CN112881576A - Detection method of fingerprint of honeysuckle, mulberry leaf and chrysanthemum beverage - Google Patents
Detection method of fingerprint of honeysuckle, mulberry leaf and chrysanthemum beverage Download PDFInfo
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- CN112881576A CN112881576A CN201911198889.8A CN201911198889A CN112881576A CN 112881576 A CN112881576 A CN 112881576A CN 201911198889 A CN201911198889 A CN 201911198889A CN 112881576 A CN112881576 A CN 112881576A
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Abstract
The invention discloses a method for detecting fingerprint of honeysuckle, mulberry leaf and chrysanthemum drink, which comprises the following steps: step 1, preparing a test solution of the honeysuckle, mulberry and chrysanthemum beverage; step 2, preparation of a mixed reference solution: step 3, respectively and precisely sucking the mixed reference solution and the test solution to be injected into a liquid chromatograph, and recording a chromatogram; step 4, exporting the fingerprint instrument of the florists dendranthema drink obtained in the step 3, importing the fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of florists dendranthema drink as common peaks; generating a control fingerprint of the honeysuckle mulberry chrysanthemum drink by using an average value calculation method; and 5, comparing the fingerprint of the honeysuckle mulberry chrysanthemum drink with the fingerprint of the mixed standard substance, and identifying the main components. The fingerprint of the honeysuckle flower mulberry chrysanthemum drink provided by the invention can comprehensively and objectively characterize the quality of the honeysuckle flower mulberry chrysanthemum drink. The fingerprint detection method provided by the invention has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like.
Description
Technical Field
The invention relates to a detection method of traditional Chinese medicines, in particular to a detection method of fingerprint spectra of honeysuckle, mulberry leaf and chrysanthemum drink.
Background
The honeysuckle, mulberry and chrysanthemum tea is a recuperation tea beverage containing the theory of traditional Chinese medicine, which is developed under the strong support of Nanjing health industry development Limited company, and is summarized by decades of clinical experience of professor Xiongbiao of traditional Chinese medicine in Nanjing. The product mainly comprises honeysuckle, mulberry leaf, chrysanthemum, mint, cassia seed and other components, has the effects of removing acnes and smoothing skin, clearing heat and eliminating dampness, diminishing inflammation and decreasing internal heat, and dispersing wind heat, effectively regulates human metabolic disorder and endocrine dyscrasia, and can remove acnes, pimples, acnes, skin rashes and the like. The traditional Chinese medicine composition is clinically used for people who relieve symptoms such as cold, fever, sore throat, headache, toothache and the like.
At present, the quality detection methods of the honeysuckle, mulberry and chrysanthemum beverage are few. The invention adopts the high performance liquid chromatography to establish the fingerprint detection method of the honeysuckle, mulberry and chrysanthemum beverage, and has important significance for ingredient identification, quality evaluation and quality standard formulation of the honeysuckle, mulberry and chrysanthemum beverage.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides the fingerprint detection method of the honeysuckle, mulberry and chrysanthemum beverage, which can objectively, comprehensively and accurately evaluate the quality of the honeysuckle, mulberry and chrysanthemum beverage and has important significance for controlling the quality of the honeysuckle, mulberry and chrysanthemum beverage and ensuring the clinical curative effect.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
a detection method of fingerprint spectrum of honeysuckle flower mulberry chrysanthemum drink is characterized by comprising the following steps:
precisely weighing different batches of flos Lonicerae flos Mori and flos Chrysanthemi beverage samples, respectively, placing in round bottom bottles, adding methanol solution, reflux extracting, filtering, placing filtrate in volumetric flask, adding methanol to constant volume, and filtering with 0.45 μm microporous membrane to obtain sample solution;
step 2, preparation of mixed reference solution:
precisely weighing chlorogenic acid, rutin, luteolin, cryptochlorogenic acid and aurantio-obtusin reference substance, placing in a volumetric flask, adding ethanol to desired volume, shaking to obtain mixed reference substance solution;
step 3, precisely absorbing the test solution and the reference solution respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;
and 5, comparing the reference fingerprint spectrum of the florida chrysanthemum drink obtained in the step 3 with the mixed reference substance fingerprint spectrum, and identifying that the No. 1 peak in the fingerprint spectrum of the florida chrysanthemum drink is chlorogenic acid, the No. 4 peak is rutin, the No. 5 peak is galuteolin, the No. 7 peak is cryptochlorogenic acid and the No. 17 peak is aurantio-obtusin.
