CN106706766B - A kind of measuring method of the newly-increased detection ingredient of Chinese medicine Tang blade that treating AIDS - Google Patents
A kind of measuring method of the newly-increased detection ingredient of Chinese medicine Tang blade that treating AIDS Download PDFInfo
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Abstract
The present invention provides a kind of measuring methods of the newly-increased detection ingredient of Chinese medicine Tang blade for treating AIDS.The measuring method of the newly-increased detection ingredient of the present invention are as follows: (1) oldenlandia diffusa in Tang's blade, myrobalan's ingredient are identified using thin layer chromatography.(2) using under the conditions of the same high performance liquid chromatography, by the gradient elution of mobile phase, gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ are measured simultaneously.Every Tang's blade is not less than 0.45mg not less than 0.25mg, picroside Ⅱ not less than 0.5mg, liquiritin not less than 0.9mg, chlorogenic acid containing gallic acid.The present invention is confirmed by full inspection project, is illustrated that former detection method can be added in the newly-increased detection project of the present invention, is further increased the detection kind and method of drug, the batch production measurement for being conducive to Chinese medicine Tang blade is conducive to improve product quality.Measuring method of the present invention is reliable and stable, specificity is strong, there is biggish application value.
Description
Technical field
The present invention relates to compound Chinese medicinal preparations, and in particular to treats the survey of the newly-increased detection ingredient of Chinese medicine Tang blade of AIDS
Determine method.
Background technique
Both at home and abroad with many experiments of Treating AIDS with Traditional Chinese Medicine and clinical study results discovery, traditional Chinese medicine is to AIDS
Treatment, have enhance and stablize body's immunity；For example, improving the symptom body of patient treating certain opportunistic infections
Sign, improves the quality of living and extending life etc. has biggish effect.
Chinese medicine Tang blade is by geranium wilfordii, Radix Astragali, black nightshade, honeysuckle, common bombax flower, myrobalan, oldenlandia diffusa, pomegranate
Skin, glutinous rice root, water chestnut, PERICARPIUM TRICHOSANTHIS, radix bupleuri, elscholtiza, Radix Glycyrrhizae, Caulis Spatholobi, safflower, ginkgo leaf, purslane, Radix picrorrhizae and scorpio two
Ten Herbs raw material is made.
Chinese patent 200410030849 discloses a kind of Chinese patent drug compound preparation for treating AIDS.Section is passed through in the invention
The basic research such as, stringent traditional Chinese medical theory, drug interaction, drug matching, drug toxicology and drug effect, clinical research.
The Chinese patent drug compound preparation has effects that nourishing qi and blood, clearing heat and detoxicating, activating microcirculation and removing stasis medicinal, dehumidifying resolving sputum, is helped by QI invigorating eliminating evil
Just, the immune function for enhancing HIV/AIDS patient, delays the duplication of inhibition of HIV, embodies the whole conditioning of traditional Chinese medicine, consolidates and dispel
Evil theory, compensates for the deficiency of Western medicine HARRT therapy, while can be used as the auxiliary treatment means of AIDS patient, valence again
Lattice are cheap, convenient to take.
Chinese patent 200910048798.6 discloses a kind of quality standard and detection for treating Chinese medicine treating AIDS Tang blade
Method identifies scorpio ingredient in Tang's blade sample using microscopic features；Old stork in Tang's blade sample is identified using thin layer chromatography
The ingredients such as grass, granatum, glutinous rice root, Radix Glycyrrhizae, Radix picrorrhizae, radix bupleuri, radix scutellariae, ginkgo leaf；It is quantitatively surveyed using high performance liquid chromatography
Determine the Huang of the chlorogenic acid of contained honeysuckle and luteolin content in Tang's blade sample, the carthamus tinctorius yellow colour A content of safflower, Radix Astragali
Stilbene glucoside content, but the measuring method is to carry out respectively, thus operation is more cumbersome, is unfavorable for batch samples
Detection, and other compositions are unable to control.In addition, the replicative cycle of human immunodeficiency virus mainly includes absorption, merges, is inverse
Transcription, integration, transcription, translation, budding and maturation etc., HIV-1 integrase are that HIV replicates required enzyme, the nothing again in host
The component of functional equivalence therewith is one of promising target of anti-HIV-1 medicines.HIV-1 integrase inhibitor can blocking virus it is whole
It closing, IN inhibitor and protease or/and reverse transcriptase inhibitor combination have important clinical value in terms for the treatment of H1V/AIDS, and
And also there is biggish help for solving current drug resistance problems.However since its complete crystal diffraction structure is still unclear
Chu affects the development of corresponding inhibitor.Mi-JeongAhn et al. has found the gallic acid and other three kinds of galla turcicas in myrobalan
Acyl glucose has the function of inhibiting hiv integrase (Mi-JeongAhn, Chul Young Kim, Ji Suk Lee et
al.Inhibition of HIV-1integrase by galloylglucoses from terminaliachebula and
flavonal glycoside gallates from euphorboapekinensis[J].Planta Med.2002,68:
457-459.).But myrobalan is not detected in the detection of existing Chinese medicine Tang blade, therefore, it is necessary to be further improved, is increased related
The measuring method of ingredient.
Summary of the invention
Technical problem to be solved by the present invention lies in overcome above-mentioned shortcoming, researching and designing specificity is strong, stability
The good measuring method for detecting the Multiple components content of Chinese medicine Tang blade simultaneously using high performance liquid chromatography.
The present invention provides a kind of measuring methods of the newly-increased detection ingredient of Chinese medicine Tang blade for treating AIDS.
The measuring method of the newly-increased detection ingredient of the present invention are as follows:
(1) oldenlandia diffusa in Tang's blade, myrobalan's ingredient are identified using thin layer chromatography.
(2) using under the conditions of the same high performance liquid chromatography, by the gradient elution of mobile phase, by gallic acid,
Chlorogenic acid, liquiritin, picroside Ⅱ are measured simultaneously.
Specifically, the method for the present invention includes the following steps:
(1) thin-layer chromatography chromatography identifies:
(1), oldenlandia diffusa identifies: Chinese medicine Tang blade sample is taken, it is finely ground, and add water 10-60ml directly to dissolve, in centrifuging and taking
Clear liquid；Or ethyl alcohol 10-60ml ultrasound or reflux, filtrate is added to be evaporated, be preferably directly dissolved in water, residue adds water 5-50ml to dissolve；
Ethyl acetate or chloroform or n-butanol 5-50ml is added, preferentially selects ethyl acetate, less preferred 2 times of extraction 1 to 2 is collected
Solvent layer is evaporated in 100 DEG C of water-baths, and residue adds 1-5ml ethyl acetate or dehydrated alcohol or methanol, preferential that methanol dissolution is selected to make
For test sample；It separately takes oldenlandia diffusa control medicinal material appropriate, adds water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds water to
50ml operates with test sample from being added ethyl acetate or chloroform or n-butanol in right amount, control medicinal material solution is made, photograph is thin
Layer chromatography (one VIB of Chinese Pharmacopoeia version in 2010) test, draws each 5-10 μ l of above two solution, is put respectively in same silicon
On glue G lamellae, with hexamethylene-chloroform-ethyl acetate-glacial acetic acid (10:2.5:4:0.25) or petroleum ether-benzene-acetic acid
Ethyl ester-glacial acetic acid (5:10:3.5:0.25) be solvent, preferably hexamethylene-chloroform-ethyl acetate-glacial acetic acid (10:
2.5:4:0.25), it is unfolded, takes out, dry, it is clear to spot development to smoke in ammonia steam, sets and inspects under daylight；Sample chromatogram
In, in position corresponding with reference medicine chromatography, show the principal spot of same color；
(2), myrobalan identifies: taking myrobalan control medicinal material 1g, adds water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds water
To 50ml, identifies test sample under item with oldenlandia diffusa from being added ethyl acetate or chloroform or n-butanol in right amount and operates,
Control medicinal material solution is made；According to thin-layered chromatography (one VIB of Chinese Pharmacopoeia version in 2010) test, draw control medicinal material solution and
Above-mentioned (1) oldenlandia diffusa identifies each 5-10 μ l of test solution under item, is put respectively on same silica gel g thin-layer plate, with two
Chloromethanes-glacial acetic acid-water (15:5:0.5) lower layer or toluene-glacial acetic acid-water (15:5:0.5) lower layer are solvent, preferably dichloro
Methane-glacial acetic acid-water (15:5:0.5) lower layer is unfolded, and takes out, dries, and spray is heated with the colour developing of 10% sulfuric acid ethyl alcohol at 105 DEG C
To clear spot, (25 ± 2 DEG C) of room temperature cooling half an hour are set in taking-up, set and inspect under daylight, in sample chromatogram, with comparison medicine
Wood color composes corresponding position, shows the spot of same color；
(2) high effective liquid chromatography for measuring:
Take Tang's blade sample appropriate, it is finely ground, 0.5g is taken, it is accurately weighed, it sets in stuffed conical flask, precision plus 50% or more first
Alcohol 25ml, close plug, weighed weight are ultrasonically treated 30 minutes, are taken out, are put to room temperature (25 ± 2 DEG C), then weighed weight, use methanol
The weight for supplying less loss, shakes up, filtration, is test solution, with gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ control
Product, chromatographic column are filler with octadecylsilane chemically bonded silica, and acid, mobile phase choosing is added with organic solvent and water in mobile phase
From acetonitrile: water: phosphoric acid or methanol: water: phosphoric acid, ratio are acetonitrile-water-phosphoric acid (6-17:94-83:0.3) or methanol-water-phosphoric acid
(20-40:80-60:0.3), preferably acetonitrile-water-phosphoric acid (6-17:94-83:0.3), Detection wavelength 263nm-293nm are accurate
Reference substance solution and test solution are drawn, liquid chromatograph is injected, according to high performance liquid chromatography Chinese Pharmacopoeia version one in 2010
Portion annex VID measurement.
