CN108152416A - The finger print measuring method of active constituent in Chinese mugwort acid extract - Google Patents
The finger print measuring method of active constituent in Chinese mugwort acid extract Download PDFInfo
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- CN108152416A CN108152416A CN201810003324.9A CN201810003324A CN108152416A CN 108152416 A CN108152416 A CN 108152416A CN 201810003324 A CN201810003324 A CN 201810003324A CN 108152416 A CN108152416 A CN 108152416A
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Abstract
The invention discloses a kind of finger print measuring methods of active constituent in Chinese mugwort acid extract, it solves the problems such as prior art cannot reflect the inherent quality of Chinese mugwort acid extract, cannot control effectively to its quality, it is characterised in that：The assay method is using Chinese mugwort acid extract as raw material, it is analyzed using high performance liquid chromatography, gradient elution is carried out by eluent system of the phosphate aqueous solution of acetonitrile -0.2%, the finger-print of test solution as obtained by high performance liquid chromatograph is tested with Chinese mugwort acid extract standard finger-print through similarity evaluation, employing fingerprint TuPu method controls the quality of Chinese mugwort acid extract comprehensively, it is ensured that the quality and health-care efficacy of Chinese mugwort acid extract product.The present invention has the characteristics that precision is high, stability is good, reproducible, and simultaneously its quality of overall monitor can be effectively separated to Chinese mugwort acid extract active constituent.
Description
Technical field：
The invention belongs to product quality control method, particularly with regard to a kind of fingerprint image of active constituent in Chinese mugwort acid extract
Compose assay method.
Background technology：
Chinese mugwort acid extract is Chinese mugwortArtemisia argyi Levl. et Van. var. argyi cv. QiaiIt is processed
Manufactured extract.It takes Chinese mugwort medicinal material appropriate, 12 times of amount water is added to impregnate 3 hours, are extracted 2 hours through steam distillation, collection is begged
End essential oil.The dregs of a decoction add 6 times of amount water to decoct 1 hour, and the filtration of 300 mesh filter clothes, it is 1.02 ~ 1.03 that filtrate, which is concentrated into relative density,（60
℃）, concentrate adds 95% ethyl alcohol to stand 10 hours to alcohol content up to 80%, the filtration of 400 mesh filter clothes, and filtrate recycling ethanol is to opposite
Density is 0.90 ~ 1.01（60℃）, most ethyl alcohol is waved, is dry, pulverize at 60 DEG C ~ 80 DEG C to get Chinese mugwort acid extract.Chinese mugwort acid
Extract has the pharmacological actions such as antibacterial, anti-inflammatory, removing toxic substances, has to Escherichia coli, staphylococcus aureus, pneumococcus and virus
Stronger inhibiting effect；There are obvious curative effects to acute laryngopharyngitis and skin disease.
But current China not yet performs the organoleptic indicator of Chinese mugwort acid extract, physical and chemical index and microbiological indicator related rule
It is fixed, fail the inherent quality for reflecting Chinese mugwort acid extract, it is impossible to control effectively to its quality.
Finger-print, can be comprehensive based on chemical composition system research in sample as a kind of comprehensive identification means
Reflect the type and content of contained chemical composition in sample, available for progress such as the authenticities, characteristic and Optimality to sample
Evaluation is one of main method of sample quality control, as one of most effective technology of internationally recognized quality control.
Invention content：
The purpose of the present invention is exactly the one kind provided for variation and the difference of thoroughly evaluating Chinese mugwort acid extract active constituent
The finger print measuring method of Chinese mugwort acid extract active constituent.
The technical scheme is that：It is analyzed using high performance liquid chromatography, with the phosphate aqueous solution of acetonitrile -0.2%
Gradient elution is carried out for eluent system, finger-print and the Chinese mugwort acid of the test solution as obtained by high performance liquid chromatograph carry
Object standard finger-print is taken to be tested through similarity evaluation, employing fingerprint TuPu method is to Chinese mugwort acid
The quality of extract is controlled comprehensively, it is ensured that the quality and health-care efficacy of Chinese mugwort acid extract product are as follows：
(a) preparation of reference substance solution：Take chlorogenic acid reference substance appropriate, it is accurately weighed, add methanol that every 1mL is made containing chlorogenic acid
The solution of 0.06mg is to get reference substance solution.
