CN104330489A - Quality control method for capsules with functions of dispelling wind and removing toxicity - Google Patents
Quality control method for capsules with functions of dispelling wind and removing toxicity Download PDFInfo
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Abstract
The invention provides a detection method for capsules with functions of dispelling wind and removing toxicity. The detection method is used for detecting hastatoside, verbenalin, polydatin, forsythiaside A, verbascoside, glabrolide monoammonium salt and rheum emodin in the capsules simultaneously with a high-efficiency liquid chromatography, wherein the high-efficiency liquid chromatography is performed under following conditions: a chromatographic column adopts octadecyl-silane-bonded silica gel as a filling material; mobile phases comprise a mobile phase A which is acetonitrile and a mobile phase B which is an acidic aqueous solution, and gradient elution is carried out; the flow velocity is 1.0ml/min; the column temperature is 30 DEG C; the detection wavelength is 210nm-400nm; and the number of theoretical plates is calculated according to rheum emodin peaks, and is not less than 5000. According to the detection method provided by the invention, the content of seven active components such as hastatoside, verbenalin, polydatin, forsythiaside A, verbascoside, glabrolide monoammonium salt and rheum emodin in the capsules can be detected simultaneously, so that the active components of the capsules with functions of dispelling wind and removing toxicity can be comprehensively represented. The detection method has the characteristics of high precision, stability and repeatability.
Description
Technical field
The present invention relates to Pharmaceutical Analysis detection technique field, relate to a kind of detection method of dispelling wind detoxicating capsule, particularly relate to the detection method adopting high performance liquid chromatography (HPLC) simultaneously to detect the various ingredients of described dispelling wind detoxicating capsule.
Background technology
Modern medicines have stable uniform, safety, effective three large characteristics, and wherein stable uniformity is that drug safety is effectively basic.And medicine stable uniformity is mainly derived from the whole-process control of medicine preparation technology and scientific evaluation two links of drug standard.Chinese medicine be one by multicomponent, the multifactor complex system formed, the diversity of its chemical composition and complicacy are the difficult points of quality control.Choose and cure mainly with prescription function the chemical composition be associated, and adopt the modern analysis detection techniques such as HPLC to carry out Accurate Determining and evaluate being called one of focus that recent traditional Chinese medicine quality is studied.
Dispelling wind detoxicating capsule is the large kind of Chinese medicine that Anhui Jiren Pharmacy Co., Ltd. produces, the compound Chinese medicinal preparation be made up of giant knotweed, Radix Isatidis, reed rhizome, the capsule of weeping forsythia, radix bupleuri, Verbena officinalis, Radix Glycyrrhizae, field pennycress 8 taste medicinal material, belong to wind-heat syndrome for acute upper respiratory infection, disease sees heating, feels sick, pharyngalgia, headache, nasal obstruction, flows turbid tears, coughs etc., clinical efficacy is remarkable.Dispelling wind detoxicating capsule operative norm is State Food and Drug Administration standard YBZ00652009, the content of tlc scanning determination archen is only adopted in this standard, adopt the thin-layer identification method Qualitive test capsule of weeping forsythia, Verbena officinalis and radix bupleuri, be difficult to the total quality reflecting medicine.The present invention establishes the RP-HPLC method curing mainly closely-related polygonin, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate 7 kinds of index components in Simultaneously test dispelling wind detoxicating capsule with function, thus controls medicine inherent quality comparatively fully and effectively.
Summary of the invention
The object of this invention is to provide a kind of method detecting dispelling wind detoxicating capsule, the method can the medicine activity component of complete detection dispelling wind detoxicating capsule and content thereof, thus characterize and control its inherent quality.
The object of the invention is to be achieved through the following technical solutions:
On the one hand, the invention provides a kind of detection method of dispelling wind detoxicating capsule, described dispelling wind detoxicating capsule is made up of giant knotweed, Radix Isatidis, reed rhizome, the capsule of weeping forsythia, radix bupleuri, Verbena officinalis, Radix Glycyrrhizae, field pennycress, described detection method comprises and adopts high performance liquid chromatography to detect polygonin, archen, halberd verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate in described dispelling wind detoxicating capsule simultaneously, wherein, the condition of described high performance liquid chromatography comprises:
Chromatographic column: take octadecylsilane chemically bonded silica as packing material;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.1% volume fraction aqueous formic acid, carries out gradient elution;
Described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 65min, mobile phase A is 10 ~ 30%, and Mobile phase B is 90% ~ 70%;
65 ~ 85min, mobile phase A is 30% ~ 80%, and Mobile phase B is 70% ~ 20%;
85 ~ 87min, mobile phase A is 80% ~ 10%, and Mobile phase B is 20% ~ 90%;
87 ~ 100min, mobile phase A is 10% ~ 10%, and Mobile phase B is 90% ~ 90%.
The condition of described high performance liquid chromatography also comprises:
Flow velocity: 1.0ml/min;
Column temperature: 30 DEG C;
Determined wavelength: 210nm ~ 400nm;
Theoretical cam curve is pressed archen peak and is calculated, and should be not less than 6000.
