CN103575842B - Radix Astragali principal ingredient detection method in the clear sheet of ephritis - Google Patents
Radix Astragali principal ingredient detection method in the clear sheet of ephritis Download PDFInfo
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Abstract
The present invention relates to Radix Astragali principal ingredient detection method in the clear sheet of Chinese medicine ephritis, the method comprises the following steps: step 1, the preparation of reference substance solution, step 2, the preparation of need testing solution, step 3, by reference substance solution and need testing solution injecting chromatograph, obtain chromatogram, step 4, calculate the content of Astragaloside IV in test sample according to chromatogram with external standard two-point method logarithmic equation.
Description
Technical field
The present invention relates to Radix Astragali principal ingredient detection method in the clear sheet of the detection method of Chinese medicine preparation, particularly ephritis.
Technical background
The clear sheet of ephritis is a kind of Chinese medicine preparation for the treatment of ephritis, be stored by the Radix Astragali, motherwort, cogongrass rhizome, pivot, fringed pink, field thistle, pyrrosia lingua, charred RADIX SANGUISORBAE, Rumex madaio and Radix Glycyrrhizae make.
Motherwort in the clear sheet side of ephritis, property is bitter, pungent and be slightly cold, and has inducing diuresis for removing edema, a merit of invigorating blood circulation of detoxifying, and is monarch drug in a prescription in side.Cogongrass rhizome is sweet, cold, can cooling blood and hemostasis, again can reducing fever and causing diuresis, heat conduction descending, be suitable for bladder damp-heat and pent up and the blood urine that causes.We are monarch drug in a prescription with itself and motherwort altogether, the blood of absurd row can be made to be attributed in arteries and veins and fall blood urine.Pivot stores and fringed pink Papillary, is good at damp-heat in lower-JIAO and promoting diuresis for stranguria clearly; Field thistle is kind controls the inferior the puckery red pain of the caused urine of close-burning heat, blood pouring hematuria; Pyrrosia lingua energy supernatant lung heat, under walk bladder and inducing diuresis for treating strangurtia; Garden burnet is longer than treatment lower-Jiao syndrome, takes the heresy that all medicine heat-clearing parts of the body cavity below the umbilicus, housing the bladder, kidneys and bowels are burning hot, crack part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels extravasated blood in we; Rumex madaio can treat hemoptysis caused by blood-head, haematemesis, hematuria, hemorrhoid blood, uterine bleeding, and the card such as traumatism and bleeding.Radix Astragali merit defends solid table, inducing diuresis for removing edema, holder sore myogenic in tonifying Qi and lifting yang, benefit.The clear sheet of ephritis has obvious effect for reducing blood urine caused by the reasons such as acute glomerulonephritis, chronic glomerulonephritis, invisible glomerulonephritis, HSPN, can reduce and alleviate the bad reaction in glucocorticoid treatment process.
For making this medicine production standard, reach determined curative effect, stay-in-grade object.The present invention, through research, establishes a kind of method detecting effective constituent Astragaloside IV in invention formulation, and the method adopts HPLC method to carry out assay to effective constituent Astragaloside IV wherein.The method precision, sensitivity, stability are all good, guarantee " safe, homogeneous, stable, effective, controlled " of product quality.
Summary of the invention
The invention provides the detection method of Radix Astragali principal ingredient in the clear sheet of a kind of Chinese medicine ephritis, this detection method comprises, and carries out assay to the effective ingredient Astragaloside IV in medicine, and this assay method adopts HPLC method.
Detection method of the present invention, comprises the following steps:
Step 1, the preparation of reference substance solution,
Step 2, the preparation of need testing solution,
Step 3, by reference substance solution and need testing solution injecting chromatograph, obtains chromatogram,
Step 4, calculates the content of Astragaloside IV in test sample according to chromatogram,
Wherein, chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent, and mobile phase is acetonitrile-aqueous solution, and detecting device adopts evaporative light-scattering detector.
According to the present invention, the volume ratio of mobile phase acetonitrile-water is 1:0.538-9.
According to the present invention, the wherein preparation of reference substance solution described in step 1, method is as follows: get Astragaloside IV reference substance and add methyl alcohol and make solution.Preferably, method is as follows: get Astragaloside IV reference substance and add methyl alcohol and make every 1ml containing the solution of 0.2-0.6mg.