Every Tang's blade is not less than 0.5mg not less than 0.9mg, chlorogenic acid containing gallic acid, liquiritin is not less than 0.25mg,
Picroside Ⅱ is not less than 0.45mg.
The present invention is further changed on the basis of detection method (patent No. 20091004878.6) of former Chinese medicine Tang blade
Into increasing identification and assay project.
Gallic acid is content very high one in a kind of polyphenol compound and Tang's blade being widely present in nature
Kind ingredient.The medicinal materials such as myrobalan, geranium wilfordii, granatum in Tang's grass tablet recipe contain gallic acid, have and inhibit HIV-1 integration
The effect of enzyme, and then directly inhibition of HIV can be blocked to replicate, while gallic acid has very strong bacteriostatic activity, causes to HIV
Complication infection have therapeutic effect.Therefore, gallic acid is one of important effective component of Tang's blade, and content should be given
With control.
Liquiritin and picroside Ⅱ are respectively the index ingredient of Radix Glycyrrhizae and Radix picrorrhizae, control its content to represent Radix Glycyrrhizae
With the quality of Radix picrorrhizae.
For Chinese medicine compound prescription, with the method for indentification by TLC, it can be determined that whether product contains taste medicinal material, thin
The presence or absence of layer chromatography spot can determine that the true and false of medicinal material used, size, the depth of color of spot can reflect to a certain extent
The superiority and inferiority of medicinal material used out.Therefore it in order to ensure the quality of compound patent medicine, answers as much as possible to medicinal material contained in product
Identified, thus the method for the present invention on the basis of Tang's blade existing detection method by research increase oldenlandia diffusa and
The thin-layer identification method of two taste medicinal material of myrobalan.Under the conditions of the same high performance liquid chromatography, washed by the gradient of mobile phase
It is de-, it is under same chromatographic condition while to be measured by gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ.
The present invention improves and improves the detection of Chinese medicine Tang blade, increases the following contents:
(1) thin layer of oldenlandia diffusa identifies in Tang's blade sample, is in feature chromatogram, sees Fig. 1.
(2) thin layer of myrobalan identifies in Tang's blade sample, is in feature chromatogram, sees Fig. 2.
(3) the high performance liquid chromatography measurement of gallic acid in Tang's blade sample, chlorogenic acid, liquiritin, picroside Ⅱ, often
The gallic acid content of piece must not be less than 0.9mg, 0.50mg must not be less than in terms of chlorogenic acid containing honeysuckle, containing Radix Glycyrrhizae with liquiritin
Must not count less than 0.25mg, 0.45mg must not be less than in terms of picroside Ⅱ containing Radix picrorrhizae, see Fig. 3.
The present invention illustrates that former detection method can be added in the newly-increased detection project of the present invention by full inspection project confirmatory test,
The detection kind and method for further increasing drug are conducive to improve product quality.
The advanced chromatographic technique of the present invention can be effectively controlled Chinese medicine Tang blade activity index ingredient, establish production
The quality control standard of product.Measuring method of the invention increases the identification of two taste medicine ingredients, and uses high performance liquid chromatography
Multiple a ingredients can be measured simultaneously, be conducive to the batch production measurement of Chinese medicine Tang blade.Measuring method of the present invention is reliable and stable, special
One property is strong, there is biggish application value.
Detailed description of the invention
Fig. 1 be Tang's blade sample in oldenlandia diffusa thin-layer chromatogram: set and inspected under daylight, point sample sequence from a left side to
The right side, 1-is oldenlandia diffusa blank negative sample, and 2-be Tang's blade sample of embodiment 1, and 3-be Tang's blade of embodiment 2
Sample, 4-be Tang's blade sample of embodiment 3, and 5-be oldenlandia diffusa control medicinal material.
Fig. 2 is the thin-layer chromatogram of myrobalan in Tang's blade sample: setting and inspects under daylight, from left to right, 1-is point sample sequence
Myrobalan's blank negative sample, 2-be Tang's blade sample of embodiment 4, and 3-be Tang's blade sample of embodiment 5, and 4-be implementation
Tang's blade sample of example 6,5-be myrobalan's control medicinal material,
Fig. 3 is the high-efficient liquid phase chromatogram of 7 Tang's blade of embodiment
The appearance serial number situation marked by peak retention time is as follows, and from from left to right, 1-is gallic acid, and 2-be green
Ortho acid, 3-be liquiritin, and 4-be picroside Ⅱ.
Fig. 4 is the high-efficient liquid phase chromatogram of 4 gallic acid of embodiment, chlorogenic acid, liquiritin, picroside Ⅱ standard items
The appearance serial number situation marked by peak retention time is as follows, and from from left to right, 1-is gallic acid, and 2-be green
Ortho acid, 3-be liquiritin, and 4-be picroside Ⅱ.
Fig. 5 be lack honeysuckle, Radix Glycyrrhizae, Radix picrorrhizae Tang's blade high-efficient liquid phase chromatogram
By the appearance serial number that peak retention time marks, from left to right, 1-is gallic acid.
Specific embodiment
The Chinese medicine Tang blade that following embodiment uses produces (commercial product) by Shanghai Baisuixing Pharmaceutical Co., Ltd, various
Reagent and control medicinal material are commercially available.
Embodiment 1
The identification of oldenlandia diffusa in Tang's blade: taking Tang's blade sample 1, sample 2, sample 3 respectively (is at the age of one hundred years old by Shanghai
The production of row pharmaceutcal corporation, Ltd, commercial product) 10 each (every slice weight 0.4g, it is finely ground, add water 50ml, ultrasonic 30min, is centrifuged,
Supernatant is taken, chloroform (agents useful for same is commercially available) 50ml is added, is extracted 1 time, chloroform layer is collected, is evaporated, it is residual
Slag adds the dissolution of 1.5ml methanol as test sample.Separately take oldenlandia diffusa control medicinal material (examining and determine institute purchased from Products in China)
0.5g adds water 20ml, is heated to reflux 1.5 hours, and filtering, filtrate adds water to 50ml, with for examination from being added chloroform 50ml
Product operation, is made control medicinal material solution.It is tested according to thin-layered chromatography (one VIB of Chinese Pharmacopoeia version in 2010), draws above-mentioned two
Kind of each 10 μ l of solution, puts respectively on same silica gel g thin-layer plate, with hexamethylene-chloroform-ethyl acetate-glacial acetic acid (10:
2.5:4:0.25) it is solvent, is unfolded, takes out, dry, it is clear to spot development to smoke in ammonia steam, sets and inspects under daylight.For examination
In product chromatography, in position corresponding with reference medicine chromatography, the principal spot of same color is shown.
