Construction method of fingerprint of gout compound decoction and its fingerprint and application

Abstract

The present application discloses a method for constructing a fingerprint of a gout compound decoction and its fingerprint and application, and belongs to the field of analytical detection technology. The method for constructing the fingerprint comprises the following steps: taking a test solution prepared from a gout compound decoction; performing HPLC-SPD detection on the test solution to obtain a fingerprint of the gout compound decoction, wherein the high performance liquid chromatography detection conditions are: using a C18 chromatographic column; using an aqueous solution containing 0.1% to 0.2% phosphoric acid as mobile phase A, and acetonitrile as mobile phase B for gradient elution; the flow rate is 0.8 to 1 mL / min, the column temperature is 25 ° C, the injection volume is 5 to 10 μL, and the diode array detector is scanned at 190-400 nm full band. The present application conducts a comprehensive and systematic analysis of the chemical components in the gout compound decoction, providing a basis for in-depth research on quality control and the basis of pharmacological substances.

Description

Technical Field

[0001] The present application belongs to the technical field of traditional Chinese medicine analysis and detection, and specifically relates to a method for constructing a fingerprint spectrum of a gout compound decoction, its fingerprint spectrum, and application. Background Art

[0002] The Chinese patent application with application number 2021111405783 discloses a traditional Chinese medicine composition for the treatment of gout, which has been clinically verified to have good uric acid-lowering, anti-inflammatory and other effects, and laboratory pharmacodynamic verification has shown that it does have good anti-hyperuricemia and anti-inflammatory effects. As a new type of compound preparation, the gout compound traditional Chinese medicine composition has a complex chemical composition and contains more than 10 single drug ingredients. At present, the material basis and mechanism of action of the compound still need in-depth study. It is worth noting that due to differences in the origin of medicinal materials, differences in intermediate links such as decoction preparation technology, the safety, stability and effectiveness of the compound preparation may fluctuate. Therefore, establishing an effective quality control method to comprehensively reflect the types and contents of compounds in the compound preparation, and then achieving quality control of the gout compound preparation, has become the primary task of promoting the research and clinical transformation of the compound preparation.

[0003] Common quality control methods, such as thin layer chromatography (TLC) identification of a single indicator and content determination of a single ingredient, are unable to comprehensively assess the overall quality of a TCM compound. TCM fingerprints can more comprehensively reflect the types and quantities of chemical components contained in TCM, thereby providing an overall description and evaluation of the drug's quality. By comparing TCM atlases from different batches / sources with reference atlases and combining similarity evaluation, a reasonable assessment of the quality and authenticity of the sample can be made. Furthermore, fingerprints, combined with the linear regression equation of a standard, can also provide specific content information for certain indicator components, enabling quality control of TCM compounds to not only achieve qualitative analysis but also control the absolute content of representative components, thereby further improving the overall quality control level of TCM compounds.

[0004] Therefore, it is very necessary to develop a method for constructing the fingerprint of the above-mentioned gout compound decoction. Summary of the Invention

[0005] In view of this, the primary purpose of this application is to provide a method for constructing a fingerprint of a gout compound decoction, which can comprehensively and accurately achieve quality control of the gout compound decoction, ensure its efficacy, and promote the quality control, clinical research and application of the gout compound decoction.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] One aspect of the present application discloses a method for constructing a fingerprint of a gout compound decoction, comprising the following steps:

[0008] A test solution of a gout compound decoction is obtained by decocting and concentrating a traditional Chinese medicine composition for treating gout; the traditional Chinese medicine composition is composed, by weight, of 5-100 parts of cinnamon twig, 5-100 parts of red peony root, 5-100 parts of anemarrhena, 5-100 parts of phellodendron, 7-100 parts of siler, 5-100 parts of gentiana macrophylla, 7-50 parts of aconite root, 5-100 parts of radix polygoni multiflori, 5-100 parts of radix aconiti lobatae, 5-100 parts of achyranthes bidentatae, 5-100 parts of codonopsis pilosula, 10-80 parts of poria peel, and 5-50 parts of liquorice;

[0009] The test solution is subjected to HPLC-SPD detection to obtain the fingerprint of the gout compound decoction, wherein the high performance liquid chromatography detection conditions are:

[0010] A chromatographic column filled with octadecyl bonded silica gel was used; an aqueous solution containing 0.1% to 0.2% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution; the gradient elution program was as follows: 0 to 5 min, 95% A; 5 to 10 min, 95% A→85% A; 10 to 40 min, 85% A→80% A; 40 to 50 min, 80% A→65% A; 50 to 55 min, 65% A→35% A; 55 to 58 min, 35% A→95% A; 58 to 65 min, 95% A; the flow rate was 0.8 to 1 mL / min, the column temperature was 25°C, the injection volume was 5 to 10 μL, and the diode array detector was used for full-band scanning at 190-400 nm.

