CN103823016B - Detecting method of DanTianJingShu oral preparation - Google Patents
Detecting method of DanTianJingShu oral preparation Download PDFInfo
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Landscapes
- Medicines Containing Plant Substances (AREA)
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 a kind of red sky neck relax the detection method of oral formulations, belong to the field of medicine technology；
Background technology
Vertebral artery type of cervical spondylosis is because vertebral artery and suffers oppression or stimulate and cause produced by its blood supply insufficiency a series of
Symptom.Cervical vertebra is the spinal segments that activity is maximum, is easily generated strain, and with advancing age and the accumulation of damage and occur
Cervical vertebra degeneration.In Patients with Cervical Spondylopathy, about 70% has vertebral artery to get involved, and more than 50 years old is dizzy, Vertebral Artery Ischemic Disease person,
More than 50% Vertebral-basilar artery caused with cervical spondylosis is got involved relevant.Tending to have " cervical vertebra dizziness " clinically, " Vertebral artery compression is combined
Close disease and " wait diagnosis, be also called " the inclined Vertebral Artery Ischemic Disease of neck ".Vertebral artery type of cervical spondylosis is by various mechanicalnesses and power
Sexual factor causes vertebral artery to be stimulated or oppresses, thus angiostenosis, warpage and cause with vertebra basilar arterial blood supply one by one not
It is all the syndrome of cardinal symptom.The main dynamic sexual factor of its pathogenesis and vascular factor.Along with human lives's rhythm
Accelerate, the increase of operating pressure so that suffer from vertebral artery type of cervical spondylosis (CSA) people get more and more, and age of onset under
Fall trend, has had a strong impact on the quality of life of people.The neck preparation that relaxes in red sky derives from decoction " disappearing dizzy drink ", by Rhizoma Gastrodiae, Radix Salviae Miltiorrhizae etc.
Seven taste Chinese medicine compositions, are Guiyang College of Traditional Chinese Medicine whole nation bone injury well-known doctor Shen Fengjun professor's Empirical formula of 35 years, have suppressing the hyperactive liver the kidney invigorating, live
Effect of blood blood stasis dispelling；Be clinically used for treating caused by liver and kidney deficiency, qi depression to blood stasis and vertebral artery type of cervical spondylosis (vertiginous type) effect that causes is good
Good；In order to preferably control the quality of this medicine, relax the detection method of preparation to this end, the invention provides red sky neck.
Summary of the invention
The technical problem to be solved is, it is provided that a kind of red sky neck relaxes the detection method of oral formulations；Described
Detection method can measure red sky neck and relax the content of gastrodine in oral formulations, moreover it is possible to the Radix Salviae Miltiorrhizae in oral disposition preparation, Ramulus Uncariae Cum Uncis, Du
Secondary, Rhizoma Chuanxiong and Fructus Gardeniae differentiate, detection method of the present invention is convenient to operate, reproducible, reliable results；
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that realization:
A kind of red sky neck relaxes oral formulations, calculates according to components by weight percent, by Radix Salviae Miltiorrhizae 200-700 part, Rhizoma Gastrodiae 200-700 part,
Ramulus Uncariae Cum Uncis 200-700 part, Concha Haliotidis 600-1200 part, Cortex Eucommiae 300-800 part, Rhizoma Chuanxiong 200-700 part, Fructus Gardeniae 200-700 part and auxiliary
Material is prepared from；
Specifically, the neck oral formulations that relaxes in described red sky calculates according to composition by weight, by Radix Salviae Miltiorrhizae 500 parts, 500 parts of Rhizoma Gastrodiae,
Ramulus Uncariae Cum Uncis 500 parts, Concha Haliotidis 900 parts, the Cortex Eucommiae 625 parts, Rhizoma Chuanxiong 500 parts, Fructus Gardeniae 500 parts and adjuvant are prepared from；
Its preparation method is: weigh each medicine according to formula, by water boiling and extraction, filters, and filtrate is concentrated into thick paste, dry
Dry, pulverize, add adjuvant and be prepared as oral formulations according to common process；
Specifically, its preparation method is: weigh each medicine according to formula, adds 6-12 times amount soak by water 1-3 time, every time
0.5-3 hour, filtering, filtrate is concentrated into thick paste, dry, pulverize, and adds adjuvant and is prepared as oral formulations according to common process；
Described detection method includes that in oral disposition preparation, the content of gastrodine is measured, Radix Salviae Miltiorrhizae in oral disposition preparation,
Ramulus Uncariae Cum Uncis, the Cortex Eucommiae, Rhizoma Chuanxiong and Fructus Gardeniae carry out indentification by TLC respectively.