The result is shown in Figure 1 of embodiment 1
Embodiment 2: the identification of myrobalan in Tang's blade: (Tang's blade sample is with embodiment 1)
Myrobalan's control medicinal material (examining and determine institute purchased from Products in China) 1g is taken, adds water 20ml, is heated to reflux 1.5 hours, mistake
Filter, filtrate add water to 50ml, identify test sample under item with 1 oldenlandia diffusa of embodiment from being added chloroform 50ml and operate,
Control medicinal material solution is made.According to thin-layered chromatography (one VIB of Chinese Pharmacopoeia version in 2010) test, draw control medicinal material solution and
1 oldenlandia diffusa of embodiment identifies each 10 μ l of test solution under item, is put respectively on same silica gel g thin-layer plate, with dichloro
Methane-glacial acetic acid-water (15:5:0.5) lower layer is solvent, is unfolded, and takes out, dries, and spray is developed the color with 10% sulfuric acid ethyl alcohol,
105 DEG C are heated to clear spot, and taking-up sets (25 ± 2 DEG C) of room temperature cooling half an hour, sets and inspect under daylight.In sample chromatogram,
In position corresponding with reference medicine chromatography, the spot of same color is shown.
2 result of embodiment is shown in Fig. 2.
The assay of gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ in 3 Tang's blade of embodiment:
Take Tang's blade sample (to be produced by Shanghai Baisuixing Pharmaceutical Co., Ltd, commercial product) in right amount, it is finely ground, it takes
1g, it is accurately weighed, it sets in stuffed conical flask, precision plus 50% methanol 50ml, close plug, weighed weight is ultrasonically treated 30 minutes, takes
Out, it puts to room temperature (25 ± 2 DEG C), then weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter, be test solution,
Gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ reference substance (examining and determine institute purchased from Products in China):
It is suitable that precision weighs gallic acid reference substance, chlorogenic acid reference substance, liquiritin reference substance, picroside Ⅱ reference substance
Amount, adds methanol to be configured to every milliliter of 0.05mg containing gallic acid respectively, chlorogenic acid 0.045mg, liquiritin 0.02mg, picroside
The mixed solution of 0.05mg to get.
Chromatographic column is filler with octadecylsilane chemically bonded silica, mobile phase be acetonitrile-water-phosphoric acid (6-17:94-83:
0.3) gradient elution, Detection wavelength 263nm, precision draw reference substance solution and test solution, inject liquid chromatograph, shine
The one annex VID measurement of version in 2010 of high performance liquid chromatography Chinese Pharmacopoeia.
Experimental result: 1.30mg containing gallic acid, 0.80mg containing chlorogenic acid, 0.41mg containing liquiritin in every Tang's blade,
0.60mg containing picroside Ⅱ.
Embodiment 4
Tang's blade (lot number 130502, for Shanghai Baisuixing Pharmaceutical Co., Ltd production commercial product) in gallic acid,
Chlorogenic acid, liquiritin, picroside Ⅱ assay methodology validation, (number in following each table is the sample of same lot number
Product)
Standard items examine and determine institute purchased from Products in China, and agents useful for same is from commercially available.
(1) accuracy (sample recovery rate)
A, gallic acid
The gallic acid sample recovery rate data of 1 Tang's blade sample of table
The gallic acid rate of recovery is between 95%~105%, and RSD is less than 2%, within the acceptable range, shows
The content assaying method accuracy of gallic acid is good.
B, chlorogenic acid
The chlorogenic acid sample recovery rate data of 2 Tang's blade sample of table
The chlorogenic acid rate of recovery is between 95%~105%, and RSD is less than 2%, within the acceptable range, shows green
The content assaying method accuracy of ortho acid is good.
C, liquiritin
The liquiritin sample recovery rate data of 3 Tang's blade sample of table
The liquiritin rate of recovery is between 95%~105%, and RSD is less than 2%, within the acceptable range, shows sweet
The content assaying method accuracy of careless glycosides is good.
D, picroside Ⅱ
The picroside Ⅱ sample recovery rate data of 4 Tang's blade sample of table
The picroside Ⅱ rate of recovery is between 95%~105%, and RSD is less than 2%, within the acceptable range, table
The content assaying method accuracy of bright picroside Ⅱ is good.
(2) precision (repeatability)
Precision weighs 6 parts of Tang's blade sample, according to Tang's blade Syrups by HPLC gallic acid, chlorogenic acid, liquiritin,
The requirement operation repetitive of assay under picroside Ⅱ item calculates 6 parts of sample sizes and is as follows:
The repeated data of 5 Tang's blade sample of table
Gallic acid average content 0.3301%, chlorogenic acid average content in the parallel six parts of samples of Tang's blade are
0.1993%, the average content of liquiritin is 1.0352%, and picroside average content is 0.1569%, and four RSD do not surpass
3% is crossed, within the acceptable range.
(3) specificity
A, the preparation of honeysuckle, Radix Glycyrrhizae, Radix picrorrhizae blank negative sample
Honeysuckle, Radix Glycyrrhizae, three taste medicinal material of Radix picrorrhizae are not added when preparation, takes the medicinal material of 100 Tang's blade formula ratios of preparation,
It being prepared in following ratio, geranium wilfordii 10%, Radix Astragali 10%, black nightshade 10%, common bombax flower 5%, myrobalan 3%, oldenlandia diffusa 7%,
Granatum 3%, glutinous rice root 10%, water chestnut 7%, PERICARPIUM TRICHOSANTHIS 3%, Caulis Spatholobi 9%, safflower 3%, ginkgo leaf 3%, purslane 7%,
Radix bupleuri 5%, elscholtiza 3%, scorpio 2% add 10 times of amount water, decoct 1 hour after boiling, and filtrate is collected in filtering；The dregs of a decoction continuously add
10 times of amount water carry out second and decoct, and decoct 1 hour, filter after boiling；Filtrate is boiled and is concentrated into sticky state by merging filtrate
When, it is transferred to vacuum oven, setting temperature is 60 degree, and vacuum degree -0.1Mpa is dried under reduced pressure 30 hours, extract powder is collected,
As honeysuckle, Radix Glycyrrhizae, Radix picrorrhizae blank negative sample.
B, specificity measures
0.5g honeysuckle, Radix Glycyrrhizae, Radix picrorrhizae blank negative sample is taken to prepare according to test sample under Tang's blade content determination item
It is required that preparing and detecting with method, it there are no chromatographic peak interference at chlorogenic acid, liquiritin, picroside Ⅱ retention time, show
The content assaying method specificity is good.Detailed map is shown in Fig. 4-5.
In addition to gallic acid, there is not chlorogenic acid, Radix Glycyrrhizae in retention time identical as standard items map (Fig. 4) position
Glycosides, the corresponding chromatographic peak interference of picroside Ⅱ, illustrates that the specificity of detection method meets the requirements.
(4) linear
Precision weighs gallic acid reference substance about 12.33mg, chlorogenic acid reference substance about 10.35mg, liquiritin picroside
II reference substance about 11.04mg, respectively into 25ml volumetric flask, with methanol constant volume to scale, i.e. respectively gallic acid reference substance
Stock solution, chlorogenic acid reference substance stock solution, picroside Ⅱ reference substance stock solution.Precision pipettes gallic acid pair respectively
According to product stock solution, chlorogenic acid reference substance stock solution, each 1ml, 2ml of picroside Ⅱ reference substance stock solution, 3ml, 5ml,
8ml, with methanol constant volume to scale, crosses 0.45 μm of organic film respectively to numbering in five 25ml volumetric flasks for being 1,2,3,4,5,
Gallic acid, chlorogenic acid, the picroside Ⅱ mixing reference substance of as five concentration gradients.
