CN102028883B - Standard fingerprint spectrum of Chinese medicinal composition, and measurement method and application thereof - Google Patents
Standard fingerprint spectrum of Chinese medicinal composition, and measurement method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of analysis of traditional Chinese medicines, and discloses a standard fingerprint spectrum of a Chinese medicinal composition, and a measurement method and application thereof. The standard fingerprint spectrum is a high performance liquid chromatography spectrum; six main chromatographic peaks are shown in the spectrum; the No. 3 peak is the chromatographic peak which has the same peak retention time as ferulic acid serving as a reference object; and compared with the No. 3 peak, the other five chromatographic peaks have the relative retention time of 0.23+/-10 percent, 0.27+/-10 percent, 1.20+/-8 percent, 1.65+/-10 percent and 1.80+/-10 percent respectively. The standard fingerprint spectrum can be used for quality control over the Chinese medicinal composition comprising szechuan lovage rhizome and tall gastrodia tuber serving as raw medicinal materials; and the similarity of the fingerprint spectrum of a qualified Chinese medicinal composition to the standard fingerprint spectrum is not less than 0.80. The standard fingerprint spectrum can completely and effectively control the product quality; a similarity evaluation system is adopted; and the invention has the advantages of convenience and high speed in operation and objective and accurate result.
Description
Technical field
The invention belongs to the Analysis of Chinese Traditional Medicine field, relate to the standard finger-print of Chinese medicinal composition preparation, the invention still further relates to assay method and the application of this standard finger-print in this composition quality is controlled of this standard finger-print.
Background technology
The Chinese medicine composition that the present invention relates to is after being made up according to a certain percentage by Ligusticum wallichii, rhizoma Gastrodiae two flavor medicines, the Chinese medicine preparation that the technique that adopts water extraction to get to combine with alcohol extracting is prepared into, has the effect of invigorating blood circulation flat liver, removing obstruction in channels to relieve pain, headache due to obstruction of collaterals by blood stasis or liver-yang hyperactivity with the passing of time, fixed pain, or have that dizzy hypochondriac pain, insomnia are irritated concurrently, dark tongue quality or ecchymosis is arranged; Angioneurotic headache.In order more comprehensively, effectively to control the quality control level of this Chinese medicine composition, and international joint, allow clinical drug safety and curative effect guarantee more to some extent, need to adopt more advanced quality control method to this Chinese medicine composition.
Because Chinese medicine and preparation thereof are the multi-component complex system, therefore estimating its quality should adopt and adapt with it, can provide the detection method of enriching authentication information, but the methods such as existing micro-discriminating, physics and chemistry discriminating and assay are not enough to all address this problem.Traditional Chinese medicine fingerprint refers to that some Chinese crude drug or Chinese medicine preparation after suitably processing, adopt certain analysis means, the chromatogram that can indicate its chemical feature or the spectrogram that obtain.Traditional Chinese medicine fingerprint is a kind of comprehensive, and quantifiable identification of means can reflect comparatively all sidedly kind and the quantity of contained chemical composition in Chinese medicine and preparation thereof, and then drug quality be carried out integral body describe and estimate.So traditional Chinese medicine fingerprint has become at present comparatively advanced in the world traditional Chinese medicine quality control device.This Chinese medicine composition is not also openly reported as the finger-print of quality control standard.
All point out in FDA (Food and Drug Adminstration) (FDA) autonomic drug product industrial directory, WHO herbal medicine evaluation guide, if the active component of herbal medicinal product can not differentiate, can pass through finger-print (fingerprint spectrum) proves the consistent of product quality.The European Community also points out in herbal medicine quality guideline note, herbal medicine and preparation thereof be with integral body as active principle, therefore, the quality stability of herbal medicine only depends on and measures known effective constituent is inadequate, should show the various compositions that it is contained by finger-print.India herbal medicine allusion quotation, British Herbal Pharmacopoeia, German medicinal plant association, Canada is medicinal and fragrant plant association etc., also accepts to guarantee with finger-print the consistance of the herbal products quality that effective constituent is not bright.National Drug Administration has required to have relevant finger-print to traditional Chinese medicine at present, the quality that improves traditional Chinese medicine has been played key effect, but the Chinese medicine of other formulations is not still had Compulsory Feature.
Traditional Chinese medicine fingerprint comprises Chemistry for Chinese Traditional Medicine finger-print, protein fingerprint spectrum, DNA fingerprinting and biological effect finger-print etc.Usually the finger-print of saying refers to chemical fingerprint, be Chinese crude drug, Chinese medical extract or Chinese patent drug after suitable processing, adopt certain analysis means, chromatogram or the spectrum that can indicate this Chinese medicine main chemical compositions characteristic that obtain.