According to the present invention, the wherein preparation of need testing solution described in step 2, method is as follows: get detection sample, adds the ultrasonic process of ammoniacal liquor, centrifugal supernatant, crosses C18 post, methanol-eluted fractions, filters, to obtain final product.Preferably, method is as follows: detect sample porphyrize, get 0.5-3g, add 2-8% ammoniacal liquor 10-50ml, ultrasonic more than process 20min, centrifugal, gets supernatant 5-25ml, is splined on C18, and 10-20ml washes, and discards, methanol-eluted fractions, and miillpore filter filters, and to obtain final product.
According to one of embodiment of the present invention, wherein the weight of C18 is no less than 150mg.
According to another embodiment of the present invention, wherein the aperture of miillpore filter is 0.22-0.45 μm.
According to the present invention, wherein the method for step 3 is as follows: get reference substance solution 10-20 μ l respectively, need testing solution 10-20 μ l, and injection liquid chromatography, obtains chromatogram, calculates Astragaloside content with external standard two-point method logarithmic equation.
The object of the invention is the content of the effective ingredient Astragaloside IV detected in the clear sheet of Chinese medicine ephritis, and whether reaches production requirement in process of production by the clear sheet of content understanding ephritis of this effective constituent, plays the effect controlling product quality.
According to detection method of the present invention, in gained test sample, the content of every sheet Astragaloside IV must not be less than 0.15mg.
The present invention finds a kind of suitable method of quality control to determine the quality of product, and this method of quality control is comparatively perfect, strong operability, favorable reproducibility, and measurement deviation is little, good stability, and precision is high.
Accompanying drawing explanation
Fig. 1 Astragaloside IV typical curve.
Embodiment
By the following examples, further illustrate the present invention, but not as restriction of the present invention.
The methodological study of experimental example 1 Determination of Astragaloside
Content assaying method of the present invention, obtained by screening, screening process is as follows:
1, instrument and reagent
Instrument high performance liquid chromatograph: Waters2695 sampling system, Waters2424ELSD detecting device; WatersEmpower chromatographic work station; MettlerAE240 (100,000/) balance (plum Teller-Tuo benefit Shanghai Instrument Ltd.).
Reagent Astragaloside IV is purchased from Chinese medicine biological standardization institute (lot number 110781-200613)
Acetonitrile is chromatographically pure; Finished product sample and negative controls (being provided by our company).
2, the selection of chromatographic condition
Chromatographic column: AgilentZORBAXSB-C
18, 5 μm, 250 × 4.6mm; Mobile phase: acetonitrile: water=35:65; Column temperature: 30 DEG C; Flow velocity 1ml/min; ELSD detecting device.Under this chromatographic condition, in Astragaloside IV and sample, other Component seperation is good, and number of theoretical plate is greater than 2000 by Astragaloside IV reference substance peak.
3, the preparation of need testing solution
Get the clear sheet of ephritis under weight differential item, porphyrize, mixing, get 1.5g, accurately weighed, put in triangular flask, precision adds 4% ammoniacal liquor 20ml, ultrasonic process 20min, centrifugal, precision measures supernatant 10ml, is splined on processed good C18(250mg) on, 5ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.45um) filters, and to obtain final product.
4, blank test
Get the negative sample of the scarce Radix Astragali, by need testing solution preparation method operation, sample introduction measures in accordance with the law, record chromatogram.As a result, at the identical retention time place of reference substance without absorption peak.Show that the composition of noiseless Astragaloside IV in sample exists.
5, the investigation of linear relationship
Precision takes Astragaloside IV reference substance 3.90mg, puts in 10ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, in contrast product solution; The each 2 μ l of the above-mentioned reference substance solution of accurate absorption, 5 μ l, 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, injection liquid chromatography respectively, Astragaloside IV peak area is measured by above-mentioned chromatographic condition, with the natural logarithm of reference substance sample size for horizontal ordinate, with the natural logarithm value of peak area for ordinate, make the typical curve of Astragaloside IV.(see Fig. 1)
Table 1 Determination of Astragaloside linear test
Measurement result shows, its coefficient R=0.9995, regression equation Y=1.4284X+4.2118.Astragaloside IV reference substance is good linear relationship (see figure 1) within the scope of the μ g of sample size 0.78 μ g ~ 19.50.
