CN113866334A - Method for establishing fingerprint of pinellia ternate heart-fire purging decoction - Google Patents
Method for establishing fingerprint of pinellia ternate heart-fire purging decoction Download PDFInfo
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- CN113866334A CN113866334A CN202111403622.5A CN202111403622A CN113866334A CN 113866334 A CN113866334 A CN 113866334A CN 202111403622 A CN202111403622 A CN 202111403622A CN 113866334 A CN113866334 A CN 113866334A
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- mobile phase
- heart
- decoction
- pinellia ternate
- fire
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Library & Information Science (AREA)
- Engineering & Computer Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of medicine detection, and particularly discloses a method for establishing a fingerprint of pinellia ternate heart-fire purging decoction, which adopts an HPLC method to carry out detection according to the following conditions: a chromatographic column: octadecyl bonded silica gel chromatographic column; a DAD detector with a detection wavelength of 268-272 nm; mobile phase A: methanol, mobile phase B is 0.04-0.06% triethylamine buffer solution, gradient elution. The fingerprint of the pinellia ternate heart-fire purging decoction is obtained through one-time determination and construction, the chemical information provided by the fingerprint established by the method is rich, 29 common characteristic peaks are detected, 8 known peaks belong to, the quality of the pinellia ternate heart-fire purging decoction can be effectively represented, the quality of the medicinal materials can be comprehensively monitored, and a guarantee is provided for the production and clinical application of the pinellia ternate heart-fire purging decoction; the method has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like, and has good practicability.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for establishing a fingerprint of pinellia ternate heart-fire purging decoction.
Background
The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which can mark chemical characteristics of certain traditional Chinese medicinal materials or traditional Chinese medicine preparations by adopting a certain analysis means after the traditional Chinese medicinal materials or the traditional Chinese medicine preparations are properly processed. It can comprehensively reflect the types and the quantity of chemical components contained in the medicinal materials, effectively reflect the integrity and the comprehensive action of the Chinese medicinal components, and has been widely applied to the aspects of Chinese medicinal analysis and identification, quality control and the like due to the characteristics of rapidness, accuracy and the like.
The pinellia ternate heart-fire purging decoction is an ancient classic and famous prescription from Shang Han miscellaneous diseases treatise of Sheng Zhang Zhongjing in medical science, is also one of representative prescriptions of Wumen medical pies, and consists of pinellia ternate, dried ginger, scutellaria baicalensis, coptis chinensis, codonopsis pilosula, honey-fried licorice root and Chinese date. As one of the outstanding representatives of the traditional Chinese medicine formulas reserved in the traditional Chinese medicine inheritance and development process, the traditional Chinese medicine formula has wide application in modern clinic, and is mainly used for treating diseases such as reflux esophagitis, various gastritis, gastroptosis, gastric mucosa prolapse, digestive tract tumor, enteritis, ulcer, irritable bowel syndrome and the like. At present, the Chinese pharmacopoeia does not contain the prescription, and although there are research reports that part of components in the pinellia ternate heart-fire clearing decoction are subjected to chemical identification, the pinellia ternate heart-fire clearing decoction has more medicinal taste and complex components, and the qualitative and quantitative analysis of individual components still cannot completely reflect the comprehensive information of the pinellia ternate heart-fire clearing decoction. In addition, due to the differences in geographical climate conditions, harvesting time, pretreatment modes and the like, the chemical component contents of the same traditional Chinese medicine in different production areas are greatly different. Therefore, establishing a fingerprint of the pinellia ternate decoction for purging fire has great significance for controlling the quality of the pinellia ternate decoction for purging fire.