[0011] Another aspect of the present application discloses a fingerprint of a compound gout decoction, obtained using the construction method described above;

[0012] The fingerprint spectrum identified 8 characteristic peaks, namely, loganic acid at peak 9, phellodendronine at peak 11, gentiopicroside at peak 17, mangiferin at peak 18, paeoniflorin at peak 21, berberine at peak 27, cinnamic acid at peak 28, and glycyrrhizic acid monoamine salt at peak 29.

[0013] Taking berberine, peak 27, as the reference peak, the relative retention times of each peak were: loganinic acid, peak 9, 0.32±10%, phellodendronine, peak 11, 0.35±10%, gentiopicroside, peak 17, 0.43±10%, mangiferin, peak 18, 0.43±10%, paeoniflorin, peak 21, 0.53±10%, cinnamic acid, peak 28, 1.11±10%, and glycyrrhizic acid monoamine salt, peak 29, 1.14±10%.

[0014] Another aspect of the present application discloses the application of the fingerprint obtained by the construction method described above in the quality control of the gout compound decoction.

[0015] Beneficial effects of this application:

[0016] This application is aimed at the traditional Chinese medicine composition for treating gout disclosed in the Chinese patent application with application number 2021111405783, and constructs its fingerprint for the first time. It abandons the problem of single evaluation index of traditional Chinese medicine compound and selects a variety of reference ingredients. The fingerprint of the gout compound obtained has high separation and good peak shape. Each characteristic peak is well identified, which can effectively reflect the difference in the content of ingredients in different samples and more comprehensively reflect the overall quality of the sample. It can accurately and comprehensively evaluate the quality of the traditional Chinese medicine composition for treating gout, and is suitable for the control of the treatment of traditional Chinese medicine composition products for treating gout.

[0017] The fingerprint of the gout compound established in this application combined with the multi-component content determination method is of great significance for the quality control of the composition and is conducive to its standardized production and clinical application.

[0018] This application conducts similarity evaluation by comparing the similarity between the sample spectrum and the standard spectrum, thereby achieving quality control of the sample more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The fingerprints of 10 batches of gout compound decoction in Example 1 and the generated control maps are shown.

[0020] Figure 2 The chromatogram of the mixed reference solution in Example 1 is shown.

[0021] Figure 3 The chromatograms of samples using different flow rates in Example 4 are shown.

[0022] Figure 4 The chromatograms are of samples with different phosphoric acid concentrations added in Example 4.

[0023] Figure 5 The chromatograms of the samples using different mobile phases in Comparative Example 1 are shown.

[0024] Figure 6 The chromatograms of the samples at different column temperatures in Comparative Example 2 are shown. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the embodiments of the present application. The technical solutions in the embodiments described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the embodiments of the present application to obtain other embodiments without creative work, and these embodiments are also within the scope of protection of the present application.

[0026] The first aspect of the present application discloses a method for constructing a fingerprint of a gout compound decoction, comprising the following steps:

[0027] A test solution of a gout compound decoction is obtained by decocting and concentrating a traditional Chinese medicine composition for treating gout; the traditional Chinese medicine composition is composed, by weight, of 5-100 parts of cinnamon twig, 5-100 parts of red peony root, 5-100 parts of anemarrhena, 5-100 parts of phellodendron, 7-100 parts of siler, 5-100 parts of gentiana macrophylla, 7-50 parts of aconite root, 5-100 parts of radix polygoni multiflori, 5-100 parts of radix aconiti lobatae, 5-100 parts of achyranthes bidentatae, 5-100 parts of codonopsis pilosula, 10-80 parts of poria peel, and 5-50 parts of liquorice;

[0028] The test solution is subjected to HPLC-SPD detection to obtain the fingerprint of the gout compound decoction, wherein the high performance liquid chromatography detection conditions are:

