CN101040915A - Method for preparing a Shuanhuanglian injection and the component detecting method - Google Patents
Method for preparing a Shuanhuanglian injection and the component detecting method Download PDFInfo
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Abstract
The invention provides a method for preparing an injection, comprising that extracting respectively the scutellaria root, honeysuckle flower and capsule of weeping forsythia, then preparing the injection. The invention also provides a component check method of the injection, for improving the quality control level, assure the treatment effect, and support the product detection.
Description
Technical field
The present invention relates to a kind of preparation method and component detection method thereof of SHUANGHUANGLIAN ZHUSHEYE, belong to the field of Chinese medicines.
Background technology
SHUANGHUANGLIAN ZHUSHEYE is stated from the 11 in preparation of the Sanitation Ministry medicine standard Chinese traditional patent formulation (WS3-B-2104-96), is made up of Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae three flavor medicines.Be used for heating, cough, pharyngalgia that affection due to external wind and heat causes.Be applicable to upper respiratory tract infection, pneumonia, tonsillitis, pharyngitis of virus and bacterial infection etc.Flos Lonicerae is sweet cold in the side, and fragrance is evacuated, and kind lung heat clearing is monarch drug through pathogenic heat; The Radix Scutellariae bitter cold, kind lung heat clearing fire and part of the body cavity above the diaphragm housing the heart and lungs excess-heat; The Fructus Forsythiae hardship is slightly cold, and the part of the body cavity above the diaphragm housing the heart and lungs wind heat of loosing has the merit of heat-clearing and toxic substances removing, is ministerial drug.Three medicines use, and play relieving the exterior syndrome with drugs of pungent in flavor and cool in nature altogether, the merit of heat-clearing and toxic substances removing.
Patent documentation 99106224.8 discloses a kind of production technology of refining injection ' Shuanghuanglian ', comprising: 1. Flos Lonicerae and Fructus Forsythiae crude drug are decocted; 2. gained filtrate concentrates back adding ethanol; 3. solution left standstill; 4. recovery ethanol adds deionized water, transfers pH; 5. leave standstill, ultrafiltration membrance filter concentrates; 6. in concentrated solution, add ethanol; 7. leave standstill, reclaim ethanol; 8. solution filters, and obtains extractum; 9. baicalin powder that above-mentioned extractum, conventional method are obtained and water for injection mix, heated and boiled; 10. filtering with microporous membrane obtains SHUANGHUANGLIAN ZHUSHEYE.Wherein, effective ingredient of honeysuckle-chlorogenic acid is met the high temperature destructible, so adopt Flos Lonicerae and Fructus Forsythiae to carry altogether, the yield of Flos Lonicerae is lower.
And in the existing SHUANGHUANGLIAN ZHUSHEYE quality standard Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae three flavor medicines have all been carried out qualitative identification, but only set up the content assaying method of Radix Scutellariae; And its prescription composition Flos Lonicerae is that monarch drug, Fructus Forsythiae are ministerial drug, in prescription, also play an important role, it is the important indicator composition of reflection medicine inherent quality, thereby the inventor thinks should increase the content assaying method of other 2 kinds of compositions on the basis of this quality standard, remedy the deficiency of proper mass control method, the control level of improving the quality of products, the curative effect of assurance product.
Summary of the invention
The preparation method that the purpose of this invention is to provide a kind of SHUANGHUANGLIAN ZHUSHEYE.
Another object of the present invention provides the component detection method of SHUANGHUANGLIAN ZHUSHEYE, and this method has improved the controllable quality level, guarantees the curative effect of product.
In order to realize the object of the invention, the preparation method of a kind of SHUANGHUANGLIAN ZHUSHEYE of the present invention is prepared by following steps:
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, adding 4～16 times of water soaked 30～180 minutes in 60～85 ℃ of insulations, soak 2～3 times, gradation is filtered, and collects filtrate and is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add ethanol to containing alcohol amount 75～80%, left standstill 12～24 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.12～1.23 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2～6 times of amounts, stir gently, left standstill 12～24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add ethanol while stirring, reach 80～85% to containing the alcohol amount, left standstill 12～24 hours, and got supernatant adjust pH 8.0～10.0, left standstill 24～36 hours, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.12～1.23 (80 ℃ ± 5 ℃ surveys), gets the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, decocting 3 times for the first time, adds 4～12 times of water gagings, soaks 30 minutes, and heated and boiled 1.0～3.0 hours is filtered; Each 2～8 times of water gaging of second, third time boiled 1.0～2.0 hours, merged medicinal liquid and were concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add ethanol, precipitate 12～24 hours to containing alcohol amount 75～80%, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2～6 times of amounts, stir, left standstill 12～24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add ethanol while stirring, make and contain alcohol amount and reach 80～85%, left standstill 12～24 hours, and got supernatant adjust pH 8.0～10.0, left standstill 24～36 hours, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.12～1.23 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 2～12 times of water gagings, decoct at least 2 times, each 1～3 hour, filter merging filtrate, be concentrated into 3～6 times of crude drug amounts, leave standstill leaching supernatant adjust pH to 1.0 after 1～3 hour～3.0,70～80 ℃ insulation 1～3 hour, left standstill 12～20 hours, filter, precipitation adds 5～10 times of water gagings, regulates pH value to 6.0～8.0, add equivalent ethanol again, stirring makes dissolving, filters filtrate adjust pH to 1.0～3.0,60～70 ℃ are incubated 30～60 minutes, left standstill 12～20 hours, and filtered, it is 6.0～8.0 that precipitation is washed till pH value with ethanol, reclaim ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 1～3 times of amount (in baicalin dry product weight) below 40 ℃, soaked into 4～10 hours, add the water for injection of 4～8 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir after 2～10 minutes, adjust pH to 6.0～7.0, the medicinal charcoal of adding 0.5% boiled 30～60 minutes, and is to be cooled after 60～80 ℃, filter with ethanol, filtrate adjust pH to 1.0～3.0,60 ± 5 ℃ insulation 30～60 minutes was left standstill 4～10 hours, abandoning supernatant, precipitate, is drained to pH＞4.0 with 95% washing with alcohol, dry baicalin;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin add the injection dilute with water respectively with Flos Lonicerae, Fructus Forsythiae equivalent extract, add baicalin behind the mixing and stir, dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15～30 minutes and is cooled to below 70 ℃ adjust pH 6.8～7.5, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 10～40 ℃, fine straining, fill was sterilized 30 minutes for 115～118 ℃, promptly.
Its preparation method is preferably:
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, first pass adds 10 times of water gagings, soaks 1 hour in 80 ℃ of insulations, add 10 times of water gagings second time, soaked 40 minutes in 80 ℃ of insulations, gradation is filtered, and collects filtrate and is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% above ethanol to containing alcohol amount 75%, left standstill 12 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5～9.0, got supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys).Get the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, first pass adds 8 times of water gagings, soaks 30 minutes, and heated and boiled 1.5 hours is filtered; Second, third boiled 1.0 hours all over each 6 times of water gaging, merged medicinal liquid and was concentrated into density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% above ethanol and measure 75%, precipitate 12 hours to containing alcohol, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5～9.0, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 8 times of water gagings, decocted 2 hours, filter, add 6 times of water gagings, decocted 1 hour, merging filtrate is concentrated into 4～5 times of crude drug amounts, leaves standstill leaching supernatant 2mol/L hydrochloric acid adjust pH to 1.0～2.0 after 1 hour, 80 ℃ are incubated 1 hour, leave standstill 12 hours, filter, precipitation adds 6～8 times of water gagings, regulate pH value to 6.5～7.5 with 40% sodium hydroxide solution, add equivalent ethanol again, stir and make dissolving, filter, filtrate was left standstill 12 hours with 2mol/L hydrochloric acid adjust pH to 1.0～2.0,60 ℃ insulation 30 minutes, filter, it is 6.5～7.5 that precipitation is washed till pH value with ethanol, reclaims ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 2 times of amounts (in baicalin dry product weight) below 40 ℃, soak into more than 4 hours, add the water for injection of 4 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir and slowly add 10% sodium hydroxide solution after 2 minutes, adjust pH to 6.5～7.0, the medicinal charcoal of adding 0.5% boiled 30 minutes, to be cooled after 60～80 ℃, the 90% above ethanol that adds 0.5 times of amount of medicine liquid volume, filtered while hot, filtrate is used 10%HCl adjust pH to 1.5～2.0,60 ± 5 ℃ are incubated 30 minutes, leave standstill more than 4 hours, abandoning supernatant, precipitate with 95% washing with alcohol to pH＞4.0, drain the dry baicalin that gets;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin.Flos Lonicerae, Fructus Forsythiae equivalent extract are added the injection dilute with water respectively, add behind the mixing baicalin stir make dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15 minutes and is cooled to below 70 ℃ adjust pH 6.8～7.5, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 10～40 ℃, fine straining, fill was sterilized 30 minutes for 115～118 ℃, promptly.
