CN101491630A - Quality control method of Fenqing Wulin pill - Google Patents
Quality control method of Fenqing Wulin pill Download PDFInfo
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- CN101491630A CN101491630A CNA2008100566339A CN200810056633A CN101491630A CN 101491630 A CN101491630 A CN 101491630A CN A2008100566339 A CNA2008100566339 A CN A2008100566339A CN 200810056633 A CN200810056633 A CN 200810056633A CN 101491630 A CN101491630 A CN 101491630A
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Abstract
The invention discloses a quality control method for clear wulin pill. The method adopts the thin-layer qualitative detection of rheum officinale, berberine hydrochloride, Gardenoside and aristolochic acid, and also adopts the qualitative detection of the content of rheum emodin. Therefore, the method guarantees the safety and efficacy of medicines, improves the quality of products, and is convenient for standardization production of the products.
Description
Technical field
The present invention relates to a kind of method of quality control of Chinese medicine, the present invention relates to a kind of method of quality control of FENQINGWULIN WAN particularly.
Background technology
FENQINGWULIN WAN prescription derives from that " one one 393 pages of Chinese Pharmacopoeia versions in 2005, its function cures mainly and is clearing away heat-fire, inducing diuresis for treating stranguria syndrome.Be used for the stranguria due to the damp invasion of lower energizer, disease is seen yellowish or reddish urine, urine urgency-frequency, the puckery pain of urethra scorching hot.This FENQINGWULIN WAN prescription is as follows:
Caulis Akebiae 80g Semen Plantaginis (salt stir-fry) 40g Radix Scutellariae 80g
Poria 40g Polyporus 40g Cortex Phellodendri 40g
Radix Et Rhizoma Rhei 120g
Hold 40g Herba Dianthi 40g
Rhizoma Anemarrhenae 40g Rhizoma Alismatis 40g Fructus Gardeniae 40g
Radix Glycyrrhizae 20g Talcum 80g
Preparation method is:
More than 14 flavors, except that Talcum, all the other Caulis Aristolochiae Manshuriensis etc. 13 flavor is ground into fine powder, sieves mixing.Use water pill, drying is broken into the impalpable powder coating with Pulvis Talci, polishing, and drying, promptly.
Method of quality control is:
Get this product, put microscopically and observe: the dendritic agglomerate of irregular branch is colourless, meets chloral hydrate liquid and dissolves; Hyphae colorless or light brown, diameter 4～6 μ m.The mycelia bonding is agglomerating, colourless mostly; Prism of calcium oxalate regular octahedron shape, diameter 32～60 μ m.Plant Intradermal chrotoplast surface and see rectangle like, the wall wavy is one group with several cells, and zyklopisch is arranged slightly.Phloem fiber is faint yellow, fusiformis, and wall thickness, the hole ditch is thin.The fibre bundle foresythia, peripheral cell contains prism of calcium oxalate, forms crystalline cellulose, and the wall lignify of crystal cell thickens.Parenchyma cell contains prism of calcium oxalate around the fibre bundle, forms crystalline cellulose.Calcium oxalate cluster crystal is big, diameter 60～140 μ m.Needle-like calcium oxalate crystal bunchy or be dispersed in, long 26～110 μ m.Parenchyma cell contains calcium oxalate cluster crystal around the fibre bundle, forms crystalline cellulose, and crystal cell vertically embarks on journey.The leaf epidermal cell periclinal wall has the cutin strain line, pore inequality, 3 of subsidiary cells; The all visible palisade tissue of last lower epidermis.The parenchyma cell similar round has oval pit, integrated pit group; The wavy bending of endodermis cell anticlinal wall, thicker, lignify has the thin pore ditch of dredging.Plant skin stone cell yellow or light brown, how broken, complete person long polygon, rectangle or irregular shape, wall thickness has big circular pit, the cell brownish red.
Though this prescription has the method for quality control of this FENQINGWULIN WAN, but be only limited to microscopical identification, this method can only be controlled product quality from the cellular level of crude drug, can not control product quality from the level of effective ingredient, this can influence the quality and the curative effect of product, be unfavorable for the standardized production of medicine and enhance productivity, also do not meet the trend of modern Chinese medicine development.
Summary of the invention
The purpose of this invention is to provide and a kind of described FENQINGWULIN WAN is carried out the method for quality control,, improve the quality of products, be convenient to standardization, the modern production of medicine to guarantee the safety and the effectiveness of medicine.
In order to achieve the above object, the invention provides a kind of method of quality control of FENQINGWULIN WAN, this method comprises at least a detection in following a, b, c, d, five kinds of detections of e:
A. the qualitative detection of Radix Et Rhizoma Rhei:
Get this product, the supersound process that adds diethyl ether filters, and filtrate concentrating made need testing solution;
Other gets the Radix Et Rhizoma Rhei control medicinal material, adds diethyl ether to extract to make control medicinal material solution.
