CN101040951A - Method of controlling the quality of Ynxingchao dropping pills compound - Google Patents
Method of controlling the quality of Ynxingchao dropping pills compound Download PDFInfo
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Abstract
The invention discloses a quality control method of composite houttuynia cordata drop, which uses high-effect liquid spectrum method to test the baicalin content of houttuynia cordata, uses thin layer spectrum to qualitatively check the methyl nonyl ketone of houttuynia cordata, the baicalin of baikal skullcap root, the phillyrin of capsule of weeping forsythia, the chlorogenic acid of honeysuckle and the arginine of isatic root, to control the quality of drop and make product safe, effective, stable and controllable.
Description
Technical field
The present invention relates to a kind of FUFANG YUXINGCAO DIWAN method of quality control, specifically control the quality of FUFANG YUXINGCAO DIWAN by thin layer chromatography qualitative method and high performance liquid chromatogram quantitative method.
Background technology
Upper respiratory tract infection, acute tonsillitis, pharyngitis, chronic bronchitis are commonly encountered diseases, frequently-occurring disease clinically, clinical treatment to use antimicrobial chemistry medicine for seeing more, but cure the symptoms, not the disease, use these medicines to cause fastbacteria to produce on the contrary repeatedly, more toxic and side effects also makes to use and is restricted simultaneously.Though traditional form of Chinese drug can overcome the untoward reaction that chemical medicine brings, the defective of medicine-releasing system imperfection of itself and quality instability, poor controllability is restricted clinical application.FUFANG YUXINGCAO DIWAN has been taken into account the two advantage, has overcome their deficiency, is a modern Chinese medicine that utilizes modern technologies and advanced and applicable technology to transform the Chinese medicine dosage form, especially its quality control index is reached a higher level.
Used Herba Houttuyniae, Radix Scutellariae, Radix Isatidis, Fructus Forsythiae, Flos Lonicerae in this prescription, by its physicochemical property and pharmacology pharmacodynamic be studies show that, measuring the clear and definite effective active composition of its structure is a preferred version as quality control index.
Herba Houttuyniae (Herba houttuyniae) is the dry aerial parts of saururaceae plant houttuynia cordata Houttuynia cordataThunb..
Its chemical constituent is mainly:
1, it is about 0.05% that volatile ingredient contains volatile oil, and effective ingredient is aldehyde ketone chemical compound, decanoylacetaldehyde (houttuynine sodium bisulfite Decanoylacetaldehyde), lauryl aldehyde (Lauraldehyde) in the oil; And contain aldehyde ketone chemical compound methyln nonyl ketone (2-undecanone), sesquiterpene people's compound Flos Caryophylli alkene (caryophyllene), monoterpene chemical compound australene (α-pinene), camphene (camphene) etc.;
2, flavone compound contains Quercetin (quercetin), Quercitroside (quercitrin), isoquercitrin (isoquercitrin) etc.; Also contain in addition organic acid and fatty acid, inorganic salts or the like (cloudy strong. modernization of cmm and clinical practice, I, 457 pages.)。
CH
3CH
2CH
2CH
2CH
2CH
2CH
2CH
2CH
2-C-CH
3Methylnonanone
[3]
The assay of Herba Houttuyniae is mainly measured total aldehyde ketone chemical compound, decanoylacetaldehyde, australene etc., research main measuring methods in recent years have colorimetry, volumetric method, infrared spectrometry, gas chromatography, titrimetry etc. (cloudy strong. Chinese medicine modern study and clinical practice, I, 458 pages).
Radix Scutellariae (Radix scutellariae) is the dry root of labiate Radix Scutellariae Scutellariabaicalensis Georgi..