The Chemistry for Chinese Traditional Medicine finger-print can be divided into the Chinese medicine spectrum fingerprint, as infrared spectrum (IR), ultraviolet spectrum (UV), X ray diffracting spectrum etc.; Chromatographic fingerprinting is as thin-layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), Capillary Electrophoresis collection of illustrative plates (CE) etc.; The wave spectrum finger-print is as mass spectrum (MS), nuclear magnetic resonance spectrum (NMR) etc.; DNA fingerprinting; And the multi-dimensional information characteristics spectrum (characteristic fingerprint of muhrdimension andmuhrdata) that adopts the coupling of various modern analytical instrument to obtain, as HPLC/MS, HPLC/Ms/MS, GC/MS, CE/MS etc.
Although the traditional Chinese medicine fingerprint research method is more, but the chromatographia method should be main stream approach, especially TLC, HPLC and GC chromatographic technique, having become the analysis means of everybody confessed three kinds of routines, is that present Study of Traditional Chinese Medicine finger-print should top-priority method.
Traditional Chinese medicine fingerprint has two effects: the one, by the characteristic of finger-print, can effectively differentiate the true and false or the place of production of Chinese crude drug: the 2nd, by the area at finger-print principal character peak or the formulation of ratio, can effectively control the quality of product, guarantee the relatively consistent of product quality.
The Production and application of traditional Chinese medicine fingerprint generally includes: (1) gathers representative traditional Chinese medicine sample, takes suitable method to prepare need testing solution, and selects suitable standard standard items (object of reference), carries out the preferred of analytical approach; (2) according to the analytical approach of determining, the traditional Chinese medicine sample of certain batch is carried out compartment analysis, the multiple batches of data of gained are processed, obtain standard finger-print; (3) with sample according to making after identical method carries out sample preparation, compartment analysis with standard finger-print, finger-print collection of illustrative plates and the standard finger-print of gained are carried out similarity relatively, and general similarity reaches and namely can be considered this sample more than 80% is qualified.The calculating of similarity and evaluation method usually adopt related coefficient and are harmonious coefficient or Cosin method.The unit roots such as China Central South University, Zhejiang University go up method according to this, have worked out corresponding computer software and have been used for completing similarity evaluation, and " similarity evaluation " wherein recommended with Chinese Pharmacopoeia Commission used more.
Summary of the invention
The purpose of this invention is to provide the standard finger-print as a kind of Chinese medicine composition of quality control standard.
Another object of the present invention is to provide the assay method of this Chinese medicinal composition standard fingerprint.
A further object of the invention is to provide the application of this Chinese medicinal composition standard fingerprint in the Chinese medicine preparation quality control.
The objective of the invention is to realize by following measures:
The Chinese medicinal composition standard fingerprint that the present invention relates to, this finger-print is the high-efficient liquid phase chromatogram spectrum, present 6 main chromatographic peaks in this collection of illustrative plates, wherein No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other 5 chromatographic peaks is respectively: No. 1 peak 0.23 ± 10%, No. 2 peaks 0.27 ± 10%, No. 4 peaks 1.20 ± 8%, No. 5 peaks 1.65 ± 10%, No. 6 peaks 1.80 ± 10%.
Above-mentioned standard finger-print, it is characterized in that described Chinese medicine composition prepares by following method: get Ligusticum wallichii 784g, rhizoma Gastrodiae 196g, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filter, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I; Dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges above-mentioned clear cream I and cream II clearly, adds appropriate amount of auxiliary materials, makes required Chinese medicine preparation.This Chinese medicine preparation includes but are not limited to the liquid preparation of the solid pharmaceutical preparation of capsule, soft capsule, tablet, pill, dripping pill, granule and oral liquid, parenteral solution.
The assay method of described Chinese medicine composition product standard finger-print, the method adopts high performance liquid chromatography, and its chromatographic condition is:
Chromatographic column adopting octadecylsilane chemically bonded silica chromatographic column;
Mobile phase is 0.1% phosphoric acid-methanol aqueous solution system,, wherein mobile phase A is 0.1% phosphoric acid solution, Mobile phase B is methyl alcohol, adopts gradient elution;
Flow velocity is 1mL/min;
Column temperature is 30 ℃;
Detecting device adopts UV-detector, and the detection wavelength is 276nm;
Number of theoretical plate is pressed the peak calculating of object of reference forulic acid, should be not less than 6000.
It is that 25～100% methyl alcohol is made solvent that the assay method of described Chinese medicinal composition standard fingerprint, the solution that wherein is used for high-performance liquid chromatogram determination adopt concentration.
