CN103823016A - Detecting method of DanTianJingShu oral preparation - Google Patents
Detecting method of DanTianJingShu oral preparation Download PDFInfo
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- CN103823016A CN103823016A CN201410083023.3A CN201410083023A CN103823016A CN 103823016 A CN103823016 A CN 103823016A CN 201410083023 A CN201410083023 A CN 201410083023A CN 103823016 A CN103823016 A CN 103823016A
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Abstract
The invention discloses a detecting method of a DanTianJingShu oral preparation. The DanTianJingShu oral preparation is prepared from the root of red-rooted salvia, rhizoma gastrodiae, uncaria rhynchophylla, concha haliotidis, eucommia ulmoides, ligusticum wallichii, fructus gardenia and auxiliary materials. The detecting method comprises the following steps: measuring the content of the rhizoma gastrodiae in the oral preparation, and carrying out thin-layer chromatography identification on the root of red-rooted salvia, the uncaria rhynchophylla, the eucommia ulmoides, the ligusticum wallichii and the fructus gardenia in the oral preparation. The detecting method is accurate, high in sensitivity, good in repeatability, and stable in detecting result, and can control the quality of the DanTianJingShu oral preparation effectively.
Description
Technical field
The present invention relates to the detection method of the easypro oral formulations of a kind of red day neck, belong to the field of medicine technology;
Background technology
Vertebral artery type of cervical spondylosis is because vertebral artery suffers oppression or stimulates and cause the series of symptoms that its blood supply insufficiency produces.Cervical vertebra is the spinal segments of activity maximum, easily produces strain, and with advancing age and the accumulation of damage and cervical vertebra degeneration occurs.In Patients with Cervical Spondylopathy, approximately 70% has vertebral artery to get involved, more than 50 years old dizzy, Vertebral Artery Ischemic Disease person, and the vertebra basal arteries that more than 50% cause with cervical spondylopathy are got involved relevant.Easily have clinically " cervical vertebra is dizzy ", diagnosis such as " Vertebral artery compression syndromes ", is called again " the inclined to one side Vertebral Artery Ischemic Disease of neck ".Vertebral artery type of cervical spondylosis is caused vertebral artery to be stimulated or oppressed by various mechanicalnesses and dynamic property factor, thus hemadostewnosis, warpage and cause take vertebra one by one basilar arterial blood supply not entirely as the syndrome of cardinal symptom.Its pathogenesis mainly contains dynamic property factor and vascular factor.Along with the quickening of human lives's rhythm, the increase of working pressure, the people of (CSA) is more and more to make to suffer from vertebral artery type of cervical spondylosis, and age of onset is on a declining curve, has had a strong impact on people's quality of life.Red day neck preparation that relaxes derives from decoction " dizzy drink disappearing ", is made up of the seven taste Chinese medicines such as rhizoma Gastrodiae, the red sage root, be the whole nation bone fracture well-known doctor Shen Fengjun professor of Guiyang College of Traditional Chinese Medicine 35 years through proved recipe, there is flat liver kidney tonifying, effect promoting blood circulation and removing blood stasis; The clinical vertebral artery type of cervical spondylosis (vertiginous type) that is used for the treatment of caused by liver and kidney deficiency, qi depression to blood stasis and cause is respond well; In order better to control the quality of this medicine, for this reason, the invention provides the detection method of the easypro preparation of red day neck.
