CN109470801A - The method for building up and its standard finger-print of a kind of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas and application - Google Patents
The method for building up and its standard finger-print of a kind of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas and application Download PDFInfo
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Abstract
The invention discloses a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas and its standard finger-print and applications, this method includes that (1) takes rhizoma dioscoreae nipponicae medicinal powder, and methanol impregnates, ultrasonic extraction, and by supernatant filtering with microporous membrane, filtrate is as test solution；(2) efficient liquid phase chromatographic analysis is carried out, chromatographic column is XBridge C18 (250mm × 4.6mm, 5 μm)；Flow velocity is 1.0ml/min；Sample volume is 10 μ l；Detection wavelength is 208nm；Column temperature is 30 DEG C；Mobile phase is acetonitrile (A)-water (B), and gradient elution program is 0~5min 5%A, 5~15min 5%~15%A, 15~55min 15%~45%A, 55~65min 45%~100%A, 65~80min 100%A；Test solution sample introduction is drawn, the HPLC finger-print of rhizoma dioscoreae nipponicae medicinal material is obtained.The rhizoma dioscoreae nipponicae standard finger-print that the present invention establishes has 21 common characteristic peaks, can be with the quality of effective monitoring rhizoma dioscoreae nipponicae medicinal material using standard finger-print.
Description
Technical field
The invention belongs to Chinese medicine detection technique fields, and in particular to a kind of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas is built
Cube method and its standard finger-print and application.
Background technique
Rhizoma dioscoreae nipponicae also known as dioscorea nipponica, dioscorea japonica, ground joist, chicken bone, dragon Bi are Dioscoreaceae plant dioscorea nipponica
The dry rhizome of (Dioscorea nipoonica Makino), many years herbaceous species plant.The rhizome of rhizoma dioscoreae nipponicae mainly contains
Steroid saponin constituents, aerial part mainly contain the ingredients such as luxuriant and rich with fragrance class and flavone compound.Stop with relaxing tendons and activating collaterals, wind-dispelling
Gout, treatment arthritis with fixed pain caused by dampness disease, arthroncus and other effects.Modern pharmacological studies have shown that it is with hypoglycemic, adjusting immunity, the improvement heart
Vascular function, antitumor and other effects.
2015 editions Chinese Pharmacopoeias are determined the content of Dioscin in Dioscorea nipponica, and using the height of its content as
Evaluate rhizoma dioscoreae nipponicae quality good or not standard.But for treating from differential diagnosis in tcm, medication use flavour of a drug rather than certain a few chemistry at
Point, it is the mass action result of pharmaceutical decocting piece or patent medicine prescription internal substance group.Substance number, the substance that these substances group is included
Amount and composition ratio difference, can all influence effect.Therefore, the amount of one or several compounds is only used to determine as index
The conclusion of quality of medicinal material seems and excessively limits to.
Efficient liquid-phase chromatograph finger print atlas technology has the characteristics that whole, macroscopic view, fuzzy analysis, can be anti-to greatest extent
Answer the type and quantity of chemical component contained by Chinese medicine.By the characteristic of finger-print, it can effectively identify the true and false or production of sample
Ground；By the area or limited proportion of its principal character peak, the quality of sample can be effectively controlled.At present there is not yet closing in document
It is reported in the research of rhizoma dioscoreae nipponicae finger-print, therefore, scientificlly and effectively to control its inherent quality, it is necessary to establish rhizoma dioscoreae nipponicae
Finger-print.
Summary of the invention
An object of the present invention is to provide a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas.
The second object of the present invention is to provide the rhizoma dioscoreae nipponicae standard finger-print established by the above method.
The third object of the present invention is to provide the application of above-mentioned rhizoma dioscoreae nipponicae standard finger-print.