Wherein, described detection method comprises prepares reference substance solution by following steps: get polygonin reference substance, archen reference substance, halberd verbenalin reference substance, verbenalin reference substance, forsythiaside A reference substance, acteoside reference substance, mono-ammonium glycyrrhizinate reference substance, add methyl alcohol dissolving and make every 1ml containing polygonin 119.2 μ g, archen 53.2 μ g, halberd leaf verbenalin 96.0 μ g, verbenalin 89.2 μ g, forsythiaside A 58.8 μ g, acteoside 211.2 μ g, the solution of mono-ammonium glycyrrhizinate 206.0 μ g, as mixing reference substance solution.
And, described detection method also comprises and prepares need testing solution by following steps: get dispelling wind detoxicating capsule content 1.0g, accurately weighed, is placed in 50ml round-bottomed flask, add 25ml 70% ethanol water, weigh, refluxing extraction 2h, weighs after cooling, weightlessness is supplied with 70% ethanolic solution, shake up, filter, get subsequent filtrate as need testing solution.
Measure: accurate absorption mixing reference substance solution and each 10 μ l of need testing solution inject high performance liquid chromatograph, measure according to following chromatographic condition:
Adopt said method can to measure the multi-target ingredient in dispelling wind detoxicating capsule sample to be measured, the polygonin of product to be measured, archen, halberd verbenalin, verbenalin, forsythiaside A, acteoside, monoammonium glycyrrhizinate content scope should be following ranges: polygonin 5.4 ~ 12.1mg/g, archen 2.9 ~ 7.3mg/g, forsythiaside A 1.2 ~ 5.6mg/g, acteoside 1.7 ~ 2.2mg/g, halberd verbenalin 2.6 ~ 3.7mg/g, verbenalin 3.2 ~ 4.6mg/g, Radix Glycyrrhizae ester mono-ammonium 4.7 ~ 7.7mg/g.
It is below detailed description of the present invention.
Chromatographic condition
This experiment is investigated and be compared for different flow phase system, comprise methanol-water, acetonitrile-water, methyl alcohol-0.1% formic acid solution, acetonitrile-0.1% formic acid solution, result shows, with acetonitrile-0.1% formic acid solution system for mobile phase, adopt gradient elution method, Component seperation to be measured in sample is better, and theoretical cam curve meets the requirements, therefore selects acetonitrile-0.1% formic acid water as mobile phase.Because the polarity difference of each index components is comparatively large, the method for gradient elution must be adopted to carry out compartment analysis.In experiment, investigated different gradient, finally determined the condition of gradient elution of dispelling wind detoxicating capsule: acetonitrile is mobile phase A, 0.1% formic acid is Mobile phase B, 0 ~ 65min, and mobile phase A is 10 ~ 30%, and Mobile phase B is 90% ~ 70%; 65 ~ 85min, mobile phase A is 30% ~ 80%, and Mobile phase B is 70% ~ 20%; 85 ~ 87min, mobile phase A is 80% ~ 10%, and Mobile phase B is 20% ~ 90%; 87 ~ 100min, mobile phase A is 10% ~ 10%, and Mobile phase B is 90% ~ 90%.
Need testing solution adopts diode array detector (DAD) to carry out full wavelength scanner in 210 ~ 400nm wavelength coverage, and carries out com-parison and analysis to the chromatogram under each wavelength.First prepare polygonin, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate reference substance solution respectively with methanol solution, carry out UV scanning, result is known, halberd leaf verbenalin, verbenalin have absorption maximum at 240nm place, polygonin has absorption maximum at 306nm place, forsythiaside A, acteoside have absorption maximum at 330nm place, and mono-ammonium glycyrrhizinate, archen have absorption maximum at 250nm place.Contrast need testing solution HPLC chromatogram under 230nm, 250nm, 330nm wavelength respectively known: under 230nm determined wavelength, baseline wander; Under 330nm determined wavelength, the absorbance of halberd leaf verbenalin, verbenalin, mono-ammonium glycyrrhizinate, archen is all lower; Under 250nm determined wavelength, 7 kinds of compositions to be measured all have higher absorbance, and degree of separation is good, meets quantitative measurement requirement.Therefore select 250nm as determined wavelength.
The preparation of mixing reference substance solution
Accurately weighed polygonin, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate reference substance in right amount, dissolve with methyl alcohol and are diluted to scale, being mixing reference substance solution respectively.
Prepared by need testing solution
This experiment with the content of polygonin, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate for paper examines index, preferred need testing solution preparation method.To comprise ultrasonic, backflow and surname extraction 3 kinds of extracting modes investigate, result show adopt refluxing extraction time extract yield the highest.Separately comprise Extraction solvent to refluxing extraction condition, extraction time, extraction time investigate, experimental result shows, when adopting 70% alcohol reflux to extract 2h, extraction efficiency is the highest.Finally be defined as getting dispelling wind detoxicating capsule content 1.0g, accurately weighed, be placed in 50ml round-bottomed flask, add 25ml 70% ethanol water, weigh, refluxing extraction 2h, weighs after cooling, supplies weightlessness with 70% ethanolic solution, shake up, filter, get subsequent filtrate as need testing solution.
