CN101028474B - Method for inspecting the quality of Chinese preparation with Yang-and kidney tonifying functions - Google Patents
Method for inspecting the quality of Chinese preparation with Yang-and kidney tonifying functions Download PDFInfo
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Abstract
A quality test method for the Chinese medicine 'Bushen Kangle' features that the wolfberry extract and emodin are qualitatively discriminated and the content of icariin is measured by high-effect liquid-phase chromatography.
Description
One, technical field:
The invention belongs to the Chinese traditional patent formulation formulation art, relate to a kind of quality determining method specifically with tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness.
Two, background technology:
The Chinese medicine preparation made from Herba Epimedii, Testis et Pentis Canis, Hippocampus, Radix Rehmanniae Preparata etc. is a kind of to have tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness and has the tonifying YANG kidney tonifying, strongly invigorating QI and blood, lick the smart marrow of giving birth to, keep fit the function of brain-strengthening, be used for the treatment of geromorphism, sexual hypofunction, waist and leg ache, fatigue and weak, insomnia forgetfulness, symptoms such as absentminded, effect is obvious.Yet the Chinese medicinal ingredients more complicated contains many unknown compositions.Herba Epimedii is a monarch drug among the we, and its master contains flavone compound, and icariin is its Main Ingredients and Appearance.Therefore, how to control content Determination of Icariin, to strengthen the quality controllability of this product, imperative.Can effectively control the product quality of the Chinese medicine preparation of treatment at present, detection method that can be easy to operate is not also reported again.
Three, summary of the invention:
The invention provides a kind of quality determining method, to overcome the product quality that can not effectively control this Chinese medicine preparation that prior art exists and the shortcoming of detection method inconvenient operation with tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness.
For achieving the above object, the technical solution used in the present invention is:
A kind of quality determining method with tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness, 65% and all the other Radix Polygoni Multiflori Preparatas, Semen arachidis hypogaeae, Carapax Et Plastrum Testudinis (scalding), Fructus Corni (system), Cortex Cinnamomi, Fructus Lycii, Testis et Pentis Canis (system), Radix Rehmanniae Preparata, Cortex Phellodendri (system), Radix Dipsaci, Fructus Schisandrae Chinensis (system), Placenta Hominis, the Cortex Eucommiae, Radix Ginseng, Fructus Alpiniae Oxyphyllae (system), Hippocampus (system) ten Six-elements of getting the Herba Epimedii total amount are ground into fine powder, sieve, mixing, standby.The medicated powder by sieve does not decoct with water secondary with the residue Herba Epimedii, and 2 hours for the first time, 1.5 hours for the second time, collecting decoction filtered, and relative density was 1.00～1.05 clear paste when filtrate was concentrated into 60 ℃.Get above-mentioned fine powder and clear paste and an amount of refined honey stirs, granulate, drying, mixing incapsulates, and makes 1000, that is, and its quality determining method: may further comprise the steps:
(1) gets this product content 10g, add methanol 40ml, supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add water 10ml makes dissolving, add hydrochloric acid 1ml again, put in the water-bath and heated 30 minutes, immediately cooling, with ether extraction 2 times, each 20ml merges ether solution, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get the emodin reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 10 μ l, each 10 μ l of control medicinal material and reference substance solution, put respectively on same silica gel g thin-layer plate, upper solution with 30～60 ℃ of petroleum ether-Ethyl formate-formic acid of 15: 5: 1 is developing solvent, launches, and takes out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show identical fluorescent orange speckle, put in the ammonia steam smoked after, speckle becomes redness;
(2) get this product content 9g, add water 100ml, heated and boiled 30 minutes is put coldly, centrifugal, gets supernatant, extracts 2 times with the ethyl acetate jolting, each 30ml, and combined ethyl acetate liquid is concentrated into about 0.5ml, as need testing solution; Other gets Fructus Lycii control medicinal material 0.5g, shines medical material solution in pairs with legal system; According to the thin layer chromatography test, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with ethyl acetate-chloroform of 3: 2: 1-formic acid, launches, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;
(3) icariin assay
It is an amount of to get the icariin reference substance, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.06mg, promptly gets reference substance solution;
Get this product content under the content uniformity item, porphyrize is got about 1g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 70% ethanol 25ml that adds, close plug claims to decide weight, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 70% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution;
Use high effective liquid chromatography for measuring: be filler with the octadecylsilane chemically bonded silica; With 30: 70 acetonitrile-waters was mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the icariin peak should be not less than 1500; Accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing inject high performance liquid chromatograph and measure content Determination of Icariin in the calculation sample.