To achieve the above object, The technical solution adopted by the invention is as follows: a kind of rhizoma dioscoreae nipponicae high performance liquid chromatography fingerprint image
The method for building up of spectrum, comprising the following steps:
(1) preparation of test solution: taking rhizoma dioscoreae nipponicae medicinal material, 50~100 meshes is smashed it through, after precision weighs sieving
Rhizoma dioscoreae nipponicae powder 1g is placed in tool plug container, 30ml methanol is added and impregnates, and ultrasonic extraction filters extracting solution, in triplicate, closes
And extracting solution is concentrated under reduced pressure, concentrate constant volume to 10ml, with filtering with microporous membrane, obtained filtrate i.e. test solution；
(2) efficient liquid phase chromatographic analysis: the test solution that step (1) is obtained injects high performance liquid chromatograph, with second
Nitrile is mobile phase A, and water is that Mobile phase B carries out gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column XBridge
C18,250mm × 4.6mm, 5 μm；Flow velocity is 1.0ml/min；Sample volume is 10 μ l；Detection wavelength is 208nm；Column temperature is 30 DEG C；
Gradient elution program be 0~5min 5%A, 5~15min 5%~15%A, 15~55min 15%~45%A, 55~
65min 45%~100%A, 65~80min100%A；Test solution is analyzed with this condition, obtains the HPLC of rhizoma dioscoreae nipponicae
Finger-print；
(3) determination of standard finger-print: having determined 21 common characteristic peaks, and No. 18 chromatographic peaks are Dioscin peak, protects
Staying the time is 62.591, is control peak with No. 18 chromatographic peaks, calculates the relative retention time of other 20 common characteristic peaks, successively
Are as follows: No. 1 peak relative retention time is that 0.591, No. 2 peak relative retention time is that 0.649, No. 3 peak relative retention time is
0.653, it is that 0.690, No. 6 peak relative retention time is that No. 4 peak relative retention times, which are 0.674, No. 5 peak relative retention time,
0.697, it is that 0.750, No. 9 peak relative retention time is that No. 7 peak relative retention times, which are 0.735, No. 8 peak relative retention time,
0.756, it is 0.769, No. 12 peak relative retention time that No. 10 peak relative retention times, which are 0.763, No. 11 peak relative retention time,
It is that be that 0.8201, No. 15 peak is opposite retain 0.7991, No. 14 peak relative retention time for 0.784, No. 13 peak relative retention time
Time is that 0.851, No. 16 peak relative retention time is that be that 0.994, No. 19 peak is opposite protect 0.959, No. 17 peak relative retention time
It is 1.229, No. 21 peak relative retention times is 1.237 that stay the time, which be 1.145, No. 20 peak relative retention times, above-mentioned shared spy
Sign peak constitutes the fingerprint characteristic of rhizoma dioscoreae nipponicae, the standard finger-print as rhizoma dioscoreae nipponicae.
Preferably, in step (1), methanol soaking time is 10min.
Preferably, in step (1), ultrasonic power 250W, frequency 400kHz, ultrasonic time 30min.
Preferably, in step (1), the aperture of the miillpore filter is 45 μm.
The rhizoma dioscoreae nipponicae HPLC finger-print that the present invention establishes can be used for rhizoma dioscoreae nipponicae quality of medicinal material control as standard finger-print
System, to be further ensured that its clinical application safety.
Compared with prior art, the invention has the following beneficial effects:
1. rhizoma dioscoreae nipponicae HPLC finger-print of the invention shares 21 common characteristic peaks, these characteristic peaks constitute rhizoma dioscoreae nipponicae
Fingerprint characteristic, can be used as the standard finger-print of rhizoma dioscoreae nipponicae.
2. rhizoma dioscoreae nipponicae finger-print of the invention can be used for rhizoma dioscoreae nipponicae after more batches of samples are verified as standard finger-print
In quality control, for evaluating and controlling the quality of rhizoma dioscoreae nipponicae medicinal material on the whole, avoids and only measure one, two chemical component
And determine the one-sidedness of rhizoma dioscoreae nipponicae total quality.
3. the method for building up of the efficient liquid-phase chromatograph finger print atlas of rhizoma dioscoreae nipponicae of the present invention has, repeatability is strong, precision is high
It is reliable, easy to the advantages of grasping with stability, suitable for the true and false of rhizoma dioscoreae nipponicae medicinal material, the place of production and quartile length, and to its clinic
Safe medication is of great significance.
Detailed description of the invention
Fig. 1 is the standard finger-print of rhizoma dioscoreae nipponicae medicinal material provided by the invention, is from left to right labelled with respectively in figure shared
Characteristic peak 1 to 21；
Fig. 2 is the finger-print of the rhizoma dioscoreae nipponicae medicinal material of 11 batches of different sources provided by the invention, and S1 to S11 is respectively in figure
The finger-print of 11 batches of rhizoma dioscoreae nipponicaes, R are standard finger-print.