[0029] A chromatographic column filled with octadecyl bonded silica gel was used; an aqueous solution containing 0.1% to 0.2% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution; the gradient elution program was as follows: 0 to 5 min, 95% A; 5 to 10 min, 95% A→85% A; 10 to 40 min, 85% A→80% A; 40 to 50 min, 80% A→65% A; 50 to 55 min, 65% A→35% A; 55 to 58 min, 35% A→95% A; 58 to 65 min, 95% A; the flow rate was 0.8 to 1 mL / min, the column temperature was 25°C, the injection volume was 5 to 10 μL, and the diode array detector was used for full-band scanning at 190-400 nm.

[0030] In this application, when content, flow rate, volume, concentration or other parameters are expressed as ranges, preferred ranges or a series of upper preferred values ​​and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when a range of "5 to 10" is disclosed, the described range should be understood to include the ranges "5 to 7", "5 to 8", "6 to 8", "6 to 10", etc. When a numerical range is described in this application, unless otherwise stated, the range should include its end values ​​and all integers and fractions within the range.

[0031] In some specific examples, the preparation of the test solution includes the following steps:

[0032] Accurately measure 1 mL of Gout Compound Decoction, add solvent to make up to 10 mL, mix well, centrifuge, take the supernatant and filter to prepare the test solution.

[0033] In some specific examples, the preparation of the gout compound decoction includes the following steps:

[0034] According to the mass ratio, the Chinese medicine composition for treating gout was taken, without crushing and sieving, and 10 times the amount of water was added and soaked for half an hour, and the mixture was decocted twice, and the filtrate was combined to obtain a gout compound decoction.

[0035] As a preferred example, the gout compound decoction is composed of the following components in parts by mass: 100 parts of cinnamon twig, 100 parts of red peony root, 100 parts of anemarrhena, 100 parts of phellodendron, 70 parts of siler, 100 parts of gentiana, 50 parts of aconite root, 100 parts of radix polygoni multiflori, 100 parts of achyranthes bidentata, 100 parts of codonopsis pilosula, 80 parts of poria peel and 50 parts of liquorice.

[0036] In some specific examples, the solvent used to prepare the test solution is methanol.

[0037] In some specific examples, the specifications of the chromatographic column are an inner diameter of 4.6 mm, a column length of 250 mm, and a filler particle size of 5 μm; as a preferred example, the chromatographic column is a ShimNex CS C18 chromatographic column.

[0038] It is understood that the construction method also includes the preparation of a mixed reference solution, and the preparation of the mixed reference solution includes the following steps:

[0039] Take reference substances loganinic acid, phellodendrine, gentiopicroside, mangiferin, paeoniflorin, berberine, cinnamic acid, and glycyrrhizic acid monoamine salt respectively, dissolve them in solvent, and prepare mother solution for storage;

[0040] Accurately measure the mother solution of each reference substance and mix them evenly to prepare a mixed reference substance solution;

[0041] In the mixed reference solution, the concentrations of each reference substance are: loganin 111.11 μg / mL, phellodendronine 155.4 μg / mL, gentiopicroside 111.11 μg / mL, mangiferin 83.25 μg / mL, paeoniflorin 155.4 μg / mL, berberine 83.25 μg / mL, cinnamic acid 83.25 μg / mL, and glycyrrhizic acid monoamine salt 111.11 μg / mL.

[0042] In some specific examples, the construction method further includes the step of generating a fingerprint of the gout compound decoction:

[0043] The obtained chromatogram was imported into the 2012 version of the Chinese medicine fingerprint similarity evaluation software. The S1 batch was used as the reference chromatogram, the time window width was set to 0.1 min, and the median method was used to perform multi-point correction and full-spectrum peak matching to generate the fingerprint of the gout compound decoction. Among them, the combined medicinal materials in the S1 batch were authentic medicinal materials.

[0044] In some specific examples, the construction method further includes a step of substance quantification:

[0045] The external standard method was used to detect the peak area of ​​paeoniflorin at a wavelength of 230 nm, the peak area of ​​loganic acid, mangiferin, berberine, and glycyrrhizic acid monoamine salt at 258 nm, the peak area of ​​cinnamic acid at 270 nm, and the peak area of ​​phellodendronine and gentiopicroside at 284 nm, to achieve quantification of the corresponding substances in the sample.