By the accurate each 10 μ l of mixing reference substance for drawing above-mentioned five concentration gradients respectively, liquid chromatograph is injected, is measured,
Peak area carries out linear regression with reference substance content is mixed, and calculates regression coefficient and the intercept of regression equation, slope are as follows:
Gallic acid, chlorogenic acid, liquiritin, the picroside Ⅱ linear data of 6 Tang's blade sample of table
Embodiment 5
The assay of gallic acid, chlorogenic acid in Tang's blade:
(1) chromatographic condition and system suitability:
Using octadecylsilane chemically bonded silica as filler；It is flowing with 0.3% phosphoric acid solution using acetonitrile as mobile phase A
Phase B, the regulation according to the form below carry out gradient elution.Detection wavelength is 293nm.Theoretical cam curve must not calculate by gallic acid peak
Lower than 5000.
(2) preparation of reference substance solution: precision weighs gallic acid reference substance, chlorogenic acid reference substance (purchased from Chinese biological
Product examines and determine institute) in right amount, add methanol that every 1ml is made and contains gallic acid 0.05mg respectively, the mixed solution of chlorogenic acid 0.045mg,
To obtain the final product.
(3) preparation of test solution: taking Tang's blade (for the commercial product of Shanghai row medicine company production at the age of one hundred years old) 20, accurate
It is weighed, it is finely ground, more 0.5g is taken, it is accurately weighed, it sets in stuffed conical flask, methanol 25ml, close plug is added in precision, and weighed weight surpasses
Sonication (power 150W, frequency 60kHz) 30 minutes, lets cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter
Cross, take subsequent filtrate to get.
(4) measuring method: it is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed to get.
(5) experimental result: 1.29mg containing gallic acid, 0.81mg containing chlorogenic acid in every Tang's blade.
Embodiment 6
Take 2 bottles of Tang's blade sample (equal with Tang's blade sample hereinafter for the commercial product of Shanghai row medicine company production at the age of one hundred years old
It is commercial product) full inspection is carried out according to following standard.
1) microscopical characters:
Tang blade sample powder 0.01g is taken, microscopically observation is set, body wall fragment is faint yellow to yellow, there is reticular texture
And round trichopore；It can be seen that sepia bristle.
2) thin layer identifies:
A, Radix Glycyrrhizae identifies: 10, Tang's blade sample is taken, it is finely ground, and add 25ml water to dissolve, sets heating and refluxing extraction 30 in water-bath
Minute, it lets cool, is centrifuged, takes supernatant, extracted 1 time with the shaking of 25ml ether, discard ether solution, then with the water saturated positive fourth of 25ml
Alcohol extracting 1 time, water washing 1 time be saturated with the n-butanol of equivalent, n-butanol liquid is evaporated, and residue adds methanol 1ml to make to dissolve, from
The heart takes supernatant as test solution.Another extracting liquorice control medicinal material, is made in the same way of control medicinal material solution.Extracting liquorice glycosides pair again
According to product 1mg, add methanol 1ml that reference substance solution is made.It is tried according to thin-layered chromatography (one VI B of annex of China's coastal port)
It tests, it is each appropriate to draw above-mentioned three kinds of solution, is put respectively on same silica gel thin-layer plate, with chloroform-acetate-methanol-
10 DEG C of water (15:40:22:10) lower layer's solution arranged below；Or with suitable solvent, be unfolded, take out, dry, spray with
10% ethanol solution of sulfuric acid, it is clear to be heated to spot development, sets inspect under daylight and ultraviolet lamp respectively.In sample chromatogram,
On position corresponding with control medicinal material and reference substance chromatography, the spot and fluorescence spot of same color are shown.
B, glutinous rice root identifies: 10, Tang's blade sample is taken, it is finely ground, and add ethyl acetate 10ml, ultrasonic treatment is extracted 30 minutes,
Centrifugation, takes supernatant to be evaporated, residue adds dehydrated alcohol 1ml to make to dissolve, as test solution.Glutinous rice root control medicinal material separately is taken,
It is made in the same way of control medicinal material solution.It is tested according to thin-layered chromatography (one VI B of annex of China's coastal port), draws above-mentioned two
Kind each 1 μ l of solution puts respectively on same silica gel thin-layer plate, is solvent with toluene-ethyl acetate (4:1), is unfolded, take out,
It dries, with 2% paradime thylaminobenzaldehyde, 40% sulfuric acid solution, it is clear to be heated to spot development for spray, sets daylight and ultraviolet respectively
It is inspected under light lamp.In sample chromatogram, on position corresponding with reference medicine chromatography, the spot and fluorescent spot of same color are shown
Point.
C, Radix picrorrhizae identifies: 10, Tang's blade sample is taken, it is finely ground, and add water to make to moisten, adds methanol 25ml, heating and refluxing extraction
15 minutes, centrifugation took supernatant to be evaporated, and residue adds the water saturated n-butanol of 25ml to make to dissolve in right amount, full with the n-butanol of equivalent
The water washing of sum 1 time, n-butanol liquid is evaporated, and residue adds methanol 1ml to make to dissolve, and centrifugation takes supernatant as test solution.Separately
Picroside-Ⅱ reference substance 1mg is taken, adds methanol 1ml that reference substance solution is made.According to thin-layered chromatography (China's coastal port
One VI B of annex) test, it is each appropriate to draw above two solution, is put respectively on same silica gel thin-layer plate, with ethyl acetate-
The suitable solvent such as lower layer's solution after 10 DEG C of alcohol-water (8:2:1) is arranged below, is unfolded, and takes out, dries, and sprays with 2% pair
The colour developing of 40% sulfuric acid solution of dimethylaminobenzaldehyde.105 DEG C to be heated to spot development clear.In sample chromatogram, with compare
On the corresponding position of product chromatography, the spot of same color is shown.
D, geranium wilfordii, granatum identify: 5, Tang's blade sample is taken, finely ground, solubilization matchmaker 50ml sets boiling water bath heating extraction,
It lets cool, is centrifuged, takes supernatant, add ethyl acetate shaking to extract twice, each 20ml, combined ethyl acetate layer is evaporated, and residue adds
Ethyl acetate 1ml makes to dissolve, as test solution.Geranium wilfordii control medicinal material, each 0.5g of granatum control medicinal material separately are taken, respectively
It is made in the same way of control medicinal material solution.It is tested according to thin-layered chromatography (one VI B of annex of China's coastal port), draws above-mentioned three
Kind solution is each appropriate, is put respectively on same silica gel thin-layer plate, is unfolded with chloroform-acetic ether-methanoic acid (4:5:1)
Agent is unfolded, and takes out, dries, set and inspect under ultraviolet lamp.In sample chromatogram, in position corresponding with reference substance medicinal material chromatography
On, show the spot of same color.
E, radix bupleuri identifies: 10, Tang's blade sample is taken, it is finely ground, and add water 25ml, sets heating and refluxing extraction in water-bath, let cool,
Centrifugation, takes supernatant, is extracted 1 time with the shaking of 25ml ether, discards ether solution, then extracted with the water saturated n-butanol shaking of 25ml
It 1 time, is washed with the ammonia solution of equivalent, then water washing 1-3 times be saturated with the n-butanol of equivalent, n-butanol liquid is evaporated, and residue adds
Methanol 1ml makes to dissolve, and centrifugation takes supernatant as test solution.Separately radix bupleuri control medicinal material 0.5g is taken to be made in the same way of comparison medicine
Material solution.It is tested according to thin-layered chromatography (one VI B of annex of China's coastal port), it is each appropriate to draw above two solution,
It is put respectively on same silica gel thin-layer plate, lower layer's solution after being placed with toluene-chloroform-methanol (5:5:1) is solvent, exhibition
It opens, takes out, dry, spray is with the color developing agent of 2% paradime thylaminobenzaldehyde, 40% sulfuric acid solution, and colour developing, 105 DEG C are heated to spot
Colour developing is clear, sets inspect under daylight and ultraviolet lamp respectively.In sample chromatogram, in position corresponding with reference medicine chromatography
On, show the principal spot and fluorescence principal spot of same color.
