CN103076424B - Oxytropis medical material and detection method of oxytropis medical material preparation - Google Patents
Oxytropis medical material and detection method of oxytropis medical material preparation Download PDFInfo
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- CN103076424B CN103076424B CN201210574043.1A CN201210574043A CN103076424B CN 103076424 B CN103076424 B CN 103076424B CN 201210574043 A CN201210574043 A CN 201210574043A CN 103076424 B CN103076424 B CN 103076424B
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Abstract
The invention relates to an oxytropis medical material and a detection method of oxytropis medical material preparation. The detection method comprises the steps of one or a plurality of the following identification or detection method(s): qualitatively identifying oxytropis of the oxytropis medical material or the oxytropis medical material preparation by a thin-layer chromatography; and qualitatively identifying the content of 2', 4'-dihydroxy chalcone in the oxytropis medical material or the oxytropis medical material preparation by a high-performance liquid chromatography. Effective detection of the oxytropis medical material or the oxytropis medical material preparation can be realized by adopting the detection method.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, be specifically related to the detection method of a kind of medicinal material and preparation thereof.
Background technology
78 editions, six provinces and regions " Tibetan medicine standard " record: whin is the dry herb of pulse family whorlleaf oxytropis herb Oxytropis chiliophylla Royle or falcate crazyweed herb Oxytropis falcata Bunge.Its source is identical, cold in nature with the source of " Chinese Pharmacopoeia " 2010 editions annex, bitter, and poisonous, function is recorded under curing mainly item: clearing heat and detoxicating, and myogenic heals sore, and weak thready is stopped blooding, defaecation.Be used for the treatment of plague, nosotoxicosis, grasserie, constipation, anthrax; External application for curing sore furuncle pain.And under " Qinghai Province's Tibetan medicine standard " falcate crazyweed herb (cowherb reaches the summer) item, source is defined as " the dry herb that this product is legume falcate crazyweed herb Oxytropis falcata Bunge ", compared with " Tibetan medicine ministerial standard " 95 editions whins (cowherb reaches the summer), its property, taste are same, and function adds under curing mainly item " knurl of boning ".
The widespread use in Tibetan medicine of whin medicinal material and preparation thereof, but in primary standard, source is only had to its detection method, proterties, and micro-qualitative etc., Comparison between detecting methods is simple, lacks norm controlling such as quantifications grade.Now in the art still not about whin medicinal material and the TLC distinguish of preparation thereof and the report of detection method of content.
Summary of the invention
The object of this invention is to provide the detection method of a kind of whin, described method is applicable to detect whin medicinal material and the preparation containing whin medicinal material, there is favorable reproducibility, specificity is strong, effectively can detect whin medicinal material and the advantage such as quality containing whin medicinal material preparation, thus make whin medicinal material and the quality of the pharmaceutical preparations containing whin medicinal material stable, safe, controlled.
To achieve these goals, technical scheme of the present invention is:
A detection method for whin, described method is applicable to detect whin medicinal material and the preparation containing whin medicinal material, comprises following discriminating and/or assay method:
By thin-layered chromatography, Qualitive test is carried out to whin;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured.
Particularly, in above-mentioned detection method, the Qualitive test of whin comprises:
1a) prepare whin need testing solution I, 2 ', 4 '-dihydroxy chalcone reference substance solution I, not containing the negative sample solution I of whin;
2a) test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, respectively aspiration step 1a) the whin need testing solution I, 2 ' that obtains, 4 '-dihydroxy chalcone reference substance solution I, negative sample solution I, and point sample is on same silica gel g thin-layer plate;
3a) with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate for developping agent, by step 2a) thin layer plate that obtains be placed on saturated in expansion cylinder, launch, take out, dry, spray is with aluminium choride ethanolic solution, and wind is clear to spot development; Inspect under putting ultraviolet lamp; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
Contained by whin, the assay of 2 ', 4 '-dihydroxy chalcone comprises:
1b) prepare whin need testing solution II, 2 ', 4 '-dihydroxy chalcone reference substance solution II, not containing the negative sample solution II of whin;
2b) test according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex NI D, respectively aspiration step 1b) the whin need testing solution II, 2 ' that obtains, 4 '-dihydroxy chalcone reference substance solution II, negative sample solution II, injection liquid chromatography, measures;
3b) contain whin with 2 ', 4 '-dihydroxy chalcone meter with the whin medicinal material often containing 1 weight portion in the whin medicinal material dry product of every 1 weight portion or preparation, 0.0004 weight portion must not be less than.
