CN106442843A - Quality check method of children's granules for clearing heat from throat - Google Patents
Quality check method of children's granules for clearing heat from throat Download PDFInfo
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- CN106442843A CN106442843A CN201610501400.XA CN201610501400A CN106442843A CN 106442843 A CN106442843 A CN 106442843A CN 201610501400 A CN201610501400 A CN 201610501400A CN 106442843 A CN106442843 A CN 106442843A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a quality check method of children's granules for clearing heat from throat. 1000g of the children's granules are prepared from the following crude drugs by quantity parts: 390.6g of mulberry leaves, 390.6g of radix isatidis, 260.4g of rhizoma belamcandae, 260.4g of radix scrophulariae, 260.4g of liquorice, 2.5mL of dementholized peppermint oil and 433-550g of soluble starch. The quality check method of the children's granules for clearing heat from throat comprises the following items: character: the product is the granules, is claybank to tawny and has fragrant smell and slightly sweet and slightly bitter taste; identification: a thin layer chromatography is adopted for identification; check: the check complies with the following various relevant provisions for the granules (Chinese Pharmacopoeia (General Principles 0104)); determination of extract: the determination comprises content determination of dilute ethanol extract; content determination: the content determination comprises content determination of tectoridin. The method is taken as a standard for checking the children's granules for clearing heat from throat, so that the stability of the preparation can be excellently ensured, thereby ensuring the curative effect of the preparation.
Description
Technical field
The present invention relates to a kind of drug quality inspection method is and in particular to a kind of be used for acute pharyngitises and upper respiratory tract infection
The little Qingyan Granules detection method for quality of the treatment having sore throat causing.
Background technology
A kind of medicine of the treatment having sore throat causing for acute pharyngitises and upper respiratory tract infection of little Qingyan Granules, tool
There are nourishing YIN and moistening the lung, the effect of detumescence sore-throat relieving.It is mainly used in the treatment having sore throat that acute pharyngitises and upper respiratory tract infection cause, right
Inside and outside all evil invade throat, such as diseases caused by exogenous pathogenic factor, wind heat cold-damp, pathogenic warmth seasonal noxious pathogen, internal injury diet, pungent decoct, inside accumulate expectorant heat etc. and fight stagnation of QI
Blood, pent up into poison, the throat illness of initiation, symptoms include red swelling and pain of throat, dysphagia, extensively fester, breathing is unfavorable, and itching throat is coughed
Cough, hoarseness, the disease such as fever with chills.
Little Qingyan Granules are made up of the crude drug of following quantity number：Folium Mori 390.6 g, Radix Isatidis 390.6 g, Rhizoma Belamcandae
260.4 g, Radix Scrophulariae 260.4 g, Radix Glycyrrhizae 260.4 g, Oleum menthae 2.5 mL, soluble starch 433 ~ 550 g；Make 1000
g.
Preparation method is：By prescription five tastes decoction pieces, plus the water of 10 times amount（Decoct for the first time and add water 12.5 times）, soak 1 hour, decoct
Boil 3 times, 0.5 hour every time.Collecting decoction, filtration.It is 1.35 ~ 1.40 that filtrate normal pressure is concentrated into relative density（60 ℃）Thick
Extractum, as 80 DEG C of drying under reduced pressure.Dry cream is ground into fine powder（Cross 60 mesh sieves）Mix homogeneously with soluble starch 433 ~ 550 g,
95 % appropriate amount of ethanol soft materials, 14 mesh sieves are pelletized, 60 DEG C of dryings, and 12 mesh sieve granulate spray into Oleum menthae 2.5 mL, mix, system
Become 1000 g granules, subpackage, obtain final product.
Currently without the quality determining method being suitable for little Qingyan Granules, in order to ensure stability and the curative effect of preparation, need
Quality testing to this medicine is studied.
Content of the invention
The technical problem to be solved provides one kind to be used for little Qingyan Granules detection method for quality, little to ensure this
The stability of Qingyan Granules and curative effect.
Technical scheme is as follows：
Little Qingyan Granules detection method for quality, this little Qingyan Granules 1000g is made up of the crude drug of following quantity number：Folium Mori
390.6 g, Radix Isatidis 390.6 g, Rhizoma Belamcandae 260.4 g, Radix Scrophulariae 260.4 g, Radix Glycyrrhizae 260.4 g, Oleum menthae 2.5 mL, can
Soluble starch 433 ~ 550 g；
Its quality determining method includes following items：
Character：This product is granule, brown color to sepia, gas fragrance, taste micro-sweet, slightly bitter；
Differentiate：Differentiated using thin layer chromatography；
Check：Meet relevant every regulation under granule item（《Chinese Pharmacopoeia》（General rule 0104））；
Determination of extractives：Measure including Diluted Alcohol extract content；
Assay：Assay including belamcandin.