Preferably, in the above method for detecting fingerprint of honeysuckle-mulberry-chrysanthemum beverage, step 1, the method for preparing the sample solution of honeysuckle-mulberry-chrysanthemum beverage comprises: putting 5g of 8 batches of floret mulberry chrysanthemum beverage samples into a 100mL round-bottomed bottle, adding 50mL of methanol solution, carrying out reflux extraction for 2h, filtering, putting the filtrate into a 100mL volumetric flask, adding methanol to a constant volume, and filtering through a 0.45-micrometer microporous filter membrane to obtain a sample solution;
preferably, in the method for detecting fingerprint of honeysuckle, mulberry and chrysanthemum beverage, step 2, the preparation of the mixed reference solution: precisely weighing chlorogenic acid, rutin, galuteolin, cryptochlorogenic acid and aurantio-obtusin reference substance, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up, and preparing into mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0902mg/mL rutin, 0.0628mg/mL galuteolin, 0.0489mg/mL cryptochlorogenic acid and 0.1044mg/mL aurantio-obtusin.
Preferably, in the above method for detecting fingerprint of honeysuckle, mulberry and chrysanthemum beverage, in step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A, mobile phase: acetonitrile and 0.05% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 280nm, column temperature of 30 ℃, flow rate of 1mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
preferably, the method for detecting fingerprint of honeysuckle, mulberry and chrysanthemum drink is characterized in that the fingerprint contains 17 peaks.
The honeysuckle-mulberry-chrysanthemum beverage is prepared from 30 parts of honeysuckle, 35 parts of mulberry leaves, 10 parts of chrysanthemum, 10 parts of mint and 15 parts of cassia seeds.
Optimizing fingerprint detection conditions:
1. in the aspect of preparation optimization of sample solution
According to the invention, through experimental comparison of different extraction methods (such as ultrasonic extraction, reflux extraction and percolation) and different extraction solvents (methanol, water, 70% ethanol aqueous solution, 85% ethanol aqueous solution, 95% ethanol and absolute ethanol), the results show that the difference components of the spectrogram obtained by reflux extraction are relatively comprehensive, the separation degree is good, so that the reflux extraction method is adopted; the investigation of the extraction solvent finds that the chromatogram map of the methanol extract has the most information content and the highest component content; therefore, methanol is selected for extraction.
2. In the aspect of optimizing chromatographic conditions
According to the invention, a diode array detector is adopted to inspect the detection wavelength, chromatograms at positions of 230nm, 254nm, 280nm, 284nm and 300nm are extracted, and when the detection wavelength is 280nm, the information content contained in the chromatograms is most comprehensive and the base line is stable, so that 280nm is selected as the detection wavelength;
the flow rates (1mL/min, 0.8mL/min, 0.7mL/min, 0.6mL/min and 0.5mL/min) are screened, and because isomers and other components with extremely similar polarities exist in the components of the florets/chrysanthemum beverage, the components cannot be separated at a high flow rate, so that the separation effect is better at a low flow rate, and finally, the substances with similar polarities are separated under gradient conditions of a flow rate of 1mL/min for multiple times and the like.
The invention compares the elution effects of 5 different elution systems of methanol-water, acetonitrile-0.1% formic acid, acetonitrile and 0.05% phosphoric acid water, and acetonitrile-0.1% phosphoric acid water under different gradients. As a result, the acetonitrile and the 0.05% phosphoric acid water are finally selected as the mobile phase, because the components in the double-flower mulberry chrysanthemum drink can be well separated by using the acetonitrile and the 0.05% phosphoric acid water as the mobile phase.
0-10 min, 5-5% acetonitrile volume; 5-20% acetonitrile in 10-25 min;
20-30% acetonitrile volume in 25-50 min; 30-50% acetonitrile volume in 50-80 min;
when the gradient elution is carried out for 80-100 min and the volume of acetonitrile is 50-50%, the compounds can achieve the best separation degree, and have the best precision, repeatability, stability and precision when being examined by methodology.
Has the advantages that:
1. according to the structural property characteristics of active ingredients contained in the honeysuckle flower-mulberry-chrysanthemum drink, the optimal mobile phase composition is screened out through a large number of experiments, and analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column, column temperature and the like are verified through a plurality of experiments.
2. The fingerprint of the honeysuckle mulberry chrysanthemum drink established by the method can effectively represent the quality of the honeysuckle mulberry chrysanthemum drink, objectively reflect the front and back sequence and the mutual relation of each fingerprint characteristic peak, pay attention to the overall facial features, avoid the one-sidedness of determining the quality of the honeysuckle mulberry chrysanthemum drink due to the determination of individual chemical components, and reduce the possibility of manual treatment for reaching the quality standard.
