the scope of 8.048 μ g/mL ~ 40.240 μ g/mL, absorbance (A) is good with reference substance concentration (C) linear relationship.The results are shown in Table 10.
Table 10 linear relationship is investigated result
Figure BDA00002541818100201
5. the assay of general flavone in sample
Get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content fine powder, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50mL, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25mL puts in 100mL measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0mL, puts in 25mL measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0mL ", is measured absorbance in accordance with the law, reads the content (μ g/mL) of rutin in need testing solution from typical curve, calculates, and to obtain final product.
6. precision test
Precision measures control substance of Rutin solution 3.0mL and puts in 25mL measuring bottle, and the method under the preparation of sighting target directrix curve, from " adding water to 5.0mL ", is measured absorbance in accordance with the law, METHOD FOR CONTINUOUS DETERMINATION 6 times.Result shows, instrument precision is good.The results are shown in Table 11.
Table 11 Precision test result
Figure BDA00002541818100202
7. replica test
Precision takes with a collection of Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 1g of content fine powder, accurately weighed, put in the conical flask of tool plug, totally 6 parts, prepare sample solution by the method under the assay item of general flavone in sample, measure absorbance, the content of general flavone in calculation sample in accordance with the law.Result shows, this analytical approach repeatability is good.The results are shown in Table 12.
Table 12 replica test result
Figure BDA00002541818100211
8. recovery test
Precision takes the Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 0.5g of content fine powder with a collection of known general flavone content, totally 6 parts, each precision adds control substance of Rutin reference substance, prepare sample solution by the method under the assay item of general flavone in sample, measure absorbance in accordance with the law, the content of general flavone in calculation sample, calculate recovery rate.Result shows, this assay method measurement result is accurate.The results are shown in Table 13.
Table 13 recovery test result
9. sample determination
Get three batches of Tibetan medicinal composition rheumatism Sialon capsules, measure and calculate general flavone content.The results are shown in Table 14.
Table 14 sample determination result
Figure BDA00002541818100213
Experimental example 4: the assay experiment of Indian Herba Swertiae bimaculatae
1. instrument, reagent and test sample
Instrument: Agilent 1200 type high performance liquid chromatographs; Shimadzu AuW220D electronic balance.
Reference substance: Swertiamarin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 0785-200203.
Sample: rheumatism Sialon capsule, 0.3g/ grain (Qinghai gold is scolded Tibetan medicine medicine company incorporated company), lot number: 20110601,20110602,20110603.
2. detect the selection of wavelength
Get Swertiamarin reference substance solution, in 190 ~ 400nm wavelength coverage, scan, Swertiamarin has absorption maximum at 243nm wavelength place, therefore be detection wavelength according to the selected 243nm of ultraviolet absorpting spectrum.
3. the selection of mobile phase
Methanol-water system, methyl alcohol-volume parts are studied respectively than under 1% glacial acetic acid aqueous solution system mobile phase condition, the separating effect of Swertiamarin chromatographic peak, found that take methyl alcohol-volume parts than 1% glacial acetic acid aqueous solution system as mobile phase, the peak shape of Swertiamarin chromatographic peak is better, methyl alcohol-volume parts is than 1% glacial acetic acid aqueous solution in the time that volume parts ratio is 25:75, and Swertiamarin can reach good chromatographic resolution effect.
4. the preparation of reference substance solution
Get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1mL containing 45 μ g, to obtain final product.
5. the preparation of need testing solution
By method under assay item, need testing solution is detected.Press the method under the preparation of need testing solution, investigated respectively under Different Extraction Method, different solvents, different extraction time condition the extraction effect of Swertiamarin.Determine extracting method take the content of Swertiamarin in every gram of medicine as index, extract solvent, extraction time.The results are shown in Table 24, table 25 and table 26.
Table 15 extracting method is investigated test findings
Figure BDA00002541818100221
Table 16 extracts solvent and investigates test findings
Figure BDA00002541818100222
Table 17 extraction time is investigated test findings
Figure BDA00002541818100223
Result shows: the content of the measured Swertiamarin of refluxing extraction is the highest, therefore selective extraction method is refluxing extraction; The content of methyl alcohol and the measured Swertiamarin of alcohol extract is basically identical, considers methyl alcohol toxicity, therefore selective extraction solvent is ethanol; Extract 1 hour and the content of 1.5 hours measured Swertiamarins basically identical, therefore the selective extraction time is 1 hour.Preferably concrete grammar is as follows:
Get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content fine powder, accurately weighed, put in round-bottomed flask, precision adds ethanol 50mL, close plug, weighed weight, refluxing extraction 1 hour, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, get subsequent filtrate 25mL, be evaporated to dry, residue add water about 20mL make dissolve, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate liquid, is evaporated to dryly, and residue adds ethanol and dissolves and be settled in 5mL volumetric flask, shake up, filter, get subsequent filtrate, to obtain final product.