Disclosure of Invention
Aiming at the problems, the invention provides a method for establishing a fingerprint of pinellia ternate heart-fire clearing decoction.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for establishing fingerprint of pinellia ternate heart-fire purging decoction adopts an HPLC method, and comprises the following steps:
(1) preparing a test solution and a mixed reference solution:
extracting raw materials of the pinellia ternate heart-fire purging decoction to prepare a test solution;
taking baicalin, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, jateorhizine hydrochloride, hanbaikal skullcap root glycoside, baicalein, hanbaikal, 6-gingerol and ginsenoside Rb1Ginsenoside Re and ginsenoside Rg1Preparing a mixed reference substance solution by using a solvent according to the liquiritin and ammonium glycyrrhizinate reference substances;
(2) detecting the mixed reference substance solution and the test substance solution, wherein the chromatographic conditions are as follows:
a chromatographic column: octadecyl bonded silica gel chromatographic column;
a detector: a DAD detector;
detection wavelength: 268nm to 272 nm;
mobile phase A: methanol, mobile phase B: 0.04 to 0.06 percent of triethylamine buffer solution;
the elution mode is gradient elution, and the elution procedure of the gradient elution is as follows:
0-8 min, 3% of mobile phase A and 97% of mobile phase B;
8-15 min, 3% → 10% mobile phase A, 97% → 90% mobile phase B;
15-28 min, 10% → 23% mobile phase A, 90% → 77% mobile phase B;
28-53 min, 23% of mobile phase A and 77% of mobile phase B;
53-73 min, 23% → 29% mobile phase A, 77% → 71% mobile phase B;
73-80 min, 29% → 38% mobile phase A, 71% → 62% mobile phase B;
80-85 min, 38% of mobile phase A and 62% of mobile phase B;
85-107 min, 38% → 53% mobile phase A, 62% → 47% mobile phase B;
107-120 min, 53% → 71% mobile phase A, 47% → 29% mobile phase B;
120-130 min, 71% of mobile phase A and 29% of mobile phase B.
Compared with the prior art, the method for establishing the fingerprint of the pinellia ternate heart-fire purging decoction provided by the invention adopts an HPLC method, adopts a gradient elution mode under a specific mobile phase system, and utilizes a DAD detector to realize the purpose of simultaneously measuring various index components in the semi-summer heart-fire purging decoction. The fingerprint spectrum established by the method is rich in chemical information, 29 common characteristic peaks are detected, 8 known peaks belong to the fingerprint spectrum, the quality of the pinellia ternate heart-fire clearing decoction medicinal material can be effectively represented, the HPLC characteristic spectrum of the pinellia ternate heart-fire clearing decoction to be detected is compared with the standard fingerprint spectrum established by the invention, the quality of the medicinal material can be comprehensively monitored, and the guarantee is provided for the production and clinical application of the pinellia ternate heart-fire clearing decoction preparation; the method has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like, and has good practicability.
The raw materials for the pinellia ternate heart-fire purging decoction comprise pinellia ternate, rhizoma zingiberis, scutellaria baicalensis, coptis chinensis, codonopsis pilosula, honey-fried licorice root and Chinese date.
Optionally, the solvent used in the preparation of the mixed control solution is methanol.
Preferably, the pH of the triethylamine buffer solution is 2.1-2.3, the flow rate is 0.9-1.1 mL/min, and the column temperature is 28-32 ℃.
Further preferably, the pH of the triethylamine buffer solution is 2.2, the flow rate is 1.0mL/min, and the column temperature is 30 ℃.
Preferably, the injection volume is 5. mu.L.
The conditions of pH value, flow rate, column temperature and the like of the preferred mobile phase can reduce band tailing and improve peak shape, thereby being beneficial to improving the separation degree of each component.
Preferably, the detection wavelength is 270 nm.
The optimal detection wavelength can avoid the interference of a large amount of impurities in the traditional Chinese medicine extract on the detection as much as possible, and the detection baseline is stable, the number and the shape of the peaks are good, and the detection sensitivity is improved.
Preferably, the column has a size of 250mm 4.6mm and a packing diameter of 5 μm.
Further preferably, the chromatographic column is Thermo Hypersil GLOD-C18.
The optimal specification and model of the chromatographic column can ensure that the peak shape, the separation degree and the detection sensitivity of each component are good, and the baseline interference is small.
