CN102944625A - Method for establishing carthamus tinctorius fingerprint by using high performance liquid chromatography and standard fingerprint of method - Google Patents
Method for establishing carthamus tinctorius fingerprint by using high performance liquid chromatography and standard fingerprint of method Download PDFInfo
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- CN102944625A CN102944625A CN2012104825555A CN201210482555A CN102944625A CN 102944625 A CN102944625 A CN 102944625A CN 2012104825555 A CN2012104825555 A CN 2012104825555A CN 201210482555 A CN201210482555 A CN 201210482555A CN 102944625 A CN102944625 A CN 102944625A
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Abstract
The invention relates to a method for establishing a traditional Chinese medicinal material fingerprint and particularly relates to a method for establishing a carthamus tinctorius fingerprint by using a high performance liquid chromatography and a standard fingerprint of the method. The method includes preparing a test solution; evenly mixing carthamus tinctorius powders with water, and subjecting the mixture to ultrasonic vibration and filtering by using a millipore filter film; and manufacturing the fingerprint, and injecting the test solution into a high performance liquid chromatograph, wherein the chromatographic conditions are that a chromatographic column filler is Phenomenex C18, and the specification of the chromatographic column filler is 250*4.6mm, 5 mu m; the column temperature is 25-35 DEG C; and the mobile phase is B phase acetonitrile and an A phase phosphoric acid water solution with mass percentage of 0.1-0.2 %, the flow velocity is 0.8-1.2 mL/min, the detection wavelength is 370nm, and the sample quantity is 20 mu L. The carthamus tinctorius fingerprint is suitable for identifying and controlling authenticity, production places and qualities of carthamus tinctorius medicines.
Description
Technical field
The present invention relates to the method for building up of Fingerprint of Chinese medicine materia, the method for building up of flos carthami high performance liquid chromatography (HPLC) finger-print especially, and the resulting flos carthami standard finger-print of method thus belong to the Pharmaceutical Analysis technical field.
Background technology
Safflower is the flower of feverfew safflower.Safflower is annual herb, and extensively cultivate on the ground such as China northeast, North China, northwest and Shandong, Zhejiang, Guizhou, Sichuan, Tibet.Safflower is warm in nature, and flavor is hot, activating blood to promote menstruation, blood stasis removing analgesic.Be used for through close, dysmenorrhoea, lochia, abdominal mass lump in the abdomen, traumatic injury.When ancients often have what blood in vivo, often add the safflower tate.Bundle is boiled available one day twice bubble pin, applicable various varication, and peripheral neuritis, blood circulation is bad, lower limb numbness or the congestion such as livid purple.Comparison of ingredients is complicated in the safflower, contains red and pigment yellow: carthamin, front carthamin, carthamus tinctorius yellow colour A and B, the bright glucoside A of safflower.Contain again polyphenol components: chlorogenic acid, with coffee acid, catechol, Jiao's property catechol, DOPA.Also contain volatile ingredient more than 80 and plant, and the safflower polysaccharide etc.Because be subjected to the impact of the place of production, weather and ecologic environment etc., the contained composition of different flos carthamis also exists different." Chinese pharmacopoeia only limits to the mensuration of carthamus tinctorius yellow colour A and Kaempferol content to the quality control of flos carthami, can not system, intactly reflect the inherent quality of safflower.Control safflower and prescribed preparation effect thereof, just can not be only for 1,2 chemical constitution wherein, must be controlled its material group integral body.
Traditional Chinese medicine fingerprint refers in particular to collection of illustrative plates or the image of each component colony common characteristic of Chinese medicine that Chinese crude drug or Chinese patent drug obtain through modern analytical techniques such as spectrum or chromatograms.At present finger-print has become the discriminating herbal species of generally acknowledging both at home and abroad and has estimated one of effective means of traditional Chinese medicine quality.
Traditional Chinese medicine fingerprint has passed through the research of many decades, used to comprise many modern analysis and test means such as ultraviolet, infrared, gas phase, high efficiency liquid phase, thin layer, nuclear magnetic resonance, scanning electron microscope, computer image analysis, electrophoretic techniques, isoenzyme analysis method, Protocols in Molecular Biology, Clustering Analysis Technology, for identification and the constituent analysis of Chinese medicine accumulated a large amount of data.But from the angle of Chinese medicine total quality control, also reach far away the purpose that effectively reflects and control the total quality of Chinese medicine.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.And, the present finger-print that does not also have flos carthami.
There is the defective of the deficiency of Quality Control Technology in existing flos carthami method of quality control.
Summary of the invention
The method for building up that the purpose of this invention is to provide a kind of flos carthami finger-print.