Summary of the invention
Technical matters to be solved by this invention is, the detection method that provides a kind of red day neck to relax oral formulations; Described detection method can be measured the relax content of Gastrodin in oral formulations of red day neck, and the red sage root, yncaria stem with hooks, the bark of eucommia, Ligusticum wallichii and cape jasmine in can also oral disposition preparation be differentiated, detection method convenient operation of the present invention is reproducible, reliable results;
For solving the problems of the technologies described above, the present invention adopts following technical scheme to realize:
The easypro oral formulations of a kind of red day neck, calculate according to components by weight percent, be prepared from by red sage root 200-700 part, rhizoma Gastrodiae 200-700 part, yncaria stem with hooks 200-700 part, shell of seaear 600-1200 part, bark of eucommia 300-800 part, Ligusticum wallichii 200-700 part, cape jasmine 200-700 part and auxiliary material;
Specifically, the easypro oral formulations of described red day neck calculates according to composition by weight, is prepared from by 500 parts of the reds sage root, 500 parts, rhizoma Gastrodiae, 500 parts of yncaria stems with hooks, 900 parts of the shells of seaear, 625 parts of the barks of eucommia, 500 parts of Ligusticum wallichiis, 500 parts of cape jasmines and auxiliary material;
Its preparation method is: take each medicine according to formula, by water boiling and extraction, filter, filtrate is concentrated into thick paste, dry, pulverize, then adds auxiliary material to be prepared into oral formulations according to common process;
Specifically, its preparation method is: take each medicine according to formula, add 6-12 times of water gaging to decoct 1-3 time, each 0.5-3 hour, filters, and filtrate is concentrated into thick paste, dry, pulverize, then adds auxiliary material to be prepared into oral formulations according to common process;
Described detection method comprises that the content of Gastrodin in oral disposition preparation measures, and the red sage root, yncaria stem with hooks, the bark of eucommia, Ligusticum wallichii and the cape jasmine in oral disposition preparation carries out respectively thin-layer chromatography discriminating.
In above-mentioned detection method, the content assaying method of described Gastrodin is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.05% phosphoric acid solution=1-10:99-90 as mobile phase; Detection wavelength is 220nm; Number of theoretical plate calculates and should be not less than 5000 by Gastrodin peak;
The preparation of reference substance solution: precision takes rhizoma Gastrodiae reference substance, with mobile phase dissolving, to obtain final product;
The preparation of need testing solution: take preparation medicine, accurately weighed, the ultrasonic 5-20min of adding distil water makes to dissolve, and lets cool, and filters, and gets subsequent filtrate and get final product;
Determination method: draw respectively reference substance solution and need testing solution, inject high performance liquid chromatograph, measure, to obtain final product.
Specifically, the content assaying method of described Gastrodin is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.05% phosphoric acid solution=8:92 as mobile phase; Flow velocity 1.0mLmin
-1, detection wavelength is 220nm; Number of theoretical plate calculates and should be not less than 5000 by Gastrodin peak;
The preparation of reference substance solution: precision takes at the rhizoma Gastrodiae reference substance of 80 ℃ of drying under reduced pressure 1h appropriate, adds mobile phase and makes the solution of every 1mL containing 30 μ g, to obtain final product;
The preparation of need testing solution: take preparation medicine 0.2g, accurately weighed, to 50mL volumetric flask, the appropriate ultrasonic 10min of adding distil water makes to dissolve, and lets cool, and is settled to scale, filters, and gets subsequent filtrate and get final product;
Determination method: draw respectively the each 10L of reference substance solution and need testing solution, inject high performance liquid chromatograph, measure, to obtain final product.