In above-mentioned detection method, the content assaying method of described gastrodine is: survey according to Chinese Pharmacopoeia high performance liquid chromatography
Fixed；
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica as filler；With methanol: 0.05%
Phosphoric acid solution=1-10:99-90 is flowing phase；Detection wavelength is 220nm；Number of theoretical plate is calculated should be not less than by gastrodine peak
5000；
The preparation of reference substance solution: precision weighs Rhizoma Gastrodiae reference substance, with flowing phased soln, to obtain final product；
The preparation of need testing solution: weigh preparation medicine, accurately weighed, add the ultrasonic 5-20min of distilled water and make dissolving, put
Cold, filter, take subsequent filtrate and get final product；
Algoscopy: draw reference substance solution and need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Specifically, the content assaying method of described gastrodine is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring；
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica as filler；With methanol: 0.05%
Phosphoric acid solution=8:92 is flowing phase；Flow velocity 1.0mL min-1, detection wavelength is 220nm；Number of theoretical plate is pressed gastrodine peak and is calculated
5000 should be not less than；
The preparation of reference substance solution: the Rhizoma Gastrodiae reference substance that precision weighs at 80 DEG C of drying under reduced pressure 1h is appropriate, adds flowing and makes mutually
Become every 1mL solution containing 30 μ g, to obtain final product；
The preparation of need testing solution: weigh preparation medicine 0.2g, accurately weighed, to 50mL volumetric flask, add distilled water and fit
Measure ultrasonic 10min and make dissolving, let cool, be settled to scale, filter, take subsequent filtrate and get final product；
Algoscopy: draw reference substance solution and each 10L of need testing solution respectively, inject high performance liquid chromatograph, measure, i.e.
?.
In above-mentioned detection method, the thin-layer identification method of described Ramulus Uncariae Cum Uncis is: get it filled thing 3g, adds methanol 50mL, reflux, extract,
1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes dissolving, adds strong aqua ammonia and adjusts pH value to 9, and chloroform extracts 3 times, each 25mL,
Combined chloroform liquid, is evaporated, and residue adds methanol 2mL makes dissolving, as sample test liquid；Another hook taking rattan control medicinal material 2.5g, by sample
Product test liquid preparation method prepares control medicinal material solution；Test according to pharmacopeia annex thin layer chromatography；Draw above-mentioned three kinds of solution each
10 μ L, put on the same silica gel g thin-layer plate with 0.5%CMC-Na as adhesive, after ammonia cylinder is saturated, with chloroform: acetic acid respectively
Ethyl ester=5: 3 is developing solvent, launches, takes out, dry；Spray with improvement bismuth potassium iodide test solution colour developing；In test sample chromatograph, with right
According on the corresponding position of medical material chromatograph, show the speckle of same color.
In above-mentioned detection method, the thin-layer identification method of described Rhizoma Chuanxiong is: get it filled thing 3g, adds methanol 50mL, reflux, extract,
1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes dissolving, and chloroform extracts 3 times, each 25mL, and combined chloroform liquid is evaporated, residue
Add methanol 2mL and make dissolving, as sample test liquid；Separately taking Rhizoma Chuanxiong control medicinal material 3g, it is right to prepare by sample test liquid preparation method
According to medical material solution；Test according to pharmacopeia annex thin layer chromatography；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is on the silica gel g thin-layer plate of adhesive, after ammonia cylinder is saturated, with chloroform: ethyl acetate=10: 3 as developing solvent, launches,
Take out, dry；Observe under 365nm uviol lamp；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious identical
The fluorescence speckle of color.
In above-mentioned detection method, the thin-layer identification method of described Fructus Gardeniae is: get it filled thing 3g, adds acetone 50mL, 60 DEG C of water
Bath backflow 1h, filters, and filtrate is evaporated, and residue adds methanol 2mL makes dissolving, as sample need testing solution；Separately take Fructus Gardeniae comparison medicine
Material 2.5g, prepares control medicinal material solution by sample test liquid preparation method；Test according to pharmacopeia annex thin layer chromatography；Draw above-mentioned
Three kinds of each 10 μ L of solution, put respectively in same silica gel G F with 0.5%CMC-Na as adhesive254On lamellae, with toluene: chlorine
Imitative: ethyl acetate: methanol: formic acid=2: be developing solvent at 3: 4: 2: 2, launches, takes out, dry；After observing under 254nm uviol lamp, spray
With 10% sulphuric acid ethanol test solution, at 105 DEG C, it is dried to colour developing；In test sample chromatograph, in position corresponding with control medicinal material chromatograph
On, the speckle of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of described Radix Salviae Miltiorrhizae is: get it filled thing 3g, adds methanol 50mL, reflux, extract,
1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes dissolving, and ether extracts 3 times, each 25mL, merges ether solution, is evaporated, residue
Add methanol 2mL and make dissolving, as sample test liquid；Separately taking Radix Salviae Miltiorrhizae control medicinal material 3g, it is right to prepare by sample test liquid preparation method
According to medical material solution；Test according to pharmacopeia annex thin layer chromatography；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is silica gel G F of adhesive254On lamellae, with toluene: chloroform: ethyl acetate: methanol: formic acid=2: 3: 4: 1: 2 for exhibition
Open agent, launch, take out, dry；After observing under 254nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；
In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.