More specifically, the preparation method of the need testing solution of whin described in detection method I, 2 ', 4 '-dihydroxy chalcone reference substance solution I, negative sample solution I comprises:
Get whin medicinal material or the preparation containing whin medicinal material, add methyl alcohol or absolute ethyl alcohol, ultrasonic process, take out, let cool, filter, filtrate concentrates, as whin need testing solution I; Separately get 2 ', 4 '-dihydroxy chalcone, add methyl alcohol or 2 ', 4 '-dihydroxy chalcone reference substance solution I made by absolute ethyl alcohol; In prescription ratio and preparation technology, preparation not containing the negative sample of whin, and is made not containing the negative sample solution I of whin by the compound method of above-mentioned need testing solution;
The preparation method of described whin need testing solution II, 2 ', 4 '-dihydroxy chalcone reference substance solution II, negative sample solution II comprises:
Get described whin medicinal material or the preparation containing whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol, close plug, weighed weight, ultrasonic process, takes out, lets cool, weighed amount again, supply less loss weight with 70% ethanol, shake up, filter, get subsequent filtrate, as whin need testing solution II; Separately get 2 ', 4 '-dihydroxy chalcone reference substance, weighed, add 70% ethanol and make 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution II of whin by the compound method of above-mentioned need testing solution.
Detection method of the present invention, comprises following discriminating and/or assay method:
By thin-layered chromatography, Qualitive test is carried out to whin:
Get whin medicinal material 0.1-10 weight portion or be equivalent to the preparation containing 0.1-10 weight portion whin medicinal material, add methyl alcohol or absolute ethyl alcohol 10-60 parts by volume, ultrasonic process 1-40 minute, take out, let cool, filter, filtrate is concentrated into about 0.1-10 parts by volume, as whin need testing solution I; Separately get 2 ', 4 '-two base chalcone reference substance 0.1-10 weight portion, adds methyl alcohol or absolute ethyl alcohol 1-10 parts by volume, makes 2 ', 4 '-dihydroxy chalcone reference substance solution I; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution I of whin by the compound method of above-mentioned need testing solution; Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, draw each 0.001-0.03 parts by volume of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate, volume ratio 1 ~ 15: 0.5 ~ 10: 0.5 ~ 10 is developping agent, saturated 0-40 minute in expansion cylinder put by thin layer plate, launch, take out, dry, spray is with 0.5-6% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development; Inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured:
Get whin medicinal material 0.1-10 weight portion or be equivalent to the preparation containing 0.1-10 weight portion whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 30%-ethanol, close plug, weighed weight, ultrasonic process 1-60 minute, take out, let cool, more weighed weight, the weight of less loss is supplied with 30%-absolute ethyl alcohol, shake up, filter, get subsequent filtrate as whin need testing solution II; Get 2 ', 4 '-dihydroxy chalcone reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1 parts by volume containing 2 ', 4 '-dihydroxy chalcone reference substance 0.0001-0.01 weight portion, as 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution II by the compound method of above-mentioned need testing solution; Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-0.1% formic acid solution of 20 ~ 80: 80 ~ 20 is for mobile phase with long-pending ratio; Determined wavelength is 365 ± 10nm; Number of theoretical plate calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone peak; Accurate absorption whin need testing solution, 2 ' respectively, each 0.005 ~ 0.02 parts by volume of 4 '-dihydroxy chalcone reference substance solution, negative sample solution, injection liquid chromatography, measure, often to contain whin with 2 ' containing whin medicinal material 1 weight portion in every 1 weight portion of whin medicinal material dry product or preparation, 4 '-dihydroxy chalcone meter, must not be less than 0.0004 weight portion.
Further, above-mentioned detection method, comprises following discriminating and/or assay method:
By thin-layered chromatography, Qualitive test is carried out to whin:
Get whin medicinal material 1 weight portion or be equivalent to the preparation containing 1 weight portion whin medicinal material, add methyl alcohol or absolute ethyl alcohol 20 parts by volume, ultrasonic process 15 minutes, take out, let cool, filter, filtrate is concentrated into about 2 parts by volume, as whin need testing solution I; Separately get 2 ', 4 '-dihydroxy chalcone reference substance, add methyl alcohol or absolute ethyl alcohol and make the solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone reference substance 1mg, as 2 ', 4 '-dihydroxy chalcone reference substance solution I; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution I by the compound method of above-mentioned need testing solution; Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, draw each 0.01 parts by volume of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate, volume ratio 7: 4: 3 is developping agent, thin layer plate to be put in expansion cylinder saturated 20 minutes, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development; Inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured:
Get whin medicinal material 1 weight portion or be equivalent to the preparation containing 1 weight portion whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50 parts by volume, close plug, weighed weight, ultrasonic process 30 minutes, take out, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, get subsequent filtrate as whin need testing solution II; Get 2 ', 4 '-dihydroxy chalcone reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1 parts by volume containing 2 ', 4 '-dihydroxy chalcone reference substance 0.02 weight portion, as 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution II by the compound method of above-mentioned need testing solution; Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-0.1% formic acid solution of 60: 40 is for mobile phase with long-pending ratio; Determined wavelength is 365nm; Number of theoretical plate calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone peak; Respectively accurate draw whin need testing solution and, 2 ', each 0.01 parts by volume of 4 '-dihydroxy chalcone reference substance solution, negative sample solution, injection liquid chromatography, measure, often to contain whin with 2 ' containing whin medicinal material 1 weight portion in every 1 weight portion of whin medicinal material dry product or preparation, 4 '-dihydroxy chalcone meter, must not be less than 0.0004 weight portion.
