CN101285812A - Antivirus compound formulation quality control method - Google Patents
Antivirus compound formulation quality control method Download PDFInfo
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- CN101285812A CN101285812A CNA2008100266546A CN200810026654A CN101285812A CN 101285812 A CN101285812 A CN 101285812A CN A2008100266546 A CNA2008100266546 A CN A2008100266546A CN 200810026654 A CN200810026654 A CN 200810026654A CN 101285812 A CN101285812 A CN 101285812A
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Abstract
The invention provides a quality control method for antiviral compound preparation. The method uses high efficiency liquid chromatography to perform qualitative detection and quantitative detection for mangiferin and forsythin at the same time. The chromatographiccondition is as follows: eighteen carbon bonding phase silica gel column with 4.6mmx250mm, 5mu m is as a stationary phase, mixed solution formed through mixing acetonitrile and 0.05mol/L of monopotassium phosphate with 0.1 percent of phosphoric acid is as a mobile phase, velocity of flow is between 0.8 and 1.0 ml per minute, and detection wavelength is 277nm. The quality control method for antiviral compound preparation can perform the qualitative detection and quantitative detection for the mangiferin and the forsythin in the antiviral compound preparation at the same time, which not only can effectively control the quality of the preparation, but also can significantly reduce operating procedure, shorten analysis time and save organic solvent.
Description
Technical field
The present invention relates to field of traditional Chinese, be specifically related to the method for quality control of the method for quality control of Chinese medicine preparation, particularly antivirus compound formulation.
Background technology
" Ministry of Health of the People's Republic of China's new drug become a full member standard " WS3-49 (X-39)-92 (z) discloses a kind of by gypsum, Radix Isatidis, reed rhizome, glutinous rehmannia, root tuber of aromatic turmeric, the wind-weed, grass-leaved sweetflag, the method of quality control of the antiviral oral liquor that Pogostemon cablin and the capsule of weeping forsythia nine flavor Chinese medicines process water extract-alcohol precipitations prepare, concrete steps are: test according to thin-layered chromatography (57 pages of appendix of Chinese Pharmacopoeia nineteen ninety version), draw need testing solution and forsythin reference substance solution (1mg/ml) 1 μ l and 2 μ l respectively, put respectively on same silica G F-254 thin layer plate, with volume ratio is that chloroform-methanol-glacial acetic acid of 85: 10: 5 is a developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show identical aubergine spot.Simultaneously scan light source: deuterium lamp, wavelength: λ s=280nm, λ according to chromatography (57 pages of thin layer chromatography scannings of appendix of Chinese Pharmacopoeia nineteen ninety version)
R=320nm measures test sample and reference substance absorbance log integrated value, presses two points external standard method and calculates, and every 1ml contains phillyrin (C
27H
34O
11) must not be less than 3.0 μ g.This method can only be carried out qualitative and detection by quantitative to the forsythin in the described oral liquid.
The application for a patent for invention that State Intellectual Property Office disclosed one " a kind of quality determining method of antiviral oral liquor " on 02 25th, 2004 (publication number is CN1477395), this application disclose the capsule of weeping forsythia, the discriminating of grass-leaved sweetflag medicinal material and the content assaying method of sarsasapogenin and forsythin.Disclosing " a kind of method of controlling an antiviral oral liquid " publication number on 04 4th, 2007 is CN1939524) application for a patent for invention, this application discloses a kind of content with Patchoulicalcohol in the gas chromatography determination antiviral oral liquor.
From said method as can be known, the method of quality control of antiviral oral liquor all lacks the detection of the mangiferin in the wind-weed in the prior art, and the mangiferin in the wind-weed is the important component that this oral liquid plays the heat-clearing effect, have effects such as antiviral, anti-oxidant, if it is not up to standard to lack this composition or content, be difficult to guarantee the drug effect of this oral liquid, but still do not have the method that detects mangiferin in this oral liquid at present.
Summary of the invention
The purpose of this invention is to provide a kind of control " Ministry of Health of the People's Republic of China's new drug become a full member standard " WS3-49 (X-39)-92 (z) the antiviral oral liquor of putting down in writing and according to the new method of other quality of the pharmaceutical preparations of the prescription preparation of this oral liquid, this method can be carried out qualitative and detection by quantitative simultaneously to mangiferin in the described antivirus compound formulation and forsythin.
The present invention realizes that the technical scheme of above-mentioned purpose is:
The method of quality control of antivirus compound formulation, this method is made up of following steps:
(1) preparation of reference substance solution: mangiferin and forsythin mix makes the reference substance solution that contains mangiferin 0.08～0.15mg/ml and forsythin 0.04～0.08mg/ml with dissolve with methanol;
The preparation of need testing solution: when antivirus compound formulation is solid pharmaceutical preparation, add 20～30 times of methyl alcohol, ultrasonic Extraction 20～40min, filter, filter residue adds 5～10 times of water-soluble separating, with 10～15 times of water-saturated n-butanol extractions 2～3 times, merge n-butanol layer, water bath method, residue becomes every ml soln to be equivalent to the former preparation of 0.2～0.3g with 50% dissolve with methanol, shakes up; When antivirus compound formulation is liquid preparation, with 10～15 times of water-saturated n-butanol extractions 2～3 times, merge n-butanol layer, water bath method, the gained dry becomes every ml soln to be equivalent to 3～5g dry with 50% dissolve with methanol, shakes up;
(2) be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, mixing material with acetonitrile and the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% (v/v) phosphoric acid is the moving phase wash-out then, flow velocity is 0.8～1.0ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample; In the described moving phase, the volume percent content of acetonitrile is 10～30%, and the volume percent content of potassium dihydrogen phosphate that contains the 0.05mol/L of 0.1% (v/v) phosphoric acid is 70～90%;
(3) relatively with the chromatogram of the chromatogram of test sample and reference substance solution: with the characteristic peak that first peak retention time is identical in the reference substance chromatogram promptly be the characteristic peak of mangiferin, the characteristic peak identical with second peak retention time in the reference substance chromatogram promptly is the characteristic peak of forsythin; And press external standard method according to the peak area at these two peaks respectively and calculate mangiferin and forsythin content.
In the said method, contain mangiferin 0.1172mg and forsythin 0.0480mg in described every milliliter of reference substance solution.