6. system suitability
Press method under assay item, accurate Swertiamarin reference substance solution, need testing solution, the each 10 μ L of negative control solution of drawing respectively, injection liquid chromatography, records chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, Swertiamarin is adjacent chromatographic peak is all greater than 1.5, and negative control does not disturb.The results are shown in accompanying drawing 10, Figure 11, Figure 12.
7. the preparation of typical curve and the investigation of linear relationship
Precision measures Swertiamarin reference substance stock solution solution (90.42 μ g/mL) 1mL, 3mL, 5mL, 8mL, 10mL, put respectively in 10mL volumetric flask, ethanol is diluted to scale, shake up, each accurate sample introduction 10 μ L, take peak area A as ordinate, reference substance concentration C is horizontal ordinate, carry out linear regression, obtain Swertiamarin regression equation: A=6.3486C+0.4797, related coefficient: R=0.9999.Result shows, Swertiamarin is within the scope of 9.042 μ g/mL ~ 90.42 μ g/mL, and peak area A and reference substance concentration C linear relationship are good.The results are shown in Table 27.
Table 18 linear relationship is investigated result
Figure BDA00002541818100231
8. precision test
The accurate Swertiamarin reference substance solution 10 μ L that draw respectively, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, this instrument precision is good.The results are shown in Table 28.
Table 19 Precision test result
Figure BDA00002541818100232
9. stability test
After prepared by need testing solution, the accurate 10 μ L that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 8 hours, records peak area and calculates relative standard deviation.Result shows, in need testing solution, Swertiamarin measurement result in 8 hours is stable.The results are shown in Table 29.
Table 20 stability test result
Figure BDA00002541818100233
Figure BDA00002541818100241
10. replica test
Precision takes with a collection of Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 1g of content fine powder, accurately weighed, put in the conical flask of tool plug, totally 6 parts, prepare need testing solution by the method under the preparation of need testing solution, accurate 10 μ L, injection liquid chromatography, the content of Swertiamarin in calculation sample drawn respectively.Result shows, this analytical approach repeatability is good.The results are shown in Table 30.
Table 21 replica test result
Figure BDA00002541818100242
11. recovery tests
Precision takes with a collection of Tibetan medicinal composition rheumatism Sialon capsule (product batch number: 20110601) the about 0.5g of content fine powder, 6 parts, each precision adds Swertiamarin reference substance, measures its content, calculate recovery rate totally.Result shows, this assay method measurement result is accurate.The results are shown in Table 31.
Table 22 recovery test result
12. sample determinations
Get three batches of Tibetan medicinal composition rheumatism Sialon capsules, measure and calculate Swertiamarin content.The results are shown in Table 32.
Table 23 sample determination result
Figure BDA00002541818100251
Accompanying drawing explanation
Fig. 1 is the thin layer identification color spectrogram of shellac in rheumatism Sialon capsule, and wherein, a is control medicinal material, and a' is test sample, a " is negative control;
Fig. 2 is the thin layer identification color spectrogram of the banksia rose in rheumatism Sialon capsule, and wherein, b is control medicinal material, and b' is test sample, b " is negative control;
Fig. 3 is the thin layer identification color spectrogram of ZANGQIANCAO in rheumatism Sialon capsule, and wherein, c is control medicinal material, and c' is test sample, c " is negative control;
Fig. 4 is the thin layer identification color spectrogram of Chinese juniper in rheumatism Sialon capsule, and wherein, d is control medicinal material, and d' is test sample, d " is negative control;
Fig. 5 is the thin layer identification color spectrogram of rheumatism Sialon capsule medium green suede wormwood artemisia, and wherein, e is control medicinal material, and e' is test sample, e " is negative control;
Fig. 6 is the thin layer identification color spectrogram of cloves in rheumatism Sialon capsule, and wherein, f is control medicinal material, and f ' is test sample, f " is negative control;
Fig. 7 is the thin layer identification color spectrogram of cardamom in rheumatism Sialon capsule, and wherein, g is control medicinal material, and g' is test sample, g " is negative control;
Fig. 8 is aristolochic acid A contrast high-efficient liquid phase chromatogram;
Fig. 9 is that in rheumatism Sialon capsule, aristolochic acid A checks test sample high-efficient liquid phase chromatogram;
Figure 10 is Swertiamarin contrast high-efficient liquid phase chromatogram;
Figure 11 is Swertiamarin assay test sample high-efficient liquid phase chromatogram in rheumatism Sialon capsule;
Figure 12 is that rheumatism Sialon capsule lacks Indian Herba Swertiae bimaculatae negative control high-efficient liquid phase chromatogram;
Wherein, the horizontal ordinate of Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12 is time (min), and ordinate is absorbance (mAU).
