CN102928538A - Radix salvia miltiorrhiza fingerprint establishment method and standard fingerprint - Google Patents
Radix salvia miltiorrhiza fingerprint establishment method and standard fingerprint Download PDFInfo
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- CN102928538A CN102928538A CN2012104828125A CN201210482812A CN102928538A CN 102928538 A CN102928538 A CN 102928538A CN 2012104828125 A CN2012104828125 A CN 2012104828125A CN 201210482812 A CN201210482812 A CN 201210482812A CN 102928538 A CN102928538 A CN 102928538A
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Abstract
The invention relates to a traditional Chinese medicine (TCM) fingerprint establishment method, in particular to a radix salvia miltiorrhiza high-performance liquid chromatograph (HPLC) fingerprint establishment method and a radix salvia miltiorrhiza standard fingerprint obtained by adopting the method, provides a TCM fingerprint establishment method and a standard fingerprint, and aims to solve the problem that TCM substance identification and composition analysis can not effectively reflect and control the total body mass of TCM at present. The method comprises the following steps: firstly, preparation of a test solution; and secondly, high-performance liquid chromatograph analysis. The radix salvia miltiorrhiza fingerprint can be obtained by adopting the method. The method is used in the technical field of medicine analysis.
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 red rooted salvia high performance liquid chromatography (HPLC) finger-print especially, and the resulting red rooted salvia standard finger-print of method thus.Belong to the Pharmaceutical Analysis technical field.
Background technology
The red sage root has another name called red ginseng, radix salviae miltiorrhizae, red etc.Main effect is promoting blood circulation for regulating menstruation, stasis-dispelling and pain-killing, the cool blood carbuncle that disappears, the relieving restlessness that clears away heart-fire, nourishing blood and tranquilization.The red sage root master contains fat-soluble Diterpenoids from bulbus and water miscible phenolic acid composition, also contains flavonoids, triterpenes, other compositions such as sterol.The clinical treatment that is mainly used in cardiovascular and cerebrovascular disease also is widely used for treating chronic liver disease, chronic renal insufficiency, infantile viral myocarditis, vasculitis, chorionitis, Hemorrhagic fever, anaphylactoid purpura, schizophrenia, pneumonia, vascular headache, rhinitis etc.Because be subjected to the impact of the place of production, weather and ecologic environment etc., the contained composition of different red rooted salvias also exists different." Chinese pharmacopoeia only limits to the mensuration of Tanshinone I I A and content of danshinolic acid B to the quality control of red rooted salvia, can not system, intactly reflect the inherent quality of the red sage root.Control the red sage root and prescribed preparation effect thereof, just can not be only for 1,2 chemical composition 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 easily obtains approval in the world, 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.
Summary of the invention
The present invention will solve in the identification of existing Chinese medicine and the constituent analysis effectively to reflect and to control the problem of the total quality of Chinese medicine, thus method for building up and the standard finger-print thereof of the red rooted salvia finger-print that provides.
The method for building up of red rooted salvia finger-print of the present invention carries out according to the following steps:
One, the preparation of need testing solution: be that 1g: 100～300mL mixes red rooted salvia powder and water by mass volume ratio, add hot reflux 45～60min, get the supernatant filtering with microporous membrane after the cooling, filtrate is as need testing solution;
Two, efficient liquid phase chromatographic analysis: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, take the quality percentage composition as the A phase as 1% glacial acetic acid aqueous solution, acetonitrile is that B carries out the eluent gradient wash-out mutually, analyzes need testing solution, obtains the finger-print of red rooted salvia; Wherein, described high-efficient liquid phase chromatogram condition is: the chromatographic resolution column packing is YMC-PackC18, specification is 150 * 4.6mm, 5 μ m, column temperature is 25 ℃～35 ℃, mobile phase be the A phase with B mutually, flow velocity 0.8～1.2mL/min, the need testing solution sample size is 10 μ l, detects wavelength 290nm in the high performance liquid chromatograph.
Utilize the method for building up of red rooted salvia finger-print to obtain the red rooted salvia finger-print.
The present invention has following significant advantage and purposes:
(1) red rooted salvia 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 easily realized;
(3) stability of method and reappearance are all relatively good;
(4) be suitable for discriminating and control to the red rooted salvia true and false, the place of production and quality.
Description of drawings
Fig. 1 is the standard finger-print of red rooted salvia provided by the invention, and wherein 1 to 18 is red rooted salvia common characteristic peak.