The component detection method of SHUANGHUANGLIAN ZHUSHEYE of the present invention, this method comprises the assay of any two or three composition in Radix Scutellariae, Flos Lonicerae and the Fructus Forsythiae.
The component detection method of SHUANGHUANGLIAN ZHUSHEYE of the present invention comprises the assay of described Radix Scutellariae, Flos Lonicerae and Fructus Forsythiae:
(1) assay of Radix Scutellariae is measured according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; Methanol-glacial acetic acid-water with volume ratio 40-60: 0.5-2: 60-40 is mobile phase, is preferably 48: 1: 52; The detection wavelength is 276nm, and number of theoretical plate calculates by the baicalin peak and is not less than 1000.
The preparation precision of reference substance solution takes by weighing the baicalin reference substance, adds 40～60% methanol or ethanol and makes the solution that every 1ml contains 0.2mg, preferably adopts 50% methanol or ethanol, promptly.
The preparation precision of need testing solution is measured this product 1ml, puts in the 50ml measuring bottle, adds 40～60% methanol or ethanol dilution to scale, preferably adopts 50% methanol or ethanol, shakes up, promptly.
Accurate respectively need testing solution and each the 8 μ l of reference substance solution of drawing of algoscopy inject chromatograph of liquid respectively, measure peak area respectively, and external standard method is calculated content, promptly.
The every 1ml of this product contains Radix Scutellariae with baicalin (C
21H
18O
11) meter, be 6.0～12.0mg.
(2) Flos Lonicerae is measured according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; Methanol-water-glacial acetic acid with volume ratio 10-30: 90-70: 0.1-2 is a mobile phase, is preferably 20: 80: 1; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 2000.
It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds water (methanol, ethanol) and makes the solution that every 1ml contains 40 μ g, promptly.
The preparation precision of need testing solution is measured this product 6ml, puts in the brown measuring bottle of 50ml, and water (methanol, ethanol) is diluted to scale, shakes up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The every 1ml of this product contains Flos Lonicerae with chlorogenic acid (C
16H
18O
9) meter, be 0.05～2.0mg.
(3) Fructus Forsythiae is measured according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; Acetonitrile-water with volume ratio 10-40: 90-60 is a mobile phase, is preferably 25: 75; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the phillyrin peak should be not less than 2000.
It is an amount of that the phillyrin reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 30～90% methanol or ethanol and makes the solution that every 1ml contains 60 μ g, preferably adopts 50% methanol or ethanol, promptly.
The preparation precision of need testing solution is measured this product 10ml, puts on the neutral alumina post (100～120 order 6g, internal diameter 1cm), with 30～90% ethanol 40ml eluting, preferably adopt 70% methanol or ethanol, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, preferably adopt 50% methanol or ethanol, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The every 1ml of this product contains Fructus Forsythiae with phillyrin (C
29H
36O
15) meter, be 0.05～0.4mg.
The method of quality control of a kind of SHUANGHUANGLIAN ZHUSHEYE of the present invention also can comprise the content assaying method of Flos Lonicerae and Fructus Forsythiae, the content assaying method of Radix Scutellariae and Flos Lonicerae, or the content assaying method of Radix Scutellariae and Fructus Forsythiae.
The preparation method of SHUANGHUANGLIAN ZHUSHEYE of the present invention adopts three kinds of raw materials effective component extracting respectively, Flos Lonicerae is changed into hot water soak precipitate with ethanol more earlier, has avoided the destruction of effective ingredient of honeysuckle, and the yield of chlorogenic acid is increased.The component detection method of SHUANGHUANGLIAN ZHUSHEYE of the present invention has improved the controllable quality level, guarantees the curative effect of product.Both helped manufacturer and administration section to product monitoring, also can provide better guarantees for medical department and patient's treatment.
Description of drawings
Fig. 1-1 is a Flos Lonicerae negative fluid chromatogram;
SHUANGHUANGLIAN ZHUSHEYE reference colour spectrogram when Fig. 1-2 detects for Flos Lonicerae;
Fig. 2-1 is a Fructus Forsythiae negative fluid chromatogram;
SHUANGHUANGLIAN ZHUSHEYE chromatogram when Fig. 2-2 detects for Fructus Forsythiae.
The specific embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 'Shuang Hualian ' injection liquid and preparation method thereof
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, first pass adds 10 times of water gagings, soaks 1 hour in 80 ℃ of insulations, add 10 times of water gagings second time, soaked 40 minutes in 80 ℃ of insulations, gradation is filtered, and collects filtrate and is concentrated into relative density 1.20 ± 0.02 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% ethanol to containing alcohol amount 75%, left standstill 12 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.18 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.20 ± 0.02 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5, got supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys).Get the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, first pass adds 8 times of water gagings, soaks 30 minutes, and heated and boiled 1.5 hours is filtered; Second, third boiled 1.0 hours all over each 6 times of water gaging, merged medicinal liquid and was concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% ethanol, precipitate 12 hours to containing alcohol amount 75%, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 9.0, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 8 times of water gagings, decocted 2 hours, filter, add 6 times of water gagings, decocted 1 hour, merging filtrate is concentrated into 5 times of crude drug amounts, leaves standstill leaching supernatant 2mol/L hydrochloric acid adjust pH to 1.0 after 1 hour, 80 ℃ are incubated 1 hour, leave standstill 12 hours, filter, precipitation adds 8 times of water gagings, regulate pH value to 7.5 with 40% sodium hydroxide solution, add equivalent ethanol again, stir and make dissolving, filter, filtrate is used 2mol/L hydrochloric acid adjust pH to 1.0, and 60 ℃ are incubated 30 minutes, left standstill 12 hours, filter, it is 7.5 that precipitation is washed till pH value with ethanol, reclaims ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 40 ℃ of 2 times of amounts (in baicalin dry product weight), soaked into 4 hours, the water for injection that adds 40 ℃ of 4 times of amounts (in baicalin dry product weight) again, stir and slowly add 10% sodium hydroxide solution after 2 minutes, adjust pH to 6.5, the medicinal charcoal of adding 0.5% boiled 30 minutes, to be cooled after 60 ℃, 90% ethanol that adds 0.5 times of amount of medicine liquid volume, filtered while hot, filtrate is used 10%HCl adjust pH to 1.5,60 ± 5 ℃ are incubated 30 minutes, left standstill 4 hours, abandoning supernatant, precipitate with 95% washing with alcohol to pH＞4.0, drain the dry baicalin that gets;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin.Flos Lonicerae, Fructus Forsythiae equivalent extract are added the injection dilute with water respectively, add behind the mixing baicalin stir make dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15 minutes and is cooled to 70 ℃, adjust pH 6.8, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 40 ℃, fine straining, fill was sterilized 30 minutes for 118 ℃, promptly.