3. The method for detecting the fingerprint of the honeysuckle, the mulberry leaf and the chrysanthemum beverage provided by the invention has the advantages of simplicity, convenience, good stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a chromatogram of a mixed control.
FIG. 2 is a comparison fingerprint of the honeysuckle, mulberry leaf and chrysanthemum beverage of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The instruments and reagents used in the examples were as follows:
experimental equipment
1.1 instruments
A high-performance liquid chromatography system with full-wave-band scanning (200- & 800nm) of Shimadzu corporation in Japan comprises a full-automatic online degassing system, a full-automatic sample introduction system promience SIL-20A, a diode array detector SPD-M20A, an automatic temperature control column oven CTO-20A, a KQ3200DB type numerical control ultrasonic cleaner (ultrasonic instruments Co., Ltd., Kunshan city) and a BP121S electronic analysis balance (SARTORIUS).
1.2 drugs and reagents
The honeysuckle flower mulberry chrysanthemum beverage is prepared from Nanjing health industry development Limited company; chlorogenic acid control (batch No. 170422) was purchased from Nanjing Senega Biotech, Inc.; rutin control (batch No. 100080-; the luteolin control (batch No. 720-; cryptochlorogenic acid control (batch 160824) was purchased from Douglas Biotech, Inc.; orange obtusin control (batch No. 111900-201605) was purchased from national institute for food and drug testing; methanol (analytically pure); petroleum ether (analytically pure); diethyl ether (analytical grade); phosphoric acid (analytically pure); acetonitrile (chromatographically pure).
Embodiment 1 a method for detecting fingerprint of honeysuckle, mulberry leaf and chrysanthemum beverage, comprising the following steps:
taking 5g of 8 batches of honeysuckle mulberry chrysanthemum beverage samples (prepared from 30 parts of honeysuckle, 35 parts of mulberry leaf, 10 parts of chrysanthemum, 10 parts of mint and 15 parts of cassia seed) and placing the samples in a 100mL round-bottom bottle, adding 50mL of methanol solution, carrying out reflux extraction for 2h, filtering, placing filtrate in a 100mL volumetric flask, adding methanol to a constant volume, and filtering through a 0.45-micron microporous filter membrane to obtain a test solution;
step 2, preparation of mixed reference solution:
precisely weighing chlorogenic acid, rutin, galuteolin, cryptochlorogenic acid and aurantio-obtusin reference substance, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up, and preparing into mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0902mg/mL rutin, 0.0628mg/mL galuteolin, 0.0489mg/mL cryptochlorogenic acid and 0.1044mg/mL aurantio-obtusin.
Step 3, precisely absorbing the 8 batches of the florists chrysnthum beverage test solution in the step 1 and the reference solution in the step 2 respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram; the liquid chromatography conditions were: a chromatographic column: YMC-Pack ODS-A, mobile phase: acetonitrile and 0.05% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 280nm, column temperature of 30 ℃, flow rate of 1mL/min, sample injection volume: 10 μ L, gradient elution procedure as in Table 1 below:
TABLE 1 gradient elution procedure
Example 2 forensic study of fingerprint detection methods:
1. study of precision
Taking honeysuckle flower-mulberry leaf-chrysanthemum beverage samples (prepared from 30 parts of honeysuckle flower, 35 parts of mulberry leaf, 10 parts of chrysanthemum, 10 parts of mint and 15 parts of cassia seed), wherein the sample number is S1 sample solution prepared by the method in the embodiment 1, analyzing the sample solution according to the detection method in the embodiment 1, carrying out parallel sample injection for 6 times, wherein the sample injection amount is 10 mu L, and analyzing the peak areas and the retention time of common peaks of sample HPLC fingerprint spectra and calculating the RSD value by taking chlorogenic acid, rutin, luteolin and obtusin as reference peaks, wherein the results are shown in Table 2.
TABLE 2 precision study of peak area and retention time
2. Stability study
Taking a honeysuckle-mulberry-chrysanthemum beverage sample (prepared from 30 parts of honeysuckle, 35 parts of mulberry leaf, 10 parts of chrysanthemum, 10 parts of mint and 15 parts of cassia seed), wherein the sample number is S1 sample solution prepared by the method in the embodiment 1, analyzing according to the detection method in the embodiment 1, carrying out sample injection analysis at different time periods of 0 hour, 2 hour, 6 hour, 12 hour, 18 hour and 24 hour, wherein the sample injection amount is 10 mu L, and the results are shown in Table 3 by analyzing the peak area and the retention time of the common peak of the HPLC fingerprint of the sample and calculating the RSD value.