F, ginkgo leaf identifies: 10, Tang's blade sample is taken, it is finely ground, and add 50% acetone 100ml, be heated to reflux, let cool, filters
It crosses, filtrate boils off acetone, and aqueous shakes extraction with ethyl acetate in right amount, and acetic acid ethyl fluid is evaporated, and 5% ethyl alcohol of residue makes molten
Solution, by polyamide column (60H, internal diameter 1.5cm, long 10cm), with 5% ethanol elution, eluent is concentrated into 25ml, lets cool, and uses
Ethyl acetate shaking is extracted 1 time, is evaporated, residue acetone 1ml makes to dissolve, as test solution.Ginkgo leaf control is separately taken to mention
Object 5mg is taken, adds acetone 1ml that reference substance solution is made.It is tried according to thin-layered chromatography (one annex VI B of Chinese Pharmacopoeia version in 2000)
It tests, it is each appropriate to draw above two solution, is put respectively on same silica gel thin-layer plate, with toluene-ethyl acetate-acetone-methanol
(10:5:5:0.6) is solvent, is unfolded, and takes out, dries, and heats about 30 minutes at 105 DEG C, sets and inspect under ultraviolet lamp.For examination
In product chromatography, at the position corresponding to the chromatogram of the reference substance, the fluorescence spot of same color is shown.
G, Radix Astragali identifies: taking Astragaloside IV reference substance 1mg, adds methanol 1ml that reference substance solution is made.According to thin-layered chromatography
(one VI B of annex of China's coastal port) test draws reference substance solution and identifies each 2 μ of test solution under (E) item
L puts respectively on same silica gel thin-layer plate, with ethyl acetate, alcohol and water (8:2:1) solvent, is unfolded, takes out, dry, spray
With 2% paradime thylaminobenzaldehyde, 40% sulfuric acid solution color developing agent, it is clear that spot development is heated at 105 DEG C.Sample chromatogram
In, at the position corresponding to the chromatogram of the reference substance, show the spot of same color.
H, oldenlandia diffusa identifies: 5, Tang's blade sample is taken, it is finely ground, and add water 25ml directly to dissolve, centrifuging and taking supernatant,
Filtrate is evaporated, and residue is dissolved in water；N-butanol 25ml is added, extracts 1 time, collects solvent layer, is evaporated, residue adds 1ml methanol molten
Solution is used as test sample；It separately takes oldenlandia diffusa control medicinal material appropriate, adds water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds
Water operates with test sample from n-butanol to 50ml, control medicinal material solution is made, according to thin-layered chromatography (Chinese Pharmacopoeia 2010 years
One VIB of version) test, each 5 μ l of above two solution is drawn, is put respectively on same silica gel g thin-layer plate, with hexamethylene-trichlorine
Methane-ethyl acetate-glacial acetic acid (10:2.5:4:0.25) is solvent, is unfolded, and takes out, dries, and is smoked in ammonia steam aobvious to spot
Color is clear, sets and inspects under daylight；In sample chromatogram, in position corresponding with reference medicine chromatography, the main spot of same color is shown
Point.
I, myrobalan identifies: taking myrobalan control medicinal material 1g, adds water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds water to
50ml identifies test sample under item with oldenlandia diffusa from being added n-butanol in right amount and operates, control medicinal material solution is made；According to thin layer
Chromatography (one VIB of Chinese Pharmacopoeia version in 2010) test, draws control medicinal material solution and above-mentioned (H) oldenlandia diffusa identifies item
Under each 5 μ l of test solution, put respectively on same silica gel g thin-layer plate, with methylene chloride-glacial acetic acid-water (15:5:0.5)
Lower layer is solvent, is unfolded, and takes out, dries, and spray is heated to clear spot at 105 DEG C, taking-up is set with the colour developing of 10% sulfuric acid ethyl alcohol
It (25 ± 2 DEG C) of room temperature cooling half an hour, sets and is inspected under daylight.In sample chromatogram, in position corresponding with reference medicine chromatography,
The spot of aobvious same color.
3) assay:
A, gallic acid, the chlorogenic acid of honeysuckle, the liquiritin of Radix Glycyrrhizae, Radix picrorrhizae picroside Ⅱ assay:
20, Tang's blade sample is taken, it is finely ground, 2g is taken, it is accurately weighed, it sets in stuffed conical flask, precision plus methanol 25ml are close
Plug, weighed weight are ultrasonically treated 30 minutes, are taken out, are put to room temperature (25 ± 2 DEG C), then weighed weight, supply less loss with methanol
Weight shakes up, filtration, is test solution, gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ reference substance are (purchased from China
Biological products assay institute): precision weighs gallic acid reference substance, chlorogenic acid reference substance, liquiritin reference substance, picroside Ⅱ pair
It is appropriate according to product, add methanol to be configured to every milliliter of 0.05mg containing gallic acid respectively, chlorogenic acid 0.045mg, liquiritin 0.02mg, recklessly
The mixed solution of coptis glycosides 0.05mg to get.
Chromatographic column is filler with octadecylsilane chemically bonded silica, mobile phase be acetonitrile-water-phosphoric acid (6-17:94-83:
0.3) gradient elution, Detection wavelength 293nm, precision draw reference substance solution and test solution, inject liquid chromatograph, shine
The one annex VID measurement of version in 2010 of high performance liquid chromatography Chinese Pharmacopoeia.Every Tang blade 1.2mg containing gallic acid, containing gold
Honeysuckle flower 0.80mg in terms of chlorogenic acid, containing Radix Glycyrrhizae 0.3mg in terms of liquiritin, containing Radix picrorrhizae 0.4mg in terms of picroside Ⅱ.
B, the galuteolin assay of honeysuckle:
Galuteolin: taking 5, Tang's blade sample, finely ground, takes about 3g, accurately weighed, sets in stuffed conical flask, and precision is added
70% ethyl alcohol 50ml, weighed weight are ultrasonically treated about 1 hour, let cool, then weighed weight, the weight of less loss is supplied with 70% ethyl alcohol
Amount, shakes up, and filters, takes subsequent filtrate, is test solution.With galuteolin reference substance, chromatographic column is bonded with octadecylsilane
Silica gel is filler, and mobile phase is acetonitrile mobile phase A, with 0.5% glacial acetic acid solution Mobile phase B, by 0~30 minute, A:B →
10:90 30~55 minutes, A:B → 30:70, carries out gradient elution；Detection wavelength is 350nm.Precision draw reference substance solution with
Test solution injects liquid chromatograph, measurement.Every Tang's blade in terms of galuteolin 5 μ g containing honeysuckle.
C, the Determination of Astragaloside of Radix Astragali:
5, Tang's blade sample is taken, it is finely ground, about 3g is taken, it is accurately weighed, it sets in triangular pyramidal bottle, 50% methanol is added in precision
50ml, close plug, weighed weight are ultrasonically treated 40 minutes, let cool, again weighed weight, supply less loss weight with methanol, shake up,
Filtration, precision measure subsequent filtrate 40ml, water bath method, and residue adds water 10ml, and low-grade fever makes to dissolve, the n-butanol shaking being saturated with water
It extracts 4 times, each 40ml, merges n-butanol liquid, sufficiently washed 2 times with ammonia solution, each 40ml, discard ammoniacal liquor, n-butanol liquid
It is evaporated, residue adds water 5ml to make to dissolve, and lets cool, by D101 type large pore resin absorption column (internal diameter 1.5cm, long 12cm), with water
50ml elution discards aqueous, then is eluted with 40% ethyl alcohol 30ml, discards eluent, elutes after with 70% ethyl alcohol 80ml, collection is washed
De- liquid, is evaporated, is dissolved and be transferred in 5ml measuring bottle with methanol, adds methanol to scale, shakes up, be test solution.With Radix Astragali first
Glycosides reference substance, chromatographic column are filler with octadecylsilane chemically bonded silica, and mobile phase is acetonitrile-water (32:68)；Detector is
Evaporative light-scattering, precision draw 10 μ l of reference substance solution, 20 μ l, and 20 μ l of test solution injects liquid chromatograph, measure, with
External standard two-point method logarithmic equation calculates.Every Tang's blade in terms of Astragaloside IV 6 μ g containing Radix Astragali.