Wherein, differentiate to include：
（1）Take invention formulation to prepare need testing solution, take Radix Isatidis control medicinal material to prepare control medicinal material solution, according to thin layer chromatography
Method is tested, and draws need testing solution and control medicinal material solution and puts respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-
Water is developing solvent, launches, and takes out, hot blast drying, and spray, with ninhydrin solution, is heated to spot development clear；In test sample chromatograph,
On the position corresponding with control medicinal material chromatograph, the speckle of aobvious same color；
（2）Take invention formulation to prepare need testing solution, take Rhizoma Belamcandae control medicinal material to prepare control medicinal material solution, according to thin layer chromatography
Test, draws need testing solution and control medicinal material solution is put respectively in same silica gel G F254On lamellae, with chloroform-first
Alcohol-glacial acetic acid is developing solvent, launches, and takes out, dries, put and inspect under ultra-violet lamp；In test sample chromatograph, with comparison medicine wood color
Compose on corresponding position, the fluorescence spot of aobvious same color.
Wherein, determination of extractives is carried out in accordance with the following steps：Take test sample accurately weighed, in conical flask, add Diluted Alcohol,
Close plug, weighed weight, standing, after 1 hour, connects reflux condensing tube, is heated to seething with excitement, and keeps micro-boiling 1 hour；After letting cool, take
Inferior pyramidal bottle, close plug, more weighed weight, supply the weight of less loss, shake up with Diluted Alcohol, and with filter filtration is dried, precision measures
Subsequent filtrate, puts and is dried to the evaporating dish of constant weight, after being evaporated, is dried 3 hours in water-bath, in dislocation exsiccator, cools down 30
Minute, accurately weighed weight rapidly.
Wherein, the content of belamcandin is according to high effective liquid chromatography for measuring；With octadecyl silane as filler；With
Acetonitrile -0.1 % phosphate aqueous solution is mobile phase, gradient elution；Flow velocity 1 mL min-1；25 DEG C of column temperature；Detection wavelength 265
nm.
By above method, the quality standard of little Qingyan Granules can be made, thus realizing the matter to little Qingyan Granules
Amount detection, the experiment proved that, in this way for the little Qingyan Granules of standard detection, can be very good to ensure the stability of preparation, from
And ensure its curative effect.
Brief description
Fig. 1 is that in little Qingyan Granules, Radix Isatidis thin layer differentiates 1. negative test samples（Scarce chromatogram of Radix Isatidis）；2. Radix Isatidis pair
According to medical material；3rd, 4,5 little Qingyan Granules test sample（Lot number：20110201,20110202,20110203）；
Fig. 2 is that in little Qingyan Granules, Rhizoma Belamcandae thin layer differentiates 1. negative test samples（Scarce Rhizoma Belamcandae medical material）；2. Rhizoma Belamcandae control medicinal material；3、4、
5 little Qingyan Granules test samples（Lot number：20110201,20110202,20110203）；
Fig. 3 is that in little Qingyan Granules, Folium Mori thin layer differentiates 1. negative test samples（Scarce Folium Mori medical material）；2. Folium Mori control medicinal material；3、4、
5 little Qingyan Granules test samples（Lot number：20110201,20110202,20110203）；
Fig. 4 is that in little Qingyan Granules, Radix Scrophulariae thin layer differentiates 1. negative test samples（Scarce Radix Scrophulariae medical material）；2. Radix Scrophulariae control medicinal material；3、4、
5 little Qingyan Granules test samples（Lot number：20110201,20110202,20110203）；
Fig. 5 is that in little Qingyan Granules, Radix Glycyrrhizae thin layer differentiates 1. negative test samples（Scarce licorice medicinal materials）；2. Radix Glycyrrhizae control medicinal material；3、4、
5 little Qingyan Granules test samples（Lot number：20110201,20110202,20110203）；
Fig. 6 is that in little Qingyan Granules, Oleum menthae thin layer differentiates 1. negative test samples（Scarce Oleum menthae）；2. Oleum menthae；3、4、
5 little Qingyan Granules test samples（Lot number：20110201,20110202,20110203）；
Fig. 7 is high-efficient liquid phase chromatogram:A tectoridin reference substance；
Fig. 8 is high-efficient liquid phase chromatogram:B granule extracting solution；
Fig. 9 is negative extractum contrast solution chromatogram；
Figure 10 is negative granules solution chromatogram；
Figure 11 is that result of the test investigated by belamcandin linear relationship；
Figure 12 is the precision test HPLC collection of illustrative plates of belamcandin assay in granule；
Figure 13 is the stability test HPLC collection of illustrative plates of belamcandin assay in granule；
Figure 14 is the replica test HPLC collection of illustrative plates of belamcandin assay in granule；
Figure 15 is the recovery test HPLC collection of illustrative plates top half of belamcandin assay in granule；
Figure 16 is the recovery test HPLC collection of illustrative plates the latter half of belamcandin assay in granule；
Figure 17 is granule chromatogram (358 nm)；
Figure 18 is control substance of Rutin chromatogram；
Figure 19 is harpagoside reference substance superposition granule sample chromatogram figure.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not subject to
Any restriction of specific embodiment, but be defined in the claims.
