CN101231273A - Method for determining fingerprint pattern of creat extract - Google Patents
Method for determining fingerprint pattern of creat extract Download PDFInfo
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- CN101231273A CN101231273A CNA2008100734601A CN200810073460A CN101231273A CN 101231273 A CN101231273 A CN 101231273A CN A2008100734601 A CNA2008100734601 A CN A2008100734601A CN 200810073460 A CN200810073460 A CN 200810073460A CN 101231273 A CN101231273 A CN 101231273A
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Abstract
The invention relates to a finger-print measuring method of a creat extract. Firstly, a creat is distilled with ethanol to prepare the creat extract, then the creat extract is weighted minutely, methyl alcohol solution is added, the creat extract is dipped in and performed ultrasonic processing, and is positioned at the cold place, methyl alcohol is added to the scale, the creat extract is shaken up evenly and is filtered with a microporous filtering film to be used as an on-approval sample; a chromatographic column taking octadecyl silane linking silica gel as fillings is adopted; gradient elution is adopted, a mobile phase A is of acetonitrile, and a mobile phase B is of 0.1 percent of phosphoric acid solution; the detecting wavelength is 254 nm; the on-approval sample solution is extracted minutely and poured into a liquid chromatograph, a finger-print atlas is obtained through measuring according to a high performance liquid chromatography. The finger-print measuring method of the creat extract of the invention is simple and convenient, is easy to master, and the stability, the precision and the reproduction quality are all fine, thereby being used for the quality control of the creat extract, and ensuring the validity, the security, the stability and the equality of the relevant products of the creat extract.
Description
Technical field
The present invention relates to a kind of method of medicinal plant determining fingerprint pattern, particularly the foundation of Andrographis Paniculata finger print measuring method.
Background technology
Herba Andrographitis is the dry aerial parts of acanthaceous plant Herba Andrographitis Andrographis paniculata (Burm.f.) Nees, is traditional Chinese medicine material commonly used, and the nature and flavor hardship is cold.The thoughts of returning home, lung, large intestine, urinary bladder channel.Have effects such as clearing heat and detoxicating, cool blood, detumescence, be usually used in cold, fever, abscess of throat, aphthae, pertussis labor is coughed, dysentery, heat is drenched puckery pain, the carbuncle sore that swells, venomous snake bite.Recent study finds that Herba Andrographitis has physiologically active widely, except antibacterial and anti-inflammation functions, antitumor in addition, enhance immunity, antiviral, prevent and treat effect such as angiocardiopathy, be widely used in clinical.
Andrographis Paniculata is processed through extracting by Herba Andrographitis, and it is the raw material of the intermediate of conventional Chinese medicine prescribed preparation chuanxinlian tablet, CHUANXINLIAN JIAONANG, lotus sesame sulfathiazole etc. and some cosmetics, veterinary drug.Set up the effective quality evaluating method of Andrographis Paniculata, guarantee the steady quality of Andrographis Paniculata, be directly connected to validity, security, stability and the homogeneity of Andrographis Paniculata Related product.
Open source literature report, the main effective constituent of Herba Andrographitis is the diterpenoid-lactone constituents, the many content with higher andrographolide of content in this constituents and Dehydro and drographolide of Herba Andrographitis medicinal material and compound preparation thereof are as quality control index.Yet certain single component content is measured the drug action that can not reflect Chinese medicine fully.Fingerprint pattern technology is the effective way that reflects the Chinese medicine mechanism of action at present, is one of gordian technique of the modernization of Chinese medicine, is traditional Chinese medicine quality control state-of-the-art technology.Therefore, we are applied to the technology of finger-print in the quality control of Andrographis Paniculata.We have set up the HPLC finger print measuring method of Andrographis Paniculata, for the quality control of Andrographis Paniculata provides effective ways.