Embodiment
Below in conjunction with embodiment, the present invention is described in detail, but is not limited to these concrete embodiment recording.The rheumatism Sialon capsule that embodiment 1 detects is that Qinghai gold is scolded the production of Tibetan medicine medicine company incorporated company.The rheumatism Sialon particle that embodiment 2 detects is the particle by rheumatism Sialon capsule bulk drug formulated.
Embodiment 1: the quality determining method of rheumatism Sialon capsule
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon capsule 's content 10g, add ethyl acetate 20ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2ml, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 5: 5: 0.5: 0.1 dimethylbenzene-methylene chloride-acetone-formic acid is developping agent, launch, take out, dry, put respectively under daylight and inspect; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon capsule 's content 3g, the 15ml that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 8: 1 methylene chloride-cyclohexanes as developping agent, launch, take out, dry, spray is with 1% vanillic aldehyde sulfuric acid solution, and hot blast blows to spot and develops the color in clear test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon capsule 's content 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon capsule 's content 5g, add methyl alcohol 10ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1ml, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 4:160～90, ℃ sherwood oil-acetone is developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon capsule 's content 10g, add volume ratio 1% hydrochloric acid solution 30ml, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract 3 times, combined dichloromethane liquid, evaporate to dryness, adds methyl alcohol and dissolves to 2ml, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, drip again two ammoniacal liquor as developping agent take volume ratio 10:4:1 normal hexane-ethyl acetate-methyl alcohol, launch, take out, dry, spray is with rare bismuth potassium iodide solution, experience under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon capsule 's content 10g, add 60-90 ℃ of sherwood oil 50ml ultrasonic, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 1ml as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 60～90 ℃ of petroleum ether-ethyl acetates of volume ratio 8:2 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon capsule 's content 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take volume ratio 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay:
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50ml measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, puts respectively in 25ml measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0ml, add weight volume parts than 5% sodium nitrite test solution 1ml, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium test solution 10ml, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50ml, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25ml puts in 100ml measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0ml, puts in 25ml measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0ml ", is measured absorbance in accordance with the law, reads the content (μ g/ml) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g.
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1ml containing 45 μ g, to obtain final product;
The preparation of need testing solution: get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in round-bottomed flask, precision adds ethanol 50ml, close plug, weighed weight, refluxing extraction 1 hour, let cool, weighed weight again, supply the weight of less loss with ethanol, shake up, filter, get subsequent filtrate 25ml, be evaporated to dry, residue add water about 20ml make dissolve, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate liquid, be evaporated to dry, residue adds ethanol and dissolves and be settled in 5ml volumetric flask, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g.
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40:60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 500ng, to obtain final product;
The preparation of need testing solution: get Tibetan medicinal composition rheumatism Sialon capsule 's content 10g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20ml to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20ml, after extraction, alkali lye uses volume parts to regulate pH2～3 than 5% hydrochloric acid, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1ml volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon capsule of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.
