CN108776183B - Quality control method of rhubarb and liquorice decoction - Google Patents
Quality control method of rhubarb and liquorice decoction Download PDFInfo
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Abstract
The invention discloses a quality control method of rhubarb licorice decoction, which screens out the best mobile phase composition through a large number of experiments, and adopts UHPLC-TQ-MS/MS method for detection under the analysis conditions of elution program, flow rate, chromatographic column and detector detection parameters, etc., and the detection method can simultaneously detect anthraquinone and saponin components in rhubarb licorice decoction and extract thereof, and total 17 active compounds. The experimental result shows that the method has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of the rhubarb licorice decoction, and has important significance for controlling the quality and ensuring the clinical curative effect.
Description
Technical Field
The invention relates to a quality control method of traditional Chinese medicinal materials, in particular to a quality control method of rhubarb and liquorice decoction.
Background
The rhubarb and licorice herb pair is a common laxative pair used in the medicine family. The rhubarb with bitter and cold property can clear away heat in the stomach, and has strong and good actions, and the licorice root with sweet and mild property is combined with the rhubarb to relieve the purgation of the rhubarb, so the rhubarb is left in the stomach to clean the house, and the rhubarb can be prevented from damaging the middle of the stomach due to bitter and cold property, so that the rhubarb can tonify the middle and cure the disease. The two herbs are combined and reinforced mutually, and the licorice root relieves the property of rhubarb with bitter and cold property of descending and descending, so that the medicine can direct adverse flow of qi to arrest vomiting without hurting stomach qi, and can clear stomach heat.
The rhubarb contains various anthraquinone components, and the liquorice contains various saponin components, so that the detection method has limited effective components, and cannot comprehensively detect the effective components in the rhubarb and liquorice medicine pair. Therefore, in order to fully control the clinical safety of the rhubarb and licorice medicine and the preparation thereof and maintain the benefits of patients, it is necessary to research and design a detection method capable of accurately and comprehensively detecting the active ingredients of the rhubarb and licorice medicine and the preparation thereof on the basis of the prior art.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects in the prior art, and through a large number of experimental screenings and detection by adopting ultra-high performance liquid chromatography, the detection method can simultaneously detect 17 active compounds including anthraquinone and steroid in rhubarb licorice decoction. The method has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of the rhubarb and liquorice decoction and the preparation thereof, and has important significance for controlling the quality and ensuring the curative effect.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a quality control method of rhubarb and liquorice decoction comprises the following steps:
(1) preparation of sample solution
Taking a licorice root medicinal material, adding 8-16 times of water for reflux extraction, then adding a rhubarb medicinal material, adding 8-16 times of water of the total weight of the medicinal materials, performing reflux extraction, filtering to obtain a rhubarb licorice root water extract, centrifuging, sucking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a sample solution;
(2) preparation of control solutions
Precisely weighing 17 reference substances including rhein, emodin, aloe-emodin, chrysophanol, physcion, rhein-8-O-beta-D-glucoside, emodin-8-O-beta-D-glucoside, sennoside A, sennoside B, glycyrrhizic acid, glycyrrhetinic acid, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, licochalcone A and glabridin, respectively dissolving with methanol to obtain reference substance stock solutions, quantitatively sucking the reference substance stock solutions to obtain mixed reference substance stock solutions, and filtering with a 0.22 μm filter membrane to obtain mixed reference substance solutions;
(4) quantitative analysis
Respectively carrying out ultra high performance liquid chromatography detection on the sample solution prepared in the step (1) and the mixed reference solution prepared in the step (2) to respectively obtain UHPLC-TQ-MS/MS chromatograms of the sample and the reference, and then carrying out qualitative and quantitative analysis.