The method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas of the present invention may further comprise the steps:
One, the preparation of need testing solution: take by weighing the flos carthami powder, place tool plug container, the ratio that in mass volume ratio is 1g:100 ~ 300mL mixes flos carthami powder and water, at ambient temperature, ultrasonic concussion 45min, get the supernatant filtering with microporous membrane, collect filtrate as need testing solution;
Two, the making of finger-print: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, is that take the quality percentage composition as 0.2% phosphate aqueous solution A phase, acetonitrile are that B carries out gradient elution mutually;
Wherein, high-efficient liquid phase chromatogram condition is:
Chromatographic column filler Phenomenex C18, specification is 250 * 4.6mm, 5 μ m; Column temperature is 25 ℃ ~ 35 ℃; Mobile phase be B phase acetonitrile with A mutually the quality percentage composition be 0.1～0.2% phosphate aqueous solution, flow velocity 0.8 ~ 1.2mL/min detects wavelength 370nm, sample size 20 μ L;
Analyze with this understanding need testing solution, obtain the finger-print of flos carthami.
The present invention adopts the finger-print of the flos carthami that the method for building up of flos carthami efficient liquid-phase chromatograph finger print atlas obtains.
The present invention comprises following beneficial effect:
(1) flos carthami is done as a whole treating, can be found out nuance between the different medicinal materials by its total peak relatively;
(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 flos carthami true and false, the place of production and quality, remedied the deficiency of existing Quality Control Technology, the quality control of flos carthami is improved and science more.
Description of drawings
Fig. 1 is the standard finger-print of flos carthami provided by the invention; Wherein, 1 to 10 is flos carthami common characteristic peak.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the combination in any between each embodiment.
Embodiment one: the method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas of present embodiment may further comprise the steps:
One, the preparation of need testing solution: take by weighing the flos carthami powder, place tool plug container, the ratio that in mass volume ratio is 1g:100 ~ 300mL mixes flos carthami powder and water, at ambient temperature, ultrasonic concussion 45min, get the supernatant filtering with microporous membrane, collect filtrate as need testing solution;
Two, the making of finger-print: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, is that take the quality percentage composition as 0.2% phosphate aqueous solution A phase, acetonitrile are that B carries out gradient elution mutually;
Wherein, high-efficient liquid phase chromatogram condition is:
Chromatographic column filler Phenomenex C18, specification is 250 * 4.6mm, 5 μ m; Column temperature is 25 ℃ ~ 35 ℃; Mobile phase be B phase acetonitrile with A mutually the quality percentage composition be 0.1～0.2% phosphate aqueous solution, flow velocity 0.8 ~ 1.2mL/min detects wavelength 370nm, sample size 20 μ L;
Analyze with this understanding need testing solution, obtain the finger-print of flos carthami.
Present embodiment comprises following beneficial effect:
(1) flos carthami is done as a whole treating, can be found out nuance between the different medicinal materials by its total peak relatively;
(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 flos carthami true and false, the place of production and quality, remedied the deficiency of existing Quality Control Technology, the quality control of flos carthami is improved and science more.
Embodiment two: what present embodiment and embodiment one were different is: in the described eluent gradient elution process, eluent A, B phase transformation turn to: 0 ~ 30min, B mutually 15% ~ 25%; 30 ~ 40min, B phase 25% ~ 50%; 40 ~ 50min, B phase 50% ~ 80%; 50 ~ 60min, B phase 80%.Other is identical with embodiment one.
Embodiment three: what present embodiment was different from embodiment one or two is: described in the step 1 is that the ratio of 1g:200mL mixes flos carthami powder and water in mass volume ratio.Other is identical with embodiment one or two.
Embodiment four: what present embodiment was different from one of embodiment one to three is: the filtering with microporous membrane described in the step 1 adopts 0.45 μ m miillpore filter to filter.Other is identical with one of embodiment one to three.
Embodiment five: the finger-print of the flos carthami that the method for building up of present embodiment employing flos carthami efficient liquid-phase chromatograph finger print atlas obtains.
Present embodiment comprises following beneficial effect:
(1) flos carthami is done as a whole treating, can be found out nuance between the different medicinal materials by its total peak relatively;
(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 flos carthami true and false, the place of production and quality, remedied the deficiency of existing Quality Control Technology, the quality control of flos carthami is improved and science more.
Embodiment six: what present embodiment and embodiment five were different is: described collection of illustrative plates has 10 common characteristic peaks, retention time is respectively: 8.1min, 9.7min, 10.5min, 10.9min, 12.9min, 13.7min, 14.6min, 22.0min, 24.3min and 38.2min, these common characteristic peaks have consisted of the fingerprint characteristic of flos carthami, can be used as the standard finger-print of flos carthami.Other is identical with embodiment five.