Embodiment 1：
The little Qingyan Granules of prescription, are made up of following crude drug：Folium Mori 390.6 g, Radix Isatidis 390.6 g, Rhizoma Belamcandae 260.4 g
, Radix Scrophulariae 260.4 g, Radix Glycyrrhizae 260.4 g, Oleum menthae 2.5 mL, soluble starch 433 ~ 550g, make 1000 g.
Preparation method is by prescription five tastes decoction pieces, plus the water of 10 times amount（Decoct for the first time and add water 12.5 times）, soak 1 hour, decoct 3
Secondary, 0.5 hour every time.Collecting decoction, filtration.It is 1.35 ~ 1.40 that filtrate normal pressure is concentrated into relative density（60 ℃）Thick leaching
Cream, as 80 DEG C of drying under reduced pressure.Dry cream is ground into fine powder（Cross 60 mesh sieves）Mix homogeneously with soluble starch 433 ~ 550 g, 95
% appropriate amount of ethanol soft material, 14 mesh sieves are pelletized, 60 DEG C of dryings, and 12 mesh sieve granulate spray into Oleum menthae 2.5 mL, mix, make
1000 g granules, subpackage, obtain final product.
Character this product is granule, brown color to sepia, gas fragrance, taste micro-sweet, slightly bitter.
Differentiate
（1）Take this preparation 0.5 g, plus Diluted Alcohol 20 mL, it is heated to reflux 20 minutes, filtration, filtrate is evaporated, and residue adds Diluted Alcohol 1
ML makes dissolving, as need testing solution.Separately take Radix Isatidis control medicinal material 0.5 g, be made in the same way of control medicinal material solution.According to《China
Pharmacopeia》Thin layer chromatography（General rule 0502）Test, draws each 5 ~ 10 μ L of above two solution, puts respectively in same silica gel G thin layer
On plate, with n-butyl alcohol-glacial acetic acid-water（19:5:5）For developing solvent, launch, take out, hot blast drying, spray with ninhydrin solution,
105 DEG C to be heated to spot development clear.In test sample chromatograph, on the position corresponding with control medicinal material chromatograph, show identical face
The speckle of color.
（2）Take this preparation 1 g, plus methanol 10 mL, it is heated to reflux 30 minutes, filtration, filtrate is concentrated into 1.5 mL, as
Need testing solution.Separately take Rhizoma Belamcandae control medicinal material 1 g, be made in the same way of control medicinal material solution.According to《Chinese Pharmacopoeia》Thin layer chromatography（Logical
Then 0502）Test, draws each 5 μ L of above two solution, puts respectively in same silica gel G F254On lamellae, with chloroform-first
Alcohol-glacial acetic acid（8:2:0.1）For developing solvent, launch, take out, dry, put ultra-violet lamp（254 nm）Under inspect.Test sample chromatograph
In, on position corresponding with reference substance medical material chromatograph, the fluorescence spot of aobvious same color.
Inspection should meet relevant every regulation under granule item（《Chinese Pharmacopoeia》（General rule 0104））.