According to thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same lamellae, be developing solvent with the upper solution of petroleum ether (30～60 ℃)-Ethyl formate-formic acid, launch, take out, dry, daylight is observed down.In the test sample chromatograph, with the corresponding position of Radix Et Rhizoma Rhei control medicinal material chromatograph on show the speckle of same color.
B. the qualitative detection of berberine hydrochloride:
Get the medicinal residues after extraction this product under a item, volatilize ether, add the methanol supersound process, filter, filtrate evaporate to dryness, residue add methanol as need testing solution.
Other gets berberine hydrochloride and adds methanol and make reference substance solution.
According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put on same lamellae, be developing solvent with ethyl acetate-acetone-formic acid-water, launch, take out, dry, put under the ultra-violet lamp that wavelength is 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color.
C. the qualitative detection of jasminoidin:
Get the jasminoidin reference substance, add methanol and make reference substance solution;
Get need testing solution under the b item as need testing solution;
According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put on same lamellae, put respectively on same lamellae, with ethyl acetate-formic acid-water is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to speckle colour developing clear.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
D. the qualitative detection of Aristolochic Acid
Get the Aristolochic Acid reference substance, the accurate title, decide, and adds ethanol preparation and become reference substance solution;
Get this product, add ethanol water-bath reflux, extract,, filter, filtrate water bath method, residue add ethanol makes dissolving, as need testing solution;
According to the thin layer chromatography test, draw above-mentioned two kinds of solution respectively, put respectively on same lamellae, be developing solvent with toluene-ethyl acetate water-formic acid upper solution, launch, take out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Aristolochic Acid reference substance chromatograph on do not have the speckle of same color.
E. the detection by quantitative of emodin:
It is an amount of that precision takes by weighing the emodin reference substance, adds methanol and make every solution, promptly.
Precision is got this product, adds 5% methanol hydrochloride solution, and accurate the title decide gross weight, and reflux 2 hours is put coldly, with 5% methanol hydrochloride solution benefit weight loss, shakes up filtration, promptly.
Draw reference substance solution and need testing solution respectively, inject chromatograph of liquid, measure, promptly.
Preferably, the method for quality control of a kind of FENQINGWULIN WAN of the present invention, this method comprises at least a detection in following a, b, c, d, five kinds of detections of e:
A. the qualitative detection of Radix Et Rhizoma Rhei:
Get this product, porphyrize takes by weighing 2g, the 30ml that adds diethyl ether, and supersound process 10 minutes filters, and filtrate is concentrated into 1ml as need testing solution;
Other gets Radix Et Rhizoma Rhei control medicinal material 0.1g, and the 1ml that adds diethyl ether dipping 2 hours is got supernatant medical material solution in contrast;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, upper solution with petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry, daylight is observed down.In the test sample chromatograph, with the corresponding position of Radix Et Rhizoma Rhei control medicinal material chromatograph on show the speckle of same color;
B. the qualitative detection of berberine hydrochloride:
Get the medicinal residues after extraction this product under a item, volatilize ether, add methanol 30ml supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add methanol 1ml as need testing solution;
Other gets berberine hydrochloride and adds methanol and make solution that every 1ml contains 1mg product solution in contrast;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 1～2 μ l of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water (5: 5: 1: 1) be developing solvent, launch, take out, dry, put under the ultra-violet lamp that wavelength is 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color;
C. the qualitative detection of jasminoidin:
Get the jasminoidin reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of need testing solution under reference substance solution and the b item, put respectively on same silica gel g thin-layer plate, with ethyl acetate-formic acid-water (10: 0.5: 0.5) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
D. the qualitative detection of Aristolochic Acid:
Get this product, porphyrize takes by weighing 10g, adds ethanol 50ml, and water-bath reflux, extract, 1h filters, and filtrate water bath method, residue add ethanol 1ml makes dissolving, as need testing solution;
Get the Aristolochic Acid reference substance, the accurate title, decide, and adds the solution that ethanol is made 0.2mg/ml, in contrast product solution;
According to the thin layer chromatography test, draw above-mentioned reference substance solution 2 μ l, sample solution 5 μ l, put respectively on same lamellae, with 20: 10: 1: the toluene-ethyl acetate-water of 1 ratio-formic acid upper solution was developing solvent, launched, and took out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Aristolochic Acid reference substance chromatograph on do not have the speckle of same color;
E. the detection by quantitative of emodin:
With octadecylsilane chemically bonded silica is filler; Methanol-0.1% phosphoric acid mixed solution with 84: 20 ratios is a mobile phase; The detection wavelength is 254nm; Flow velocity: 1.0ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the emodin peak should be not less than 2000;
It is an amount of that precision takes by weighing the emodin reference substance, adds methanol and make the solution that every 1ml contains 15 μ g, promptly gets reference substance solution;
Get this product, be ground into fine powder, get powder 0.4g, the accurate title, decide, put in the tool plug conical flask, precision adds 5% methanol hydrochloride solution 50ml, and gross weight, reflux 2 hours decided in accurate title, put coldly, mend weight loss, shake up filtration, promptly get need testing solution with 5% methanol hydrochloride solution;
Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
The every gram of this product contains Radix Et Rhizoma Rhei with emodin (C
15H
10O
5) meter, must not be less than 0.50mg.