Compared with prior art, dispelling wind detoxicating capsule detection method provided by the invention has following good effect:
First, dispelling wind detoxicating capsule content involved in the present invention is 8 taste Chinese medicine compositions, complex chemical composition contained by each medicinal material, mutually interference is caused to each composition during content detection, when adopting HPLC to detect, causing other compositions to disturb each index components, making content results poor repeatability, so the chromatographic condition of mobile phase strictly must be controlled, each index components just can be made to obtain good characteristic peak.Experiment proves, only has practicable, the specific chromatographic condition adopting the present invention to obtain through great many of experiments, just can obtain the characteristic peak of the effective constituent polygonin of this medicine, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate, thus achieve effective separation of characteristic peak.
Secondly, method of the present invention can carry out content detection to the principle active component polygonin of dispelling wind detoxicating capsule such as dispelling wind detoxicating capsule, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate simultaneously, thus realize detecting the maximum possible chemical composition of dispelling wind detoxicating capsule, be conducive to the monitoring in all directions to its quality, to make the method for quality control of dispelling wind detoxicating capsule more perfect.
Further, the present invention has that method is easy, stable, precision is high, favorable reproducibility, is easy to the feature grasped.Under same test condition, achieve the assay of polygonin in dispelling wind detoxicating capsule, archen, halberd leaf verbenalin, verbenalin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate simultaneously.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 is the HPLC figure of mixing reference substance,
Wherein 1 is halberd verbenalin, and 2 is verbenalin, and 3 is polygonin, and 4 is forsythiaside A, and 5 is acteoside, and 6 is mono-ammonium glycyrrhizinate, and 7 is archen;
Fig. 2 is the HPLC figure of dispelling wind detoxicating capsule content,
Wherein 1 is halberd verbenalin, and 2 is verbenalin, and 3 is polygonin, and 4 is forsythiaside A, and 5 is acteoside, and 6 is mono-ammonium glycyrrhizinate, and 7 is archen;
Fig. 3 to 5 is respectively the HPLC figure of the condition of gradient elution 1 to 3 of test in embodiment 5;
Fig. 6 is dispelling wind detoxicating capsule UPLC/Q-TOF and Analysis on Biological Activity.
Embodiment
The technological means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with specific embodiments and the drawings, set forth the present invention further, but following embodiment being only the preferred embodiments of the present invention, and not all.Based on the embodiment in embodiment, those skilled in the art under the prerequisite not making creative work obtain other embodiment, all belong to the protection domain of this patent.
The reference substance source used in following embodiment is as follows:
Halberd leaf verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate, archen reference substance are purchased from National Institute for Food and Drugs Control, and lot number is respectively 110713-201208,110737-201212,111575-201302,111810-201306,111530-201305,110731-201310,110756-201310.
The test sample dispelling wind detoxicating capsule used in following embodiment is prepared according to State Food and Drug Administration standard YBZ00652009 for Anhui Jiren Pharmacy Co., Ltd., has fully focused on the place of production attribute of each medicinal material in prescription, collecting season feature, process control features in preparation process.Obtained batch number sees the following form 1:
The sample table of the dispelling wind detoxicating capsule adopted in table 1 embodiment
Other Instruments and reagent:
Adopt Dionex liquid chromatograph, comprise quaternary pump, online degasser, automatic sampler, diode array detector (DAD), column oven, Chromeleon workstation; Electronic balance: Sartovius (100,000/balance); METTLER TOLEDO (ten thousand/balance); Ultrasound Instrument: AUTOSCIENCE (AS3120).
Acetonitrile (chromatographically pure) is purchased from Concord, Tianjin Science and Technology Ltd.; Formic acid (top grade is pure) is purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.; 95% ethanol (analyzing pure) is purchased from Tianjin Kai Xin chemical industry company limited; Methyl alcohol (top grade is pure) is purchased from Concord, Tianjin Science and Technology Ltd.; It magnetic pure water.
embodiment 1 adopts HPLC to set up the multicomponent assay method of dispelling wind detoxicating capsule content
The preparation of mixing reference substance solution: accurately weighed halberd leaf verbenalin 4.80mg, verbenalin 4.46mg, polygonin 5.96mg, forsythiaside A 2.94mg, acteoside 10.56mg, mono-ammonium glycyrrhizinate 10.30mg, archen 2.66mg respectively, respectively to 25ml measuring bottle, methyl alcohol dissolved dilution is to scale, shake up, as storing solution.Precision measures in each 5ml to the 10ml measuring bottle of reference substance storing solution, with methanol dilution to scale, shakes up, obtains mixing reference substance solution.
Prepared by need testing solution: dispelling wind detoxicating capsule content is placed in mortar grind into fine powder, take 1.000g medicinal powder, be placed in 100ml round-bottomed flask, add 25ml 70% ethanol water, weigh, refluxing extraction 2h, weigh after cooling, supply weightlessness with 70% ethanolic solution, filter, as need testing solution.