Determination of extractives
Take test sample 2 g, accurately weighed, put in 100 mL conical flasks, accurate plus Diluted Alcohol 50 mL, close plug, weighed weight, quiet
After putting 1 hour, connect reflux condensing tube, be heated to seething with excitement, and keep micro-boiling 1 hour.After letting cool, take off conical flask, close plug, then
Weighed weight, supplies the weight of less loss, shakes up with Diluted Alcohol, and with filter filtration is dried, precision measures subsequent filtrate 25 mL, puts
It is dried to the evaporating dish of constant weight, after water-bath is evaporated, in 105 DEG C of dryings 3 hours, in dislocation exsiccator, cool down 30 points
Clock, accurately weighed weight rapidly.This product extract content containing Diluted Alcohol is not less than 43 %.
Assay
Belamcandin shines《Chinese Pharmacopoeia》High performance liquid chromatography（General rule 0512）Measure.
Chromatographic condition and system suitability are with octadecyl silane as filler（Yi Lite Hypersil BDS
C18（250 mm × 4.6 mm, 5 μm））；With acetonitrile -0.1 % phosphate aqueous solution as mobile phase（Table 1）, gradient elution；Flow velocity 1
mL·min-1；25 DEG C of column temperature；Detection wavelength 265 nm；Number of theoretical plate is calculated by belamcandin and should be not less than 3000.
The preparation precision of reference substance solution weighs tectoridin reference substance 10.56 mg being dried to constant-quality, plus 50% first
Alcohol dissolves in right amount, is settled to 50 mL, is configured to 0.2112 mg mL-1Reference substance solution, obtain final product.
The preparation precision of need testing solution weighs little Qingyan Granules 1 g in conical flask, plus 25 mL 50 % methanol shakes up,
Stand 30 min, weighed weight, supersound extraction 60 min, mend weight, filtration.
Algoscopy：Accurate absorption reference substance solution and each 5 μ L of need testing solution, inject chromatograph of liquid respectively, measure,
Obtain final product.
This product contains belamcandin and must not be less than 0.22%.
Indication nourishing YIN and moistening the lung, the effect of detumescence sore-throat relieving.It is mainly used in the pharynx that acute pharyngitises and upper respiratory tract infection cause
The treatment of larynx pain, invades throat to inside and outside all heresies, such as diseases caused by exogenous pathogenic factor, wind heat cold-damp, pathogenic warmth seasonal noxious pathogen, and internal injury diet is pungent pan-fried,
Inside accumulate expectorant heat etc. fight stagnation of QI blood, pent up into poison, the throat illness of initiation, symptoms include red swelling and pain of throat, dysphagia, extensively burst
Rotten, breathing is unfavorable, and itching throat is coughed, hoarseness, the disease such as fever with chills.
Embodiment 2：
1. differentiate
This product is made up of the Chinese medicine of the five flavours material such as Folium Mori, Radix Isatidis, extracted, refined form, composition is more complicated.We are in each taste
The composition of medical material has carried out the experimental study of discrimination method, establishes the thin-layer identification method of Radix Isatidis, Radix Scrophulariae, Rhizoma Belamcandae.
（1）The discriminating of Radix Isatidis：Take this preparation 0.5 g, plus Diluted Alcohol 20 mL, it is heated to reflux 20 minutes, filtration, filtrate
It is evaporated, residue adds Diluted Alcohol 1 mL makes dissolving, as need testing solution.Take negative simulation preparation 0.5 g of scarce Radix Isatidis, same to method
Prepare negative control solution.Separately take Radix Isatidis control medicinal material 0.5 g, be made in the same way of control medicinal material solution.According to《Chinese Pharmacopoeia》Thin
Layer chromatography（General rule 0502）Test, draws each 5 ~ 10 μ L of above-mentioned three kinds of solution, puts respectively on same silica gel g thin-layer plate, with
N-butyl alcohol-glacial acetic acid-water（19:5:5）For developing solvent, launch, take out, hot blast drying, spray, with ninhydrin solution, adds at 105 DEG C
Heat is clear to spot development.In test sample chromatograph, on the position corresponding with control medicinal material chromatograph, the speckle of aobvious same color
Point, negative control is noiseless.Verify, the method is reproducible through test agent in 3 batches, therefore taken in quality standard draft text
（See Fig. 1）.