Preferably, the method for preparing the test solution comprises the following steps:
step a, weighing the raw materials according to the prescription amount, adding water, boiling with strong fire, decocting with slow fire for 30-35 min, filtering, and continuously decocting the obtained filtrate with slow fire for 50-55 min to obtain pinellia ternate heart-fire-purging decoction;
step b, carrying out vacuum freeze drying on the pinellia ternate heart-fire clearing decoction to obtain a pinellia ternate heart-fire clearing decoction reference substance;
and c, taking the pinellia ternate heart-fire purging reference substance, adding water to dissolve, centrifuging and filtering to obtain a test solution.
The pinellia ternate heart-fire clearing decoction is prepared from 7 medicinal materials, a water extract contains a large amount of impurity components such as saccharides, amino acids and the like, and the preparation method can reduce the interference of impurities and ensure that the peak shape of a chromatographic peak, the response value of the chromatographic peak and the separation degree of each detected component are good.
Preferably, in the step a, the addition amount of the water is 7-9 times of the total amount of the raw materials.
Exemplarily, in the step a, the decoction is decocted by slow fire until the liquid medicine is 1200mL, and after filtration, the obtained filtrate is continuously decocted by slow fire until the liquid medicine is 600 mL.
Preferably, in step b, the vacuum freeze-drying comprises the following specific steps: the pinellia ternate heart-fire clearing decoction is pre-frozen for 20 to 25 hours at the temperature of between 15 ℃ below zero and 25 ℃ below zero, and then is subjected to vacuum freeze drying for 70 to 75 hours at the temperature of between 75 ℃ below zero and 85 ℃ below zero.
Illustratively, the specific steps of step b are: and subpackaging the pinellia ternate heart-fire clearing decoction into 20mL penicillin bottles, subpackaging 5mL each penicillin bottle, pre-freezing the penicillin bottles at-15 to-25 ℃ for 20 to 25 hours, then carrying out vacuum freeze drying at-75 to-85 ℃ for 70 to 75 hours, taking out, sealing and capping to obtain the pinellia ternate heart-fire clearing decoction reference substance.
Illustratively, the specific steps of step c are: precisely transferring 5mL of water into a penicillin bottle, sealing, ultrasonically dissolving, transferring 4mL of water into a 10mL centrifuge tube, centrifuging, collecting the supernatant, filtering, and collecting the subsequent filtrate to obtain the test solution.
Preferably, the concentration of the test solution is 0.451 g/mL.
The concentration of the sample solution refers to the amount of crude drug contained in a unit volume.
The preferable concentration of the test solution can make the chromatographic peak area of each component in the test solution closer to the corresponding peak area obtained by the reference solution, thereby making the comparison result more accurate.
Preferably, the mixed reference solution contains baicalin, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, jateorhizine hydrochloride, baicalin, baicalein, hancein, 6-gingerol, and ginsenoside Rb1Ginsenoside Re and ginsenoside Rg1The concentrations of glycyrrhizin and ammonium glycyrrhizinate were 0.1 mg/mL respectively-1。
The concentration of the reference solution is preferably selected to facilitate the peak shape of each component in the reference solution, thereby facilitating peak assignment confirmation.
The modern method has great significance in developing and researching Chinese patent medicines, and provides an objective and integral evaluation basis for the quality of the Chinese patent medicines. The invention adopts HPLC method to carry out fingerprint construction on the semi-xiaxia xiexin decoction, is beneficial to comprehensively monitoring the quality of the pinellia ternate xiexin decoction, effectively ensures the stability, consistency and controllability of the quality of the pinellia ternate xiexin decoction, and provides reference for development, quality control and clinical application of the semi-xiaexin decoction in future.