The mensuration of multi-target ingredient: draw above-mentioned mixing reference substance solution and dispelling wind detoxicating capsule need testing solution injection liquid chromatography, according to high effective liquid chromatography for measuring, wherein chromatographic determination condition comprises:
Chromatographic column: Unitary C18 (200mm × 4.6mm, 5 μm);
Determined wavelength: 230nm; Column temperature: 30 DEG C; Flow velocity: 1.0ml/min; Sample size 10 μ l;
Acetonitrile-0.1% aqueous formic acid gradient elution, condition of gradient elution sees the following form 2:
Table 2 assay gradient
Mixing reference substance solution, need testing solution HPLC chromatogram are shown in Fig. 1 and Fig. 2 respectively.
the investigation of need testing solution preparation method in embodiment 2 multicomponent detection method
1, extracting mode is investigated
Take dispelling wind detoxicating capsule content 3 parts, add appropriate 70% ethanol, adopt ultrasonic respectively, backflow, surname extraction, filter, obtain need testing solution, the cubage carrying out testing Related Component with determined condition the results are shown in Table 3.
Table 3 extracting mode investigates result
Contrast three kinds of extracting modes, can find out that refluxing extraction is most effective, therefore selects reflux extraction method.
2, Extraction solvent is investigated
Parallelly take 3 parts of 1.000g dispelling wind detoxicating capsule medicinal powder, add respectively 25ml50%, 70%, 90% ethanolic solution, refluxing extraction 2h, filters, obtains need testing solution, and the cubage carrying out testing Related Component with determined condition the results are shown in Table 4.
Table 4 Extraction solvent investigates result
Experimental result shows, the highest as extraction efficiency during Extraction solvent with 70% ethanol, and can obtain the gem-pure chromatographic peak of lines.Therefore determine to select the ethanol water of 70% as Extraction solvent.
3, extraction time is investigated
Parallelly take 3 parts of 1.000g dispelling wind detoxicating capsule medicinal powder, add 25ml 70% ethanol, respectively refluxing extraction 1h, 1.5h, 2h, filter, obtain need testing solution, the cubage carrying out testing Related Component with determined condition the results are shown in Table 5.
Table 5 extraction time investigates result
Experimental result shows, along with the increase of extraction time, extraction efficiency is obviously improved, so the extraction time selecting 2h is top condition.
4, extraction time is investigated
Parallelly take 3 parts of 1.000g dispelling wind detoxicating capsule medicinal powder, add 25ml 70% ethanol, respectively refluxing extraction 1,2,3 times, each 2h, filter, obtain need testing solution, the cubage carrying out testing Related Component with determined condition the results are shown in Table 6.
Table 6 extraction time investigates result
Note: "-" expression does not detect.
Experimental result shows, extraction time does not make significant difference to extraction efficiency, so selective extraction number of times is 1 time.
the methodological study of embodiment 3 multicomponent detection method
(1) precision test
Adopt the operation identical with embodiment 1 and condition, sample thief is about 1.000g, accurately weighed, by need testing solution, preparation method processes, accurate absorption need testing solution 10 μ l, inject high performance liquid chromatograph, continuous sample introduction 6 times under above-mentioned chromatographic condition, halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen RSD value are respectively 2.53%, 2.18%, 2.76%, 2.87%, 2.19%, 1.33%, 1.97%.Explanation precision is good.
(2) study on the stability
Adopt the operation identical with embodiment 1 and condition, sample thief is about 1.000g, by need testing solution preparation extraction, respectively at 0,1,3,6,9,12,24h sample introduction, measure under above-mentioned chromatographic condition.As a result, halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen RSD value are respectively 2.04%, 2.68%, 1.96%, 2.37%, 2.15%, 2.08%, 2.23%.Illustrate that need testing solution places 24h internal stability at ambient temperature good.
(3) linear relationship is determined
Draw respectively three reference substance storing solutions 0.5,1,2, in 3ml to 10ml measuring bottle, draw halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen reference substance storing solution certain volume methanol dilution become some concentration; Respectively measure 10 μ L injection liquid chromatographies, under the chromatographic condition of embodiment 1, measure peak area.
With reference substance peak area absorption value for ordinate (Y), take sample size as horizontal ordinate (X) drawing standard curve, calculate regression equation, as shown in table 7.
Table 7 each reference substance typical curve summary sheet
(4) replica test
Adopt the operation identical with embodiment 1 and condition, get same sample lots and be about 1.000g, accurately weighed 6 parts, after carrying out extraction process by need testing solution preparation method, measure each sample solution 10 μ L, inject high performance liquid chromatograph, measure, result halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen RSD value are respectively 2.04%, 2.68%, 1.96%, 2.37%, 2.15%, 2.08%, 2.23%, and repeatability is good.
(5) recovery test
The sample getting known content is about 0.500g, accurately weighed 9 parts, add a certain amount of reference substance solution by 80%, 100%, 120% precision of sample determination amount respectively, operate by need testing solution preparation method, measure under above-mentioned chromatographic condition, calculate the recovery.The average recovery rate of result halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen is followed successively by 99.44%, 97.85%, 98.65%, 96.55%, 100.23%, 98.54% and 99.78%, and its RSD value is followed successively by 1.85%, 2.15%, 2.35%, 2.56%, 1.77%, 1.69% and 2.01%.
the multicomponent content detection of embodiment 4 dispelling wind detoxicating capsule content
14 batches of dispelling wind detoxicating capsule contents are measured to the content of its halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen.