6, precision test
The above-mentioned reference substance solution 10 μ l of accurate absorption, repeats sample introduction 5 times, measures, and record peak area, asks relative standard deviation RSD.The results are shown in Table 2.Result illustrates that this law precision is good.
Table 2 precision test
7, stability test
(lot number: 20101201) at room temperature, measures respectively at certain hour after preparation, each sample introduction 10 μ l, record peak area the same need testing solution of accurate absorption.The results are shown in Table 15-3.Result shows that need testing solution is basicly stable in 12 hours.
Table 3 stability test
8, reappearance test
Precision takes 6 parts, sample, and (lot number: 20101201), by need testing solution preparation method operation, sample introduction measures in accordance with the law, calculates content, asks relative standard deviation RSD.Result shows, this law measures has good reappearance, the results are shown in Table 15-4.
Table 4 Astragaloside IV reappearance is tested
9, determination of recovery rates
Adopt application of sample absorption method, precision measures Astragaloside IV reference substance solution (Astragaloside IV concentration is 1.116mg/ml) 0.5ml, put respectively in triangular flask, volatilize methyl alcohol, the accurate same sample lots (lot number: 20101201) (Astragaloside content is 0.574mg/g) about 0.75g, is placed in triangular flask, operates by need testing solution preparation method taking known content respectively, sample introduction measures in accordance with the law, calculates.Result shows, this law has the good recovery.The results are shown in Table 5.
Table 5 Astragaloside IV determination of recovery rates result
10, sample determination
Get this product three batches, according to need testing solution preparation method preparation, accurate absorption need testing solution 20 μ l, measures by above-mentioned chromatographic condition, the content of Astragaloside IV in calculation sample respectively.The results are shown in Table 6.
Table 6 three batches of ephritis clear sheet Determination of Astragaloside results
According to the present invention, the every sheet of this product presses Astragaloside IV (C containing the Radix Astragali
44h
68o
4) must not count and be less than 0.15mg.
In the present invention, the preparation of the clear sheet of ephritis can see Chinese patent CN201010261828.4.
Embodiment 1
1. the clear sheet of ephritis is prepared Radix Astragali 400g motherwort 400g cogongrass rhizome 200g pivot and is stored 200g fringed pink 200g field thistle 200g pyrrosia lingua 80g charred RADIX SANGUISORBAE 60g Rumex madaio 200g Radix Glycyrrhizae 20g.The Radix Astragali, add 6 times amount soak by water 1 hour, decocting liquid filters and is concentrated into thick paste, vacuum drying, obtains the dry cream of Astragalus Root P.E.Rumex madaio, field thistle, add 10 times amount 65% alcohol refluxs and extract, decocting liquid is concentrated into thick paste, vacuum drying, obtains the dry cream of Rumex madaio Herba Cirsii extract.Motherwort, cogongrass rhizome, pivot store, fringed pink, pyrrosia lingua, charred RADIX SANGUISORBAE, Radix Glycyrrhizae seven flavor medicine material add 6 times amount soak by water, and decocting liquid is concentrated into 1.1(60 DEG C), add 3 times amount ethanol, leave standstill more than 12 hours, filter, filtrate recycling ethanol is to thick paste, vacuum drying, obtains the dry cream of seven taste extracts.Above three kinds of dried cream powders, add microcrystalline cellulose, sodium carboxymethyl starch, dolomol, compressing tablet film coating, to obtain final product.
2. ephritis clear sheet Radix Astragali effective constituent qualitative determination
This product is Film coated tablets, aobvious brown after removing film-coating; Mildly bitter flavor, gas is micro-.
Get this product 1.5g, put in 25ml measuring bottle, add methyl alcohol 20ml, ultrasonic 20min makes to leach, filter, water bath method, the residue 20ml that adds water makes dissolving, and loading is to C18 solid phase extraction column (the internal diameter 1cm that pre-service is good, the heavy 2.0g of C18 filler, its granularity is 50 ~ 100 orders) on, first use water 20ml wash-out, discard eluent; Use 20ml50% methanol-eluted fractions again, eluent discards, and finally uses methanol-eluted fractions, collects eluent, is settled to 5ml, shakes up, as need testing solution; Separately get linarin reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast; Test according to thin-layered chromatography (annex VI B), draw each 5 μ l of above-mentioned solution, put respectively in same sodium carboxymethyl cellulose be on bonding agent silica gel H (efficiently) thin layer plate, with ethyl acetate-formic acid-water (8: 1: 1) for developping agent, launch, take out, dry, spray, with 2% aluminium choride ethanolic solution, is dried, is inspected under putting uviol lamp under (365nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
3. Determination of Astragaloside method in the clear sheet of ephritis
According to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, with acetonitrile-water (35:65) for mobile phase; Evaporative light-scattering detector detects.Number of theoretical plate calculates should be not less than 4000 by Astragaloside IV peak.