Drawings
FIG. 1 is a chromatogram at different detection wavelengths of 2.6.2 in example 1;
FIG. 2 is a chromatogram of example 1 under a different gradient elution sequence at 2.6.3;
FIG. 3 is a chromatogram of 20 batches of the sample solution of ban Xia Xiexin decoction in example 2;
FIG. 4 is the HPLC characteristic spectrum of 20 batches of ban Xia Xie Xin Tang in example 2, which is automatically matched by the mean value method to generate the standard fingerprint spectrum of ban Xia Xie Xin Tang;
FIG. 5 is a chromatogram of the precision test item in example 3;
FIG. 6 is a chromatogram of the reproducibility test item in example 3;
FIG. 7 is a chromatogram of the stability test item of example 3;
FIG. 8 is a chromatogram of the test solutions of the decoction pieces of each single herb and the decoction of ban Xia Xie Xin.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1. Reagent
1.1 crude drug decoction pieces
Pinellia tuber decoction pieces (batch No. Y-241-181201-53, Su Xihe county of Gansu province); scutellaria decoction pieces (lot 2018121303, produced in shanxi Cheng county); rhizoma Coptidis decoction pieces (batch No. 2018121311, Sichuan Chengdu); decoction pieces of dried ginger (lot No. 2018121306, Yunnan wenshan of origin); ginseng decoction pieces (lot No. 2018121316, black dragon river, iron force of origin); honey-fried licorice root decoction pieces (lot No. 2018121308, inner Mongolia civil Texiu Right flag of producing area); fructus Jujubae decoction pieces (batch No. 2018121319, Hebei Cangzhou, origin).
1.2 reference substance
Baicalin (batch No. 110715-201821, purity 95.4%), wogonin (batch No. 112002-201702, purity 98.5%), berberine hydrochloride (batch No. 110713-201814, purity 86.7%), palmatine hydrochloride (batch No. 110732-201611, purity 86.8%), coptisine hydrochloride (batch No. 112026-201802, purity 94.0%), jatrorrhizine hydrochloride (batch No. 110733-201609, purity 89.5%), baicalein (batch No. 111595-201808, purity 97.9%), wogonin (batch No. 111514-201706), 6-gingerol (batch No. 111833-201705, purity 96.8%), ginsenoside1(batch No. 110703-201832, purity 92.4%), ginsenoside Re (batch No. 110754-201827, purity 93.4%), ginsengSaponin Rb1(batch No. 110704-201827 with a purity of 91.2%), liquiritin (batch No. 111610-201607 with a purity of 93.1%), ammonium glycyrrhizinate (batch No. 110731-201720 with a purity of 97.7%), all of which are purchased from China institute for testing and testing food and drug.
2. Preparation of the solution
2.1 preparation of test solutions
S1, weighing 55.2g of rhizoma pinelliae preparata, 41.4g of radix scutellariae, 13.8g of coptis chinensis, 41.4g of dried ginger, 36g of Chinese date, 41.4g of liquorice and 41.4g of ginseng in a ceramic pot, adding 2000mL of water, boiling with strong fire (1800W), continuously decocting with slow fire (800W) for about 31min until the liquid medicine is 1200mL while keeping the micro-boiling state, filtering with a layer of 300-mesh nylon cloth while hot, and decocting the filtrate with slow fire (800W) for about 54min until the liquid medicine is 600mL to obtain a rhizoma pinelliae heart-purging decoction;
s2, subpackaging the pinellia ternate heart-fire clearing decoction into 20mL penicillin bottles, subpackaging 5mL each penicillin bottle, placing the penicillin bottles into a refrigerator at the temperature of-20 ℃ for pre-freezing for 24h, controlling a vacuum freeze dryer to run at the temperature of-80 ℃ for 30min, placing the penicillin bottles into the vacuum freeze dryer, drying for 72h, taking out, sealing and capping to obtain a pinellia ternate heart-fire clearing decoction reference substance;
s3, precisely adding 5mL of water into a penicillin bottle, sealing, dissolving by ultrasonic wave (250W, 40KHz) for 15min, taking out, cooling, shaking up, taking 4mL into a 10mL centrifuge tube, centrifuging at 1000r/min for 10min, taking supernatant, filtering through a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain the test solution.
2.2 preparation of Mixed control solutions
Taking baicalin, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, jateorhizine hydrochloride, hanbaikal skullcap root glycoside, baicalein, hanbaikal, 6-gingerol and ginsenoside Rb1Ginsenoside Re and ginsenoside Rg1The liquiritin and ammonium glycyrrhizinate reference substances are precisely weighed and dissolved by adding methanol to respectively prepare the reference substances with the concentration of 0.1 mg/mL-1Mixed control solution of (4).