Whin material medicine of the present invention or to have treatment containing the preparation of whin medicinal material clearing heat and detoxicating, myogenic heals the effect of sore, weak thready hemostasis, defaecation.Be used for the treatment of plague, nosotoxicosis, grasserie, constipation, anthrax; The purposes of sore furuncle pain medicine is controlled in external application.
Detection method of the present invention establishes whin medicinal material or the thin-layer identification method containing whin medicinal material preparation, and specificity is strong, and reappearance is better; Also add the content assaying method of whin medicinal material containing 2 ', 4 '-dihydroxy chalcone contained by whin in the preparation of whin medicinal material in addition, this detection method specificity is strong, and negative noiseless.Described detection method is through many people repeatedly repeatable operation, and result accurately, favorable reproducibility, specificity be strong, meets accurate, easy, sensitive, principle fast, effectively can detect the quality of medicinal material and preparation thereof.
Accompanying drawing explanation
Fig. 1 is the thin-layer chromatogram of whin of the present invention;
Wherein, from left to right 1-3 road is whin test sample, 4 roads are 2 ', 4 '-dihydroxy chalcone reference substance, 5 roads are negative sample.
Fig. 2 is 2 ', 4 '-dihydroxy chalcone chromatogram contained by whin of the present invention;
Wherein, A is 2 ', 4 '-dihydroxy chalcone reference substance, B is whin test sample, C is negative sample, D is whin medicinal material.
Fig. 3 is the peak area of 2 ', 4 '-dihydroxy chalcone and the linear relationship chart of reference substance concentration.
Embodiment
Following experimental example and embodiment further illustrate but do not limit the present invention.
The reagent below used and instrument, except special instruction, be common commercially available.
Whin medicinal material: pick up from unexpected rival river, Qinghai, through being accredited as legume falcate crazyweed herb Oxytropis falcata Bunge.
2 ', 4 '-dihydroxy chalcone reference substance: purity 98%, purchased from American Alfa company.
Negative sample: in prescription ratio and preparation technology, preparation is not containing the negative sample of whin.
Experimental example 1:
the TLC distinguish of whin
(1) instrument
Electronic scales, tool plug conical flask, transfer pipet, ultrasound wave extraction apparatus, filter paper, sample applicator, silica G plate, chromatography cylinder, uviol lamp, spray bottle.
(2) reference substance
2 ', 4 '-dihydroxy chalcone, purity 98%, purchased from American Alfa company.
(3) reagent
Methyl alcohol, ethanol, formic acid, ethyl acetate, sherwood oil, normal hexane.
(4) method of inspection:
Extraction solvent: ethanol, methyl alcohol, 80% methyl alcohol, ethyl acetate.
Extracting method: jolting and ultrasonic.
Developping agent: sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (7: 4: 3), ethyl acetate-methenyl choloride-sherwood oil (60 ~ 90 DEG C) (7: 2: 3).
Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Repetition test in the above conditions, the specificity thin-layer identification method finally determining whin is as follows:
Get whin medicinal material 1g, add methyl alcohol 20ml, ultrasonic process 15 minutes, take out, let cool, filter, filtrate is concentrated into about 2ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution of whin by the compound method of above-mentioned need testing solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (7: 4: 3) for developping agent, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development, inspects under putting ultraviolet lamp (365nm), in test sample chromatogram, with on 2 ', 4 '-dihydroxy chalcone reference substance chromatogram relevant position, show the spot of same color.
In negative sample chromatogram, with on 2 ', 4 '-dihydroxy chalcone reference substance chromatogram relevant position, without obviously disturbing.
experimental example 2: the assay of whin 2 ', 4 '-dihydroxy chalcone contained by whin medicinal material
(1) instrument
Electronic balance, conical flask, volumetric flask, transfer pipet, syringe, beaker, funnel, filter paper, filter, high performance liquid chromatograph.
(2) reference substance
2 ', 4 '-dihydroxy chalcone, purity 98%, purchased from American Alfa company.
(3) reagent
Methyl alcohol, acetonitrile, ethanol, absolute ethyl alcohol, formic acid, water.
(4) method of inspection:
Extraction solvent: absolute ethyl alcohol, 70% ethanol, 30% ethanol.
Extraction time: ultrasonic 10 minutes, 40 minutes, 60 minutes.