Its main component is a flavonoid.Flavone noroxylin (baicalein), baicalin (baicalin), 5,6-dihydroxy-flavone-7-O-glucoside (5,6-dihydroxy-7-O-glucoside-flavone), chrysin (chrysin), 5,7,2 '-trihydroxy-flavone (5,7,2 '-trihydrox-y-flavone), 5,7,2 ', 3 '-kaempferol (5,7,2 ', 3 '-tetraflavone), nor-wogonin (nor-wogonin), wogonin (Wogonin), wogonoside (Wogonoside), neobaicalein I (skullcapflavoneI), neobaicalein II (skullcapflavone II) etc.; Flavanone 7,2 ', 6 '-trihydroxy-5-melonia flavone (7,2 ', 6 '-trihydroxy-5-methoxyflavanone) etc.; Chalcone 7,2 ', 6 '-trihydroxy-5-methoxyl group-chalcone (7,2 ', 6 '-trihydroxy-5-methoxychalcone) etc.; Flavanonol 3,5,7,2 ', 6 '-penta hydroxy group flavanonol (3,5,7,2 ', 6 '-pentahydroxy flavanonll) etc.; Flavonol 3,5,7,2 ', 6 '-pentahydroxyflavone alcohol (3,5,7,2 ', 6 '-pentahydroxyflavanonol) etc.; Also contain phenethanol glucoside, aminoacid, volatilization wet goods (cloudy strong. Chinese medicine modern study and clinical practice, I, 560 pages).Baicalin R=H neobaicalein I R1=R2=H baicalin R=gluA neobaicalein II R1=R2=OCH3
[7]Wogonin R=H wogonoside R=gluA
[7]The assay of Radix Scutellariae mainly contain colorimetry, dual-wavelength ultraviolet spectrophotometry, thin layer chromatography-colorimetry, thin layer chromatography scanning, high performance liquid chromatography etc. (cloudy strong. Chinese medicine modern study and clinical practice, I, 560 pages).Radix Isatidis (Radix isatidis) is the dry root of cruciferae isatis Isatisindigotica Fort..Contain total amino acids 3.8% in the Radix Isatidis, arginine, glutamic acid, tyrosine, proline, valine, leucine, γ-An Jidingsuan etc. are arranged in the aminoacid, and protein, resin-like thing etc.Indigo, indirubin, 1-Hydrogen thiocyanate-2-hydroxyl-3-butylene, left-handed 5-vinyl oxazole pyridine-2 thioketone, couroupitine A, qingdainone, adenosine, uridnine, the fast cry of certain animals of inferior Huang, uracil, Palmic acid, salicylic acid, cupreol, clionasterol, daucosterol etc., still contain in addition sucrose 20.3%, resin etc. (Miao Ming three Li Zhen states. modern practical Chinese medicine quality control technology, 617 pages).
Fructus Forsythiae (Fructus forsythiae) is the dry fruit of Oleaceae plant Fructus Forsythiae Forsythia suspensa (Thunb.) Vahl.
Its main chemical compositions is:
1, volatile component mainly is present in the seed, content average 3.8%.Its hydro carbons has australene (α-pinene), camphene (camphene), nopinene (β-pinene), p-ymene, limonene, γ, terpinene, β-phellandrene, myrcene, β-ocimene etc.; Aldoketones has Camphora (camphor), geranial (geranial); The alcohol ester ethers has Borneolum Syntheticum (borneol) etc.
2, phenethanol glycoside forsythoside (forsythoside) A, B, C, D, forsythol (forsythol) secondly is.Contain in addition in addition compositions such as triterpenes, Coumarins (cloudy strong. Chinese medicine modern study and clinical practice, I, 356-357 page or leaf).The assay of Fructus Forsythiae mainly contain thin layer chromatography scanning, bounce technique etc. (cloudy strong. Chinese medicine modern study and clinical practice, I, 357 pages).Flos Lonicerae (Floslonicerae) is the dry flower of caprifoliaceae plant Radix Ophiopogonis Lonicera japonica Thunb., Flos Lonicerae Lonicera hypoglauca Miq., Flos Lonicerae Lonicera confusa DC. or hair style Radix Ophiopogonis Lonicera dasystyla Rehd. or the flower that band is just opened.
Its main chemical compositions is:
Contain 30 multiple components in the Flos Lonicerae in the volatile oil, mainly contain linalool (linalool), (-)-suitable-2,6,6-trimethyl-2-vinyl-5-hydroxy tetrahydro pyrans [(-)-cis-2,6,6-trimethyl-2-ethenyl-5-hydroxytetrapyrane], pinene (pinene), 1-hexene (1-hexene), suitable-3-hexenol-1 (cis-3-hexenol-1) etc., contain luteolin (luteolin), inositol (inositol) in addition in the alabastrum.Isolate chlorogenic acid (chlorogenicacid) and isochlorogenic acid (isochlorogenic acid) etc. (cloudy strong. Chinese medicine modern study and clinical practice, I, 449 pages).
In the Flos Lonicerae assay mainly contain volumetric method, colorimetry, thin layer chromatography speckle quantitative method, gas chromatography, ultraviolet spectrophotometry, high-efficient liquid phase technique etc. (cloudy strong. Chinese medicine modern study and clinical practice, I, 450-452 page or leaf).
Summary of the invention
The object of the present invention is to provide a kind of method of quality control of FUFANG YUXINGCAO DIWAN.Quality control method of the present invention has adopted the modern technologies thin layer chromatography to differentiate the methylnonanone of Herba Houttuyniae in the FUFANG YUXINGCAO DIWAN, the baicalin in the Radix Scutellariae, the arginine in the Radix Isatidis, the phillyrin in the Fructus Forsythiae, the chlorogenic acid in the Flos Lonicerae.