Specific embodiment
Invention is further described in detail in the following with reference to the drawings and specific embodiments.
Embodiment 1: with the finger-print of a batch rhizoma dioscoreae nipponicae medicinal material
1, test apparatus: 2695 high performance liquid chromatograph of WATERS；WATERS 2998PAD UV detector is containing online true
Empty degasser, high pressure binary gradient pump, thermostated autosampler, column oven.
2, reagent: acetonitrile (chromatographically pure), ultrapure water.
3, high-efficient liquid phase chromatogram condition: chromatographic column: XBridge C18 (250mm × 4.6mm, 5 μm)；Flow velocity is 1.0ml/
min；Sample volume is 10 μ l；Detection wavelength is 208nm；Column temperature is 30 DEG C；Mobile phase is acetonitrile (A)-water (B), is washed using gradient
De- mode is 0~5min, 5%A；5~15min, 5%~15%A；15~55min, 15%~45%A；55~65min,
45%~100%A；65~80min, 100%A.
4, the preparation of test solution: taking rhizoma dioscoreae nipponicae medicinal material, 50~100 meshes is smashed it through, after precision weighs sieving
Rhizoma dioscoreae nipponicae powder 1g is placed in tool plug container, 30ml methanol is added and impregnates 10min, is 250W in power, frequency is 400kHz's
Under the conditions of ultrasonic extraction 30min, filter extracting solution, in triplicate, combined extract be concentrated under reduced pressure, concentrate constant volume to 10ml,
With 0.45 μm of filtering with microporous membrane, obtained filtrate i.e. test solution.
5, efficient liquid phase chromatographic analysis
Precision draws test solution and injects high performance liquid chromatograph, is divided under above-mentioned high-efficient liquid phase chromatogram condition
Analysis, obtains the finger-print of rhizoma dioscoreae nipponicae, as shown in Figure 1.The finger-print has 21 common characteristic peaks, and No. 18 chromatographic peaks are Chinese yam
Saponin(e peak, retention time 62.591 are control peak with No. 18 chromatographic peaks, calculate the opposite reservation of other 20 common characteristic peaks
Time, successively are as follows: No. 1 peak relative retention time is that 0.591, No. 2 peak relative retention time is 0.649, No. 3 peak with respect to when retaining
Between for 0.653, No. 4 peak relative retention time be 0.674, No. 5 peak relative retention time be 0.690, No. 6 peak relative retention time
It is 0.735, No. 8 peak relative retention time for 0.697, No. 7 peak relative retention time is that 0.750, No. 9 peak relative retention time is
0.756, it is 0.769, No. 12 peak relative retention time that No. 10 peak relative retention times, which are 0.763, No. 11 peak relative retention time,
It is that be that 0.8201, No. 15 peak is opposite retain 0.7991, No. 14 peak relative retention time for 0.784, No. 13 peak relative retention time
Time is that 0.851, No. 16 peak relative retention time is that be that 0.994, No. 19 peak is opposite protect 0.959, No. 17 peak relative retention time
It is 1.229, No. 21 peak relative retention times is 1.237 that stay the time, which be 1.145, No. 20 peak relative retention times,.
6, methodological study
6.1 method reappearance tests
It takes with a collection of 6 parts of medicinal material of rhizoma dioscoreae nipponicae, it is molten to prepare test sample according to the preparation method of above-mentioned rhizoma dioscoreae nipponicae test solution
Liquid carries out determining fingerprint pattern under above-mentioned high-efficient liquid phase chromatogram condition, respectively the relative retention time to shared peak and opposite
Peak area is counted, and the results are shown in Table 1, and RSD% is no more than 3%, and display this method reproducibility is good.
1 reproducible test results of table (n=6)
6.2 finger-print precision tests
Take the test solution of method reappearance test a, under above-mentioned high-efficient liquid phase chromatogram condition, continuous sample introduction 5
It is secondary, the relative retention time and relative peak area at shared peak are counted respectively, the results are shown in Table 2, and RSD% is no more than
3%, method precision of the invention is good as the result is shown.