Mobile phase: methanol-water (30: 70), acetonitrile-0.1% aqueous formic acid (60: 40)
Select above Extraction solvent, extraction time and mobile phase to test respectively, result is as follows:
Repetition test in the above conditions, the method finally determining 2 ', 4 '-dihydroxy chalcone assay contained by whin medicinal material is as follows:
Get whin medicinal material 1g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate as whin need testing solution; Get 2 ', 4 '-dihydroxy chalcone reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone reference substance 0.02g, product solution in contrast; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution of whin by the compound method of above-mentioned need testing solution; Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-0.1% formic acid solution of 60: 40 is for mobile phase with long-pending ratio; Determined wavelength is 365nm.Number of theoretical plate calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone peak.Accurate absorption need testing solution, reference substance solution, each 0.01ml of negative sample solution respectively, injection liquid chromatography, measures, and contains whin with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 10 μ g with the every 1g of whin medicinal material dry product.
experimental example 3: contained by whin medicinal material, whin 2 ', 4 '-dihydroxy chalcone assay-methodology is examined
examine
(1) instrument and reagent
Aglient 1100 high performance liquid chromatograph (Agilent company of the U.S.): G1314A UV-vis detector, G1314A binary pump, Agilent1100 chromatographic work station.SK8200HP ultrasonic cleaner (power 250W, frequency 40kHz) (Shanghai High Kudos Science Instrument Co., Ltd.).
2 ', 4 '-dihydroxy chalcone reference substance (purchased from American Alpha Co., Ltd); Whin medicinal material (being provided by Gansu Qizheng Tibetan Medicine Co., Ltd); Acetonitrile (chromatographically pure); Methyl alcohol (analyzing pure); Formic acid (chromatographically pure).
(2) chromatographic condition
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
Mobile phase: acetonitrile-0.1% formic acid solution (60: 40);
Flow velocity: 1.0ml/min;
Determined wavelength: 365nm;
Column temperature: 30 DEG C.
(3) chromatographic system employment and suitability test (E & ST)
Under above chromatographic condition, better, theoretical cam curve calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone for 2 ', 4 '-dihydroxy chalcone and all the other impurity peaks separating effects.
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product;
The preparation of need testing solution: get whin medicinal material and be about 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product;
The preparation of negative sample solution: get not containing the negative sample 0.5g of whin medicinal material, be made in the same way of negative sample solution, for subsequent use.
(5) negative interference test: in each 10 μ l injection liquid chromatographies of accurate absorption reference substance solution, need testing solution and negative sample solution, from chromatogram, on the position corresponding to reference substance solution chromatographic peak retention time, the chromatographic peak that need testing solution has identical retention time occurs, and negative sample solution at this wavelength without absorption, noiseless to the assay of whin 2 ', 4 '-dihydroxy chalcone contained by whin medicinal material.Chromatogram A, B, C, D as shown in Figure 2.
(6) linear relationship is investigated
Get 2 ', 4 '-dihydroxy chalcone reference substance 13.72mg, put in 50ml volumetric flask, shake up, as storing solution.In the above-mentioned stock solution 0.1ml of accurate absorption, 0.6ml, 1ml, 2ml, 4ml, 6ml, 8ml to 25ml volumetric flask, add 70% ethanol and be diluted to scale, shake up, precision measures 10 μ l injection liquid chromatography respectively, measure peak area, 2 ', 4 '-dihydroxy chalcone between 1.0976 ~ 87.808 μ g/ml in the concentration of peak area and reference substance be good linear relationship, its regression equation is y=0.02224x-0.07977 (r=0.99992).The results are shown in Table 1 and Fig. 3.
Table 12 ', 4 '-dihydroxy chalcone concentration and peak area result
(7) precision is investigated
Get whin medicinal material 0.5g, by sample solution preparation method preparation, by continuous sample introduction under chromatographic condition 6 times, calculate the RSD value of 2 ', 4 '-dihydroxy chalcone peak area.The results are shown in Table 2.
Table 2 Precision Experiment result
Result shows, continuous 6 sample introductions, and 2 ', 4 '-dihydroxy chalcone peak area relative standard deviation value is no more than 1%, illustrates that instrument precision is good.
(8) repeatability is investigated
Get whin medicinal material and be about 0.5g, 5 parts, by sample solution preparation method preparation, by continuous sample introduction under chromatographic condition, the RSD value of 2 ', 4 '-dihydroxy chalcone content in calculation sample.The results are shown in Table 3.
Table 3 repeated experiment result
Result shows, in sample, 2 ', 4 '-dihydroxy chalcone content relative standard deviation value is no more than 1%, and the reappearance of illustrative experiment method is good.
(9) study on the stability
Get whin medicinal material 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, refluxing extraction 30 minutes, takes out, lets cool, weighed amount again, supplies less loss weight with 70% ethanol, shakes up, filter, get subsequent filtrate and get final product, 0,2,4,8,16,24 hour difference sample introduction after the production, calculate the RSD value of 2 ', 4 '-dihydroxy chalcone content.The results are shown in Table 4.