[0046] The second aspect of the present application discloses a fingerprint of a compound gout decoction, obtained by the construction method described above;

[0047] By comparing with the mixed reference substance, the fingerprint spectrum identified 8 characteristic peaks, namely, loganic acid at peak 9, phellodendronine at peak 11, gentiopicroside at peak 17, mangiferin at peak 18, paeoniflorin at peak 21, berberine at peak 27, cinnamic acid at peak 28, and glycyrrhizic acid monoamine salt at peak 29;

[0048] Since berberine has a sharp peak shape, a high chromatographic peak response value, and a high degree of separation from other peaks, berberine is selected as the reference peak. Therefore, in this application, peak 27 berberine is used as the reference peak, and the relative retention times of the peaks are: peak 9 loganic acid 0.32±10%, peak 11 phellodendronine 0.35±10%, peak 17 gentiopicroside 0.43±10%, peak 18 mangiferin 0.43±10%, peak 21 paeoniflorin 0.53±10%, peak 28 cinnamic acid 1.11±10%, peak 29 glycyrrhizic acid monoamine salt 1.14±10%.

[0049] The third aspect of the present application discloses the application of the fingerprint obtained by the construction method described above in the quality control of the gout compound decoction.

[0050] In specific application, the fingerprint spectrum construction method described in the first aspect of this application is used for sample injection detection, and the similarity software of the traditional Chinese medicine fingerprint spectrum is imported to generate a control fingerprint spectrum. The control fingerprint spectrum is used as a reference. If the similarity between the sample to be tested and the control spectrum is greater than 0.9, it indicates that the quality is qualified.

[0051] The technical solution of the present application is further illustrated below with reference to specific embodiments. It should be noted that the following specific embodiments are only for illustrative purposes and do not limit the scope of the present application in any way.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0053] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0054] The traditional Chinese medicine composition for treating gout described in this application is the one described in the Chinese patent application with application number 2021111405783, and the preparation method of the compound gout decoction is as disclosed in the Chinese patent application with application number 2021111405783: according to mass parts, take 100 parts of cinnamon twig, 100 parts of red peony root, 100 parts of anemarrhena, 100 parts of phellodendron, 70 parts of saposhnikovia, 100 parts of gentiana, 50 parts of aconite root, 100 parts of radix polygoni multiflori, 100 parts of Achyranthes bidentata, 100 parts of Codonopsis pilosula, 80 parts of Poria peel and 50 parts of liquorice, without crushing and sieving, add 10 times the amount of water and soak for half an hour, decoct twice, combine the filtrate, and obtain the compound gout decoction.

[0055] The origin information of the compatibility of medicinal materials in 10 batches of Gout Compound Decoction is shown in Table 1:

[0056] Table 1 Origin information of the medicinal materials used in 10 batches of gout compound decoctions

[0057]

[0058] Note: All the combined medicinal materials in batch S1 are authentic medicinal materials.

[0059] The reagents and instrument information involved in the examples in this application are as follows:

[0060] 1. Reagents:

[0061] Table 2 Reference substance information

[0062]

[0063] Acetonitrile (chromatographic grade, Merck, USA), methanol (chromatographic grade, Merck, USA), phosphoric acid (chromatographic grade, Merck, USA), and water were ultrapure water.

[0064] 2. Instruments:

[0065] LC-40D (Shimadzu Corporation, Japan) (diode array detector SPD-M40); chromatographic column ShimNex CS C18 (250 mm × 4.6 mm, 5 μm, Shimadzu Corporation, Japan); balance (Sartorius, Germany); ultrasonic cleaning machine (XM-P15H, Xiaomei Ultrasonic Instrument Co., Ltd.).

[0066] Example 1 Establishment of fingerprint of gout compound decoction

[0067] 1.1 Preparation of test solution

[0068] Accurately measure 1 mL of Gout Compound Decoction, add methanol to make up to 10 mL, mix well, centrifuge at 12000 r / min for 15 min, take the supernatant, and filter to obtain the test solution.