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, first pass adds 4 times of water gagings, soaked 80 minutes in 60 ℃ of insulations, add 8 times of water gagings second time, soaked 60 minutes in 80 ℃ of insulations, add 12 times of water gagings the 3rd time, soaked 30 minutes in 85 ℃ of insulations, gradation is filtered, and collects filtrate and is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 95% ethanol to containing alcohol amount 80%, left standstill 18 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 6 times of amounts, stir gently, left standstill 24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 90%, left standstill 18 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 9.0, got supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys).Get the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, first pass adds 12 times of water gagings, soaks 40 minutes, and heated and boiled 3 hours is filtered; Second, third boiled 2 hours all over each 8 times of water gaging, merged medicinal liquid and was concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% ethanol, precipitate 20 hours to containing alcohol amount 80%, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2 times of amounts, stir gently, left standstill 24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 90%, left standstill 18 hours, the siphon supernatant left standstill 36 hours with 20% sodium hydroxide solution adjust pH 8.0, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.20～1.23 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 2 times of water gagings, decocted 1 hour, filter, add 8 times of water gagings, decocted 2 hours, add 10 times of water gagings, decocted merging filtrate 1 hour, be concentrated into 3 times of crude drug amounts, leave standstill leaching supernatant 2mol/L hydrochloric acid adjust pH to 2.0 after 1 hour, 70 ℃ are incubated 3 hours, left standstill 20 hours, and filtered, precipitation adds 5 times of water gagings, regulate pH value to 6.0 with 40% sodium hydroxide solution, add equivalent ethanol again, stir and make dissolving, filter, filtrate is used 2mol/L hydrochloric acid adjust pH to 2.0, and 70 ℃ are incubated 60 minutes, left standstill 20 hours, filter, it is 7.0 that precipitation is washed till pH value with ethanol, reclaims ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 3 times of amounts (in baicalin dry product weight) below 40 ℃, soaked into 10 hours, add the water for injection of 6 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir and slowly add 10% sodium hydroxide solution after 6 minutes, adjust pH to 7.0, the medicinal charcoal of adding 0.5% boiled 40 minutes, to be cooled after 70 ℃, the 90% above ethanol that adds 0.5 times of amount of medicine liquid volume, filtered while hot, filtrate is used 10%HCl adjust pH to 1.0,60 ± 5 ℃ are incubated 60 minutes, left standstill 10 hours, abandoning supernatant, precipitate with 95% washing with alcohol to pH＞4.0, drain the dry baicalin that gets;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin.Flos Lonicerae, Fructus Forsythiae equivalent extract are added the injection dilute with water respectively, add behind the mixing baicalin stir make dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15 minutes and is cooled to below 70 ℃ adjust pH 7.5, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 30 ℃, fine straining, fill was sterilized 60 minutes for 118 ℃, promptly.
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, first pass adds 6 times of water gagings, soaks 1 hour in 70 ℃ of insulations, add 16 times of water gagings second time, soaked 30 minutes in 85 ℃ of insulations, gradation is filtered, and collects filtrate and is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% ethanol to containing alcohol amount 78%, left standstill 24 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2 times of amounts, stir gently, left standstill 20 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 24 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5, got supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys).Get the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, first pass adds 4 times of water gagings, soaks 60 minutes, and heated and boiled 1 hour is filtered; Second, third boiled 1.5 hours all over each 2 times of water gaging, merged medicinal liquid and was concentrated into relative density 1.22～1.25 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 95% ethanol, precipitate 24 hours to containing alcohol amount 78%, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 6 times of amounts, stir gently, left standstill 20 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 24 hours, the siphon supernatant left standstill 30 hours with 20% sodium hydroxide solution adjust pH 10.0, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.12～1.16 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 12 times of water gagings, decocted 2 hours, filter, add 6 times of water gagings, decocted 3 hours, merging filtrate is concentrated into 6 times of crude drug amounts, leaves standstill leaching supernatant 2mol/L hydrochloric acid adjust pH to 3.0 after 1 hour, 75 ℃ are incubated 2 hours, leave standstill 18 hours, filter, precipitation adds 10 times of water gagings, regulate pH value to 8.0 with 40% sodium hydroxide solution, add equivalent ethanol again, stir and make dissolving, filter, filtrate is used 2mol/L hydrochloric acid adjust pH to 1.5, and 65 ℃ are incubated 40 minutes, left standstill 18 hours, filter, it is 8.0 that precipitation is washed till pH value with ethanol, reclaims ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 1 times of amount (in baicalin dry product weight) below 40 ℃, soaked into 6 hours, add the water for injection of 8 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir and slowly add 10% sodium hydroxide solution after 10 minutes, adjust pH to 7.0, the medicinal charcoal of adding 0.5% boiled 60 minutes, to be cooled after 80 ℃, the 90% above ethanol that adds 0.5 times of amount of medicine liquid volume, filtered while hot, filtrate is used 10%HCl adjust pH to 3.0,60 ± 5 ℃ are incubated 40 minutes, left standstill 6 hours, abandoning supernatant, precipitate with 95% washing with alcohol to pH＞4.0, drain the dry baicalin that gets;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin.Flos Lonicerae, Fructus Forsythiae equivalent extract are added the injection dilute with water respectively, add behind the mixing baicalin stir make dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15 minutes and is cooled to below 70 ℃ adjust pH 7.0, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 10 ℃, fine straining, fill was sterilized 40 minutes for 115 ℃, promptly.
Embodiment 4
SHUANGHUANGLIAN ZHUSHEYE by embodiment 1 preparation detects three kinds of compositions.
1) assay of Flos Lonicerae
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).With the octadecylsilane chemically bonded silica is filler; With methanol-water-glacial acetic acid (20: 80: 1) is mobile phase; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 2000.It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds water and makes the solution that every 1ml contains 40 μ g, is reference substance solution.Precision is measured this product 6ml, puts in the brown measuring bottle of 50ml, is diluted with water to scale, shakes up, and promptly gets need testing solution.Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure.The every 1ml of SHUANGHUANGLIAN ZHUSHEYE contains Flos Lonicerae with chlorogenic acid (C among the present invention
16H
18O
9) meter, be 1.1mg.
2) assay of Fructus Forsythiae
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).With the octadecylsilane chemically bonded silica is filler; With acetonitrile-water (25: 75) is mobile phase; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the phillyrin peak should be not less than 2000.It is an amount of to get the phillyrin reference substance, and accurate the title decides, and adds 50% ethanol and makes the solution that every 1ml contains 60 μ g, is reference substance solution.Precision is measured this product 10ml, puts on the neutral alumina post (100～120 order 6g, internal diameter 1cm), with 70% ethanol 40ml eluting, collect eluent, be concentrated into driedly, it is an amount of that residue adds 50% ethanol, warmly makes dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, shake up, promptly get need testing solution.Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure.The every 1ml of SHUANGHUANGLIAN ZHUSHEYE contains Fructus Forsythiae with phillyrin (C among the present invention
29H
36O
15) meter, be 0.2mg.
3) assay of Radix Scutellariae
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).With the octadecylsilane chemically bonded silica is filler; With methanol-glacial acetic acid-water (48: 1: 52) is mobile phase; The detection wavelength is 276nm, and number of theoretical plate calculates by the baicalin peak and is not less than 1000.Precision takes by weighing the baicalin reference substance, adds 50% methanol and makes the solution that every 1ml contains 0.2mg, promptly gets reference substance solution.Precision is measured this product 1ml, puts in the 50ml measuring bottle, adds 50% methanol and is diluted to scale, shakes up, and promptly gets need testing solution.Accurate respectively need testing solution and each 8 μ l of reference substance solution of drawing inject chromatograph of liquid respectively, measure peak area respectively, and external standard method is calculated content, promptly.The every 1ml of this product contains Radix Scutellariae with baicalin (C
21H
18O
11) meter, be 8.8mg.
