CN110850019A - Method for detecting fingerprint of Minggu Shuqing drink - Google Patents
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Abstract
The invention discloses a method for detecting a fingerprint of Minggu Shuqing drink, which comprises the following steps: step 1, preparing a Minggu Shuqing beverage test solution; 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 Minggu Shuqing drink obtained in the step 3, importing the Minggu Shuqing drink into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of Minggu Shuqing drinks as common peaks; generating a contrast fingerprint of the Minggu Shuqing drink by using an average value calculation method; and 5, comparing the fingerprint of the Minggu Shuqing drink obtained in the step 3 with the spectrum of the mixed standard substance to identify the main components. The fingerprint of the Minggu Shuqing drink provided by the invention can comprehensively and objectively represent the quality of the Minggu Shuqing drink; and has the advantages of simple and stable method, 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 a fingerprint of Minggu Shuqing drink.
Background
Minggu Shuqing is a recuperation tea drink which is developed under the strong support of Nanjing Chiense Tang and is built by the theory of traditional Chinese medicine, and is summarized by decades of clinical experience of professor of Xiyingbusao of traditional Chinese medicine in Nanjing. The product mainly comprises the components of hawthorn, cassia seed, kudzu root, chrysanthemum, lotus leaf, raw Pu' er tea, medlar and the like, has the effects of reducing blood pressure, improving eyesight, removing dampness and reducing blood fat, is beneficial to conditioning the body of a hypertensive, is suitable for conditioning the blood pressure, removing dampness and reducing blood fat, can effectively protect heart and cerebral vessels, improves myocardial ischemia, and simultaneously can reduce blood pressure, nourish liver to improve eyesight, digest food retention, remove fat and reduce weight and lighten the body. Through the vigorous popularization and use of the south medical hall in recent years, the medicine has received the pursuit of consumers, and the effect is obvious.
At present, the quality detection methods of the Minggu Shuqing drink are few. The invention adopts the high performance liquid chromatography to establish the fingerprint detection method of the Minggu Shuqing drink, and has important significance for ingredient identification, quality evaluation and quality standard formulation of the Minggu Shuqing drink.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides a fingerprint detection method of Minggu Shuqing drink, which can objectively, comprehensively and accurately evaluate the quality of Minggu Shuqing drink and has important significance for controlling the quality of Minggu Shuqing drink 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 method for detecting a fingerprint of Minggu Shuqing drink is characterized by comprising the following steps:
step 1, preparing a Minggu Shuqing beverage test solution:
taking different batches of Minggu Shuqing beverage, respectively and precisely weighing Minggu Shuqing beverage traditional Chinese medicine samples, placing in a round-bottomed bottle, adding methanol solution for extraction, filtering, placing filtrate in a volumetric flask, adding methanol for constant volume, and filtering through a 0.45 mu m microporous filter membrane to obtain a test sample solution;
precisely weighing chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin reference substance, placing in a volumetric flask, adding ethanol to desired volume to scale, 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 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 Mingguanshuqing beverage test solution obtained in the step 3, and importing the Mingguanshuqing beverage test solution into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of Minggu Shuqing drinks as common peaks, and identifying 17 common chromatographic peaks; generating a comparison fingerprint of the Mingguanshuqing drink by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak;
step 5, comparing the fingerprint of the Ming Ge Shuqing drink obtained in the step 3 with the mixed standard substance, and identifying that the No. 2 peak in the Ming Ge Shuqing drink is chlorogenic acid; peak 11 is cryptochlorogenic acid; the 14 th peak is puerarin; peak 19 is aurantio-obtusin.
As a preferred scheme, the method for detecting the fingerprint of the Mingguanshuqing drink comprises the following steps of 1: taking 5g of 8 batches of Minggu Shuqing beverage samples, placing the samples in a 100mL round-bottomed bottle, adding 50mL of methanol solution, extracting for 2 hours at the temperature of 75 ℃ in a cable manner, filtering, placing the filtrate in 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.
As a preferred scheme, the method for detecting the fingerprint of the Minggu Shuqing beverage comprises the following steps of 2, preparation of a mixed reference solution: precisely weighing chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin reference substance, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up, and making into mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0489mg/mL cryptochlorogenic acid, 0.0521mg/mL puerarin and 0.1044mg/mL aurantio-obtusin.
