CN102119997B - Method for establishing HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and standard finger-print thereof - Google Patents
Method for establishing HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and standard finger-print thereof Download PDFInfo
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- CN102119997B CN102119997B CN2010101065857A CN201010106585A CN102119997B CN 102119997 B CN102119997 B CN 102119997B CN 2010101065857 A CN2010101065857 A CN 2010101065857A CN 201010106585 A CN201010106585 A CN 201010106585A CN 102119997 B CN102119997 B CN 102119997B
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Abstract
The invention provides a method for establishing an HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and a standard finger-print thereof. The method comprises the following steps of: preparing a test article solution; preparing a reference substance solution; selecting chromatographic conditions; and measuring with a high performance liquid chromatograph to obtain the finger-print of ophiopogon japonicus. 16 batches of HPLC finger-prints of ophiopogon japonicus are compared to determine the common finger-print characteristics of ophiopogon japonicus, thereby obtaining the standard finger-print. The method provided by the invention is simple, convenient, accurate and reliable, and has the characteristics of good reproducibility, multiple characteristic peaks and the like. The established standard finger-print can effectively show the quality of ophiopogon japonicus.
Description
Technical field
The present invention relates to the construction method of Chinese crude drug finger-print, especially the construction method and the standard finger-print thereof of river medicinal material tuber of dwarf lilyturf high performance liquid chromatography (HPLC) finger-print.Belong to the Pharmaceutical Analysis technical field.
Background technology
Traditional Chinese medicine fingerprint refers in particular to the collection of illustrative plates or the image of each component population characteristic of Chinese medicine that the Chinese medicine sample obtains through modern analytical techniques such as spectrum or chromatograms.Traditional Chinese medicine fingerprint at present, admitted by numerous international medical mechanism as a kind of comprehensive, quantifiable quality control method, become one of effective method of Chinese patent drug, natural drug quality control.FDA (Food and Drug Adminstration) is in " FDA is about the guide of vegetable products " formulated in 1996, and requiring provides corresponding finger-print to article and autonomic drug product in the middle of plant material, the autonomic drug.Medicinal and the fragrant plant association of British Herbal Pharmacopoeia, India herbal medicine allusion quotation and Canada, German medicinal plant association also accept chromatographic fingerprinting.China national drug regulatory department has issued " technical requirement (provisional) of the finger-print research of traditional Chinese medicine " in 2000, explicitly call for traditional Chinese medicine of newly declaring and the traditional Chinese medicine that has gone on the market are carried out the finger-print standard.
Traditional Chinese medicine fingerprint has passed through the research of many decades; Many modern analysis means of testing such as comprising ultraviolet, infrared, gas phase, high performance liquid chromatogram, thin layer, nuclear magnetic resonance, scanning electron microscope, computer image analysis, electrophoretic techniques, isoenzyme analysis method, Protocols in Molecular Biology, cluster analysis technology have been used, for the material of Chinese medicine is identified and constituent analysis has accumulated lot of data.But see from the angle of Chinese medicine total quality control, also reach the purpose that reflects effectively and control the total quality of Chinese medicine far away.High performance liquid chromatography (HPLC) finger-print obtains approval in the world easily, comprises that many countries of the U.S., Britain, France, Canada, Germany, Japan and India more are ready to accept the HPLC finger-print.Therefore, setting up corresponding HPLC finger-print for Chinese crude drug, is the effective means of present stage control Chinese medicine inherent quality.
Be the dried root of Liliaceae Ophiopogon per nnial herb Ophiopogon tuber of dwarf lilyturf japonicus (Thunb.) Ker-Gawl. the tuber of dwarf lilyturf, and Sichuan is one of its maximum main producing region.Be usually used in illnesss such as dryness of the lung dry cough, cough due to consumptive disease, thirsty, the vexed insomnia of Tianjin wound, interior heat are quenched one's thirst, the dry constipation of intestines on the tcm clinical practice.Contain the number of chemical composition tuber of dwarf lilyturf, since report in 1936 contains vitamin a compound in the piece root tuber of dwarf lilyturf, successively from the tuber of dwarf lilyturf, isolated compounds such as carbohydrate, saponin(e, flavones; At present, " Chinese pharmacopoeia is single to the quality assessment mode of the tuber of dwarf lilyturf, is not enough to system, intactly shows the total quality of the medicinal material tuber of dwarf lilyturf, needs that exploration is more advanced, more effective quality control method badly.
