Establishment method of wine-fried licorice root fingerprint spectrum and fingerprint spectrum of wine-fried licorice root fingerprint spectrum

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to an establishment method of a wine-fried licorice root fingerprint spectrum and a fingerprint spectrum thereof. According to the method, the wine-fried licorice root test solution and the mixed reference substance solution are prepared and determined by high performance liquid chromatography, the fingerprint spectrum of the wine-fried licorice root is established, 11 common peaks achieving effective separation are provided, the quality stability of the wine-fried licorice root can be ensured, the influence of component fluctuation on the drug effect is avoided, and the method is suitable for large-scale popularization and application. Data support is provided for the production process of the honey-fried licorice root with wine, and the consistency of different batches of products is ensured; 15 batches of wine-fried licorice roots are detected at the same time, the result shows that the relative retention time RSD of chromatographic peaks at the same position is 1.01% or less, and it is indicated that the fingerprint spectrum provided by the invention has good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for establishing a fingerprint spectrum of wine-roasted licorice and the fingerprint spectrum thereof. Background Art

[0002] Licorice is the dried root and rhizome of the leguminous plants licorice, licorice inflated fruit or licorice glabra, which are often used in antioxidant, antiviral and other drugs. The inventors previously studied the processing of wine-fried licorice and found that the content of total flavonoids in wine-fried licorice is better than that of raw products, making it a better drug raw material, and wine-fried licorice can be produced in a quantitative manner.

[0003] However, there are few literatures on the components of wine-fried licorice. At present, there are studies on making licorice into licorice slices and measuring their extracts, and using high performance liquid chromatography to establish a fingerprint of licorice. However, 1. It focuses on honey-fried licorice and cannot achieve quality control of wine-fried licorice; 2. It does not specify the components and their contents in licorice, and cannot provide support for further exploration of its efficacy, and there may be problems with efficacy being affected by fluctuations in components.

[0004] Therefore, it is of great significance to establish a method for determining the components and contents in wine-fried licorice to make the components of wine-fried licorice more controllable. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for establishing a fingerprint of wine-fried licorice and its fingerprint. The method of the present invention can not only measure the flavonoid components in wine-fried licorice, but also measure the triterpenoid components in wine-fried licorice, and determine the contents of 11 components, which is conducive to comprehensive monitoring of the quality of wine-fried licorice and provides a theoretical basis for the quality evaluation of wine-fried licorice.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a method for establishing a fingerprint spectrum of wine-fried licorice is provided, comprising the following steps:

[0008] S1, prepare the test solution: take the wine-roasted licorice sample powder and sieve it, add ethanol solution, weigh and record the weight, then perform ultrasonic treatment, and then filter and take the filtrate as the test solution;

[0009] S2, using high performance liquid chromatography to test the sample solution obtained in S1, and generating a fingerprint of liquorice based on the obtained chromatographic data according to the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition";

[0010] The chromatographic conditions of the high performance liquid chromatography are:

[0011] Column: Agilent Eclipse XDB-C 18 ;

[0012] Detection wavelength: 248~370nm;

[0013] Mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B);

[0014] The elution method is gradient elution, and the gradient elution program is shown in Table 1;

[0015] Table 1 Gradient elution program

[0016]

[0017]

[0018] Preferably, in S1, the volume concentration of ethanol is 70%; the ultrasonic temperature is 60° C., the ultrasonic power is 420 W, and the ultrasonication is performed for 30 min.

[0019] Preferably, in S1, after ultrasonic treatment, the sample is weighed again. If the weight is inconsistent with the weight before ultrasonic treatment, 70% ethanol is added to make up to the weight before ultrasonic treatment, and the sample is filtered through a 0.22 μm microporous filter membrane after being shaken well.

[0020] Preferably, in S2, the chromatographic conditions further include:

[0021] Flow rate: 1.0 mL min -1 ;

[0022] Column temperature: 20°C;

[0023] Column specifications: 250mm×4.6mm, 5μm;

[0024] Injection volume: 10 μL;

[0025] Wavelength: 276nm (0~20min), 360nm (20~30min), 276nm (30~35min), 370nm (35~48min), 248nm (48~55min).

[0026] Preferably, the method for establishing the fingerprint spectrum of liquorice further comprises preparing a mixed reference solution, and the preparation method of the mixed reference solution comprises the following steps:

[0027] Eleven reference substances, including licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid, were respectively dissolved in methanol and ultrasonically dissolved to obtain respective solutions, and the solutions were mixed to obtain a mixed reference substance solution.