Open source literature has been reported the research of some Herba Andrographitis medicinal materials fingerprints at present, for example:
[autograph] GAP Herba Andrographitis medicinal material HPLC finger-print research [author] Zhu Chen Gall does not build rosy clouds [periodical name] Chinese Pharmaceutical Journal .2004,39 (10) .-737-739[digests] purpose provides new fundamental research data for the research of GAP Herba Andrographitis quality of medicinal material.Method RP-HPLC sets up the Herba Andrographitis finger-print, and carries out the comparison of different planting bases (place of production) and commodity medicinal material, different growing stages, different medicinal part finger-prints, adopts computer assisted similarity evaluation software to carry out similarity evaluation.The result is under selected chromatographic condition, and 226nm detects the fingerprint characteristic of 15 peaks formation Herba Andrographitis medicinal materials under the wavelength, and each batch sample finger-print all has above-mentioned feature, and there is some difference but the relative content distributional difference of characteristic peak causes the chromatogram general picture.The area of computer aided similarity evaluation, 5 GAP base product Herba Andrographitis medicinal material fingerprint characteristics are comparatively identical each other, have consistance preferably, and the commodity medicinal material then differs greatly.The different growing stages medicinal materials fingerprint also has consistance preferably, and most characteristic peak content present with the dynamic accumulative process that increases growth period, and different parts then is much higher than stem in the content distribution leaf of characteristic peak.Conclusion is carried out GAP and is had great importance for the stability that guarantees quality of medicinal material, and the method for being set up can reflect Herba Andrographitis medicinal material multicomponent feature more all sidedly, and carries out the quality of medicinal material evaluation.
The Chinese natural drug .2005 of chromatographic fingerprinting [author] Zhang Zunjian [1] Dong Haijuan [1] Yu Jing [2] of [autograph] Herba Andrographitis medicinal material [periodical name], 3 (6) .-373-376[digests] purpose: set up the HPLC/UV chromatographic fingerprinting of Herba Andrographitis medicinal material, and the medicinal material in the different places of production is compared.Method: use Shimadzu VP-ODS post, 0.1% formic acid acetonitrile and 0.2% formic acid carry out gradient elution, and flow velocity is 1.0ml/min, detect wavelength 254nm, and column temperature is a room temperature.Result: obtain all Herba Andrographitis medicinal material HPLC/UV finger-prints preferably of degree of separation, reappearance, indicated 9 total peaks.Use the HPLC/MS technology its 7 main chromatographic peaks have been carried out preliminary ownership, and the chromatographic fingerprinting of different places of production medicinal material has been carried out similarity evaluation.Conclusion: the foundation of Herba Andrographitis medicinal materials fingerprint, for its quality control provides foundation.
Comparative studies [author] Dong Haijuan [1] Zhang Zunjian [1] Yu Jing [2] of [autograph] different place of production Herba Andrographitis medicinal material chromatographic fingerprinting [periodical name] Chinese patent drug .2006,28 (3) .-321-324[digests] purpose: set up the HPLC finger-print of Herba Andrographitis medicinal material, for its total quality evaluation and control provide reference.Method: adopt the HPLC/UV method, measured 23 batches of Herba Andrographitis medicinal material samples in 10 different places of production.Chromatographic condition: Shimadzu ODS post, moving phase are 0.1% formic acid acetonitrile and 0.2% formic acid, gradient elution, and flow velocity 1.0ml/min detects wavelength 254nm, and living temperature is room temperature.The methods such as cluster analysis, Nonlinear Mapping and similarity calculating of using have been carried out qualitative, quantitative evaluation to the gained finger-print.The result: set up simple to operate, degree of separation is good, the Herba Andrographitis medicinal materials fingerprint detection method of favorable reproducibility, different places of production medicinal materials fingerprint has certain difference.Conclusion: methods such as cluster analysis, Nonlinear Mapping and similarity calculating can realize qualitative, the quantitative evaluation to finger-print, and the chromatographic fingerprinting of being set up can be used for the discriminating and the quality assessment of Herba Andrographitis medicinal material.
The document of above-mentioned open report mainly is the research to the Herba Andrographitis medicinal materials fingerprint, and the present invention is the foundation to the finger print measuring method of Andrographis Paniculata.Andrographis Paniculata is processed through extracting by Herba Andrographitis, is the raw material of the intermediate of conventional Chinese medicine prescribed preparation chuanxinlian tablet, CHUANXINLIAN JIAONANG, lotus sesame sulfathiazole etc. and some cosmetics, veterinary drug.The finger print measuring method of Andrographis Paniculata is not seen as yet bibliographical information and patented claim.