[0069] 1.2 Preparation of mixed reference solution

[0070] Loganinic acid, phellodendronine, gentiopicroside, mangiferin, paeoniflorin, berberine, cinnamic acid, and glycyrrhizic acid monoamine salt were taken respectively, dissolved in methanol, and the mother liquor was prepared for storage; the mother liquor of each reference substance was accurately measured and mixed to prepare a mixed reference substance solution, the mass concentration of each reference substance in the mixed reference substance solution was 111.11 μg / mL for loganinic acid, 155.4 μg / mL for phellodendronine, 111.11 μg / mL for gentiopicroside, 83.25 μg / mL for mangiferin, 155.4 μg / mL for paeoniflorin, 83.25 μg / mL for berberine, 83.25 μg / mL for cinnamic acid, and 111.11 μg / mL for glycyrrhizic acid monoamine salt.

[0071] 1.3 Chromatographic conditions and HPLC-SPD detection

[0072] The chromatographic column was ShimNex CS C18 (250 mm × 4.6 mm, 5 μm), mobile phase A was an aqueous solution containing 0.1% phosphoric acid, mobile phase B was acetonitrile, and the gradient elution program was shown in Table 3. The flow rate was 1 mL / min, the column temperature was 25°C, the injection volume was 10 μL, and the SPD detector scanned the entire 190-400 nm band to obtain comprehensive sample information. By comparing the UV absorption of the substances at different wavelengths, the overall sample response at a wavelength of 230 nm was high, especially for the eight indicative quantitative components, and the chromatographic peak separation was moderate. Therefore, the chromatograms at a wavelength of 230 nm were selected for presentation in the subsequent results of this application.

[0073] Table 3 Gradient elution program

[0074]

[0075] 1.4 Fingerprint establishment and similarity evaluation

[0076] Take 10 batches of gout compound decoction in Table 1, prepare the test solution according to the method under "1.1", record the chromatogram of the sample according to the method under "1.3", and uniformly import the chromatograms of the 10 batches of gout compound decoction into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for data analysis. Use the S1 chromatogram as the reference spectrum, set the time window width to 0.1min, use the median method, perform multi-point correction and full spectrum peak matching, and generate the gout compound decoction fingerprint and the reference fingerprint, see Figure 1 , a total of 29 characteristic peaks were matched.

[0077] Prepare the mixed reference solution according to the method under "1.2" and test it according to the method under "1.3" to obtain the chromatogram of the mixed reference solution, such as Figure 2 As shown in the figure, based on the chromatogram of the mixed reference solution, peaks 9, loganinic acid (Rt = 16.58 min), 11, phellodendronine (Rt = 17.95 min), 17, gentiopicroside (Rt = 22.01 min), 18, mangiferin (Rt = 22.44 min), 21, paeoniflorin (Rt = 25.06 min), 27, berberine (Rt = 51.36 min), 28, cinnamic acid (Rt = 57.63 min), and 29, glycyrrhizic acid monoamine salt (Rt = 58.82 min) were further identified. Berberine had a sharp peak shape, high chromatographic response, and good resolution from other peaks, so it was selected as the reference peak.

[0078] Furthermore, the similarity evaluation results of the 10 batches of samples are shown in Table 4.

[0079] Table 4 Similarity evaluation results of 10 batches of samples

[0080]

[0081] Through similarity evaluation, it was found that the similarities of the chromatograms of each batch to the control chromatogram were 0.995, 0.991, 0.981, 0.99, 0.999, 0.991, 0.995, 0.987, 0.998, and 0.998, respectively, all greater than 0.9, indicating that the fingerprint spectrum constructed using the method of the present application is of stable quality and can reflect its fingerprint characteristics.

[0082] Example 2 Methodological Verification of the Fingerprint Construction Method of Gout Compound Decoction

[0083] 2.1 Precision experiment

[0084] Prepare the test solution from the same batch of samples (Batch S2 in Table 1) according to the method in "1.1." Repeat the injection six times according to the method in "1.3." Record the chromatograms. Using berberine as the reference peak (S peak), calculate the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks. See Tables 5 and 6 for the results.

[0085] Table 5 Precision test results of fingerprint of Gout Compound Decoction (relative retention time)

[0086]

[0087] Table 6 Fingerprint precision test results of gout compound decoction (relative peak area)

[0088]

[0089] It can be seen from Tables 5 and 6 that the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks range from 0.07% to 0.17% and from 0.86% to 2.79%, respectively, indicating that the fingerprint construction method in this application has good precision.