The present invention is achieved like this, and implementation step is as follows:
One, the detection method of methylnonanone in the Herba Houttuyniae
1, the preparation of test sample
Get FUFANG YUXINGCAO DIWAN 30g, add water 500ml, press determination of volatile oil Division A League Matches of French Football method (2000 editions appendix XD of Chinese Pharmacopoeia) in determinator scale part topped up with water, add the 2ml ethyl acetate, extracted volatile oil 4 hours, put cold, divide and get the cerinic acid methacrylate layer, as need testing solution.
2, the preparation of reference substance solution
Other gets the methylnonanone reference substance, adds ethyl acetate to making the solution that every ml contains 0.1ml, in contrast product solution.
3, detection method
Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw test sample dissolution fluid 6 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (7～9: 3～1) be developing solvent, launch, take out, dry, spray is with the dinitrophenylhydrazine test solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the reference substance chromatograph on the relevant position, show the speckle of same color.
Two, the detection method of baicalin in the Radix Scutellariae
1, the preparation of test sample
Get FUFANG YUXINGCAO DIWAN 2g, add warm water 20ml, ultrasonic 10 minutes, transfer PH1.5 ~ 2,80 ℃ insulation 1h, centrifugal, abandoning supernatant, precipitation washing secondary, residue adds methanol 2ml makes dissolving, as test liquid.
2, the preparation of reference substance solution
Other gets the baicalin reference substance, adds methanol and makes the solution that every ml contains 0.5mg, in contrast product solution.
3, detection method
Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw test liquid 1 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-water (5～8: 0.5～2: 1～3) be developing solvent, launch, take out, dry, spray is with 2% ferric chloride ethanol liquid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with reference substance chromatograph relevant position on show the speckle of same color.
Three, the detection method of phillyrin in the Fructus Forsythiae
1, the preparation of test sample
Get FUFANG YUXINGCAO DIWAN 30g, add water 100ml, PH1.5 ~ 2,80 ℃ insulation 1h is transferred in warm dissolving, and is centrifugal, get supernatant, (60,40,40ml), mother solution is with ethyl acetate extraction three times (50,50 for ether extraction three times, 50ml), merge ethyl acetate extraction liquid, evaporate to dryness, the 10ml water dissolution is added in D
101On the macroporous resin column (the macroporous resin amount is column length 2/3 amount for internal diameter 1.6cm, length 18cm), use the 100ml water elution, reuse 25%-50% ethanol 50ml eluting is collected eluent (standby), continue with 50%-80% ethanol 80ml eluting, collect eluent, evaporate to dryness adds the 10ml water dissolution, it is an amount of to add neutral alumina, mix thoroughly, drying under reduced pressure adds neutral alumina post (200 ~ 300 orders, 5g, internal diameter 1.5 ~ 2cm, dry column-packing), 50%-80% ethanol elution 100ml, collect eluent, evaporate to dryness, residue add 2ml methanol makes dissolving, as test liquid.
2, the preparation of reference substance solution
Other gets the forsythin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.
3, detection method
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw test liquid 3 μ l, reference substance solution 1 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with chloroform-methanol (5: 1), launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color.
Four, the detection method of chlorogenic acid in the Flos Lonicerae
1, the preparation of need testing solution
Get 25%-50% ethanol elution under the discriminating (three), water bath method, dissolve with methanol, standardize solution are in the 5ml measuring bottle, as test liquid.
2, the preparation of reference substance solution
Other gets the chlorogenic acid reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.
3, detection method
According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw test liquid 2 μ l, reference substance solution 2 μ l put respectively on same polyamide film, are developing solvent with acetic acid, launch, and take out, and dry, and put under the 365nm fluorescence and inspect.In the test sample chromatograph, with reference substance chromatograph relevant position on, show the fluorescence speckle of same color.
Five, arginic detection in the Radix Isatidis
1, the preparation of need testing solution
Get this product 5g, add the warm dissolving of water 150ml, be added in (internal diameter 2cm, long 25cm) on the hydro-strong acidic cation exchange resin post, add water 300ml flushing (1ml/ minute), be washed till near colourless, reuse 0.25N-0.7N ammonia 400ml eluting is collected eluent (1ml/ minute), water bath method, residue adds the dissolving of 1ml Diluted Alcohol, as need testing solution.
2, the preparation of reference substance solution
Other gets the arginine reference substance, adds Diluted Alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.
3, detection method
Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw test liquid, each 1 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-water (19: 5: 5) is developing solvent, launch, take out, dry, spray is with ninhydrin solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, showing the same color speckle with the stratographic relevant position of reference substance.