Summary of the invention
The invention provides the method that from Herba Andrographitis, prepares Andrographis Paniculata and the finger print measuring method of Andrographis Paniculata.The Andrographis Paniculata for preparing can be used as the raw material of the intermediate of conventional Chinese medicine prescribed preparation chuanxinlian tablet, CHUANXINLIAN JIAONANG, lotus sesame sulfathiazole etc. and some cosmetics, veterinary drug.Finger print measuring method by Andrographis Paniculata can be controlled the quality of Andrographis Paniculata effectively, thereby guarantees validity, security, stability and the homogeneity of Andrographis Paniculata Related product.
The technical solution adopted for the present invention to solve the technical problems is:
1. with the Herba Andrographitis alcohol extract, prepare Andrographis Paniculata;
2. the preparation of need testing solution: precision takes by weighing Andrographis Paniculata, adds methanol solution, soaks, and sonicated is put coldly, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, is test sample;
3. chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase A is an acetonitrile, and Mobile phase B is a phosphoric acid solution; The detection wavelength is 254nm;
4. measure: the accurate need testing solution of drawing injects liquid chromatograph, according to high effective liquid chromatography for measuring, obtains finger-print.
The preparation method of the Andrographis Paniculata in the above the 1st step: the aerial part of getting dry Herba Andrographitis, be ground into meal, extract secondary with 85% ethanol (volumetric concentration) hot dipping, each 2 hours, add 8 times of amount 85% ethanol (volume ratio) at every turn, merge extract, filter, it is 1.30～1.32 (60 ℃) that filtrate decompression recycling ethanol below 80 ℃ is concentrated into relative density, vacuum drying below 80 ℃, promptly.
Methanol solution in the above the 2nd step is the aqueous solution of 40% methyl alcohol (volume ratio).
The preparation of the middle need testing solution in the above the 3rd step: precision takes by weighing Andrographis Paniculata (crossing sieve No. four) 0.5g, put in the 25mL measuring bottle, add 40% methyl alcohol (volume ratio) 20mL, soak 1h, sonicated (power 160W, frequency 40kHz) 20min, put coldly, add 40% methyl alcohol (volume ratio), shake up to scale, filter with miillpore filter (0.45 μ m), be the test sample sample;
Mobile phase B in the above the 3rd step is 0.1% phosphate aqueous solution.
Chromatographic condition described in the above the 4th step: the detection wavelength is 254nm; Column temperature: 25 ℃; Sample size: 5 μ l; Moving phase: A is acetonitrile mutually, and B is 0.1% phosphoric acid solution mutually, and flow velocity is 1.0mL/min.Adopt gradient elution, the gradient elution program is as follows:
0～18 minute, mobile phase A was that 23.5% acetonitrile, B are 76.5% 0.1% phosphoric acid solution;
18～40 minutes, mobile phase A was that 38% acetonitrile, B are 62% 0.1% phosphoric acid solution;
40～55 minutes, mobile phase A was that 46% acetonitrile, B are 54% 0.1% phosphoric acid solution;
55～60 minutes, mobile phase A was that 24% acetonitrile, B are 76% 0.1% phosphoric acid solution.
The configuration proportion of the above A phase acetonitrile and mobile B solution is to prepare by volume.
The finger print measuring method of the above Andrographis Paniculata is measured in conjunction with the requirement of finger-print with reference to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test Waters XTerra
TMRP
18Chromatographic column (4.6mm * 250mm, 5 μ m) chromatographic column; With the acetonitrile is mobile phase A, is Mobile phase B with 0.1% phosphoric acid solution, carries out gradient elution in proportion, and flow velocity is 1.0mL/min; The detection wavelength is 254nm.Number of theoretical plate calculates by the andrographolide peak should be not less than 8000, calculates by the Dehydro and drographolide peak and should be not less than 70000.
The preparation precision of object of reference solution takes by weighing andrographolide and the Dehydro and drographolide reference substance is an amount of, adds methyl alcohol and makes the solution that every 1mL contains 0.1mg, shakes up, promptly.
Accurate respectively object of reference solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure the record chromatogram.