[0028] Preferably, the mass concentrations of solutions of licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid are 0.6 mg / ml, 0.22 mg / ml, 0.20 mg / ml, 0.04 mg / ml, 0.01 mg / ml, 0.22 mg / ml, 0.01 mg / ml, 0.015 mg / ml, 0.8 mg / ml, 0.04 mg / ml and 0.02 mg / ml, respectively.

[0029] Preferably, the mass concentrations of apigenin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, licorice, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid in the mixed reference solution are: 0.300 mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.200mg·mL -1 , 0.200mg·mL -1 , 0.220mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.680mg·mL -1 , 0.200mg·mL -1 and 0.200 mg mL -1 .

[0030] Preferably, the mixed reference solution is tested using the same HPLC conditions as the test solution to obtain a chromatogram of the mixed reference solution, and the chromatogram of the mixed reference solution is compared with the wine-roasted licorice fingerprint to identify the chromatographic peaks in the wine-roasted licorice fingerprint.

[0031] In another aspect of the present invention, a fingerprint spectrum of liquorice prepared by the above method is provided, wherein the glycyrrhizic acid peak is used as a reference peak, and the fingerprint spectrum of liquorice prepared by the above method includes 11 common peaks, and the relative retention time RSD of the 11 common peaks is between 0.06% and 1.01%.

[0032] Preferably, the 11 common peaks are peaks 10, 11, 18, 20, 23, 25, 32, 35, 36, 49, and 58, which are respectively licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, and glycyrrhetinic acid.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention provides a method for establishing a fingerprint spectrum of wine-fried licorice and establishes a fingerprint spectrum of wine-fried licorice, which has 11 common peaks that are effectively separated, can ensure the quality stability of wine-fried licorice, avoid the influence of component fluctuation on drug efficacy, provide data support for the production process of wine-fried licorice, and ensure the consistency of different batches of products;

[0035] (2) The present invention tested 15 batches of wine-fried licorice, and the results showed that the relative retention time RSD of the chromatographic peaks at the same position was within 1.01% or less, indicating that the fingerprint provided by the present invention has good reproducibility;

[0036] (3) The method provided by the present invention can not only detect the flavonoid components in wine-fried licorice, but also detect the triterpenoid components in wine-fried licorice, and determine the contents of 11 components, which is conducive to comprehensive monitoring of the quality of wine-fried licorice and provides a theoretical basis for the quality evaluation of wine-fried licorice. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The HPLC fingerprints of wine-fried licorice (JZ1-JZ15) and the reference fingerprint (R), wherein S1-15 is JZ1-JZ15;

[0038] Figure 2 It is a chromatogram of a mixed reference substance, wherein, peak 10-apiosyl liquiritin, peak 11-liquiritin, peak 18-apiosyl isoliquiritin, peak 20-isoliquiritin, peak 23-licorice chalcone B, peak 25-liquiritigenin, peak 32-spiny licorice chalcone, peak 35-isoliquiritigenin, peak 36-glycyrrhizic acid, peak 49-licorice chalcone A, peak 58-glycyrrhetinic acid;

[0039] Figure 3 It is the chromatogram of Comparative Example 1;

[0040] Figure 4 It is the chromatogram of Comparative Example 2. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0042] Example 1

[0043] 1.1 Instruments and reagents

[0044] Agilent 1260 high performance liquid chromatograph (Agilent, USA); EX224ZH 1 / 10,000 electronic balance (Ohaus Instrument Co., Ltd., Changzhou); KQ-700DE numerical control ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); FW135 crusher (Tianjin Test Instrument Co., Ltd.). Licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, glycyrrhetinic acid (purity ≥ 98%) were purchased from Nanjing Puyi; rice wine was purchased from Zhejiang Guyue Longshan Shaoxing Wine Co., Ltd., acetonitrile, phosphoric acid, and methanol were all chromatographically pure, and water was purified water. Licorice medicinal materials were produced in Gansu and purchased from Longxi County Baibao Pharmaceutical Co., Ltd., with sample numbers G1 to G15.

[0045] 1.2 Methods

[0046] 1.2.1 Preparation of processed liquorice products

[0047] Wine-roasted licorice: Add rice wine to the raw licorice slices and mix well. Seal and let it soak for 1.5 hours. Place in a preheated frying container, fry at 70℃ for 5 minutes, and let cool.