Six, high effective liquid chromatography for measuring content of baicalin
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-water-phosphoric acid (47: 53: 0.2) mobile phase; The detection wavelength is 240-340nm.Number of theoretical plate is pressed the baicalin peak and is calculated, and should be not less than 2300.
The preparation precision of reference substance solution takes by weighing at 40 ℃ of-90 ℃ of baicalins of drying under reduced pressure 3-6 hour an amount of, adds 30%70% methanol and makes the solution that every 1ml contains 20 μ g-40 μ g, promptly.
This product 1.5g-3.0g is got in the preparation of need testing solution, grinds, and gets 0.1g-1.0g, the accurate title, decide, and adds the heat of solution of 2ml-20ml water temperature, adds 0.5ml-5ml 6mol/L hydrochloric acid solution, shake up, 40 ℃-90 ℃ are incubated 0.5-2 hour, and placement is spent the night, centrifugal, the supernatant that inclines, precipitation adds 70% ethanol ultrasonic dissolution, be transferred in the 50ml measuring bottle, add 50%-90% ethanol to scale, shake up, filter, discard filtrate just, the accurate subsequent filtrate 1ml-4ml that draws, water bath method adds 30%-70% ethanol gradation dissolving, and be transferred in the 10ml measuring bottle, add 30%70% ethanol to scale, shake up, promptly.
Accurate respectively reference substance solution and each 20ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures promptly.
This product contains Radix Scutellariae to contain baicalin (C
21H
18O
11) meter, must not be less than 2.0%.
Advantage of the present invention is: quality control method of the present invention has adopted the modern technologies thin layer chromatography to differentiate the methylnonanone of Herba Houttuyniae in the FUFANG YUXINGCAO DIWAN, the baicalin in the Radix Scutellariae, the arginine in the Radix Isatidis, the phillyrin in the Fructus Forsythiae, the chlorogenic acid in the Flos Lonicerae, and determined condition and method with the high effective liquid chromatography for measuring content of baicalin by a large amount of experiments, control the FUFANG YUXINGCAO DIWAN method for quality thereby set up with highly sensitive, favorable reproducibility, qualitative, quantitative that degree of accuracy is high.
The specific embodiment
Embodiment 1
Herba Houttuyniae 1100g Radix Scutellariae 333g Radix Isatidis 330g
Fructus Forsythiae 116g Flos Lonicerae 116g
Get the water logging bubble 5 hours that Herba Houttuyniae, Radix Isatidis, Fructus Forsythiae, Flos Lonicerae add 12 times of amounts, distillation, volatile oil device is in addition preserved, and is standby; Medicinal residues filter, and filtrate [I] is deposited in addition; The water that medicinal residues add 10 times of amounts continues to decoct 2 times, each 1 hour, collecting decoction filtered, merge with filtrate [I], be concentrated in the time of 60 ℃ and survey relative density about 1.15, put coldly, add ethanol and make and contain the alcohol amount and reach 70%, placed 24 hours, filter, filtrate recycling ethanol also continues to be condensed into fluid extract, and is standby; Get Radix Scutellariae, add the decocting that 10-12 doubly measures and boil secondary, each 2 hours, filter, merging filtrate is regulated pH value 1-3 with hydrochloric acid, and 80 ℃ are incubated 1 hour, left standstill 24 hours, filter, precipitation is washed with ethanol, and reuse is washed to PH5-6, the decompression cold drying is pulverized, and gets the Radix Scutellariae extract powder, join in the above-mentioned fluid extract, add volatile oil, mixing, get the PEG-6000 fusion, medicine is joined in the fused solution substrate medicine and substrate composition 1: 3.In following system of 85 ℃ of keeping warm modes, be condensing agent with liquid Paraffin or dimethicone, chilling temperature-5 ℃ is collected drop pill, wiping, promptly.
(1) gets this product 30g, add water 500ml, press determination of volatile oil Division A League Matches of French Football method (2000 editions appendix XD of Chinese Pharmacopoeia), add the 2ml ethyl acetate, extracted volatile oil 4 hours, put coldly, divide and get the cerinic acid methacrylate layer, as need testing solution in determinator scale part topped up with water.Other gets the methylnonanone reference substance, adds ethyl acetate to making the solution that every ml contains 0.1ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw test sample dissolution fluid 6 μ l, reference substance solution 4 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with normal hexane-ethyl acetate (9: 1), launches, take out, dry, spray is with the dinitrophenylhydrazine test solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the reference substance chromatograph on the relevant position, show the speckle of same color.