The assay method of described Chinese medicinal composition standard fingerprint, the need testing solution that wherein is used for high-performance liquid chromatogram determination adopts 50% methyl alcohol to make solvent, and object of reference solution adopts 50% methyl alcohol to make solvent.
The assay method of described Chinese medicinal composition standard fingerprint, wherein the preparation of object of reference solution is that to get forulic acid appropriate, accurately weighed, adds 50% methyl alcohol and makes the solution that every 1mL contains 20 μ g, and get final product.
The assay method of described standard finger-print, the preparation process of need testing solution is as follows:
This composition capsule, tablet, pill, dripping pill, granule: get this Chinese medicine composition content, mixing, porphyrize is got approximately 1g, puts in conical flask, and precision adds 50% methyl alcohol 25mL, and ultrasonic 30min filters, and gets subsequent filtrate as need testing solution, and get final product;
This composition soft: get this Chinese medicine composition content, mixing is got approximately 1g, puts in conical flask, and precision adds 75% methyl alcohol 30mL, and ultrasonic 30min filters, and gets subsequent filtrate as need testing solution, and get final product;
This composition oral liquid: get this Chinese medicine composition, mixing is got approximately 10mL, and to flask, precision adds 95% methyl alcohol 50mL, and refluxing extraction 30min filters, and gets subsequent filtrate as need testing solution, and get final product.
This composite injection: get approximately 10mL of parenteral solution, to flask, add 95% methyl alcohol 50mL, fully stir, filter, get subsequent filtrate as need testing solution, and get final product.
The assay method of described standard finger-print, wherein gradient elution is sequentially:
Time (min) mobile phase A (%) Mobile phase B (%)
0～5 85 15
5～55 85～5 15～95
55～60 5 95
The assay method of described standard finger-print, wherein sample size is 2 μ L～50 μ L.
The application of described standard finger-print in the quality control of this Chinese medicine composition.
Adopt above-mentioned finger-print to sample detection, the standard of specification product is: the finger-print of qualified Chinese medicine composition and the similarity of standard finger-print are not less than 0.80.The method of comparative sample finger-print and standard finger-print similarity is to adopt Cosin method or correlation coefficient process, and it adopts corresponding computer software is the similarity evaluation of Chinese Pharmacopoeia Commission's regulation.
If no special instructions, capsule of the present invention refers to the hard shell capsules in appendix IL capsule of Pharmacopoeia of People's Republic of China version in 2005.
Beneficial effect of the present invention:
1, the present invention has set up the standard finger-print under the 276nm wavelength, the quality information that can comprehensively reflect this composition need not more more complicated collection of illustrative plates or more complicated quality control index and just can reach the purpose of controlling fully and effectively this composite preparation product quality.
2, to the identification of survey finger-print, the similarity evaluation that adopts Chinese Pharmacopoeia Commission to provide, easy to operate, quick; And, with this similarity result that draws, preparation finger to be estimated, conclusion is comparatively objective, accurate.
the present invention is with reference to the requirement of traditional Chinese medicine finger-print, the Chinese medicine composition finger-print that the present invention relates to is studied, process is to test sample preparation method's investigation and the instrument of mensuration finger-print, chromatographic column, mobile phase, the conditions such as detection wavelength are carried out the preferred of system, set up the determining fingerprint pattern condition and carried out methodological study, on the basis to many batches of this Chinese medicine composition finger-print testing results, accumulation data gradually, standard finger-print has been proposed, as this product finger-print standard, thereby be able to more comprehensively, effectively control the purpose of the quality of the pharmaceutical preparations.
identification to measured finger-print, the present invention adopts Chinese Pharmacopoeia Commission to provide similarity evaluation as this Chinese medicine composition fingerprint similarity software for calculation, through test of many times research, and by comparing with the method for calculating relative retention time and relative peak area, drawn evaluation conclusion is basically identical, use similarity evaluation to estimate the similarity of finger-print, easy to operate, fast, with its similarity result that draws, preparation finger is estimated, conclusion is comparatively objective, accurately.
Research and explanation to finger print measuring method of the present invention:
Take the capsule of Chinese medicine composition of the present invention as example, the detection method of finger-print of the present invention is carried out methodological study:
1. the selection of object of reference solution
Select forulic acid as object of reference.