Outstanding substantive distinguishing features of the present invention and obvious improvement are:
1. the many content with higher andrographolide of content in the diterpenoid-lactone constituents and Dehydro and drographolide of Herba Andrographitis medicinal material and compound preparation thereof are as quality control index.Yet certain single component content is measured the drug action that can not reflect Chinese medicine fully.Fingerprint pattern technology is the effective way that reflects the Chinese medicine mechanism of action at present, is one of gordian technique of the modernization of Chinese medicine, is traditional Chinese medicine quality control state-of-the-art technology.Therefore, we are applied to the technology of finger-print in the quality control of Andrographis Paniculata.
2. the Andrographis Paniculata HPLC finger-print of the present invention's foundation is being represented Herba Andrographitis part pharmacologically active, can characterize the quality of Andrographis Paniculata effectively.
3. with the HPLC finger-print of Andrographis Paniculata means, both avoided judging the one-sidedness of Herba Andrographitis total quality that having reduced again was the possibility of the artificial processing of requisite quality because of only measuring one, two chemical constitution as its quality of control.The present invention provides effective ways for quality complete, that accurately estimate Andrographis Paniculata, thereby the quality of its further product and curative effect are guaranteed.
4. to have method easy in the present invention, is easy to grasp, stable, precision height, the characteristics of favorable reproducibility.
Embodiment
Embodiment 1
The preparation of Andrographis Paniculata: the aerial part of getting dry Herba Andrographitis, be ground into meal, extract secondary with volume ratio 85% ethanol hot dipping, each 2 hours, add 8 times of amount 85% ethanol at every turn, merge extract, filter, it is 1.30～1.32 (60 ℃) that filtrate decompression recycling ethanol below 80 ℃ is concentrated into relative density, vacuum drying below 80 ℃, promptly.
Embodiment 2
The finger print measuring method of Andrographis Paniculata: get the Andrographis Paniculata that the foregoing description 1 obtains, pulverized sieve No. four, precision takes by weighing 0.5g, put in the 25mL measuring bottle, add volume ratio 40% methyl alcohol 20mL, soak 1h, sonicated (power 160W, frequency 40kHz) 20min, put coldly, add 40% methyl alcohol, shake up to scale, filter with miillpore filter (0.45 μ m), be the test sample sample; Other precision takes by weighing andrographolide and the Dehydro and drographolide reference substance is an amount of, adds methyl alcohol and makes the solution that every 1mL contains 0.1mg, shakes up, as object of reference solution; Accurate respectively object of reference solution and each 5 μ l of need testing solution of drawing inject liquid chromatograph, measure.
The selection of the chromatographic column of embodiment 2
Once selected Lichrospher C for use
18Post (4.6mm * 250mm, 5 μ m), Waters μ BONDAPAK
TMC
18Chromatographic column (PIN27324) and Waters XTerra
TMRP
18Chromatographic column (4.6mm * 250mm, 5 μ m) is carried out experiment sieving, relatively degree of separation, go out peak number, peak shape and post and imitate, Waters XTerra as a result
TMRP
18(4.6mm * 250mm, 5 μ m) are comparatively suitable for chromatographic column, so select its chromatographic column as finger-print research.
The selection of the moving phase of embodiment 2
Once selected many groups such as methanol-water, methyl alcohol-phosphoric acid solution and acetonitrile-phosphoric acid solution flow phase system in the process of the test, carried out gradient elution with different gradients with different ratios.The result is good with the gradient elution of acetonitrile-0.1% phosphoric acid solution (volume ratio), and each chromatographic peak separating effect and shape at 254nm on the chromatogram are better, so select its moving phase as finger-print research.
The selection of the detection wavelength of embodiment 2
With reference to relevant document, once selected for use 225nm and 254nm respectively for detecting wavelength.With 225nm is that though it is more to detect peak number, separating effect is bad, the baseline injustice when detecting wavelength; And be when detecting wavelength with 254nm, though the peak number that detects lack 2 than the peak number that detects at 225nm, its degree of separation, peak shape and post are imitated and to go out the stability at peak comparatively satisfied, so selection 254nm is the detection wavelength.