(2) get this product 2g, add warm water 20ml, ultrasonic 10 minutes, transfer PH1.5 ~ 2,80 ℃ insulation 1h, centrifugal, abandoning supernatant, precipitation washing secondary, residue adds methanol 2ml makes dissolving, as test liquid.Other gets the baicalin reference substance, adds methanol and makes the solution that every ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw test liquid 1 μ l, reference substance solution 5 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with n-butyl alcohol-glacial acetic acid-water (7: 1: 2), launches, take out, dry, spray is with 2% ferric chloride ethanol liquid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with reference substance chromatograph relevant position on show the speckle of same color.
(3) get this product 30g, add water 100ml, PH1.5 ~ 2,80 ℃ insulation 1h is transferred in warm dissolving, and is centrifugal, get supernatant, (60,40,40ml), mother solution is with ethyl acetate extraction three times (50,50 for ether extraction three times, 50ml), merge ethyl acetate extraction liquid, evaporate to dryness, the 10ml water dissolution is added in D
101On the macroporous resin column (the macroporous resin amount is column length 2/3 amount for internal diameter 1.6cm, length 18cm), use the 100ml water elution, reuse 30% ethanol 50ml eluting is collected eluent (standby), continue with 70% ethanol 80ml eluting, collect eluent, evaporate to dryness adds the 10ml water dissolution, it is an amount of to add neutral alumina, mix thoroughly, drying under reduced pressure adds neutral alumina post (200 ~ 300 orders, 5g, internal diameter 1.5 ~ 2cm, dry column-packing), 70% ethanol elution 100ml, collect eluent, evaporate to dryness, residue add 2ml methanol makes dissolving, as test liquid.Other gets the forsythin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw test liquid 3 μ l, reference substance solution 1 μ l, putting respectively on same silica gel g thin-layer plate, is developing solvent with chloroform-methanol (5: 1), launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color.
(4) get 30% ethanol elution under the discriminating (3), water bath method, dissolve with methanol, standardize solution are in the 5ml measuring bottle, as test liquid.Other gets the chlorogenic acid reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw test liquid 2 μ l, reference substance solution 2 μ l put respectively on same polyamide film, are developing solvent with acetic acid, launch, and take out, and dry, and put under the 365nm fluorescence and inspect.In the test sample chromatograph, with reference substance chromatograph relevant position on, show the fluorescence speckle of same color.
(5) get this product 5g, add the warm dissolving of water 150ml, be added in (internal diameter 2cm, long 25cm) on the hydro-strong acidic cation exchange resin post, add water 300ml flushing (1ml/ minute), be washed till near colourless, reuse 0.5N ammonia 400ml eluting is collected eluent (1ml/ minute), water bath method, residue adds the dissolving of 1ml Diluted Alcohol, as need testing solution.Other gets the arginine reference substance, adds Diluted Alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw test liquid, each 1 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-water (19: 5: 5) is developing solvent, launch, take out, dry, spray is with ninhydrin solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, showing the same color speckle with the stratographic relevant position of reference substance.
Embodiment 2
Herba Houttuyniae 1166g Radix Scutellariae 290g Radix Isatidis 290g
Fructus Forsythiae 126g Flos Lonicerae 126g
Get the water logging bubble 4 hours that Herba Houttuyniae, Radix Isatidis, Fructus Forsythiae, Flos Lonicerae add 10 times of amounts, distillation, volatile oil device is in addition preserved, and is standby; Medicinal residues filter, and filtrate [I] is deposited in addition; The water that medicinal residues add 10 times of amounts continues to decoct 2 times, each 1.5 hours, collecting decoction filtered, merge with filtrate [I], be concentrated in the time of 60 ℃ and survey relative density about 1.15, put coldly, add ethanol and make and contain the alcohol amount and reach 70%, placed 48 hours, filter, filtrate recycling ethanol also continues to be condensed into fluid extract, and is standby; Get Radix Scutellariae, add the decocting that 10-12 doubly measures and boil secondary, each 2.5 hours, filter, merging filtrate suitably concentrates the back and regulates 1.5,80 ℃ of insulations of pH value 1 hour with hydrochloric acid, leaves standstill 24 hours, filter, precipitation is washed with ethanol, and reuse is washed to PH5-6, the decompression cold drying is pulverized, and gets the Radix Scutellariae extract powder, join in the above-mentioned fluid extract, add volatile oil, mixing, get PEG-6000 and PGE4000 mixed melting, medicine is joined in the fused solution substrate, medicine and substrate composition are advisable at 1: 4.In following system of 80 ℃ of keeping warm modes, be condensing agent with liquid Paraffin or dimethicone, chilling temperature-3 ℃ is collected drop pill, wiping, promptly.