FENQINGWULIN WAN method of quality control of the present invention is convenient to standardization, the modern production of medicine, helps enhancing productivity, and has guaranteed the safety and the effectiveness of medicine.
In order to understand the present invention better, the present invention will be described in detail in conjunction with the specific embodiment now.
The specific embodiment
Following experimental example and embodiment further specify but are not limited to the present invention
The qualitative checking method research of experimental example 1 Radix Et Rhizoma Rhei
1, ether supersound extraction time preferred in the preparation of detection method a sample solution of the present invention:
Get totally 5 parts of FENQINGWULIN WAN (pressing embodiment 1 prescription and method preparation) 5g, porphyrize with ether supersound extraction different time, filters, and measures wherein emodin content, the results are shown in following table:
Table 1 ether ultrasonic time optimization experiment result
| Ultrasonic time (min) | 5 | 10 | 15 | 20 |
| Emodin content (mg) | 63.3 | 95.2 | 95.6 | 95.7 |
As can be seen from Table 1, the ultrasonic 10min of ether just can extract emodin wherein fully, so the preferred ultrasonic time of the preparation of this laboratory sample solution is 10min.
2, developing solvent consumption proportion preferred among the detection method a of the present invention:
Get need testing solution, with each 5 μ l point of control medicinal material solution of method preparation on different silica gel g thin-layer plates, using petroleum ether (30～60 ℃)-Ethyl formate-formic acid proportioning respectively is that 25: 5: 1,20: 5: 1,15: 5: 1,10: 5: 1,5: 5: 1 developing solvent launches, take out, dry, put that daylight is following to be inspected, observe the unfolded effect of each speckle of test sample on each lamellae, the results are shown in following table:
Table 2 developing solvent consumption proportion optimization experiment result
| The developing solvent proportioning | 25∶5∶1 | 20∶5∶1 | 15∶5∶1 | 10∶5∶1 | 5∶5∶1 |
| Launch effect | Very poor | Difference | Good | Relatively poor | Hangover appears |
Developing solvent proportioning as can be seen from Table 2 is 15: 5: 1 o'clock, and it is best that need testing solution launches effect, and appearance hangover, principal spot separate phenomenons such as bad.
3, sample solution point sample amount preferred among the detection method a of the present invention:
Get need testing solution 1 μ l, 2 μ l, 3 μ l, 4 μ l, reference substance solution 4 μ l, point is on same silica gel g thin-layer plate, with petroleum ether (30～60 ℃)-Ethyl formate-formic acid proportioning is that 15: 5: 1 developing solvent launches, take out, dry, put that daylight is following to be inspected, observe the effect of test sample principal spot colour developing on the lamellae, the results are shown in following table:
Table 3 sample solution point sample amount optimization experiment result
| The point sample amount | 1μl | 2μl | 3μl | 4μl |
| Effect | Test sample is at corresponding reference substance position immaculate | Test sample is good at corresponding reference substance position speckle color developing effect | Test sample is good at corresponding reference substance position speckle color developing effect | Test sample separates bad at corresponding reference substance position speckle |
Test sample point sample amount is when 2～3 μ l as can be seen from Table 3, and color developing effect is good on lamellae, is fit to test requirements document.
4, negative control test
Get the negative sample that lacks Radix Et Rhizoma Rhei, prepare negative control solution, launch the back and corresponding speckle on the reference substance solution correspondence position, do not occur, illustrate that selected monitoring experiment specificity is strong according to need testing solution preparation method among the above-mentioned detection method a.
The qualitative checking method research of experimental example 2. berberine hydrochloride
1. the methanol supersound extraction time is preferred in the preparation of detection method b sample solution of the present invention:
Get the medicinal residues behind the ether extraction in the experimental example 1, volatilize ether, add methanol 30ml supersound process different time, filter, measure wherein content of berberine hydrochloride, the results are shown in following table:
Table 4 methanol ultrasonic time optimization experiment result
| Ultrasonic time (min) | 10 | 20 | 30 | 40 |
| Content of berberine hydrochloride (mg) | 10.3 | 15.9 | 19.2 | 19.5 |
As can be seen from Table 4, the ultrasonic 30min of ether just can extract berberine hydrochloride wherein fully, is 30min so this tests preferred ultrasonic time.