The system suitability test of embodiment 2
According to above result of study, work out chromatographic condition and be: Waters XTerraTMRP18 chromatographic column (4.6mm * 250mm, 5 μ m); With the acetonitrile is mobile phase A, is Mobile phase B with 0.1% phosphoric acid solution, carries out gradient elution by table 1, and flow velocity is 1.0mL/min; The detection wavelength is 254nm; Column temperature: 25 ℃; Sample size: 5 μ L.
Table 1 moving phase condition and gradient condition
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0～18 18～40 40～55 55～60 | 23.5 38 46 24 | 76.5 62 54 76 |
Detect 10 batches of Andrographis Paniculatas with the chromatographic condition of working out, press andrographolide peak theory of computation plate number, the result should be not less than 8000 so work out number of theoretical plate by the calculating of andrographolide peak all more than 8000; Press Dehydro and drographolide peak theory of computation plate number, the result should be not less than 70000 so work out number of theoretical plate by the calculating of Dehydro and drographolide peak all more than 70000.
The demarcation of embodiment 2 with reference to the peak
Get each 5 μ L of object of reference solution and test liquid, inject liquid chromatograph, record chromatogram (seeing Fig. 1～2) in the test sample chromatogram, is with reference to the peak with the peak on the retention time relevant position, Dehydro and drographolide peak in the object of reference solution.
The writing time of embodiment 2
Record 60min chromatogram, 35min does not go out the peak later on, and Andrographis Paniculata becomes to tell the peak complete in the prompting 35min.
The methodological study of embodiment 2
(1) stability test
Accurate each the 5 μ L of same sample (9#) solution that draw, sample introduction is measured once at regular intervals, measures altogether 6 times.The RSD of the relative retention time at each total peak and peak area ratio is all less than 3% (seeing Table 2～3) as a result; Use similarity software for calculation (" working specification of chromatographic fingerprints of Chinese materia medica similarity evaluation system (the version 2 004A) " that Chinese Pharmacopoeia Commission is recommended, down together.) calculate similarity, each finger-print similarity is 1.000, meets the requirement of chromatographic fingerprinting investigative technique.
The relative retention time at each total peak of table 2 stability test
| Stability test | Relative retention time | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 (0h) 9-1 (4h) 9-1 (9h) 9-1 (11h) 9-1 (20h) 9-1 (22h) mean value RSD% | 0.248 0.249 0.248 0.237 0.248 0.248 0.248 1.86 | 0.289 0.289 0.290 0.290 0.289 0.290 0.290 0.19 | 0.326 0.327 0.327 0.328 0.326 0.326 0.326 0.25 | 0.380 0.381 0.382 0.382 0.380 0.380 0.380 0.26 | 0.458 0.459 0.460 0.461 0.458 0.458 0.459 0.28 | 0.534 0.535 0.536 0.536 0.534 0.534 0.535 0.18 | 0.616 0.617 0.617 0.618 0.615 0.615 0.616 0.20 | 0.667 0.668 0.670 0.671 0.667 0.667 0.668 0.26 | 0.729 0.736 0.736 0.737 0.735 0.735 0.735 0.39 | 1 1 1 1 1 1 1 0 |
The relative peak area at each total peak of table 3 stability test
| Stability test | Relative peak area | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 (0h) 9-1 (4h) 9-1 (9h) 9-1 (11h) 9-1 (20h) 9-1 (22h) mean value RSD% | 0.045 0.043 0.044 0.045 0.045 0.045 0.044 1.88 | 0.090 0.089 0.090 0.090 0.091 0.091 0.090 0.83 | 0.017 0.018 0.017 0.018 0.018 0.017 0.018 3.13 | 0.454 0.456 0.456 0.455 0.459 0.454 0.456 0.41 | 0.247 0.245 0.246 0.247 0.244 0.248 0.246 0.60 | 0.080 0.079 0.080 0.080 0.080 0.080 0.080 0.51 | 0.055 0.054 0.053 0.053 0.055 0.052 0.054 2.26 | 0.100 0.101 0.100 0.101 0.102 0.102 0.101 0.89 | 0.024 0.024 0.024 0.025 0.023 0.024 0.024 2.64 | 1 1 1 1 1 1 1 0 |
(2) precision test
Accurate each the 5 μ L of same sample (9#) solution that draw, continuous respectively 6 sample introductions are measured.The RSD of the relative retention time at each total peak and peak area ratio is all less than 3% (seeing Table 4～5) as a result; Use the similarity software for calculation to calculate similarity, each finger-print similarity is 1.000, meets the requirement of chromatographic fingerprinting investigative technique.