Preferably, in the quality control method of rhubarb licorice decoction, the parameters of the ultra-high performance liquid chromatography used in the step (3) are set as follows:
the instrument comprises the following steps: waters ACQUITY UPLC system; a Tetons mass spectrometer detector;
a chromatographic column: thermo Scientific Hypersil Gold C18Chromatography column, 100mm × 2.1mm, 1.9 μm; column temperature: 35 ℃; sample introduction volume: 2 mu L of the solution; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; flow rate: 0.4 ml/min-1(ii) a Gradient elution;
the mass spectrum conditions are as follows: an ion source: ESI ion source; ion source temperature: 150 ℃; the scanning mode is as follows: a multi-reaction detection mode; capillary voltage: 3.0 kV; temperature of the desolventizing gas: 150 ℃; flow rate of desolventizing agent: 1000 L.h-1(ii) a Taper hole gas flow: 30 L.h-1(ii) a Collision gas flow rate: 0.15 mL/min-1。
Preferably, in the quality control method of the rhubarb licorice decoction, the gradient elution procedure is as follows: 0-2 min, A: 95% -95%, B: 5% -5%; 2-4 min, A: 95% -65%, B: 5% -35%; 4-8 min, A: 65% -5%, B: 35% -95%; 8-13 min, A: 5% -5%, B: 95 to 95 percent; 13-14 min, A: 5% -95%, B: 95% -5%; 14-15 min, A: 95% -95%, B: 5 to 5 percent.
Preferably, in the quality control method of rhubarb licorice decoction, the mixed reference solution is diluted step by 90% methanol according to a step-by-step dilution method in step (3), 5 reference solutions with different concentration levels are prepared and respectively subjected to ultra performance liquid chromatography detection, the peak areas of 17 components in each reference solution are measured, and the concentration of each component is taken as the transverse directionDrawing a standard curve of 17 components by taking the coordinates and the peak area as vertical coordinates, performing linear analysis by adopting a regression method, and calculating the linear correlation coefficient r of each reference substance2(ii) a And LOQ and LOD were measured as shown in the following table
Preferably, in the quality control method of rhubarb licorice decoction, the preparation method of the reference substance solution in the step (2) is as follows:
precisely weighing rhein, emodin, aloe-emodin, chrysophanol, physcion, rhein-8-O-beta-D-glucoside, emodin-8-O-beta-D-glucoside, sennoside A, sennoside B, glycyrrhizic acid, glycyrrhetinic acid, liquiritin, isoliquiritin, liquiritigenin, licochalcone A and glabridin control each 5mg or so, dissolving with 90% methanol to obtain rhein 108.6 μ g/mL-1Emodin, 106.0. mu.g.mL-1Aloe-emodin 220.0 microgram/mL-1Chrysophanol 90.0. mu.g/mL-1Emodin methyl ether 130.0 μ g/mL-1rhein-8-O-beta-D-glucoside 1347.0 mug/mL-1emodin-8-O-beta-D-glucoside 408.3 mug/mL-1Sennoside A126.0. mu.g/mL-1Sennoside B245.0. mu.g/mL-11353.0 mug/mL glycyrrhizic acid-1721.4 mug/mL glycyrrhetinic acid-1Glycyrrhiza glycoside 505.7. mu.g/mL-13320.0 mug/mL isoliquiritigenin-13233.0 mug/mL liquiritigenin-13553.0 mug/mL isoliquiritigenin-12127.0 μ g/mL of licochalcone-12647.0 mug/mL glabridin-1The control stock solution of (a); then quantitatively sucking each control stock solution to obtain mixed control stock solution, wherein rhein (1), emodin (2), aloe-emodin (3), chrysophanol (4), physcion (5), rhein-8-O-beta-D-glucoside (6)emodin-8-O-beta-D-glucoside (7), sennoside A (8), sennoside B (9), glycyrrhizic acid (10), glycyrrhetinic acid (11), liquiritin (12), isoliquiritin (13), liquiritigenin (14), isoliquiritigenin (15), licochalcone A (16) and glabridin (17) respectively have the content of 15.404 mu g/mL-1、15.035μg·mL-1、15.603μg·mL-1、12.766μg·mL-1、18.440μg·mL-1、19.106μg·mL-1、14.479μg·mL-1、17.872μg·mL-1、17.376μg·mL-1、19.191μg·mL-1、15.349μg·mL-1、14.346μg·mL-1、23.546μg·mL-1、22.929μg·mL-1、25.199μg·mL-1、15.085μg·mL-1、23.005μg·mL-1。
Preferably, in the quality control method of rhubarb licorice decoction, the preparation method of the sample solution in the step (1) is as follows:
firstly, taking a licorice root medicinal material, adding 8 times of water for reflux extraction for 1 hour, then adding a rhubarb medicinal material, adding 8 times of water of the total weight of the medicinal materials, reflux extracting for 2 times, combining extracting solutions, filtering to obtain a rhubarb and licorice root water extract, centrifuging, sucking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a sample solution. The weight ratio of the rhubarb to the liquorice is 4:1, 4:2, 4:3, 4:4, 3:4, 2:4 or 1: 4.