As a preferred scheme, in the above method for detecting a fingerprint of ming ge shu qing drink, 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, diode array detector, detection wavelength: 325nm, column temperature 30 ℃, flow rate 1mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
| procedure for measuring the movement of a moving object | Time (min) | Acetonitrile volume (%) |
| 1 | 0 | 5 |
| 2 | 10 | 5 |
| 3 | 25 | 20 |
| 4 | 50 | 30 |
| 5 | 75 | 50 |
。
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 (ultrasonic, reflux, Soxhlet extraction, percolation and the like) and different extraction solvents (methanol, water, 70% ethanol aqueous solution, 85% ethanol aqueous solution, 95% ethanol and absolute ethanol), the results show that the components of a spectrogram difference obtained by Soxhlet extraction for 2 hours at the temperature of 75 ℃ by adopting methanol as the extraction solvent are relatively comprehensive, the separation degree is good, so that the Soxhlet extraction for 2 hours at the temperature of 75 ℃ 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, 284nm, 300nm and 325nm are extracted, and when the detection wavelength is 325nm, the information content contained in the chromatograms is most comprehensive and the base line is stable, so 325nm 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 in the Ming Geshuqing drink, 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 the gradient conditions of multiple flow rates of 1mL/min 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 Mingguanshuqing drink can be well separated by using the acetonitrile and the 0.05% phosphoric acid water as the mobile phase.
Has the advantages that:
1. according to the structural property characteristics of active ingredients contained in the Mingguanshuqing 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 Minggu Shuqing drink established by the method can effectively represent the quality of Minggu Shuqing drink, objectively embody 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 judging the quality of Minggu Shuqing 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 Minggu Shuqing drink provided by the invention has the advantages of simple method, good stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a chromatogram of a mixed control of the present invention.
FIG. 2 is a comparison fingerprint of the Mingguanshuqing drink sample 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
Minggu Shuqing sample is from Nanjing health industry development Limited company; chlorogenic acid control (batch No. 170422) was purchased from Nanjing Senega Biotech, Inc.; puerarin reference (lot No. 100080-201308) was purchased from national institute for food and drug testing; 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 a fingerprint of a Minggu Shuqing drink, comprising the following steps:
step 1, preparing a Minggu Shuqing beverage test solution:
putting 8 batches of Minggu Shuqing traditional Chinese medicine samples (each batch comprises 15g of hawthorn, 10g of cassia seed, 25g of kudzuvine root, 15g of chrysanthemum, 10g of lotus leaf, 15g of raw Pu' er tea and 10g of medlar) into a 1000mL round-bottomed bottle, adding 500mL of methanol solution, performing Soxhlet extraction (continuous reflux extraction method) at the temperature of 75 ℃ for 2 hours, filtering, putting the filtrate into a 1000mL volumetric flask, adding methanol to a constant volume, and passing through a 0.45-micrometer microporous filter membrane to obtain a sample solution;
precisely weighing chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin reference substance, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up to obtain mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0489mg/mL cryptochlorogenic acid, 0.0521mg/mL puerarin and 0.1044mg/mL aurantio-obtusin;
respectively and precisely sucking 8 batches of the Mingguanshuqing beverage test solution and the reference solution, 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, diode array detector, detection wavelength: 325nm, column temperature 30 ℃, flow rate 1mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
| procedure for measuring the movement of a moving object | Time (min) | Acetonitrile volume (%) |
| 1 | 0 | 5 |
| 2 | 10 | 5 |
| 3 | 25 | 20 |
| 4 | 50 | 30 |
| 5 | 75 | 50 |
Step 4, exporting the fingerprints of the 8 batches of the Mingguanshuqing beverage test solution obtained in the step 3, and introducing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of 8 batches of Minggu Shuqing drinks as common peaks; generating a comparison fingerprint of the Mingguanshuqing drink by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; the result shows that 17 common peaks in the Pushuqing drink are shown in figure 2, and the liquid chromatogram of the reference solution is shown in figure 1.
And 5, comparing the obtained fingerprint spectrum of the Ming Ge Shuqing drink with the spectrum of the mixed standard substance, identifying the main components, and comparing the chromatographic peaks 2, 11, 14 and 19 in the Ming Ge Shuqing drink to respectively obtain: chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin.
Wherein the chlorogenic acid retention time is 22.91min, the cryptochlorogenic acid retention time is 39.00min, the puerarin retention time is 51.94min, and the aurantio-obtusin retention time is 70.54 min.
Example 2 forensic study of fingerprint detection methods:
1. study of precision
The Mingguanshuqing drink (the traditional Chinese medicine composition comprises 15g of hawthorn, 10g of cassia seed, 25g of kudzuvine root, 15g of chrysanthemum, 10g of lotus leaf, 15g of raw Pu' er tea and 10g of medlar) is prepared into a test solution with the serial number of S1 to be tested according to the method of the embodiment 1, the sample is parallelly injected for 6 times according to the detection and analysis of the chromatographic condition of the embodiment 1, the sample injection amount is 10 mu L, chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin are taken as reference peaks, and the result is shown in a table 1 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 1 precision Studies of Peak area and Retention time
2. Stability study
The Mingguanshuqing beverage (the traditional Chinese medicine composition comprises 15g of hawthorn, 10g of cassia seed, 25g of kudzuvine root, 15g of chrysanthemum, 10g of lotus leaf, 15g of raw Pu' er tea and 10g of medlar) is prepared into a test solution with the serial number of S1 according to the method of the embodiment 1, the test solution is analyzed according to the detection method of the embodiment 1, sample injection analysis is carried out at different time periods of 0 hour, 2 hour, 6 hour, 12 hour, 18 hour and 24 hour, the sample injection amount is 10 mu L, chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin are taken as reference peaks, the peak area and the retention time of the common peak of the HPLC fingerprint spectrum of a sample are analyzed, and the RSD value is calculated, the result is shown in Table 2, the chromatographic peak of the test solution of the Mingguanshuqing beverage is almost.