(b) preparation of test solution：Chinese mugwort acid extract 0.25g is taken, it is accurately weighed, it puts in conical flask with cover, it is accurate
50% methanol 25mL, close plug are added in, weighed weight is ultrasonically treated（Power 250W, frequency 40KHZ）It 30 minutes, lets cool to room temperature,
Weighed weight again is supplied the weight of less loss with 50% methanol, is shaken up, and filtration, precision measures subsequent filtrate 5mL, puts in 10mL measuring bottles,
50% methanol is added to shake up to scale, use miillpore filter（0.45μm）Filtration, takes subsequent filtrate to get test solution.
(c) chromatographic condition is set：It is filler with octadecylsilane chemically bonded silica, mobile phase A is acetonitrile, and Mobile phase B is
Mass percent is 0.2% phosphate aqueous solution；Using gradient elution mode, elution program such as following table：
Gradient elution program
| Time（Minute） | Mobile phase A（%） | Mobile phase B（%） |
| 0-12 | 10→20 | 90→80 |
| 12-25 | 20→25 | 80→75 |
| 25-50 | 25→65 | 75→35 |
| 50-60 | 65→10 | 35→90 |
Detection wavelength 330nm, flow velocity 0.8mL/min, 30 DEG C of column temperature；Number of theoretical plate should be not less than by the calculating of chlorogenic acid peak
5000。
(d) acquisition of standard finger-print：Precision draws 10 μ L of reference substance solution and test solution, according to the color of setting
Spectral condition injects high performance liquid chromatograph, by the chromatographic peak of reference substance chlorogenic acid, determines green in test solution chromatogram
Ortho acid peak obtains the standard finger-print of Chinese mugwort acid extract according to the method for chromatogram methodological study.
(e) chromatography：By the finger-print of the test solution obtained by every batch of and Chinese mugwort acid extract standard fingerprint
Collection of illustrative plates compares through similarity evaluation, to determine the quality of Chinese mugwort acid extract.
The standard finger-print of the obtained Chinese mugwort acid extract of the present invention detects 6 characteristic fingerprint peaks, and No. 5 peaks are green
Ortho acid,
No. 1 peak：Retention time：3.827 ± 0.2min,
No. 2 peaks：Retention time：4.887 ± 0.2 min,
No. 3 peaks：Retention time：8.847 ± 0.2min,
No. 4 peaks；Retention time：10.550 ± 0.2min,
No. 5 peaks：Retention time：11.693±0.2min.
No. 6 peaks：Retention time：12.257±0.2min.
Beneficial effects of the present invention：The present invention is using high performance liquid chromatography to the fingerprint of Chinese mugwort acid extract active constituent
Collection of illustrative plates is studied, by the finger-print of the test solution of gained and Chinese mugwort acid extract standard finger-print, through Chinese medicine
Chromatographic fingerprinting similarity evaluation system test.It has the following advantages：
1. the chromatographic process established has the advantages that precision is high, stability is good, reproducible, meet the requirement of finger-print,
Chinese mugwort acid extraction of substance quantity measuring method, the quality of overall monitor Chinese mugwort acid extract can be used as；
2. Chinese mugwort acid extract active constituent can be effectively separated, the finger-print of Chinese mugwort acid extract is constructed, is filled up
The blank that Chinese mugwort acid extract is studied in active constituent separation and finger-print；
3. the research to a certain ingredient is no longer limited to the quality evaluation of Chinese mugwort acid extract, but from the overall evaluation, it should
The quality of Chinese mugwort acid extract is controlled comprehensively with Fingerprints, it is ensured that the health-care efficacy of Chinese mugwort acid extract and beg
The quality and safety of the sour extract product of Chinese mugwort provides commenting for a kind of effective product quality detection method and technology innovation
Valency platform.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Description of the drawings：
Fig. 1 is the chromatogram of reference substance chlorogenic acid；
Fig. 2 is the standard finger-print of Chinese mugwort acid extract；
Fig. 3 is ten crowdes of Chinese mugwort acid extract collection of illustrative plates stacking charts.
Specific embodiment：
The finger print measuring method of active constituent in Chinese mugwort acid extract, it is characterised in that：Using high performance liquid chromatography into
Row analysis carries out gradient elution, as obtained by high performance liquid chromatograph by eluent system of the phosphate aqueous solution of acetonitrile -0.2%
The finger-print of test solution is with Chinese mugwort acid extract standard finger-print through chromatographic fingerprints of Chinese materia medica similarity evaluation system
System experiment, employing fingerprint TuPu method control the quality of Chinese mugwort acid extract comprehensively, it is ensured that Chinese mugwort acid extract product
Quality and health-care efficacy.In following embodiments, chlorogenic acid is bought in Nat'l Pharmaceutical ＆ Biological Products Control Institute, lot number 110753-
201314；Methanol is chromatographically pure, is bought in U.S. world company.It is as follows：
(a) preparation of reference substance solution：Take chlorogenic acid reference substance appropriate, it is accurately weighed, add methanol that every 1mL is made containing chlorogenic acid
The solution of 0.06mg is to get reference substance solution.