In the said method, when anti-virus formulation to be measured is solid pharmaceutical preparation, the preparation method of described need testing solution is preferably: get anti-virus formulation and add 25 times of dissolve with methanol, ultrasonic Extraction 40min filters, and filter residue adds 5 times of water-soluble separating, with 10 times of water-saturated n-butanol extractions 4 times, merge n-butanol layer, water bath method, gained dry become every ml soln to be equivalent to the former preparation of 0.2g with 50% dissolve with methanol to shake up.
In the said method, the content of acetonitrile is 20% in the described moving phase, and the content of potassium dihydrogen phosphate that contains the 0.05mol/L of 0.1% phosphoric acid is 80%.
The inventive method can also be in conjunction with thin-layered chromatography, forsythin and mangiferin are being carried out quantitatively and on the basis of qualitative detection, respectively to the sarsasapogenin in the described antivirus compound formulation, arginine, grass-leaved sweetflag effective constituent and in hundred autumns one or more compositions in the alcohol carry out qualitative detection.Sarsasapogenin, arginine, grass-leaved sweetflag effective constituent and in hundred autumns the thin-layered chromatography qualitative detection of alcohol can be this area method commonly used, concrete grammar is as follows:
The qualitative checking method of sarsasapogenin:
(1) get anti-virus formulation and add 4～8 times of absolute ethyl alcohols, ultrasonic Extraction 20～40min filters, filtrate adds 0.1～0.2 times 36.5% hydrochloric acid, and evaporate to dryness behind reflux 1～2h adds 2～3 times of water-soluble separating, add 4～5 times of chloroform extractions 2～3 times, chloroform extraction liquid discards alkali lye with 4～5 times of 0.5%NaOH solution washings, uses 4～5 times of water washings again, discard water liquid, with the chloroform solution evaporate to dryness, residue adds methanol constant volume to every ml soln and is equivalent to the former preparation of 4～6g, as need testing solution then;
(2) get the sarsasapogenin reference substance in addition, add methyl alcohol and make the solution that every 1ml contains 1mg, as positive control solution;
(3) drawing each 5 μ l of need testing solution and positive reference substance solution, put respectively on same silica gel g thin-layer plate, is 9: 1 toluene and acetone mixture developping agent with volume ratio, launch, take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to be heated to the spot colour developing at 105 ℃;
(4) result judges: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, illustrate in this test sample and contain sarsasapogenin.
Arginic qualitative checking method:
(1) get anti-virus formulation and add 10～15 times of alcohol reflux 20～40min, filter, the extract evaporate to dryness, residue becomes every ml soln to be equivalent to the former preparation of 1～2g with dissolve with methanol, as test sample liquid;
(2) get the arginine reference substance in addition and add methyl alcohol and make the solution that every 1ml contains 0.5mg, as positive reference substance solution;
(3) draw each 5 μ l of need testing solution and positive reference substance solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with 0.5% carboxymethylcellulose sodium solution, be that the potpourri of 8: 2: 2 normal butyl alcohol, glacial acetic acid and water is a developping agent with volume ratio, with the sulfuric acid controlled humidity is 58%, presaturation 30min launches 8～9cm, takes out, dry, launch 8～9cm with same developping agent secondary again, take out, dry, spray develops the color with 5% ninhydrin solution, and 105 ℃ are dried by the fire to clear spot;
(4) result judges: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, illustrate in this test sample and contain arginine.
The qualitative checking method of grass-leaved sweetflag effective constituent:
(1) get anti-virus formulation and add 4～6 times of water-soluble separating after, add 2～3 times of petroleum ether extractions 2～3 times, merge petroleum ether layer, water bath method, residue becomes every milliliter to be equivalent to the former preparation of 8～12g with acetic acid ethyl dissolution, as test sample liquid;
(2) get grass-leaved sweetflag medicinal material fine powder in addition and add 30～50 times of petroleum ether dissolutions, ultrasonic Extraction 20～40min filters, and gets the filtrate evaporate to dryness, and residue becomes every milliliter to be equivalent to 8～12g crude drug with acetic acid ethyl dissolution, as positive control medicinal material solution;
(3) draw each 5 μ l of need testing solution and positive reference substance solution, put in same silica G F respectively
254On the thin layer plate, be that 8: 2 60～90 ℃ of sherwood oils and the potpourri of ethyl acetate are developping agent, launch, take out, dry, put under the 254nm uviol lamp and inspect with volume ratio;
(4) result judges: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, the effective constituent that contains grass-leaved sweetflag in this test sample is described.
The qualitative checking method of alcohol in hundred autumns:
(1) get anti-virus formulation and add 8～10 times of water-soluble separating after, with 3～5 times of extracted with diethyl ether 2～3 times, get the ether layer evaporate to dryness, residue becomes every ml soln to be equivalent to 4～6g crude drug with dissolve with methanol, as need testing solution;
(2) get pure reference substance in hundred autumns in addition, add methyl alcohol and make every milliliter of solution that contains 0.5mg, as positive reference substance solution;
(3) drawing each 5 μ l of need testing solution and positive reference substance solution, put respectively on same silica gel g thin-layer plate, is that 10: 3 cyclohexane and ethyl acetate mixed liquor is developping agent with volume ratio, launches 8～9cm; Take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to dry by the fire to the spot colour developing at 105 ℃;
(4) result judges: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, illustrate and contain alcohol in hundred autumns in this test sample.
In order further to guarantee the accuracy of qualitative detection, above-mentioned thin-layer chromatography qualitative checking method can also add negative control, when promptly detecting every kind of composition need testing solution, positive control solution and negative control solution are carried out thin-layer chromatography simultaneously, chromatogram result should be: in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, no corresponding spot in the negative control product chromatogram.Described negative control solution is the methanol solution that lacks with the anti-virus compound extract of the corresponding medicinal material of branch that is detected as, and the preparation method is as follows:
The negative control product that lack the wind-weed:
(1) get Pogostemon cablin 2.9 weight portions, grass-leaved sweetflag meal 2.5 weight portions, CO packs into
2In the supercritical extraction reactor, extracted 2 hours down for 55 ℃, collect volatile oil, stay the dregs of a decoction standby in pressure 28MPa, temperature;
(2) get gypsum 5.7 weight portions, reed rhizome meal 6.1 weight portions, add 8～10 times in water, 80 ℃ are extracted 2～3 times down, each 1～2 hour, filter, merge extract, temperature be 100 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(3) getting the capsule of weeping forsythia 4.6 weight portions, Radix Isatidis 12.9 weight portions, root tuber of aromatic turmeric 2.5 weight portions, glutinous rehmannia 3.2 weight portions and step (1) the gained dregs of a decoction mixes, extract 2～3 times with 8～10 times of 80% alcohol heating reflux, each 1～2 hour, filter, merge extract, with ethanol extract temperature be 60 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(4) get the clear cream of volatile oil, step (2) and (3) gained of step (1) gained, be prepared into granule or oral liquid according to a conventional method.