2.3 preparation of Single-ingredient decoction piece test solution
Taking pinellia ternate, dried ginger, scutellaria baicalensis, coptis chinensis, codonopsis pilosula, honey-fried licorice root or Chinese date in a single decoction piece according to the prescription amount, and preparing a corresponding single decoction piece test solution according to the preparation method of the test solution.
2.4 preparation of pinellia ternate heart-fire-purging decoction-yin control test solution:
preparing negative samples without pinellia ternate, rhizoma zingiberis, scutellaria baicalensis, coptis chinensis, codonopsis pilosula, honey-fried licorice root or Chinese date medicinal decoction pieces according to the formula of the pinellia ternate heart-fire clearing decoction, and preparing corresponding negative sample solutions according to the preparation method of the test solution.
2.5 High Performance Liquid Chromatography (HPLC)) conditions:
a chromatographic column: thermo Hypersil GLOD C18(4.6 mm. times.250 mm, 5 μm);
mobile phase: methanol (a) -0.05% triethylamine buffer solution (ph2.2 adjusted with phosphoric acid) (B);
flow rate: 1.0mL/min-1；
Column temperature: 30 ℃;
sample introduction amount: 5 mu L of the solution;
detection wavelength: 270nm, gradient elution conditions are shown in Table 1.
TABLE 1 gradient elution conditions
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 3 | 97 |
| 8 | 3 | 97 |
| 15 | 10 | 90 |
| 28 | 23 | 77 |
| 53 | 23 | 77 |
| 73 | 29 | 71 |
| 80 | 38 | 62 |
| 85 | 38 | 62 |
| 107 | 53 | 47 |
| 120 | 71 | 29 |
| 130 | 71 | 29 |
And (3) determination: precisely sucking 5 μ L of each of the sample solution and the mixed reference solution, injecting into high performance liquid chromatograph, measuring by the high performance liquid chromatography, and recording chromatogram.
Note: the glycyrrhizic acid reference substance is ammonium glycyrrhizinate corresponding to glycyrrhizic acid in the test solution.
2.6 optimization of chromatographic conditions
2.6.1 Mobile phase species investigation
The effect of separating methanol (a) -water (B), acetonitrile (a) -water (B), methanol-0.1% phosphoric acid water (ph2.20), acetonitrile-0.1% phosphoric acid water (ph2.20), methanol-0.05% phosphoric acid water (ph2.40), acetonitrile-0.05% phosphoric acid water (ph2.40), methanol-0.5% formic acid water (ph2.35), acetonitrile-0.5% formic acid water (ph2.35), methanol-0.1% formic acid water (ph2.69), acetonitrile-0.1% formic acid water (ph2.69), and methanol-0.05% triethylamine buffer solution was examined for each of the whole formula using different mobile phases.
Chromatographic conditions are as follows: chromatographic column Thermo Hypersil GLOD C18(4.6 mm. times.250 mm, 5 μm); detection wavelength: 270 nm; flow rate: 1.0 mL/min; the sample injection amount is 10 mu L; the column temperature was 30 ℃ and the gradient is shown in Table 1.
Injecting 5 μ L of sample solution into high performance liquid chromatograph, measuring by the high performance liquid chromatography, recording chromatogram, and comparing and analyzing to find that the mobile phase can not reach the peak number, peak shape and resolution under the condition of methanol-0.05% triethylamine buffer solution (pH 2.2). Therefore, methanol-0.05% triethylamine buffer solution was chosen as the mobile phase.
2.6.2 selection of detection wavelength
Through comparative analysis of 260nm, 270nm, 280nm and 3 detection wavelengths, the 270nm detection wavelength can comprehensively reflect chemical components in the pinellia ternate heart-fire clearing decoction, and each spectral peak is well separated, and the base line is stable, so 270nm is selected as the fingerprint spectrum measurement wavelength of the pinellia ternate heart-fire clearing decoction. The other chromatographic conditions were the same as in the case of 2.5 High Performance Liquid Chromatography (HPLC)). The chromatogram at each wavelength is shown in FIG. 1.