[0048] Add 10g of yellow wine to every 100g of raw licorice slices. Take an appropriate amount of wine-fried licorice slices and crush them into powder, pass through a No. 4 pharmacopoeia sieve, and set aside. Take 0.5g of each portion, and take 15 portions, with the corresponding numbers being wine-fried licorice 1 (JZ1) to wine-fried licorice (JZ15).

[0049] 1.2.2 Preparation of mixed reference solution

[0050] Appropriate amounts of 11 reference substances, including apigenin liquiritin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, liquiritigenin, licorice chalcone spinosa, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, and glycyrrhetinic acid, were accurately weighed and dissolved in methanol by ultrasonication to prepare the substances with mass concentrations of 0.6 mg / ml, 0.22 mg / ml, 0.20 mg / ml, 0.04 mg / ml, 0.01 mg / ml, 0.22 mg / ml, 0. 0.01 mg / ml, 0.015 mg / ml, 0.8 mg / ml, 0.04 mg / ml and 0.02 mg / ml solutions were prepared, and the above solutions were mixed to obtain a mixed reference solution, and the mass concentrations of apigenin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid in the mixed reference solution were kept at 0.300 mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.200mg·mL -1, 0.200mg·mL -1 , 0.220mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.680mg·mL -1 , 0.200mg·mL -1 and 0.200 mg mL -1 Filter the mixed reference solution through a microporous filter (0.22 μm) and set aside.

[0051] 1.2.3 Preparation of test solution

[0052] Take 0.5g of powder of wine-roasted licorice samples (JZ1-JZ15) respectively, add 10mL of 70% ethanol accurately, and weigh them respectively: 80.44g, 80.42g, 80.41g, 80.40g, 80.43g, 80.44g, 80.41g, 80.42g, 80.44g, 80.45g, 80.43g, 80.41g, 80.42g, 80.40g and 80.46g, then ultrasonicate for 30min respectively, take out, cool, and weigh again, the masses are: 80.43g, 80.41g, 80.40g, 80.39g, 80.41g, 80.42g, 80.40g, 80.40g, 80.42g, 80.44g, 80.42g, 80.39g, 80.41g, 80.39g, 80.45g, the weight of 15 samples decreased to varying degrees, which was inconsistent with the weight before ultrasonic treatment. 70% ethanol was used to make up the weight to the pre-ultrasonic weight, shaken well, and filtered with 0.22μm microporous filter membrane respectively. The filtrate was taken as the test solution and numbered JZR1 to JZR15 respectively.

[0053] 1.2.4 High performance liquid chromatography

[0054] The chromatographic conditions of HPLC are:

[0055] Column: Agilent Eclipse XDB-C 18 (250 mm × 4.6 mm, 5 μm);

[0056] Detection wavelength: 276nm (0-20min), 360nm (20-30min), 276nm (30-35min), 370nm (35-48min), 248nm (48-55min);

[0057] Mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B);

[0058] The elution method is gradient elution, and the gradient elution program is shown in Table 1 in the Summary of the Invention;

[0059] Flow rate: 1.0 mL min -1 ;

[0060] Column temperature: 20°C;

[0061] Injection volume: 10 μL.

[0062] The test solution in 1.2.3 was tested using the above HPLC conditions, and the fingerprint of liquorice was generated from the obtained chromatographic data according to the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition";

[0063] The mixed reference solution in 1.2.2 is tested using the above-mentioned HPLC conditions to obtain a chromatogram of the mixed reference solution, and the chromatogram of the mixed reference solution is compared with the wine-roasted licorice fingerprint to identify the chromatographic peaks in the wine-roasted licorice fingerprint.

[0064] Taking the glycyrrhizic acid peak as the reference peak, the wine-roasted licorice fingerprint includes 11 common peaks, namely peaks 10, 11, 18, 20, 23, 25, 32, 35, 36, 49, and 58, which are licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, and glycyrrhetinic acid, respectively.

[0065] Example 2

[0066] Precision experiment

[0067] Take the wine-fried licorice test solution (No. JZR1) in Example 1, and measure it according to the conditions of 1.2.4 in Example 1, inject 6 times continuously, and record the HPLC chromatogram. Take the glycyrrhizic acid peak as the reference peak, calculate the relative peak area and relative retention time of each common peak and the reference peak, the results are shown in Table 2 and Table 3, and the RSD of the relative retention time of each chromatographic peak is less than 0.52%, and the RSD of the relative peak area is less than 1.09%, indicating that the instrument has good precision.