The negative control product that lack Radix Isatidis:
(1) get Pogostemon cablin 2.9 weight portions, grass-leaved sweetflag meal 2.5 weight portions, CO packs into
2In the supercritical extraction reactor, extracted 2 hours down for 55 ℃, collect volatile oil, stay the dregs of a decoction standby in pressure 28MPa, temperature;
(2) get the wind-weed 2.5 weight portions, gypsum 5.7 weight portions, reed rhizome meal 6.1 weight portions, add 8～10 times in water, 80 ℃ are extracted 2～3 times down, each 1～2 hour, filter, merge extract, temperature be 100 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(3) getting the capsule of weeping forsythia 4.6 weight portions, root tuber of aromatic turmeric 2.5 weight portions, glutinous rehmannia 3.2 weight portions and step (1) the gained dregs of a decoction mixes, extract 2～3 times with 8～10 times of 80% alcohol heating reflux, each 1～2 hour, filter, merge extract, with ethanol extract temperature be 60 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(4) the clear cream of getting volatile oil, step (2) and (3) gained of step (1) gained is prepared into oral liquid or granule according to a conventional method.
The negative control product that lack grass-leaved sweetflag:
(1) get Pogostemon cablin 2.9 weight portions, CO packs into
2In the supercritical extraction reactor, extracted 2 hours down for 55 ℃, collect volatile oil, stay the dregs of a decoction standby in pressure 28MPa, temperature;
(2) get the wind-weed 2.5 weight portions, gypsum 5.7 weight portions, reed rhizome meal 6.1 weight portions, add 8～10 times in water, 80 ℃ are extracted 2～3 times down, each 1～2 hour, filter, merge extract, temperature be 100 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(3) getting the capsule of weeping forsythia 4.6 weight portions, Radix Isatidis 12.9 weight portions, root tuber of aromatic turmeric 2.5 weight portions, glutinous rehmannia 3.2 weight portions and step (1) the gained dregs of a decoction mixes, extract 2～3 times with 8～10 times of 80% alcohol heating reflux, each 1～2 hour, filter, merge extract, with ethanol extract temperature be 60 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(4) the clear cream of getting volatile oil, step (2) and (3) gained of step (1) gained is prepared into oral liquid or granule according to a conventional method.
The negative control product that lack Pogostemon cablin:
(1) get grass-leaved sweetflag 2.5 weight portions, CO packs into
2In the supercritical extraction reactor, extracted 2 hours down for 55 ℃, collect volatile oil, stay the dregs of a decoction standby in pressure 28MPa, temperature;
(2) get the wind-weed 2.5 weight portions, gypsum 5.7 weight portions, reed rhizome meal 6.1 weight portions, add 8～10 times in water, 80 ℃ are extracted 2～3 times down, each 1～2 hour, filter, merge extract, temperature be 100 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(3) getting the capsule of weeping forsythia 4.6 weight portions, Radix Isatidis 12.9 weight portions, root tuber of aromatic turmeric 2.5 weight portions, glutinous rehmannia 3.2 weight portions and step (1) the gained dregs of a decoction mixes, extract 2～3 times with 8～10 times of 80% alcohol heating reflux, each 1～2 hour, filter, merge extract, with ethanol extract temperature be 60 ℃, vacuum tightness be-0.08～-0.04MPa under concentrating under reduced pressure become clear cream, making its density under 50 ℃ is 1.05～1.15g/ml;
(4) the clear cream of getting volatile oil, step (2) and (3) gained of step (1) gained is prepared into oral liquid or granule according to a conventional method.
The inventive method be applicable to " Ministry of Health of the People's Republic of China's new drug become a full member standard " WS3-49 (X-39)-92 (z) the antiviral oral liquor of putting down in writing and the various preparations that prepare according to the prescription " gypsum, Radix Isatidis, reed rhizome, glutinous rehmannia, root tuber of aromatic turmeric, the wind-weed, grass-leaved sweetflag, Pogostemon cablin and the capsule of weeping forsythia " of this oral liquid.
The present invention has set up the high-efficiency liquid chromatography method for detecting of effective constituent in the antivirus compound formulation, can carry out qualitative and detection by quantitative simultaneously to important indicator forsythin and mangiferin among this side, not only control the quality of this square preparation effectively, also significantly reduced operation steps, shortened analysis time, saved organic solvent; The method of the invention also can with arginine in detecting Radix Isatidis, in the wind-weed sarsasapogenin, known method such as alcohol and grass-leaved sweetflag effective constituent combines in hundred autumns in the Pogostemon cablin, more fully reflects the inherent quality of antivirus compound formulation.
Description of drawings
Fig. 1 is the high performance liquid chromatogram collection of illustrative plates that detects the reference substance of antiviral granule agent with embodiment 1 method, and wherein No. 1 peak is a mangiferin, and No. 2 peaks are forsythin.
Fig. 2 is the high performance liquid chromatogram collection of illustrative plates that detects the test sample of antiviral granule agent with embodiment 1 method, and wherein No. 1 peak is a mangiferin, and No. 2 peaks are forsythin.
Fig. 3 is the canonical plotting that detects the mangiferin content of antiviral granule agent with embodiment 1 method.
Fig. 4 is the canonical plotting that detects the forsythin content of antiviral granule agent with embodiment 1 method.
Fig. 5 is the high performance liquid chromatogram collection of illustrative plates that detects the reference substance of antiviral oral liquid with embodiment 2 methods, and wherein No. 1 peak is a mangiferin, and No. 2 peaks are forsythin.
Fig. 6 is the high performance liquid chromatogram collection of illustrative plates that detects the test sample of antiviral oral liquid with embodiment 2 methods, and wherein No. 1 peak is a mangiferin, and No. 2 peaks are forsythin.