Table 4 stability experiment result
Result shows, in sample solution after the production 24 hours, 2 ', 4 '-dihydroxy chalcone is stablized.
(10) average recovery experiment
Precision takes the whin medicinal material 0.25g of known content, divide 3 groups, totally 12 parts, be placed in the tool plug triangular flask of 150ml, accurately respectively add 2 ', 4 '-dihydroxy chalcone reference substance solution 0.945ml that concentration is 0.2744mg/ml, 1.180ml, 1.415ml, namely 2 ' is added by 80%, 100%, 120% amount of known content, 4 '-dihydroxy chalcone reference substance solution, by need testing solution preparation method preparation, calculates average recovery.The results are shown in Table 5.
Table 5 average recovery experimental result
(11) sample size measures
Get the whin medicinal material 0.5g of different batches, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, refluxing extraction 30 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 70% ethanol, shake up, filter, get subsequent filtrate and get final product.The results are shown in Table 6.
Table 6 sample size measurement result
According to above-mentioned multiple batches of whin medicinal material measurement result, contain whin with the amount of 2 ', 4 '-dihydroxy chalcone between 0.45 ~ 4.48mg with the every 1g of whin medicinal material dry product, in tentative whin medicinal material, whin is with 2 ', 4 '-dihydroxy chalcone (C
21h
220
11) gauge, every 1g must not be less than 0.40mg.
Following embodiment all can realize the effect of above-mentioned experimental example.
Containing various preparations of whin medicinal material and preparation method thereof in embodiment 1-9:
Ointment containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, and is ground into fine powder, adds whiteruss, glycerine and appropriate emulsifying agent and water etc. and stirs cream processed at about 80 DEG C, add muscone, stir, make 5000g, to obtain final product in time being cooled to 38 DEG C.
Tablet containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, and is ground into fine powder, boiling twice, 1 hour first time, second time 1.5 hours, considered, merging filtrate, concentrated, dry, mix with muscone and appropriate amount of starch, granulate, dry, compressing tablet, to obtain final product.
Pill containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other are ground into fine powder, sieve, and mixing, adds suitable quantity of water pill with muscone, to obtain final product.
Granule containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, and is ground into fine powder, boiling twice, and 1 hour first time, second time 1.5 hours, considered, merging filtrate, concentrated, dry, mixed with muscone and appropriate amount of starch, granulated, dry, to obtain final product.
Powder containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, eight tastes such as all the other whins, are ground into fine powder, sieve, mixing, add muscone's mixing, to obtain final product.
Aerosol containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, be ground into fine powder, add 50% alcohol reflux twice, 1 hour first time, second time 1.5 hours, considered, merging filtrate, muscone adds 50% alcohol dipping and extracts 3 times, each 7 days, merging filtrate, filling, sealing, pours propellant appropriate, to obtain final product.
Cataplasm containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes; be ground into fine powder, boiling twice, 1 hour first time; second time 1.5 hours, considered, merging filtrate; concentrated; drying, mixes with muscone, and gelatin adds suitable quantity of water to be made fully swelling; add other matrix and extract powder; mixing, in 50 DEG C ~ 60 DEG C water-baths, heating makes melting, is slowly stirred to evenly; coat while hot on nonwoven fabrics; coating thickness is about 0.5mm, places slaking 4 ~ 6h, and when tearing after cream cover upper protective film, adhesion is degree again; cover diaphragm, to obtain final product.
Spray containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, and is ground into fine powder, adds 50% alcohol reflux twice, 1 hour first time, second time 1.5 hours, considered, merging filtrate, muscone adds 50% alcohol dipping and extracts 3 times, each 7 days, merging filtrate, filling, sealing, to obtain final product.
Emplastrum containing whin medicinal material:
Get whin 100g, sub-rheum officinale 50g, myrobalan's (stoning) 100g, terminaliae billericae,fructus 100g,
Emblic 100g, styrax 35g, wide muscle rattan 150g, muscone 25g, Aconitum Szechenyianum Gay 75g.Above nine tastes, except muscone, all the other whins etc. eight taste mixes, be ground into fine powder, boiling twice, 1 hour first time, second time 1.5 hours, considered, merging filtrate, be concentrated into appropriate, mix with muscone, add by rubber, in the matrix that rosin etc. are made, make coating, carry out painting cream; Lid lining, section, to obtain final product.
[embodiment 1] detects containing the ointment of whin medicinal material
(1) TLC distinguish
Get the ointment 10g containing whin medicinal material, add methyl alcohol 50ml, ultrasonic process 30 minutes, take out, let cool, filter, filtrate is concentrated into about 2ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (7: 4: 3) for developping agent, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development, inspects under putting ultraviolet lamp (365nm); In test sample chromatogram, with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (60: 40); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 6g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate and get final product.