2 accuracy test result (n=5) of table
6.3 finger-print stability tests
It takes the test solution of method reappearance test a, under above-mentioned high-efficient liquid phase chromatogram condition, carries out fingerprint
Map measurement investigates stability of the test solution in 0/4/8/12/48 hour, respectively to the relative retention time at shared peak
It is counted with relative peak area, the results are shown in Table 3, and RSD% is no more than 3%, and test solution is small 48 as the result is shown
When interior stabilization.
3 stability experiment result (n=5) of table
| Peak number | Relative retention time | RSD (%) | Relative peak area | RSD (%) |
| 1 | 0.591 | 1.98 | 0.171 | 2.21 |
| 2 | 0.651 | 2.32 | 3.179 | 1.35 |
| 3 | 0.653 | 1.97 | 0.224 | 2.57 |
| 4 | 0.674 | 1.43 | 0.109 | 1.18 |
| 5 | 0.690 | 2.05 | 0.230 | 1.85 |
| 6 | 0.697 | 2.64 | 0.19 | 1.52 |
| 7 | 0.735 | 2.65 | 9.134 | 2.33 |
| 8 | 0.750 | 2.50 | 2.777 | 2.82 |
| 9 | 0.756 | 1.68 | 1.043 | 2.74 |
| 10 | 0.763 | 1.77 | 0.154 | 1.43 |
| 11 | 0.769 | 1.93 | 0.837 | 1.77 |
| 12 | 0.784 | 1.69 | 0.150 | 2.74 |
| 13 | 0.799 | 1.63 | 0.132 | 2.87 |
| 14 | 0.820 | 1.67 | 0.164 | 1.68 |
| 15 | 0.85 | 1.58 | 1.133 | 1.88 |
| 16 | 0.959 | 1.76 | 0.187 | 1.87 |
| 17 | 0.994 | 1.42 | 1.90 | 1.65 |
| 18 | 1.000 | 0.0 | 1.000 | 0.0 |
| 19 | 1.145 | 1.68 | 1.278 | 1.87 |
| 20 | 1.229 | 1.35 | 0.073 | 1.62 |
| 21 | 1.236 | 1.47 | 0.285 | 1.33 |
Embodiment 2: the finger-print of 11 batches of rhizoma dioscoreae nipponicae medicinal materials of different sources
(1) 11 batches of rhizoma dioscoreae nipponicaes from different sources such as Jilin, Anhui, Hebei, Liaoning the determination of standard finger-print: are fetched
Medicinal material carries out efficient liquid phase chromatographic analysis by the testing conditions of embodiment 1, obtains the HPLC finger-print of 11 batches of samples, such as Fig. 2
It is shown.Compared by the analysis of the HPLC finger-print of the 11 batch rhizoma dioscoreae nipponicaes to different sources, carries out similarity evaluation, it is true altogether
21 common characteristic peaks are determined, No. 18 chromatographic peaks are Dioscin peak, and retention time 62.591 is control with No. 18 chromatographic peaks
Peak calculates the relative retention time of other 20 common characteristic peaks, successively are as follows: No. 1 peak relative retention time is 0.591, No. 2 peak
It is 0.653, No. 4 peak relative retention time is 0.674, No. 5 peak phase that relative retention time, which is 0.649, No. 3 peak relative retention time,
It is 0.690, No. 6 peak relative retention time to retention time be 0.697, No. 7 peak relative retention time is that 0.735, No. 8 peak is opposite
It is 0.756, No. 10 peak relative retention time is that 0.763, No. 11 peak is opposite that retention time, which is 0.750, No. 9 peak relative retention time,
It is 0.784, No. 13 peak relative retention time is 0.7991, No. 14 peak phase that retention time, which is 0.769, No. 12 peak relative retention time,
It is 0.8201, No. 15 peak relative retention time to retention time be 0.851, No. 16 peak relative retention time is 0.959, No. 17 peak
It is 1.145, No. 20 peak relative retention times is 1.229, No. 21 that relative retention time, which is 0.994, No. 19 peak relative retention time,
Peak relative retention time is 1.237, and above-mentioned common characteristic peaks constitute the fingerprint characteristic of rhizoma dioscoreae nipponicae, and the standard as rhizoma dioscoreae nipponicae refers to
Line map.As seen from Figure 2, the standard of the rhizoma dioscoreae nipponicae with 21 common characteristic peaks can be clearly obtained by this method
Finger-print provides good foundation for the quality of overall evaluation rhizoma dioscoreae nipponicae.