Table 2 stability study peak area and retention time
3. Repetitive studies
Six samples (compound composition of 15g of hawthorn, 10g of cassia seed, 25g of kudzuvine root, 15g of chrysanthemum, 10g of lotus leaf, 15g of raw puerh and 10g of medlar) are weighed parallelly and precisely, 6 parts of the same sample solution to be tested are prepared according to the method of the embodiment 1, the sample amount is 10 mu L according to the chromatographic condition of the embodiment 1, chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin are taken as reference peaks, the peak area and the retention time of the common peaks of the HPLC fingerprint of the samples are analyzed, the RSD value is calculated, and the result is shown in a table 3.
TABLE 3 repeated study of peak area and retention time
The experimental results show that the fingerprint detection method for the Minggu Shuqing 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 Minggu Shuqing 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 method for detecting a fingerprint of Minggu Shuqing drink is characterized by comprising the following steps:
step 1, preparing a Minggu Shuqing beverage test solution:
taking different batches of Minggu Shuqing beverage, respectively and precisely weighing Minggu Shuqing beverage traditional Chinese medicine samples, placing in a round-bottomed bottle, adding methanol solution for extraction, filtering, placing filtrate in a volumetric flask, adding methanol for constant volume, and filtering through a 0.45 mu m microporous filter membrane to obtain a test sample solution;
step 2, preparation of mixed reference solution:
precisely weighing chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin reference substance, placing in a volumetric flask, adding ethanol to desired volume to scale, 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 Mingguanshuqing beverage test solution obtained in the step 3, and importing the Mingguanshuqing beverage test solution into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of Minggu Shuqing drinks as common peaks, and identifying 17 common chromatographic peaks; generating a comparison fingerprint of the Mingguanshuqing drink by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak;
step 5, comparing the fingerprint of the Ming Ge Shuqing drink obtained in the step 3 with the mixed standard substance to identify 2-chlorogenic acid in the Ming Ge Shuqing drink; peak 11 is cryptochlorogenic acid; the 14 th peak is puerarin; peak 19 is aurantio-obtusin.
2. The method for detecting the fingerprint of the Mingguanshuqing drink according to claim 1, wherein the preparation method of the Mingguanshuqing drink test solution in the step 1 is as follows: taking 5g of 8 batches of Minggu Shuqing traditional Chinese medicine samples, placing the samples in a 100mL conical flask, adding 50mL of methanol solution, performing cable extraction at 75 ℃ for 2 hours, filtering, placing the filtrate in a 100mL volumetric flask, adding methanol to a constant volume, and filtering through a 0.45-micrometer microporous membrane to obtain a test solution.
3. The method for detecting the fingerprint of Mingguanshuqing drink according to claim 1, wherein the step 2 of preparing the mixed reference solution comprises the following steps: precisely weighing chlorogenic acid, cryptochlorogenic acid, puerarin and aurantio-obtusin reference substance, placing in a volumetric flask, fixing the volume to scale with ethanol, shaking up, and making into mixed reference substance solution containing 0.0674mg/mL chlorogenic acid, 0.0489mg/mL cryptochlorogenic acid, 0.0521mg/mL puerarin and 0.1044mg/mL aurantio-obtusin.
4. The method for detecting the Mingguanshuqing fingerprint 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: 325nm, column temperature 30 ℃, flow rate 1mL/min, sample injection volume: 10 μ L.
5. The method for detecting the Mingguanshuqing fingerprint according to claim 4, wherein the gradient elution procedure is as follows:
。
6. The method for detecting the fingerprint of the Mingguanshuqing drink according to claim 1, wherein the Mingguanshuqing drink is prepared from 15 parts of hawthorn, 10 parts of cassia seed, 25 parts of kudzuvine root, 15 parts of chrysanthemum, 10 parts of lotus leaf, 15 parts of raw Pu' er tea and 10 parts of medlar.
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| CN112255324B (en) * | 2020-08-31 | 2022-11-29 | 广州泽力医药科技有限公司 | Method for establishing fingerprint of wolfberry fruit and kudzuvine root solid beverage and fingerprint thereof |
| CN112748201A (en) * | 2020-12-28 | 2021-05-04 | 南京海昌中药集团有限公司 | Method for detecting fingerprint of radix puerariae assorted Chinese herbal tea |
| CN115356423A (en) * | 2022-08-11 | 2022-11-18 | 广东唯纳健康产业集团有限公司 | Content detection method of composite artichoke concentrated beverage |
| CN115356423B (en) * | 2022-08-11 | 2023-08-04 | 广东唯纳健康产业集团有限公司 | Method for detecting content of compound globe artichoke concentrated beverage |
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