Embodiment
Embodiment one: the method for building up of the red rooted salvia finger-print of present embodiment is realized according to the following steps:
One, the preparation of need testing solution: be that 1g: 100～300mL mixes red rooted salvia powder and water by mass volume ratio, add hot reflux 45～60min, get the supernatant filtering with microporous membrane after the cooling, filtrate is as need testing solution;
Two, efficient liquid phase chromatographic analysis: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, take the quality percentage composition as the A phase as 1% glacial acetic acid aqueous solution, acetonitrile is that B carries out the eluent gradient wash-out mutually, analyzes need testing solution, obtains the finger-print of red rooted salvia; Wherein, described high-efficient liquid phase chromatogram condition is: the chromatographic resolution column packing is YMC-PackC18, specification is 150 * 4.6mm, 5 μ m, column temperature is 25 ℃～35 ℃, mobile phase be the A phase with B mutually, flow velocity 0.8～1.2mL/min, the need testing solution sample size is 10 μ l, detects wavelength 290nm in the high performance liquid chromatograph.
Embodiment two: what present embodiment and embodiment one were different is: the filtering with microporous membrane described in the step 1 adopts 0.45 μ m miillpore filter to filter.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment was different from embodiment one or two is: in the eluent gradient elution process described in the step 2, eluent A, B phase transformation turn to: with gradient elution mode 0min～50min, acetonitrile 0%～30%.Other step and parameter are identical with embodiment one or two.
Embodiment four: present embodiment utilizes the method for building up of red rooted salvia finger-print to obtain the red rooted salvia finger-print.
Embodiment five: what present embodiment and embodiment four were different is: collection of illustrative plates has 18 common characteristic peaks, retention time is respectively: 7.1min, 9.0min, 10.1min, 10.9min, 14.4min, 19.9min, 22.9min, 27.2min, 30.6min, 34.4min, 34.8min, 36.4min, 37.3min, 38.3min, 38.9min, 41.7min, 44.5min and 45.4min, these common characteristic peaks have consisted of the fingerprint characteristic of red rooted salvia, can be used as the standard finger-print of red rooted salvia.Other step and parameter are identical with embodiment four.
Present embodiment has following significant advantage and purposes:
(1) red rooted salvia 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 easily realized;
(3) stability of method and reappearance are all relatively good;
(4) be suitable for discriminating and control to the red rooted salvia true and false, the place of production and quality.
By following verification experimental verification beneficial effect of the present invention:
One, the preparation of need testing solution: precision takes by weighing red rooted salvia powder 0.5g, places container, adds water 100mL, adds hot reflux 60min, gets supernatant after the cooling with 0.45 μ m filtering with microporous membrane, and filtrate is as need testing solution;
Two, efficient liquid phase chromatographic analysis: chromatographic resolution column packing 5 μ m YMC-Pack C18 150 * 4.6mm; Mobile phase 1% glacial acetic acid aqueous solution-acetonitrile; Adopt gradient elution mode 0min～50min, acetonitrile 0%～30%; Flow velocity 1.0mL/min; Detect wavelength 290nm; 30 ℃ of column temperatures; Sample size 10 μ L; Analyze with this understanding need testing solution, obtain the finger-print of red rooted salvia.
Three, according to method provided by the invention, 10 batches of red rooted salvias have been set up the HPLC finger-print, by analyzing relatively, 18 common characteristic peaks have been determined, retention time is respectively: 7.1min, 9.0min, 10.1min, 10.9min, 14.4min, 19.9min, 22.9min, 27.2min, 30.6min, 34.4min, 34.8min, 36.4min, 37.3min, 38.3min, 38.9min, 41.7min, 44.5min, 45.4min, these common characteristic peaks have consisted of the fingerprint characteristic of red rooted salvia, can be used as the standard finger-print of red rooted salvia.
Instrument and medicine that this test is used: 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 red rooted salvias all are purchased from Chinese herbal medicine market; Other reagent is chromatographically pure or analyzes pure.
The standard finger-print of the red rooted salvia that this test obtains as shown in Figure 1.As seen from Figure 1, can clearly obtain having the standard finger-print of the red rooted salvia at 18 common characteristic peaks by this method, for the quality of overall evaluation red rooted salvia provides good foundation.