In above-mentioned detection method, the thin-layer identification method of described yncaria stem with hooks is: the thing 3g that gets it filled, adds methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, the 30mL that adds water makes to dissolve, add strong aqua to adjust pH value to 9, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Another hook taking rattan control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=5: 3 as developping agent, launch, take out, dry; Spray is with the colour developing of improvement bismuth potassium iodide test solution; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of described Ligusticum wallichii is: the thing 3g that gets it filled, add methyl alcohol 50mL, and refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get Ligusticum wallichii control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=10: 3 as developping agent, launch, take out, dry; Under 365nm uviol lamp, observe; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of described cape jasmine is: the thing 3g that gets it filled, add acetone 50mL, and 60 ℃ of water-bath backflow 1h, filter, filtrate evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample need testing solution; Separately get cape jasmine control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 2: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray, with 10% sulfuric acid ethanol test solution, is dried to colour developing at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of the described red sage root is: the thing 3g that gets it filled, add methyl alcohol 50mL, and refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merge ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get red sage root control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 1: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of the described bark of eucommia is: the thing 3g that gets it filled, add methyl alcohol 50mL, and refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merge ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get bark of eucommia control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, take toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developping agent, launch, take out, dry; After observing under 365nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Inventor has carried out a large amount of experiments, is below the research of detection method of the present invention
Experimental example: detection method research
1 instrument and reagent
1.1 instruments: Japanese Shimadzu LC-20A type liquid chromatograph, SPD-20A type UV-visible detector (Japanese Shimadzu), HT-20 type column oven; JA2003 type electronic balance (the above flat instrument and meter of current chart company limited); Auy220 type electronic balance (Japanese Shimadzu); Section leads the desk-top ultrasonic cleaner of SK8210HP type (Shanghai High Kudos Science Instrument Co., Ltd.); Bark of eucommia F-6210 vacuum drying chamber (Shanghai Qi Xin scientific instrument company limited); WBZ-2 micro-wave vacuum case (Kweiyang novel Microwave Industry Ltd); The automatic pure water distiller of SZ-96 (Shanghai Yarong Biochemical Instrument Plant); LRH-150S constant temperature and humidity incubator (Guangdong Medical Apparatus and Instruments Factory); ZB-1C Intelligent disintegration tester (Precision Instrument Factory, Tianjin Univ.); TDL-5A low speed automatic centrifuge (Hunan Xing Ke Science and Technology Ltd.); WD-A stability somascope (Tianjin Pharmacopoeia Standard Instrument Factory); DC-B-1 Intelligent box type resistance furnace (Beijing original creation Science and Technology Ltd.).
1.2 reagents: Gastrodin (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 0807-200104); In prescription, ingredients, all purchased from Kweiyang medicinal material main office, all meets the examination criteria of pharmacopeia; Silica G, silica GF254 (Qingdao Haiyang chemical industry), hplc grade methanol (Hanbon Sci. & Tech. Co., Ltd.), chromatographic grade second eyeball (Hanbon Sci. & Tech. Co., Ltd.), water is ultrapure water, it is pure that other reagent are analysis.
Within red day, neck relieving capsule is prepared by the following method: get red sage root 500g, rhizoma Gastrodiae 500g, yncaria stem with hooks 500g, shell of seaear 900g, bark of eucommia 625g, Ligusticum wallichii 500g, cape jasmine 500g; Add 8 times of water gagings to decoct 3 times, each 1.5 hours, filter, filtrate is concentrated into thick paste, and micro-wave vacuum is pulverized, and adds starch, encapsulated, makes 1000, and every 0.45g, to obtain final product.
2. differentiate
The thin layer of 2.1 reds sage root is differentiated
Get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, and each 25mL, merges ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid.Separately get red sage root control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution.With the standby negative control solution that lacks the red sage root of legal system.Test according to pharmacopeia appendix VI B thin-layered chromatography.Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene-chloroform-ethyl acetate-methyl alcohol-formic acid (2: 3: 4: 1: 2) as developping agent, launch, take out, dry.After observing under 254nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing.Through three batch samples experiments, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.Negative control is noiseless (thin-layer chromatogram is shown in Fig. 1, Fig. 2).
The thin layer of 2.2 yncaria stems with hooks is differentiated
Get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, the 30mL that adds water makes to dissolve, and adds strong aqua to adjust pH value to 9, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid.Another hook taking rattan control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution.With the standby negative control solution that lacks yncaria stem with hooks of legal system.Test according to pharmacopeia appendix VI B thin-layered chromatography.Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform-ethyl acetate (5: 3) as developping agent, launch, take out, dry.Spray is with the colour developing of improvement bismuth potassium iodide test solution.Through three batch samples tests, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.Negative control is noiseless (thin-layer chromatogram is shown in Fig. 3).
The thin layer of 2.3 Ligusticum wallichiis is differentiated
Get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, chloroform extraction 3 times, and each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid.Separately get Ligusticum wallichii control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution.With the standby negative control solution that lacks Ligusticum wallichii of legal system.Test according to pharmacopeia appendix VI B thin-layered chromatography.Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform-ethyl acetate (10: 3) as developping agent, launch, take out, dry.Under 365nm uviol lamp, observe.Through three batch samples tests, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescent spot point spot of aobvious same color.Negative control is noiseless (thin-layer chromatogram is shown in Fig. 4).