Adopt the operation identical with embodiment 1 and condition preparation to mix reference substance solution and need testing solution, accurately measure each 10 μ l injection liquid chromatographies, record peak area, calculates content by external standard method.Result table 8.
Each component content measurement result (mg/g) in table 8 dispelling wind detoxicating capsule
Multicomponent detection method provided by the invention may be used for the quality controlling dispelling wind detoxicating capsule.This detection method is adopted to detect the content of halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen in dispelling wind detoxicating capsule product, if its content is then specification product within the limits prescribed.
the choice and optimization of condition of gradient elution in embodiment 5 chromatographic detection method
Because dispelling wind detoxicating capsule is large compound, flavour of a drug are many, complicated component, and easily cause interference between composition when detecting.Before determining chromatographic test strip part of the present invention, carry out repeatedly comparative experiments.Wherein the determination of condition of gradient elution comprises:
Determining using acetonitrile-0.05% volume fraction aqueous formic acid as elution flow after phase, under other process and the identical situation of method of the present invention, attempting multiple different condition of gradient elution, such as:
Condition of gradient elution 1: acetonitrile (A)-0.1% aqueous formic acid (B), 0 ~ 10min, 5%A ~ 15%A; 10 ~ 35min, 15%A ~ 30%A; 35 ~ 60min, 30% ~ 80%A; 60 ~ 70min, 80% ~ 100%A; 70 ~ 72min, 100% ~ 5%A; 72 ~ 85min, 5% ~ 5%A; Chromatogram is shown in Fig. 3;
Condition of gradient elution 2: acetonitrile (A)-0.1% aqueous formic acid (B), 0 ~ 50min, 15% ~ 30%A; 50 ~ 70min, 30% ~ 80%A; 70 ~ 72min, 80% ~ 15%A; 72 ~ 85min, 15% ~ 15%A; Chromatogram is shown in Fig. 4;
Condition of gradient elution 3: acetonitrile (A)-0.1% aqueous formic acid (B), 0 ~ 65min, 10% ~ 30%A; 65 ~ 85min, 30% ~ 80%A; 85 ~ 87min, 80% ~ 10%A; 87 ~ 100min, 10% ~ 10%A; Chromatogram is shown in Fig. 5.
Experimental result shows, under the elution requirement except gradient 3, halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen all can not be made well to be separated simultaneously.Therefore, from a large amount of condition of gradient elution experimental studies, optimize condition of gradient elution of the present invention, halberd verbenalin can be made, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen is well separated simultaneously, noiseless with the adjacent chromatographic peak of each composition, and have good reappearance, also show that elution requirement of the present invention becomes swarming interference without other through negative controls test, condition of gradient elution of the present invention can simultaneously to the principle active component halberd verbenalin of dispelling wind detoxicating capsule such as dispelling wind detoxicating capsule, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen carries out content detection, effectively characterize the multifarious inherent quality of traditional Chinese medicine ingredients on the whole.
the correlation analysis of embodiment 6 multicomponent content and drug effect
This place adopts the directed method knocked out, and select and cure mainly with dispelling wind detoxicating capsule function the extracorporeal anti-inflammatory pharmacological model matched, spectrum effect relationship dispelling wind detoxicating capsule finger-print and extracorporeal anti-inflammatory drug action having been carried out to dispelling wind detoxicating capsule is studied.
1, laboratory sample preparation
UPLC/Q-TOF sample preparation: get 0.2g dispelling wind detoxicating capsule powder in 50ml conical flask, add 12ml 70% methyl alcohol, after ultrasonic 1h, the centrifugal 15min of 12000rpm, gets supernatant and analyze for UPLC/Q-TOF.
Cell experiment sample preparation: get 1.0g dispelling wind detoxicating capsule powder in 50ml conical flask, add 12ml 70% methyl alcohol, after ultrasonic 1h, the centrifugal 15min of 12000rpm, gets supernatant and connect peak for UPLC.Sample is after UPLC is separated, by eluent by within every 30 seconds, being that one section (about 200 μ L mobile phase) is directly collected in (specification: every pore volume is 2.2ml) in 96 deep-well plates, then be put in 50 DEG C of vacuum drying chambers and volatilize mobile phase, residue directly adds appropriate cell culture medium, ultrasonic 20min redissolves, and finally nutrient culture media is joined experiment Analysis in cell screening system.
2, UPLC chromatographic condition
Liquid-phase chromatographic column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 100mm); Mobile phase: acetonitrile (A), 0.1% (V/V) formic acid ultrapure water (B), flow rate of mobile phase: 0.4ml/min; Column temperature: 35 DEG C; Sample introduction 4 μ l (when connecing peak sample introduction 10 μ l); Experiment adopts gradient elution mode, and wash-out table is in table 9.