Condition optimization experiment
According to the invention, conditions such as a mobile phase, a gradient elution program, a flow rate and the like are examined, so that all components can be separated in a short time and a good peak shape can be obtained. The discovery shows that 17 active ingredients in the rhubarb and the liquorice can be well separated by 0.1 percent of formic acid water-acetonitrile, and the tailing phenomenon of aromatic acid ingredients can be reduced by adding formic acid; the high-proportion organic phase is better separated than the active ingredients in the rhubarb and the liquorice by optimizing the gradient elution program, and the elution gradient is determined; the flow rate of the mobile phase is 0.4 ml/min-1The separation effect and peak shape of each component are excellentAt a flow rate of 0.2 ml/min-1. The mass spectrum parameters of 17 compounds are optimized by adopting a full-scan mode (positive ion and negative ion modes), the instrument selects a detection mode in terms of analyzing the signal intensity and sensitivity of each component in the positive ion and negative ion modes, and the taper hole voltage and the capillary voltage are automatically optimized by software. UHPLC-TQ-MS/MS chromatogram of 17 active ingredients in radix et rhizoma Rhei-Glycyrrhrizae radix obtained after optimization of chromatography and mass spectrum conditions is shown in figure 1.
Has the advantages that: compared with the prior art, the quality control method of the rhubarb and liquorice soup provided by the invention has the following advantages:
according to the structure and property characteristics of 17 active compounds with different structures and properties, the optimal mobile phase composition, elution program, flow rate, chromatographic column, detector detection parameters and other analysis conditions are screened out through a large number of experiments according to different anthraquinone and saponin 2 components in the rhubarb licorice decoction. Multiple experiments prove that the method can simultaneously detect 17 compounds of anthraquinone and saponin 2 different components, has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of the rhubarb and liquorice decoction and the preparation thereof, and has important significance for controlling the quality and ensuring the clinical curative effect.
Drawings
FIG. 1 is a multi-reaction detection mode (MRM) chromatogram of UHPLC-TQ-MS/MS of 17 components in DAHUANGGANCAO decoction.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1
1. A quality control method of rhubarb and liquorice decoction comprises the following steps:
instruments and reagents
1.1 instruments
WatersACQUITY UHPLC System, available from Waters corporation, USA; tetons mass spectrometryA tester, available from Waters corporation, usa; MassLynxTMMass Spectrometry workstation software, available from Waters, USA; ultrapure water instruments, purchased from Nanjing Yipu Yida science and technology development Limited; a mocofuge 22R Centrifuge type desktop Centrifuge, available from boya, beijing, innovative technologies development ltd; an ultrasonic cleaning instrument, purchased from Kunshan standing grain ultrasonic instruments, Inc.;
1.2 drugs and reagents
Rhubarb (produced in Gansu, batch No. 20160319), liquorice (produced in Ningxia, batch No. 151108), rhubarb (Tanggute radix et rhizoma Rhei), liquorice (Ural liquorice) and according with pharmacopoeia standards.
The chemical standard products of rhein (1), emodin (2), aloe-emodin (3), chrysophanol (4), physcion (5), rhein-8-O-beta-D-glucoside (6), emodin-8-O-beta-D-glucoside (7), sennoside A (8), sennoside B (9), glycyrrhizic acid (10), glycyrrhetinic acid (11), liquiritin (12), isoliquiritin (13), liquiritigenin (14), isoliquiritigenin (15), licochalcone A (16) and glabridin (17) are purchased from Dougesi Biotech limited company, and the purity is more than 98%. Methanol, chromatographically pure, available from Merck reagent, germany; acetonitrile, chromatographically pure, commercially available from Merck reagent, germany.