The thin layer of 2.4 barks of eucommia is differentiated
Get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, and each 25mL, merges ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid.Separately get bark of eucommia control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution.With the standby negative control solution that lacks the bark of eucommia of legal system.Test according to pharmacopeia appendix VI B thin-layered chromatography.Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, take toluene-chloroform-ethyl acetate-formic acid (5: 1: 1: 0.5) as developping agent, launch, take out, dry.After observing under 365nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing.Through three batch samples experiments, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.Negative noiseless (thin-layer chromatogram is shown in Fig. 5, Fig. 6).
The thin layer of 2.5 cape jasmines is differentiated
Get capsule 's content 3g, add acetone 50mL, 60 ℃ of water-bath backflow 1h, filter, filtrate evaporate to dryness, and residue adds methyl alcohol 2mL to be made to dissolve, as sample need testing solution.Separately get cape jasmine control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution.With the standby negative control solution that lacks cape jasmine of legal system.Test according to pharmacopeia appendix VI B thin-layered chromatography.Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene-chloroform-ethyl acetate-methyl alcohol-formic acid (2: 3: 4: 2: 2) as developping agent, launch, take out, dry.After observing under 254nm uviol lamp, spray, with 10% sulfuric acid ethanol test solution, is dried to colour developing at 105 ℃.Through three batch samples experiments, in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.Negative control is noiseless (thin-layer chromatogram is shown in Fig. 7, Fig. 8).
3. gastrodin content method for measuring research
Measure according to high performance liquid chromatography (appendix VI D of " Chinese Pharmacopoeia " version in 2010).
3.1 chromatographic condition
Chromatographic column be Agilent HC-C18 post (250mm × 4.6mm, 5 μ m); Mobile phase is methyl alcohol: 0.05% phosphoric acid solution (8: 92), flow velocity 1.0mLmin
-1, detect wavelength 220nm, sample size 10 μ L, column temperature is room temperature.
The preparation of 3.2 reference substance solution
Learn from else's experience 80 ℃ and be dried to the Gastrodin reference substance 3.50mg of constant weight, put in 25mL volumetric flask, accurately weighed, add mobile phase and dissolve, be diluted to scale, shake up, obtain 0.14mgmL
-1reference substance mother liquor, the accurate 2mL mother liquor of drawing is put in 10mL volumetric flask, is diluted to scale, obtains 0.028mgmL
-1reference substance solution.
3.3 extract the investigation of solvent
Precision takes red day neck relieving capsule content 1g, accurately weighed, adds respectively various solvent 100mL in following table, accurately weighed, ultrasonic extraction 30min, lets cool, add corresponding solvent to supply weight, divided by distilled water as outside solvent, all the other sample liquid, filter respectively accurate subsequent filtrate 10mL, the evaporate to dryness drawn, add appropriate mobile phase and dissolve, be transferred in 10mL volumetric flask, be settled to respectively scale, filter, subsequent filtrate is for assay.The results are shown in Table 1.
Table 1 extracts the investigation of solvent
Result shows: distilled water is extracted into dissolving as extracting solvent with ultrasonic, and the time is short, and method is simple, and content is high.Because peak area is larger, therefore adopt and reduce sampling amount, reduce solvent use amount and reach sample peak area and standard items peak area similar purpose, to reduce the error of assay.
The investigation of 3.4 extraction times
According to 5.3 result, get red day neck relieving capsule content 0.2g, accurately weighed after to 50mL volumetric flask, add the ultrasonic different time of distilled water, let cool, be settled to scale, filter, get subsequent filtrate for assay.Result and method are in table 2.
The investigation of table 2 extraction time
Result shows, the sample content of Gastrodin in ultrasonic 10min to 30min that adds water does not have conspicuousness to change, and according to the observation, when sample ultrasonic 10min, is dissolved in completely in solvent.Therefore selecting ultrasonic extraction time is 10min.