In above-mentioned detection method, the thin-layer identification method of the described Cortex Eucommiae is: get it filled thing 3g, adds methanol 50mL, reflux, extract,
1h, filters, and filtrate water-bath volatilizes, and the 30mL that adds water makes dissolving, and ether extracts 3 times, each 25mL, merges ether solution, is evaporated, residue
Add methanol 2mL and make dissolving, as sample test liquid；Separately taking Cortex Eucommiae control medicinal material 3g, it is right to prepare by sample test liquid preparation method
According to medical material solution；Test according to pharmacopeia annex thin layer chromatography；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is on the silica gel g thin-layer plate of adhesive, with toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developing solvent, exhibition
Open, take out, dry；After observing under 365nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；Test sample color
In spectrum, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.
Inventor has carried out substantial amounts of experiment, the following is the research of detection method of the present invention
Experimental example: detection method research
1 instrument and reagent
1.1 instruments: Japan's Shimadzu LC-20A type chromatograph of liquid, SPD-20A type UV-visible detector (the day island proper
Tianjin), HT-20 type column oven；JA2003 type electronic balance (above current chart level instruments and meters company limited)；Auy220 type electronics sky
Flat (Japan's Shimadzu)；Section leads the desk-top ultrasonic cleaner of SK8210HP type (Shanghai High Kudos Science Instrument Co., Ltd.)；Cortex Eucommiae F-
6210 vacuum drying ovens (Shanghai Qi Xin scientific instrument company limited)；WBZ-2 microwave vacuum drying case (Kweiyang novelty Microwave Industry
Co., Ltd)；SZ-96 automatic pure water distillator (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 electricity
Resistance stove (Beijing original creation Science and Technology Ltd.).
1.2 reagents: gastrodine (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 0807-200104)；Each taste medicine in prescription
Material is purchased from Kweiyang medical material head office, all meets the examination criteria of pharmacopeia；Silica gel G, silica GF254 (Qingdao Haiyang chemical industry), color
Spectrum level methanol (Hanbon Sci. & Tech. Co., Ltd.), chromatographic grade second eyeball (Hanbon Sci. & Tech. Co., Ltd.), water is ultra-pure water, its
He is analytical pure by reagent.
Red sky neck relieving capsule is prepared by the following method: take Radix Salviae Miltiorrhizae 500g, Rhizoma Gastrodiae 500g, Ramulus Uncariae Cum Uncis 500g, Concha Haliotidis 900g,
Cortex Eucommiae 625g, Rhizoma Chuanxiong 500g, Fructus Gardeniae 500g；Adding 8 times amount soak by water 3 times, each 1.5 hours, filter, filtrate is concentrated into thick
Cream, microwave vacuum drying, pulverize, add starch, encapsulated, make 1000, every 0.45g, to obtain final product.
2. differentiate
The thin layer of 2.1 Radix Salviae Miltiorrhizaes differentiates
Taking capsule 's content 3g, add methanol 50mL, reflux, extract, 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes molten
Solving, ether extracts 3 times, each 25mL, merges ether solution, is evaporated, and residue adds methanol 2mL makes dissolving, as sample test liquid.Separately
Take Radix Salviae Miltiorrhizae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method.The feminine gender lacking Radix Salviae Miltiorrhizae with method preparation is right
According to solution.Test according to pharmacopeia annex VI B thin layer chromatography.Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is silica gel G F of adhesive254On lamellae, with toluene-chlorofonn-ethylacetate-methyl alcohol-formic acid (2: 3: 4: 1: 2) it is
Developing solvent, launches, and takes out, dries.After observing under 254nm uviol lamp, spray and show with 2% ferric chloride-1% iron potassuim cyanide test liquid
Color.Test through three batch samples, in test sample chromatograph, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.
Negative control is noiseless (thin-layer chromatogram is shown in Fig. 1, Fig. 2).
The thin layer of 2.2 Ramulus Uncariae Cum Uncis differentiates
Taking capsule 's content 3g, add methanol 50mL, reflux, extract, 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes molten
Solving, add strong aqua ammonia and adjust pH value to 9, chloroform extracts 3 times, each 25mL, and combined chloroform liquid is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid.Another hook taking rattan control medicinal material 2.5g, prepares control medicinal material solution by sample test liquid preparation method.
The negative control solution of Ramulus Uncariae Cum Uncis is lacked with method preparation.Test according to pharmacopeia annex VI B thin layer chromatography.Draw above-mentioned three kinds of solution each 10
μ L, puts respectively on the same silica gel g thin-layer plate with 0.5%CMC-Na as adhesive, after ammonia cylinder is saturated, with chloroform-acetic acid second
Ester (5: 3) is developing solvent, launches, and takes out, dries.Spray with improvement bismuth potassium iodide test solution colour developing.Test through three batch samples, test sample
In chromatograph, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.Negative control is noiseless (thin layer chromatography
Figure is shown in Fig. 3).