2 method of experiment
2.1 preparation of control solutions
The 17 control samples were precisely weighed to about 5mg each, and dissolved in 90% methanol to give rhein (1, 108.6. mu.g. mL)-1) Emodin (2, 106.0. mu.g. mL)-1) Aloe-emodin (3, 220.0. mu.g/mL)-1) Chrysophanol (4, 90.0. mu.g. mL)-1) Emodin methyl ether (5, 130.0. mu.g. mL)-1) rhein-8-O-beta-D-glucoside (6, 1347.0 mu g. mL)-1) emodin-8-O-beta-D-glucoside (7, 408.3 mu g. mL)-1) Sennoside A (8, 126.0. mu.g. mL)-1) Sennoside B (9, 245.0. mu.g. mL)-1) Glycyrrhizic acid (10, 1353.0 mug. multidot.mL)-1) Glycyrrhetinic acid (11, 721.4. mu.g/mL)-1) Glycyrrhizin (12, 505.7. mu.g. mL)-1) Isoliquiritin (13, 3320.0 mug. multidot.mL)-1) Glycyrrhizin (14, 3233.0 mug. multidot.mL)-1) Isoliquiritigenin (15, 3553.0 mug. multidot.mL)-1) Licochalcone (16, 2127.0. mu.g. mL)-1) Glabridin (17, 2647.0. mu.g. mL)-1) Control stock solution of (4). Quantitatively sucking each control stock solution to obtain mixed control stock solution, wherein the contents of rhein (1), emodin (2), aloe-emodin (3), chrysophanol (4), physcion (5), rhein-8-O-beta-D-glucoside (6), emodin-8-O-beta-D-glucoside (7), sennoside A (8), sennoside B (9), glycyrrhizic acid (10), glycyrrhetinic acid (11), liquiritin (12), isoliquiritin (13), liquiritigenin (14), isoliquiritigenin (15), licochalcone A (16) and glabridin (17) are 15.404 μ g/mL respectively-1、15.035μg·mL-1、15.603μg·mL-1、12.766μg·mL-1、18.440μg·mL-1、19.106μg·mL-1、14.479μg·mL-1、17.872μg·mL-1、17.376μg·mL-1、19.191μg·mL-1、15.349μg·mL-1、14.346μg·mL-1、23.546μg·mL-1、22.929μg·mL-1、25.199μg·mL-1、15.085μg·mL-1、23.005μg·mL-1。
2.2 preparation of test solutions
Firstly, taking a licorice root medicinal material, adding 8 times of water for reflux extraction for 1 hour, then adding a rhubarb medicinal material, adding 8 times of water of the total weight of the medicinal materials, reflux extracting for 2 times, combining extracting solutions, filtering to obtain a rhubarb and licorice root water extract, centrifuging, sucking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a sample solution. The weight ratio of the rhubarb to the liquorice is 4: 1.
2.3 chromatographic conditions
A chromatographic column: thermo Scientific Hypersil Gold C18Chromatography column (100 mm. times.2.1 mm, 1.9 μm); column temperature: 35 ℃; sample introduction volume: 2 mu L of the solution; mobile phase: a (0.1% formic acid), B (acetonitrile); flow rate: 0.4 ml/min-1(ii) a Gradient elution: 0-1 min, A: 95% -95%, B: 5% -5%; 2-4 min, A: 95% -65%, B: 5% -35%; 4-8 min, A: 65% -5%, B: 35% -95%; 8-13 min, A: 5% -5%, B: 95 to 95 percent; 13-14 min, A: 5 to 95 percent of the total weight of the mixture,B：95％～5％；14～15min，A：95％～95％，B：5％～5％。
2.4 Mass Spectrometry conditions
An ion source: ESI ion source; ion source temperature: 150 ℃; the scanning mode is as follows: multiple reaction detection mode (MRM); capillary voltage: 3.0 kV; temperature of the desolventizing gas: 150 ℃; flow rate of desolventizing agent: 1000 L.h-1(ii) a Taper hole gas flow: 30 L.h-1(ii) a Collision gas flow rate: 0.15 mL/min-1. The retention time and mass spectral parameters of the components are shown in Table 1.