Content of baicalin is measured and is measured according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of People's Republic of China version in 2000 D).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-water-phosphoric acid (47: 53: 0.2) mobile phase; The detection wavelength is 280nm.Number of theoretical plate is pressed the baicalin peak and is calculated, and should be not less than 2500.
The preparation precision of reference substance solution takes by weighing and adds 50% methanol in right amount at 4 hours baicalin of 60 ℃ of drying under reduced pressure and make the solution that every 1ml contains 30ug, promptly.
This product 2.5g is got in the preparation of need testing solution, grinds, and gets 0.5g, the accurate title, decide, and adds the heat of solution of 10ml water temperature, adds 1.5ml 6mol/L hydrochloric acid solution, shake up, 80 ℃ are incubated 1 hour, and placement is spent the night, centrifugal, the supernatant that inclines, precipitation adds 70% ethanol ultrasonic dissolution, be transferred in the 50ml measuring bottle, add 70% ethanol to scale, shake up, filter, discard filtrate just, the accurate subsequent filtrate 2ml that draws, water bath method adds 50% ethanol gradation dissolving, and be transferred in the 10ml measuring bottle, add 50% ethanol to scale, shake up, promptly.
Accurate respectively reference substance solution and each 20ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures promptly.
Test 1 chromatographic condition chromatographic column: Shimpack VP-ODS Φ 4.6 * 150mm; Mobile phase: methanol-water-phosphoric acid (47: 53: 0.2); Detect wavelength: 280nm; Column temperature: room temperature; Flow: 1ml/min.It is 280nm that wavelength is selected the maximum absorption wavelength of baicalin, so select 280nm for detecting wavelength; Determining of mobile phase, as mobile phase, baicalin reference substance appearance time is about 11 minutes with methanol-water-phosphoric acid (47: 53: 0.2).In the sample, baicalin can reach baseline separation with other components, and separating degree is=13.756, and negative control is noiseless.
Test the experiment of 2 system suitabilitys.
1. number of theoretical plate (n), tailing factor (T) are got need testing solution 20ul with the mensuration of separating degree (R), inject chromatograph of liquid, record n=6124 (40830 * 0.15), T=1.02, R=13.756, number of theoretical plate is not less than 2000 through repeatedly measuring and being decided to be with reference to relevant document.
2. replica test: baicalin reference substance solution (29ug/ml) continuous sample introduction 6 times (20ul quantitatively encircles), results peaks area integral value is comparatively stable, and RSD＜2% sees Table 1:
Table 1 baicalin replica test result
| NO | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area (A) | 2353298 | 2368719 | 2391229 | 2390385 | 2396071 | 2445802 |
| X＝2390917.33 RSD＝1.32％ | ||||||
Experiment shows: the baicalin retention time is consistent with reference substance in the test sample, with this understanding, all can reach baseline separation with other components, and the system suitability experiment meets the requirements fully.
Test the preparation of 3 standard curves and the investigation of linear relationship
Accurate absorption baicalin reference substance solution (69.6ug/ml) 2ul, 4ul, 8ul, 12ul, 16ul, 20ul measure peak area by above-mentioned chromatographic condition in accordance with the law, with the peak area is vertical coordinate, baicalin amount (ug) is an abscissa drawing standard curve, and it is that Y=1525581.739X-6616.553 r=0.99996 (n=6) the results are shown in Table 2 that experimental data must return agenda through rectilinear regression.
Table 2 standard curve is investigated the result
| Sample size (ul) | 2 | 4 | 8 | 12 | 16 | 20 |
| Baicalin (ug) peak area integrated value | 0.1392 211401 | 0.2784 412823 | 0.5568 844636 | 0.8352 1258415 | 1.1136 1700789 | 1.3920 2115427 |
| Y＝1525581.739 X-6616.553 r＝0.99996(n＝6) | ||||||
Experiment shows: the baicalin reference substance has good linear relationship with peak area in 0.1392ug ~ 1.3920ug scope.
Test the test of 4 precision
Accurate sample introduction 20ul (test liquid) repeats sample introduction 6 times, the results are shown in Table 3, relative standard deviation, RSD=1.0%.
Table 3 Precision test result
| NO | 1 | 2 | 3 | 4 | 5 | 6 |
| The peak area integrated value | 3842590 | 3871931 | 3912517 | 3839881 | 3888552 | 3938071 |
| Statistical procedures X=3882257 RSD=1.0% | ||||||
Experimental result shows: this law precision is better.
Test the test of 5 repeatability
Get 6 parts in the sample of same lot number, the method for pressing under the text assay item is measured the test sample content of baicalin, the results are shown in Table 4, its relative standard deviation RSD=1.64%.