The preparation of 3.5 need testing solutions
Take red day neck relieving capsule content 0.2g, accurately weighed, to 50mL volumetric flask, the appropriate ultrasonic 10min of adding distil water makes to dissolve, and lets cool, and is settled to scale, filters, and gets subsequent filtrate for assay.
The investigation of 3.6 mobile phases
According to list of references, select second eyeball: 0.05% phosphoric acid solution, methyl alcohol: 0.05% phosphoric acid solution, methyl alcohol: 0.1% glacial acetic acid, methyl alcohol: water is mobile phase, result shows with methyl alcohol: when 0.05% phosphoric acid solution (8: 92), can obtain good peak type, Gastrodin peak separates enough completely with other adjacent peak energy.Investigate the impact of different temperatures on test, result shows that 20 ℃-30 ℃ appearance times on target peak and content do not have the impact of conspicuousness simultaneously.
3.7 specificities are investigated
Get red day neck relieving capsule sample 0.2g, accurately weighed, by need testing solution, preparation method prepares negative control solution, the accurate Gastrodin reference substance solution of drawing, the each 10 μ L of negative need testing solution, inject high performance liquid chromatograph, and result shows, in test sample peak, Gastrodin peak energy enough reaches baseline separation, and disturb without other peaks the adjacent both sides at peak, and degree of separation is 23.777, negative test sample, not occurring peak with corresponding position, Gastrodin peak, shows that the specificity of method is good.The results are shown in Figure 9-Figure 11.
3.8 linearities and scope
The accurate reference substance mother liquor of drawing is diluted to concentration respectively: 7 μ gmL
-1, 14 μ gmL
-1, 21 μ gmL
-1, 28 μ gmL
-1, 35 μ gmL
-1, 42 μ gmL
-1, the accurate each 10 μ L of above-mentioned solution that draw inject high performance liquid chromatographs and measure peak area respectively, and take the quality of reference substance as horizontal ordinate, peak area is ordinate, linear regression, obtaining regression equation is y=1986.8x+6870.5, R=0.9999.Result shows that Gastrodin is good linear relationship within the scope of 70～420ng.The results are shown in Table 3, linear relationship is shown in Figure 12.
The investigation of table 3 range of linearity
3.9 precision test
Accurate absorption concentration is 7 μ gmL
-1, 28 μ gmL
-1, 42 μ gmL
-1gastrodin reference substance solution 10 μ L, continuous sample introduction 5 times respectively, with calculated by peak area RSD value, RSD is respectively 0.46%, 0.91%0.63%, shows that the precision of instrument is good.The results are shown in Table 4.
Table 4 Precision Experiment
3.10 repeatability is investigated
Get 80%, 100%, 120% each 3 parts of content 0.2g of red day neck relieving capsule, accurately weighed, prepare 9 parts of 3 concentration need testing solutions by the preparation method of need testing solution, filter, subsequent filtrate is for assay.Calculate content, the results are shown in Table 5.
Table 5 repeatability is investigated
3.11 study on the stability
Get the content 0.2g of red day neck relieving capsule, accurately weighed, by preparation method's processing sample of need testing solution, respectively at 0,2,4,6,8,12h working sample content, and to calculate RSD be 1.09%.Result shows, stablizes, in table 6 after sample preparation in 12 hours.
Table 6 study on the stability
3.12 the recovery is investigated
Take totally 9 parts of the content 0.1g of red day neck relieving capsule of known content, be divided into 3 groups, every group 3 parts, take the content in sample as standard, according to the principle of 80%, 100%, 120% reference substance that adds standard, precision adds the Gastrodin standard solution that content is suitable, prepares 9 parts of 3 concentration sample solutions by the preparation method of need testing solution, filter, subsequent filtrate is for assay.Calculate recovery rate.The results are shown in Table 7.
The investigation of table 7 recovery
Result shows that the recovery of sample is that 100.5%, RSD is 0.58%, and the method recovery is good.