Table 9 eluent gradient wash-out table
3, Q-TOF experiment condition
This experiment uses Waters Premier mass spectrometer, and positive and negative two kinds of Mode scans measure, and instrument parameter is as follows:
Adopt electric spray ion source; V model; Capillary voltage holotype 3.0kV, negative mode 2.5kV; Taper hole voltage 30V; Ion source temperature 110 DEG C; Desolventizing temperature degree 350 DEG C; Desolventizing nitrogen flow 600L/h; Taper hole airshed 50L/h; Detector voltage holotype 1900V, negative mode 2000V; Sample frequency 0.1s, interval 0.02s; Mass number sensing range 100 ~ 1500Da; Internal reference calibration solution adopts LEK acetate ([M+H]
+=555.2931, [M-H]
-=553.2775).
4, cell culture processes
Human embryonic kidney epithelial cells (293T) is purchased from Shanghai Bai Li Bioisystech Co., Ltd.Cell culture condition: cultivate (containing 1% dual anti-and 10% hyclone) with DMEM HIGH GLUCOSE complete medium and be incubated at 37 DEG C, 5%CO
2cO
2incubator.
4.1 cell recovery
(1) from liquid nitrogen container, take out cell rapidly and put into 37 DEG C of water-baths immediately, shake cryopreservation tube makes its fast melt.
(2) cell is immediately after centrifugal 1000rpm, 3min, abandoning supernatant.
(3) in white cell precipitation, add 1ml nutrient culture media, mix centrifugal, abandoning supernatant.
(4) 25m is transferred to after adding 5ml complete culture solution dissolution precipitation
2in culture flask, " cross " method pats culture flask wall concussion mixing, puts into 37 DEG C, 5%CO
2cultivate in incubator.
(5) liquid is changed after cell chulture 6h, to remove the metabolic waste and dead cell etc. of frozen middle generation.
4.2 cells change liquid and go down to posterity
(1) determined to change liquid frequency by cell growth status, within general one to two days, change liquid once.
(2) operate simple and easy when changing liquid, discard old training base, add new complete medium.
(3) when Growth of Cells to 90%, discard old training base, add 1ml pancreatin (25m
2culture flask) in 37 DEG C, 5%CO
2incubator digestion 1min.
(4) add 1ml complete culture solution and stop trypsinization reaction, rob repeatedly to blow and beat at the bottom of culture flask with moving liquid attached cell is suspended.
(5) transitional cell suspending liquid is in centrifuge tube, abandoning supernatant after the centrifugal 3min of 1000rpm.
(6) in cell precipitation, add 1ml complete culture solution mixing cell, and transfer to the 25m being added with 4ml complete medium
2in culture flask, in 37 DEG C, 5%CO
2continue in incubator to cultivate.
5, luciferase reporter gene plasmid transient cotransfection
By 293T cell chulture in 96 orifice plates, when Growth of Cells merges to 50-70%, the mixed liquor of transfection reagent PEI (1mg/ml), internal reference luciferase reporter plasmid Renilla (9.6ng/ hole) and NF-κ B luciferase reporter plasmid pGL4.32 (100ng/ hole) is transfected in cell, wherein PEI:pGL4.32=8:1 (mass ratio), after plasmid transfection 24h, add after serum-free medium cultivates 12h synchronization cell and can be used for subsequent experimental.
6, cell experiment grouping and administration
The blank group (Control) of this Setup Experiments, model group (Model produces inflammation with the TNF-α irritation cell of 10ng/ml), positive drug group (Dex, 10
-5the dexamethasone of mol/L) and dispelling wind detoxicating capsule administration group.After each group of medicine preincubate 2h, add TNF-α modeling 6h, collect lysate and be used for NF-κ B fluoroscopic examination.
7, NF-κ B luciferase assays
After administration completes, discard cell culture fluid, PBS cleans cell twice, discards PBS, and every hole adds 20 μ l cell pyrolysis liquids and shake 30min on oscillator, and cell pyrolysis liquid is used for NF-κ B fluoroscopic examination, and concrete operation step is as follows:
(1) (Dual-GloTM Luciferase substrate fully mixes with Dual-GloTM Luciferase damping fluid equal-volume to prepare firefly luciferase detection reagent,-20 DEG C of preservations are stand-by) and internal reference Luciferase Assay Reagent (Dual-GloTM Stop & Glo substrate and Dual-GloTM Stop & Glo damping fluid mix with the volume ratio of 50:1, need matching while using).
(2) get 15 μ l cell pyrolysis liquids, add 20 μ l firefly luciferases and detect reagent, gently after mixing, detect with Modulus fluorescence detector and record NF-κ B fluorescent value.
(3) add 20 μ l internal reference Luciferase Assay Reagent again, mix gently, detect with Modulus fluorescence detector and record Renilla fluorescent value.
(4) calculate luciferase reporter gene active, represent (relative fluorescence ratio=NF-κ B fluorescent value/internal reference Renilla fluorescent value) with relative ratio.
8, active material configuration qualification
For identifying the structure of 19 labels, experiment have employed UPLC/Q-TOF and has carried out first mass spectrometric and second order ms experimental analysis to sample.Measured [M+H] that can obtain material by the positive and negative pattern analysis of first mass spectrometric
+[M-H]
-the information of quasi-molecular ion peak, carry out Elemental Composition by the accurate mass number of four after radix point to analyse, then the molecular fragment information in secondary spectrogram is utilized, analyze the cracking rule of its fragment, consult a large amount of pertinent literatures, accurate mass number and secondary molecules fragment and list of references and compound library are analysed and compared, referring again to uv absorption wavelength and the relative retention time information at peak, considers the structural information analyzing and then determine peak.