Embodiment 2: the quality determining method of rheumatism Sialon particle
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon particle 10g, add ethyl acetate 20ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2ml, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 5: 5: 0.5: 0.1 dimethylbenzene-methylene chloride-acetone-formic acid is developping agent, launch, take out, dry, put respectively under daylight and inspect; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon particle 3g, the 15ml that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 8: 1 methylene chloride-cyclohexanes as developping agent, launch, take out, dry, spray is with 1% vanillic aldehyde sulfuric acid solution, and hot blast blows to spot and develops the color in clear test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon particle 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon particle 5g, add methyl alcohol 10ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1ml, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 4:160～90, ℃ sherwood oil-acetone is developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon particle 10g, add volume ratio 1% hydrochloric acid solution 30ml, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract 3 times, combined dichloromethane liquid, evaporate to dryness, adds methyl alcohol and dissolves to 2ml, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, drip again two ammoniacal liquor as developping agent take volume ratio 10:4:1 normal hexane-ethyl acetate-methyl alcohol, launch, take out, dry, spray is with rare bismuth potassium iodide solution, experience under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon particle 10g, add 60-90 ℃ of sherwood oil 50ml ultrasonic, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 1ml as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 60～90 ℃ of petroleum ether-ethyl acetates of volume ratio 8:2 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon particle 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take volume ratio 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay:
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50ml measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, puts respectively in 25ml measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0ml, add weight volume parts than 5% sodium nitrite test solution 1ml, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium test solution 10ml, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon particle 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50ml, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25ml puts in 100ml measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0ml, puts in 25ml measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0ml ", is measured absorbance in accordance with the law, reads the content (μ g/ml) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon particle of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g.
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1ml containing 45 μ g, to obtain final product;
The preparation of need testing solution: get Tibetan medicinal composition rheumatism Sialon particle 1g, accurately weighed, put in round-bottomed flask, precision adds ethanol 50ml, close plug, weighed weight, refluxing extraction 1 hour, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, get subsequent filtrate 25ml, be evaporated to dry, residue add water about 20ml make dissolve, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate liquid, is evaporated to dryly, and residue adds ethanol and dissolves and be settled in 5ml volumetric flask, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon particle of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g.
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40:60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 500ng, to obtain final product;
The preparation of need testing solution: get Tibetan medicinal composition rheumatism Sialon particle 10g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20ml to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20ml, after extraction, alkali lye uses volume parts to regulate pH2～3 than 5% hydrochloric acid, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1ml volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product; Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon particle of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.

Claims (4)

1. a bulk drug consists of Bailey Myospalax Born 3.2 weight portions, myrobalan's 36.3 weight portions, safflower 21.1 weight portions, cardamom 28.5 weight portions, ichor cream 10.6 weight portions, Indian Herba Swertiae bimaculatae 10.6 weight portions, sword bean 10.6 weight portions, caudate sweetleaf leaf 10.6 weight portions, ZANGQIANCAO 10.6 weight portions, shellac 10.6 weight portions, Chinese juniper 10.6 weight portions, borneol 3.2 weight portions, Tabasheer 10.6 weight portions, cloves 4.2 weight portions, nutmeg 6.3 weight portions, tsaoko 9.0 weight portions, agalloch eaglewood 5.3 weight portions, Santalum album 9.5 weight portions, santal 6.1 weight portions, green suede wormwood artemisia 6.1 weight portions, common bombax flower 11.3 weight portions, the banksia rose 9.5 weight portions, Cuminum celery 9.5 weight portions, banksia rose birthwort 5.3 weight portions, Chinese cassia tree 9.5 weight portions, spiral shell operculum 6.1 weight portions, the stem of noble dendrobium 6.1 weight portions, rhizoma nardostachyos 8.2 weight, parmelia saxatilis 14.7 weight portions, russian fenugreek herb 5.3 weight portions, terminaliae billericae,fructus 5.3 weight portions, the quality determining method of the rheumatism Sialon preparation of emblic 6.3 weight portions, is characterized in that, the method comprises one or more in following discriminating and/or assay:
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon solid pharmaceutical preparation 5～15g, add ethyl acetate 10～20mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into 1～2mL, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 5～10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take dimethylbenzene-methylene chloride-acetone-formic acid of volume ratio 4～6:4～6:0.5:1 as developping agent, launch, take out, dry, put respectively under daylight and inspect, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon solid pharmaceutical preparation 2～5g, the 10～20mL that adds methylene chloride, ultrasonic processing 20～40 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 5～10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the methylene chloride-cyclohexane of volume ratio 5～8:1～2 as developping agent, launch, take out, dry, spray is take volume ratio as 1% vanillic aldehyde sulfuric acid solution, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon solid pharmaceutical preparation 5～15g, the 50～150mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5～1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～5 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 7～9:3～1 as developping agent, launch, take out, dry, spray is take volume ratio as 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, add methyl alcohol 10～20mL, ultrasonic processing 20～40 minutes, filters, and filtrate is concentrated into approximately 1～2mL, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the boiling range of volume ratio 2～6:1～2 as 60～90 ℃ of sherwood oil-acetone are as developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, add volume ratio 1% hydrochloric acid solution 20～30mL, ultrasonic processing 30～40 minutes, filter, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol dissolves about 2mL, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, drip again two ammoniacal liquor as developping agent take normal hexane-ethyl acetate-methyl alcohol of volume ratio 10～8:4～5:1, launch, take out, dry, spray, with rare bismuth potassium iodide solution, is inspected under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon solid pharmaceutical preparation 5～10g, adding boiling range is that 60-90 ℃ of sherwood oil 50～80mL is ultrasonic, and ultrasonic processing 30～40 minutes filters, and filtrate is concentrated into 1～2mL as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take sherwood oil (60～90 ℃)-ethyl acetate of volume ratio 8～9:2～1 as developping agent, launch, take out, dry, spray is take volume ratio as 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon solid pharmaceutical preparation 10～20g, the 80～100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw each 2～3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8～9:2～1 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50mL measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, puts respectively in 25mL measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 8-10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15-20 minute, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon preparation fine powder 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50mL, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25mL puts in 100mL measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0mL, puts in 25mL measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0mL ", is measured absorbance in accordance with the law, reads the content (μ g/mL) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon preparation of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g;
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:70-75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1mL containing 45 μ g, to obtain final product;
The preparation of need testing solution: get the about 1g of Tibetan medicinal composition rheumatism Sialon preparation fine powder, accurately weighed, put in round-bottomed flask, precision adds ethanol 50mL, close plug, weighed weight, refluxing extraction 1 hour, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, get subsequent filtrate 25mL, be evaporated to dry, residue add water about 20mL make dissolve, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate liquid, is evaporated to dryly, and residue adds ethanol and dissolves and be settled in 5mL volumetric flask, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon preparation of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g;
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40-45:55-60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1mL containing 500ng, to obtain final product;
The preparation of need testing solution: get the about 10g of Tibetan medicinal composition rheumatism Sialon preparation fine powder, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25mL, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20mL to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20mL, after extraction, alkali lye uses volume parts than 5% salt acid for adjusting pH 2～3, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1mL volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon preparation of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.
2. the quality determining method of rheumatism Sialon preparation as claimed in claim 1, preparation wherein refers to the bulk drug of getting the claims 1, technique routinely adds conventional auxiliary material to be prepared into clinical acceptable any formulation.
3. the quality determining method of rheumatism Sialon preparation as claimed in claim 1 or 2, is characterized in that, the method comprises one or more in following discriminating and/or content assaying method:
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon capsule 's content 10g, add ethyl acetate 20mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2mL, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 5: 5: 0.5: dimethylbenzene-methylene chloride-acetone-formic acid of 0.1 is developping agent, launch, take out, dry, put respectively under daylight and inspect; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon capsule 's content 3g, the 15mL that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the volume ratio methylene chloride-cyclohexane of 8: 1 as developping agent, launch, take out, dry, spray is with 1% vanillic aldehyde sulfuric acid solution, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon capsule 's content 10g, the 100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8.5:1.5 as developping agent, launch, take out, dry, spray is with 1% ethanol solution of sulfuric acid, it is clear that hot blast blows to spot colour developing, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon capsule 's content 5g, add methyl alcohol 10mL, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1mL, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, boiling range take volume ratio as 4:1 as 60～90 ℃ of sherwood oil-acetone be developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon capsule 's content 10g, add volume