Table 4 precision is tested the relative retention time at each total peak
| The precision test | Relative retention time | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 (1) 9-1 (2) 9-1 (3) 9-1 (4) 9-1 (5) 9-1 (6) mean value RSD% | 0.248 0.248 0.248 0.248 0.236 0.248 0.246 1.99 | 0.289 0.289 0.289 0.290 0.290 0.289 0.289 0.18 | 0.326 0.327 0.327 0.327 0.328 0.326 0.327 0.23 | 0.380 0.380 0.380 0.382 0.382 0.380 0.381 0.27 | 0.458 0.459 0.459 0.460 0.461 0.458 0.459 0.25 | 0.534 0.535 0.535 0.536 0.536 0.534 0.535 0.17 | 0.616 0.616 0.616 0.617 0.618 0.615 0.616 0.17 | 0.667 0.668 0.668 0.669 0.670 0.667 0.668 0.17 | 0.729 0.736 0.736 0.736 0.737 0.735 0.735 0.40 | 1 1 1 1 1 1 1 0 |
Table 5 precision is tested the relative peak area at each total peak
| The precision test | Relative peak area | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 (1) 9-1 (2) 9-1 (3) 9-1 (4) 9-1 (5) 9-1 (6) mean value RSD% | 0.045 0.045 0.045 0.044 0.044 0.044 0.044 1.23 | 0.089 0.090 0.090 0.090 0.091 0.091 0.090 0.83 | 0.017 0.018 0.018 0.017 0.018 0.017 0.018 3.13 | 0.457 0.454 0.457 0.456 0.455 0.460 0.457 0.45 | 0.249 0.248 0.245 0.246 0.247 0.244 0.247 0.76 | 0.080 0.080 0.081 0.080 0.081 0.080 0.080 0.64 | 0.052 0.054 0.054 0.053 0.055 0.051 0.053 2.77 | 0.100 0.101 0.101 0.100 0.101 0.102 0.101 0.75 | 0.024 0.025 0.024 0.024 0.023 0.025 0.024 3.11 | 1 1 1 1 1 1 1 0 |
(3) reappearance test
Get same sample (9#), prepare 6 parts of need testing solutions, sample introduction is measured respectively.The RSD of the relative retention time at each total peak and peak area ratio is all less than 3% (seeing Table 6～7) as a result; Use the similarity software for calculation to calculate similarity, each finger-print similarity is 1.000, meets the requirement of chromatographic fingerprinting investigative technique.
Table 6 reappearance is tested the relative retention time at each total peak
| The reappearance test | Relative retention time | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 9-2 9-3 9-4 9-5 9-6 mean value RSD% | 0.234 0.233 0.233 0.239 0.237 0.237 0.236 1.07 | 0.278 0.277 0.277 0.282 0.281 0.280 0.279 0.77 | 0.311 0.310 0.310 0.316 0.315 0.314 0.313 0.85 | 0.383 0.383 0.383 0.387 0.387 0.386 0.385 0.53 | 0.445 0.444 0.445 0.447 0.448 0.446 0.446 0.33 | 0..527 0.527 0.526 0.529 0.530 0.529 0.528 0.29 | 0.602 0.602 0.605 0.603 0.605 0.604 0.604 0.23 | 0.654 0.653 0.654 0.656 0.658 0.656 0.655 0.28 | 0.728 0.728 0.728 0.729 0.731 0.730 0.729 0.17 | 1 1 1 1 1 1 1 0 |
Table 7 reappearance is tested the relative peak area at each total peak
| The reappearance test | Relative peak area | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 9-1 9-2 9-3 9-4 9-5 9-6 mean value RSD% | 0.046 0.047 0.047 0.047 0.046 0.047 0.047 1.11 | 0.081 0.080 0.080 0.080 0.080 0.080 0.080 0.51 | 0.015 0.016 0.016 0.015 0.016 0.016 0.016 3.30 | 0.447 0.448 0.445 0.444 0.447 0.446 0.446 0.33 | 0.244 0.245 0.240 0.243 0.245 0.245 0.244 0.81 | 0.080 0.081 0.082 0.081 0.081 0.081 0.081 0.78 | 0.058 0.056 0.056 0.057 0.056 0.056 0.057 1.48 | 0.090 0.090 0.090 0.091 0.090 0.090 0.090 0.45 | 0.022 0.021 0.021 0.022 0.022 0.022 0.022 2.38 | 1 1 1 1 1 1 1 0 |
The mensuration of the sample of embodiment 2
Get 10 batches of embodiment 1 Andrographis Paniculatas, 2 parts every batch, make the Andrographis Paniculata test liquid, measure the record chromatogram by the chromatographic condition of working out.