D, the hydroxyl radical carthamin yellow carthamus A assay of safflower:
Take Tang's blade sample appropriate, it is finely ground, about 2g is taken, it is accurately weighed, it sets in stuffed conical flask, 25% methanol is added in precision
50ml, weighed weight are ultrasonically treated 40 minutes, let cool, then weighed weight, the weight of less loss is supplied with 25% methanol, is shaken up, and filter
It crosses, takes subsequent filtrate, be test solution.With hydroxyl radical carthamin yellow carthamus A reference substance, chromatographic column octadecylsilane chemically bonded silica
For filler, mobile phase is -0.7% phosphoric acid solution of methanol-acetonitrile (26:2:72), and Detection wavelength 403nm, precision is drawn molten
Liquid and test solution inject liquid chromatograph, measurement.Every Tang's blade 0.08mg in terms of carthamus tinctorius yellow colour A containing safflower.
By above-mentioned full inspection project confirmatory test, illustrate that the newly-increased detection project of the present invention can supplement the former detection side of addition
Method further increases the detection quality of drug, is conducive to improve product quality.
Embodiment 7
Take 2 bottles of Tang's blade sample (equal with Tang's blade sample hereinafter for the commercial product of Shanghai row medicine company production at the age of one hundred years old
It is commercial product) full inspection is carried out according to following standard.
Microscopical characters:
Scorpio microscopic features identify: taking Tang blade sample 0.05g, set microscopically observation, body wall fragment is faint yellow to yellow
Color has reticular texture and round trichopore；Sometimes visible sepia bristle.
1) thin layer identifies:
A, Radix Glycyrrhizae identifies: 5, Tang's blade sample is taken, it is finely ground, and add 50ml ethyl alcohol to dissolve, ultrasonic extraction 30 minutes, lets cool, from
The heart takes supernatant, is extracted 3 times with ether shaking, and each 50ml discards ether solution, then the n-butanol being saturated with water extracts 3 times,
Each 50ml merges n-butanol extracting liquid, and water washing 3 times be saturated with the n-butanol of equivalent, n-butanol liquid is evaporated, and residue adds first
Alcohol 0.5ml makes to dissolve, and centrifugation takes supernatant as test solution.Another extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material
Solution.Extracting liquorice glycosides reference substance 2mg again adds methanol 1ml that reference substance solution is made.According to thin-layered chromatography (Chinese Pharmacopoeia 2005
One VI B of annex of version) test, above-mentioned each 5 μ l of three kinds of solution is drawn, is put respectively on same silica gel thin-layer plate, with chloroform-
10 DEG C of acetate-methanol-water (15:40:22:10) lower layer's solution arranged below is solvent, is unfolded, and takes out, dries, and is sprayed
With 2% paradime thylaminobenzaldehyde, 40% sulfuric acid solution, 105 DEG C to be heated to spot development clear, sets and examines under ultraviolet lamp 365nm
Depending on.In sample chromatogram, on position corresponding with control medicinal material and reference substance chromatography, the fluorescence spot of same color is shown.
B, glutinous rice root identifies: 5, Tang's blade sample is taken, it is finely ground, and add methanol 50ml, ultrasonic treatment is extracted 45 minutes, centrifugation,
Supernatant is taken to be evaporated, residue adds methanol 1ml to make to dissolve, as test solution.Glutinous rice root control medicinal material 2g separately is taken, is made in the same way of
Control medicinal material solution.It is tested according to thin-layered chromatography (one VI B of annex of China's coastal port), it is each to draw above two solution
5 μ l put respectively on same silica gel thin-layer plate, are solvent with benzene-chloroform-methanol (5:5:1), are unfolded, take out, dry, spray
With 2% paradime thylaminobenzaldehyde, 40% sulfuric acid solution, 105 DEG C to be heated to spot development clear, sets inspect under daylight respectively.For
In test product chromatography, on position corresponding with reference medicine chromatography, the spot of same color is shown.
C, Radix picrorrhizae identifies: 5, Tang's blade sample is taken, it is finely ground, and add ethyl alcohol to make to moisten, adds ethyl alcohol 50ml, ultrasonic extraction 30
Minute, centrifugation takes supernatant to be evaporated, and residue adds water saturated n-butanol 25ml to make to dissolve, the water being saturated with the n-butanol of equivalent
Washing 3 times, n-butanol liquid is evaporated, and residue adds methanol 2ml to make to dissolve, and centrifugation takes supernatant as test solution.It separately takes recklessly yellow
Even-II reference substance 1mg of glycosides, adds methanol 2ml that reference substance solution is made.According to thin-layered chromatography, (China's coastal port one attached
Record VI B) test, draw each 5 μ l of above two solution, put respectively on same silica gel thin-layer plate, with toluene-chloroform-methanol (5:
5:1) it is solvent, is unfolded, take out, dry, spray is with the color developing agents such as 10% sulfuric acid solution, colour developing.105 DEG C are heated to spot development
Clearly.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the spot of same color is shown.
D, geranium wilfordii, granatum identify: 10, Tang's blade sample is taken, it is finely ground, and add water 100ml, ultrasonic extraction 30 minutes, puts
Cold, centrifugation takes supernatant, adds ethyl acetate shaking to extract twice, each 40ml, combined ethyl acetate layer is evaporated, and residue adds second
Acetoacetic ester 1ml makes to dissolve, as test solution.Geranium wilfordii control medicinal material, each 2g of granatum control medicinal material separately are taken, respectively same method
Control medicinal material solution is made.It is tested according to thin-layered chromatography (one VI B of annex of China's coastal port), above-mentioned three kinds of absorption molten
Each 5 μ l of liquid puts respectively on same silica gel thin-layer plate, is solvent with benzene-chloroform-methanol-glacial acetic acid (5:5:2:0.5), opens up
Open, take out, dry, set smoked in iodine vapor it is clear to spot development.In sample chromatogram, corresponding with reference substance medicinal material chromatography
On position, the spot of same color is shown.
E, radix bupleuri identifies: 10, Tang's blade sample is taken, it is finely ground, and add water 50ml, ultrasonic extraction 30 minutes, let cool, is centrifuged, takes
Supernatant is extracted 3 times, each 50ml with ether shaking, discards ether solution, then shaken and extracted with the water saturated n-butanol of equivalent
3 times, merge n-butanol extracting liquid, washed with the ammonia solution of equivalent, then water washing 3 times be saturated with the n-butanol of equivalent, positive fourth
Alcohol liquid is evaporated, and residue adds methanol 1ml to make to dissolve, and centrifugation takes supernatant as test solution.Separately take the same method of radix bupleuri control medicinal material
Control medicinal material solution is made.It tests, draws above two molten according to thin-layered chromatography (one VI B of annex of China's coastal port)
Each 5 μ l of liquid puts respectively on same silica gel thin-layer plate, is solvent with ethyl acetate, alcohol and water (8:2:1), is unfolded, take out,
It dries, spray is with color developing agents such as 10% sulfuric acid solutions, colour developing, and 105 DEG C to be heated to spot development clear, sets and inspects under daylight.For examination
In product chromatography, on position corresponding with reference medicine chromatography, the principal spot of same color is shown.
F, ginkgo leaf identifies: 10, Tang's blade sample is taken, it is finely ground, and add 50% acetone 100ml, be heated to reflux, let cool, filters
It crosses, filtrate boils off acetone, and aqueous is shaken with ethyl acetate 50ml and extracted, and acetic acid ethyl fluid is evaporated, and 25% ethyl alcohol of residue makes molten
Solution, by polyamide column (90H, internal diameter 1.5cm, long 10cm), with 10% ethanol elution, eluent is concentrated into, and is let cool, and uses 50ml
Ethyl acetate shaking is extracted 3 times, is evaporated, residue makes to dissolve with 2ml acetone, as test solution.Ginkgo leaf control is separately taken to mention
Object 2g is taken, adds 1ml acetone in proper that reference substance solution is made.According to thin-layered chromatography (one annex VI B of Chinese Pharmacopoeia version in 2000)
Test, draws each 1 μ l of above two solution, is put respectively on same silica gel thin-layer plate, with ethyl acetate-butanone-methanol-water
(5:3:1:1) is solvent, is unfolded, and takes out, dries, set and inspect under ultraviolet lamp.In sample chromatogram, with reference substance chromatography
On corresponding position, the fluorescence spot of same color is shown.