The assay of 3.13 ten batches of samples
Get each 2 parts of the content 0.2g of red day neck relieving capsule of 10 different lot numbers, accurately weighed, by preparation method's processing sample of need testing solution, filter subsequent filtrate confession assay.Calculate content, the results are shown in Table 8.
Table 8 sample size is measured (n=2)
According to the known optimum determining method of measuring gastrodin content in red day neck relieving capsule of above experiment be: adopt HPLC method, Agilent HC-C
18; Flow velocity: 1.0mLmin
-1; Mobile phase: methyl alcohol-0.05% phosphoric acid solution (8:92); Column temperature: room temperature; Sample size: 10 μ L; Detect wavelength: 220nm; Using distilled water as extracting the ultrasonic Gastrodin extracting in red sky neck relieving capsule content for solvent.Institute's construction method is easy fast, result is accurate, reproducible, can be used as the content assaying method of Gastrodin in red day neck relieving capsule.
Beneficial effect of the present invention is: the invention provides the detection method of the easypro oral formulations of red day neck, in comprising writing out a prescription, monarch drug in a prescription Gastrodin in Gastrodia eleta Bl. is index, adopts the content of high effective liquid chromatography for measuring Gastrodin; And thin-layer chromatography (TLC) discrimination method of the red sage root, Ligusticum wallichii, yncaria stem with hooks, cape jasmine and the bark of eucommia in medicine; Described detection method is accurate, highly sensitive, reproducible, testing result is stable, can effectively control the relax quality of oral formulations of red day neck, both be more conducive to the monitoring to product quality of manufacturer and supervisory and management department, also can provide better guarantee for medical department and patient's treatment.
Accompanying drawing explanation
Fig. 1 is the fluorescent thin-layer chromatography figure of the red sage root of the present invention
Fig. 2 is the colour developing thin-layer chromatogram of the red sage root of the present invention
Fig. 3 is the thin-layer chromatogram of yncaria stem with hooks of the present invention
Fig. 4 is the thin-layer chromatogram of Ligusticum wallichii of the present invention
Fig. 5 is the fluorescent thin-layer chromatography figure of the bark of eucommia of the present invention
Fig. 6 is the colour developing thin-layer chromatogram of the bark of eucommia of the present invention
Fig. 7 is the fluorescent thin-layer chromatography figure of cape jasmine of the present invention
Fig. 8 is the colour developing thin-layer chromatogram of cape jasmine of the present invention
Fig. 9 is the high-efficient liquid phase chromatogram of Gastrodin standard items of the present invention
Figure 10 is the high-efficient liquid phase chromatogram of the negative test sample of the present invention
Figure 11 is the high-efficient liquid phase chromatogram of sample of the present invention
Figure 12 is Gastrodin canonical plotting of the present invention
Below in conjunction with embodiment, the present invention is further illustrated;
Embodiment
Embodiment 1: the content assaying method of Gastrodin in red day neck relieving capsule
Within red day, neck relieving capsule is prepared like this: get red sage root 500g, rhizoma Gastrodiae 500g, yncaria stem with hooks 500g, shell of seaear 900g, bark of eucommia 625g, Ligusticum wallichii 500g, cape jasmine 500g; Add 8 times of water gagings to decoct 3 times, each 1.5 hours, filter, filtrate is concentrated into thick paste, and micro-wave vacuum is pulverized, and adds starch, encapsulated, makes 1000, and every 0.45g, to obtain final product.
The content assaying method of Gastrodin is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.05% phosphoric acid solution=8:92 as mobile phase; Flow velocity 1.0mLmin
-1, detection wavelength is 220nm; Number of theoretical plate calculates and should be not less than 5000 by Gastrodin peak;
The preparation of reference substance solution: precision takes at the rhizoma Gastrodiae reference substance of 80 ℃ of drying under reduced pressure 1h appropriate, adds mobile phase and makes the solution of every 1mL containing 30 μ g, to obtain final product;
The preparation of need testing solution: take capsule 's content 0.2g, accurately weighed, to 50mL volumetric flask, the appropriate ultrasonic 10min of adding distil water makes to dissolve, and lets cool, and is settled to scale, filters, and gets subsequent filtrate and get final product;
Determination method: draw respectively the each 5-10L of reference substance solution and need testing solution, inject high performance liquid chromatograph, measure, to obtain final product.