TABLE 1 component Retention time and Mass Spectrometry parameters
2.5 methodological considerations
2.5.1 Linear relationship, quantitative Limit (LOD), Limit of detection (LOQ)
Diluting the reference stock solution with 90% methanol by stepwise dilution method to obtain reference solutions with different concentration levels, measuring the peak areas of 17 components in each reference solution according to chromatography and mass spectrometry detection methods under items 2.3 and 2.4, drawing standard curves of the 17 components with each component concentration as abscissa and peak area as ordinate, performing linear analysis by regression method, and calculating linear correlation coefficient (r) of each reference2) (ii) a The concentrations of the control samples at signal-to-noise ratios of 3 and 10 were LOQ and LOD, respectively. As in table 2 below:
TABLE 2 Standard Curve and Linear Range of ingredients and LOQ, LOD
2.5.2 precision, repeatability, stability
Precision investigation: and (3) measuring the peak areas of 17 components in each reference solution by using a part of reference solution according to the chromatographic and mass spectrometric detection methods, repeatedly injecting samples for 3 times every day, continuously injecting samples for 6 times within 3 days and the same day, and evaluating the precision of the samples in the day and in the day by using the Relative Standard Deviation (RSD) of the peak areas of the components.
And (3) repeatability inspection: preparing 6 parts of a test sample solution with the ratio of rhubarb to liquorice being 1:1 in parallel, measuring the peak area of each component in the test sample according to the chromatographic and mass spectrometric detection method, and evaluating the repeatability of the instrument by evaluating the Relative Standard Deviation (RSD) of the peak area of each component.
And (3) stability investigation: sample solutions with the ratio of rhubarb to liquorice being 1:1 are respectively injected at 1, 2, 4, 8, 12 and 24 hours, the content change of 17 compounds in the sample is measured at different time, and the RSD value of the peak area of the 17 compounds is used for evaluating the stability of the sample.
2.5.3 recovery test
Adding control solutions with the contents of 80%, 100% and 120% to a sample with the known content of 17 components, measuring sample peak areas of the sample solution according to the chromatographic and mass spectrometric detection methods, obtaining sample concentration according to a standard curve, and calculating sample recovery rate, wherein the sample recovery rate is (standard sample concentration-sample concentration)/scalar. The result shows that the recovery rate of 17 active ingredients in the rhubarb and the liquorice is within the range of 98.16 to 100.64 percent, and the accuracy is good.
2.6 statistical analysis
All results were processed using SPSS 22.0 software and statistically different analyzed using Dunnett's method in ANOVA, with P <0.05 indicating statistically significant differences and all results expressed as mean ± standard deviation (mean ± SD).
3. Results of the experiment
As shown in Table 3, the precision, repeatability and stability of the invention show that the RSD is less than 3%, the precision is good, and the recovery rate of 17 active ingredients in rhubarb and licorice is in the range of 98.16-100.64%.