(2d) determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 0.6mg containing whin.
[embodiment 2] is containing the detection of the tablet of whin medicinal material
(1) TLC distinguish
Get the tablet powder 5g containing whin medicinal material, add absolute ethyl alcohol 50ml, ultrasonic process 15 minutes, take out, let cool, filter, filtrate is concentrated into about 6ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (10: 6: 5) for developping agent, launch, take out, dry, spray is with 3% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development, inspects under putting ultraviolet lamp (365nm); In test sample chromatogram, with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (50: 50); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product powder and be about 5g, accurately weighed, put in tool plug conical flask, precision adds 100% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply the weight of less loss with 100% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 20 μ g containing whin.
[embodiment 3] detects containing the pill of whin medicinal material
(1) TLC distinguish
Get this product 2g, add methyl alcohol 30ml, ultrasonic process 20 minutes, take out, let cool, filter, filtrate is concentrated into about 3ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 15 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (5: 1: 1) for developping agent, launch, take out, dry, spray is with 4% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (40: 60); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 5g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 50ml, close plug, weighed weight, ultrasonic process 25 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 80% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.02ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 12 μ g containing whin.
[embodiment 4] detects containing the granule of whin medicinal material
(1) TLC distinguish
Get this product 4g, add absolute ethyl alcohol 20ml, ultrasonic process 30 minutes, take out, let cool, filter, filtrate is concentrated into about 4ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 30 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (15: 8: 8) for developping agent, launch, take out, dry, spray is with 5% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (20: 80); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 4g, accurately weighed, put in tool plug conical flask, precision adds 50% ethanol 40ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 50% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.005ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 10 μ g containing whin.
[embodiment 5] detects containing the powder of whin medicinal material
(1) TLC distinguish
Get this product 1g, add methyl alcohol 10ml, ultrasonic process 35 minutes, take out, let cool, filter, filtrate is concentrated into about 1ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (6: 4: 3) for developping agent, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (80: 20); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 4g, accurately weighed, put in tool plug conical flask, precision adds 60% ethanol 30ml, close plug, weighed weight, ultrasonic process 20 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 60% ethanol, shake up, filter, get subsequent filtrate and get final product.
Determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 8 μ g weight portions containing whin.
[embodiment 6] detects containing the aerosol of whin medicinal material
(1) TLC distinguish
Get this product 10g, add absolute ethyl alcohol 50ml, ultrasonic process 10 minutes, take out, let cool, filter, filtrate is concentrated into about 5ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (10: 6: 5) for developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (70: 30); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 10g, accurately weighed, put in tool plug conical flask, precision adds 90% ethanol 40ml, close plug, weighed weight, ultrasonic process 20 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 90% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 14 μ g containing whin.
[embodiment 7] detects containing the cataplasm of whin medicinal material
(1) TLC distinguish
Get this product 10g, add methyl alcohol 60ml, ultrasonic process 40 minutes, take out, let cool, filter, filtrate is concentrated into about 3ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (10: 5: 4) for developping agent, launch, take out, dry, spray is with 6% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (45: 55); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 10g, accurately weighed, put in tool plug conical flask, precision adds 50% ethanol 60ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 50% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 15 μ g containing whin.
[embodiment 8] detects containing the spray of whin medicinal material
(1) TLC distinguish
Get this product 8g, add methyl alcohol 50ml, ultrasonic process 30 minutes, take out, let cool, filter, filtrate is concentrated into about 3ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (6: 4: 4) for developping agent, launch, take out, dry, spray is with 0.5% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (35: 65); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 8g, accurately weighed, put in tool plug conical flask, precision adds absolute ethyl alcohol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, takes out, lets cool, more weighed amount, supply less loss weight with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.05ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 10 μ g containing whin.
[embodiment 9] detects containing the emplastrum of whin medicinal material
(1) TLC distinguish
Get this product 10g, add absolute ethyl alcohol 60ml, ultrasonic process 30 minutes, take out, let cool, filter, filtrate is concentrated into about 3ml, as need testing solution; Separately get 2 ', 4 '-dihydroxy chalcone appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI B), draw each 15 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-n-hexane-ethyl acetate (7: 4: 5) for developping agent, launch, take out, dry, spray is with 0.8% aluminium choride ethanolic solution, it is clear that hot blast blows to spot development, inspects, in test sample chromatogram under putting ultraviolet lamp (365nm), with on reference substance chromatogram relevant position, show the spot of same color.
(2) assay
Chromatographic condition
Mobile phase: acetonitrile-0.1% formic acid solution (55: 45); Flow velocity: 1.0ml/min; Determined wavelength: 365nm; Column temperature: 25 DEG C;
Chromatographic system employment and suitability test (E & ST)
Octadecyl silane is filling agent; Chromatographic column: Agilent C18 (4.6 × 250mm, 5.0 μm, Agilent company);
The preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get 2 ', 4 '-dihydroxy chalcone appropriate, adds 70% ethanol and makes the reference substance solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone 20 μ g, to obtain final product.