G, Radix Astragali identifies: taking Astragaloside IV reference substance 2mg, adds 1ml methanol that reference substance solution is made.According to thin-layered chromatography
(one VI B of annex of China's coastal port) test draws reference substance solution and identifies each 5 μ of test solution under (E) item
L puts respectively on same silica gel thin-layer plate, with ethyl acetate, alcohol and water (8:2:1) solvent, is unfolded, takes out, dry, spray
With 10% sulfuric acid solution, it is clear that spot development is heated at 105 DEG C.In sample chromatogram, in position corresponding with reference substance chromatography
It sets, shows the spot of same color.
H, the identification of oldenlandia diffusa: taking 10, Tang's blade sample, finely ground, adds ethyl alcohol 50ml ultrasound 30 minutes, and filtrate is steamed
Dry, residue adds 50ml water to dissolve；50ml chloroform is added, extracts 2 times, collects solvent layer, is evaporated, residue adds the anhydrous second of 1ml
Alcohol dissolution is used as test sample；Oldenlandia diffusa control medicinal material 5g separately is taken, adds water 20ml, is heated to reflux 1.5 hours, is filtered, filtrate
50ml is added water to, is operated from being added chloroform with test sample, control medicinal material solution is made, according to thin-layered chromatography (middle traditional Chinese medicines
One VIB of allusion quotation version in 2010) test, each 10 μ l of above two solution is drawn, is put respectively on same silica gel g thin-layer plate, with stone
Oily ether-benzene-ethyl acetate-glacial acetic acid (5:10:3.5:0.25) is solvent, is unfolded, and takes out, dries, smoked in ammonia steam to spot
Point colour developing is clear, sets and inspects under daylight；In sample chromatogram, in position corresponding with reference medicine chromatography, same color is shown
Principal spot.
I, the identification of myrobalan: taking myrobalan control medicinal material 1g, add water 20ml, is heated to reflux 1.5 hours, and filtering, filtrate adds water
To 50ml, identifies test sample under item with oldenlandia diffusa from adding chloroform and operate, control medicinal material solution is made；According to thin layer color
Spectrometry (one VIB of Chinese Pharmacopoeia version in 2010) test, draws control medicinal material solution and above-mentioned (H) oldenlandia diffusa identifies under item
Each 10 μ l of test solution, put respectively on same silica gel g thin-layer plate, be with toluene-glacial acetic acid-water (15:5:0.5) lower layer
Solvent is unfolded, and takes out, dries, and spray is heated to clear spot at 105 DEG C with the colour developing of 10% sulfuric acid ethyl alcohol, and room temperature is set in taking-up
It (25 ± 2 DEG C) cooling half an hour, sets and is inspected under daylight.In sample chromatogram, in position corresponding with reference medicine chromatography, phase is shown
With the spot of color.
2) assay:
A, the picroside assay of gallic acid, the chlorogenic acid of honeysuckle, the liquiritin of Radix Glycyrrhizae, Radix picrorrhizae:
20, Tang's blade sample is taken, it is finely ground, 2g is taken, it is accurately weighed, it sets in stuffed conical flask, precision plus methanol 100ml are close
Plug, weighed weight are ultrasonically treated 45 minutes, are taken out, are put to room temperature (25 ± 2 DEG C), then weighed weight, supply less loss with methanol
Weight shakes up, filtration, is test solution, gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ reference substance are (purchased from China
Biological products assay institute): precision weighs gallic acid reference substance, chlorogenic acid reference substance, liquiritin reference substance, picroside Ⅱ pair
It is appropriate according to product, add methanol to be configured to every milliliter of 0.1mg containing gallic acid respectively, chlorogenic acid 0.09mg, liquiritin 0.04mg, it is recklessly yellow
Even glycosides 0.1mg mixed solution to get.
Chromatographic column is filler with octadecylsilane chemically bonded silica, mobile phase be acetonitrile-water-phosphoric acid (6-17:94-83:
0.3) gradient elution, Detection wavelength 275nm, precision draw reference substance solution and test solution, inject liquid chromatograph, shine
The one annex VID measurement of version in 2010 of high performance liquid chromatography Chinese Pharmacopoeia.Every Tang blade 1.2mg containing gallic acid, containing gold
Honeysuckle flower 0.8mg in terms of chlorogenic acid, containing Radix Glycyrrhizae 0.3mg in terms of liquiritin, containing Radix picrorrhizae 0.4mg in terms of picroside Ⅱ.
B, the galuteolin assay of honeysuckle:
Galuteolin: taking 10, Tang's blade sample, finely ground, takes about 3g, accurately weighed, sets in stuffed conical flask, and precision adds
Enter 70% ethyl alcohol 50ml, weighed weight is ultrasonically treated about 1 hour, lets cool, then weighed weight, supply less loss with 70% ethyl alcohol
Weight shakes up, and filtration takes subsequent filtrate, is test solution.With galuteolin reference substance, chromatographic column octadecylsilane key
Conjunction silica gel is filler, and mobile phase is acetonitrile mobile phase A, with 0.5% glacial acetic acid solution Mobile phase B, by 0~30 minute, A:B →
10:90 30~55 minutes, A:B → 30:70, carries out gradient elution；Detection wavelength is 350nm.Precision draw reference substance solution with
Test solution injects liquid chromatograph, measurement.Testing result: every Tang's blade in terms of galuteolin 7 μ g containing honeysuckle.
C, the Determination of Astragaloside of Radix Astragali:
10, Tang's blade sample is taken, it is finely ground, about 3g is taken, it is accurately weighed, it sets in triangular pyramidal bottle, methanol is added in precision
50ml, close plug, weighed weight are ultrasonically treated 40 minutes, let cool, again weighed weight, supply less loss weight with methanol, shake up,
Filtration, precision measure subsequent filtrate 40ml, water bath method, and residue adds water 10ml, and low-grade fever makes to dissolve, the n-butanol shaking being saturated with water
It extracts 4 times, each 40ml, merges n-butanol liquid, sufficiently washed 2 times with ammonia solution, each 40ml, discard ammoniacal liquor, n-butanol liquid
It is evaporated, residue adds water 5ml to make to dissolve, and lets cool, by D101 type large pore resin absorption column (internal diameter 1.5cm, long 12cm), with water
50ml elution discards aqueous, then is eluted with 40% ethyl alcohol 30ml, discards eluent, elutes after with 70% ethyl alcohol 80ml, collection is washed
De- liquid, is evaporated, is dissolved and be transferred in 5ml measuring bottle with methanol, adds methanol to scale, shakes up, be test solution.With Radix Astragali first
Glycosides reference substance, chromatographic column are filler with octadecylsilane chemically bonded silica, and mobile phase is acetonitrile-water (32:68)；Detector is
Evaporative light-scattering, precision draw 10 μ l of reference substance solution, 20 μ l, and 20 μ l of test solution injects liquid chromatograph, measure, with
External standard two-point method logarithmic equation calculates.Testing result: every Tang's blade in terms of Astragaloside IV 5 μ g containing Radix Astragali.
D, the hydroxyl radical carthamin yellow carthamus A assay of safflower:
10, Tang's blade sample is taken, it is finely ground, about 2g is taken, it is accurately weighed, it sets in stuffed conical flask, 25% methanol is added in precision
50ml, weighed weight are ultrasonically treated 40 minutes, let cool, then weighed weight, the weight of less loss is supplied with 25% methanol, is shaken up, and filter
It crosses, takes subsequent filtrate, be test solution.With hydroxyl radical carthamin yellow carthamus A reference substance, chromatographic column octadecylsilane chemically bonded silica
For filler, mobile phase is -0.7% phosphoric acid solution of methanol-acetonitrile (26:2:72), and Detection wavelength 403nm, precision is drawn molten
Liquid and test solution inject liquid chromatograph, measurement.Every Tang's blade of testing result is containing safflower with hydroxyl radical carthamin yellow carthamus A
Count 0.07mg.