The selection at the total peak of embodiment 2
According to 10 batch sample measurement results, each batch sample finger-print chromatographic peak has all gone out in 35min.We mainly are to andrographolide constituents finger-print research, and chromatographic retention 5 minutes is solvent and impurity peaks as last, so deduct the chromatographic peak of retention time before 5 minutes when selecting total peak.The chromatogram of comprehensive 10 batch samples, after 5min, it is that 10 batch samples are common that 10 peaks are arranged, and wherein No. 6 peaks are the andrographolide peak, and No. 10 peaks are the Dehydro and drographolide peak.10 peak total areas of each batch sample account for the total peak area ratio all greater than 90%, determine that therefore these 10 peaks are total fingerprint peaks.
The foundation of the Andrographis Paniculata finger-print examination criteria of embodiment 2
Adopt the working specification (version 2 004A) of the chromatographic fingerprints of Chinese materia medica similarity evaluation system of Chinese Pharmacopoeia Commission's recommendation, adopt averaging method, by 20 finger-prints generation reference fingerprint (see figure 3)s of 10 batch samples.
With the contrast spectrogram is reference, each sample finger-print compares with the contrast spectrogram, calculates different batches Andrographis Paniculata finger-print similarity value, and the similarity between each batch extract and the reference fingerprint is all greater than 0.90 as a result, average 0.942, RSD is 3.28% (seeing Table 8).
10 batches of Andrographis Paniculata determining fingerprint patterns of table 8 result (n=2)
| Numbering | Similarity (contrast) | Mean value | RSD％ |
| 1-1 1-2 2-1 2-2 3-1 3-2 4-1 4-2 5-1 5-2 6-1 6-2 7-1 7-2 8-1 8-2 9-1 9-2 10-1 10-2 | 0.959 0.904 0.912 0.968 0.972 0.970 0.958 0.964 0.913 0.913 0.960 0.959 0.905 0.903 0.965 0.965 0.911 0.911 0.970 0.974 | 0.942 | 3.28％ |
According to above test findings, and in conjunction with the technical requirement of traditional Chinese medicine fingerprint research, tentative this product finger-print should be consistent with reference fingerprint, and the similarity value must not be lower than 0.90.
Description of drawings
Fig. 1 is the embodiment of the invention 2 said object of reference figure.
Fig. 2 is the embodiment of the invention 2 said Andrographis Paniculata finger-prints.
Fig. 3 is the embodiment of the invention 2 said Andrographis Paniculata reference fingerprints.Numbering 1～10 among the figure is total peak, and 6 and 10 are respectively andrographolide and Dehydro and drographolide with reference to the peak.
Claims (6)
1. the finger print measuring method of an Andrographis Paniculata is characterized in that:
(a) with the Herba Andrographitis alcohol extract, the preparation Andrographis Paniculata;
(b) preparation of need testing solution: precision takes by weighing Andrographis Paniculata, adds methanol solution, soaks, and sonicated is put coldly, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, is test sample;
(c) chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase A is an acetonitrile, and Mobile phase B is a phosphoric acid solution; The detection wavelength is 254nm;
(d) measure: the accurate need testing solution of drawing injects liquid chromatograph, according to high effective liquid chromatography for measuring, obtains finger-print.
2. the finger print measuring method of Andrographis Paniculata according to claim 1, it is characterized in that: the preparation method of Andrographis Paniculata gets the aerial part of dry Herba Andrographitis, be ground into meal, extract secondary, each 2 hours with volume ratio 85% ethanol hot dipping, add 8 times of 85% ethanol at every turn, merge extract, filter, filtrate when decompression recycling ethanol is concentrated into 60 ℃ below 80 ℃ relative density be 1.30～1.32, vacuum drying below 80 ℃, promptly.