（2）The discriminating of Rhizoma Belamcandae：Take this preparation 1 g, plus methanol 10 mL, it is heated to reflux 30 minutes, filtration, filtrate is concentrated into
1.5 mL, as need testing solution.Take negative simulation preparation 1 g of scarce Rhizoma Belamcandae, prepare negative control solution with method.Separately take Rhizoma Belamcandae
Control medicinal material 1 g, is made in the same way of control medicinal material solution.According to《Chinese Pharmacopoeia》Thin layer chromatography（General rule 0502）Test, in absorption
State each 5 μ L of two kinds of solution, put respectively in same silica gel G F254On lamellae, with chloroform-methanol-glacial acetic acid（8:2:0.1）
For developing solvent, launch, take out, dry, put ultra-violet lamp（254 nm）Under inspect.In test sample chromatograph, with reference substance medical material
On the corresponding position of chromatograph, the fluorescence spot of aobvious same color, negative control is noiseless.Verify through test agent in 3 batches, the method
Reproducible, therefore taken in quality standard draft text（See Fig. 2）.
（3）The discriminating of Folium Mori：Take this preparation 2 g, plus petroleum ether（60~90℃）30 mL, are heated to reflux 30 minutes, discard
Petroleum ether liquid, medicinal residues volatilize, plus ethanol 30 mL, supersound process 20 minutes, filtration, and filtrate is evaporated, and residue heating water 10 mL puts
It is stirred to dissolve in 60 DEG C of water-baths, filtration, filtrate is evaporated, and residue adds methanol 1 mL makes dissolving, as need testing solution.Take scarce Mulberry
Negative simulation preparation 2 g of leaf, prepares negative control solution with method.Separately take Folium Mori control medicinal material 2 g, be made in the same way of control medicinal material
Solution.According to《Chinese Pharmacopoeia》Thin layer chromatography（General rule 0502）Test, draws each 5 μ L of above-mentioned three kinds of solution, puts in same respectively
On silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid（5:2:1）Upper solution be developing solvent, put and use developing solvent presaturation
In the expansion cylinder of 10 minutes, launch, about to 8 cm, to take out, dry, put ultra-violet lamp（365 nm）Under inspect.Because the method repeats
Property is not good, wouldn't list quality standard draft in（See Fig. 3）.
（4）The discriminating of Radix Scrophulariae：Take this preparation 2 g, plus methanol 25 mL, soak 1 hour, be heated to reflux 30 minutes, filtration,
Filtrate is evaporated, and residue 25 mL that add water make dissolving, is extracted 2 times with the shaking of water saturated n-butyl alcohol, 30 mL every time, merges n-butyl alcohol
Liquid, is evaporated, and residue adds methanol 5 mL makes dissolving, as need testing solution.Take negative simulation preparation 2 g of scarce Radix Scrophulariae, same to legal system
Standby negative control solution.Separately take Radix Scrophulariae control medicinal material 2 g, be made in the same way of control medicinal material solution.According to《Chinese Pharmacopoeia》Thin layer chromatography
Method（General rule 0502）Test, draws each 5 μ L of above-mentioned three kinds of solution, puts respectively on same silica gel g thin-layer plate, with chloroform-
Methanol-water（12:4:1）Lower floor's solution be developing solvent, put with the developing solvent presaturation expansion cylinder of 15 minutes, launch, take out,
Dry, with 5 % vanillin-sulfuric acid solution, it is clear that hot blast is blown to spot development for spray.Because the method repeatability is not good, matter wouldn't be listed in
Amount draft standard（See Fig. 4）.
（5）The discriminating of Radix Glycyrrhizae：Take this preparation 5 g, plus n-butyl alcohol 25 mL, it is heated to reflux 30 minutes, filtration, filtrate is evaporated,
Residue adds methanol 5 mL makes dissolving, as need testing solution.Take negative simulation preparation 5 g of scarce Radix Glycyrrhizae, negative right with method preparation
According to solution.Another extracting liquorice control medicinal material 5 g, is made in the same way of control medicinal material solution.According to《Chinese Pharmacopoeia》Thin layer chromatography（General rule
0502）Test, draws each 5 ~ 10 μ L of above-mentioned three kinds of solution, puts respectively on same silica gel g thin-layer plate, with ethyl acetate-first
Acid-glacial acetic acid-water（15:1:1:2）For developing solvent, launch, take out, dry, spray, with 10 % ethanol solution of sulfuric acid, adds at 105 DEG C
Heat is clear to spot development, puts ultra-violet lamp（365 nm）Under inspect.Because the method repeatability is not good, quality standard wouldn't be listed in
Draft（See Fig. 5）.