The present invention draws by test, and every 0.25g of this product contains Herba Epimedii with icariin (C
33H
40O
15) meter, must not be less than 0.30mg.
The dosage form of described Chinese medicine preparation comprises pill, granule, capsule, tablet regular dosage form.
The present invention is with respect to the advantage of prior art: the invention provides the qualitative identification to Fructus Lycii, emodin, made best test sample preparation method, found suitable developing solvent, can carry out effective qualitative identification to said preparation.The invention provides the content Determination of Icariin assay method in addition, adopt high performance liquid chromatography that icariin is carried out assay, make best test sample preparation method and mobile phase compound method, improved the content Determination of Icariin accuracy in detection greatly.Advantages such as this method has sensitivity, and is accurate help on the market true and false quality to this Chinese medicine preparation and differentiate.
Four, specific embodiment:
Below embodiment by the kidney invigorating 'Xiaochuankangle ' capsule quality determining method further specify useful effect of the present invention:
65% and all the other Radix Polygoni Multiflori Preparatas, Semen arachidis hypogaeae, Carapax Et Plastrum Testudinis (scalding), Fructus Corni (system), Cortex Cinnamomi, Fructus Lycii, Testis et Pentis Canis (system), Radix Rehmanniae Preparata, Cortex Phellodendri (system), Radix Dipsaci, Fructus Schisandrae Chinensis (system), Placenta Hominis, the Cortex Eucommiae, Radix Ginseng, Fructus Alpiniae Oxyphyllae (system), Hippocampus (system) ten Six-elements of getting the Herba Epimedii total amount are ground into fine powder, sieve, mixing, standby.The medicated powder by sieve does not decoct with water secondary with the residue Herba Epimedii, and 2 hours for the first time, 1.5 hours for the second time, collecting decoction filtered, and relative density was 1.00～1.05 clear paste when filtrate was concentrated into 60 ℃.Get above-mentioned fine powder and clear paste and an amount of refined honey stirs, granulate, drying, mixing incapsulates, and makes 1000, that is and, its quality determining method may further comprise the steps:
(1) gets this product content 10g, add methanol 40ml, supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add water 10ml makes dissolving, add hydrochloric acid 1ml again, put in the water-bath and heated 30 minutes, immediately cooling, with ether extraction 2 times, each 20ml merges ether solution, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get the emodin reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 10 μ l, each 10 μ l of control medicinal material and reference substance solution, put respectively on same silica gel g thin-layer plate, upper solution with 30～60 ℃ of petroleum ether-Ethyl formate-formic acid of 15: 5: 1 is developing solvent, launches, and takes out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show identical fluorescent orange speckle, put in the ammonia steam smoked after, speckle becomes redness;
(2) get this product content 9g, add water 100ml, heated and boiled 30 minutes is put coldly, centrifugal, gets supernatant, extracts 2 times with the ethyl acetate jolting, each 30ml, and combined ethyl acetate liquid is concentrated into about 0.5ml, as need testing solution; Other gets Fructus Lycii control medicinal material 0.5g, shines medical material solution in pairs with legal system; According to the thin layer chromatography test, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with ethyl acetate-chloroform of 3: 2: 1-formic acid, launches, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;
(3) icariin assay
It is an amount of to get the icariin reference substance, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.06mg, promptly gets reference substance solution;
Get this product content under the content uniformity item, porphyrize is got about 1g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 70% ethanol 25ml that adds, close plug claims to decide weight, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 70% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution;
Use high effective liquid chromatography for measuring: be filler with the octadecylsilane chemically bonded silica; With 30: 70 acetonitrile-waters was mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the icariin peak should be not less than 1500; Accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing inject high performance liquid chromatograph and measure content Determination of Icariin in the calculation sample.
Repeat above-mentioned steps once, the meansigma methods of icariin content is the 0.38mg/ grain in the calculation sample.
With the described quality determining method repetition test of embodiment 9 times, draw icariin content (mg/ grain) meansigma methods, the results are shown in following table:
Laboratory report:
1. instrument and reagent
Instrument: Agilent1100 type high performance liquid chromatograph, G1311A type quaternary gradient pump, G1314A type UV-detector, G2170AA type chem workstation.
Reagent: acetonitrile is a chromatorgaphy reagent, and water is ultra-pure water, and other reagent is analytical pure.
