Establishment, fingerprint and application of a UPLC fingerprint of a traditional Chinese medicine compound

Abstract

The present invention uses the water-soluble part of Licorice Decoction for Purging the Heart to prepare a test solution, takes Licorice, Scutellaria, Dry Ginger, Jujube, Pinellia and Coptis chinensis reference substances and mixes them to form a reference solution, uses a 0.4% volume fraction of formic acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B, injects samples under UPLC chromatographic conditions to obtain a chromatogram, and obtains a UPLC fingerprint by system analysis. The present invention purifies the water-soluble part from the whole recipe of Licorice Decoction for Purging the Heart and establishes a corresponding UPC fingerprint. The results show that the UPLC fingerprint provided by the present invention has excellent reproducibility and accuracy, has 46 common peaks, can comprehensively detect its efficacy, can accurately and efficiently detect its drug effect when used for anti-inflammatory and antibacterial efficacy detection, and controls the accuracy of the efficacy of Licorice Decoction for anti-inflammatory and antibacterial use.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical ingredient detection, and in particular to the establishment, fingerprint and application of a UPLC fingerprint of a traditional Chinese medicine compound. Background Art

[0002] Gancao Xiexin Decoction is a Traditional Chinese Medicine formula composed of licorice, scutellaria, dried ginger, pinellia, jujube, and coptis root. It has the effects of invigorating qi and soothing the stomach, relieving fatigue and stopping vomiting. It is commonly used clinically for acute and chronic gastrointestinal inflammation, Behçet's syndrome, and other conditions. To effectively monitor its quality, its ingredients must be identified and their active components measured. Fingerprinting technology has become a highly efficient and convenient method for detecting drug components.

[0003] Ultra-performance liquid chromatography (UPLC) performs liquid chromatography analysis on traditional Chinese medicine (TCM) extracts. By identifying specific peaks and their combinations that characterize specific components of a TCM, a unique fingerprint is formed, enabling quality testing or monitoring of TCM preparations. UPLC leverages the theories and principles of HPLC (high-performance liquid chromatography) to increase analytical throughput, sensitivity, and chromatographic peak capacity. It also leverages the advantages of small-particle columns, which are not available in traditional HPLC, achieving new levels of chromatographic resolution.

[0004] Based on this, chromatography and mass spectrometry are combined to combine the separation ability of chromatography with the qualitative function of mass spectrometry to achieve more accurate quantitative and qualitative analysis of complex mixtures. By complementing each other's advantages, drug quality can be more accurately detected. Summary of the Invention

[0005] The purpose of the present invention is to provide a UPLC fingerprint of Licorice Decoction for Purging the Heart. After extraction and purification of Licorice Decoction for Purging the Heart, a UPLC fingerprint of its water-soluble part is established to determine the effectiveness and stability of the medicinal components in Licorice Decoction for Purging the Heart, ensuring that it can effectively detect the corresponding medicinal effects.

[0006] The object of the present invention is to provide a method for establishing the UPLC fingerprint of the above-mentioned Licorice Xiexin Decoction.

[0007] The present invention aims to provide an application of the UPLC fingerprint of the Gancao Xiexin Decoction.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for establishing a UPLC fingerprint of a traditional Chinese medicine compound, characterized by comprising the following steps:

[0010] (1) Preparation of test solution

[0011] According to the prescription of Gancao Xiexin Decoction, 20g of licorice, 15g of scutellaria, 15g of dried ginger, 12g of jujube, 13g of pinellia, and 5g of coptis were taken, and 16 batches were weighed. The sample mother liquor was extracted by semi-bionic extraction method. 20mL of the sample mother liquor was added to 20mL of ethyl acetate solvent, and the extraction bottle was shaken for 5min and allowed to stand for 10min. The lower layer solution was extracted three times in succession, and the three extracts were combined. The extract was rotary evaporated to dryness at 55°C and diluted to 20mL with 50% methanol solution to obtain the water-soluble sample solution, which was filtered with a 0.45μm filter membrane to obtain the test solution;

[0012] (2) Preparation of reference solution

[0013] Liquorice inoside, baicalin, wogonin, coptisine hydrochloride, glycyrrhizin, ammonium glycyrrhizate, and berberine hydrochloride were mixed and dissolved in methanol to a concentration of 1.0 mg / mL as the reference solution;

[0014] (3) UPLC chromatographic conditions

[0015] Chromatographic column Shim-pack Scepter C18-200 (3 μm, 4.6×250 mm), gradient elution: mobile phase A is 0.4% formic acid aqueous solution, mobile phase B is acetonitrile, 0-5 min, the volume percentage of mobile phase B increases from 2% to 6%; 5-10 min, the volume percentage of mobile phase B increases from 6% to 8%; 10-15 min, the volume percentage of mobile phase B increases from 8% to 15%; 15-20 min, the volume percentage of mobile phase B increases from 15% to 20%; 20-30 min, the volume percentage of mobile phase B increases from 20% to 23%; 30-40 min, the volume percentage of mobile phase B increases from 10% to 25%. From 23% to 32%; from 40 to 60 minutes, the volume percentage of mobile phase B increased from 32% to 35%; from 60 to 80 minutes, the volume percentage of mobile phase B increased from 35% to 50%; from 80 to 85 minutes, the volume percentage of mobile phase B increased from 50% to 80%; from 85 to 90 minutes, the volume percentage of mobile phase B increased from 80% to 98%; from 90 to 95 minutes, the volume percentage of mobile phase B decreased from 98% to 60%; from 95 to 100 minutes, the volume percentage of mobile phase B decreased from 60% to 35%; from 100 to 105 minutes, the volume percentage of mobile phase B decreased from 35% to 2%;

[0016] (4) Establishment of UPLC fingerprint

[0017] Sixteen batches of water-soluble fraction samples were injected according to chromatographic conditions, and the UPLC data (CDF format data) were recorded. The original data were imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) software, and common peak matching was performed using the multi-point correction method to establish fingerprint spectra. S9 was used as the reference spectrum, and the control spectrum generation method was the median method, with a time window width of 0.5 and a spectrum spacing of 400. Fingerprint spectra of 16 batches of water-soluble fraction samples were obtained.