2. the developing solvent consumption proportion is preferred among the detection method b of the present invention:
Get need testing solution, with each 2 μ l point of control medicinal material solution of method preparation on different silica gel g thin-layer plates, be 3: 5: 1 with ethyl acetate-acetone-formic acid-water proportioning respectively: 1,4: 5: 1: 1,5: 5: 1: 1,6: 5: 1: 1,7: 5: 1: 1 developing solvent launches, take out, dry, put under the outer light modulation (365nm) and inspect, observe the unfolded effect of each speckle of test sample on each lamellae, the results are shown in following table:
Table 5 developing solvent consumption proportion optimization experiment result
| The developing solvent proportioning | 3∶5∶1∶1 | 4∶5∶1∶1 | 5∶5∶1∶1 | 6∶5∶1∶1 | 7∶5∶1∶1 |
| Launch effect | Principal spot is difficult to separate | Difference | Good | Relatively poor | Hangover appears |
Developing solvent proportioning as can be seen from Table 5 is 5: 5: 1: 1 o'clock, it is best that need testing solution launches effect, occur hangover, etc. phenomenon.
3, sample solution point sample amount preferred among the detection method b of the present invention:
Get need testing solution 1 μ l, 2 μ l, 3 μ l, 4 μ l, reference substance solution 2 μ l, point is on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water proportioning is 5: 5: 1: 1 developing solvent launches, take out, dry, put under the uviol lamp (365nm) and inspect, observe the effect of test sample principal spot colour developing on the lamellae, the results are shown in following table:
Table 6 sample solution point sample amount optimization experiment result
| The point sample amount | 1μl | 2μl | 3μl | 4μl |
| Effect | Test sample shows in corresponding reference substance position | Test sample shows at corresponding reference substance position speckle | Test sample shows at corresponding reference substance position speckle | Test sample is at corresponding reference substance position speckle branch |
| Chromatic effect is good | Chromatic effect is good | Mottle point is bigger, separates bad | From bad |
Test sample point sample amount is when 1～2 μ l as can be seen from Table 6, and color developing effect is good on lamellae, is fit to test requirements document.
4, negative control test
Get the negative sample that lacks Cortex Phellodendri, prepare negative control solution, launch the back and corresponding speckle on the reference substance solution correspondence position, do not occur, illustrate that selected monitoring experiment specificity is strong according to above-mentioned need testing solution preparation method.
The qualitative detection experimental study of experimental example 3. jasminoidins
1, developing solvent consumption proportion preferred among the detection method c of the present invention:
Get 2 following need testing solutions of experimental example, with each 5 μ l point of control medicinal material solution of method preparation on different silica gel g thin-layer plates, it with ethyl acetate-formic acid-water proportioning respectively 10: 2: 0.5,10: 1.5: 0.5,10: 0.5: 0.5,10: 0.3: 0.5 developing solvent expansion, take out, dry, spray is with 10% ethanol solution of sulfuric acid, the unfolded effect of each speckle of test sample on each lamellae is observed in 105 ℃ of heating, the results are shown in following table:
Table 7 developing solvent consumption proportion optimization experiment result
| The developing solvent proportioning | 10∶2∶0.5、 | 10∶1.5∶0.5 | 10∶1.0∶0.5 | 10∶0.5∶0.5 | 10∶0.3∶0.5 |
| Launch effect | Very poor | Difference | Relatively poor | Good | Hangover appears |
Developing solvent proportioning as can be seen from Table 7 is 10: 0.5: 0.5 o'clock, and it is best that need testing solution launches effect, and appearance hangover, principal spot separate phenomenons such as bad.
2, sample solution point sample amount preferred among the detection method c of the present invention:
Get 2 following need testing solution 1 μ l of experimental example, 2 μ l, 3 μ l, 4 μ l, reference substance solution 2 μ l, point is on same silica gel g thin-layer plate, with ethyl acetate-formic acid-water proportioning is that 10: 0.5: 0.5 developing solvent launches, and takes out, and dries, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, the effect of observing test sample principal spot colour developing on the lamellae the results are shown in following table:
Table 8 sample solution point sample amount optimization experiment result
| The point sample amount | 1μl | 2μl | 3μl | 4μl |
| Effect | Test sample is at corresponding reference substance position immaculate | Test sample is clear in the colour developing of corresponding reference substance position speckle | Test sample is clear in the colour developing of corresponding reference substance position speckle | Test sample separates bad at corresponding reference substance position speckle |
Test sample point sample amount is when 2～3 μ l as can be seen from Table 8, and color developing effect is good on lamellae, is fit to test requirements document.
3, negative control test
Get the negative sample that lacks Fructus Gardeniae, prepare negative control solution, launch the back and corresponding speckle on the reference substance solution correspondence position, do not occur, illustrate that selected test experience specificity is strong according to need testing solution preparation method among the above-mentioned detection method c.