Reference substance: icariin, lot number are 0737-9910, for assay usefulness, are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Test sample: the kidney invigorating 'Xiaochuankangle ' capsule, lot number: 200309009; Specification: every dress 0.25g; Packing: aluminium-plastic panel, 18 * 2 plates/box.
2, chromatographic condition
Chromatographic column: 5 μ m, the Kromasil C-18 post 100 of 250mm * 4.6mm;
Mobile phase: 30: 70 acetonitrile-water;
Flow velocity: 1.0ml/min;
Detect wavelength: 270nm.
3, extraction conditions is selected and is extracted the completeness investigation
Extract choice of Solvent: precision takes by weighing the about 1g of this product content, put in the conical flask, precision adds 70% ethanol 25ml and methanol 25ml respectively, and close plug claims to decide weight, supersound extraction is 30 minutes respectively, claim to decide weight, supply the weight that subtracts mistake, filter with the extraction solvent, get the subsequent filtrate sample introduction, record its content results and see Table 1.
The different comparisons of extracting solvent of table 1
| Tested number | Extract solvent | Content Determination of Icariin (mg/ grain) |
| 1 | Methanol | 0.3836 |
| 2 | 70% ethanol | 0.4178 |
Above result shows that therefore 70% ethanol select for use 70% ethanol as extracting solvent than the content height of methanol extraction.
The test of extraction completeness: take by weighing four parts of this product contents respectively, every part of about 1g, the accurate title, decide, put in the tool plug conical flask, add 70% ethanol 25ml, claim to decide weight, difference supersound process different time, put coldly, supply the weight that subtracts mistake, shake up with Diluted Alcohol, filter, get the subsequent filtrate sample introduction, record its content, the results are shown in Table 2.
The investigation of table 2 different extraction times
Above result shows: ultrasonic 30 minutes, content Determination of Icariin was the highest, so definite 30 minutes is the supersound extraction time.
4, methodological study
The investigation of linear relationship and scope: precision is measured icariin reference substance solution (C=0.0482mg/ml) 4,8,12,16,20 μ l, injects chromatograph of liquid respectively, measures in accordance with the law, and the result asks for an interview table 3.
Table 3 linear relationship is investigated test and result
With the sample size is abscissa, and the peak area integrated value is a vertical coordinate, the drawing standard curve, and carry out rectilinear regression, regression equation is Y=2737.6X+66.33, r=0.9990.Result of the test shows that this method sample size is good linear relationship in 0.1928～0.964 μ g scope.
The precision test: accurate icariin reference substance solution (C=0.0482mg/ml) the 10 μ l that draw, repeat sample introduction continuously 5 times, measure, write down its peak area value, the results are shown in Table 4.
The test of table 4 icariin precision
The result shows: the icariin reference substance repeats sample introduction and records relative standard deviation RSD＜2.0% of peak area integrated value for 5 times, so think that this method has good precision.
Negative blank test: get the simulation prescription of removing Herba Epimedii, make the blank sample that does not contain Herba Epimedii, make negative control solution by the need testing solution method for making.200309009) and reference substance solution (C=0.0482mg/ml) other gets need testing solution (lot number:, measure by the text method.Result of the test shows, in the test sample chromatograph, with the corresponding retention time of reference substance chromatograph position on, the single chromatographic peak of a correspondence is arranged, and in the negative control liquid chromatography, does not have corresponding peak in this retention time corresponding position and occur.Illustrate negative blank noiseless to its mensuration.
Stability test: the accurate absorption with a collection of (lot number: 200309009) need testing solution and reference substance solution (C=0.0482mg/ml), interval by 0,0.5,1,2,4 hour, inject chromatograph of liquid respectively and measure, result of the test please sees Table 5 respectively.
Table 5 stability test result
Result of the test shows that icariin is all good at 4 hours internal stabilities in icariin reference substance and the sample solution.
Replica test: get with a collection of that (lot number: 200309009) sample is 5 parts, prepares the sample test liquid by the text method, records peak area and calculates content, the results are shown in Table 6.
Table 6 sample replica test and result
Result of the test shows that the relative standard deviation RSD=1.73% of 5 replications of this method illustrates that its repeatability is better.
Average recovery test: precision take by weighing known content (the 0.386mg/ grain, sample 1.544mg/g) (lot number: 200309009) an amount of, accurate claim fixed, it is an amount of to add the icariin reference substance respectively, measure in accordance with the law,, the results are shown in Table 7 with following formula calculate recovery rate.
Table 7 sample recovery rate result of the test
Result of the test shows: this method has the higher response rate, and its average recovery rate is 98.5%, and application of sample reclaims good.