The thin layer of 2.3 Rhizoma Chuanxiongs differentiates
Taking capsule 's content 3g, add methanol 50mL, reflux, extract, 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes molten
Solving, chloroform extracts 3 times, each 25mL, and combined chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as sample test liquid.Separately
Take Rhizoma Chuanxiong control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method.The feminine gender lacking Rhizoma Chuanxiong with method preparation is right
According to solution.Test according to pharmacopeia annex VI B thin layer chromatography.Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is on the silica gel g thin-layer plate of adhesive, after ammonia cylinder is saturated, with chlorofonn-ethylacetate (10: 3) as developing solvent, and exhibition
Open, take out, dry.Observe under 365nm uviol lamp.Through three batch samples test, in test sample chromatograph, with control medicinal material chromatograph phase
On the position answered, the fluorescent spot point speckle of aobvious same color.Negative control is noiseless (thin-layer chromatogram is shown in Fig. 4).
The thin layer of 2.4 Cortexs Eucommiae differentiates
Taking capsule 's content 3g, add methanol 50mL, reflux, extract, 1h, filter, filtrate water-bath volatilizes, and the 30mL that adds water makes molten
Solving, ether extracts 3 times, each 25mL, merges ether solution, is evaporated, and residue adds methanol 2mL makes dissolving, as sample test liquid.Separately
Take Cortex Eucommiae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method.The feminine gender lacking the Cortex Eucommiae with method preparation is right
According to solution.Test according to pharmacopeia annex VI B thin layer chromatography.Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same with 0.5%
CMC-Na is on the silica gel g thin-layer plate of adhesive, with toluene-chlorofonn-ethylacetate-formic acid (5: 1: 1: 0.5) as developing solvent, and exhibition
Open, take out, dry.After observing under 365nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing.Through three lot samples
Product are tested, in test sample chromatograph, on position corresponding with control medicinal material chromatograph, and the speckle of aobvious same color.Negative noiseless
(thin-layer chromatogram is shown in Fig. 5, Fig. 6).
The thin layer of 2.5 Fructus Gardeniaes differentiates
Taking capsule 's content 3g, add acetone 50mL, 60 DEG C of water-bath backflow 1h, filter, filtrate is evaporated, and residue adds methanol
2mL makes dissolving, as sample need testing solution.Separately taking Fructus Gardeniae control medicinal material 2.5g, it is right to prepare by sample test liquid preparation method
According to medical material solution.The negative control solution of Fructus Gardeniae is lacked with method preparation.Test according to pharmacopeia annex VI B thin layer chromatography.Draw above-mentioned
Three kinds of each 10 μ L of solution, put respectively in same silica gel G F with 0.5%CMC-Na as adhesive254On lamellae, with toluene-chlorine
Imitative-acetate-methanol-formic acid (2: 3: 4: 2: 2) is developing solvent, launches, and takes out, dries.After observing under 254nm uviol lamp,
Spray, with 10% sulphuric acid ethanol test solution, is dried to colour developing at 105 DEG C.Through three batch samples test, in test sample chromatograph, with compare medicine
Wood color is composed on corresponding position, the speckle 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 (one annex VI D of " Chinese Pharmacopoeia " version in 2010).
3.1 chromatographic condition
Chromatographic column is Agilent HC-C18 post (250mm × 4.6mm, 5 μm)；Flowing is methanol mutually: 0.05% phosphoric acid is molten
Liquid (8: 92), flow velocity 1.0mL min-1, detecting 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 DEG C and be dried the gastrodine reference substance 3.50mg to constant weight, put in 25mL volumetric flask, accurately weighed, add flowing
Phased soln, is diluted to scale, shakes up, and obtains 0.14mg mL-1Reference substance mother solution, accurate 2mL mother solution of drawing puts 10mL volumetric flask
In, it is diluted to scale, obtains 0.028mg mL-1Reference substance solution.
The investigation of 3.3 Extraction solvent
Precision weighs red sky neck relieving capsule content 1g, accurately weighed, is separately added into various solvent 100mL in following table, essence
Close weighed, supersound extraction 30min, let cool, add corresponding solvent and supply weight, divided by distilled water as solvent outside, remaining sample
Product liquid, filters respectively, accurate absorption subsequent filtrate 10mL, is evaporated, adds the phased soln that flows in right amount, is transferred in 10mL volumetric flask, point
Not being settled to scale, filter, subsequent filtrate is for assay.The results are shown in Table 1.
The investigation of table 1 Extraction solvent
Result shows: distilled water is as Extraction solvent supersound extraction to dissolving, and the time is short, and method is simple, and content is high.By
Relatively big in peak area, therefore use and reduce sampling amount, reduce solvent usage amount and reach sample peak area and standard substance peak area
Similar purpose, to reduce the error of assay.