Experimental example 4 Aristolochic Acids qualitative detection
Caulis Akebiae is the easily chaotic simply medical material that uses, and mainly the section of showing as belongs to the kind confusion, the Lardizabalaceae Caulis Akebiae is arranged, the kind of the multiple not equal genus of Ranunculaceae Caulis Akebiae and Aristolochiaceae Caulis Akebiae.And the Aristolochiaceae Caulis Akebiae contains the Aristolochic Acid that can cause kidney damage, so this experiment has been carried out thin-layer qualitative detection research to whether containing Aristolochic Acid in the Caulis Akebiae.
1. detection method Aristolochic Acid extracting method of the present invention preferred:
Get totally 3 parts of FENQINGWULIN WAN (pressing embodiment 1 prescription and method preparation) 10g, porphyrize, every part adds Aristolochic Acid 2mg, after handling with distinct methods, measures the Aristolochic Acid content that is extracted, and the results are shown in following table
The assay result of table 9 Different Extraction Method Aristolochic Acid
| Method | Add ethanol 50ml, water-bath reflux, extract, 1h filters | With acetic acid moistening after, add ethanol 50ml supersound extraction 30min, filter | With acetic acid moistening after, add methanol 50ml merceration and cross liquid and filter |
| The content of Aristolochic Acid (mg) | 1.96 | 1.55 | 1.34 |
As can be seen from Table 1, the alcohol heating reflux method just can be extracted Aristolochic Acid wherein fully, so the preparation of this laboratory sample solution preferably adds ethanol 50ml, water-bath reflux, extract, 1h filters the filtrate water bath method, residue adds ethanol 1ml makes dissolving, as need testing solution.
2, thin layer chromatography condition test
Adopt the 0.6%CMC-Na silica gel g thin-layer plate, thickness of thin layer 0.3mm, 105 ℃ of activation 0.5h; Get need testing solution, with each 5 μ l point of Caulis Aristolochiae Manshuriensis control medicinal material solution of method preparation on different silica gel g thin-layer plates, use following developing solvent respectively: (1) chloroform-acetone-formic acid (7: 3: 0.1), uviol lamp (365nm) is observed down; (2) toluene-ethyl acetate-methanol-formic acid (20: 10: 1: 1), observed down by uviol lamp (365nm); (3) toluene-ethyl acetate-water-formic acid (20: 10: 1: 1) upper solution, daylight, uviol lamp (365nm) are observed down, observe the unfolded effect of each speckle of test sample on each lamellae, the results are shown in following table:
Table 10 developing solvent consumption proportion optimization experiment result
| Developing solvent and proportioning | Chloroform-acetone-formic acid (7: 3: 0.1) | Toluene-ethyl acetate-methanol-formic acid | Toluene-ethyl acetate-water-formic acid |
| (20∶10∶1∶1) | (20∶10∶1∶1) | ||
| Launch effect | Very poor | Good | Difference |
Developing solvent is that (20: 10: 1: in the time of 1), it is best that need testing solution launches effect, and appearance hangover, principal spot separate phenomenons such as bad for toluene-ethyl acetate-methanol-formic acid as can be seen from Table 2.
3, Aristolochic Acid lowest detectable limit test
Get the Aristolochic Acid reference substance, the accurate title, decide, and adds ethanol and make 0.199gL
-1Solution, product solution in contrast.Accurate 0.5,1,2,4,8, the 16 μ l that draw put respectively on same silica gel g thin-layer plate, launch with above-mentioned condition, take out, and dry, and inspect under the daylight, and 2,4,8,16 μ l show the glassy yellow speckle, and 0.5,1 μ l end detects speckle; Inspect under the ultraviolet light 365nm, 2,4,8,16 μ l show yellowish-brown fluorescence speckle, and 0.5,1 μ l end detects the fluorescence speckle.The result shows: with this law experiment, the Aristolochic Acid lowest detection is limited to 0.398 μ g.
4, controlled trial
Get FENQINGWULIN WAN (pressing embodiment 1 prescription and method preparation) 6g, the Caulis Aristolochiae Manshuriensis that Caulis Akebiae in the prescription is replaced with equivalent makes positive control drug according to embodiment 1 prescription and method, above-mentioned Aristolochic Acid solution is product solution in contrast, get above-mentioned FENQINGWULIN WAN, positive control medicinal liquid each 10 μ l, reference substance solution 2 μ l according to above-mentioned detection method, point sample is on same silica gel g thin-layer plate, launch back above-mentioned FENQINGWULIN WAN on the reference substance solution correspondence position and corresponding speckle do not occur, corresponding speckle appears in positive control drug, illustrates that selected test experience specificity is strong.