9, statistical analysis technique
Experimental result represents with the standard units of the error of the mean (SEM), and compare between group and adopt one-way analysis of variance (One-way ANOVA), compare between independent two groups and use the t method of inspection, p<0.05 is for there being significant difference.
10, result and discussion
10.1 Anti-Inflammatory Actives the selection result
This experiment carries out cell experiment to collect 80 sections of dispelling wind detoxicating capsule samples, first carries out administration concentration and gropes.Obtain the eluent that every hole about 200 μ l is dissolved with medicine when connecing peak, after volatilizing solvent, dilute one times (add 400 μ l and train base) and original content administration (add 200 μ l and train base) respectively with cell culture fluid.After found that dilution one times of administration, every section of medicine does not all have too significant antiphlogistic effects, illustrates that administration concentration is lower.And have a few segment table to reveal obvious reduction by the expression of cell NF-κ B after original content administration, therefore determine to carry out cell administration with original content.
Have significant inhibiting effect by filtering out 9 sections of 10 chromatographic peaks after original content administration altogether to NF-κ B expression, peak number is followed successively by 1,2,3,4,5,6,7,8,9, No. 10 (as Fig. 6) in chronological order, and concrete data are in table 10.
NF-κ B inhibiting rate tables of data after the administration of table 10 dispelling wind detoxicating capsule original content
10.2 Anti-Inflammatory Actives qualification result
After chemical substance carries out first mass spectrometric mensuration in dispelling wind detoxicating capsule, material quasi-molecular ion peak ([M+H] can be obtained
+or [M-H]
-) relevant information.On this basis, take quasi-molecular ion as the mensuration that parent ion carries out secondary fragment under corresponding pattern, according to second order ms structural information and the report in conjunction with pertinent literature, to in dispelling wind detoxicating capsule 10 have the chemical composition of anti-inflammatory activity to carry out identification and analysis, the qualification result of concrete composition and see table 11.
Table 11 dispelling wind detoxicating capsule anti-inflammatory activity monomer structure information table
In sum, detection method provided by the invention can adopt high performance liquid chromatography to detect halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen in dispelling wind detoxicating capsule simultaneously, therefore can characterize this product active constituents of medicine content comprehensively, thus its inherent quality of standard, while the stable uniform ensureing product, guarantee the safety and effectiveness of product.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions is only preference of the present invention; be not used for limiting the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (5)
1. the detection method of a dispelling wind detoxicating capsule, it is characterized in that, described dispelling wind detoxicating capsule is made up of giant knotweed, the capsule of weeping forsythia, Radix Isatidis, radix bupleuri, field pennycress, Verbena officinalis, reed rhizome, Radix Glycyrrhizae, described detection method comprises and adopts high performance liquid chromatography to detect halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen in described dispelling wind detoxicating capsule simultaneously, wherein, the condition of described high performance liquid chromatography comprises:
Chromatographic column: take octadecylsilane chemically bonded silica as packing material;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.1% volume fraction aqueous formic acid, carries out gradient elution;
Described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 65min, mobile phase A is 10 ~ 30%, and Mobile phase B is 90% ~ 70%;
65 ~ 85min, mobile phase A is 30% ~ 80%, and Mobile phase B is 70% ~ 20%;
85 ~ 87min, mobile phase A is 80% ~ 10%, and Mobile phase B is 20% ~ 90%;
87 ~ 100min, mobile phase A is 10% ~ 10%, and Mobile phase B is 90% ~ 90%.
2. detection method according to claim 1, is characterized in that: the condition of described high performance liquid chromatography also comprises:
Flow velocity: 1.0ml/min;
Column temperature: 30 DEG C;
Determined wavelength: 210nm ~ 400nm;
Theoretical cam curve is pressed archen peak and is calculated, and should be not less than 6000.
3. detection method according to claim 2, it is characterized in that: described detection method also comprises and prepares reference substance solution by following steps: get halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen reference substance, add methyl alcohol dissolving and make every 1ml halberd verbenalin 96 μ g, verbenalin 89.2 μ g, polygonin 119.2 μ g, forsythiaside A 58.8 μ g, acteoside 211.2 μ g, mono-ammonium glycyrrhizinate 206 μ g, the solution of archen 53.2 μ g, as mixing reference substance solution.
4. detection method according to claim 3, it is characterized in that, described method for building up also comprises and prepares need testing solution by following steps: dispelling wind detoxicating capsule content is placed in mortar grind into fine powder, takes 1.000g medicinal powder, be placed in 100ml round-bottomed flask, add 25ml70% ethanol water, weigh, refluxing extraction 2h, weigh after cooling, supply weightlessness with 70% ethanolic solution, filter, as need testing solution.