This product is containing rhizoma Gastrodiae in Gastrodin, and every must not be less than 3.00mg.
Embodiment 2: the TLC Identification of yncaria stem with hooks in red day neck relieving capsule
Within described red day, neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of yncaria stem with hooks is: get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, and adds strong aqua to adjust pH value to 9, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Another hook taking rattan control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix VI B thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=5: 3 as developping agent, launch, take out, dry; Spray is with the colour developing of improvement bismuth potassium iodide test solution; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Embodiment 3: the TLC Identification of Ligusticum wallichii in red day neck relieving capsule
Within described red day, neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of Ligusticum wallichii is: get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get Ligusticum wallichii control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix VI B thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=10: 3 as developping agent, launch, take out, dry; Under 365nm uviol lamp, observe; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
Embodiment 4: the TLC Identification of cape jasmine in red day neck relieving capsule
Within described red day, neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of cape jasmine is: get capsule 's content 3g, add acetone 50mL, 60 ℃ of water-bath backflow 1h, filter, filtrate evaporate to dryness, and residue adds methyl alcohol 2mL to be made to dissolve, as sample need testing solution; Separately get cape jasmine control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix VI B thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 2: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray, with 10% sulfuric acid ethanol test solution, is dried to colour developing at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Embodiment 5: the TLC Identification of the red sage root in red day neck relieving capsule
Within described red day, neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of the red sage root is: get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merge ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get red sage root control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix VI B thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 1: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Embodiment 6: the TLC Identification of the bark of eucommia in red day neck relieving capsule
Within described red day, neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of the bark of eucommia is: get capsule 's content 3g, add methyl alcohol 50mL, refluxing extraction 1h, filters, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merge ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get bark of eucommia control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix VI B thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, take toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developping agent, launch, take out, dry; After observing under 365nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
Claims (8)
1. the detection method of the easypro oral formulations of red day neck, it is characterized in that: the easypro oral formulations of described red day neck calculates according to components by weight percent, be prepared from by the following method by red sage root 200-700 part, rhizoma Gastrodiae 200-700 part, yncaria stem with hooks 200-700 part, shell of seaear 600-1200 part, bark of eucommia 300-800 part, Ligusticum wallichii 200-700 part, cape jasmine 200-700 part and auxiliary material: take each medicine according to formula, by water boiling and extraction, filter, filtrate is concentrated into thick paste, dry, pulverize, then add auxiliary material to be prepared into conventional oral formulations according to common process; Described detection method comprises that the content of Gastrodin in oral disposition preparation measures, and the red sage root, yncaria stem with hooks, the bark of eucommia, Ligusticum wallichii and the cape jasmine in oral disposition preparation carries out respectively thin-layer chromatography discriminating.
2. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: the content assaying method of described Gastrodin is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.05% phosphoric acid solution=1-10:99-90 as mobile phase; Detection wavelength is 220nm; Number of theoretical plate calculates and should be not less than 5000 by Gastrodin peak;
The preparation of reference substance solution: precision takes rhizoma Gastrodiae reference substance, with mobile phase dissolving, to obtain final product;
The preparation of need testing solution: take preparation medicine, accurately weighed, the ultrasonic 5-20min of adding distil water makes to dissolve, and lets cool, and filters, and gets subsequent filtrate and get final product;
Determination method: draw respectively reference substance solution and need testing solution, inject high performance liquid chromatograph, measure, to obtain final product.