The detection by quantitative research of experimental example 5 emodins
(1) stability test is got test sample (pressing embodiment 1 prescription and method preparation) and is pressed method processing under the assay, respectively at preparing the back 0,2,4,6,8,24 hour, measures in accordance with the law, sampling volume 5 μ l, the result shows that it is basicly stable in 24 hours, the results are shown in following table
Table 11 study on the stability
(2) linear relationship is investigated and to be got reference substance solution (14.8 μ g/ml) and shake up, accurate respectively 1,2.5,5,10,15, the 17.5 μ l of absorption inject high performance liquid chromatograph, measure peak area, the results are shown in following table, and drawing standard curve, show that emodin is linear between 0.0148 μ g-0.259 μ g, its regression equation is:
Area＝3689858.916*Amt+1443.748(r＝0.9995)
Table 12 linear relationship is investigated
(3) the accurate reference substance solution 5 μ l that draw of precision test repeat sample introduction 5 times, try to achieve relative standard deviation＜2%, the results are shown in following table
The test of table 13 precision
(4) replica test is pressed the text method, and (lot number: 05071101) sample is 5 parts, and every part is measured, and tries to achieve relative standard deviation＜2%, the results are shown in following table to get same lot number
Table 14 replica test
(5) recovery test takes by weighing sample (lot number: 05071101) 0.2g, the accurate title, decide, accurate emodin reference substance solution (the 25 μ g/ml) 10ml that adds, accurate 5% methanol hydrochloride solution that adds 15ml, press the preparation method operation of text need testing solution, measure its content, and calculate its response rate, measurement result sees the following form:
Table 15 recovery test
From above result of the test as can be seen, its linear relationship of content assaying method of the present invention, stability, precision, repeatability, the response rate are all good, can effectively control emodin content in the FENQINGWULIN WAN.
Embodiment 1
Take by weighing the crude drug of following weight portion (kg):
Caulis Akebiae 80 Semen Plantaginiss (salt stir-fry) 40 Radix Scutellariaes 80
Poria 40 Polyporus 40 Cortex Phellodendris 40
Radix Et Rhizoma Rhei 120
Hold 40 Herba Dianthis 40
The Rhizoma Anemarrhenae 40 Rhizoma Alismatis 40 Fructus Gardeniaes 40
Radix Glycyrrhizae 20 Talcums 80
More than 14 flavors, except that Talcum, all the other Caulis Akebiaes etc. 13 flavor is ground into fine powder, sieves mixing.Use water pill, drying is broken into the impalpable powder coating with Pulvis Talci, polishing, and drying, promptly.
Embodiment 2
Take by weighing the crude drug of following weight portion (kg):
Caulis Akebiae 80 Semen Plantaginiss (salt stir-fry) 40 Radix Scutellariaes 80
Poria 40 Polyporus 40 Cortex Phellodendris 40
Radix Et Rhizoma Rhei 120
Hold 40 Herba Dianthis 40
The Rhizoma Anemarrhenae 40 Rhizoma Alismatis 40 Fructus Gardeniaes 40
Radix Glycyrrhizae 20 Talcums 80
More than 14 flavors, except that Talcum, all the other Caulis Akebiaes etc. 13 flavor is pulverized, and crosses 100 mesh sieves, mixing.Use water pill, drying is pulverized Talcum, crosses 200 mesh sieves, coating, and polishing, drying, promptly.
Embodiment 3-6
According to embodiment 2 identical method and consumption, 4 batches of these FENQINGWULIN WAN of middle trial production.
Embodiment 7. method of quality control of the present invention:
(1) get FENQINGWULIN WAN, porphyrize takes by weighing 2g, the 30ml that adds diethyl ether, and supersound process 10 minutes filters, and filtrate is concentrated into 1ml as need testing solution.Other gets Radix Et Rhizoma Rhei control medicinal material 0.1g, and the 1ml that adds diethyl ether dipping 2 hours is got supernatant medical material solution in contrast.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, upper solution with petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry, daylight is observed down.In the test sample chromatograph, with the corresponding position of Radix Et Rhizoma Rhei control medicinal material chromatograph on show the speckle of same color.
(2) medicinal residues behind the ether extraction under present embodiment (1) item volatilize ether, add methanol 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue add methanol 1ml as need testing solution.Other gets berberine hydrochloride and adds methanol and make solution that every 1ml contains 1mg product solution in contrast.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 1～2 μ l of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water (5: 5: 1: 1) be developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color.
(3) get the jasminoidin reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of need testing solution under reference substance solution and present embodiment (2) item, put respectively on same silica gel g thin-layer plate, with ethyl acetate-formic acid-water (10: 0.5: 0.5) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the speckle colour developing clear.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(4) get the Aristolochic Acid reference substance, the accurate title, decide, and adds ethanol preparation and become 0.200gL
-1Reference substance solution;
Get FENQINGWULIN WAN, precision takes by weighing 10g and adds ethanol water-bath reflux, extract, 1h, filters, and filtrate water bath method, residue add ethanol 1ml makes dissolving, as need testing solution.
According to the thin layer chromatography test, draw above-mentioned two kinds of solution respectively, put respectively on same lamellae, with 20: 10: 1: the toluene-ethyl acetate-water of 1 ratio-formic acid upper solution is developing solvent, launches, and takes out, and dries, daylight is observed down; In the test sample chromatograph, with the corresponding position of Aristolochic Acid reference substance chromatograph on do not have the speckle of same color.