Four, methodological study
1, precision is investigated
Get same batch sample, carry out the need testing solution preparation by this test method, continuous sample introduction is measured 5 times, and data are inputted similarity evaluation, calculate, obtain the similarity data of precision matched data and precision coupling shown in table 1, table 2:
Table 1 precision matched data
The similarity data of table 2 precision coupling
Similarity is more than 0.9, and precision is good.
2, method repeatability is investigated
Get same batch sample, be prepared 6 parts of need testing solutions by this test method, sample introduction is measured respectively, and data are inputted similarity evaluation, calculates repeated matched data and repeated matching similarity data shown in table 3, table 4:
The repeated matched data of table 3
The repeated matching similarity data of table 4
Similarity is more than 0.9, and repeatability is good.
3, study on the stability
Get same batch sample, be prepared need testing solution by the text method, measure at 0,1,2,3,4,5,6,7 hour sample introduction respectively, data are inputted similarity evaluation, calculate the similarity data of Stable matching data and Stable matching shown in table 5, table 6:
Table 5 Stable matching data
The similarity data of table 6 Stable matching
Similarity has good stability more than 0.9.
4, sample tests
Get ten batches of different places of production red rooted salvias, preparation method according to need testing solution makes need testing solution (before use preparation), sample introduction 10 μ L detect, carrying out spectrogram processes, data are inputted similarity evaluation (2004A version) generate red rooted salvia 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 (5)
1. the method for building up of red rooted salvia finger-print is characterized in that the method for building up of red rooted salvia finger-print carries out according to the following steps:
One, the preparation of need testing solution: be that 1g: 100～300mL mixes red rooted salvia powder and water by mass volume ratio, add hot reflux 45～60min, get the supernatant filtering with microporous membrane after the cooling, filtrate is as need testing solution;
Two, efficient liquid phase chromatographic analysis: the need testing solution that step 1 is obtained injects high performance liquid chromatograph, take the quality percentage composition as the A phase as 1% glacial acetic acid aqueous solution, acetonitrile is that B carries out the eluent gradient wash-out mutually, analyzes need testing solution, obtains the finger-print of red rooted salvia; Wherein, described high-efficient liquid phase chromatogram condition is: the chromatographic resolution column packing is YMC-PackC18, specification is 150 * 4.6mm, 5 μ m, column temperature is 25 ℃～35 ℃, mobile phase be the A phase with B mutually, flow velocity 0.8～1.2mL/min, the need testing solution sample size is 10 μ l, detects wavelength 290nm in the high performance liquid chromatograph.
2. the method for building up of red rooted salvia finger-print 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.
3. the method for building up of red rooted salvia finger-print according to claim 1 is characterized in that in the eluent gradient elution process described in the step 2, eluent A, B phase transformation turn to: with gradient elution mode 0min～50min, acetonitrile 0%～30%.
4. the method for building up of red rooted salvia finger-print as claimed in claim 1 has obtained the red rooted salvia finger-print.
5. red rooted salvia finger-print according to claim 4, it is characterized in that described collection of illustrative plates has 18 common characteristic peaks, retention time is respectively: 7.1min, 9.0min, 10.1min, 10.9min, 14.4min, 19.9min, 22.9min, 27.2min, 30.6min, 34.4min, 34.8min, 36.4min, 37.3min, 38.3min, 38.9min, 41.7min, 44.5min and 45.4min, these common characteristic peaks have consisted of the fingerprint characteristic of red rooted salvia, can be used as the standard finger-print of red rooted salvia.
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Cited By (3)
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| CN106770037A (en) * | 2017-02-13 | 2017-05-31 | 中国药科大学 | A kind of discrimination method of datura flower medicinal material |
| CN108760923A (en) * | 2018-05-30 | 2018-11-06 | 吉林大学 | The HPLC fingerprint atlas detection methods of red-rooted-salvia-root chuanxiong-rhizome azine injecta |
| CN110646542A (en) * | 2019-09-30 | 2020-01-03 | 贵州中医药大学 | Quality detection method for salvia miltiorrhiza medicinal material |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108760923A (en) * | 2018-05-30 | 2018-11-06 | 吉林大学 | The HPLC fingerprint atlas detection methods of red-rooted-salvia-root chuanxiong-rhizome azine injecta |
| CN110646542A (en) * | 2019-09-30 | 2020-01-03 | 贵州中医药大学 | Quality detection method for salvia miltiorrhiza medicinal material |
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Application publication date: 20130213 |