2. the preparation of need testing solution
Through the experiment screening comparative analysis, determine that this Chinese medicine composition determining fingerprint pattern test sample preparation method is:
This composition capsule: get this Chinese medicine composition content under the content uniformity item, mixing, porphyrize is got approximately 1g, puts in conical flask, and precision adds 50% methyl alcohol 25mL, and ultrasonic 30min filters, and gets subsequent filtrate as need testing solution, and get final product;
3. detection method
Instrument and reagent:
Instrument: Agilent 1100 liquid chromatographies; MWD multi-wavelength UV-visible detector, the full-automatic injector of G1313A, Agilent LC chromatographic work station.
Chromatographic column: C
18, 4.6 * 250mm, 5 μ m; Mobile phase A is 0.1% phosphoric acid solution, and Mobile phase B is methyl alcohol, carries out gradient elution;
Eluting order is:
Time (min) mobile phase A (%) Mobile phase B (%)
0～5 85 15
5～55 85～5 15～95
55～60 5 95
Flow velocity is 1mL/min; The detection wavelength is 276nm, and number of theoretical plate is pressed object of reference (forulic acid) peak calculating, should be not less than 6000.
Reagent: methyl alcohol is chromatographically pure (Tedia company), and water is ultrapure water, and it is pure that all the other reagent are analysis.
Measure wavelength: in order more fully to reflect the composition in preparation, compare analysis through adopting a plurality of wavelength to measure finger-print, determine that 276nm is the detection wavelength of preparation finger, in preparation finger under this wavelength, other composition fingerprint peaks also can be embodied preferably, and each peak degree of separation is good, and baseline is steady, good reproducibility.See Fig. 1, Fig. 2.
4. stability test
Get this Chinese medicinal composition capsules finished product, prepare need testing solution by the preparation method of need testing solution in embodiment 1, measure at set intervals once its finger-print, the results are shown in Table 1, show the need testing solution stable components.
Table 1 lot number is 060712 Chinese medicinal composition capsules finished product stability similarity (wavelength 276nm)
| Time | Similarity (reference) |
| 0h | 1.000 |
| 3h | 0.998 |
| 6h | 0.997 |
| 9h | 0.998 |
| 12h | 0.998 |
| 15h | 0.997 |
5. precision test
Get this Chinese medicinal composition capsules finished product, prepare need testing solution by the preparation method of need testing solution, continuous sample introduction is measured.Measurement result sees Table 2.Calculate the similarity of rear 5 sample introduction gained finger-prints as contrast with the 1st sample introduction gained finger-print again, measurement result sees Table 3, shows that the method precision is good.
Table 2 lot number is 060712 Chinese medicinal composition capsules finished product finger-print precision investigation result (wavelength 276nm) (accounting for the retention time of total peak area 5% above main peaks)
Table 3 lot number is 060712 Chinese medicinal composition capsules finished product precision similarity (wavelength 276nm)
| The sample introduction number of times | Similarity (reference) |
| 1 | 1.000 |
| 2 | 0.998 |
| 3 | 0.999 |
| 4 | 0.997 |
| 5 | 0.998 |
| 6 | 0.998 |
6. replica test
Get this Chinese medicine composition of lot number, prepare need testing solution by the preparation method of need testing solution in embodiment 1, with legal system available test sample solution, measure in accordance with the law, measurement result sees Table 4, and result is calculated similarity, meets the technical requirement of finger-print.
Table 4 lot number is 060712 Chinese medicinal composition capsules finished product repeatability similarity (wavelength 276nm)
| Sample number into spectrum | Similarity (reference) |
| 1 | 1.000 |
| 2 | 0.997 |
| 3 | 0.998 |
| 4 | 0.998 |
| 5 | 0.997 |
| 6 | 0.999 |
According to above methodological study result, show that the method measures the finger-print of this Chinese medicine composition, precision, repeatability, stability are all better, finger-print that can this composition of Accurate Determining.
7. the mensuration of finger-print manufactures a finished product greatly
Get respectively this Chinese medicinal composition capsules finished product, measure according to the fingerprint atlas detection method that the invention described above provides, measurement result sees Table 5, gained finished product finger-print calculates with similarity software with standard finger-print respectively, and similarity meets the regulation of this Chinese medicine composition finished product finger-print as a result.Analysis result shows that consistance is preferably arranged between different batches.
10 batches of present composition capsule finished product similarities of table 5 (wavelength 276nm)
| Lot number | Similarity (standard) |
| 060712 | 0.977 |
| 060806 | 0.978 |
| 060819 | 0.975 |
| 060902 | 0.977 |
| 060916 | 0.979 |
| 060930 | 0.982 |
| 061012 | 0.975 |
| 061025 | 0.978 |
| 061116 | 0.978 |
| 061209 | 0.982 |
Description of drawings
Fig. 1: the Chinese medicine composition liquid-phase fingerprint is measured the selection of wavelength.