[0068] Table 2 Relative retention time of common peaks

[0069]

[0070] Table 3 Relative peak areas of common peaks

[0071]

[0072]

[0073] Example 3

[0074] Repeatability test

[0075] Take 6 portions of the same batch of wine-roasted licorice sample powder (No. JZ1) in Example 1, prepare according to the method in 1.2.3 in Example 1, measure according to the conditions in 1.2.4 in Example 1, and record the HPLC chromatogram. Take the glycyrrhizic acid peak as the reference peak, calculate the relative peak area and relative retention time of each common peak and the reference peak, the results are shown in Table 4 and Table 5, and the RSD of the relative retention time of each chromatographic peak is less than 0.68%, and the RSD of the relative peak area is less than 1.92%, indicating that the method has good repeatability.

[0076] Table 4 Relative retention time of common peaks

[0077]

[0078] Table 5 Relative peak areas of common peaks

[0079]

[0080]

[0081] Example 4

[0082] Stability test

[0083] Take the wine-fried licorice test solution (No. JZR1) in Example 1, place it at room temperature for 0, 2, 4, 8, 12, and 24 hours, measure it according to the conditions in 1.2.4 in Example 1, and record the HPLC chromatogram. Taking the glycyrrhizic acid peak as the reference peak, calculate the relative peak area and relative retention time of each common peak and the reference peak, and the results are shown in Table 6 and Table 7, and calculate that the RSD of the relative retention time of each chromatographic peak is less than 0.82%, and the RSD of the relative peak area is less than 1.92%, indicating that the test solution has good stability within 24 hours.

[0084] Table 6 Relative retention time of common peaks

[0085]

[0086] Table 7 Relative peak areas of common peaks

[0087]

[0088]

[0089] Example 5

[0090] 5.1 Establishment of fingerprint of wine-roasted licorice

[0091] The 15 batches of liquorice test sample solutions (JZR1-JZR15) in Example 1 were measured according to the conditions in 1.2.4 of Example 1, and the HPLC chromatograms were recorded. The chromatograms were integrated and processed, and then imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) software for analysis in CDF format. JZR1 was used as the reference spectrum, and the time window width was set to 0.5 min. The superimposed fingerprint spectrum and the reference fingerprint spectrum (R) were generated by multi-point correction, automatic peak matching and median method. The spectrum is shown in FIG. Figure 1 As shown in the results, 61 common peaks were identified in wine-fried licorice (the common peak area accounted for 94% of the total chromatographic peak area).

[0092] 5.2 Similarity Evaluation

[0093] The HPLC fingerprint of wine-fried licorice and the reference fingerprint were analyzed for similarity using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software. Results The similarities of the 15 batches of wine-fried licorice were 0.984, 0.984, 0.985, 0.985, 0.984, 0.984, 0.985, 0.980, 0.981, 0.980, 0.984, 0.980, 0.984, 0.984, 0.984, and 0.984, respectively.

[0094] 5.3 Identification and analysis of common peaks

[0095] Take the mixed reference substance solution in 1.2.2 of Example 1, and measure it according to the conditions in 1.2.4 of Example 1 to obtain a chromatogram of the mixed reference substance. The chromatogram is as shown in Figure 2 shown.

[0096] The retention time comparison method was used to identify 11 chromatographic peaks, namely peaks 10, 11, 18, 20, 23, 25, 32, 35, 36, 49, and 58, which were respectively apigenin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, and glycyrrhetinic acid. Among them, the retention time of the glycyrrhizic acid chromatographic peak was relatively moderate, the peak area was large, and the separation degree with the adjacent chromatographic peaks was very good. Therefore, glycyrrhizic acid was used as the reference peak of the fingerprint spectrum to calculate the relative retention time and relative peak area of ​​other common peaks. The relative retention time determination results of the fingerprint spectrum of 15 batches of liquor-fried licorice are shown in Table 8. Results The relative retention time RSDs of the common peaks of the liquor-fried licorice fingerprint spectrum were 0.06% to 1.01%, respectively, and it can be seen that the retention time of the common peaks is relatively stable.