5. detection method according to any one of claim 1 to 4, is characterized in that, described high performance liquid chromatography comprises the following steps:
(1) preparation mixing reference substance solution: get halberd verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen, add methyl alcohol dissolving and make every 1ml containing halberd verbenalin 96 μ g, verbenalin 89.2 μ g, polygonin 119.2 μ g, forsythiaside A 58.8 μ g, acteoside 211.2 μ g, mono-ammonium glycyrrhizinate 206 μ g, the solution of archen 53.2 μ g, as mixing reference substance solution;
(2) need testing solution is prepared: dispelling wind detoxicating capsule content is placed in mortar grind into fine powder, take 1.000g medicinal powder, be placed in 100ml round-bottomed flask, add 25ml70% ethanol water, weigh, refluxing extraction 2h, weigh after cooling, supply weightlessness with 70% ethanolic solution, filter, as need testing solution;
(3) measure: accurate absorption mixing reference substance solution and each 10 μ l of need testing solution inject high performance liquid chromatograph, measure according to following chromatographic condition:
Chromatographic column: take octadecylsilane chemically bonded silica as packing material;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.1% aqueous formic acid, carries out gradient elution;
Flow velocity: 1.0ml/min;
Column temperature: 30 DEG C;
UV detect wavelength: 250nm;
Mobile phase ratio is as follows:
0 ~ 65min, mobile phase A is 10 ~ 30%, and Mobile phase B is 90% ~ 70%;
65 ~ 85min, mobile phase A is 30% ~ 80%, and Mobile phase B is 70% ~ 20%;
85 ~ 87min, mobile phase A is 80% ~ 10%, and Mobile phase B is 20% ~ 90%;
87 ~ 100min, mobile phase A is 10% ~ 10%, and Mobile phase B is 90% ~ 90%;
Record peak area, adopts external standard method to calculate the content of verbenalin, verbenalin, polygonin, forsythiaside A, acteoside, Radix Glycyrrhizae ester mono-ammonium, archen.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105126106A (en) * | 2015-09-17 | 2015-12-09 | 安徽济人药业有限公司 | Application of TIPE2 (tumor necrosis factor-alpha induced protein 8 like-2) agonist in protection on sepsis complicated with acute lung injury |
| CN107233511A (en) * | 2017-06-14 | 2017-10-10 | 安徽济人药业有限公司 | A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes |
| CN107807183A (en) * | 2017-10-09 | 2018-03-16 | 株洲千金药业股份有限公司 | The content assaying method of polygonin in relaxing muscles and tendons rheumatism wine |
| CN111189952A (en) * | 2020-03-19 | 2020-05-22 | 安徽济人药业有限公司 | Method for measuring special content of verbena and controlling quality of verbena |
| CN114832009A (en) * | 2022-05-06 | 2022-08-02 | 安徽医科大学 | Application of pennywort glycoside in medicine for treating hepatic fibrosis |
| CN115128200A (en) * | 2022-07-26 | 2022-09-30 | 吉首大学 | HPLC (high Performance liquid chromatography) quality detection method for paulownia leaves |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197018A (en) * | 2012-01-04 | 2013-07-10 | 天士力制药集团股份有限公司 | Yangxueqingnao granule anthraquinone component detection method |
| CN103675189A (en) * | 2012-09-05 | 2014-03-26 | 亚宝药业集团股份有限公司 | Quality detection method for fructus forsythiae leaf medicinal materials |
-
2014
- 2014-10-27 CN CN201410583418.XA patent/CN104330489B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197018A (en) * | 2012-01-04 | 2013-07-10 | 天士力制药集团股份有限公司 | Yangxueqingnao granule anthraquinone component detection method |
| CN103675189A (en) * | 2012-09-05 | 2014-03-26 | 亚宝药业集团股份有限公司 | Quality detection method for fructus forsythiae leaf medicinal materials |
Non-Patent Citations (3)
| Title |
|---|
| CHAO FENG 等: "Simultaneous determination of 10 active components in traditional Chinese medicine "YIGONG" capsule by RP-HPLC–DAD", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 张海欢 等: "HPLC法测定疏风解毒胶囊中虎杖苷", 《中成药》 * |
| 杨春杰 等: "HPLC法测定疏风解毒胶囊中大黄素的含量", 《医药前沿》 * |
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| CN107233511A (en) * | 2017-06-14 | 2017-10-10 | 安徽济人药业有限公司 | A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes |
| CN107807183A (en) * | 2017-10-09 | 2018-03-16 | 株洲千金药业股份有限公司 | The content assaying method of polygonin in relaxing muscles and tendons rheumatism wine |
| CN111189952A (en) * | 2020-03-19 | 2020-05-22 | 安徽济人药业有限公司 | Method for measuring special content of verbena and controlling quality of verbena |
| CN114832009A (en) * | 2022-05-06 | 2022-08-02 | 安徽医科大学 | Application of pennywort glycoside in medicine for treating hepatic fibrosis |
| CN114832009B (en) * | 2022-05-06 | 2023-07-18 | 安徽医科大学 | Application of euphorbia pekinensis glycoside in medicine for treating liver fibrosis |
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| CN115128200B (en) * | 2022-07-26 | 2024-01-19 | 吉首大学 | HPLC quality detection method for paulownia tomentosa leaves |
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