Fig. 2: the finger-print HPLC-UV 3D figure of Chinese medicine composition.
Fig. 3: Chinese medicinal composition standard fingerprint (the detection wavelength is 276nm).
Embodiment
The invention will be further elaborated by the following examples.
Embodiment 1: determining fingerprint pattern
The preparation method of capsule is as follows:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
Method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I; Dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merge above-mentioned clear cream I and clear cream II, add appropriate silicon dioxide, vacuum drying is pulverized, sieve, incapsulate, make 1000, and get final product.Specification: every dress 0.34g
Finger-print detects:
Get this product content, mixing, porphyrize, get approximately 1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution.Separately get the forulic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution that every 1ml contains 20 μ g, in contrast product solution.
Measure instrument: Agilent 1100 liquid chromatographies according to high performance liquid chromatography (2005 editions one appendix VI D of Chinese Pharmacopoeia); MWD multi-wavelength UV-visible detector; Reagent: methyl alcohol is chromatographically pure (Tedia company), and water is ultrapure water, and it is pure that all the other reagent are analysis.Take octadecylsilane chemically bonded silica as filling agent, phenomenex Luna C
18Chromatographic column (column length is 250mm, and column internal diameter is 4.6mm, granularity 5 μ m); As mobile phase A, methyl alcohol is Mobile phase B with 0.1% phosphoric acid solution, and according to the form below carries out gradient elution; Flow velocity is per minute 1ml; The detection wavelength is 276nm; Column temperature is 30 ℃.Number of theoretical plate should be not less than 6000 by forulic acid peak calculating.
Precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography records the chromatogram in 55 minutes.
Should present 6 main chromatographic peaks in the test sample chromatogram, the chromatographic peak identical with reference substance peak retention time is No. 3 peaks, compare with No. 3 peak retention times, the relative retention time of other 5 chromatographic peaks is respectively: No. 1 peak 0.23, No. 2 peaks 0.27, No. 4 peaks 1.20, No. 5 peaks 1.65, No. 6 peaks 1.80.
The similarity software for calculation is the similarity evaluation of Chinese Pharmacopoeia Commission regulation, after measured, with the similarity of standard finger-print be 0.95.
Embodiment 2: the acquisition of standard finger-print.
10 batches of this Chinese medicinal composition capsules finished products, preparation method by need testing solution prepares need testing solution, measure (the detection wavelength is 276nm) according to embodiment 1 method, result is calculated similarity, the results are shown in Table 6, with obtaining " common pattern " as standard finger-print take these 10 batches of test sample finger-prints as the basis in similarity software, the results are shown in Figure 3.
Ten batches of similarities of this composition capsule of table 6 finished product (wavelength 276nm)
| Lot number | Similarity (standard) |
| 060712 | 0.977 |
| 060806 | 0.978 |
| 060819 | 0.975 |
| 060902 | 0.977 |
| 060916 | 0.979 |
| 060930 | 0.982 |
| 061012 | 0.975 |
| 061025 | 0.978 |
| 061116 | 0.978 |
| 061209 | 0.982 |
Embodiment 3: the determining of production quality control standard
Adopt the similarity evaluation of Chinese Pharmacopoeia Commission's regulation, version is 2004 editions.
The described 10 batches of finished product finger-prints of embodiment 2 and standard finger-print are calculated similarity, stipulate that the finger-print of this Chinese medicine composition product and standard finger-print calculate through similarity software, similarity is not less than 0.80.
The principal character of the finger-print of production quality control standard:
Present 6 main chromatographic peaks in this collection of illustrative plates, wherein No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other 5 chromatographic peaks is respectively: No. 1 peak 0.23 ± 10%, No. 2 peaks 0.27 ± 10%, No. 4 peaks 1.20 ± 8%, No. 5 peaks 1.65 ± 10%, No. 6 peaks 1.80 ± 10%.
Embodiment 4: the impact on the product fingerprint collection of illustrative plates of different auxiliary material and different dosage form.