ratio 1% hydrochloric acid solution 30mL, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH10, then with methylene chloride equal-volume extract three times, combined dichloromethane liquid, evaporate to dryness, methyl alcohol is dissolved to 2mL, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take volume ratio as 10:4:1 normal hexane-ethyl acetate-methyl alcohol, then to drip two ammoniacal liquor be developping agent, launches, take out, dry, spray, with rare bismuth potassium iodide solution, is inspected under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon capsule 's content 10g, the sherwood oil 50mL that adds boiling range and be 60-90 ℃ is ultrasonic, and ultrasonic processing 30 minutes filters, and filtrate is concentrated into 1mL as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 5 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take the boiling range 60-90 ℃ of petroleum ether-ethyl acetate of volume ratio 8:2 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon capsule 's content 10g, the 100mL that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of " Chinese Pharmacopoeia " version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take the petroleum ether-ethyl acetate of volume ratio 8.5:1.5 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50mL measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, puts respectively in 25mL measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0mL, add weight volume parts than 5% sodium nitrite test solution 1mL, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1mL, shake up, place 6 minutes, hydro-oxidation sodium test solution 10mL, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon capsule 's content 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50mL, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25mL puts in 100mL measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0mL, puts in 25mL measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0mL ", is measured absorbance in accordance with the law, reads the content (μ g/mL) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g;
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1mL containing 45 μ g, to obtain final product;
The preparation of need testing solution: get the about 1g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in round-bottomed flask, precision adds ethanol 50mL, close plug, weighed weight, refluxing extraction 1 hour, let cool, weighed weight again, supply the weight of less loss with ethanol, shake up, filter, get subsequent filtrate 25mL, be evaporated to dry, residue add water about 20mL make dissolve, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate liquid, be evaporated to dry, residue adds ethanol and dissolves and be settled in 5mL volumetric flask, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g;
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40:60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1mL containing 500ng, to obtain final product;
The preparation of need testing solution: get the about 10g of Tibetan medicinal composition rheumatism Sialon capsule 's content, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25mL, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20mL to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20mL, after extraction, alkali lye uses volume parts than 5% salt acid for adjusting pH 2～3, be extracted with ethyl acetate 5 times, each 20mL, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1mL volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon capsule of the present invention, the content of aristolochic acid A (C17H11NO7) must not be higher than 10 μ g/g.
4. the quality determining method of rheumatism Sialon preparation as claimed in claim 1 or 2, is characterized in that, the method comprises one or more in following discriminating and/or content assaying method:
Differentiate:
(1) discriminating of shellac
Get rheumatism Sialon particle 10g, add ethyl acetate 20ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2ml, as need testing solution; Separately get shellac control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 5: 5: 0.5: 0.1 dimethylbenzene-methylene chloride-acetone-formic acid is developping agent, launch, take out, dry, put respectively under daylight and inspect; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, spot and the fluorescence spot of aobvious same color;
(2) discriminating of the banksia rose
Get rheumatism Sialon particle 3g, the 15ml that adds methylene chloride, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution; Get banksia rose control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 10 μ L of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 8: 1 methylene chloride-cyclohexanes as developping agent, launch, take out, dry, spray is with 1% vanillic aldehyde sulfuric acid solution, and hot blast blows to spot and develops the color in clear test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) discriminating of Chinese juniper
Get rheumatism Sialon particle 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get Chinese juniper control medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) discriminating of ZANGQIANCAO
Get rheumatism Sialon particle 5g, add methyl alcohol 10ml, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into about 1ml, as need testing solution; Separately get ZANGQIANCAO medicinal material 0.5g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 4:160～90, ℃ sherwood oil-acetone is developping agent, launch, take out, dry, put under 365nm ultraviolet lamp and inspect; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(5) discriminating of green suede wormwood artemisia
Get rheumatism Sialon particle 10g, add volume ratio 1% hydrochloric acid solution 30ml, ultrasonic processing 30 minutes, filters, filtrate with 1% NaOH adjust alkali to pH be 10, then with methylene chloride equal-volume extract 3 times, combined dichloromethane liquid, evaporate to dryness, adds methyl alcohol and dissolves to 2ml, as need testing solution; Separately get green suede wormwood artemisia medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, drip again two ammoniacal liquor as developping agent take volume ratio 10:4:1 normal hexane-ethyl acetate-methyl alcohol, launch, take out, dry, spray is with rare bismuth potassium iodide solution, experience under daylight; In test sample chromatogram, with control medicinal material and the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;