（6）The discriminating of Oleum menthae：Take this preparation 1 g, plus dehydrated alcohol 5 mL makes dissolving, as need testing solution.Take
Negative simulation preparation 1 g of scarce Oleum menthae, prepares negative control solution with method.Separately take Oleum menthae 0.1 g, be made in the same way of
Control medicinal material solution.According to《Chinese Pharmacopoeia》Thin layer chromatography（General rule 0502）Test, draws each 5 ~ 10 μ L of above-mentioned three kinds of solution,
Put respectively in same silica gel G F254On lamellae, with toluene-ethyl acetate-（19:1）For developing solvent, launch, take out, dry, spray
With 10% ethanol solution of sulfuric acid, it is heated to spot development clearly at 105 DEG C, puts ultra-violet lamp（365 nm）Under inspect.Because of the method
Repeatability is not good, wouldn't list quality standard draft in（See Fig. 6）.
Determination of extractives
Exist with reference to the little Qingyan Granules composition prepared slices of Chinese crude drugs《Chinese Pharmacopoeia》In related medical material extractum solvent select, in conjunction with little
Properties of chemical composition contained by Qingyan Granules composition medical material（Predominantly steroid, triterpeness, flavonoid, indoles alkaloid etc.）,
Consider the alcohol extract solvent selecting Diluted Alcohol as little Qingyan Granules.
Assay method
Take test sample 2 g, accurately weighed, put in 100 mL conical flasks, accurate plus Diluted Alcohol 50 mL, close plug, weighed weight, quiet
After putting 1 hour, connect reflux condensing tube, be heated to seething with excitement, and keep micro-boiling 1 hour.After letting cool, take off conical flask, close plug, then
Weighed weight, supplies the weight of less loss, shakes up with Diluted Alcohol, and with filter filtration is dried, precision measures subsequent filtrate 25 mL, puts
It is dried to the evaporating dish of constant weight, after water-bath is evaporated, in 105 DEG C of dryings 3 hours, in dislocation exsiccator, cool down 30 points
Clock, accurately weighed weight rapidly.Computing formula is as follows：
2.2 by little 8 batches of Qingyan Granules sample carry out ethanol-soluble extractiveses mensure, as shown in the table, result table
Bright：Its ethanol-soluble extractiveses average content is 54.0%.Float downward 20% with its ethanol-soluble extractiveses average content as lower bound, quality is examined
Survey should require the content of the ethanol-soluble extractiveses of little Qingyan Granules to be not less than 43.2%.
Assay
3.1 material
3.1.1 instrument
Agilent 1100 type high performance liquid chromatograph（Agilent company of the U.S.）, Agilent liquid chromatographic system chemical work
Stand；AG135 type electronic balance（Ten thousand/, Mettler-Toledo company of Switzerland）；CSF-1B ultrasonic washing instrument（Shanghai
Ultrasonic instrument factory）；DZ-2BC II vacuum desiccator.
Reagent
Methanol（Tianjin Kermel Chemical Reagent Co., Ltd., chromatographically pure）；Phosphoric acid（Zunyi Mormal college chemical reagent factory）；Pure
Water purification（Hangzhou Wahaha Group Co., Ltd）；It is pure that remaining reagent is analysis.
Reference substance
Tectoridin reference substance (Products in China examines and determine institute, lot number 111632-200501)；Rutin (examine and determine by Products in China
Institute, lot number：100080-200707).
Formulation samples
Little Qingyan Granules are produced due to No. 2 Affiliated Hospital of Guiyang College of Traditional Chinese Medicine Pharmaceutical Department Drug Manufacturing Room.
Method and result
3.2.1 chromatographic condition
Yi Lite Hypersil BDS C18（250 mm × 4.6 mm, 5 μm）；Mobile phase acetonitrile -0.1 % phosphate aqueous solution,
Gradient elution；25 DEG C of column temperature；Detection wavelength 265 nm；Sample size 5 L.Result shows, can reach with this understanding preferably
Separating effect, separating degree is more than 1.5, and tailing factor is 0.95.Eluent gradient is shown in Table 1, and experimental result is shown in Fig. 7, Fig. 8, Fig. 9 and
Figure 10.
Prepared by reference substance solution
Precision weighs dry tectoridin reference substance 10.56 mg to constant-quality, plus 50 % methanol dissolve in right amount, are settled to 50
ML, is configured to the reference substance solution of 0.2112 mg/mL, obtains final product.
The preparation of test sample
Precision weighs extract powder and each 1g of Rhizoma Belamcandae decoction pieces in conical flask, plus 25 mL 50 % methanol shakes up, and stands 30 min,
Weighed weight, supersound extraction 60 min, mends weight, filtration.