The investigation of 3.4 extraction times
According to the result of 5.3, take red sky neck relieving capsule content 0.2g, accurately weighed after in 50mL volumetric flask, add
The ultrasonic different time of distilled water, lets cool, and is settled to scale, filters, and takes subsequent filtrate for assay.Result and method are shown in Table 2.
The investigation of table 2 extraction time
Result shows, the sample content of gastrodine in ultrasonic 10min to 30min that adds water does not has significance to change, according to sight
Examine, be completely dissolved in a solvent during sample ultrasonic 10min.Therefore selecting the supersound extraction time is 10min.
The preparation of 3.5 need testing solutions
Weigh red sky neck relieving capsule content 0.2g, accurately weighed, to 50mL volumetric flask, add distilled water the most ultrasonic
10min makes dissolving, lets cool, and is settled to scale, filters, and takes subsequent filtrate for assay.
The investigation of 3.6 flowing phases
According to list of references, selection second eyeball: 0.05% phosphoric acid solution, methanol: 0.05% phosphoric acid solution, methanol: 0.1% ice
Acetic acid, methanol: water is flowing phase, and result shows with methanol: can obtain good peak type during 0.05% phosphoric acid solution (8: 92),
Gastrodine peak is enough with other adjacent peak energy completely separable.Simultaneously investigate different temperatures on test impact, result show 20 DEG C-
30 DEG C of appearance times on target peak and content do not have the impact of significance.
3.7 specificities are investigated
Take red sky neck relieving capsule sample 0.2g, accurately weighed, prepare negative control solution by need testing solution preparation method,
Accurate gastrodine reference substance solution of drawing, the negative each 10 μ L of need testing solution, inject high performance liquid chromatograph, result shows, supplies
In test product peak, gastrodine peak energy enough reaches baseline separation, and the adjacent both sides at peak are disturbed without other peaks, and separating degree is 23.777, cloudy
Is there is not peak with corresponding position, gastrodine peak in property test sample, shows that the specificity of method is good.Result is shown in Fig. 9-Figure 11.
3.8 linear and scopes
Accurate reference substance mother solution of drawing is diluted to concentration respectively: 7 μ g mL-1, 14 μ g mL-1, 21 μ g mL-1, 28 μ g
mL-1, 35 μ gm L-1, 42 μ g mL-1, precision measures peak in drawing each 10 μ L injection high performance liquid chromatographs of above-mentioned solution respectively
Area, with the quality of reference substance as abscissa, peak area is vertical coordinate, linear regression, and obtaining regression equation is y=1986.8x+
6870.5, R=0.9999.Result show gastrodine in the range of 70～420ng in good linear relationship.The results are shown in Table 3, line
Sexual relationship is shown in Figure 12.
The investigation of table 3 range of linearity
3.9 precision test
Accurate concentration of drawing is 7 μ g mL-1, 28 μ g mL-1, 42 μ g mL-1Gastrodine reference substance solution 10 μ L, respectively
Continuous sample introduction 5 times, with calculated by peak area RSD value, RSD is respectively 0.46%, 0.91%0.63%, shows that the precision of instrument is good
Good.The results are shown in Table 4.
Table 4 Precision Experiment
3.10 repeatability is investigated
Take the 80% of content 0.2g, 100%, 120% each 3 parts of red sky neck relieving capsule, accurately weighed, by need testing solution
Preparation method prepare 9 parts of 3 concentration need testing solutions, 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
Take the content 0.2g of red sky neck relieving capsule, accurately weighed, process sample by the preparation method of need testing solution, point
Not in 0,2,4,6,8,12h measure sample sizes, and to calculate RSD be 1.09%.Result shows, after sample treatment in 12 hours
Stable, it is shown in Table 6.
Table 6 study on the stability
3.12 the response rate is investigated
Weigh the content 0.1g totally 9 parts of the red sky neck relieving capsule of known content, be divided into 3 groups, often group 3 parts, with in sample
Content be standard, according to add the 80% of standard, 100%, 120% the principle of reference substance, it is accurate that to add content suitable
Gastrodine standard solution, prepares 9 parts of 3 concentration samples solution by the preparation method of need testing solution, filters, and subsequent filtrate is for containing
It is fixed to measure.Calculate the response rate.The results are shown in Table 7.
The investigation of table 7 response rate
Result shows that the response rate of sample is 100.5%, and RSD is 0.58%, and the method response rate is good.
The assay of 3.13 ten batch sample
Take each 2 parts of the content 0.2g of the red sky neck relieving capsule of 10 different lot numbers, accurately weighed, by need testing solution
Preparation method processes sample, filters, and subsequent filtrate is for assay.Calculate content, the results are shown in Table 8.