Fig. 7 is the canonical plotting that detects the mangiferin content of antiviral oral liquid with embodiment 2 methods.
Fig. 8 is the canonical plotting that detects the forsythin content of antiviral oral liquid with embodiment 2 methods.
Fig. 9 detects the thin-layer chromatogram that the antiviral granule agent detects sarsasapogenin with embodiment 3 methods, and wherein 1,2 is test liquid, and 3,4 for lacking the negative test liquid of the wind-weed, and 5 is sarsasapogenin.
Figure 10 detects the antiviral granule agent with embodiment 3 methods to detect arginic thin-layer chromatogram, and wherein 1,2,3 is test liquid, and 4 is arginine, and 5 for lacking the negative test liquid of Radix Isatidis.
Figure 11 detects the thin-layer chromatogram that the antiviral granule agent detects the grass-leaved sweetflag medicinal material with embodiment 3 methods, and wherein 1,2 is test liquid, and 3,4 is grass-leaved sweetflag control medicinal material test liquid, and 5,6 for lacking the negative test liquid of grass-leaved sweetflag.
Figure 12 detects the thin-layer chromatogram that the antiviral granule agent detects alcohol in hundred autumns with embodiment 3 methods, and wherein 1,2,3 is test liquid, and 4 is alcohol in hundred autumns, and 5 for lacking the negative test liquid of Pogostemon cablin.
Embodiment
The used anti-virus formulation of following examples is:
1, antiviral oral liquor is the antiviral oral liquor that the Guangzhou Xiangxue Pharmaceutical Co produces, and lot number is 060403;
2, the antiviral granule agent is self-control, and the preparation method is as follows:
(1) extract: get Pogostemon cablin 2.9Kg, grass-leaved sweetflag 2.5Kg, CO packs into
2In the supercritical extraction reactor, in 55 ℃ of pressure 28MPa, temperature extraction 2 hours down, collect volatile oil 198g, standby;
The dregs of a decoction and capsule of weeping forsythia 4.6Kg, Radix Isatidis 12.9Kg, root tuber of aromatic turmeric 2.5Kg, glutinous rehmannia 3.2Kg add 8 times of amount 80% ethanol, heating and refluxing extraction 2 times, and each 2 hours, emit alcohol extract through horizontal screw unloading filter centrifugal machine, merge alcohol extract twice, standby;
Get wind-weed 2.5Kg, gypsum 5.7Kg, reed rhizome 6.1Kg meal, the water that adds 8 times of amounts extracts 2 times down at 80 ℃, and each 2 hours, emit the water extract through horizontal screw unloading filter centrifugal machine, merge the water extract twice, standby;
(2) concentrate: being 100 ℃ and 60 ℃, vacuum tightness in temperature respectively with water extract and alcohol extract is concentrating under reduced pressure under the-0.06MPa, merging, must about 11800ml (50 ℃ time survey its density be 1.12g/ml) concentrate, measure its paste-forming rate simultaneously, standby;
(3) drying: in concentrate, add the dextrin that is equivalent to its dried cream amount 30%, the dissolving mixing, obtaining medicinal extract 12000ml (50 ℃ time survey its density be 1.15g/ml), is that 155～165 ℃, air outlet temperature are that 100 ℃, pressure are to carry out spray drying under the 1bar condition promptly to get 9.27Kg tawny dried powder at intake air temperature;
(4) preparation of inclusion compound: get step (1) gained volatile oil and dilute with absolute ethyl alcohol in 1: 1 ratio, splash in the beta-schardinger dextrin-saturated solution of 6 times of amount volatile oil weight, stirred 2 hours in 50 ℃, stirring rate is 200rpm, left standstill 24 hours, suction filtration, the control temperature promptly gets 1.39Kg white powder inclusion compound carrying out vacuum drying below 60 ℃, and inclusion rate is 70.2%;
(5) granulate: the tawny dried powder of above-mentioned gained is mixed the dextrin mixing of 1.5 times of amounts of back general assembly (TW) with volatile oil clathrate compound and both, 95% ethanol of adding 30% is wetting, and the system softwood sieves, the 40 ℃ of heat-wind circulate dryings of particle that sieve, whole grain;
(6) can, sterilization: the dried particles can becomes the 2.5g/ bag, and sterilization gets final product.
Example 1
The detection of mangiferin and forsythin in the antiviral granule agent
1, the preparation of reference substance solution: precision takes by weighing mangiferin, forsythin is an amount of, puts in the 50ml volumetric flask, adds methyl alcohol to scale, makes mangiferin concentration 0.1172mg/ml, forsythin concentration is 0.0480mg/ml solution, in contrast product solution.
The preparation of need testing solution: get this product 2g, precision is weighed, and puts in the tool plug Erlenmeyer flask, add methyl alcohol 50ml, ultrasonic 40min filters, residue adds water 10ml dissolving, with water saturation extracting n-butyl alcohol 4 times (20ml * 4), merge n-butanol layer, water bath method, residue 50% dissolve with methanol, constant volume shakes up in the 10ml measuring bottle, and is standby.
2, be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, be that 20%: 80% the acetonitrile and the mixing material of the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% phosphoric acid are the moving phase wash-out with volume ratio then, flow velocity is 1ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample;
3, Fig. 1 is the chromatogram of reference substance, and the peak that wherein average retention time is 15.6min is the characteristic peak of mangiferin, and the peak that average retention time is 32.6min is the characteristic peak of forsythin; Fig. 2 is the chromatogram of test sample, is that 15.5min and 32.4min have two peaks respectively in average retention time, and is consistent with the retention time of mangiferin and forsythin in the reference substance chromatogram, is respectively the characteristic peak of mangiferin and forsythin.Fig. 3 is the typical curve according to the mangiferin content of external standard method drafting, curvilinear equation is y=1146.2x+70.62, γ=0.9998, Fig. 4 is the typical curve according to the forsythin content of external standard method drafting, curvilinear equation is 1506.7x-6.34, γ=0.9999, wherein y is a peak area, x is a component content to be measured; According to the peak area and the typical curve equation of the characteristic peak of mangiferin among Fig. 2 and forsythin, can get that the content of mangiferin and forsythin is respectively 0.608mg/g and 0.124mg/g in the said preparation.