[assay]
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.1% phosphoric acid (84: 20) is mobile phase; The detection wavelength is 254nm; Flow velocity: 1.0ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the emodin peak should be not less than 2000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the emodin reference substance, adds methanol and make the solution that every 1ml contains 15 μ g, promptly.
It is an amount of that this product is got in the preparation of need testing solution, is ground into fine powder, gets powder 0.4g, and accurate the title decides, put in the tool plug conical flask, precision adds 5% methanol hydrochloride solution 50ml, and gross weight, reflux 2 hours decided in accurate title, put coldly, mend weight loss, shake up filtration, promptly with 5% methanol hydrochloride solution.
Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The every gram of this product contains Radix Et Rhizoma Rhei with emodin (C
15H
10O
5) meter, must not be less than 0.50mg.
The prepared FENQINGWULIN WAN of embodiment of the invention 1-6 has been carried out above-mentioned quality control, proves the complete quality control standard according to the invention of the prepared pill of embodiment 1-6.
The emodin content measurement result of table 16 embodiment 1-6
In addition, also ratio and the preparation technology with the prescription crude drug made the negative sample that does not contain Radix Et Rhizoma Rhei, Fructus Gardeniae and Cortex Phellodendri respectively, prepared negative control solution according to the preparation method for test agent in the method for quality control, on silica gel g thin-layer plate, speckle does not appear in described 3 kinds of negative samples on the relevant position; And ratio and preparation technology with the prescription crude drug have made the positive control drug that replaces Caulis Akebiae with Caulis Aristolochiae Manshuriensis, on silica gel g thin-layer plate, and the speckle of the sour same color of demonstration and horse pocket spirit on same position, and FENQINGWULIN WAN is not seen speckle in this position; Through repetition test, therefore this method of quality control specificity, good reproducibility as the quality control qualitative checking method of FENQINGWULIN WAN, carry out quality control to FENQINGWULIN WAN.
Claims (2)
1, a kind of method of quality control of FENQINGWULIN WAN, this method comprise at least a detection in following a, b, c, d, five kinds of detections of e:
A. the qualitative detection of Radix Et Rhizoma Rhei:
Get this product, the supersound process that adds diethyl ether filters, and filtrate concentrating made need testing solution;
Other gets the Radix Et Rhizoma Rhei control medicinal material, adds diethyl ether to extract to make control medicinal material solution;
According to thin layer chromatography test, draw above-mentioned two kinds of solution each, put respectively on same lamellae, be developing solvent with the upper solution of petroleum ether (30～60 ℃)-Ethyl formate-formic acid, launch, take out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Radix Et Rhizoma Rhei control medicinal material chromatograph on show the speckle of same color;
B. the qualitative detection of berberine hydrochloride:
Get the medicinal residues after extraction this product under a item, volatilize ether, add the methanol supersound process, filter, filtrate evaporate to dryness, residue add methanol as need testing solution;
Other gets berberine hydrochloride and adds methanol and make reference substance solution;
According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put on same lamellae, be developing solvent with ethyl acetate-acetone-formic acid-water, launch, take out, dry, put under the ultra-violet lamp that wavelength is 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color;
C. the qualitative detection of jasminoidin:
Get the jasminoidin reference substance, add methanol and make reference substance solution;
Get need testing solution under the b item as need testing solution;
According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put on same lamellae, put respectively on same lamellae, with ethyl acetate-formic acid-water is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to speckle colour developing clear; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
D. the qualitative detection of Aristolochic Acid
Get the Aristolochic Acid reference substance, the accurate title, decide, and adds ethanol preparation and become reference substance solution;
Get this product, add ethanol water-bath reflux, extract,, filter, filtrate water bath method, residue add ethanol makes dissolving, as need testing solution;
According to the thin layer chromatography test, draw above-mentioned two kinds of solution respectively, put respectively on same lamellae, be developing solvent with toluene-ethyl acetate-water-formic acid upper solution, launch, take out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Aristolochic Acid reference substance chromatograph on do not have the speckle of same color;
E. the detection by quantitative of emodin:
It is an amount of that precision takes by weighing the emodin reference substance, adds methanol and make reference substance solution, promptly;
Precision is got this product, adds 5% methanol hydrochloride solution, and accurate the title decide gross weight, and reflux 2 hours is put coldly, with 5% methanol hydrochloride solution benefit weight loss, shakes up filtration, promptly;
Draw reference substance solution and need testing solution respectively, inject chromatograph of liquid, measure, promptly.