TABLE 3 precision, repeatability, stability and sample recovery experiments
The detection method provided by the invention can be used for simultaneously and accurately detecting 17 compounds, and the quality control method of the rhubarb licorice decoction provided by the invention is high in precision, high in sensitivity, high in stability and accuracy, capable of objectively, comprehensively and accurately evaluating the quality of the rhubarb licorice decoction, and has important significance for accurately controlling the quality of the rhubarb licorice decoction.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A quality control method of rhubarb and liquorice decoction is characterized by comprising the following steps:
(1) preparation of sample solution
Taking a licorice root medicinal material, adding 8-16 times of water for reflux extraction, then adding a rhubarb medicinal material, adding 8-16 times of water of the total weight of the medicinal materials, performing reflux extraction, filtering to obtain a rhubarb licorice root water extract, centrifuging, sucking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a sample solution;
(2) preparation of control solutions
Precisely weighing rhein, emodin, aloe-emodin, chrysophanol, physcion, rhein-8-O-beta-D-glucoside, emodin-8-O-beta-D-glucoside, sennoside A, sennoside B, glycyrrhizic acid, glycyrrhetinic acid, liquiritin, isoliquiritin, liquiritigenin, licochalcone A and glabridin control each 5mg or so, dissolving with 90% methanol to obtain rhein 108.6 μ g/mL-1Emodin 106.0. mu.g/mL-1Aloe-emodin 220.0 microgram/mL-1Chrysophanol 90.0. mu.g/mL-1Emodin methyl ether 130.0 μ g/mL-1rhein-8-O-beta-D-glucoside 1347.0 mug/mL-1emodin-8-O-beta-D-glucoside 408.3 mug/mL-1Sennoside A126.0. mu.g/mL-1Sennoside B245.0. mu.g/mL-11353.0 mug/mL glycyrrhizic acid-1721.4 mug/mL glycyrrhetinic acid-1Glycyrrhiza glycoside 505.7. mu.g/mL-13320.0 mug/mL isoliquiritigenin-13233.0 mug/mL liquiritigenin-13553.0 mug/mL isoliquiritigenin-12127.0 μ g/mL of licochalcone-12647.0 mug/mL glabridin-1The control stock solution of (a);
then quantitatively sucking each control stock solution to obtain mixed control stock solution, wherein the contents of rhein, emodin, aloe-emodin, chrysophanol, physcion, rhein-8-O-beta-D-glucoside, emodin-8-O-beta-D-glucoside, sennoside A, sennoside B, glycyrrhizic acid, glycyrrhetinic acid, liquiritin, isoliquiritigenin, licochalcone A, and glabridin are 15.404 μ g/mL respectively-1、15.035 μg·mL-1、15.603 μg·mL-1、12.766 μg·mL-1、18.440 μg·mL-1、19.106 μg·mL-1、14.479 μg·mL-1、17.872 μg·mL-1、17.376 μg·mL-1、19.191 μg·mL-1、15.349 μg·mL-1、14.346 μg·mL-1、23.546 μg·mL-1、22.929 μg·mL-1、25.199 μg·mL-1、15.085 μg·mL-1、23.005 μg·mL-1；
(3) Quantitative analysis
Respectively carrying out ultra-high performance liquid chromatography detection on the sample solution prepared in the step (1) and the mixed reference stock solution prepared in the step (2) to respectively obtain UHPLC-TQ-MS/MS chromatograms of the sample and the reference, and then carrying out qualitative and quantitative analysis;
the ultra-high performance liquid chromatography parameters used in the step (3) are set as follows:
the instrument comprises the following steps: waters ACQUITY UPLC system; a Tetons mass spectrometer detector;
a chromatographic column: thermo Scientific Hypersil Gold C18Chromatography column, 100mm × 2.1mm, 1.9 μm; column temperature: 35 ℃; sample introduction volume: 2 mu L of the solution; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; flow rate:0.4 ml·min-1(ii) a Gradient elution, the gradient elution procedure is: 0-2 min, A: 95% -95%, B: 5% -5%; 2-4 min, A: 95% -65%, B: 5% -35%; 4-8 min, A: 65% -5%, B: 35% -95%; 8-13 min, A: 5% -5%, B: 95% -95%; 13-14 min, A: 5% -95%, B: 95% -5%; 14-15 min, A: 95% -95%, B: 5% -5%;
the mass spectrum conditions are as follows: an ion source: ESI ion source; ion source temperature: 150 ℃; the scanning mode is as follows: a multi-reaction detection mode; capillary voltage: 3.0 kV; temperature of the desolventizing gas: 150 ℃; flow rate of desolventizing agent: 1000 L.h-1(ii) a Taper hole gas flow: 30 L.h-1(ii) a Collision gas flow rate: 0.15 mL/min-1。
2. The quality control method of rhubarb licorice root decoction according to claim 1, wherein the preparation method of the sample solution in the step (1) is as follows:
taking a licorice root medicinal material, adding 8 times of water for reflux extraction for 1 hour, then adding a rhubarb medicinal material, adding 8 times of water of the total weight of the medicinal materials, reflux extracting for 2 times, combining extracting solutions, filtering to obtain a rhubarb and licorice root water extract, centrifuging, sucking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a sample solution, wherein the weight ratio of rhubarb to licorice root is 4:1, 4:2, 4:3, 4:4, 3:4, 2:4 or 1: 4.
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