Summary of the invention
The method for building up that the purpose of this invention is to provide a kind of river medicinal materials fingerprint tuber of dwarf lilyturf, and the resulting river of method medicinal material tuber of dwarf lilyturf standard finger-print thus.For realizing this purpose; The present invention is through the research to river medicinal material tuber of dwarf lilyturf efficient liquid-phase chromatograph finger print atlas; Proposed a kind of better river quality of medicinal material tuber of dwarf lilyturf control method, remedied the deficiency of existing Quality Control Technology, the quality control of the river medicinal material tuber of dwarf lilyturf is improved and science more.
The method for building up of river of the present invention medicinal material tuber of dwarf lilyturf efficient liquid-phase chromatograph finger print atlas comprises the steps:
(1) preparation of need testing solution: precision takes by weighing medicinal powder weighing sample tuber of dwarf lilyturf 3g to tool plug container, adds 95% ethanol water 50ml, and ultrasonic concussion (20 ℃, 90min); Membrane filtration, filtrating rotary evaporation to 2-3ml (50 ℃, 80r/min) separate with 40ml is water-soluble, be transferred in the centrifuge tube; Extracted with diethyl ether 4 times, each 20ml is with ether layer rotary evaporation to 3～5ml; Methanol constant volume is crossed the disposable filter membrane of 0.45 μ m to 10ml, and filtrating is as need testing solution.
(2) efficient liquid phase chromatographic analysis: chromatography column is Symmetry-C
18(250 * 4.6mm, 5 μ m); Moving phase is methyl alcohol-0.2% aqueous acetic acid, gradient elution, and elution program is: 0min → 20min → 38min → 67min → 90min, methyl alcohol 15% → 32% → 67% → 74% → 100%; Flow velocity: 1.0mL/min detects wavelength: 280nm, sample size: 15 μ L, column temperature: 30 ℃.Analyze need testing solution with this understanding, obtain the finger-print of the river medicinal material tuber of dwarf lilyturf.
According to method provided by the invention, the 16 batches of river medicinal materials tuber of dwarf lilyturf have been set up the HPLC finger-print, through analyzing relatively, confirmed 25 common characteristic peaks.
The retention time of characteristic peak is respectively: 5.2min, 17.2min, 18.1min, 23.4min, 32.4min, 36.5min, 41.4min, 48.6min, 51.2min, 52.7min, 55.6min, 57.0min, 64.4min, 67.1min, 68.0min, 69.6min, 76.7min, 77.2min, 78.1min, 78.7min, 79.3min, 80.0min, 80.9min, 83.3min, 87.0min; These common characteristic peaks have constituted the fingerprint characteristic of the river medicinal material tuber of dwarf lilyturf, can be used as the standard finger-print of the river medicinal material tuber of dwarf lilyturf.
The present invention has following significant advantage and purposes:
(1) the river medicinal material tuber of dwarf lilyturf is done as a whole treating, can find out the nuance between the different medicinal materials through comparing its total peak;
(2) test sample is simple for production, and chromatographic condition is realized easily;
(3) stability of method and reappearance are all relatively good;
(4) be suitable for discriminating and control to the river medicinal material tuber of dwarf lilyturf true and false, the place of production and quality.
Description of drawings
Fig. 1 is the standard finger-print of the river provided by the invention medicinal material tuber of dwarf lilyturf.From left to right marked No. 1 to 25, its common characteristic peak among the figure respectively.
Embodiment
The preferred embodiments of the present invention are following:
(1) instrument and medicine: use Agilent 1101 high performance liquid chromatographs; Totally 16 batches of the river medicinal materials tuber of dwarf lilyturf all are collected in Mianyang City Santai County, Sichuan Province, the genuine producing region tuber of dwarf lilyturf, river; Other reagent is chromatographically pure or analyzes pure.
(2) precision takes by weighing medicinal powder weighing sample tuber of dwarf lilyturf 3g to tool plug container, adds 95% ethanol water 50ml, and ultrasonic concussion (20 ℃, 90min); Membrane filtration, filtrating rotary evaporation to 2-3ml (50 ℃, 80r/min) separate with 40ml is water-soluble, be transferred in the centrifuge tube; Extracted with diethyl ether 4 times, each 20ml is with ether layer rotary evaporation to 3～5ml; Methanol constant volume is crossed the disposable filter membrane of 0.45 μ m to 10ml, and filtrating is as need testing solution.
(3) efficient liquid phase chromatographic analysis: the chromatographic resolution column packing is 5 μ m Symmetry-C
18(250 * 4.6mm); Moving phase is methyl alcohol-0.2% aqueous acetic acid, gradient elution, and elution program is: 0min → 20min → 38min → 67min → 90min, methyl alcohol 15% → 32% → 67% → 74% → 100%; Flow velocity: 1.0mL/min detects wavelength: 280nm, sample size: 15 μ L, column temperature: 30 ℃.Analyze need testing solution with this understanding, obtain the finger-print of the river medicinal material tuber of dwarf lilyturf.