5, assay in the sample
Accurate respectively reference substance solution and the sample solution drawn pressed method under the text assay item, measures.The results are shown in Table 8.
Icariin assay result (n=2) in table 8 sample
6, content Determination of Icariin is measured in the epimedium herb
Get the epimedium herb of separate sources, carry out assay by the text method, the result asks for an interview table 9.
Table 9 epimedium herb assay result (n=2)
7, content limit determines
In ten batch samples, the measured result of icariin, high-load is the 0.598mg/ grain, minimum is the 0.327mg/ grain, epimedium herb is pressed regulation of Chinese Pharmacopoeia version in 2000, its content must not be lower than 0.50%, and the minimum content of three batches of separate sources medical materials of actual measurement is 0.505%, is up to 0.603%, consider the prescription consumption, factor affecting such as process loss and content uniformity so tentative every of this product contains Herba Epimedii in icariin, must not be less than 0.30mg.
Claims (2)
1. quality determining method with tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness, 65% and all the other Radix Polygoni Multiflori Preparatas, Semen arachidis hypogaeae, Carapax Et Plastrum Testudinis, Fructus Corni, Cortex Cinnamomi, Fructus Lycii, Testis et Pentis Canis, Radix Rehmanniae Preparata, Cortex Phellodendri, Radix Dipsaci, Fructus Schisandrae Chinensis, Placenta Hominis, the Cortex Eucommiae, Radix Ginseng, Fructus Alpiniae Oxyphyllae, Hippocampus ten Six-elements of getting the Herba Epimedii total amount are ground into fine powder, sieve, mixing, standby; The medicated powder by sieve does not decoct with water secondary with the residue Herba Epimedii, and 2 hours for the first time, 1.5 hours for the second time, collecting decoction filtered, and relative density was 1.00～1.05 clear paste when filtrate was concentrated into 60 ℃; Get above-mentioned fine powder and clear paste and an amount of refined honey stirs, granulate, drying, mixing gets product, and it is characterized in that: detection method may further comprise the steps:
(1) gets this product content 10g, add methanol 40ml, supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add water 10ml makes dissolving, add hydrochloric acid 1ml again, put in the water-bath and heated 30 minutes, immediately cooling, with ether extraction 2 times, each 20ml merges ether solution, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Radix Polygoni Multiflori control medicinal material 1g, shines medical material solution in pairs with legal system; Get the emodin reference substance again, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned need testing solution 10 μ l, each 10 μ l of control medicinal material and reference substance solution, put respectively on same silica gel g thin-layer plate, upper solution with 30～60 ℃ of petroleum ether-Ethyl formate-formic acid of 15: 5: 1 is developing solvent, launches, and takes out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show identical fluorescent orange speckle, put in the ammonia steam smoked after, speckle becomes redness;
(2) get this product content 9g, add water 100ml, heated and boiled 30 minutes is put coldly, centrifugal, gets supernatant, extracts 2 times with the ethyl acetate jolting, each 30ml, and combined ethyl acetate liquid is concentrated into about 0.5ml, as need testing solution; Other gets Fructus Lycii control medicinal material 0.5g, shines medical material solution in pairs with legal system; According to the thin layer chromatography test, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with ethyl acetate-chloroform of 3: 2: 1-formic acid, launches, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;
(3) icariin assay
It is an amount of to get the icariin reference substance, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.06mg, promptly gets reference substance solution;
Get this product content under the content uniformity item, porphyrize is got about 1g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 70% ethanol 25ml that adds, close plug claims to decide weight, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 70% ethanol, shake up, filter, get subsequent filtrate, promptly get need testing solution;
Use high effective liquid chromatography for measuring: be filler with the octadecylsilane chemically bonded silica; With 30: 70 acetonitrile-waters was mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the icariin peak should be not less than 1500; Accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing inject high performance liquid chromatograph and measure content Determination of Icariin in the calculation sample.
2. a kind of quality determining method with tonifying YANG kidney benefiting functions Chinese medicine preparation the kidney invigorating health and happiness as claimed in claim 1, it is characterized in that: the every 0.25g of described Chinese medicine preparation contains Herba Epimedii in icariin, must not be less than 0.30mg.
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| CN109856254B (en) * | 2018-01-11 | 2022-11-18 | 广西中医药大学 | Method for establishing ethyl acetate part HPLC fingerprint spectrum of longan leaf |
| CN110389177A (en) * | 2018-04-18 | 2019-10-29 | 四川济生堂药业有限公司 | A kind of detection method of pharmaceutical composition |
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