Basic process is with embodiment 4, and different is three kinds of conditions in the composition detection process:
In the assay of Radix Scutellariae, be mobile phase with methanol-glacial acetic acid of 40: 2: 60-water; Preparation reference substance solution and need testing solution, solvent for use is 40% ethanol.
In the assay of Flos Lonicerae, be mobile phase with methanol-water-glacial acetic acid of 10: 90: 2;
In the assay of Fructus Forsythiae, be mobile phase with 10: 90 acetonitrile-waters, preparation reference substance solution solvent for use is 30% methanol, need testing solution eluting 30% methanol, 90% methanol of dissolving usefulness.
Basic process is with embodiment 4, and different is three kinds of conditions in the composition detection process:
In the assay of Radix Scutellariae, be mobile phase with methanol-glacial acetic acid of 60: 0.5: 40-water; Preparation reference substance solution and need testing solution, solvent for use is 60% ethanol.
In the assay of Flos Lonicerae, be mobile phase with methanol-water-glacial acetic acid of 30: 70: 0.1;
In the assay of Fructus Forsythiae, be mobile phase with 40: 60 acetonitrile-waters, preparation reference substance solution solvent for use is 90% methanol, need testing solution eluting 90% methanol, 30% methanol of dissolving usefulness.
For product composition detect also can be only at the content assaying method of content assaying method, Radix Scutellariae and the Flos Lonicerae of Flos Lonicerae and Fructus Forsythiae, or the content assaying method of Radix Scutellariae and Fructus Forsythiae.
Experimental example 1 Flos Lonicerae assay and methodological study
(1) instrument and reagent
Instrument: high performance liquid chromatograph Agilent 1200, chromatographic column: Agilent ZORBAXEclipse XDB-C18 4.6 * 150mm 5 μ.
Reference substance: chlorogenic acid (derive from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 110753-200413, use for assay).
Sample lot number: 20060801,20060802,20060803
Reagent: methanol is chromatographically pure, and glacial acetic acid is an analytical pure.
(2) chromatographic condition
With the octadecylsilane chemically bonded silica is filler; With methanol-water-glacial acetic acid (10-30: 90-70: 0.1-2) be mobile phase; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 2000.
(3) preparation of need testing solution
Precision is measured this product 6ml, puts in the brown measuring bottle of 50ml, and water (methanol, ethanol) is diluted to scale, shakes up, promptly.
(4) preparation of reference substance solution
It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds water (methanol, ethanol) and makes the solution that every 1ml contains 40 μ g, promptly.
(5) range of linearity test
The accurate title, decided chlorogenic acid reference substance 10.28mg, puts to add water (methanol, ethanol) in the brown measuring bottle of 100ml and dissolve and be diluted to scale.Therefrom take out in the brown measuring bottle of 4ml to 10ml and be diluted to scale.The above-mentioned solution of accurate absorption each 2 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l inject chromatograph of liquid, the record chromatogram.With the peak area integrated value is vertical coordinate Y, and (μ g) is abscissa X with sample size, carries out linear regression, and the result shows the chlorogenic acid sample size in 0.08-0.82 μ g scope, and sample size and peak area are good linear relationship, the results are shown in Table 1.
The test of table 1 range of linearity
| Sample size (μ g) | 0.08 | 0.21 | 0.41 | 0.62 | 0.82 |
| Peak area | 244.2 | 615.4 | 1240.8 | 1860.3 | 2490.2 |
| Regression equation | Y＝-9.801+3037.34X r＝0.9999 | ||||
(6) precision test
The accurate reference substance solution 10 μ l that draw, continuous sample introduction 6 times is measured peak area in accordance with the law, and RSD=0.16% is up to specification as a result, sees Table 2.
The test of table 2 precision
| The sample introduction number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 1240.3 | 1244.0 | 1238.1 | 1240.8 | 1241.1 | 1242.5 |
(7) stability test
Get same need testing solution and preparing back 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours injection chromatograph of liquid respectively, its peak area there is no significant change (RSD=0.14%) as a result, illustrate in the need testing solution 24 hours more stablely, the results are shown in Table 3.
Table 3 stability test
| Standing time (hour) | 0 | 2 | 4 | 6 | 8 | 24 |
| Peak area | 1161.1 | 1159.3 | 1160.4 | 1159.7 | 1156.9 | 1157.6 |
(8) specificity test
Get other medical materials that do not contain Flos Lonicerae and prepare negative controls by production technology, get 6ml and put in the brown measuring bottle of 50ml, water (methanol, ethanol) is diluted to scale, shakes up, promptly.Inject chromatograph of liquid, measure, the result (sees Fig. 1-1,1-2) to sample determination is noiseless.
(9) replica test
Table 4 replica test
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | RSD(％) | |
| Content | 0.324 | 0.326 | 0.321 | 0.325 | 0.322 | 0.32 | 0.6 |
(10) recovery test
Precision is measured totally 6 parts of No. 5 need testing solution 6ml under the replica test item, and to brown measuring bottle, the accurate chlorogenic acid reference substance 2.2762mg that adds adds water (methanol, ethanol) and stirs and make dissolving, and is diluted to scale, shakes up, and filters, and gets subsequent filtrate promptly.Inject chromatograph of liquid, measure calculate recovery rate and RSD% value respectively.The results are shown in Table 5.
Table 5 recovery test
| Sample number | Sample total content (mg) | Sample theoretical amount (mg) | Add reference substance amount (mg) | Record reference substance amount (mg) | The response rate (%) |
| 1 | 4.2213 | 1.9324 | 2.2762 | 2.2889 | 100.56 |
| 2 | 4.2050 | 1.9324 | 2.2762 | 2.2726 | 99.84 |
| 3 | 4.2143 | 1.9324 | 2.2762 | 2.2819 | 100.25 |
| 4 | 4.2291 | 1.9324 | 2.2762 | 2.2967 | 100.90 |
| 5 | 4.2343 | 1.9324 | 2.2762 | 2.3019 | 101.13 |
| 6 | 4.2241 | 1.9324 | 2.2762 | 2.2917 | 100.68 |
The average recovery rate of chlorogenic acid is 100.56%, RSD=0.46%; The result shows the response rate ideal of this method.
(11) sample size is measured
Precision is measured sample (lot number: 20060801,20060802,20060803), every part of 6ml is put in the brown measuring bottle of 50ml, and water (methanol, ethanol) is diluted to scale, shakes up, promptly respectively.Inject chromatograph of liquid, measure respectively, the results are shown in Table 6.
Table 6 sample size is measured
| The sample lot number | Average content (%) |
| 20060801 | 0.32 |
| 20060802 | 0.36 |
| 20060803 | 0.33 |
Experimental example 2 Fructus Forsythiae assay and methodological studies
(1) instrument and reagent
Instrument: high performance liquid chromatograph Agilent 1200 types, chromatographic column: Agilent ZORBAXEclipse XDB-C184.6 * 150mm 5 μ.
Reference substance: phillyrin (derive from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 110821-200609, use for assay).
Sample lot number: 20060801,20060802,20060803
Reagent: acetonitrile is a chromatographically pure.
(2) chromatographic condition
With the octadecylsilane chemically bonded silica is filler; With acetonitrile-water (10-40: 90-60) be mobile phase; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the phillyrin peak should be not less than 2000.
(3) preparation of reference substance solution
It is an amount of to get the phillyrin reference substance, and accurate the title decides, and adds 30～90% methanol or ethanol and makes the solution that every 1ml contains 60 μ g, promptly.
(4) preparation of need testing solution
Precision is measured this product 10ml, put on the neutral alumina post (100～120 order 6g, internal diameter 1cm), with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, shake up, promptly.