By following verification experimental verification beneficial effect of the present invention:
Test 1
1. instrument and medicine: use Agilent-1100 type high performance liquid chromatograph, be furnished with online degasser, quaternary gradient pump, automatic sampler, column oven, diode array detector; Totally 10 batches of flos carthamis all are purchased from Chinese herbal medicine market; Other reagent is chromatographically pure or analyzes pure.
2. the preparation of need testing solution: precision takes by weighing flos carthami powder 0.5g, places tool plug container, adds water 100mL, and ultrasonic concussion 45min gets supernatant with 0.45 μ m filtering with microporous membrane after the cooling, and filtrate is as need testing solution.
3. efficient liquid phase chromatographic analysis: chromatographic resolution column packing 5 μ m Phenomenex C18,250 * 4.6mm; Mobile phase 0.2% phosphate aqueous solution-acetonitrile; Adopt gradient elution mode 0min → 30min → 40min → 50min → 60min, acetonitrile 15% → 25% → 50% → 80% → 80%; Flow velocity 1.0mL/min; Detect wavelength 370nm; 30 ℃ of column temperatures; Sample size 20 μ l; Analyze with this understanding need testing solution, obtain the finger-print of flos carthami.
4. according to method provided by the invention, 10 batches of flos carthamis have been set up the HPLC finger-print, by analyzing relatively, 10 common characteristic peaks have been determined, retention time is respectively: 8.1min, 9.7min, 10.5min, 10.9min, 12.9min, 13.7min, 14.6min, 22.0min, 24.3min, 38.2min, these common characteristic peaks have consisted of the fingerprint characteristic of flos carthami, can be used as the standard finger-print of flos carthami.
The standard finger-print of the flos carthami that this test obtains as shown in Figure 1.As seen from Figure 1, can clearly obtain having the standard finger-print of the flos carthami at 10 common characteristic peaks by this method, for the quality of overall evaluation flos carthami provides good foundation.
5. methodological study
5.1 precision is investigated
Get same batch sample, carry out the need testing solution preparation by the method for test 1, continuous sample introduction is measured 5 times, data are inputted similarity evaluation, calculate, obtain the similarity data of precision matched data and precision coupling, the results are shown in Table 1 and table 2 shown in.
Table 1 precision matched data
The similarity data of table 2 precision coupling
By table 1 and table 2 as can be known, the standard finger-print similarity of the flos carthami of this test is more than 0.9, and precision is good.
5.2 method repeatability is investigated
Get same batch sample, prepare 6 parts of need testing solutions by the method for testing 1, sample introduction is measured respectively, data are inputted similarity evaluation, calculate, obtain repeated matched data and repeated matching similarity data, the results are shown in Table 3 and table 4 shown in.
The repeated matched data of table 3
The repeated matching similarity data of table 4
By table 3 and table 4 as can be known, similarity is more than 0.9, and repeatability is good.
5.3 study on the stability
Get same batch sample, method by test 1 is prepared need testing solution, measure at 0,1,2,3,4,5 and 6 hour sample introduction respectively, data are inputted similarity evaluation, calculate, obtain the similarity data of Stable matching data and Stable matching, the results are shown in Table 5 and table 6 shown in.
Table 5 Stable matching data
The similarity data of table 6 Stable matching
By table 5 and table 6 as can be known, similarity has good stability more than 0.9.
6. sample tests
Get ten batches with place of production flos carthami, make need testing solution (before use preparation) according to the method for test 1, sample introduction 20 μ L detect, carrying out spectrogram processes, data are inputted similarity evaluation (2004A version) generate flos carthami finger-print template, the results are shown in Table 7, table 8.
Technical parameter: contrast collection of illustrative plates generation method: median method
Time window width: 0.5
Matching way: Auto-matching
Table 7 generates the matched data of contrast spectrum
The similarity data of table 80 batch samples coupling
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. the method for building up of a flos carthami efficient liquid-phase chromatograph finger print atlas is characterized in that, may further comprise the steps:
One, the preparation of need testing solution: take by weighing the flos carthami powder, place tool plug container, the ratio that in mass volume ratio is 1g:100 ~ 300mL mixes flos carthami powder and water, at ambient temperature, ultrasonic concussion 45min, get the supernatant filtering with microporous membrane, collect filtrate as need testing solution;
Two, the making of finger-print: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, is that take the quality percentage composition as 0.2% phosphate aqueous solution A phase, acetonitrile are that B carries out gradient elution mutually;
Wherein, high-efficient liquid phase chromatogram condition is:
Chromatographic column filler Phenomenex C18, specification is 250 * 4.6mm, 5 μ m; Column temperature is 25 ℃ ~ 35 ℃; Mobile phase be B phase acetonitrile with A mutually the quality percentage composition be 0.1～0.2% phosphate aqueous solution, flow velocity 0.8 ~ 1.2mL/min detects wavelength 370nm, sample size 20 μ L;
Analyze with this understanding need testing solution, obtain the finger-print of flos carthami.