Example 2
The assay of mangiferin and forsythin in antiviral oral liquid
1, the preparation of reference substance solution: precision takes by weighing mangiferin, forsythin is an amount of, puts in the 50ml volumetric flask, adds methyl alcohol to scale, makes mangiferin concentration 0.15mg/ml, forsythin concentration is 0.04mg/ml solution, in contrast product solution.
The preparation of need testing solution: get commercially available antiviral oral liquor 20ml, add water-saturated n-butanol extraction 2 times, each 30ml discards water layer, and normal butyl alcohol liquid is put evaporate to dryness in the water-bath, and residue adds 50% dissolve with methanol, is settled to the 10ml scale, as need testing solution.
2, be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, be that 30%: 70% the acetonitrile and the mixing material of the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% phosphoric acid are the moving phase wash-out with volume ratio then, flow velocity is 0.8ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample;
3, Fig. 5 is the chromatogram of reference substance, and the peak that wherein average retention time is 9.3min is the characteristic peak of mangiferin, and the peak that average retention time is 26.2min is the characteristic peak of forsythin; Fig. 6 is the chromatogram of test sample, is that 9.3min and 26.3min have two peaks respectively in average retention time, and is consistent with the retention time of mangiferin and forsythin in the reference substance chromatogram, is respectively the characteristic peak of mangiferin and forsythin.Fig. 7 is the typical curve according to the mangiferin content of external standard method drafting, curvilinear equation is y=2330x-66.844, γ=0.9994, Fig. 8 is the typical curve according to the forsythin content of external standard method drafting, curvilinear equation is y=615.13x+13.516, γ=0.9995, wherein y is a peak area, x is a component content to be measured; According to the peak area and the typical curve equation of the characteristic peak of mangiferin among Fig. 6 and forsythin, can this oral liquid in the content of mangiferin and forsythin be respectively 0.182mg/10ml and 0.308mg/10ml.
Example 3
Sarsasapogenin, arginine, alcohol and the thin-layer chromatography detection of grass-leaved sweetflag medicinal material and the high performance liquid chromatography detection of mangiferin and forsythin in hundred autumns in the antiviral granule agent.
The qualitative detection of sarsasapogenin:
1, gets this product 5g, add the 20ml absolute ethyl alcohol, ultrasonic Extraction 40min, filter, filtrate adds hydrochloric acid 1ml, evaporate to dryness behind the reflux 1h, add water 15ml dissolving, add chloroform extraction 3 times, each 20ml, chloroform extraction liquid discards alkali lye with 0.5%NaOH solution 20ml washing, again with distilled water 20ml washing, discard water liquid, chloroform solution evaporate to dryness, residue add methanol constant volume to 1ml, as need testing solution.
2, get the sarsasapogenin reference substance in addition, add methyl alcohol and make the solution that every 1ml contains 1mg, in contrast product solution.
3, lack the preparation of the negative control product solution of the wind-weed:
(1) extract: get Pogostemon cablin 2.9Kg, grass-leaved sweetflag 2.5Kg, CO packs into
2In the supercritical extraction reactor, in 55 ℃ of pressure 28MPa, temperature extraction 2 hours down, collect volatile oil 198g, standby;
The dregs of a decoction and capsule of weeping forsythia 4.6Kg, Radix Isatidis 12.9Kg, root tuber of aromatic turmeric 2.5Kg, glutinous rehmannia 3.2Kg add 8 times of amount 80% ethanol, heating and refluxing extraction 2 times, and each 2 hours, emit alcohol extract through horizontal screw unloading filter centrifugal machine, merge alcohol extract twice, standby;
Get gypsum 5.7Kg, reed rhizome 6.1Kg meal, the water that adds 8 times of amounts extracts 2 times down at 80 ℃, and each 2 hours, emit the water extract through horizontal screw unloading filter centrifugal machine, merge the water extract twice, standby;
(2) concentrate: being 100 ℃ and 60 ℃ of vacuum tightness in temperature respectively with water extract and alcohol extract is concentrating under reduced pressure under the-0.06MPa, merging, must about 11800ml (50 ℃ time survey its density be 1.12g/ml) concentrate, measure its paste-forming rate simultaneously, standby;
(3) drying: in concentrate, add the dextrin that is equivalent to its dried cream amount 30%, the dissolving mixing, obtaining medicinal extract 12000ml (50 ℃ time survey its density be 1.15g/ml), is that 155～165 ℃, air outlet temperature are that 100 ℃, pressure are to carry out spray drying under the 1bar condition promptly to get 9.27Kg tawny dried powder at intake air temperature;
(4) preparation of inclusion compound: get step (1) gained volatile oil and dilute with absolute ethyl alcohol in 1: 1 ratio, splash in the beta-schardinger dextrin-saturated solution of 6 times of amount volatile oil weight, stirred 2 hours in 50 ℃, stirring rate is 200rpm, left standstill 24 hours, suction filtration, the control temperature promptly gets 1.39Kg white powder inclusion compound carrying out vacuum drying below 60 ℃, and inclusion rate is 70.2%;
(5) granulate: the tawny dried powder of above-mentioned gained is mixed the dextrin mixing of 1.5 times of amounts of back general assembly (TW) with volatile oil clathrate compound and both, 95% ethanol of adding 30% is wetting, and the system softwood sieves, the 40 ℃ of heat-wind circulate dryings of particle that sieve, whole grain;
(6) get above-mentioned particle 5g, add the 20ml absolute ethyl alcohol, ultrasonic Extraction 40min, filter, filtrate adds hydrochloric acid 1ml, evaporate to dryness behind the reflux 1h, add water 15ml dissolving, add chloroform extraction 3 times, each 20ml, chloroform extraction liquid discards alkali lye with 0.5%NaOH solution 20ml washing, again with distilled water 20ml washing, discard water liquid, chloroform solution evaporate to dryness, residue add methanol constant volume to 1ml, as negative control product solution.
4, according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that toluene-acetone of 9: 1 is developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, and negative control is noiseless, as shown in Figure 9.
Arginic qualitative detection:
1, get this product 2g,, filter with 20ml alcohol reflux 30min, extract evaporate to dryness, residue with methanol constant volume to 1ml, as test sample liquid.
2, get the arginine reference substance in addition and add methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.