(b) preparation of test solution：This product about 0.25g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds in 50%
Methanol 25mL, close plug, weighed weight are ultrasonically treated（Power 250W, frequency 40KHZ）It 30 minutes, lets cool to room temperature, then weighed heavy
Amount, the weight of less loss is supplied with 50% methanol, is shaken up, and is filtered, and precision measures subsequent filtrate 5mL, puts in 10mL measuring bottles, adds 50% methanol
It to scale, shakes up, uses miillpore filter（0.45μm）Filtration, takes subsequent filtrate to get test solution.
(c) chromatographic condition：Use the Agilent ZORBAX Extend-C that octadecylsilane chemically bonded silica is filler18
(250mm × 4.6mm, 5 μm) chromatographic column；Using gradient elution, mobile phase A is acetonitrile, and Mobile phase B is that mass percent is 0.2%
Phosphate buffered saline solution, it is as shown in the table for the program of gradient elution：
Gradient elution program
| Time（Minute） | Mobile phase A（%） | Mobile phase B（%） |
| 0-12 | 10→20 | 90→80 |
| 12-25 | 20→25 | 80→75 |
| 25-50 | 25→65 | 75→35 |
| 50-60 | 65→10 | 35→90 |
Detection wavelength 330nm, flow velocity 0.8mL/min, 30 DEG C of column temperature；Number of theoretical plate should be not less than by the calculating of chlorogenic acid peak
5000。
(d) acquisition of standard finger-print：Precision draws 10 μ L of reference substance solution and test solution, according to the color of setting
Spectral condition injects high performance liquid chromatograph, by the chromatographic peak of reference substance chlorogenic acid, determines green in test solution chromatogram
Ortho acid peak obtains the standard finger-print of Chinese mugwort acid extract according to the method for chromatogram methodological study.
Fig. 1 is the chromatogram of reference substance chlorogenic acid；Fig. 2 is the standard finger-print of Chinese mugwort acid extract, and Chinese mugwort acid carries
It takes and 6 characteristic fingerprint peaks is detected on the standard finger-print of object, No. 5 peaks are chlorogenic acid, as shown in the table：
The characteristic fingerprint peak of Chinese mugwort acid extract standard finger-print
| Peak serial number | Retention time (min) | Peak area (mAU*min) |
| 1 | 3.827 | 1.4131 |
| 2 | 4.887 | 0.6564 |
| 3 | 8.847 | 2.4012 |
| 4 | 10.557 | 1.6232 |
| 5 | 11.693 | 16.5029 |
| 6 | 12.257 | 3.3372 |
(e) chromatography：The quality of evaluation Chinese mugwort acid extract that finger-print can integrate, therefore by begging obtained by every batch of
The sour extract finger-print of Chinese mugwort is with Chinese mugwort acid extract standard finger-print through similarity evaluation
2004A editions are compared.The similarity of 10 batches of Chinese mugwort acid extract chromatographic peaks is calculated, similarity is not less than 0.9, illustrates that Chinese mugwort acid extracts
Amount of substance is qualified.3 crowdes of Chinese mugwort acid extract finger-print stacking charts are as shown in Figure 3.
The methodological study of the present invention：
1) precision research：Precision draws 6 μ L of test solution, and according to the chromatographic condition of setting, continuous sample introduction 5 times records it
Chromatogram.Using chlorogenic acid peak as with reference to peak, it is 1.0000 to specify its α value, investigates instrument precision.Each characteristic fingerprint peak is opposite
The RSD values of retention time and relative peak area are respectively less than 3%, meet the requirement of finger-print.
2) stability study：Precision draws 10 μ L of test solution, according to the chromatographic condition of setting, respectively 0h, 4h,
8h, 12h, it is detected for 24 hours, using chlorogenic acid peak as with reference to peak, it is 1.0000 to specify its α value, investigates test solution stability.
The relative retention time at each characteristic fingerprint peak and the RSD values of relative peak area are respectively less than 3%, meet the requirement of finger-print.
3) repetitive research：Take same batch Chinese mugwort acid extract（Lot number is 201409）5 parts of sample, it is accurately weighed, it presses
Method described in step (b) prepares test solution, is measured according to the chromatographic condition of setting, using chlorogenic acid peak as reference
Peak, it is 1.0000 to specify its α value, investigates the repeatability of method.The relative retention time and relative peak area at each characteristic fingerprint peak
RSD values be respectively less than 3%, meet the requirement of finger-print.