(5) range of linearity test
The accurate title, decided phillyrin reference substance 14.80mg, puts to add 30～90% methanol or dissolve with ethanol in the 100ml measuring bottle and be diluted to scale.Therefrom take out in 4ml to the 10ml measuring bottle and be diluted to scale.The above-mentioned solution of accurate absorption each 2 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l inject chromatograph of liquid, the record chromatogram.With the peak area integrated value is vertical coordinate Y, and (μ g) is abscissa X with sample size, carries out linear regression, and the result shows the phillyrin sample size in 0.12～1.18 μ g scope, and sample size and peak area are good linear relationship, the results are shown in Table 7.
The test of table 7 range of linearity
| Sample size (μ g) | 0.12 | 0.30 | 0.59 | 0.89 | 1.18 |
| Peak area | 71.6 | 192.4 | 376.6 | 567.2 | 750.8 |
| Regression equation | Y＝-1.97+639.11X r＝0.9999 | ||||
(6) precision test
The accurate reference substance solution 10 μ l that draw, continuous sample introduction 6 times is measured peak area in accordance with the law, and RSD=0.35% is up to specification as a result, sees Table 8.
The test of table 8 precision
| The sample introduction number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 374.7 | 376.6 | 375.2 | 376.8 | 373.4 | 374.3 |
(7) stability test
Get same need testing solution and preparing back 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours injection chromatograph of liquid respectively, its peak area there is no significant change (RSD=0.25%) as a result, illustrate in the need testing solution 24 hours more stablely, the results are shown in Table 9.
Table 9 stability test
| Standing time (hour) | 0 | 2 | 4 | 6 | 8 | 24 |
| Peak area | 1020.1 | 1018.2 | 1018.5 | 1014.0 | 1015.9 | 1013.8 |
(8) specificity test
Get other medical materials that do not contain Fructus Forsythiae, prepare negative controls by same process, precision is measured 10ml, prepares sample by the preparation method of need testing solution, injects chromatograph of liquid, and sample introduction is measured, and the result (sees Fig. 2-1,2-2) to sample determination is noiseless.
(9) replica test
(lot number: 20060801), therefrom each accurate 10ml that draws puts neutral alumina post (100～120 order 6g respectively to measure 6 parts in an amount of sample altogether, internal diameter 1cm) on, with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, shake up, promptly.Inject chromatograph of liquid, measure respectively, repeatability is good, the results are shown in Table 10.
Table 10 replica test
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | RSD(％) | |
| Content | 0.080 | 0.081 | 0.081 | 0.079 | 0.080 | 0.08 | 1.04 |
(10) recovery test
Precision is measured totally 6 parts of No. 5 need testing solution 10ml under the replica test item, and the accurate phillyrin reference substance mg that adds is after the stirring and dissolving, put respectively on the neutral alumina post (100～120 order 6g, internal diameter 1cm), with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, shake up, promptly.Inject chromatograph of liquid, measure calculate recovery rate and RSD% value respectively.The results are shown in Table 11.
Table 11 recovery test
| Sample number | Sample total content (mg) | Sample theoretical amount (mg) | Add reference substance amount (mg) | Record reference substance amount (mg) | The response rate (%) |
| 1 | 1.812 | 0.802 | 1.014 | 1.010 | 99.61 |
| 2 | 1.814 | 0.802 | 1.014 | 1.012 | 99.80 |
| 3 | 1.807 | 0.802 | 1.014 | 1.005 | 99.11 |
| 4 | 1.814 | 0.802 | 1.014 | 1.012 | 99.80 |
| 5 | 1.815 | 0.802 | 1.014 | 1.013 | 99.90 |
| 6 | 1.813 | 0.802 | 1.014 | 1.011 | 99.70 |
The average recovery rate of phillyrin is 99.64%, RSD=0.32%; The result shows that the response rate of this method is more satisfactory.
(11) sample size is measured
Precision measure sample (lot number: 20060801,20060802,20060803) each 10ml, prepare sample by the preparation method of need testing solution, inject chromatograph of liquid, measure respectively, the results are shown in Table 12.
Table 12 sample size is measured
| The sample lot number | Average content (%) |
| 20060801 | 0.08 |
| 20060802 | 0.08 |
| 20060803 | 0.09 |
SHUANGHUANGLIAN ZHUSHEYE of the present invention has heat-clearing and toxic substances removing, declares the effect of wind heat clearly.Pharmacodynamic study shows according to preparation method of the present invention and product inspection method controls product quality, and the finished product of gained is at antibacterial, antiviral, antiinflammatory, and analgesic, there is curative effect preferably aspects such as analgesia.
Concrete condition is as follows:
Bacteriostasis compares (the lumbar injection staphylococcus aureus being caused the protective effect of dead mouse) in experimental example 3 bodies
Get mice and be divided into 7 groups every group 10 at random, be i.e. blank group, SHUANGHUANGLIAN ZHUSHEYE I (by existing preparation technology's gained) high, normal, basic three dosage groups and high, normal, basic three the dosage groups of SHUANGHUANGLIAN ZHUSHEYE II (by embodiment 1 gained) by body weight.Administration group lumbar injection (last subcutaneous injection) medicinal liquid 40ml/kg, once a day, successive administration 7 days; The blank group gives water for injection with the same manner.The last time behind the administration 30min, each organize the lumbar injection staphylococcus aureus THIOGLYCOLLIC ACID salt culture fluid (37 ℃ ± 1 ℃, 24h, bacterial population about 10
8/ ml), dosage is every mice 0.5ml, observes at once, and observes continuously 7 days, record dead mouse situation every day is calculated median effective dose (ED50).The result shows; SHUANGHUANGLIAN ZHUSHEYE I and SHUANGHUANGLIAN ZHUSHEYE II all have certain protective role to the dead mouse that the lumbar injection staphylococcus aureus causes; and the protective effect of SHUANGHUANGLIAN ZHUSHEYE II is better than SHUANGHUANGLIAN ZHUSHEYE I; but both ED50 there was no significant differences (P＞0.05) see Table 13.
Bacteriostasis comparative test result in the table 13 'Shuang Hualian ' injection liquid
| Group | Dosage (gkg -1Contain crude drug) | The survival number | Survival rate | ED50±s |
| Blank | -- | 0 | 0 | -- |
| High I | 40 | 10 | 100 | 18.56±5.33 |
| Middle I | 20 | 6 | 60 | |
| Low I | 10 | 1 | 10 | |
| High II | 40 | 10 | 100 | 16.80±3.1 |
| Middle II | 20 | 7 | 70 | |
| Low II | 10 | 2 | 20 |
Experimental example 4 interior resisting virus effects are (to the influence of mice influenza virus property pneumonia) relatively
Get mice by the body weight random packet, the lumbar injection sample, the viral infection matched group all gives identical medicinal liquid volumetrical water for injection with intact animal's matched group.Except that the normal control group, mice is slightly anaesthetized with ether, infect every 0.05ml with 15 LD50 influenza virus drop noses.Begin administration or water the previous day from infecting, every day 2 times, continuous 5 days, took by weighing after the mice body weight fixingly on the 6th day, blood-letting, dissection are won full lung and are weighed, and calculate the lung exponential quantity one by one, and obtain lung index suppression ratio, the results are shown in Table 14.Lung tissue 10% formaldehyde fixed, the ethanol series dehydration, dimethylbenzene is transparent, paraffin embedding.Slice thickness 4～5 μ m, HE dyeing, ordinary optical microscope is observed the lung tissue morphological change.Press inflammatory cell infiltration grading standard, pair cell is scored, and carries out statistical procedures, organizes a t check relatively, the results are shown in Table 15.