By above-mentioned full inspection project confirmatory test, illustrate that the newly-increased detection project of the present invention can supplement the former detection side of addition
Method further increases the detection quality of drug, is conducive to improve product quality.
Claims (4)
1. a kind of measuring method of the newly-increased detection ingredient of Chinese medicine Tang blade for treating AIDS, which is characterized in that the newly-increased inspection
The measuring method for surveying ingredient includes the following steps:
(1) thin-layer chromatography chromatography identifies:
(1), oldenlandia diffusa identifies: Chinese medicine Tang blade sample is taken, it is finely ground, and add water 10-60ml directly to dissolve, centrifuging and taking supernatant
Liquid；Or ethyl alcohol 10-60ml ultrasound or reflux, filtrate is added to be evaporated, residue adds water 5-50ml to dissolve；Ethyl acetate or three chloromethanes are added
Alkane or n-butanol 5-50ml are extracted 1 to 2 time, collect solvent layer, are evaporated in 100 DEG C of water-baths, residue add 1-5ml ethyl acetate or
Dehydrated alcohol or methanol dissolution are used as test solution；It separately takes oldenlandia diffusa control medicinal material appropriate, adds water 20ml, be heated to reflux
1.5 hours, filtering, filtrate added water to 50ml, adds ethyl acetate or chloroform or n-butanol and operates with test sample, is made
Control medicinal material solution draws each 5-10 μ of above two solution according to the one VIB test of version in 2010 of thin-layered chromatography Chinese Pharmacopoeia
L, puts respectively on same silica gel g thin-layer plate, with hexamethylene-chloroform-ethyl acetate-glacial acetic acid 10:2.5:4:0.25 or
Petroleum ether-benzene-ethyl acetate-glacial acetic acid 5:10:3.5:0.25 is solvent, is unfolded, and takes out, dries, smoked in ammonia steam to spot
Point colour developing is clear, sets and inspects under daylight；In sample chromatogram, in position corresponding with reference medicine chromatography, same color is shown
Principal spot；
(2), myrobalan identifies: taking myrobalan control medicinal material 1g, adds water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds water to
50ml, after adding ethyl acetate or chloroform or n-butanol 5-50ml, same to above-mentioned steps (1) oldenlandia diffusa identifies under item
Test sample operation, is made control medicinal material solution；It is tested according to thin-layered chromatography, draws control medicinal material solution and above-mentioned steps (1) are white
Flower HERBA HEDYOTIS DIFFUSAE identifies each 5-10 μ l of test solution under item, is put respectively on same silica gel g thin-layer plate, with methylene chloride-ice
Acetic Acid-Water 15:5:0.5 lower layer or toluene-glacial acetic acid-water 15:5:0.5 lower layer are solvent, are unfolded, and take out, dry, spray with
The colour developing of 10% sulfuric acid ethyl alcohol, clear spot is heated at 105 DEG C, and taking-up sets 25 ± 2 DEG C of room temperature cooling half an hour, sets and examine under daylight
Depending in position corresponding with reference medicine chromatography, showing the spot of same color in sample chromatogram；
(2) high effective liquid chromatography for measuring:
Take Tang's blade sample appropriate, it is finely ground, 0.5g is taken, it is accurately weighed, it sets in stuffed conical flask, precision plus 50% or more methanol
25ml, close plug, weighed weight are ultrasonically treated 30 minutes, are taken out, are put to 25 ± 2 DEG C of room temperature, then weighed weight, supplied with methanol
The weight of less loss, shakes up, filtration, is test solution, with gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ preparation control
Product solution, chromatographic column are filler with octadecylsilane chemically bonded silica, and mobile phase is added sour gradient and is washed with organic solvent and water
De-, the mobile phase is selected from acetonitrile-water-phosphoric acid 6-17:94-83:0.3 or methanol-water-phosphoric acid 20-40:80-60:0.3, detection
Wavelength is 263nm-293nm, and precision draws reference substance solution and test solution, liquid chromatograph is injected, according to high-efficient liquid phase color
The one annex VID measurement of version in 2010 of spectrometry Chinese Pharmacopoeia；
Every Tang's blade is yellow not less than 0.25mg, Hu not less than 0.5mg, liquiritin not less than 0.9mg, chlorogenic acid containing gallic acid
Even glycosides II is not less than 0.45mg.
2. the measuring method of the newly-increased detection ingredient of a kind of Chinese medicine Tang blade for treating AIDS according to claim 1, special
Sign is that (one) the thin-layer chromatography chromatography identifies:
Step (1), oldenlandia diffusa identify: Chinese medicine Tang blade sample is taken, it is finely ground, and add ethyl alcohol 10-60ml ultrasound or reflux, filtrate
It is evaporated, residue adds water 5-50ml to dissolve；Ethyl acetate 5-50ml is added, extracts 2 times, collects solvent layer, is steamed in 100 DEG C of water-baths
Dry, residue adds the dissolution of 1-5ml methanol as test solution；It separately takes oldenlandia diffusa control medicinal material appropriate, adds water 20ml, heat
Reflux 1.5 hours, filtering, filtrate add water to 50ml, after adding ethyl acetate or chloroform or n-butanol 5-50ml, ibid
The operation of step (1) test sample is stated, control medicinal material solution is made, is tested according to thin-layered chromatography, draws each 5-10 μ of above two solution
L puts respectively on same silica gel g thin-layer plate, is with hexamethylene-chloroform-ethyl acetate-glacial acetic acid 10:2.5:4:0.25
Solvent is unfolded, and takes out, dries, it is clear to spot development to smoke in ammonia steam, sets and inspects under daylight；In sample chromatogram, with
The corresponding position of reference medicine chromatography shows the principal spot of same color.
3. the measuring method of the newly-increased detection ingredient of a kind of Chinese medicine Tang blade for treating AIDS according to claim 1, special
Sign is that (one) the thin-layer chromatography chromatography identifies:
Step (2), myrobalan identify: taking myrobalan control medicinal material 1g, add water 20ml, be heated to reflux 1.5 hours, filter, filtrate adds water
To 50ml, after adding ethyl acetate or chloroform or n-butanol 5-50ml, same to above-mentioned steps (1) oldenlandia diffusa identifies item
Lower test sample operation, is made control medicinal material solution；It is tested according to thin-layered chromatography, draws control medicinal material solution and above-mentioned steps (1)
Oldenlandia diffusa identifies each 5-10 μ l of test solution under item, is put respectively on same silica gel g thin-layer plate, with methylene chloride-
Glacial acetic acid-water 15:5:0.5 lower layer is solvent, is unfolded, and takes out, dries, and spray is heated with the colour developing of 10% sulfuric acid ethyl alcohol at 105 DEG C
To clear spot, 25 ± 2 DEG C of room temperature cooling half an hour are set in taking-up, set and inspect under daylight, in sample chromatogram, with control medicinal material
The corresponding position of chromatography shows the spot of same color.
4. the measuring method of the newly-increased detection ingredient of a kind of Chinese medicine Tang blade for treating AIDS according to claim 1, special
Sign is, (two) high effective liquid chromatography for measuring:
Take Tang's blade sample appropriate, it is finely ground, 0.5g is taken, it is accurately weighed, it sets in stuffed conical flask, precision plus 50% or more methanol
25ml, close plug, weighed weight are ultrasonically treated 30 minutes, are taken out, are put to 25 ± 2 DEG C of room temperature, then weighed weight, supplied with methanol
The weight of less loss, shakes up, filtration, is test solution, with gallic acid, chlorogenic acid, liquiritin, picroside Ⅱ preparation control
Product solution, chromatographic column are filler with octadecylsilane chemically bonded silica, and mobile phase is added sour gradient and is washed with organic solvent and water
De-, the mobile phase is acetonitrile-water-phosphoric acid 6-17:94-83:0.3, Detection wavelength 263nm-293nm, and precision draws control
Product solution and test solution inject liquid chromatograph, according to one annex VID of high performance liquid chromatography Chinese Pharmacopoeia version in 2010
Measurement.
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