Claims (2)
1. a kind of finger print measuring method of active constituent in Chinese mugwort acid extract, it is characterised in that：Using high-efficient liquid phase color
Spectrometry is analyzed, and is carried out gradient elution by eluent system of the phosphate aqueous solution of acetonitrile -0.2%, is passed through high performance liquid chromatograph
The finger-print of the test solution of gained is with Chinese mugwort acid extract standard finger-print through chromatographic fingerprints of Chinese materia medica similarity
Evaluation system is tested, and employing fingerprint TuPu method controls the quality of Chinese mugwort acid extract comprehensively, it is ensured that Chinese mugwort acid extracts
The quality and health-care efficacy of Tetramune, are as follows：
(a) preparation of reference substance solution：Accurately weighed chlorogenic acid reference substance adds methanol to be made every 1mL 0.06mg's containing chlorogenic acid
Solution is to get reference substance solution；
(b) preparation of test solution：Chinese mugwort acid extract 0.25g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds in
50% methanol 25mL, close plug, weighed weight by the supersound process 30 minutes of power 250W, frequency 40KHZ, are let cool to room temperature,
Weighed weight again is supplied the weight of less loss with 50% methanol, is shaken up, and filtration, precision measures subsequent filtrate 5mL, puts in 10mL measuring bottles,
50% methanol is added to shake up to scale, filtered with 0.45 μm of miillpore filter, take subsequent filtrate to get test solution；
(c) chromatographic condition is set：It is filler with octadecylsilane chemically bonded silica, mobile phase A is acetonitrile, and Mobile phase B is quality
Percentage is 0.2% phosphate aqueous solution；Using gradient elution mode, elution program such as following table：
Gradient elution program
Detection wavelength 330nm, flow velocity 0.8mL/min, 30 DEG C of column temperature；Number of theoretical plate should be not less than by the calculating of chlorogenic acid peak
5000；
(d) acquisition of standard finger-print：Precision draws 10 μ L of reference substance solution and test solution, according to the chromatostrip of setting
Part injects high performance liquid chromatograph, by the chromatographic peak of reference substance chlorogenic acid, determines the chlorogenic acid in test solution chromatogram
Peak obtains the standard finger-print of Chinese mugwort acid extract according to the method for chromatogram methodological study；
(e) chromatography：By the finger-print of the test solution obtained by every batch of and Chinese mugwort acid extract standard finger-print,
Compare through similarity evaluation, to determine the quality of Chinese mugwort acid extract.
2. the finger print measuring method of active constituent in Chinese mugwort acid extract according to claim 1, it is characterised in that：
The standard finger-print of obtained Chinese mugwort acid extract detects 6 characteristic fingerprint peaks, and No. 5 peaks are chlorogenic acid,
No. 1 peak：Retention time：3.827 ± 0.2min,
No. 2 peaks：Retention time：4.887 ± 0.2 min,
No. 3 peaks：Retention time：8.847 ± 0.2min,
No. 4 peaks；Retention time：10.550 ± 0.2min,
No. 5 peaks：Retention time：11.693 ± 0.2min,
No. 6 peaks：Retention time：12.257±0.2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115372534A (en) * | 2022-08-22 | 2022-11-22 | 北京康仁堂药业有限公司 | Folium artemisiae argyi and construction method of characteristic map of preparation of folium artemisiae argyi, characteristic map and application |
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| CN103705559A (en) * | 2013-01-07 | 2014-04-09 | 中国医学科学院药用植物研究所 | Folium artemisiae argyi extract extracted by ethyl acetate and preparation and detection methods and application thereof |
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2018
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Patent Citations (3)
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| CN101550120A (en) * | 2008-04-02 | 2009-10-07 | 天津天士力制药股份有限公司 | Effective component of artemisia leaves and preparation method thereof |
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| CN103705559A (en) * | 2013-01-07 | 2014-04-09 | 中国医学科学院药用植物研究所 | Folium artemisiae argyi extract extracted by ethyl acetate and preparation and detection methods and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115372534A (en) * | 2022-08-22 | 2022-11-22 | 北京康仁堂药业有限公司 | Folium artemisiae argyi and construction method of characteristic map of preparation of folium artemisiae argyi, characteristic map and application |
| CN115372534B (en) * | 2022-08-22 | 2023-10-24 | 北京康仁堂药业有限公司 | Construction method of characteristic spectrum of mugwort leaf and preparation thereof, characteristic spectrum and application |
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