Table 14 SHUANGHUANGLIAN ZHUSHEYE is to the influence (n=10) of influenza virus infecting mouse pneumonia (lung index)
| Group | Dosage (g/kg contains crude drug) | The lung exponential quantity (x ± s) | Suppression ratio (%) |
| Infect matched group | -- | 1.60±0.20 | -- |
| The normal control group | -- | 0.99±0.09 ** | -- |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 1.38±0.11 ** | 13.11 |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 1.36±0.20 ** | 14.65 |
| Among the | 10 | 1.40±0.17 * | 12.14 |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 1.46±0.13 | 9.55 |
Annotate: compare with infecting matched group,
*P＜0.01,
*P＜0.05
Table 15 SHUANGHUANGLIAN ZHUSHEYE is to the influence (n=10) of influenza virus infecting mouse pneumonia (virus is observed)
| Group | Dosage (g/kg contains crude drug) | The lung exponential quantity (x ± s) | Suppression ratio (%) |
| Infect matched group | -- | 2.70±0.65 | -- |
| The normal control group | -- | 0.05±0.18 ** | -- |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 1.85±0.72 ** | 31.06 |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 1.75±0.60 ** | 33.35 |
| Among the | 10 | 2.00±0.57 * | 26.84 |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 2.10±0.63 * | 23.10 |
Annotate: compare with infecting matched group,
*P＜0.01,
*P＜0.05
By table 14,15 results as seen, infecting back mouse lung exponential quantity obviously increases, the inflammation scoring value obviously increases, the pneumonia that in the dosage range of SHUANGHUANGLIAN ZHUSHEYE under this experimental condition the influenza virus infecting mouse is caused has the obvious suppression effect, the lung exponential quantity obviously reduces, the lung tissue lesion degree obviously alleviates, and scoring value obviously reduces.And under the same dose condition, the effect of SHUANGHUANGLIAN ZHUSHEYE II is better than SHUANGHUANGLIAN ZHUSHEYE I.
Experimental example 5 antiinflammatory actions are (influence that lumbar injection acetic acid induced mice abdominal cavity capillary permeability is increased) relatively
Get 50 mices, male and female half and half are divided into 5 groups at random by body weight.The medication group is pressed 20ml/kg body weight lumbar injection medicinal liquid, and the blank group gives the water for injection with volume.Be administered once every day, continuous 7 days, in the last administration after 30 minutes, the blue normal saline solution 0.1ml/10g of equal tail vein injection 0.5% ivens of each mice body weight, and lumbar injection 0.6% acetic acid normal saline solution 0.2ml/ only takes off cervical vertebra and puts to death behind the 20min immediately, open the abdominal cavity, with 6ml normal saline washing abdominal cavity, collect cleaning mixture and centrifugal, get supernatant and survey trap in the 590nm place.The result by table 16 as seen, SHUANGHUANGLIAN ZHUSHEYE can significantly suppress increasing of lumbar injection acetic acid induced mice abdominal cavity capillary permeability, with blank group ratio, 20g/kg, 10g/kg (containing crude drug) group, difference highly significant (P＜0.01) is dependency with dosage.The action intensity of SHUANGHUANGLIAN ZHUSHEYE II is similar to SHUANGHUANGLIAN ZHUSHEYE I.
The influence that table 16 SHUANGHUANGLIAN ZHUSHEYE increases lumbar injection acetic acid induced mice abdominal cavity capillary permeability
| Group | Dosage (gkg -1Contain crude drug) | Trap |
| The blank group | -- | 0.115±0.020 |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 0.076±0.031 ** |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 0.074±0.050 ** |
| Among the | 10 | 0.085±0.033 * |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 0.091±0.027 |
Annotate: with blank group ratio,
*P＜0.01,
*P＜0.05
Experimental example 6 refrigeration functions relatively
Get the qualified rabbit of pre-thermometric, before test day is measured administration on request, behind the rectal temperature, press the body temperature random packet.Administration group intravenous injection medicinal liquid, the blank group gives water for injection, and the administration volume is 4ml/kg.Except that the blank group, each organizes after the administration intravenous injection bacterial endotoxin 50ED/kg immediately, surveys rectal temperature once every 30min later on, with body temperature that different time is surveyed and administration precursor using warming therapy difference, for the fervescence value (body temperature descend 0.4 ℃ in interior person with 0), the results are shown in Table 17.
The influence (n=6) that table 17 SHUANGHUANGLIAN ZHUSHEYE raises to rabbit body temperature due to the bacterial endotoxin
| Group | Dosage (g/kg contains crude drug) | Body weight (kg) | Basal body temperature (℃) | High fever (℃) | The highest intensification value (℃) |
| Model group | -- | 2.55±0.21 | 38.38±0.34 | 39.92±0.31 | 1.54±0.22 |
| The blank group | -- | 2.40±0.19 | 38.40±0.22 | 38.70±0.30 | 0.30±0.16 ** |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 2.34±0.28 | 38.64±0.23 | 3.36±0.26 | 1.02±0.20 ** |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 2.30±0.24 | 38.50±0.26 | 39.55±0.35 | 1.05±0.23 ** |
| Among the | 10 | 2.35±0.18 | 38.37±0.20 | 39.47±0.20 | 1.10±0.21 ** |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 2.38±0.16 | 38.42±0.30 | 39.72±0.31 | 1.30±0.19 * |
Annotate: compare with model group,
*P＜0.01,
*P＜0.05
The result shows that when (containing crude drug), the rabbit body temperature rising that bacterial endotoxin is caused has good refrigeration function to the SHUANGHUANGLIAN ZHUSHEYE dosage, with model group comparing difference highly significant (P＜0.01) at 20g/kg, 10g/kg.The refrigeration function of SHUANGHUANGLIAN ZHUSHEYE II is similar to SHUANGHUANGLIAN ZHUSHEYE I.
Experimental example 7 analgesic activities are (writhing method) relatively
50 mices are divided into 5 groups at random, and the medication group is pressed 20ml/kg body weight lumbar injection medicinal liquid, and the blank group gives the water for injection with volume.Once a day, continuous 5 days, administration in the morning in the 5th day was after 45 minutes, lumbar injection 0.6% acetic acid 0.2ml/ only, each Mus is turned round the body number of times in the observed and recorded 15 minutes, the result carries out statistical procedures.
The influence of table 18 SHUANGHUANGLIAN ZHUSHEYE Dichlorodiphenyl Acetate induced mice pain
| Group | Dosage (g/kg contains crude drug) | Turn round the body number of times |
| The blank group | -- | 25.0±7.0 |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 17.9±4.1 * |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 15.5±3.2 * |
| Among the | 10 | 18.2±3.5 * |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 19.0±5.6 |
Annotate: with blank group ratio,
*P＜0.05
As seen from Table 18, the analgesic activity of SHUANGHUANGLIAN ZHUSHEYE II is better than SHUANGHUANGLIAN ZHUSHEYE I under the Isodose.
Experimental example 8 antitussive actions relatively
Get 50 of mices and be divided into 5 groups at random, the medication group is pressed 20ml/kg body weight lumbar injection medicinal liquid, and the blank group gives the water for injection with volume.Successive administration 5 days, once a day, after the last administration 1 hour, utilize spray 26% ammonia 3 seconds of ultrasound atomizer, observe and the cough latent period (promptly stopping to be sprayed to the time of cough) of record mice and mouse cough number of times in 2 minutes (with its abdominal muscle shrink, magnify mouth and hear to cough be as the criterion), the results are shown in Table 19.
Table 19 SHUANGHUANGLIAN ZHUSHEYE is to the influence of ammonia induced mice cough
| Group | Dosage (g/kg contains crude drug) | Incubation period | The cough number of times |
| The blank group | -- | 4.9±1.5 | 33.2±7.6 |
| SHUANGHUANGLIAN ZHUSHEYE I | 20 | 8.8±2.2 ** | 21.2±6.0 * |
| SHUANGHUANGLIAN ZHUSHEYE II height | 20 | 9.0±1.9 ** | 20.0±5.2 * |
| Among the | 10 | 7.9±2.5 * | 24.6±6.5 * |
| SHUANGHUANGLIAN ZHUSHEYE II is low | 5 | 6.2±2.3 | 26.3±5.4 |
Annotate: compare with the blank group,
*P＜0.01,
*P＜0.05
As seen from Table 19, but SHUANGHUANGLIAN ZHUSHEYE significant prolongation ammonia induced mice cough latent period also can obviously reduce the mouse cough number of times.The antitussive effect of SHUANGHUANGLIAN ZHUSHEYE II slightly is better than SHUANGHUANGLIAN ZHUSHEYE I.