2.6.3 determination of gradient condition
Chromatographic conditions are as follows: column Thermo Hypersil GLOD C18(4.6 mm. times.250 mm, 5 μm); the detection wavelength is 270 nm; the flow rate is 1 mL/min; the sample injection amount is 5 mu L; the column temperature was 30 ℃.
Elution was performed with different gradients as in tables 2-4, and the results are shown in FIG. 2.
TABLE 2 method 1 mobile phase elution gradient
TABLE 3 method 2 mobile phase elution gradient
| Time/min | Methanol/% of | 0.05% triethylamine buffer (pH2.2 adjusted with phosphoric acid)/%) |
| 0 | 3 | 97 |
| 8 | 3 | 97 |
| 20 | 23 | 77 |
| 45 | 23 | 77 |
| 55 | 29 | 71 |
| 65 | 38 | 62 |
| 80 | 38 | 62 |
| 120 | 71 | 29 |
| 130 | 71 | 29 |
TABLE 4 method 3 mobile phase elution gradient
The chromatograms under different gradient elution conditions are analyzed and compared, and it can be seen from the graphs that the separation degree between the peaks is obviously superior to that of the gradients of the methods 1 to 3 according to the number and the form of the peaks under the preferable gradient elution condition of the invention. Therefore, the conditions for fingerprint construction of pinellia ternate heart-fire clearing decoction are selected from the gradient elution sequence in the table 1.
Example 2
The embodiment provides an HPLC fingerprint of pinellia ternate heart-fire clearing decoction, which comprises the following specific steps:
the pinellia ternate heart-fire purging decoction fingerprint prepared in example 1 is adoptedConstruction method of chromatogram HPLC fingerprint chromatogram of 20 batches of pinellia ternate decoction for purging stomach fire is established. And respectively preparing baicalin, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, jateorhizine hydrochloride, baicalin, baicalein, hanbaicalein, 6-gingerol, and ginsenoside Rb1Ginsenoside Re and ginsenoside Rg1And liquiritin and ammonium glycyrrhizinate, detecting according to the HPLC conditions, and attributing the peak in the test solution as a positioning peak by using the obtained chromatogram.
HPLC finger print of 20 batches of pinellia ternate heart-fire clearing decoction is shown in figure 3, and finger print of 20 batches of samples is synthesized by using a Chinese medicine chromatography finger print similarity evaluation system (2012 edition) of the national pharmacopoeia committee to generate the HPLC standard finger print of the pinellia ternate heart-fire clearing decoction consisting of 29 common characteristic peaks, which is shown in figure 4. Wherein, the No. 4 peak is coptisine, the No. 6 peak is jateorhizine, the No. 9 peak is berberine, the No. 10 peak is palmatine, the No. 14 peak is baicalin, the No. 19 peak is hancein, the No. 22 peak is baicalein, and the No. 26 peak is glycyrrhizic acid.
Wherein, the similarity data of 20 samples to the comparison fingerprint is shown in Table 5.
TABLE 520 comparison of fingerprint similarity of test solutions in batches
As can be seen from the above table, the similarity of the fingerprints of 20 batches of the pinellia ternate heart-fire purging decoction is 0.960-0.996, and the similarity is more than 0.900, which indicates that the quality of each batch of samples has better consistency.
Example 3
And (3) verification of methodology:
precision, repeatability and stability test
Precision: taking the same sample solution prepared under item 2.1, determining according to the conditions of high performance liquid chromatography in example 1, continuously injecting samples for 6 times, recording a chromatogram, introducing the chromatogram into a 2012 version of evaluation system for similarity of traditional Chinese medicine chromatogram fingerprint, removing the baicalin peak, performing full spectrum peak matching in a mode of a median method and a time width of 0.1, calculating the similarity, wherein the result is shown in table 6, and the chromatogram is shown in fig. 5. The result shows that the similarity results are all larger than 0.990, and the precision of the instrument is good.