Blank assay
Take negative sample 1 g of scarce Rhizoma Belamcandae, prepare negative control solution by under " 3.2.3 " item, by content assaying method test, cloudy
In the HPLC collection of illustrative plates of property sample, do not present and tectoridin reference substance retention time identical chromatographic peak, show that this law specificity is good
Good.
Belamcandin measures
Accurate absorption reference substance solution and each 5 μ L of need testing solution, inject chromatograph of liquid respectively, measure, and are pressed outer with peak area
Mark method calculates.
Methodological study
3.2.6.1 linear relationship is investigated
Accurate absorption tectoridin reference substance solution, prepares series concentration reference substance solution.I.e. 42.24,84.48,126.72,
168.96,211.20ug mL-1, measure peak area according to chromatographic condition under " 3.2.1 " item, repeat sample introduction 3 times respectively, with peak area
Meansigma methodss（A）To sample size（X）Make linear regression, obtaining regression equation is：Y=4147.19044X+ 5.142816, r=
0.99989.Result shows, in 42.24 ~ 211.20 μ g range and peak area is in good linear relation to belamcandin sample size, sees
Table 3, Figure 11.
Precision test
Accurate absorption same concentration tectoridin reference substance solution 5 L, according to chromatographic condition under " 3.2.3 " item, injects efficient liquid chromatography
Instrument, repeats sample introduction 6 times, measures peak area.RSD is 0.49%, and result shows, instrument precision is good, is shown in Table 4, Figure 12.
Stability test
Take same extract powder about 1 g, accurately weighed, according to legal system available test sample solution below " 3.2.3 " item, according under " 3.2.1 " item
Chromatographic condition, measures respectively at 0,2,4,6,8,10,12 h sample introduction, each sample introduction 5 L, calculates the RSD of belamcandin peak area
For 0.36%, result shows, little Qingyan Granules agent need testing solution is stable in 12h, is shown in Table 5, Figure 13.
Replica test
Take 6 parts of granule, every part of about 1 g, accurately weighed, legal system available test sample solution below photograph " 3.2.3 " item, shine " 3.2.1 "
Under, chromatographic condition measures in accordance with the law, and the RSD calculating belamcandin content is 0.83%, and result shows, the repeatability that this law measures is good
Good, it is shown in Table 6, Figure 14.
Average recovery is tested
Take 9 parts of little Qingyan Granules measuring content, every part of about 1 g, accurately weighed, plus 0.8 respectively, 1.0,1.2 times amount Rhizoma Belamcandae
Glycosides reference substance, according to legal system available test sample solution below " 3.2.3 " item, measures according to chromatographic condition under " 3.2.1 " item, calculating is penetrated in accordance with the law
Dry glycosides average recovery rate is respectively 103.4%, 99.60%, and 100.2%, RSD are respectively 1.9%, 1.6%, 2.5%, and result shows, this
Method accuracy of measurement is higher, is shown in Table 7, Figure 15, Figure 16.
Water decocting process assay index selects
This prescription is made up of Folium Mori, Rhizoma Belamcandae, Radix Scrophulariae etc., and wherein Mulberry is monarch drug, all accounts for the 39.06% of prescription, Rhizoma Belamcandae accounts for 26.06%.
Initially attempt to from rutin in Folium Mori, in Radix Scrophulariae harpagoside be measurement index, result wherein content be less than ten thousand/, all not
Therefore can finally determine that belamcandin is quality control index as quality control index（See Figure 17, Figure 18, Figure 19）.
The selection of chromatographic condition
3.4.1 the selection of chromatographic column
It is respectively adopted：Yi Lite Hypersil BDS C18（250 mm × 4.6 mm, 5 μm）With Agilent SB-C18
（250 mm × 4.6 mm, 5 μm）Chromatographic column is analyzed testing, and result shows Yi Lite Hypersil BDS C18（250
Mm × 4.6 mm, 5 μm）Chromatographic column separating effect preferably, therefore selects this chromatographic column.
The selection of mobile phase
It has been respectively adopted acetonitrile -0.03% phosphate aqueous solution, acetonitrile -0.1% phosphate aqueous solution carries out gradient elution as mobile phase,
When result shows that mobile phase is, chromatographic peak separating effect and chromatographic peak profile preferably, therefore select acetonitrile -0.1% phosphate aqueous solution conduct
Mobile phase.