1, chewable tablets:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I, dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges above-mentioned clear cream I and cream II clearly, adds appropriate amount of auxiliary materials (as silicon dioxide, dextrin, starch, phosphate, carbonate, sweet mellow wine, sorbierite, sorbic acid or sylvite, xylitol, maltose, glucose, fructose, dextran, glycocoll, starch, sucrose, one or more in auxiliary material of the dilution such as lactose or mannitol) mix, drying is pulverized, and obtains fine powder and appropriate bonding agent (solid binder such as PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, the macromolecular material polyvinylpyrrolidone, sodium carboxymethyl cellulose, methylcellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium alginate etc. or liquid adhesive such as ethanol, starch slurry, dextrin slurry etc. one or more) mix particle processed, add appropriate flavouring (as sweet mellow wine, sucrose, xylitol, stevioside, maltitol, menthol, four butanols, orange oil, cassia oil, various essence etc. one or more), lubricant is (as dolomol, talcum powder, sodium stearyl fumarate, magnesium laurylsulfate etc. one or more), disintegrant is (as sodium carboxymethyl starch, Ac-Di-Sol, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, pregelatinized starch, microcrystalline cellulose, soybean polyoses, alginic acid, process agar etc. one or more) whole grain, total mixed, be pressed into 1000, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.24, No. 2 peaks 0.28, No. 4 peaks 1.22, No. 5 peaks 1.75, No. 6 peaks 1.87.With the similarity of standard finger-print be 0.90.
2, lozenge:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I, dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges above-mentioned clear cream I and cream II clearly, adds appropriate amount of auxiliary materials (as silicon dioxide, dextrin, starch, phosphate, carbonate, sweet mellow wine, sorbierite, sorbic acid or sylvite, xylitol, maltose, glucose, fructose, dextran, glycocoll, starch, sucrose, lactose, the dilution auxiliary material that mannitol and other pharmacies are commonly used etc. one or more) mix, drying is pulverized, obtain fine powder with appropriate flavouring (as mannitol, sucrose, xylitol, stevioside, maltitol, menthol, four butanols, orange oil, cassia oil, various essence etc. one or more) mix, add appropriate bonding agent (solid binder such as PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, the macromolecular material polyvinylpyrrolidone, sodium carboxymethyl cellulose, methylcellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium alginate etc. or liquid adhesive such as ethanol, gelatine size, starch slurry etc. one or more) particle processed, add lubricant (as dolomol, talcum powder, sodium stearyl fumarate etc. one or more) whole grain, total mixed, be pressed into 1000, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.23, No. 2 peaks 0.28, No. 4 peaks 1.28, No. 5 peaks 1.54, No. 6 peaks 1.65.With the similarity of standard finger-print be 0.90.
3, oral disintegrating tablet:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I, dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges above-mentioned clear cream I and cream II clearly, adds appropriate amount of auxiliary materials (as silicon dioxide, dextrin, starch, phosphate, carbonate, sweet mellow wine, sorbierite, sorbic acid or sylvite, xylitol, maltose, glucose, fructose, dextran, glycocoll, starch, sucrose, lactose, the dilution auxiliary material that mannitol and other pharmacies are commonly used etc. one or more) mix, drying is pulverized, and obtains fine powder and adds appropriate flavouring (as mannitol, sucrose, xylitol, stevioside, maltitol, menthol, four butanols, orange oil, cassia oil, various essence etc. one or more), lubricant is (as dolomol, talcum powder, sodium stearyl fumarate, magnesium laurylsulfate etc. one or more), disintegrant is (as sodium carboxymethyl starch, Ac-Di-Sol, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, process agar etc. one or more) direct pressing becomes 1000, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.25, No. 2 peaks 0.28, No. 4 peaks 1.16, No. 5 peaks 1.45, No. 6 peaks 1.69.With the similarity of standard finger-print be 0.90.
4, oral liquid:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
Method for making: above two flavors, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filter, filtrate recycling ethanol also is condensed into the clear cream that relative density is 1.27 (55～60 ℃); Dregs of a decoction boiling secondary, each 1 hour, collecting decoction filters, and filtrate shortens the clear cream that relative density is 1.27 (55～60 ℃) deeply into, merge with above-mentioned clear cream, join in boiling water thermal treatment appropriate time, refrigeration, filter, add the agent of the tender flavor of auxiliary material as sucrose, honey, xylitol, stevioside, maltitol etc. one or more; Antiseptic such as sorbic acid or sylvite, benzoic acid and propionic acid etc. one or more; Solubilizer such as tween series etc., antioxidant such as sodium bisulfite, sodium thiosulfate etc. one or more), filter, 1000mL is made in embedding, sterilization, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.22, No. 2 peaks 0.26, No. 4 peaks 1.19, No. 5 peaks 1.55, No. 6 peaks 1.76.With the similarity of standard finger-print be 0.90.