2, the method for quality control of a kind of FENQINGWULIN WAN as claimed in claim 1, this method comprise at least a detection in following a, b, c, d, five kinds of detections of e:
A. the qualitative detection of Radix Et Rhizoma Rhei:
Get this product, porphyrize takes by weighing 2g, the 30ml that adds diethyl ether, and supersound process 10 minutes filters, and filtrate is concentrated into 1ml as need testing solution;
Other gets Radix Et Rhizoma Rhei control medicinal material 0.1g, and the 1ml that adds diethyl ether dipping 2 hours is got supernatant medical material solution in contrast;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, upper solution with petroleum ether (30～60 ℃)-Ethyl formate-formic acid mixed liquor of 15: 5: 1 ratios is developing solvent, launch, take out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Radix Et Rhizoma Rhei control medicinal material chromatograph on show the speckle of same color;
B. the qualitative detection of berberine hydrochloride:
Get the medicinal residues after extraction this product under a item, volatilize ether, add methanol 30ml supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add methanol 1ml as need testing solution;
Other gets berberine hydrochloride and adds methanol and make solution that every 1ml contains 1mg product solution in contrast;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 1～2 μ l of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with 5: 5: 1: the ethyl acetate-acetone of 1 ratio-formic acid-water was developing solvent, launch, take out, dry, put under the ultra-violet lamp that wavelength is 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color;
C. the qualitative detection of jasminoidin:
Get the jasminoidin reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution;
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 2～3 μ l of need testing solution under reference substance solution and the b item, put respectively on same silica gel g thin-layer plate, ethyl acetate-formic acid-water with 10: 0.5: 0.5 ratios is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
D. the qualitative detection of Aristolochic Acid:
Get this product, porphyrize takes by weighing 10g, adds ethanol 50ml, and water-bath reflux, extract, 1h filters, and filtrate water bath method, residue add ethanol 1ml makes dissolving, as need testing solution;
Get the Aristolochic Acid reference substance, the accurate title, decide, and adds the solution that ethanol is made 0.2mg/ml, in contrast product solution;
According to the thin layer chromatography test, draw above-mentioned reference substance solution 2 μ l, sample solution 5 μ l, put respectively on same lamellae, with 20: 10: 1: the toluene-ethyl acetate-water of 1 ratio-formic acid upper solution was developing solvent, launched, and took out, dry, daylight is observed down; In the test sample chromatograph, with the corresponding position of Aristolochic Acid reference substance chromatograph on do not have the speckle of same color;
E. the detection by quantitative of emodin:
With octadecylsilane chemically bonded silica is filler; Methanol-0.1% phosphoric acid mixed solution with 84: 20 ratios is a mobile phase; The detection wavelength is 254nm; Flow velocity: 1.0ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the emodin peak should be not less than 2000;
It is an amount of that precision takes by weighing the emodin reference substance, adds methanol and make the solution that every 1ml contains 15 μ g, promptly gets reference substance solution;
Get this product, be ground into fine powder, get powder 0.4g, the accurate title, decide, put in the tool plug conical flask, precision adds 5% methanol hydrochloride solution 50ml, and gross weight, reflux 2 hours decided in accurate title, put coldly, mend weight loss, shake up filtration, promptly get need testing solution with 5% methanol hydrochloride solution;
Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
The every gram of this product contains Radix Et Rhizoma Rhei with emodin (C
15H
10O
5) meter, must not be less than 0.50mg.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961528A (en) * | 2012-12-10 | 2013-03-13 | 青岛中科润美润滑材料技术有限公司 | Traditional Chinese medicine composition for treating postpartum stranguria |
| CN103536839A (en) * | 2013-10-31 | 2014-01-29 | 金占荣 | Medicament for treating cystitis and preparation method thereof |
| CN103623149A (en) * | 2013-12-10 | 2014-03-12 | 马家庆 | Traditional Chinese medicine for treating gonococcal urethritis |
| CN115308352A (en) * | 2022-08-19 | 2022-11-08 | 江阴天江药业有限公司 | Quality control method of mollissima sample |
-
2008
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961528A (en) * | 2012-12-10 | 2013-03-13 | 青岛中科润美润滑材料技术有限公司 | Traditional Chinese medicine composition for treating postpartum stranguria |
| CN103536839A (en) * | 2013-10-31 | 2014-01-29 | 金占荣 | Medicament for treating cystitis and preparation method thereof |
| CN103536839B (en) * | 2013-10-31 | 2015-07-08 | 金占荣 | Medicament for treating cystitis and preparation method thereof |
| CN103623149A (en) * | 2013-12-10 | 2014-03-12 | 马家庆 | Traditional Chinese medicine for treating gonococcal urethritis |
| CN103623149B (en) * | 2013-12-10 | 2015-08-19 | 马家庆 | A kind of Chinese medicine for the treatment of gonococcal urethritis |
| CN115308352A (en) * | 2022-08-19 | 2022-11-08 | 江阴天江药业有限公司 | Quality control method of mollissima sample |
| CN115308352B (en) * | 2022-08-19 | 2023-11-10 | 江阴天江药业有限公司 | Quality control method of herba Aristolochiae Mollissimae sample |
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