[0090] 2.2 Repeatability Experiment

[0091] From the same sample batch (Batch S1 in Table 1), prepare six parallel test solutions according to the method in "1.1." Inject and test according to the method in "1.3." Record the chromatograms. Using berberine as the reference peak (S peak), calculate the relative retention times and relative peak area RSDs for the common peaks. See Tables 7 and 8 for the results.

[0092] Table 7 Repeatability test results of fingerprint of gout compound decoction (relative retention time)

[0093]

[0094] Table 8 Repeatability test results of fingerprint of gout compound decoction (relative peak area)

[0095]

[0096] According to Tables 7 and 8, the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks were in the range of 0.15%-0.26% and 1.10%-3.76%, respectively, indicating that the method had good repeatability.

[0097] 2.3 Stability test

[0098] Prepare the test solution from the same batch of samples (Batch S1 in Table 1) according to the method in "1.1." Inject and test the solution at 0, 3, 6, 12, 24, and 30 hours according to the method in "1.3." Record the chromatograms. Using berberine as the reference peak (S peak), calculate the relative retention times and relative peak area RSDs for the common peaks. See Tables 9 and 10 for the results.

[0099] Table 9 Results of stability test of fingerprint of Gout Compound Decoction (relative retention time)

[0100]

[0101] Table 10 Results of stability test of fingerprint of compound gout decoction (relative peak area)

[0102]

[0103] From the test results in Tables 9 and 10, it can be seen that the RSD values ​​of the relative retention time and relative peak area of ​​the common peak ranged from 0.06% to 0.43% and 1.89% to 3.47%, respectively, indicating that the sample remained stable within 30 h.

[0104] Example 3 Determination of the content of multiple index components in the gout compound decoction

[0105] 3.1 Linear regression equation

[0106] Accurately pipette the mixed reference solution under "1.2" and perform gradient dilution to obtain mixed reference solutions of different concentrations (see Table 11). Inject and test according to the method under "1.3". Record the chromatograms of mixed reference solutions of different concentrations and calculate their peak areas. Use the reference concentration as the horizontal axis and the corresponding peak area as the vertical axis to draw the linear regression equation of the reference solution. The results are shown in Table 12.

[0107] Table 11 Mixed reference solutions of different concentrations (μg / mL)

[0108]

[0109] Table 12 Results of linear relationship investigation of 8 characteristic components

[0110]

[0111] 3.2 Determination of characteristic component content

[0112] Take 10 batches of standard gout compound decoction in Table 1, prepare test solution according to the method under "1.1", and inject and test according to the chromatographic conditions under "1.3", and record the chromatogram.

[0113] According to the external standard method, the peak area of ​​paeoniflorin at a wavelength of 230 nm, the peak area of ​​loganic acid, mangiferin, berberine, and glycyrrhizic acid monoamine salt at 258 nm, the peak area of ​​cinnamic acid at 270 nm, and the peak area of ​​phellodendronine and gentiopicroside at 284 nm were statistically analyzed. According to the standard curve equation listed in Table 12, the peak areas were substituted into the formula to calculate the mass fractions of loganic acid, gentiopicroside, glycyrrhizic acid monoamine salt, mangiferin, berberine, cinnamic acid, phellodendronine, and paeoniflorin in 10 batches of gout compound decoction. The results are shown in Table 13.

[0114] Table 13 Mass contents of eight ingredients in 10 batches of standard gout compound decoctions

[0115]

[0116] The results in Table 13 show that the mass fractions of loganinic acid, gentiopicroside, glycyrrhizic acid monoamine salt, mangiferin, berberine, cinnamic acid, phellodendronine, and paeoniflorin in the 10 samples ranged from 25.2 to 49.19 μg / g, 39.36 to 68.82 μg / g, 3.85 to 6.71 μg / g, 3.87 to 10.04 μg / g, 3.29 to 5.68 μg / g, 4.43 to 9.75 μg / g, 2.59 to 4.87 μg / g, and 9.38 to 30.03 μg / g, respectively. Among them, loganinic acid, gentiopicroside, and paeoniflorin were relatively high, with differences in content between batches, which may be related to the compatibility of medicinal materials from different sites.

[0117] The fingerprint construction method of this application can be used to determine the content of index components in gout compound prescriptions. Eight characteristic components were selected as index components for content determination. This method has good repeatability, accuracy, and stability, and can simultaneously determine the content of multiple index components. Combined with the fingerprint method, it can achieve more comprehensive quality control of gout compound prescriptions.