The preparation of reference substance solution gets Astragaloside IV reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product.
The clear sheet of ephritis under weight differential item is got in the preparation of need testing solution, porphyrize, mixing, get 1.5g, accurately weighed, put in triangular flask, precision adds 4% ammoniacal liquor 20ml, ultrasonic process 20min, centrifugal, precision measures supernatant 10ml, is splined on processed good C18(250mg) on, 10ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.45 μm) filters, and to obtain final product.
Determination method is accurate respectively draws reference substance solution 10 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, calculates, to obtain final product with external standard two-point method logarithmic equation.
The every sheet of this product must not be less than 0.15mg containing Astragaloside IV.
Embodiment 2
The preparation of the clear sheet of ephritis and the qualitative determination of Radix Astragali effective constituent are with embodiment 1.
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, with acetonitrile-water (10:90) for mobile phase; Evaporative light-scattering detector detects.Number of theoretical plate calculates should be not less than 4000 by Astragaloside IV peak.
The preparation of reference substance solution gets Astragaloside IV reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.2mg, to obtain final product.
The clear sheet of ephritis under weight differential item is got in the preparation of need testing solution, porphyrize, mixing, get 0.5g, accurately weighed, put in triangular flask, precision adds 2% ammoniacal liquor 15ml, ultrasonic process 25min, centrifugal, precision measures supernatant 5ml, is splined on processed good C18(200mg) on, 10ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.22 μm) filters, and to obtain final product.
Determination method is accurate respectively draws reference substance solution 10 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, calculates, to obtain final product with external standard two-point method logarithmic equation.
Embodiment 3
The preparation of the clear sheet of ephritis and the qualitative determination of Radix Astragali effective constituent are with embodiment 1.
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, with acetonitrile-water (65:35) for mobile phase; Evaporative light-scattering detector detects.Number of theoretical plate calculates should be not less than 4000 by Astragaloside IV peak.
The preparation of reference substance solution gets Astragaloside IV reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.6mg, to obtain final product.
The clear sheet of ephritis under weight differential item is got in the preparation of need testing solution, porphyrize, mixing, get 3g, accurately weighed, put in triangular flask, precision adds 6% ammoniacal liquor 40ml, ultrasonic process 28min, centrifugal, precision measures supernatant 15ml, is splined on processed good C18(300mg) on, 10ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.45 μm) filters, and to obtain final product.
Determination method is accurate respectively draws reference substance solution 10 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, calculates, to obtain final product with external standard two-point method logarithmic equation.
Embodiment 4
The preparation of the clear sheet of ephritis and the qualitative determination of Radix Astragali effective constituent are with embodiment 1.
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, with acetonitrile-water (20:80) for mobile phase; Evaporative light-scattering detector detects.Number of theoretical plate calculates should be not less than 4000 by Astragaloside IV peak.
The preparation of reference substance solution gets Astragaloside IV reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.5mg, to obtain final product.
The clear sheet of ephritis under weight differential item is got in the preparation of need testing solution, porphyrize, mixing, get 2g, accurately weighed, put in triangular flask, precision adds 8% ammoniacal liquor 10ml, ultrasonic process 30min, centrifugal, precision measures supernatant 20ml, is splined on processed good C18(150mg) on, 10ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.22 μm) filters, and to obtain final product.
Determination method is accurate respectively draws reference substance solution 10 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, calculates, to obtain final product with external standard two-point method logarithmic equation.
Embodiment 5
The preparation of the clear sheet of ephritis and the qualitative determination of Radix Astragali effective constituent are with embodiment 1.
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, with acetonitrile-water (13:88) for mobile phase; Evaporative light-scattering detector detects.Number of theoretical plate calculates should be not less than 4000 by Astragaloside IV peak.
The preparation of reference substance solution gets Astragaloside IV reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.3mg, to obtain final product.