3, the negative control solution that lacks Radix Isatidis:
(1) extract: get Pogostemon cablin 2.9Kg, grass-leaved sweetflag 2.5Kg, CO packs into
2In the supercritical extraction reactor, in 55 ℃ of pressure 28MPa, temperature extraction 2 hours down, collect volatile oil 198g, standby;
The dregs of a decoction and capsule of weeping forsythia 4.6Kg, Radix Isatidis 12.9Kg, root tuber of aromatic turmeric 2.5Kg, glutinous rehmannia 3.2Kg add 8 times of amount 80% ethanol, heating and refluxing extraction 2 times, and each 2 hours, emit alcohol extract through horizontal screw unloading filter centrifugal machine, merge alcohol extract twice, standby;
Get gypsum 5.7Kg, reed rhizome 6.1Kg meal, the water that adds 8 times of amounts extracts 2 times down at 80 ℃, and each 2 hours, emit the water extract through horizontal screw unloading filter centrifugal machine, merge the water extract twice, standby;
(2) concentrate: being 100 ℃ and 60 ℃ of vacuum tightness in temperature respectively with water extract and alcohol extract is concentrating under reduced pressure under the-0.06MPa, merging, must about 11800mL (50 ℃ time survey its density be 1.12g/mL) concentrate, measure its paste-forming rate simultaneously, standby;
(3) drying: in concentrate, add the dextrin that is equivalent to its dried cream amount 30%, the dissolving mixing, obtaining medicinal extract 12000mL (50 ℃ time survey its density be 1.15g/mL), is that 155～165 ℃, air outlet temperature are that 100 ℃, pressure are to carry out spray drying under the 1bar condition promptly to get 9.27Kg tawny dried powder at intake air temperature;
(4) preparation of inclusion compound: get step (1) gained volatile oil and dilute with absolute ethyl alcohol in 1: 1 ratio, splash in the beta-schardinger dextrin-saturated solution of 6 times of amount volatile oil weight, stirred 2 hours in 50 ℃, stirring rate is 200rpm, left standstill 24 hours, suction filtration, the control temperature promptly gets 1.39Kg white powder inclusion compound carrying out vacuum drying below 60 ℃, and inclusion rate is 70.2%;
(5) granulate: the tawny dried powder of above-mentioned gained is mixed the dextrin mixing of 1.5 times of amounts of back general assembly (TW) with volatile oil clathrate compound and both, 95% ethanol of adding 30% is wetting, and the system softwood sieves, the 40 ℃ of heat-wind circulate dryings of particle that sieve, whole grain;
(6) get above-mentioned particle 2g,, filter with 20ml alcohol reflux 30min, extract evaporate to dryness, residue with methanol constant volume to 1ml, as negative control product solution.
4, according to thin-layered chromatography, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with 0.5% carboxymethylcellulose sodium solution, with volume ratio is that normal butyl alcohol-glacial acetic acid-waterborne layer of 8: 2: 2 is a developping agent, is 58% with the sulfuric acid controlled humidity, presaturation 30min, launch about 8cm, take out, dry, launch about 8cm with same developping agent secondary again, take out, dry, spray is with the colour developing of 5% ninhydrin solution, and 105 ℃ are dried by the fire to clear spot, in test sample chromatogram and the reference substance chromatogram, the spot that shows same color, and negative control immaculate on the relevant position, as shown in figure 10.
The detection of grass-leaved sweetflag effective constituent:
1, get this product 5g, add water 20ml dissolving after, add petroleum ether extraction 2 times, each 30ml merges petroleum ether layer, water bath method, residue is settled to 1ml with ethyl acetate, as test sample liquid.
2, get grass-leaved sweetflag medicinal material fine powder 0.5g in addition, add sherwood oil 20ml ultrasonic Extraction 30min, filter, get the filtrate evaporate to dryness, residue is settled to 1ml with ethyl acetate, in contrast medicinal material solution.
3, the negative control solution that lacks grass-leaved sweetflag:
(1) extract: get Pogostemon cablin 2.9Kg, CO packs into
2In the supercritical extraction reactor, in 55 ℃ of pressure 28MPa, temperature extraction 2 hours down, collect volatile oil 198g, standby;
The dregs of a decoction and capsule of weeping forsythia 4.6Kg, Radix Isatidis 12.9Kg, root tuber of aromatic turmeric 2.5Kg, glutinous rehmannia 3.2Kg add 8 times of amount 80% ethanol, heating and refluxing extraction 2 times, and each 2 hours, emit alcohol extract through horizontal screw unloading filter centrifugal machine, merge alcohol extract twice, standby;
Get gypsum 5.7Kg, reed rhizome 6.1Kg meal, the water that adds 8 times of amounts extracts 2 times down at 80 ℃, and each 2 hours, emit the water extract through horizontal screw unloading filter centrifugal machine, merge the water extract twice, standby;
(2) concentrate: being 100 ℃ and 60 ℃, vacuum tightness in temperature respectively with water extract and alcohol extract is concentrating under reduced pressure under the-0.06MPa, merging, must about 11800mL (50 ℃ time survey its density be 1.12g/mL) concentrate, measure its paste-forming rate simultaneously, standby;
(3) drying: in concentrate, add the dextrin that is equivalent to its dried cream amount 30%, the dissolving mixing, obtaining medicinal extract 12000mL (50 ℃ time survey its density be 1.15g/mL), is that 155～165 ℃, air outlet temperature are that 100 ℃, pressure are to carry out spray drying under the 1bar condition promptly to get 9.27Kg tawny dried powder at intake air temperature;
(4) preparation of inclusion compound: get step (1) gained volatile oil and dilute with absolute ethyl alcohol in 1: 1 ratio, splash in the beta-schardinger dextrin-saturated solution of 6 times of amount volatile oil weight, stirred 2 hours in 50 ℃, stirring rate is 200rpm, left standstill 24 hours, suction filtration, the control temperature promptly gets 1.39Kg white powder inclusion compound carrying out vacuum drying below 60 ℃, and inclusion rate is 70.2%;
(5) granulate: the tawny dried powder of above-mentioned gained is mixed the dextrin mixing of 1.5 times of amounts of back general assembly (TW) with volatile oil clathrate compound and both, 95% ethanol of adding 30% is wetting, and the system softwood sieves, the 40 ℃ of heat-wind circulate dryings of particle that sieve, whole grain;
(6) get above-mentioned particle 10g, add water 20ml dissolving after, add petroleum ether extraction 2 times, each 30ml merges petroleum ether layer, water bath method, residue is settled to 1ml with ethyl acetate, as negative control product solution.