[0118] Example 4 Durability Test

[0119] 4.1 Different flow rates

[0120] Take the sample of batch S1 in Table 1, and inject and test it according to the method under "1.3" in Example 1 and record the chromatogram. The only difference is that the flow rate in the chromatographic conditions is set to 0.8 mL / min. Figure 3 .

[0121] The results showed that except for the changes in the elution time of some characteristic peaks, the overall number of peaks and peak areas remained almost unchanged. Therefore, when the flow rate was set at 0.8-1 mL / min, there was almost no effect on the chromatographic separation of the gout compound decoction.

[0122] 4.2 Different phosphoric acid addition concentrations

[0123] Take the sample from batch S1 in Table 1, and inject and test it according to the method under "1.3" in Example 1 and record the chromatogram, except that the phosphoric acid concentration in the chromatographic conditions is 0.2%. Figure 4 .

[0124] The results showed that compared with the 0.1% phosphoric acid concentration, when the phosphoric acid concentration was set to 0.2%, the overall peak number and time of the gout compound decoction were almost unaffected. Therefore, the 0.2% phosphoric acid concentration is also applicable to the separation of the gout compound.

[0125] Comparative Example 1

[0126] This comparative example provides another method for constructing a fingerprint of a gout compound decoction, which is carried out according to the chromatographic conditions in Example 1, with the only difference being that the mobile phase B is replaced by methanol from acetonitrile.

[0127] Take the sample of batch S1, and inject the sample according to the method under "1.3" in Example 1 and the chromatographic conditions in Comparative Example 1 and record the chromatogram. The results are as follows: Figure 5 As shown in .

[0128] like Figure 5 As shown, when the mobile phase is 0.1% phosphoric acid / methanol, the overall peak elution time is delayed and the separation of the substances is poor. However, when using 0.1% phosphoric acid / acetonitrile, the peak elution time of the target peak is shifted earlier and the peak shape is sharper. For this sample, acetonitrile has a greater elution capacity than methanol, making the use of 0.1% phosphoric acid / acetonitrile more conducive to the separation of the substances in the sample.

[0129] Comparative Example 2

[0130] This comparative example provides another method for constructing a fingerprint of a gout compound decoction, which is performed with reference to the chromatographic conditions in Example 1, with the only difference being that the column temperatures are set to 30°C and 35°C, respectively.

[0131] Take the sample of batch S1, and inject the sample according to the method under "1.3" in Example 1 and the chromatographic conditions in Comparative Example 2, and record the chromatogram. The results are as follows: Figure 6 As shown in .

[0132] Focusing on peaks 16, 17, 20, and 21, the researchers found that fewer peaks eluted at 35°C than at 30°C. Peak 21 showed some signs of elution at 30°C. However, further lowering the column temperature to 25°C effectively eluted peaks 17 (gentiopicroside) and 21 (peoniflorin). Therefore, appropriately lowering the column temperature can improve the resolution of the gout compound components.

[0133] In summary, this application established fingerprints for 10 batches of gout compound decoctions from different origins. The similarity between the patterns of each batch and the characteristic pattern was greater than 0.9, reflecting its fingerprint characteristics. Similarity evaluation can be used to assess the quality and quality of samples. In addition, based on qualitative analysis, the representative ingredients in the compound were used as controls to accurately determine the content of loganinic acid, gentiopicroside, glycyrrhizic acid monoamine salt, mangiferin, berberine, cinnamic acid, phellodendronine, and peony in the samples, further achieving more accurate and comprehensive control of the quality of the gout compound.