Table 4 reproducible test results
| NO | 1 | 2 | 3 | 4 | 5 | 6 |
| Content of baicalin (%) | 2.40 | 2.45 | 2.42 | 2.37 | 2.44 | 2.35 |
| Statistical procedures X=2.40% RSD=1.64% | ||||||
Experiment shows: this method repeatability is better.
Test 6 stability tests
Get need testing solution, at room temperature, 1 hour sample introduction is once measured peak area at interval, the results are shown in Table 5, and experiment shows that baicalin is stable in 5 hours of being surveyed, relative standard deviation RSD=2.41%.
Table 5 stability test result
| Standing time (minute) | 0 | 30 | 60 | 90 | 120 | 150 |
| The peak area integrated value | 2901661 | 3052576 | 2982866 | 3031602 | 3895144 | 2900347 |
| Statistical procedures X=2960699.33 RSD=2.41% | ||||||
Test the test of 7 average recoveries
Get 6 parts in the sample (content is 2.40%) of known content, add the baicalin reference substance respectively, the method for pressing under the text assay item is measured, and average recovery rate is 97.57%, RSD=1.17%.The results are shown in Table 6, experiment shows that this law accuracy is better.
The test of table 6 average recovery
| NO | Sample volume (g) | The amount of baicalin (mg) in the sample | Add baicalin reference substance amount (mg) | Record result (mg) | The response rate (%) |
| 1 2 3 4 5 6 | 0.2047 0.2129 0.2027 0.1990 0.2632 0.2441 | 4.9128 5.1096 4.8648 4.7760 6.3168 5.8584 | 1.83 1.79 1.36 1.19 2.03 1.97 | 6.7003 6.8750 6.1925 5.9487 8.2546 7.7790 | 97.68 98.62 97.62 98.54 95.46 97.49 |
| X＝97.57％ RSD＝1.17％ | |||||
Test determining of 8 sample determinations and content limit.
Press text [assay] item method down, content of baicalin in three batch samples is measured, the results are shown in Table 7.
Table 7 sample size measurement result
| Lot number | Content of baicalin (%) | ||
| 1 | 2 | X | |
| 20001023 20001025 20001026 | 2.42 2.31 2.18 | 2.40 2.35 2.16 | 2.41 2.33 2.17 |
Experiment shows: this product content peak is 2.41%, and minimum is 2.17%.The content limit 8% of pressing baikal skullcap root decoction pieces calculates, and the theoretical content of baicalin should be not less than 2.4% in the preparation, consider produce and storage process in loss is arranged unavoidably, so content of baicalin limit in this product must not be decided to be less than 2.0% and be advisable.
Claims (3)
1, a kind of method of quality control of FUFANG YUXINGCAO DIWAN is characterized in that comprising the steps:
(1) methylnonanone in the Herba Houttuyniae detects
Get FUFANG YUXINGCAO DIWAN 30g, add water 500ml, press determination of volatile oil Division A League Matches of French Football method, in determinator scale part topped up with water, add the 2-5ml ethyl acetate, extracted volatile oil 2-5 hour, put cold, divide and get the cerinic acid methacrylate layer, as need testing solution, other gets the methylnonanone reference substance, adds ethyl acetate to making the solution that every ml contains 0.1ml, product solution in contrast, according to the thin layer chromatography test, draw test sample dissolution fluid 6 μ l, reference substance solution 4 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate is developing solvent, launches, and takes out, dry, spray is with the dinitrophenylhydrazine test solution, and it is clear that hot blast blows to the speckle colour developing, in the test sample chromatograph, with the reference substance chromatograph on the relevant position, show the speckle of same color;
(2) baicalin in the Radix Scutellariae detects
Get FUFANG YUXINGCAO DIWAN 1-3g, add warm water 10-30ml, ultrasonic 10 minutes, transfer PH1.5～2,80 ℃ insulation 1h, centrifugal, abandoning supernatant, precipitation washing secondary, residue adds methanol 2ml makes dissolving, as test liquid, other gets the baicalin reference substance, adds methanol and makes the solution that every ml contains 0.5mg, in contrast product solution, test according to thin layer chromatography, draw test liquid 1 μ l, reference substance solution 5 μ l put respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-water is that developing solvent launches, taking-up is dried, and spray is with 2% ferric chloride ethanol liquid, and it is clear that hot blast blows to the speckle colour developing, in the test sample chromatograph, with reference substance chromatograph relevant position on show the speckle of same color;
(3) phillyrin in the Fructus Forsythiae detects