5, granule:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I, dregs of a decoction boiling secondary, each 1 hour, collecting decoction, filter, it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merge above-mentioned clear cream I and clear cream II, add appropriate amount of auxiliary materials (as silicon dioxide, dextrin, starch, phosphate, carbonate, sweet mellow wine, sorbierite, sorbic acid or sylvite, xylitol, maltose, glucose, fructose, dextran, glycocoll, starch, sucrose, lactose, one or more in the dilution such as mannitol auxiliary material), dry, pulverize, add suitable bonding agent (solid binder such as PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, microcrystalline cellulose etc. or liquid adhesive such as ethanol, gelatine size, starch slurry, syrup etc. one or more), granulate, whole grain is made 1000g, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.21, No. 2 peaks 0.26, No. 4 peaks 1.22, No. 5 peaks 1.72, No. 6 peaks 1.86.With the similarity of standard finger-print be 0.90.
6, soft capsule:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I, dregs of a decoction boiling secondary, each 1 hour, collecting decoction, filter, it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merge above-mentioned clear cream I and clear cream II, add appropriate amount of auxiliary materials (as silicon dioxide, dextrin, starch, phosphate, carbonate, sweet mellow wine, sorbierite, sorbic acid or sylvite, xylitol, maltose, glucose, fructose, dextran, glycocoll, starch, sucrose, lactose, one or more in the dilution such as mannitol auxiliary material), dry, pulverize, with suitable auxiliary material (as PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, and vegetable oil series etc., antiseptic such as sorbic acid or sylvite, benzoic acid and propionic acid etc., solubilizer such as tween series etc. one or more, antioxidant such as sodium bisulfite, sodium thiosulfate etc. one or more) by any way after mixing, in incapsulating, make 1000 soft capsules, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.22, No. 2 peaks 0.27, No. 4 peaks 1.28, No. 5 peaks 1.55, No. 6 peaks 1.70.With the similarity of standard finger-print be 0.90.
7, parenteral solution:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
Method for making: above two flavors, pulverize, mix, extract secondary with 90% alcohol reflux, each 2 hours, merge extract, filtration, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I; Dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges clear cream, mixing, drying obtains intermediate.Get water for injection 1000ml, boil, add intermediate, refrigerate 48 hours, filter, get filtrate, boiled 45 minutes, refrigerate 48 hours, filter, get filtrate, add 0.5% activated charcoal, boiled 3 minutes, filter, filtrate is got in refrigeration, filtrate is got in ultrafiltration (molecular weight 30000), injects water and supplies 1000ml, embedding, sterilization, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.23, No. 2 peaks 0.26, No. 4 peaks 1.18, No. 5 peaks 1.72, No. 6 peaks 1.85.With the similarity of standard finger-print be 0.90.
8, dripping pill:
Prescription: Ligusticum wallichii 784g rhizoma Gastrodiae 196g
Method for making: above two flavors, pulverize, mix, extract secondary with 60% alcohol reflux, 1.5 hours for the first time, 6 times of amount ethanol, 1 hour for the second time, 4 times of amount ethanol, merge extract, drying under reduced pressure is concentrated into without the alcohol flavor, uses the water saturation extracting n-butyl alcohol, the extract recovered under reduced pressure is to relative density 1.30～1.35, drying under reduced pressure, adsorbs with macroporous absorbent resin with 20 times of water gaging suspendibles to without the normal butyl alcohol flavor, then wash resin column with water, again with 30% ethanol to the polymeric adsorbent wash-out, collect eluent, concentrated, drying under reduced pressure gets extract powder.With PEG-4000 and extract powder with 3～6: 1～4 ratio melting mixing, methyl-silicone oil is made cooling medium, dripping becomes dripping pill, and get final product.
By the detection method of need testing solution preparation method provided by the invention and embodiment 1, above-mentioned preparation is carried out finger-print and detect, and carry out similarity with standard finger-print and calculate, result is as follows:
No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other each chromatographic peaks is respectively: No. 1 peak 0.23, No. 2 peaks 0.28, No. 4 peaks 1.23, No. 5 peaks 1.56, No. 6 peaks 1.78.With the similarity of standard finger-print be 0.90.
get this Chinese medicine composition of above employing different dosage form and different auxiliary material, method (the detection wavelength is 276nm) with reference to embodiment 1 detects, its determining fingerprint pattern result all meets the requirement of the finger-print (the detection wavelength is 276nm) of the present invention's regulation, illustrate that different dosage form and different auxiliary material are on the not impact of finger-print of this Chinese medicine composition, the finger-print quality control standard of the present invention's regulation (is that described this collection of illustrative plates is the high-efficient liquid phase chromatogram spectrum, present 6 main chromatographic peaks in collection of illustrative plates, wherein No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times (by 1), the relative retention time of other 5 chromatographic peaks is respectively: No. 1 peak 0.23 ± 10%, No. 2 peaks 0.27 ± 10%, No. 4 peaks 1.20 ± 8%, No. 5 peaks 1.65 ± 10%, No. 6 peaks 1.80 ± 10%) can be used as the standard finger-print quality control standard of this Chinese medicine composition.Adopt the similarity evaluation of Chinese Pharmacopoeia Commission's regulation to finger-print and this Chinese medicinal composition standard fingerprint calculating similarity of this Chinese medicine composition product of above-mentioned different auxiliary material different dosage form, its result is all greater than 0.80.