(2) similarity evaluation: the efficient liquid-phase chromatograph finger print atlas established to the rhizoma dioscoreae nipponicae medicinal material of 11 batches of different sources is led
Enter similarity evaluation software (the Pharmacopoeia of People's Republic of China committee, 2004 editions) to be counted
It calculates, similarity evaluation result such as table 4.
4 11 kinds of different sources rhizoma dioscoreae nipponicae medicinal material similarities of table
Claims (5)
1. a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas, which comprises the following steps:
(1) preparation of test solution: taking rhizoma dioscoreae nipponicae medicinal material, smashes it through 50~100 meshes, and precision weighs and wears mountain after being sieved
Imperial powder 1g is placed in tool plug container, 30ml methanol is added and impregnates, and ultrasonic extraction filters extracting solution, and in triplicate, merging mentions
Liquid is taken to be concentrated under reduced pressure, concentrate constant volume to 10ml, with filtering with microporous membrane, obtained filtrate i.e. test solution；
(2) efficient liquid phase chromatographic analysis: the test solution that step (1) is obtained injects high performance liquid chromatograph, is with acetonitrile
Mobile phase A, water are that Mobile phase B carries out gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column is XBridge C18,
250mm × 4.6mm, 5 μm；Flow velocity is 1.0ml/min；Sample volume is 10 μ l；Detection wavelength is 208nm；Column temperature is 30 DEG C；Gradient
Elution program is 0~5min 5%A, 5~15min 5%~15%A, 15~55min 15%~45%A, 55~65min
45%~100%A, 65~80min 100%A；Test solution is analyzed with this condition, obtains the HPLC fingerprint image of rhizoma dioscoreae nipponicae
Spectrum；
(3) determination of standard finger-print: having determined 21 common characteristic peaks, and No. 18 chromatographic peaks are Dioscin peak, when reservation
Between be 62.591, with No. 18 chromatographic peaks be control peak, calculate the relative retention time of other 20 common characteristic peaks, successively are as follows: 1
It is 0.649, No. 3 peak relative retention time is 0.653, No. 4 that number peak relative retention time, which is 0.591, No. 2 peak relative retention time,
It is 0.690, No. 6 peak relative retention time is 0.697, No. 7 peak that peak relative retention time, which is 0.674, No. 5 peak relative retention time,
It is 0.750, No. 9 peak relative retention time is 0.756, No. 10 peak that relative retention time, which is 0.735, No. 8 peak relative retention time,
It is 0.769, No. 12 peak relative retention time is 0.784, No. 13 that relative retention time, which is 0.763, No. 11 peak relative retention time,
Peak relative retention time be 0.7991, No. 14 peak relative retention time be 0.8201, No. 15 peak relative retention time be 0.851,
No. 16 peak relative retention times are that 0.959, No. 17 peak relative retention time is that 0.994, No. 19 peak relative retention time is
1.145, it is 1.237 that No. 20 peak relative retention times, which are 1.229, No. 21 peak relative retention times, and above-mentioned common characteristic peaks are constituted
The fingerprint characteristic of rhizoma dioscoreae nipponicae, standard finger-print as rhizoma dioscoreae nipponicae.
2. a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas according to claim 1, which is characterized in that
In step (1), methanol soaking time is 10min.
3. a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas according to claim 1, which is characterized in that
In step (1), ultrasonic power 250W, frequency 400kHz, ultrasonic time 30min.
4. a kind of method for building up of rhizoma dioscoreae nipponicae efficient liquid-phase chromatograph finger print atlas according to claim 1, which is characterized in that
In step (1), the aperture of the miillpore filter is 45 μm.
5. the rhizoma dioscoreae nipponicae standard finger-print that method described in claim 1 is established answering in the control of rhizoma dioscoreae nipponicae quality of medicinal material
With.
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| CN110261525A (en) * | 2019-06-13 | 2019-09-20 | 广西中医药大学 | The method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print |
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Application publication date: 20190315 |