Table 8 sample size measures (n=2)
Understanding the optimum determining method of gastrodin content in mensuration red sky neck relieving capsule according to above experiment is: use HPLC
Method, Agilent HC-C18；Flow velocity: 1.0mL min-1；Flowing phase: methanol-0.05% phosphoric acid solution (8:92)；Column temperature: room temperature；
Sample size: 10 μ L；Detection wavelength: 220nm；Red sky neck relieving capsule content is extracted as Extraction solvent with ultrasonic using distilled water
In gastrodine.Institute construction method is easy quickly, result is accurate, reproducible, can be as the containing of gastrodine in red sky neck relieving capsule
Quantity measuring method.
The beneficial effects of the present invention is: the invention provides red sky neck and relax the detection method of oral formulations, including with place
In side, monarch drug Gastrodin in Gastrodia eleta Bl. is index, uses the content of high effective liquid chromatography for measuring gastrodine；And Radix Salviae Miltiorrhizae in medicine,
Thin layer chromatography (TLC) discrimination method of Rhizoma Chuanxiong, Ramulus Uncariae Cum Uncis, Fructus Gardeniae and the Cortex Eucommiae；Described detection method is accurate, highly sensitive, repeatability
Good, testing result is stable, can effectively control red sky neck and relax the quality of oral formulations, both be more beneficial for manufacturer and supervision and management
Department's monitoring to product quality, it is also possible to the treatment for medical department and patient provides preferably guarantee.
Accompanying drawing explanation
Fig. 1 is the fluorescent thin-layer chromatography figure of Radix Salviae Miltiorrhizae of the present invention
Fig. 2 is the colour developing thin-layer chromatogram of Radix Salviae Miltiorrhizae of the present invention
Fig. 3 is the thin-layer chromatogram of Ramulus Uncariae Cum Uncis of the present invention
Fig. 4 is the thin-layer chromatogram of Rhizoma Chuanxiong of the present invention
Fig. 5 is the fluorescent thin-layer chromatography figure of the Cortex Eucommiae of the present invention
Fig. 6 is the colour developing thin-layer chromatogram of the Cortex Eucommiae of the present invention
Fig. 7 is the fluorescent thin-layer chromatography figure of Fructus Gardeniae of the present invention
Fig. 8 is the colour developing thin-layer chromatogram of Fructus Gardeniae of the present invention
Fig. 9 is the high-efficient liquid phase chromatogram of gastrodine standard substance of the present invention
Figure 10 is the high-efficient liquid phase chromatogram of feminine gender test sample of the present invention
Figure 11 is the high-efficient liquid phase chromatogram of inventive samples
Figure 12 is gastrodine canonical plotting of the present invention
Below in conjunction with embodiment, the present invention is further illustrated；
Detailed description of the invention
Embodiment 1: the content assaying method of gastrodine in red sky neck relieving capsule
Red sky neck relieving capsule is prepared: take Radix Salviae Miltiorrhizae 500g, Rhizoma Gastrodiae 500g, Ramulus Uncariae Cum Uncis 500g, Concha Haliotidis 900g, the Cortex Eucommiae
625g, Rhizoma Chuanxiong 500g, Fructus Gardeniae 500g；Adding 8 times amount soak by water 3 times, each 1.5 hours, filter, filtrate is concentrated into thick paste, micro-
Ripple is vacuum dried, and pulverizes, and adds starch, encapsulated, makes 1000, and every 0.45g to obtain final product.
The content assaying method of gastrodine is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring；
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica as filler；With methanol: 0.05%
Phosphoric acid solution=8:92 is flowing phase；Flow velocity 1.0mL min-1, detection wavelength is 220nm；Number of theoretical plate is pressed gastrodine peak and is calculated
5000 should be not less than；
The preparation of reference substance solution: the Rhizoma Gastrodiae reference substance that precision weighs at 80 DEG C of drying under reduced pressure 1h is appropriate, adds flowing and makes mutually
Become every 1mL solution containing 30 μ g, to obtain final product；
The preparation of need testing solution: weigh capsule 's content 0.2g, accurately weighed, to 50mL volumetric flask, add distilled water
Appropriate ultrasonic 10min makes dissolving, lets cool, and is settled to scale, filters, takes subsequent filtrate and get final product；
Algoscopy: draw reference substance solution and each 5-10L of need testing solution respectively, inject high performance liquid chromatograph, measure,
Obtain.
This product is containing Rhizoma Gastrodiae in terms of gastrodine, and every must not be less than 3.00mg.
Embodiment 2: the TLC Identification of Ramulus Uncariae Cum Uncis in red sky neck relieving capsule
Described red sky neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of Ramulus Uncariae Cum Uncis is: take capsule 's content 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, adds strong aqua ammonia and adjusts pH value to 9, and chloroform extracts 3 times, each 25mL, combined chloroform liquid, steams
Dry, residue adds methanol 2mL makes dissolving, as sample test liquid；Another hook taking rattan control medicinal material 2.5g, by sample test liquid preparation side
Method prepares control medicinal material solution；Test according to pharmacopeia annex VI B thin layer chromatography；Draw each 10 μ L of above-mentioned three kinds of solution, point respectively
On the same silica gel g thin-layer plate with 0.5%CMC-Na as adhesive, after ammonia cylinder is saturated, with chloroform: ethyl acetate=5: 3 are
Developing solvent, launches, and takes out, dries；Spray with improvement bismuth potassium iodide test solution colour developing；In test sample chromatograph, with control medicinal material chromatograph
On corresponding position, the speckle of aobvious same color.