Claims (3)
1. the method for building up of the standard finger-print of a Chinese medicine composition that is comprised of Ligusticum wallichii and rhizoma Gastrodiae, is characterized in that the method adopts high performance liquid chromatography, and chromatographic condition is:
Chromatographic column adopting octadecylsilane chemically bonded silica chromatographic column;
Mobile phase adopts 0.1% phosphoric acid-methanol aqueous solution system, and wherein mobile phase A is 0.1% phosphoric acid solution, and Mobile phase B is methyl alcohol, adopts gradient elution;
Flow velocity is 1mL/min;
Column temperature is 30 ℃;
Detecting device adopts UV-detector, and the detection wavelength is 276nm;
Number of theoretical plate is pressed the peak calculating of object of reference forulic acid, should be not less than 6000;
Wherein:
Described Chinese medicine composition prepares by following method: get Ligusticum wallichii 784g, rhizoma Gastrodiae 196g pulverizes, mix, extract secondary, each 2 hours with 90% alcohol reflux, merge extract, filter, filtrate recycling ethanol and to be condensed into relative density be 1.27～1.28 clear cream I; Dregs of a decoction boiling secondary, each 1 hour, collecting decoction filtered, and it is 1.27～1.28 clear cream II that filtrate is condensed into relative density, merges above-mentioned clear cream I and cream II clearly, adds appropriate amount of auxiliary materials, makes required Chinese medicine preparation; Its formulation comprises hard shell capsules, soft capsule, tablet, pill, granule, dripping pill, oral liquid, injection;
Gradient elution is sequentially:
Time (min) mobile phase A (%) Mobile phase B (%)
0～5 85 15
5～55 85～5 15～95
55～60 5 95
The solution that is used for high-performance liquid chromatogram determination comprises need testing solution and object of reference solution, and adopting concentration is that 25～100% methyl alcohol is made solvent;
The preparation process of need testing solution is as follows:
If this Chinese medicine composition is hard shell capsules, tablet, pill, dripping pill, granule: get this Chinese medicine composition content, mixing, porphyrize is got 1g, put in conical flask, precision adds 50% methyl alcohol 25mL, ultrasonic 30min, filter, get subsequent filtrate as need testing solution, and get final product;
If this Chinese medicine composition is soft capsule: get this Chinese medicine composition content, mixing is got 1g, puts in conical flask, adds 75% methyl alcohol 30mL, and ultrasonic 30min filters, and gets subsequent filtrate as need testing solution, and get final product;
If this Chinese medicine composition is oral liquid: get this Chinese medicine composition, mixing is got 10mL, to flask, adds 95% methyl alcohol 50mL, fully stirs, and filters, and gets subsequent filtrate as need testing solution, and get final product;
If this Chinese medicine composition is parenteral solution: get parenteral solution 10mL, to flask, add 95% methyl alcohol 50mL, fully stir, filter, get subsequent filtrate as need testing solution, and get final product;
The preparation of object of reference solution is that to get forulic acid appropriate, accurately weighed, adds 50% methyl alcohol and makes the solution that every 1mL contains forulic acid 20 μ g, and get final product.
2. method according to claim 1, it is characterized in that this finger-print is the high-efficient liquid phase chromatogram spectrum, present 6 main chromatographic peaks in this collection of illustrative plates, wherein No. 3 peaks are chromatographic peaks identical with the peak retention time of object of reference forulic acid, compare with No. 3 peak retention times, the relative retention time of other 5 chromatographic peaks is respectively: No. 1 peak 0.23 ± 10%, No. 2 peaks 0.27 ± 10%, No. 4 peaks 1.20 ± 8%, No. 5 peaks 1.65 ± 10%, No. 6 peaks 1.80 ± 10%.
3. method according to claim 1, is characterized in that sample size is 2 μ L～50 μ L.
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Inventor after: Zhou Xiaofeng Inventor after: Xiao Wei Inventor after: Bi Yuan Inventor after: Hu Junhua Inventor after: Qin Jianping Inventor after: Wang Zhenzhong Inventor after: Zhang Chenfeng Inventor before: Xiao Wei Inventor before: Bi Yuan Inventor before: Hu Junhua Inventor before: Qin Jianping Inventor before: Wang Zhenzhong Inventor before: Zhang Chenfeng |
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