Get FUFANG YUXINGCAO DIWAN 30g, add water 100ml, warm dissolving, transfer PH1.5～2,80 ℃ insulation 1h, centrifugal, get supernatant, ether extraction three times, mother solution ethyl acetate extraction 2-3 time, merge ethyl acetate extraction liquid, evaporate to dryness, 10ml water dissolution, be added on the D101 macroporous resin column, use the 100ml water elution, reuse 25%-50% ethanol 50ml eluting, collect eluent, continue with 50%80% ethanol 80ml eluting, collect eluent, evaporate to dryness, add the 10ml water dissolution, it is an amount of to add neutral alumina, mixes drying under reduced pressure thoroughly, add the neutral alumina post, 50%-80% ethanol elution 100ml collects eluent, evaporate to dryness, residue adds 2ml methanol makes dissolving, as test liquid, other gets the forsythin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, product solution in contrast, according to the thin layer chromatography test, draw test liquid 3 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, with the chloroform-methanol is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear that hot blast blows to the speckle colour developing, in the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;
(4) chlorogenic acid in the Flos Lonicerae detects
Get 25%-50% ethanol elution under the discriminating (3), water bath method, dissolve with methanol, standardize solution is in the 5ml measuring bottle, as test liquid, other gets the chlorogenic acid reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution, test according to thin layer chromatography, draw test liquid 2 μ l, reference substance solution 2 μ l put respectively on same polyamide film, with acetic acid is developing solvent, launch, take out, dry, put under the 365nm fluorescence and inspect, in the test sample chromatograph, with reference substance chromatograph relevant position on, show the fluorescence speckle of same color;
(5) aminoacid in the Radix Isatidis detects
Get FUFANG YUXINGCAO DIWAN 5g, add the warm dissolving of water 150ml, be added on the hydro-strong acidic cation exchange resin post, add water 300ml flushing, be washed till near colourless reuse 0.2N-0.7N ammonia 400ml eluting, collect eluent, water bath method, residue add the dissolving of 1ml Diluted Alcohol, as need testing solution.Other gets the arginine reference substance, adds Diluted Alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography, draw test liquid, each 1 μ l of reference substance solution, putting respectively on same silica gel g thin-layer plate, is developing solvent with n-butyl alcohol-glacial acetic acid-water, launches, take out, dry, spray is with ninhydrin solution, and it is clear that hot blast blows to the speckle colour developing, in the test sample chromatograph, showing the same color speckle with the stratographic relevant position of reference substance.
2, the method for quality control of FUFANG YUXINGCAO DIWAN as claimed in claim 1 is characterized in that: contain Radix Scutellariae and be no less than 2.0%, five qualitative identification in baicalin and all be positive reaction and judge that then this product is qualified.
3, the method for quality control of FUFANG YUXINGCAO DIWAN as claimed in claim 1, it is characterized in that the test of chromatographic condition and system suitability: with octadecylsilane chemically bonded silica is filler; Methanol-water-phosphoric acid is a mobile phase; The detection wavelength is 240-340nm, and number of theoretical plate is pressed the baicalin peak and calculated, and should be not less than 2300.
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| CN101167909B (en) * | 2007-11-07 | 2010-08-11 | 马志伟 | Content determination method of haw and corium stomachium galli preparation |
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| CN1548104A (en) * | 2003-05-24 | 2004-11-24 | 毛友昌 | Compound cordate bouttuynia bolus and its prepn |
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| CN102590424A (en) * | 2012-03-06 | 2012-07-18 | 浙江国镜药业有限公司 | Quality detection method of compound houttuynia cordata mixture |
| CN104297441A (en) * | 2014-10-29 | 2015-01-21 | 内蒙古天奇中蒙制药股份有限公司 | Infrared spectroscopy on-line quality monitoring and controlling system applied to anaesthetic preparation |
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| CN114184730A (en) * | 2020-09-15 | 2022-03-15 | 桂林三金药业股份有限公司 | Quality control method of cucumber cream |
| CN115372516A (en) * | 2022-08-24 | 2022-11-22 | 浙江惠松制药有限公司 | Method for determining content of nucleoside ingredients in houttuynia cordata, scutellaria baicalensis and blue mixture intermediate |
| CN115372516B (en) * | 2022-08-24 | 2023-11-03 | 浙江惠松制药有限公司 | Method for measuring content of nucleoside components in houttuynia cordata, radix scutellariae and blue mixture intermediate |
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| CN101040951B (en) | 2011-11-02 |
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Address after: 335200 north of national highway 320, Jiaotong North Road, Yujiang District, Yingtan City, Jiangxi Province Patentee after: Jiangxi Kangenbei tianshikang Pharmaceutical Co.,Ltd. Address before: 21 Jianshe Road, Yingtan City, Jiangxi Province Patentee before: Jiangxi Herbi-Sky Co.,Ltd. |