TABLE 6 precision test of fingerprint of ban Xia Xie Xin Tang (n ═ 6)
Repeatability: taking the same batch of pinellia ternate heart-fire clearing decoction sample solution, preparing 6 parts of sample solution in parallel according to the preparation method of the sample solution under item 2.1, determining according to the conditions of the high performance liquid chromatography in example 1, recording a chromatogram, introducing the chromatogram into a 2012 version of a traditional Chinese medicine chromatography fingerprint similarity evaluation system, removing a baicalin peak, performing full spectrum peak matching in a mode of a median method and a time width of 0.1, and calculating the similarity, wherein the result is shown in table 7, and the chromatogram is shown in fig. 6. The similarity results are all larger than 0.999, which indicates that the repeatability of the sample is good.
TABLE 7 repeatability test results of fingerprint of ban Xia Xiexin Tang (n ═ 6)
| S1 | S2 | S3 | S4 | S5 | S6 | Control map | |
| S1 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 |
| S2 | 1.000 | 1.000 | 0.999 | 1.000 | 0.999 | 1.000 | 1.000 |
| S3 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S4 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Control map | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
Stability: taking the same sample solution prepared under item 2.1, detecting at 0h, 3h, 6h, 9h, 12h and 24h respectively according to the chromatographic conditions in example 1, injecting 5 microliter of sample each time, recording a chromatogram, introducing the chromatogram into 2012 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system', removing a baicalin peak, performing full spectrum peak matching in a mode of a median method and a time width of 0.1, calculating the similarity, wherein the result is shown in table 8, and the chromatogram is shown in fig. 7. The similarity results were all greater than 0.940, indicating that the samples remained stable over 24 h.
TABLE 8 stability test results of fingerprint chromatogram of ban Xia Xiexin Tang
| S1 | S2 | S3 | S4 | S5 | S6 | Control map | |
| S1 | 1.000 | 1.000 | 0.994 | 0.994 | 0.995 | 0.921 | 0.996 |
| S2 | 1.000 | 1.000 | 0.995 | 0.994 | 0.996 | 0.920 | 0.996 |
| S3 | 0.994 | 0.995 | 1.000 | 1.000 | 0.999 | 0.922 | 0.997 |
| S4 | 0.994 | 0.994 | 1.000 | 1.000 | 0.999 | 0.921 | 0.997 |
| S5 | 0.995 | 0.996 | 0.999 | 0.999 | 1.000 | 0.925 | 0.998 |
| S6 | 0.921 | 0.920 | 0.922 | 0.921 | 0.925 | 1.000 | 0.946 |
| Control map | 0.996 | 0.996 | 0.997 | 0.997 | 0.998 | 0.946 | 1.000 |
The various methodological verifications prove the reliability of the fingerprint construction method of the pinellia ternate heart-fire purging decoction provided by the invention.
Example 4
This example provides the analysis of the fingerprint spectrum of ban Xia Xiexin Tang from component sources:
the sample solution of pinellia ternate decoction for purging stomach fire, the water decoction of pinellia ternate decoction pieces, the water decoction of dried ginger decoction pieces, the water decoction of scutellaria baicalensis decoction pieces, the water decoction of coptis chinensis decoction pieces, the water decoction of codonopsis pilosula decoction pieces, the water decoction of honey-fried licorice root decoction pieces and the water decoction of Chinese date decoction pieces are detected according to the chromatographic conditions in the embodiment 1, the possible sources of common peaks in the fingerprint are assigned, the results are shown in table 9, and the chromatogram of each decoction piece is shown in fig. 8.