Table 3 stability study peak area and retention time
3. Repetitive studies
Taking honeysuckle flower-mulberry-chrysanthemum beverage samples (30 parts of honeysuckle flower, 35 parts of mulberry leaf, 10 parts of chrysanthemum flower, 10 parts of mint and 15 parts of cassia seed), precisely weighing six parts of S1 sample samples in parallel, wherein the weight of each part of honeysuckle flower-mulberry-chrysanthemum beverage traditional Chinese medicine is 5g, preparing 6 parts of the same sample solution according to the method of 2.3, taking chlorogenic acid, rutin, luteoloside, cryptochlorogenic acid and aurantio-obtusin as reference peaks according to the chromatographic conditions of example 1, analyzing the peak areas and retention time of common peaks of sample HPLC fingerprint and calculating RSD value, and the result is shown in Table 4.
TABLE 4 repeated study of peak area and retention time
The experimental results show that the fingerprint spectrum detection method for the honeysuckle flower mulberry chrysanthemum drink provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the honeysuckle flower mulberry chrysanthemum drink, and has important significance for ensuring the clinical curative effect.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (6)
1. A detection method of fingerprint spectrum of honeysuckle flower mulberry chrysanthemum drink is characterized by comprising the following steps:
step 1, preparing a test solution of the double-flower mulberry chrysanthemum beverage:
precisely weighing different batches of flos Lonicerae flos Mori and flos Chrysanthemi beverage samples, respectively, placing in round bottom bottles, adding methanol solution, reflux extracting, filtering, placing filtrate in volumetric flask, adding methanol to constant volume, and filtering with 0.45 μm microporous membrane to obtain sample solution;
step 2, preparation of mixed reference solution:
precisely weighing chlorogenic acid, rutin, luteolin, cryptochlorogenic acid and aurantio-obtusin reference substance, placing in a volumetric flask, adding ethanol to desired volume, shaking to obtain mixed reference substance solution;
step 3, precisely absorbing the test solution and the reference solution respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;
step 4, exporting the fingerprint of the honeysuckle, mulberry and chrysanthemum beverage test solution obtained in the step 3, and introducing the fingerprint into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of florets and mulberry chrysanthemum drinks as common peaks; generating a control fingerprint of the honeysuckle, the mulberry and the chrysanthemum drink by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram;
and 5, comparing the reference fingerprint spectrum of the florida chrysanthemum drink obtained in the step 3 with the mixed reference substance fingerprint spectrum, and identifying that the No. 1 peak in the fingerprint spectrum of the florida chrysanthemum drink is chlorogenic acid, the No. 4 peak is rutin, the No. 5 peak is galuteolin, the No. 7 peak is cryptochlorogenic acid and the No. 17 peak is aurantio-obtusin.
2. The method for detecting the fingerprint of the honeysuckle-mulberry-chrysanthemum beverage according to claim 1, wherein the preparation method of the honeysuckle-mulberry-chrysanthemum beverage test solution in step 1 comprises the following steps: taking 5g of 8 batches of floret mulberry chrysanthemum beverage samples, placing the samples into a 100mL conical flask, placing the conical flask into a round bottom flask, respectively adding 50mL of methanol solution, refluxing and extracting, filtering, placing the filtrate into a 100mL volumetric flask, adding methanol to a constant volume, and filtering through a 0.45 mu m microporous filter membrane to obtain a sample solution.
3. The method for detecting fingerprint of honeysuckle, mulberry leaf and chrysanthemum beverage according to claim 1, wherein the step 2 of preparing the mixed reference solution comprises the following steps: precisely weighing chlorogenic acid, rutin, galuteolin, cryptochlorogenic acid and aurantio-obtusin as reference substances, placing the reference substances into a volumetric flask, fixing the volume to scale by using ethanol, shaking up, and preparing a mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0902mg/mL rutin, 0.0628mg/mL galuteolin, 0.0489mg/mL cryptochlorogenic acid and 0.1044mg/mL aurantio-obtusin.
4. The method for detecting the fingerprint of the honeysuckle, the mulberry and the chrysanthemum beverage according to claim 1, wherein in the step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A, mobile phase: acetonitrile and 0.05% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 280nm, column temperature of 30 ℃, flow rate of 1mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
5. the method for detecting the fingerprint of the honeysuckle mulberry chrysanthemum drink according to claim 1, wherein the fingerprint contains 17 peaks.
6. The method for detecting the fingerprint of the honeysuckle-mulberry-chrysanthemum drink according to any one of claims 1 to 5, wherein the honeysuckle-mulberry-chrysanthemum drink is prepared from 30 parts of honeysuckle, 35 parts of mulberry leaves, 10 parts of chrysanthemum, 10 parts of mint and 15 parts of cassia seeds.
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Application publication date: 20210601 |