[0097] Table 8 Relative retention time determination results of fingerprints of 15 batches of wine-roasted licorice

[0098]

[0099] Example 6

[0100] 6.1 Investigation of linear relationship

[0101] Accurately pipette each reference substance solution in 1.2.2 of Example 1, place it in a 10 mL volumetric flask, dilute to volume with methanol, mix by ultrasonication, obtain reference substance solutions of different concentrations, filter through a 0.22 μm microporous membrane, measure according to the conditions in 1.2.4 of Example 1, and record the chromatogram. Take the peak area (Y) as the ordinate and the mass concentration of the chemical component (X, μg·μL -1 ) as the horizontal axis, and the standard curve was drawn. The correlation coefficient, regression equation and linear range are shown in Table 9. The results show that the 11 chemical components have a good linear relationship.

[0102] Table 9 Regression equation and linear range

[0103]

[0104] 6.2 Precision experiment

[0105] Take the mixed reference solution in Example 1, inject it continuously 6 times according to the conditions of 1.2.4 in Example 1, and record the chromatogram.

[0106] Results The RSDs of the peak areas of licorice root, licorice root, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid were 0.34%, 0.33%, 0.27%, 0.33%, 0.33%, 0.32%, 0.32%, 1.99%, 1.12%, 0.35% and 0.26% (n=6), respectively, indicating that the instrument has good precision.

[0107] 6.3 Repeatability study

[0108] Take 6 portions of the wine-roasted licorice test solution (No. JZR1) from the same batch of Example 1, measure it under the conditions of 1.2.4 in Example 1, and record the chromatogram.

[0109] Results The RSDs of the peak areas of licorice root, licorice root, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid were 0.52%, 0.41%, 0.49%, 0.40%, 0.82%, 0.73%, 0.91%, 1.40%, 0.13%, 0.35% and 0.43% (n=6), respectively, indicating that the method had good repeatability.

[0110] 6.4 Stability Study

[0111] Take the wine-roasted licorice test solution (No. JZR1) in Example 1, place it at room temperature for 0, 2, 4, 8, 12, and 24 hours, measure it according to the conditions of 1.2.4 in Example 1, and record the chromatogram.

[0112] Results The RSDs of the peak areas of licorice root, licorice root, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid were 0.99%, 0.49%, 0.11%, 0.13%, 1.45%, 0.90%, 0.58%, 0.47%, 0.10%, 1.09% and 0.52% (n=6), respectively, indicating that the test solution had good stability within 24 hours.

[0113] 6.5 Sample recovery test

[0114] Take 6 portions of liquor-roasted licorice sample powder (No. JZ1) with known content in Example 1, each portion is about 0.5g, accurately weighed, accurately add appropriate amount of apigenin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, glycyrrhetinic acid reference solution in Example 1, prepare the test solution according to the method in 1.2.3 of Example 1, measure according to the conditions in 1.2.4 of Example 1, record the chromatogram and calculate the sample recovery rate and RSD of 11 chemical components. The results are shown in Table 10, the average recovery rate of 11 chemical components is 100.26% to 103.02%, and the RSD is 0.13% to 1.40%.

[0115] Table 10 Sample recovery test results

[0116]

[0117]

[0118] 6.6 Sample content determination

[0119] Take 15 batches of wine-fried licorice samples (numbered JZ1 to JZ15) in Example 1, prepare test solutions according to the method in 1.2.3 of Example 1, measure according to the conditions in 1.2.4 of Example 1, record the chromatogram and peak area, and calculate the contents of 11 chemical components based on the dry product. The results are shown in Table 11. It can be seen that the method of the present invention can determine 11 chemical components and their contents in wine-fried licorice.

[0120] Table 11 Determination results of wine-fried licorice samples (n=3, mg·g -1 )

[0121]

[0122] Comparative Example 1

[0123] The difference from Example 1 is that in the chromatographic conditions, the mobile phase is methanol (A)-0.1% phosphoric acid aqueous solution (B), and the other steps and condition parameters are the same as those in Example 1. The mixed reference solution is measured, and the results are as follows: Figure 3 As shown by Figure 3 It can be seen that when using this mobile phase, the chromatographic peak separation is poor and the peak area is small, which is not suitable for the determination of mixed reference substances and test substances.