3. the finger print measuring method of Andrographis Paniculata according to claim 1, it is characterized in that: methanol solution is the methanol aqueous solution of volume ratio 40%.
4. the finger print measuring method of Andrographis Paniculata according to claim 1, it is characterized in that: the preparation of need testing solution is that precision takes by weighing Andrographis Paniculata, crosses sieve 0.5g No. four, place measuring bottle, add volume ratio 40% methyl alcohol 20mL, soak 1h, use power 160W, the sonication 20min that frequency 40kHz is super, put coldly, add volume ratio 40% methyl alcohol, shake up to scale, filter with miillpore filter, be the test sample sample.
5. the finger print measuring method of Andrographis Paniculata according to claim 1, it is characterized in that: Mobile phase B is volume ratio 0.1% phosphate aqueous solution.
6. the finger print measuring method of Andrographis Paniculata according to claim 1, it is characterized in that: described chromatographic condition: the detection wavelength is 254nm; Column temperature: 25 ℃; Sample size: 5 μ l; Moving phase: A is acetonitrile mutually, and B is 0.1% phosphoric acid solution by volume mutually, and flow velocity is 1.0mL/min.Adopt gradient elution, the gradient elution program is as follows:
0～18 minute, mobile phase A was that acetonitrile, the B of volume ratio 23.5% is 76.5% 0.1% phosphoric acid solution;
18～40 minutes, mobile phase A was that acetonitrile, the B of volume ratio 38% is 62% 0.1% phosphoric acid solution;
40～55 minutes, mobile phase A was that acetonitrile, the B of volume ratio 46% is 54% 0.1% phosphoric acid solution;
55～60 minutes, mobile phase A was that acetonitrile, the B of volume ratio 24% is 76% 0.1% phosphoric acid solution.
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| CNA2008100734601A CN101231273A (en) | 2008-02-03 | 2008-02-03 | Method for determining fingerprint pattern of creat extract |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013071461A1 (en) * | 2011-11-16 | 2013-05-23 | 广州白云山和记黄埔中药有限公司 | Andrographis paniculata and testing method for preparation thereof |
| CN104458632A (en) * | 2014-11-27 | 2015-03-25 | 山东大学 | Method for detecting and distinguishing traditional Chinese medicines with same quality |
| CN105699546A (en) * | 2016-04-25 | 2016-06-22 | 广西壮族自治区梧州食品药品检验所 | Method for measuring content of effective components in herba andrographitis tablets through liquid mass series method |
| CN109142588A (en) * | 2018-10-25 | 2019-01-04 | 广州白云山光华制药股份有限公司 | A kind of LianZhixiaoyan Capsule HPLC characteristic spectrum and its construction method and application |
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2008
- 2008-02-03 CN CNA2008100734601A patent/CN101231273A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013071461A1 (en) * | 2011-11-16 | 2013-05-23 | 广州白云山和记黄埔中药有限公司 | Andrographis paniculata and testing method for preparation thereof |
| CN103930118A (en) * | 2011-11-16 | 2014-07-16 | 广州白云山和记黄埔中药有限公司 | Andrographis paniculata and testing method for preparation thereof |
| CN104458632A (en) * | 2014-11-27 | 2015-03-25 | 山东大学 | Method for detecting and distinguishing traditional Chinese medicines with same quality |
| CN105699546A (en) * | 2016-04-25 | 2016-06-22 | 广西壮族自治区梧州食品药品检验所 | Method for measuring content of effective components in herba andrographitis tablets through liquid mass series method |
| CN109142588A (en) * | 2018-10-25 | 2019-01-04 | 广州白云山光华制药股份有限公司 | A kind of LianZhixiaoyan Capsule HPLC characteristic spectrum and its construction method and application |
| CN109142588B (en) * | 2018-10-25 | 2021-07-09 | 广州白云山光华制药股份有限公司 | HPLC (high performance liquid chromatography) characteristic spectrum of Lianzhi anti-inflammation capsules as well as construction method and application thereof |
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