TABLE 9 attribution of the sources of the characteristic peaks
In conclusion, the method for constructing the fingerprint of pinellia ternate heart-fire clearing decoction provided by the invention is simple to operate, the corresponding chromatographic peaks of characteristic components are completely retained, the solution stability is good, the method is high in precision and good in reproducibility, the specificity is good, and the separation degree between the chromatographic peaks in the obtained fingerprint is good. The standard fingerprint spectrum of the pinellia ternate heart-fire clearing decoction provided by the invention can effectively reflect the quality of the pinellia ternate heart-fire clearing decoction, and is favorable for comprehensively monitoring the product quality.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The method for establishing the fingerprint of the pinellia ternate heart-fire purging decoction is characterized by adopting an HPLC method and comprising the following steps of:
(1) preparing a test solution and a mixed reference solution:
extracting raw materials of the pinellia ternate heart-fire purging decoction to prepare a test solution;
taking baicalin, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, jateorhizine hydrochloride, hanbaikal skullcap root glycoside, baicalein, hanbaikal, 6-gingerol and ginsenoside Rb1Ginsenoside Re and ginsenoside Rg1Preparing a mixed reference substance solution by using a solvent according to the liquiritin and ammonium glycyrrhizinate reference substances;
(2) detecting the mixed reference substance solution and the test substance solution, wherein the chromatographic conditions are as follows:
a chromatographic column: octadecyl bonded silica gel chromatographic column;
a detector: a DAD detector;
detection wavelength: 268nm to 272 nm;
mobile phase A: methanol, mobile phase B: 0.04 to 0.06 percent of triethylamine buffer solution;
the elution mode is gradient elution, and the elution procedure of the gradient elution is as follows:
0-8 min, 3% of mobile phase A and 97% of mobile phase B;
8-15 min, 3% → 10% mobile phase A, 97% → 90% mobile phase B;
15-28 min, 10% → 23% mobile phase A, 90% → 77% mobile phase B;
28-53 min, 23% of mobile phase A and 77% of mobile phase B;
53-73 min, 23% → 29% mobile phase A, 77% → 71% mobile phase B;
73-80 min, 29% → 38% mobile phase A, 71% → 62% mobile phase B;
80-85 min, 38% of mobile phase A and 62% of mobile phase B;
85-107 min, 38% → 53% mobile phase A, 62% → 47% mobile phase B;
107-120 min, 53% → 71% mobile phase A, 47% → 29% mobile phase B;
120-130 min, 71% of mobile phase A and 29% of mobile phase B.
2. The method for establishing the fingerprint of pinellia ternate heart-fire purging decoction as claimed in claim 1, wherein the pH of the triethylamine buffer solution is 2.1-2.3, the flow rate is 0.9-1.1 mL/min, and the column temperature is 28-32 ℃.
3. The method for establishing fingerprint of ban Xia Xie Xin Tang according to claim 1 or 2, wherein the pH of the triethylamine buffer solution is 2.2, the flow rate is 1.0mL/min, and the column temperature is 30 ℃.
4. The method for establishing fingerprint of ban Xia Xie Xin Tang in claim 1, wherein the injection volume is 5 μ L.
5. The method for establishing fingerprint of ban Xia Xie Xin Tang in claim 1, wherein the detection wavelength is 270 nm.
6. The method for establishing fingerprint of ban Xia Xie Xin Tang in claim 1, wherein the size of the chromatographic column is 250mm x 4.6mm and the diameter of the packing is 5 μm.
7. The method for establishing fingerprint of pinellia ternate heart-fire removing decoction according to claim 6, wherein the chromatographic column is Thermo Hypersil GLOD-C18.
8. The method for establishing fingerprint of ban Xia Xie Xin Tang in claim 1, wherein the preparation method of the test solution comprises the following steps:
step a, weighing the raw materials according to the prescription amount, adding water, boiling with strong fire, decocting with slow fire for 30-35 min, filtering, and continuously decocting the obtained filtrate with slow fire for 50-55 min to obtain pinellia ternate heart-fire-purging decoction;
step b, carrying out vacuum freeze drying on the pinellia ternate heart-fire clearing decoction to obtain a pinellia ternate heart-fire clearing decoction reference substance;
and c, taking the pinellia ternate heart-fire purging reference substance, adding water to dissolve, centrifuging and filtering to obtain a test solution.
9. The method for establishing fingerprint of pinellia ternate heart-fire clearing decoction according to claim 8, wherein in the step a, the addition amount of water is 7-9 times of the total amount of the raw materials.
10. The method for establishing fingerprint of ban Xia Xie Xin Tang of claim 8, wherein in step b, the vacuum freeze-drying comprises the following steps: the pinellia ternate heart-fire clearing decoction is pre-frozen for 20 to 25 hours at the temperature of between 15 ℃ below zero and 25 ℃ below zero, and then is subjected to vacuum freeze drying for 70 to 75 hours at the temperature of between 75 ℃ below zero and 85 ℃ below zero.
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