[0124] Comparative Example 2

[0125] The difference from Example 1 is that in the chromatographic conditions, the gradient elution is as shown in Table 12, and the other steps and condition parameters are the same as those in Example 1. The mixed reference solution is measured, and the results are as follows: Figure 4 As shown by Figure 4 It can be seen that when this gradient elution program is used, the chromatographic peaks are incomplete and good separation cannot be achieved, making it unsuitable for the determination of mixed reference substances and test substances.

[0126] Table 12 Gradient elution program

[0127]

Claims

1. A method for establishing a fingerprint spectrum of liquorice, characterized in that: The following steps are involved: S1, prepare the test solution: take the wine-roasted licorice sample powder and sieve it, add ethanol solution, weigh and record the weight, then perform ultrasonic treatment, and then filter and take the filtrate as the test solution; S2, using high performance liquid chromatography to test the sample solution obtained in S1, and generating a fingerprint of liquorice from the obtained chromatographic data according to the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition"; The chromatographic conditions of the high performance liquid chromatography are: Column: Agilent Eclipse XDB-C 18 ; Detection wavelength: 248~370nm; Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid aqueous solution; The elution method is gradient elution, and the gradient elution program is shown in Table 1; Table 1 Gradient elution program 2. The method according to claim 1, characterized in that In S1, the volume concentration of ethanol was 70%, the ultrasonic temperature was 60°C, the ultrasonic power was 420W, and the ultrasonication lasted for 30 min.

3. The method according to claim 1, characterized in that In S1, the pore size of the microporous filter membrane during filtration is 0.22 μm.

4. The method according to claim 1, characterized in that In S1, after ultrasonic treatment, the sample is weighed again. If the weight is different from the weight before ultrasonic treatment, 70% ethanol is added to make up to the weight before ultrasonic treatment, and the sample is filtered after being shaken.

5. The method according to claim 1, characterized in that In S2, the chromatographic conditions further include: Flow rate: 1.0 mL min -1 ; Column temperature: 20°C; Column specifications: 250mm×4.6mm, 5μm; Injection volume: 10 μL; Wavelength: The wavelength of 0-20min is 276nm, the wavelength of 20-30min is 360nm, the wavelength of 30-35min is 276nm, the wavelength of 35-48min is 370nm, and the wavelength of 48-55min is 248nm.

6. The method according to claim 1, characterized in that The method further comprises preparing a mixed reference solution, wherein the preparation method of the mixed reference solution comprises the following steps: 11 reference substances, including apigenin liquiritin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, liquiritigenin, licorice chalcone spinosa, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid, were respectively dissolved in methanol, and ultrasonically dissolved to obtain respective solutions, and the solutions were mixed to obtain a mixed reference substance solution; The mixed reference solution is tested under the same HPLC conditions as the test solution to obtain a chromatogram of the mixed reference solution. The chromatogram of the mixed reference solution is compared with the wine-roasted licorice fingerprint to identify the chromatographic peaks in the wine-roasted licorice fingerprint.

7. The method according to claim 6, characterized in that The mass concentrations of solutions of licorice root, licorice root, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, liquiritigenin, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid are 0.6 mg / ml, 0.22 mg / ml, 0.20 mg / ml, 0.04 mg / ml, 0.01 mg / ml, 0.22 mg / ml, 0.01 mg / ml, 0.015 mg / ml, 0.8 mg / ml, 0.04 mg / ml and 0.02 mg / ml respectively.

8. The method according to claim 6, characterized in that The mass concentrations of apigenin, liquiritin, apigenin isoliquiritin, isoliquiritin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A and glycyrrhetinic acid in the mixed reference solution are: 0.300 mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.200mg·mL -1 , 0.200mg·mL -1 , 0.220mg·mL -1 , 0.200mg·mL -1 , 0.300mg·mL -1 , 0.680mg·mL -1 , 0.200mg·mL -1 , 0.200mg·mL -1 .

9. The fingerprint of liquorice root obtained by the method according to any one of claims 1 to 8 is characterized in that: Taking the glycyrrhizic acid peak as the reference peak, the wine-roasted licorice fingerprint includes 11 common peaks, and the 11 common peaks are peaks 10, 11, 18, 20, 23, 25, 32, 35, 36, 49, and 58, which are respectively licorice glycoside, licorice glycoside, licorice isoliquiritigenin, isoliquiritigenin, licorice chalcone B, licoricein, licorice chalcone, isoliquiritigenin, glycyrrhizic acid, licorice chalcone A, and glycyrrhetinic acid.

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