Embodiment 3: the TLC Identification of Rhizoma Chuanxiong in red sky neck relieving capsule
Described red sky neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of Rhizoma Chuanxiong is: take capsule 's content 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, and chloroform extracts 3 times, each 25mL, and combined chloroform liquid is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Rhizoma Chuanxiong control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex VI B thin layer chromatography is tested；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same be glutinous with 0.5%CMC-Na
On the silica gel g thin-layer plate of mixture, after ammonia cylinder is saturated, with chloroform: ethyl acetate=10: 3 as developing solvent, launch, take out, dry；
Observe under 365nm uviol lamp；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the fluorescence of aobvious same color
Speckle.
Embodiment 4: the TLC Identification of Fructus Gardeniae in red sky neck relieving capsule
Described red sky neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of Fructus Gardeniae is: take capsule 's content 3g, adds acetone 50mL, 60 DEG C of water-bath backflow 1h, filters,
Filtrate is evaporated, and residue adds methanol 2mL makes dissolving, as sample need testing solution；Separately take Fructus Gardeniae control medicinal material 2.5g, supply by sample
Test solution preparation method prepares control medicinal material solution；Test according to pharmacopeia annex VI B thin layer chromatography；Draw above-mentioned three kinds of solution each 10
μ L, puts respectively in same silica gel G F with 0.5%CMC-Na as adhesive254On lamellae, with toluene: chloroform: ethyl acetate: first
Alcohol: formic acid=2: be developing solvent at 3: 4: 2: 2, launches, takes out, dry；After observing under 254nm uviol lamp, spray is with 10% sulphuric acid ethanol
Test solution, is dried to colour developing at 105 DEG C；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious same color
Speckle.
Embodiment 5: the TLC Identification of Radix Salviae Miltiorrhizae in red sky neck relieving capsule
Described red sky neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of Radix Salviae Miltiorrhizae is: take capsule 's content 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, and ether extracts 3 times, each 25mL, merges ether solution, is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Radix Salviae Miltiorrhizae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex VI B thin layer chromatography is tested；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same be glutinous with 0.5%CMC-Na
Silica gel G F of mixture254On lamellae, with toluene: chloroform: ethyl acetate: methanol: formic acid=2: 3: 4: 1: 2 as developing solvent, launch,
Take out, dry；After observing under 254nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；Test sample chromatograph
In, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.
Embodiment 6: the TLC Identification of the Cortex Eucommiae in red sky neck relieving capsule
Described red sky neck relieving capsule is prepared according to the method for embodiment 1.
The thin-layer identification method of the Cortex Eucommiae is: take capsule 's content 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, and ether extracts 3 times, each 25mL, merges ether solution, is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Cortex Eucommiae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex VI B thin layer chromatography is tested；Draw each 10 μ L of above-mentioned three kinds of solution, put respectively in same be glutinous with 0.5%CMC-Na
On the silica gel g thin-layer plate of mixture, with toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developing solvent, launch, take out, dry in the air
Dry；After observing under 365nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；In test sample chromatograph, with
On the corresponding position of control medicinal material chromatograph, the speckle of aobvious same color.