3. the relax detection method of oral formulations of red day neck as claimed in claim 2, it is characterized in that: the detection method of the easypro oral formulations of red day neck as claimed in claim 1, is characterized in that: the content assaying method of described Gastrodin is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.05% phosphoric acid solution=8:92 as mobile phase; Flow velocity 1.0mLmin
-1, detection wavelength is 220nm; Number of theoretical plate calculates and should be not less than 5000 by Gastrodin peak;
The preparation of reference substance solution: precision takes at the rhizoma Gastrodiae reference substance of 80 ℃ of drying under reduced pressure 1h appropriate, adds mobile phase and makes the solution of every 1mL containing 30 μ g, to obtain final product;
The preparation of need testing solution: take preparation medicine 0.2g, accurately weighed, to 50mL volumetric flask, the appropriate ultrasonic 10min of adding distil water makes to dissolve, and lets cool, and is settled to scale, filters, and gets subsequent filtrate and get final product;
Determination method: draw respectively the each 10L of reference substance solution and need testing solution, inject high performance liquid chromatograph, measure, to obtain final product.
4. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: in described preparation, the thin-layer identification method of yncaria stem with hooks is: the thing 3g that gets it filled, adds methyl alcohol 50mL, refluxing extraction 1h, filters, and filtrate water-bath volatilizes, the 30mL that adds water makes to dissolve, add strong aqua to adjust pH value to 9, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Another hook taking rattan control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=5: 3 as developping agent, launch, take out, dry; Spray is with the colour developing of improvement bismuth potassium iodide test solution; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
5. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: in described preparation, the thin-layer identification method of Ligusticum wallichii is: the thing 3g that gets it filled, adds methyl alcohol 50mL, refluxing extraction 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, chloroform extraction 3 times, each 25mL, combined chloroform liquid, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get Ligusticum wallichii control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, after ammoniacal liquor cylinder is saturated, take chloroform: ethyl acetate=10: 3 as developping agent, launch, take out, dry; Under 365nm uviol lamp, observe; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
6. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: in described preparation, the thin-layer identification method of cape jasmine is: the thing 3g that gets it filled, adds acetone 50mL, 60 ℃ of water-bath backflow 1h, filter filtrate evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample need testing solution; Separately get cape jasmine control medicinal material 2.5g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 2: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray, with 10% sulfuric acid ethanol test solution, is dried to colour developing at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
7. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: in described preparation, the thin-layer identification method of the red sage root is: the thing 3g that gets it filled, adds methyl alcohol 50mL, refluxing extraction 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merges ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get red sage root control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively in the same silica G F take 0.5%CMC-Na as binder
254on thin layer plate, take toluene: chloroform: ethyl acetate: methyl alcohol: formic acid=2: 3: 4: 1: 2 as developping agent, launch, take out, dry; After observing under 254nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
8. the relax detection method of oral formulations of red day neck as claimed in claim 1, is characterized in that: in described preparation, the thin-layer identification method of the bark of eucommia is: the thing 3g that gets it filled, adds methyl alcohol 50mL, refluxing extraction 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes to dissolve, extracted with diethyl ether 3 times, each 25mL, merges ether solution, evaporate to dryness, residue adds methyl alcohol 2mL to be made to dissolve, as sample test liquid; Separately get bark of eucommia control medicinal material 3g, by sample test liquid, preparation method prepares control medicinal material solution; Test according to pharmacopeia appendix thin-layered chromatography; Draw the each 10 μ L of above-mentioned three kinds of solution, put respectively on the same silica gel g thin-layer plate take 0.5%CMC-Na as binder, take toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developping agent, launch, take out, dry; After observing under 365nm uviol lamp, spray is with 2% ferric trichloride-1% iron potassuim cyanide test liquid colour developing; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
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| CN111458451A (en) * | 2020-04-10 | 2020-07-28 | 河北中医学院 | Rapid multi-information thin-layer identification method for eucommia ulmoides charcoal medicinal materials, particles and standard decoction powder |
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| CN107389810A (en) * | 2017-07-03 | 2017-11-24 | 广西中医药大学 | A kind of content assaying method of strong drug composition |
| CN108693296A (en) * | 2018-05-23 | 2018-10-23 | 云南中丹红制药有限责任公司 | SHUMITONG JIAONANG and its raw material detection method |
| CN111458451A (en) * | 2020-04-10 | 2020-07-28 | 河北中医学院 | Rapid multi-information thin-layer identification method for eucommia ulmoides charcoal medicinal materials, particles and standard decoction powder |
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