4, according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put in same silica G F respectively
254On the thin layer plate, with volume ratio is that sherwood oil (60～90 ℃)-ethyl acetate of 8: 2 is developping agent, launch, take out, dry, put under the uviol lamp (254nm) and inspect, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, and negative control is noiseless, as shown in figure 11.
The qualitative detection of alcohol in hundred autumns:
1, get this product 2.5g, add water 20ml dissolving, with ether 20ml extraction 3 times, ether layer evaporate to dryness, residue with methanol constant volume to 1ml, as test sample liquid.
2, get pure reference substance in hundred autumns in addition, add methyl alcohol and make every milliliter of solution that contains 0.5mg, product solution in contrast.
3, the negative control solution that lacks Pogostemon cablin:
(1) extract: get grass-leaved sweetflag 2.5Kg, CO packs into
2In the supercritical extraction reactor, in 55 ℃ of pressure 28MPa, temperature extraction 2 hours down, collect volatile oil 198g, standby;
The dregs of a decoction and capsule of weeping forsythia 4.6Kg, Radix Isatidis 12.9Kg, root tuber of aromatic turmeric 2.5Kg, glutinous rehmannia 3.2Kg add 8 times of amount 80% ethanol, heating and refluxing extraction 2 times, and each 2 hours, emit alcohol extract through horizontal screw unloading filter centrifugal machine, merge alcohol extract twice, standby;
Get gypsum 5.7Kg, reed rhizome 6.1Kg meal, the water that adds 8 times of amounts extracts 2 times down at 80 ℃, and each 2 hours, emit the water extract through horizontal screw unloading filter centrifugal machine, merge the water extract twice, standby;
(2) concentrate: being 100 ℃ and 60 ℃ of vacuum tightness in temperature respectively with water extract and alcohol extract is concentrating under reduced pressure under the-0.06MPa, merging, must about 11800ml (50 ℃ time survey its density be 1.12g/ml) concentrate, measure its paste-forming rate simultaneously, standby;
(3) drying: in concentrate, add the dextrin that is equivalent to its dried cream amount 30%, the dissolving mixing, obtaining medicinal extract 12000ml (50 ℃ time survey its density be 1.15g/ml), is that 155～165 ℃, air outlet temperature are that 100 ℃, pressure are to carry out spray drying under the 1bar condition promptly to get 9.27Kg tawny dried powder at intake air temperature;
(4) preparation of inclusion compound: get step (1) gained volatile oil and dilute with absolute ethyl alcohol in 1: 1 ratio, splash in the beta-schardinger dextrin-saturated solution of 6 times of amount volatile oil weight, stirred 2 hours in 50 ℃, stirring rate is 200rpm, left standstill 24 hours, suction filtration, the control temperature promptly gets 1.39Kg white powder inclusion compound carrying out vacuum drying below 60 ℃, and inclusion rate is 70.2%;
(5) granulate: the tawny dried powder of above-mentioned gained is mixed the dextrin mixing of 1.5 times of amounts of back general assembly (TW) with volatile oil clathrate compound and both, 95% ethanol of adding 30% is wetting, and the system softwood sieves, the 40 ℃ of heat-wind circulate dryings of particle that sieve, whole grain;
(6) get above-mentioned particle 2g, add water 20ml dissolving, with ether 20ml extraction 3 times, ether layer evaporate to dryness, residue with methanol constant volume to 1ml, as negative control product solution.
4, according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, drawing each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, is that cyclohexane-ethyl acetate of 10: 3 is developping agent with volume ratio, launches about 10cm.Take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to dry by the fire to the spot colour developing at 105 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, and negative control is noiseless, as shown in figure 12.
The detection of mangiferin and forsythin:
1, the preparation of reference substance solution: precision takes by weighing mangiferin, forsythin is an amount of, puts in the 50ml volumetric flask, adds methyl alcohol to scale, makes mangiferin concentration 0.1172mg/ml, forsythin concentration is 0.0480mg/ml solution, in contrast product solution.
The preparation of need testing solution: get this product 2g, precision is weighed, and puts in the tool plug Erlenmeyer flask, add methyl alcohol 50ml, ultrasonic 40min filters, residue adds water 10ml dissolving, with water saturation extracting n-butyl alcohol 4 times (20ml * 4), merge n-butanol layer, water bath method, residue 50% dissolve with methanol, constant volume shakes up in the 10ml measuring bottle, and is standby.
2, be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, be that 20%: 80% the acetonitrile and the mixing material of the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% phosphoric acid are the moving phase wash-out with volume ratio then, flow velocity is 1ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample;
3, result: the chromatogram of reference substance and test sample is similar to example 1, and the average retention time of mangiferin is 15.7min, and content is 0.602mg/g, and the average retention time of forsythin is 32.6min, and content is respectively 0.119mg/g.
Example 4
Sarsasapogenin, arginine, alcohol and the thin-layer chromatography detection of grass-leaved sweetflag medicinal material and the high performance liquid chromatography detection of mangiferin and forsythin in hundred autumns in the antiviral oral liquor.
1, the discriminating of sarsasapogenin:
Get commercially available antiviral oral liquor 20ml, add evaporate to dryness behind the hydrochloric acid 1ml reflux 1h, add water 15ml dissolving, add chloroform extraction 3 times, each 20ml, chloroform extraction liquid discards alkali lye with 0.5%NaOH solution 20ml washing, again with distilled water 20ml washing, discard water liquid, chloroform solution evaporate to dryness, residue add methanol constant volume to 1ml, as need testing solution.Other gets the sarsasapogenin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that toluene-acetone of 9: 1 is developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
Arginic discriminating:
Get commercially available antiviral oral liquor 10ml, evaporate to dryness, residue with methanol constant volume to 1ml, as test sample liquid.Other gets the arginine reference substance and adds methyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.According to thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with 0.5% carboxymethylcellulose sodium solution, with volume ratio is that normal butyl alcohol-glacial acetic acid-waterborne layer of 8: 2: 2 is a developping agent, with the sulfuric acid controlled humidity is 58%, presaturation 30min launches about 8cm, takes out, dry, launch about 8cm with same developping agent secondary again, take out, dry, spray develops the color with 5% ninhydrin solution, 105 ℃ are dried by the fire to spot colour developing clearly, in test sample chromatogram and the reference substance chromatogram, show the spot of same color.