[0134] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for constructing a fingerprint of a gout compound decoction, characterized in that: The following steps are involved: A test solution of a gout compound decoction is obtained by decocting and concentrating a traditional Chinese medicine composition for treating gout; the traditional Chinese medicine composition is composed, by weight, of 5-100 parts of cinnamon twig, 5-100 parts of red peony root, 5-100 parts of anemarrhena, 5-100 parts of phellodendron, 7-100 parts of siler, 5-100 parts of gentiana macrophylla, 7-50 parts of aconite root, 5-100 parts of radix polygoni multiflori, 5-100 parts of radix aconiti lobatae, 5-100 parts of achyranthes bidentatae, 5-100 parts of codonopsis pilosula, 10-80 parts of poria peel, and 5-50 parts of liquorice; The test solution is subjected to HPLC-SPD detection to obtain the fingerprint of the gout compound decoction, wherein the high performance liquid chromatography detection conditions are: A chromatographic column filled with octadecyl bonded silica gel was used; an aqueous solution containing 0.1% to 0.2% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution; the gradient elution program was as follows: 0-5 min, 95% A; 5-10 min, 95% A→85% A; 10-40 min, 85% A→80% A; 40-50 min, 80% A→65% A; 50-55 min, 65% A→35% A; 55-58 min, 35% A→95% A; 58-65 min, 95% A; the flow rate was 0.8-1 mL / min, the column temperature was 25°C, the injection volume was 5-10 μL, and the diode array detector was used for full-band scanning at 190-400 nm. The fingerprint spectrum identified 8 characteristic peaks, namely, loganic acid at peak 9, phellodendronine at peak 11, gentiopicroside at peak 17, mangiferin at peak 18, paeoniflorin at peak 21, berberine at peak 27, cinnamic acid at peak 28 and glycyrrhizic acid monoamine salt at peak 29; Taking berberine, peak 27, as the reference peak, the relative retention times of the peaks were: loganinic acid, peak 9, 0.32±10%, phellodendronine, peak 11, 0.35±10%, gentiopicroside, peak 17, 0.43±10%, mangiferin, peak 18, 0.43±10%, paeoniflorin, peak 21, 0.53±10%, cinnamic acid, peak 28, and glycyrrhizic acid monoamine salt, peak 29, 1.14±10%.

2. The construction method according to claim 1, wherein The preparation of the test solution comprises the following steps: Accurately measure 1 mL of Gout Compound Decoction, add solvent to make up to 10 mL, mix well, centrifuge, take the supernatant and filter to prepare the test solution.

3. The construction method according to claim 1 or 2, wherein: The preparation of the gout compound decoction comprises the following steps: According to the mass ratio, the Chinese medicine composition for treating gout was taken, without crushing and sieving, and 10 times the amount of water was added and soaked for half an hour, and the mixture was decocted twice, and the filtrate was combined to obtain a gout compound decoction.

4. The construction method according to claim 2, wherein: The solvent is methanol.

5. The construction method according to claim 1, wherein: The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

6. The construction method according to claim 1, wherein: The construction method further includes preparing a mixed reference solution, and the preparation of the mixed reference solution includes the following steps: Take reference substances loganinic acid, phellodendrine, gentiopicroside, mangiferin, paeoniflorin, berberine, cinnamic acid and glycyrrhizic acid monoamine salt respectively, dissolve them in solvent, and prepare mother solution for storage; Accurately measure the mother solution of each reference substance and mix them evenly to prepare a mixed reference substance solution; In the mixed reference solution, the concentrations of each reference substance are: loganin 111.11 μg / mL, phellodendronine 155.4 μg / mL, gentiopicroside 111.11 μg / mL, mangiferin 83.25 μg / mL, paeoniflorin 155.4 μg / mL, berberine 83.25 μg / mL, cinnamic acid 83.25 μg / mL, and glycyrrhizic acid monoamine salt 111.11 μg / mL.

7. The construction method according to claim 1, wherein: The construction method further comprises the step of generating a fingerprint of the gout compound decoction: The obtained chromatogram was imported into the 2012 version of the Chinese medicine fingerprint similarity evaluation software. The S1 batch was used as the reference chromatogram, the time window width was set to 0.1 min, and the median method was used to perform multi-point correction and full-spectrum peak matching to generate the fingerprint of the gout compound decoction. Among them, the combined medicinal materials in the S1 batch were authentic medicinal materials.

8. The construction method according to claim 1, wherein: The construction method further comprises the step of substance quantification: The external standard method was used to detect the peak area of ​​paeoniflorin at a wavelength of 230 nm, the peak area of ​​loganic acid, mangiferin, berberine, and glycyrrhizic acid monoamine salt at 258 nm, the peak area of ​​cinnamic acid at 270 nm, and the peak area of ​​phellodendronine and gentiopicroside at 284 nm, to achieve quantification of the corresponding substances in the sample.

9. Application of the fingerprint obtained by the construction method according to any one of claims 1 to 8 in the quality control of a gout compound decoction.

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