(4) according to method provided by the invention; The 16 batches of river medicinal materials tuber of dwarf lilyturf have been set up the HPLC finger-print; Through analyzing relatively; 25 common characteristic peaks have been confirmed; Its retention time is respectively: 5.2min, 17.2min, 18.1min, 23.4min, 32.4min, 36.5min, 41.4min, 48.6min, 51.2min, 52.7min, 55.6min, 57.0min, 64.4min, 67.1min, 68.0min, 69.6min, 76.7min, 77.2min, 78.1min, 78.7min, 79.3min, 80.0min, 80.9min, 83.3min, 87.0min; These common characteristic peaks have constituted the fingerprint characteristic of the river medicinal material tuber of dwarf lilyturf, can be used as the standard finger-print of the river medicinal material tuber of dwarf lilyturf.
Claims (1)
1. the method for building up of a river medicinal material tuber of dwarf lilyturf efficient liquid-phase chromatograph finger print atlas, its characteristic comprise preparation, the making of finger-print and the confirming of standard finger-print of need testing solution,
The preparation method of described need testing solution is: precision takes by weighing medicinal powder weighing sample tuber of dwarf lilyturf 3g to tool plug container, adds 95% ethanol water 50ml, 20 ℃ of following ultrasonic concussion 90min, membrane filtration; Filtrating is at 50 ℃, and 80r/min rotating speed rotation down is evaporated to 2-3ml, separates with 40ml is water-soluble, is transferred in the centrifuge tube; Extracted with diethyl ether 4 times, each 20ml is with ether layer rotary evaporation to 3～5ml; Methanol constant volume is crossed the disposable filter membrane of 0.45 μ m to 10ml, and filtrating is as need testing solution;
The manufacturing process of described finger-print is: chromatography column is Symmetry-C
18250 * 4.6mm, 5 μ m; Moving phase is methyl alcohol-0.2% aqueous acetic acid, gradient elution, and elution program is: 0min → 20min → 38min → 67min → 90min, methyl alcohol 15% → 32% → 67% → 74% → 100%; Flow velocity: 1.0mL/min, detect wavelength: 280nm, sample size: 15 μ L, column temperature: 30 ℃, analyze need testing solution with this understanding, obtain the finger-print of the river medicinal material tuber of dwarf lilyturf;
Definite method of described standard finger-print is: the 16 batches of river medicinal materials tuber of dwarf lilyturf have been set up the HPLC finger-print; Through analyzing relatively; 25 common characteristic peaks have been confirmed; Retention time is respectively: 5.2min, 17.2min, 18.1min, 23.4min, 32.4min, 36.5min, 41.4min, 48.6min, 51.2min, 52.7min, 55.6min, 57.0min, 64.4min, 67.1min, 68.0min, 69.6min, 76.7min, 77.2min, 78.1min, 78.7min, 79.3min, 80.0min, 80.9min, 83.3min, 87.0min; These common characteristic peaks have constituted the fingerprint characteristic of the river medicinal material tuber of dwarf lilyturf, as the standard finger-print of the river medicinal material tuber of dwarf lilyturf.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101065857A CN102119997B (en) | 2010-01-29 | 2010-01-29 | Method for establishing HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and standard finger-print thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101065857A CN102119997B (en) | 2010-01-29 | 2010-01-29 | Method for establishing HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and standard finger-print thereof |
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| CN102119997A CN102119997A (en) | 2011-07-13 |
| CN102119997B true CN102119997B (en) | 2012-08-22 |
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| CN2010101065857A Expired - Fee Related CN102119997B (en) | 2010-01-29 | 2010-01-29 | Method for establishing HPLC (high performance liquid chromatography) finger-print of ophiopogon japonicus and standard finger-print thereof |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109541098B (en) * | 2018-11-27 | 2020-11-10 | 华润三九（雅安）药业有限公司 | Radix ophiopogonis fingerprint spectrum, construction method thereof and radix ophiopogonis quality detection method |
| CN109633012B (en) * | 2018-12-29 | 2021-11-12 | 正大青春宝药业有限公司 | Identification method of Zhejiang ophiopogon root |
| CN111024855B (en) * | 2019-12-30 | 2022-09-09 | 常熟雷允上制药有限公司 | Fingerprint detection method for traditional Chinese medicine radix ophiopogonis |
| CN112858535A (en) * | 2021-04-02 | 2021-05-28 | 中国计量大学 | Radix ophiopogonis producing area identification method |
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