Claims (1)
1. a red sky neck relaxes the detection method of oral formulations, it is characterised in that: described red sky neck relaxes oral formulations according to weight
Calculation, by Radix Salviae Miltiorrhizae 200-700 part, Rhizoma Gastrodiae 200-700 part, Ramulus Uncariae Cum Uncis 200-700 part, Concha Haliotidis 600-1200 part, the Cortex Eucommiae
300-800 part, Rhizoma Chuanxiong 200-700 part, Fructus Gardeniae 200-700 part and adjuvant are prepared from by the following method: weigh according to formula
Each medicine, by water boiling and extraction, filters, and filtrate is concentrated into thick paste, dry, pulverize, and adds adjuvant and prepares according to common process
Become traditional oral preparation；Described detection method includes that in oral disposition preparation, the content of gastrodine is measured, in oral disposition preparation
Radix Salviae Miltiorrhizae, Ramulus Uncariae Cum Uncis, the Cortex Eucommiae, Rhizoma Chuanxiong and Fructus Gardeniae carry out indentification by TLC respectively；The content assaying method of described gastrodine is: shine
Chinese Pharmacopoeia high effective liquid chromatography for measuring；
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica as filler；With methanol: 0.05% phosphoric acid
Solution=8:92 is flowing phase；Flow velocity 1.0 mL min-1, detection wavelength is 220nm；Number of theoretical plate presses the calculating of gastrodine peak should not
Less than 5000；
The preparation of reference substance solution: the Rhizoma Gastrodiae reference substance that precision weighs at 80 DEG C of drying under reduced pressure 1h is appropriate, adds flowing and makes every mutually
The 1mL solution containing 30 μ g, to obtain final product；
The preparation of need testing solution: weigh preparation medicine 0.2g, accurately weighed, to 50mL volumetric flask, add distilled water and surpass in right amount
Sound 10min makes dissolving, lets cool, and is settled to scale, filters, takes subsequent filtrate and get final product；
Algoscopy: draw reference substance solution and each 10L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product；
In described preparation, the thin-layer identification method of Ramulus Uncariae Cum Uncis is: get it filled thing 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, adds strong aqua ammonia and adjusts pH value to 9, and chloroform extracts 3 times, each 25mL, combined chloroform liquid, steams
Dry, residue adds methanol 2mL makes dissolving, as sample test liquid；Another hook taking rattan control medicinal material 2.5g, by sample test liquid preparation side
Method prepares control medicinal material solution；Test according to pharmacopeia annex thin layer chromatography；Draw each 10 μ L of above-mentioned solution, put respectively in same with
0.5%CMC-Na is on the silica gel g thin-layer plate of adhesive, after ammonia cylinder is saturated, with chloroform: ethyl acetate=5: 3 as developing solvent, exhibition
Open, take out, dry；Spray with improvement bismuth potassium iodide test solution colour developing；In test sample chromatograph, in position corresponding with control medicinal material chromatograph
On, the speckle of aobvious same color；
In described preparation, the thin-layer identification method of Rhizoma Chuanxiong is: get it filled thing 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, and chloroform extracts 3 times, each 25mL, and combined chloroform liquid is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Rhizoma Chuanxiong control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex thin layer chromatography is tested；Draw each 10 μ L of above-mentioned solution, put respectively in same with 0.5%CMC-Na as adhesive
On silica gel g thin-layer plate, after ammonia cylinder is saturated, with chloroform: ethyl acetate=10: 3 as developing solvent, launch, take out, dry；365nm
Observe under uviol lamp；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the fluorescence speckle of aobvious same color；
In described preparation, the thin-layer identification method of Fructus Gardeniae is: get it filled thing 3g, adds acetone 50mL, 60 DEG C of water-bath backflow 1h, filters,
Filtrate is evaporated, and residue adds methanol 2mL makes dissolving, as sample need testing solution；Separately take Fructus Gardeniae control medicinal material 2.5 g, by sample
Test liquid preparation method prepares control medicinal material solution；Test according to pharmacopeia annex thin layer chromatography；Draw each 10 μ L of above-mentioned solution, point
Other point is in same silica gel G F with 0.5%CMC-Na as adhesive254On lamellae, with toluene: chloroform: ethyl acetate: methanol: first
Acid=2: 3: 4: 2: 2 are developing solvent, launch, and take out, dry；After observing under 254nm uviol lamp, spray with 10% sulphuric acid ethanol test solution,
Colour developing it is dried at 105 DEG C；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color；
In described preparation, the thin-layer identification method of Radix Salviae Miltiorrhizae is: get it filled thing 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and the 30mL that adds water makes dissolving, and ether extracts 3 times, each 25 mL, merges ether solution, is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Radix Salviae Miltiorrhizae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex thin layer chromatography is tested；Draw each 10 μ L of above-mentioned solution, put respectively in same with 0.5%CMC-Na as adhesive
Silica gel G F254On lamellae, with toluene: chloroform: ethyl acetate: methanol: formic acid=2: 3: 4: 1: 2 as developing solvent, launch, take out,
Dry；After observing under 254 nm uviol lamps, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；In test sample chromatograph,
On position corresponding with control medicinal material chromatograph, the speckle of aobvious same color；
In described preparation, the thin-layer identification method of the Cortex Eucommiae is: get it filled thing 3g, adds methanol 50mL, reflux, extract, 1h, filters, filtrate water
Bath volatilizes, and 30 mL that add water make dissolving, and ether extracts 3 times, each 25mL, merges ether solution, is evaporated, and residue adds methanol 2mL to be made molten
Solve, as sample test liquid；Separately take Cortex Eucommiae control medicinal material 3g, prepare control medicinal material solution by sample test liquid preparation method；According to
Pharmacopeia annex thin layer chromatography is tested；Draw each 10 μ L of above-mentioned solution, put respectively in same with 0.5%CMC-Na as adhesive
On silica gel g thin-layer plate, with toluene: chloroform: ethyl acetate: formic acid=5: 1: 1: 0.5 as developing solvent, launch, take out, dry；365
After observing under nm uviol lamp, spray with 2% ferric chloride-1% iron potassuim cyanide test liquid colour developing；In test sample chromatograph, with compare medicine
Wood color is composed on corresponding position, the speckle of aobvious same color.
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