Discriminating in the grass-leaved sweetflag medicinal material:
Get commercially available antiviral oral liquor 20ml, add petroleum ether extraction 2 times, each 30ml merges petroleum ether layer, water bath method, and residue is settled to 1ml with ethyl acetate, as test sample liquid.Other gets grass-leaved sweetflag control medicinal material fine powder 0.5g, adds sherwood oil 20ml ultrasonic Extraction 30min, filters, and gets the filtrate evaporate to dryness, and residue is settled to 1ml with ethyl acetate, in contrast medicinal material solution.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put in same silica G F respectively
254On the thin layer plate, be that sherwood oil (60～90 ℃)-ethyl acetate of 8: 2 is developping agent, launch, take out, dry, put under the uviol lamp (254nm) and inspect with volume ratio, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
The discriminating of alcohol in hundred autumns:
Get commercially available antiviral oral liquor 20ml, with ether 20ml extraction 3 times, ether layer evaporate to dryness, residue with methanol constant volume to 1ml, as test sample liquid.Other gets pure reference substance in hundred autumns, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, be that cyclohexane-ethyl acetate of 10: 3 is developping agent with volume ratio, launch about 10cm.Take out, dry, spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, and it is clear to dry by the fire to the spot colour developing at 105 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
The assay of mangiferin and forsythin
1, the preparation of reference substance solution: precision takes by weighing mangiferin, forsythin is an amount of, puts in the 50ml volumetric flask, adds methyl alcohol to scale, makes mangiferin concentration 0.15mg/ml, forsythin concentration is 0.04mg/ml solution, in contrast product solution.
The preparation of need testing solution: get commercially available antiviral oral liquor 20ml, add water-saturated n-butanol extraction 2 times, each 30ml discards water layer, and normal butyl alcohol liquid is put evaporate to dryness in the water-bath, and residue adds 50% dissolve with methanol, is settled to the 10ml scale, as need testing solution.
2, be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, be that 30%: 70% the acetonitrile and the mixing material of the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% phosphoric acid are the moving phase wash-out with volume ratio then, flow velocity is 0.8ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample;
3, result: the chromatogram of reference substance and test sample is similar to example 2, and the average retention time of mangiferin is 9.3min, and content is 0.598mg/g, and the average retention time of forsythin is 26.6min, and content is 0.118mg/g.
Claims (4)
1, the method for quality control of antivirus compound formulation, this method is made up of following steps:
(1) preparation of reference substance solution: mangiferin and forsythin mix makes the reference substance solution that contains mangiferin 0.08～0.15mg/ml and forsythin 0.04～0.08mg/ml with dissolve with methanol;
The preparation of need testing solution: when antivirus compound formulation is solid pharmaceutical preparation, add 20～30 times of methyl alcohol, ultrasonic Extraction 20～40min, filter, filter residue adds 5～10 times of water-soluble separating, with 10～15 times of water-saturated n-butanol extractions 2～3 times, merge n-butanol layer, water bath method, the gained dry becomes every ml soln to be equivalent to the former preparation of 0.2～0.3g with 50% dissolve with methanol, shakes up; When antivirus compound formulation is liquid preparation, with 10～15 times of water-saturated n-butanol extractions 2～3 times, merge n-butanol layer, water bath method, the gained dry becomes every ml soln to be equivalent to 3～5g dry with 50% dissolve with methanol, shakes up;
(2) be the stationary phase chromatography column with 4.6mm * 250mm, 5 μ m carbon, 18 bonding phase silicagel columns; At first accurate reference substance solution 10 μ l and the need testing solution 5 μ l of drawing, inject high performance liquid chromatograph respectively, mixing material with acetonitrile and the potassium dihydrogen phosphate of the 0.05mol/L that contains 0.1% phosphoric acid is the moving phase wash-out then, flow velocity is 0.8～1.0ml/min, theoretical cam curve is calculated by mangiferin should be not less than 5000, calculates by forsythin and should be not less than 6000; Last is the ultraviolet light detection eluate of 277nm with the wavelength, and draws the chromatogram of reference substance and the chromatogram of test sample; In the described moving phase, the content of acetonitrile is 10～30%, and the content of potassium dihydrogen phosphate that contains the 0.05mol/L of 0.1% phosphoric acid is 70～90%;
(3) relatively with the chromatogram of the chromatogram of test sample and reference substance solution: with the characteristic peak that first peak retention time is identical in the reference substance chromatogram promptly be the characteristic peak of mangiferin, the characteristic peak identical with second peak retention time in the reference substance chromatogram promptly is the characteristic peak of forsythin; And press external standard method according to the peak area at these two peaks respectively and calculate mangiferin and forsythin content;
The degree of various liquid is volume percent content in the above-mentioned steps.
2, method according to claim 1, the content that it is characterized in that mangiferin in the described reference substance solution are that the content of 0.1172mg/ml and forsythin is 0.0480mg/ml.
3, method according to claim 1, the preparation method who it is characterized in that described need testing solution is: get antiviral solid pharmaceutical preparation and add 25 times of dissolve with methanol, ultrasonic Extraction 40min, filter, filter residue adds 5 times of water-soluble separating, and with 10 times of water-saturated n-butanol extractions 4 times, merges n-butanol layer, water bath method, gained dry become every ml soln to be equivalent to the former preparation of 0.2g with 50% dissolve with methanol to shake up.
4, according to claim 1,2 or 3 described methods, it is characterized in that the content of acetonitrile in the described moving phase is 20%, the content of potassium dihydrogen phosphate that contains the 0.05mol/L of 0.1% phosphoric acid is 80%.
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| CN105362283A (en) * | 2014-08-07 | 2016-03-02 | 富力 | Applications of phillyrin/phillygenin composition in preparation of drugs or health products for relieving or/and treatment of viral diseases |
| CN105362283B (en) * | 2014-08-07 | 2018-07-27 | 富力 | Forsythin/phillygenol composition is preparing the application in alleviating or/and treating the drug or health products of viral disease |
| CN104597198A (en) * | 2015-02-02 | 2015-05-06 | 上海中华药业有限公司 | Method for isolating and identifying compound through fluorescent quenching |
| CN106353446A (en) * | 2016-08-12 | 2017-01-25 | 上海黄海制药有限责任公司 | Identification and content measuring method of climacteric-syndrome-soothing granules |
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