The clear sheet of ephritis under weight differential item is got in the preparation of need testing solution, porphyrize, mixing, get 2.5g, accurately weighed, put in triangular flask, precision adds 5% ammoniacal liquor 50ml, ultrasonic process 30min, centrifugal, precision measures supernatant 25ml, is splined on processed good C18(280mg) on, 10ml washes, discard, methanol-eluted fractions is also settled in the volumetric flask of 2ml, shakes up, miillpore filter (0.45 μm) filters, and to obtain final product.
Determination method is accurate respectively draws reference substance solution 10 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, calculates, to obtain final product with external standard two-point method logarithmic equation.
Claims (8)
1. detect a method for Radix Astragali principal ingredient in the clear sheet of ephritis, comprise the following steps:
Step 1, the preparation of reference substance solution,
Step 2, the preparation of need testing solution,
Step 3, injects high performance liquid chromatograph by reference substance solution and need testing solution, obtains chromatogram,
Step 4, calculates the content of Astragaloside IV in test sample according to chromatogram,
Wherein, chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent, and mobile phase is acetonitrile-aqueous solution, and detecting device adopts evaporative light-scattering detector,
Wherein, the preparation of need testing solution described in step 2, method is as follows: detect sample porphyrize, get 0.5-3g, add 2-8% ammoniacal liquor 10-50ml, ultrasonic more than process 20min, centrifugal, get supernatant 5-25ml, be splined on C18,10-20ml washes, discard, methanol-eluted fractions, miillpore filter filters, and to obtain final product.
2. method according to claim 1, is characterized in that, the volume ratio of mobile phase acetonitrile-water is 1:0.538-9.
3. method according to claim 1, is characterized in that, wherein the preparation of reference substance solution described in step 1, and method is as follows:
Get Astragaloside IV reference substance to add methyl alcohol and make solution.
4. method according to claim 3, is characterized in that, wherein the preparation of reference substance solution described in step 1, and method is as follows:
Get Astragaloside IV reference substance to add methyl alcohol and make every 1ml containing the solution of 0.2-0.6mg.
5. method according to claim 1, wherein the weight of C18 is no less than 150mg.
6. method according to claim 1, wherein the aperture of miillpore filter is 0.22-0.45 μm.
7. method according to claim 1, is characterized in that, wherein the method for step 3 is as follows: get reference substance solution 10-20 μ l respectively, need testing solution 10-20 μ l, injection liquid chromatography, obtains chromatogram, calculates Astragaloside content with external standard two-point method logarithmic equation.
8., according to described method arbitrary in claim 1-7, wherein in gained test sample, the content of every sheet Astragaloside IV must not be less than 0.15mg.
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| CN105467059B (en) * | 2015-12-30 | 2017-10-24 | 云南理想药业有限公司 | A kind of quality determining method for the Chinese medicine composition for treating blood urine |
| CN107064325A (en) * | 2016-12-08 | 2017-08-18 | 广州中医药大学第附属医院 | A kind of method of quality control of Qige granules |
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| CN102539615A (en) * | 2012-03-20 | 2012-07-04 | 安徽中医学院第一附属医院 | Method for qualitative and quantitative detection of Qibaipingfei capsule |
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| CN102370901A (en) * | 2010-08-25 | 2012-03-14 | 天津天士力制药股份有限公司 | Pharmaceutical composition for treating nephrosis and preparing process thereof |
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Address after: 300410 2 East Road Puji River, Beichen District, Tianjin (Tsun Li modern Chinese medicine city) Patentee after: TASLY PHARMACEUTICAL GROUP Co.,Ltd. Address before: 300410 Tianjin Beichen District Huaihe road and Ting Jiang West Road intersection heaven proud of the park Legal Center Intellectual Property Department Patentee before: TASLY PHARMACEUTICAL GROUP Co.,Ltd. |
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Effective date of registration: 20180726 Address after: No. 3, Ting Jiangxi Road, Beichen District, Tianjin Patentee after: TIANJIN TASLY ST. SPECIALTY PHARMACEUTICAL CO.,LTD. Address before: 300410 2 East Road Puji River, Beichen District, Tianjin (Tsun Li modern Chinese medicine city) Patentee before: TASLY PHARMACEUTICAL GROUP Co.,Ltd. |
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