From above result of the test as seen, the main pharmacodynamics experimental result of the SHUANGHUANGLIAN ZHUSHEYE II under the Isodose all is better than SHUANGHUANGLIAN ZHUSHEYE I.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. the preparation method of a SHUANGHUANGLIAN ZHUSHEYE is characterized in that, is prepared by following steps:
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, adding 4～16 times of water soaked 30～180 minutes in 60～85 ℃ of insulations, soak 2～3 times, gradation is filtered, and collects filtrate and is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add ethanol to containing alcohol amount 75～80%, left standstill 12～24 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.12～1.23 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2～6 times of amounts, stir gently, left standstill 12～24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add ethanol while stirring, reach 80～85% to containing the alcohol amount, left standstill 12～24 hours, and got supernatant adjust pH 8.0～10.0, left standstill 24～36 hours, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.12～1.23 (80 ℃ ± 5 ℃ surveys), gets the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, decocting 3 times for the first time, adds 4～12 times of water gagings, soaks 30 minutes, and heated and boiled 1.0～3.0 hours is filtered; Each 2～8 times of water gaging of second, third time boiled 1.0～2.0 hours, merged medicinal liquid and were concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add ethanol, precipitate 12～24 hours to containing alcohol amount 75～80%, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 2～6 times of amounts, stir, left standstill 12～24 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.15～1.25 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add ethanol while stirring, make and contain alcohol amount and reach 80～85%, left standstill 12～24 hours, and got supernatant adjust pH 8.0～10.0, left standstill 24～36 hours, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.12～1.23 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 2～12 times of water gagings, decoct at least 2 times, each 1～3 hour, filter merging filtrate, be concentrated into 3～6 times of crude drug amounts, leave standstill leaching supernatant adjust pH to 1.0 after 1～3 hour～3.0,70～80 ℃ insulation 1～3 hour, left standstill 12～20 hours, filter, precipitation adds 5～10 times of water gagings, regulates pH value to 6.0～8.0, add equivalent ethanol again, stirring makes dissolving, filters filtrate adjust pH to 1.0～3.0,60～70 ℃ are incubated 30～60 minutes, left standstill 12～20 hours, and filtered, it is 6.0～8.0 that precipitation is washed till pH value with ethanol, reclaim ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 1～3 times of amount (in baicalin dry product weight) below 40 ℃, soaked into 4～10 hours, add the water for injection of 4～8 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir after 2～10 minutes, adjust pH to 6.0～7.0, the medicinal charcoal of adding 0.5% boiled 30～60 minutes, and is to be cooled after 60～80 ℃, filter with ethanol, filtrate adjust pH to 1.0～3.0,60 ± 5 ℃ insulation 30～60 minutes was left standstill 4～10 hours, abandoning supernatant, precipitate, is drained to pH＞4.0 with 95% washing with alcohol, dry baicalin;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin add the injection dilute with water respectively with Flos Lonicerae, Fructus Forsythiae equivalent extract, add baicalin behind the mixing and stir, dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15～30 minutes and is cooled to below 70 ℃ adjust pH 6.8～7.5, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 10～40 ℃, fine straining, fill was sterilized 30 minutes for 115～118 ℃, promptly.
2. the preparation method of SHUANGHUANGLIAN ZHUSHEYE according to claim 1 is characterized in that, is prepared by following steps:
1) preparation of Flos Lonicerae equivalent extract
Take by weighing Flos Lonicerae, first pass adds 10 times of water gagings, soaks 1 hour in 80 ℃ of insulations, add 10 times of water gagings second time, soaked 40 minutes in 80 ℃ of insulations, gradation is filtered, and collects filtrate and is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% above ethanol to containing alcohol amount 75%, left standstill 12 hours, filter, filtrate recycling ethanol is not distinguished the flavor of to there being ethanol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5～9.0, got supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys).Get the Flos Lonicerae equivalent extract;
2) preparation of Fructus Forsythiae equivalent extract
Take by weighing Fructus Forsythiae, first pass adds 8 times of water gagings, soaks 30 minutes, and heated and boiled 1.5 hours is filtered; Second, third boiled 1.0 hours all over each 6 times of water gaging, merged medicinal liquid and was concentrated into density 1.18～1.22 (80 ± 5 ℃ of surveys), treat that temperature reduces to below 40 ℃, add 90% above ethanol and measure 75%, precipitate 12 hours to containing alcohol, filter, filtrate recycling ethanol is to there not being the ethanol flavor; Wait to be chilled to below 40 ℃, add the purified water that cream weighs 4 times of amounts, stir gently, left standstill 12 hours, get supernatant liquid filtering, filtrate is concentrated into relative density 1.18～1.22 (80 ± 5 ℃ of surveys); Wait to be chilled to below 40 ℃, add 95% ethanol while stirring, make and contain alcohol amount and reach 85%, left standstill 12 hours, the siphon supernatant left standstill 24 hours with 20% sodium hydroxide solution adjust pH 8.5～9.0, get supernatant liquid filtering, filtrate recycling ethanol is not distinguished the flavor of to there being alcohol, and is concentrated into relative density 1.15～1.20 (80 ± 5 ℃ of surveys), gets the Fructus Forsythiae equivalent extract;
3) preparation of baicalin
Get scutellaria tablet, add 8 times of water gagings, decocted 2 hours, filter, add 6 times of water gagings, decocted 1 hour, merging filtrate is concentrated into 4～5 times of crude drug amounts, leaves standstill leaching supernatant 2mol/L hydrochloric acid adjust pH to 1.0～2.0 after 1 hour, 80 ℃ are incubated 1 hour, leave standstill 12 hours, filter, precipitation adds 6～8 times of water gagings, regulate pH value to 6.5～7.5 with 40% sodium hydroxide solution, add equivalent ethanol again, stir and make dissolving, filter, filtrate was left standstill 12 hours with 2mol/L hydrochloric acid adjust pH to 1.0～2.0,60 ℃ insulation 30 minutes, filter, it is 6.5～7.5 that precipitation is washed till pH value with ethanol, reclaims ethanol, the centrifugal precipitate that gets; Taking precipitate adds the water for injection of 2 times of amounts (in baicalin dry product weight) below 40 ℃, soak into more than 4 hours, add the water for injection of 4 times of amounts (in baicalin dry product weight) below 40 ℃ again, stir and slowly add 10% sodium hydroxide solution after 2 minutes, adjust pH to 6.5～7.0, the medicinal charcoal of adding 0.5% boiled 30 minutes, to be cooled after 60～80 ℃, the 90% above ethanol that adds 0.5 times of amount of medicine liquid volume, filtered while hot, filtrate is used 10%HCl adjust pH to 1.5～2.0,60 ± 5 ℃ are incubated 30 minutes, leave standstill more than 4 hours, abandoning supernatant, precipitate with 95% washing with alcohol to pH＞4.0, drain the dry baicalin that gets;
4) preparation of SHUANGHUANGLIAN ZHUSHEYE
Press crude drug than 1: 2: 1, respectively extracting honeysuckle equivalent extract, Fructus Forsythiae equivalent extract and baicalin.Flos Lonicerae, Fructus Forsythiae equivalent extract are added the injection dilute with water respectively, add behind the mixing baicalin stir make dissolve the SHUANGHUANLIAN concentrated wiring liquid; Silk filters, and filtering with microporous membrane adds injection and is diluted with water to 90% of full dose, the medicinal charcoal that adds recipe quantity boiled 15 minutes and is cooled to below 70 ℃ adjust pH 6.8～7.5, be settled to full dose, stir, carry out coarse filtration, the medicinal liquid after the coarse filtration is cooled to 10～40 ℃, fine straining, fill was sterilized 30 minutes for 115～118 ℃, promptly.