The preparation of need testing solution: get this product and be about 10g, accurately weighed, put in tool plug conical flask, precision adds 50% ethanol 50ml, close plug, weighed weight, ultrasonic process 40 minutes, takes out, lets cool, more weighed amount, supply less loss weight with 50% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method
Accurate absorption need testing solution and each 0.01ml of reference substance solution respectively, injection liquid chromatography, measures, and the every 1g of this product with 2 ', 4 '-dihydroxy chalcone meter, must not be less than 10 μ g containing whin.
Claims (5)
1. a detection method for whin, described method is applicable to detect whin medicinal material and the preparation containing whin medicinal material, comprises following discriminating and assay method:
By thin-layered chromatography, Qualitive test is carried out to whin;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured;
It is characterized in that:
Carry out Qualitive test by thin-layered chromatography to whin to comprise:
1a) prepare whin need testing solution I, 2 ', 4 '-dihydroxy chalcone reference substance solution I, not containing the negative sample solution I of whin;
2a) test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, respectively aspiration step 1a) the whin need testing solution I, 2 ' that obtains, 4 '-dihydroxy chalcone reference substance solution I, negative sample solution I, and point sample is on same silica gel g thin-layer plate;
3a) with sherwood oil-n-hexane-ethyl acetate for developping agent, developping agent volume ratio is 1 ~ 15:0.5 ~ 10:0.5 ~ 10, sherwood oil temperature is 60 ~ 90 DEG C, by step 2a) thin layer plate that obtains be placed on saturated in expansion cylinder, launch, take out, dry, spray is with aluminium choride ethanolic solution, and wind is clear to spot development; Inspect under putting ultraviolet lamp; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
Carry out mensuration by high performance liquid chromatography to the content of 2 ', 4 '-dihydroxy chalcone contained by whin to comprise:
1b) prepare whin need testing solution II, 2 ', 4 '-dihydroxy chalcone reference substance solution II, not containing the negative sample solution II of whin;
2b) test according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, respectively aspiration step 1b) the whin need testing solution II, 2 ' that obtains, 4 '-dihydroxy chalcone reference substance solution II, negative sample solution II, injection liquid chromatography, measures;
3b) contain whin with 2 ', 4 '-dihydroxy chalcone meter with the whin medicinal material often containing 1 weight portion in the whin medicinal material dry product of every 1 weight portion or preparation, 0.0004 weight portion must not be less than.
2. detection method as claimed in claim 1, it is characterized in that, the preparation method of described whin need testing solution I, 2 ', 4 '-dihydroxy chalcone reference substance solution I, negative sample solution I comprises:
Get whin medicinal material or the preparation containing whin medicinal material, add methyl alcohol or absolute ethyl alcohol, ultrasonic process, take out, let cool, filter, filtrate concentrates, as whin need testing solution I; Separately get 2 ', 4 '-dihydroxy chalcone, add methyl alcohol or 2 ', 4 '-dihydroxy chalcone reference substance solution I made by absolute ethyl alcohol; In prescription ratio and preparation technology, preparation not containing the negative sample of whin, and is made not containing the negative sample solution I of whin by the compound method of above-mentioned need testing solution;
The preparation method of described whin need testing solution II, 2 ', 4 '-dihydroxy chalcone reference substance solution II, negative sample solution II comprises:
Get described whin medicinal material or the preparation containing whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol, close plug, weighed weight, ultrasonic process, takes out, lets cool, weighed amount again, supply less loss weight with 70% ethanol, shake up, filter, get subsequent filtrate, as whin need testing solution II; Separately get 2 ', 4 '-dihydroxy chalcone reference substance, weighed, add 70% ethanol and make 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution II of whin by the compound method of above-mentioned need testing solution.