2. the method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas according to claim 1 is characterized in that in the described eluent gradient elution process, eluent A, B phase transformation turn to: 0 ~ 30min, B phase 15% ~ 25%; 30 ~ 40min, B phase 25% ~ 50%; 40 ~ 50min, B phase 50% ~ 80%; 50 ~ 60min, B phase 80%.
3. the method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas according to claim 1 is characterized in that described in the step 1 being that the ratio of 1g:200mL mixes flos carthami powder and water in mass volume ratio.
4. the method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas according to claim 1 is characterized in that the filtering with microporous membrane described in the step 1 adopts 0.45 μ m miillpore filter to filter.
5. the finger-print of the flos carthami that obtains of the method for building up of a kind of flos carthami efficient liquid-phase chromatograph finger print atlas as claimed in claim 1.
6. the finger-print of flos carthami according to claim 5, it is characterized in that described collection of illustrative plates has 10 common characteristic peaks, retention time is respectively: 8.1min, 9.7min, 10.5min, 10.9min, 12.9min, 13.7min, 14.6min, 22.0min, 24.3min and 38.2min, these common characteristic peaks have consisted of the fingerprint characteristic of flos carthami, can be used as the standard finger-print of flos carthami.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364501A (en) * | 2013-07-04 | 2013-10-23 | 上海大学 | Method for detecting pigment-adulterated golden orange II in red flower |
| CN105920071A (en) * | 2016-04-29 | 2016-09-07 | 中国科学院新疆理化技术研究所 | Applications of safflower extract with definite spectrum-effect relationship |
| CN106770037A (en) * | 2017-02-13 | 2017-05-31 | 中国药科大学 | A kind of discrimination method of datura flower medicinal material |
| CN108866228A (en) * | 2018-07-17 | 2018-11-23 | 成都中医药大学 | A kind of discrimination method of different sources safflower |
| CN113092602A (en) * | 2021-03-05 | 2021-07-09 | 甘肃佛阁藏药有限公司 | Fingerprint quality detection method for safflower Ruyi pill |
| CN115541728A (en) * | 2022-05-13 | 2022-12-30 | 华润三九现代中药制药有限公司 | Method for constructing characteristic spectrum of safflower medicinal material and safflower medicinal preparation and application |
-
2012
- 2012-11-23 CN CN2012104825555A patent/CN102944625A/en active Pending
Non-Patent Citations (1)
| Title |
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| 宋金春 等: "红花的HPLC指纹图谱研究", 《中国药学杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364501A (en) * | 2013-07-04 | 2013-10-23 | 上海大学 | Method for detecting pigment-adulterated golden orange II in red flower |
| CN105920071A (en) * | 2016-04-29 | 2016-09-07 | 中国科学院新疆理化技术研究所 | Applications of safflower extract with definite spectrum-effect relationship |
| CN105920071B (en) * | 2016-04-29 | 2019-07-23 | 中国科学院新疆理化技术研究所 | A kind of purposes of the safflower extract with clear spectrum effect relationship |
| CN106770037A (en) * | 2017-02-13 | 2017-05-31 | 中国药科大学 | A kind of discrimination method of datura flower medicinal material |
| CN106770037B (en) * | 2017-02-13 | 2018-02-27 | 中国药科大学 | A kind of discrimination method of datura flower medicinal material |
| CN108866228A (en) * | 2018-07-17 | 2018-11-23 | 成都中医药大学 | A kind of discrimination method of different sources safflower |
| CN108866228B (en) * | 2018-07-17 | 2022-02-15 | 成都中医药大学 | Method for identifying safflower in different producing areas |
| CN113092602A (en) * | 2021-03-05 | 2021-07-09 | 甘肃佛阁藏药有限公司 | Fingerprint quality detection method for safflower Ruyi pill |
| CN115541728A (en) * | 2022-05-13 | 2022-12-30 | 华润三九现代中药制药有限公司 | Method for constructing characteristic spectrum of safflower medicinal material and safflower medicinal preparation and application |
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Application publication date: 20130227 |