(6) discriminating of cloves
Get rheumatism Sialon particle 10g, add 60-90 ℃ of sherwood oil 50ml ultrasonic, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 1ml as need testing solution; Separately get cloves medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw two kinds of each 5 μ L of solution, put respectively on the same silica gel g thin-layer plate take sodium carboxymethyl cellulose as binder, take 60～90 ℃ of petroleum ether-ethyl acetates of volume ratio 8:2 as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(7) discriminating of cardamom
Get rheumatism Sialon particle 10g, the 100ml that adds water, by extraction by steam distillation volatile oil, volatile oil is as test sample; Get cardamom control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to appendix VI B thin-layered chromatography of Chinese Pharmacopoeia version in 2010, draw the each 3 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take volume ratio 8.5:1.5 petroleum ether-ethyl acetate as developping agent, launch, take out, dry, spray is with volume ratio 1% ethanol solution of sulfuric acid, and it is clear that hot blast blows to spot colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay:
(1) assay of general flavone
The preparation of reference substance solution: precision takes control substance of Rutin 10mg, puts in 50ml measuring bottle, adds appropriate amount of ethanol, puts low-grade fever in water-bath and makes to dissolve, and lets cool, and adds ethanol to scale, shakes up, and to obtain final product;
The preparation of typical curve: precision measures above-mentioned reference substance liquid 0.0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, puts respectively in 25ml measuring bottle, is numbered 1 to No. 6; Respectively add water to 5.0ml, add weight volume parts than 5% sodium nitrite test solution 1ml, mix, place 6 minutes, add weight volume parts than 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium test solution 10ml, add water to again scale, shake up, place 15 minutes, take No. 1 as blank; Test according to appendix V A UV-VIS spectrophotometry of " Chinese Pharmacopoeia " version in 2010, measure absorbance at 500nm wavelength place, take absorbance as ordinate, reference substance concentration is horizontal ordinate, drawing standard curve;
Determination method: get Tibetan medicinal composition rheumatism Sialon particle 1g, accurately weighed, put in the conical flask of tool plug, precision adds ethanol 50ml, close plug, weighed weight, ultrasonic processing 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, getting subsequent filtrate 25ml puts in 100ml measuring bottle, add ethanol to scale, shake up, obtain need testing solution; Precision measures need testing solution 4.0ml, puts in 25ml measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 5.0ml ", is measured absorbance in accordance with the law, reads the content (μ g/ml) of rutin in need testing solution from typical curve, calculates, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon particle of the present invention, the content of general flavone is by rutin (C 27h 30o 16) meter, must not be less than 25mg/g;
(2) assay of Indian Herba Swertiae bimaculatae
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take its silane group silica gel of octadecane as filling agent; Take volume parts than methyl alcohol-volume parts of 25:75 than 1% glacial acetic acid aqueous solution as mobile phase; Detect wavelength 243nm; Number of theoretical plate calculates and should be not less than 2500 by Swertiamarin peak;
The preparation of reference substance solution: get Swertiamarin reference substance appropriate, add ethanol and make the solution of every 1ml containing 45 μ g, to obtain final product;
The preparation of need testing solution: get Tibetan medicinal composition rheumatism Sialon particle 1g, accurately weighed, put in round-bottomed flask, precision adds ethanol 50ml, close plug, weighed weight, refluxing extraction 1 hour, lets cool, more weighed weight, the weight of supplying less loss with ethanol, shakes up, and filters, get subsequent filtrate 25ml, be evaporated to dry, residue add water about 20ml make dissolve, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate liquid, is evaporated to dryly, and residue adds ethanol and dissolves and be settled in 5ml volumetric flask, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: draw respectively the each 10 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
In Tibetan medicinal composition rheumatism Sialon particle of the present invention, the content of Indian Herba Swertiae bimaculatae is pressed Swertiamarin (C 16h 22o 10) meter, must not be less than 0.35mg/g;
(3) inspection of aristolochic acid A
Measure according to appendix VI D high performance liquid chromatography of " Chinese Pharmacopoeia " version in 2010;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than methyl alcohol-volume parts of 40:60 than 1% glacial acetic acid aqueous solution as mobile phase; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;
The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 500ng, to obtain final product;
The preparation of need testing solution: get Tibetan medicinal composition rheumatism Sialon particle 10g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, residue adds quality volume portion rate 0.5% sodium hydroxide solution 20ml to be dissolved it completely and is transferred in separating funnel, be extracted with ethyl acetate 2 times, each 20ml, after extraction, alkali lye uses volume parts than 5% salt acid for adjusting pH 2～3, be extracted with ethyl acetate 5 times, each 20ml, combined ethyl acetate extract, volatilize, dissolve and be transferred in 1ml volumetric flask with methyl alcohol, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain,
Determination method: draw respectively the each 20 μ L of reference substance solution and need testing solution, injection liquid chromatography, measures, and to obtain final product;
Aristolochic acid A (C in Tibetan medicinal composition rheumatism Sialon particle of the present invention 17h 11nO 7) content must not be higher than 10 μ g/g.
CN201210523974.9A 2012-12-07 2012-12-07 A kind of quality determining method of Tibetan medicinal composition rheumatism Sialon preparation Active CN103852555B (en)

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