3. the detection method as described in any one of claim 1-2, is characterized in that, comprises following discriminating and/or assay method:
By thin-layered chromatography, Qualitive test is carried out to whin:
Get whin medicinal material 0.1-10 weight portion or be equivalent to the preparation containing 0.1-10 weight portion whin medicinal material, add methyl alcohol or absolute ethyl alcohol 10-60 parts by volume, ultrasonic process 1-40 minute, take out, let cool, filter, filtrate is concentrated into about 0.1-10 parts by volume, as whin need testing solution I; Separately get 2 ', 4 '-two base chalcone reference substance 0.1-10 weight portion, adds methyl alcohol or absolute ethyl alcohol 1-10 parts by volume, makes 2 ', 4 '-dihydroxy chalcone reference substance solution I; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and is made not containing the negative sample solution I of whin by the compound method of above-mentioned need testing solution; Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, draw each 0.001-0.03 parts by volume of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil-n-hexane-ethyl acetate, volume ratio 1 ~ 15:0.5 ~ 10:0.5 ~ 10 are developping agent, saturated 0-40 minute in expansion cylinder put by thin layer plate, launch, take out, dry, spray is with 0.5-6% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development; Inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured:
Get whin medicinal material 0.1-10 weight portion or be equivalent to the preparation containing 0.1-10 weight portion whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 30%-absolute ethyl alcohol, close plug, weighed weight, ultrasonic process 1-60 minute, take out, let cool, more weighed weight, the weight of less loss is supplied with 30%-100%-ethanol, shake up, filter, get subsequent filtrate as whin need testing solution II; Get 2 ', 4 '-dihydroxy chalcone reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1 parts by volume containing 2 ', 4 '-dihydroxy chalcone reference substance 0.0001-0.01 weight portion, as 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution II by the compound method of above-mentioned need testing solution; Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-0.1% formic acid solution of 20 ~ 80:80 ~ 20 is for mobile phase with long-pending ratio; Determined wavelength is 365 ± 10nm; Number of theoretical plate calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone peak; Accurate absorption whin need testing solution, 2 ' respectively, each 0.005 ~ 0.02 parts by volume of 4 '-dihydroxy chalcone reference substance solution, negative sample solution, injection liquid chromatography, measure, often to contain whin with 2 ' containing whin medicinal material 1 weight portion in every 1 weight portion of whin medicinal material dry product or preparation, 4 '-dihydroxy chalcone meter, must not be less than 0.0004 weight portion.
4. detection method as claimed in claim 3, is characterized in that, comprise following discriminating and/or assay method:
By thin-layered chromatography, Qualitive test is carried out to whin:
Get whin medicinal material 1 weight portion or be equivalent to the preparation containing 1 weight portion whin medicinal material, add methyl alcohol or absolute ethyl alcohol 20 parts by volume, ultrasonic process 15 minutes, take out, let cool, filter, filtrate is concentrated into about 2 parts by volume, as whin need testing solution I; Separately get 2 ', 4 '-dihydroxy chalcone reference substance, add methyl alcohol or absolute ethyl alcohol and make the solution of every 1ml containing 2 ', 4 '-dihydroxy chalcone reference substance 1mg, as 2 ', 4 '-dihydroxy chalcone reference substance solution I; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution I by the compound method of above-mentioned need testing solution; Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VI B, draw each 0.01 parts by volume of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil-n-hexane-ethyl acetate, volume ratio 7:4:3 is developping agent, thin layer plate to be put in expansion cylinder saturated 20 minutes, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, and it is clear that hot blast blows to spot development; Inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, with on the corresponding position of 2 ', 4 '-dihydroxy chalcone reference substance chromatogram, show the spot of same color fluorescence;
By high performance liquid chromatography, the content of 2 ', 4 '-dihydroxy chalcone contained by whin is measured:
Get whin medicinal material 1 weight portion or be equivalent to the preparation containing 1 weight portion whin medicinal material, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50 parts by volume, close plug, weighed weight, ultrasonic process 30 minutes, take out, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, get subsequent filtrate as whin need testing solution II; Get 2 ', 4 '-dihydroxy chalcone reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1 parts by volume containing 2 ', 4 '-dihydroxy chalcone reference substance 0.02 weight portion, as 2 ', 4 '-dihydroxy chalcone reference substance solution II; In prescription ratio and preparation technology, configuration not containing the negative sample of whin, and makes whin negative sample solution II by the compound method of above-mentioned need testing solution; Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; Acetonitrile-0.1% formic acid solution being 60:40 with long-pending ratio is for mobile phase; Determined wavelength is 365nm; Number of theoretical plate calculates should be not less than 1000 by 2 ', 4 '-dihydroxy chalcone peak; Respectively accurate draw whin need testing solution and, 2 ', each 0.01 parts by volume of 4 '-dihydroxy chalcone reference substance solution, negative sample solution, injection liquid chromatography, measure, often to contain whin with 2 ' containing whin medicinal material 1 weight portion in every 1 weight portion of whin medicinal material dry product or preparation, 4 '-dihydroxy chalcone meter, must not be less than 0.0004 weight portion.
5. detection method as claimed in claim 1, is characterized in that, described whin medicinal material or the preparation containing whin medicinal material have for the preparation for the treatment of plague, nosotoxicosis, grasserie, constipation, anthrax; The purposes of the medicine of sore furuncle pain is controlled in external application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210574043.1A CN103076424B (en) | 2012-12-26 | 2012-12-26 | Oxytropis medical material and detection method of oxytropis medical material preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210574043.1A CN103076424B (en) | 2012-12-26 | 2012-12-26 | Oxytropis medical material and detection method of oxytropis medical material preparation |
Publications (2)
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