Column temperature is investigated
It is respectively adopted 25 DEG C and 30 DEG C of column temperatures are analyzed, the separating effect of 25 DEG C of column temperature gained preferably, therefore selects 25 DEG C of works
For chromatogram column temperature.
The selection of test sample extraction conditions
3.5.1 the selection of Extraction solvent
Methanol, 50% methanol, 70% ethanol have been respectively adopted it as Extraction solvent, supersound extraction 30 min, 45 min, 60 min.
Result shows with 50% methanol supersound extraction content highest, therefore selects 50% methanol as Extraction solvent.
The selection of extracting mode
Use 50% methanol, be respectively adopted reflux, extract, 60 min, supersound extraction 60 min.Result shows with supersound extraction 60 min
Need testing solution, therefore select supersound extraction 60 min.
The selection of extraction time
Use 50% methanol, be respectively compared supersound extraction 30 min, 45 min and 60 min.Supersound extraction 60 min belamcandin contains
Amount is higher, and peak shape is preferably, therefore selects supersound extraction 60 min.
Certainly, the concrete application example of above simply invention, the present invention also has other embodiments, all is replaced using equivalent
Change or equivalent transformation formed technical scheme, all fall within protection domain of the presently claimed invention.
Claims (4)
1. little Qingyan Granules detection method for quality, this little Qingyan Granules 1000g is made up of the crude drug of following quantity number：Folium Mori
390.6 g, Radix Isatidis 390.6 g, Rhizoma Belamcandae 260.4 g, Radix Scrophulariae 260.4 g, Radix Glycyrrhizae 260.4 g, Oleum menthae 2.5 mL, can
Soluble starch 433 ~ 550 g；
Its quality determining method includes following items：
Character：This product is granule, brown color to sepia, gas fragrance, taste micro-sweet, slightly bitter；
Differentiate：Differentiated using thin layer chromatography；
Check：Meet relevant every regulation under granule item（《Chinese Pharmacopoeia》（General rule 0104））；
Determination of extractives：Measure including Diluted Alcohol extract content；
Assay：Assay including belamcandin.
2. little Qingyan Granules detection method for quality according to claim 1 is it is characterised in that described discriminating includes：
（1）Take invention formulation to prepare need testing solution, take Radix Isatidis control medicinal material to prepare control medicinal material solution, according to thin layer chromatography
Method is tested, and draws need testing solution and control medicinal material solution and puts respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-
Water is developing solvent, launches, and takes out, hot blast drying, and spray, with ninhydrin solution, is heated to spot development clear；In test sample chromatograph,
On the position corresponding with control medicinal material chromatograph, the speckle of aobvious same color；
（2）Take invention formulation to prepare need testing solution, take Rhizoma Belamcandae control medicinal material to prepare control medicinal material solution, according to thin layer chromatography
Test, draws need testing solution and control medicinal material solution is put respectively in same silica gel G F254On lamellae, with chloroform-first
Alcohol-glacial acetic acid is developing solvent, launches, and takes out, dries, put and inspect under ultra-violet lamp；In test sample chromatograph, with comparison medicine wood color
Compose on corresponding position, the fluorescence spot of aobvious same color.
3. described little Qingyan Granules detection method for quality according to claim 1 is it is characterised in that described extractum is surveyed
Determine to carry out in accordance with the following steps：Take test sample accurately weighed, conical flask adds Diluted Alcohol, close plug, weighed weight, standing 1 is little
Shi Hou, connects reflux condensing tube, is heated to seething with excitement, and keeps micro-boiling 1 hour；After letting cool, take off conical flask, close plug, more weighed
Weight, supplies the weight of less loss, shakes up with Diluted Alcohol, and with filter filtration is dried, precision measures subsequent filtrate, puts and is dried to constant weight
Evaporating dish in, after water-bath is evaporated, be dried 3 hours, in dislocation exsiccator, cool down 30 minutes, accurately weighed weight rapidly.
4. described little Qingyan Granules detection method for quality according to claim 1 it is characterised in that：The content of belamcandin
According to high effective liquid chromatography for measuring；With octadecyl silane as filler；With acetonitrile -0.1 % phosphate aqueous solution for stream
Dynamic phase, gradient elution；Flow velocity 1 mL min-1；25 DEG C of column temperature；Detection wavelength 265 nm.
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| CN111896672A (en) * | 2020-07-31 | 2020-11-06 | 江苏弘典中药产业研究院有限公司 | Thin-layer identification method of children's Ganmaoshu granules |
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Application publication date: 20170222 |