3. the component detection method of the SHUANGHUANGLIAN ZHUSHEYE of making according to the method for claim 1 or 2 is characterized in that this method comprises the assay of any two or three composition in Radix Scutellariae, Flos Lonicerae and the Fructus Forsythiae.
4. according to the component detection method of claim 3 SHUANGHUANGLIAN ZHUSHEYE, it is characterized in that the assay of described Radix Scutellariae, Flos Lonicerae and Fructus Forsythiae comprises the following steps:
(1) assay of Radix Scutellariae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 40～60: methanol-glacial acetic acid of 0.5～2: 60～40-water is mobile phase; The detection wavelength is 276nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the baicalin peak and is not less than 1000, and precision takes by weighing the baicalin reference substance, adds 40～60% methanol or ethanol and makes the solution that every 1ml contains 0.2mg, is reference substance solution; Precision is measured this product 1ml, puts in the 50ml measuring bottle, adds 40～60% methanol or ethanol dilution to scale, shakes up, and is need testing solution; Accurate respectively need testing solution and each 8 μ l of reference substance solution of drawing inject chromatograph of liquid respectively, measure peak area respectively, and external standard method is calculated content, and the every 1ml of this product contains Radix Scutellariae in baicalin, is 6.0～12.0mg;
(2) assay of Flos Lonicerae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～30: methanol-water-glacial acetic acid of 90～70: 0.1～2 is a mobile phase; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature.Number of theoretical plate calculates by the chlorogenic acid peak and is not less than 2000; It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds water, methanol or ethanol and makes the solution that every 1ml contains 40 μ g, is reference substance solution; Precision is measured this product 6ml, puts in the brown measuring bottle of 50ml, and water, methanol or ethanol dilution shake up to scale, gets need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Flos Lonicerae in chlorogenic acid, is 0.05～2.0mg;
(3) assay of Fructus Forsythiae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～40: 90～60 acetonitrile-water is a mobile phase; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the phillyrin peak and is not less than 2000; It is an amount of to get the phillyrin reference substance, and accurate the title decides, and adds 30～90% methanol or ethanol and makes the solution that every 1ml contains 60 μ g, is reference substance solution; Precision is measured this product 10ml, put 100～120 order 6g, on the neutral alumina post of internal diameter 1cm, with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, and shake up, get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Fructus Forsythiae in phillyrin, is 0.05～0.4mg.
5. the component detection method of SHUANGHUANGLIAN ZHUSHEYE according to claim 4 is characterized in that, in the assay of Radix Scutellariae, is mobile phase with methanol-glacial acetic acid of 48: 1: 52 of volume ratio-water; In the assay of Flos Lonicerae, be mobile phase with methanol-water-glacial acetic acid of 20: 80: 1 of volume ratio; In the assay of Fructus Forsythiae, be mobile phase with 25: 75 acetonitrile-waters of volume ratio.
6. according to the component detection method of claim 3 SHUANGHUANGLIAN ZHUSHEYE, it is characterized in that the assay of described Flos Lonicerae and Fructus Forsythiae comprises the following steps:
(1) assay of Flos Lonicerae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～30: methanol-water-glacial acetic acid of 90～70: 0.1～2 is a mobile phase; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature, number of theoretical plate are calculated by the chlorogenic acid peak and are not less than 2000; It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds water, methanol or ethanol and makes the solution that every 1ml contains 40 μ g, is reference substance solution; Precision is measured this product 6ml, puts in the brown measuring bottle of 50ml, and water, methanol or ethanol dilution shake up to scale, gets need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Flos Lonicerae in chlorogenic acid, is 0.05～2.0mg;
(2) assay of Fructus Forsythiae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～40: 90～60 acetonitrile-water is a mobile phase; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the phillyrin peak and is not less than 2000; It is an amount of to get the phillyrin reference substance, and accurate the title decides, and adds 30～90% methanol or ethanol and makes the solution that every 1ml contains 60 μ g, is reference substance solution; Precision is measured this product 10ml, put 100～120 order 6g, on the neutral alumina post of internal diameter 1cm, with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, and shake up, get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Fructus Forsythiae in phillyrin, is 0.05～0.4mg.
7. according to the component detection method of claim 3 SHUANGHUANGLIAN ZHUSHEYE, it is characterized in that the assay of described Radix Scutellariae and Flos Lonicerae comprises the following steps:
(1) assay of Radix Scutellariae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 40～60: methanol-glacial acetic acid of 0.5～2: 60～40-water is mobile phase; The detection wavelength is 276nm; Flow velocity: 1ml/min; Column temperature: room temperature, number of theoretical plate are calculated by the baicalin peak and are not less than 1000, and precision takes by weighing the baicalin reference substance, adds 40～60% methanol or ethanol and makes the solution that every 1ml contains 0.2mg, is reference substance solution; Precision is measured this product 1ml, puts in the 50ml measuring bottle, adds 40～60% methanol or ethanol dilution to scale, shakes up, and is need testing solution; Accurate respectively need testing solution and each 8 μ l of reference substance solution of drawing inject chromatograph of liquid respectively, measure peak area respectively, and external standard method is calculated content, and the every 1ml of this product contains Radix Scutellariae in baicalin, is 6.0～12.0mg;
(2) assay of Flos Lonicerae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～30: methanol-water-glacial acetic acid of 90～70: 0.1～2 is a mobile phase; The detection wavelength is 324nm; Flow velocity: 1ml/min; Column temperature: room temperature, number of theoretical plate are calculated by the chlorogenic acid peak and are not less than 2000; It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds water, methanol or ethanol and makes the solution that every 1ml contains 40 μ g, is reference substance solution; Precision is measured this product 6ml, puts in the brown measuring bottle of 50ml, and water, methanol or ethanol dilution shake up to scale, gets need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Flos Lonicerae in chlorogenic acid, is 0.05～2.0mg.
8. according to the component detection method of claim 3 SHUANGHUANGLIAN ZHUSHEYE, it is characterized in that the assay of described Radix Scutellariae and Fructus Forsythiae comprises the following steps:
(1) assay of Radix Scutellariae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 40～60: methanol-glacial acetic acid of 0.5～2: 60～40-water is mobile phase; The detection wavelength is 276nm; Flow velocity: 1ml/min; Column temperature: room temperature, number of theoretical plate are calculated by the baicalin peak and are not less than 1000, and precision takes by weighing the baicalin reference substance, adds 40～60% methanol or ethanol and makes the solution that every 1ml contains 0.2mg, is reference substance solution; Precision is measured this product 1ml, puts in the 50ml measuring bottle, adds 40～60% methanol or ethanol dilution to scale, shakes up, and is need testing solution; Accurate respectively need testing solution and each 8 μ l of reference substance solution of drawing inject chromatograph of liquid respectively, measure peak area respectively, and external standard method is calculated content, and the every 1ml of this product contains Radix Scutellariae in baicalin, is 6.0～12.0mg;
(2) assay of Fructus Forsythiae
With the octadecylsilane chemically bonded silica is filler; With volume ratio 10～40: 90～60 acetonitrile-water is a mobile phase; The detection wavelength is 278nm; Flow velocity: 1ml/min; Column temperature: room temperature; Number of theoretical plate calculates by the phillyrin peak and is not less than 2000; It is an amount of to get the phillyrin reference substance, and accurate the title decides, and adds 30～90% methanol or ethanol and makes the solution that every 1ml contains 60 μ g, is reference substance solution; Precision is measured this product 10ml, put 100～120 order 6g, on the neutral alumina post of internal diameter 1cm, with 30～90% ethanol 40ml eluting, collect eluent, be concentrated into dried, residue add 30～90% methanol or ethanol an amount of, warmly make dissolving, be transferred in the 5ml measuring bottle, and be diluted to scale, and shake up, get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, and the every 1ml of this product contains Fructus Forsythiae in phillyrin, is 0.05～0.4mg.
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