[0018] Furthermore, the mother liquor of the sample was extracted in the above step (1) by decocting it three times. The specific extraction conditions were as follows: the pH of the first decoction was 4.0 and the decoction was performed for 1.0 h; the pH of the second decoction was 7.0 and the decoction was performed for 0.5 h; the pH of the third decoction was 8.0 and the decoction was performed for 0.5 h; the decoction liquid-to-material ratio was 1:10; the extraction temperature was 95°C and the reflux extraction was performed; and the pH value was 0.1 mol·L -1 HCl and 0.1 mol·L -1 Adjust with NaOH.

[0019] Furthermore, in step (3), the UPLC chromatography conditions were 277 nm for detection and 0.3 mL min for flow rate. -1 , injection volume 3 μL, column temperature 15 °C.

[0020] Further, the water-soluble fraction samples were subjected to mass spectrometry analysis. The UPLC fingerprint of the water-soluble fraction of Gancao Xiexin Decoction established in step (4) had 46 common peaks, and 23 compound components were analyzed. These 23 compounds were classified into categories, and it was found that they were mainly ketones, acids, glycosides, flavonoids, and alkaloids. Among them, the common peaks 11, 12, 17, 18, 34, 36, 38, and 40 were attributed to Glycyrrhiza uralensis, 19, 23, 24, 28, 29, 32, 36, and 39 were attributed to Scutellaria baicalensis, 22, 30, and 35 were attributed to Coptis chinensis, 15 was attributed to Pinellia ternata, 5, 21, and 31 were attributed to Jujube, among which peak No. 4 was phenylalanine, peak No. 5 was adenosine, peak No. 6 was glycyrrhizic acid, and peak No. 8 was 5,7-dihydroxy-6-methoxyflavone- Peak 10 is sucrose, Peak 11 is formononetin, Peak 12 is liquiritigenin-4'-O-β-D-glucose-(1→6)-O-β-D-glucoside, Peak 17 is liquiritigenin, Peak 21 is cyclic adenosine monophosphate, Peak 22 is palmatine hydrochloride, Peak 23 is norwogonin-7-O-β-D-glucuronide, Peak 24 is baicalin, peak 25 is magnolamine, peak 28 is 5,7,8-trihydroxyflavone-7-O-glucoside, peak 29 is scutellarin, peak 30 is coptisine, peak 31 is jujube benzyl glycoside, peak 32 is wogonin, peak 35 is berberine, peak 36 is glycyrrhizin, peak 38 is ural glycyrrhizin P, and peak 40 is ammonium glycyrrhizate.

[0021] Furthermore, combined with previous studies and reference maps, 13 of the 20 anti-inflammatory active ingredient groups in the water-soluble fraction samples were analyzed, namely 5,7-dihydroxy-6-methoxyflavonoid-7-O-glucuronide, sucrose, glycyrrhizin-4'-O-β-D-glucose-(1→6)-O-β-D-glucoside, glycyrrhizin, norwogonoside-7-O-β-D-glucuronide, baicalin, magnolamine, 5,7,8- Trihydroxyflavone-7-O-glucoside, baicalin, coptisine, wogonoside, berberine and ammonium glycyrrhizate; there are 25 antibacterial active ingredient groups, and 14 of them were analyzed, namely sucrose, spinulosin, glycyrrhizin, cyclic adenosine monophosphate, palmatine hydrochloride, baicalin, magnolamine, 5,7,8-trihydroxyflavone-7-O-glucoside, magnolamine, baicalin, coptisine, wogonoside, glycyrrhizin and ural glycyrrhizin P.

[0022] The application of the above fingerprint spectrum is characterized by: being used in the quality detection of Licorice Decoction for Purging Heart, specifically in the detection of the anti-inflammatory and antibacterial efficacy of Licorice Decoction for Purging Heart.

[0023] The present invention has the following technical effects:

[0024] The present invention purifies the water-soluble parts from the whole recipe of Licorice Decoction for Purging the Heart and establishes the corresponding UPC fingerprint. The results show that the UPLC fingerprint provided by the present invention has excellent reproducibility and accuracy, and has 46 common peaks, which can comprehensively detect its efficacy. When used for anti-inflammatory and antibacterial efficacy testing, it can accurately and efficiently detect its drug effects, and accurately control the efficacy of Licorice Decoction for anti-inflammatory and antibacterial use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Chromatogram of the water-soluble fraction sample of Gancao Xiexin Decoction.

[0026] Figure 2 : Chromatogram of the reference solution mixed with 7 components.

[0027] Figure 3 : Fingerprint of water-soluble fraction samples of Gancao Xiexin Decoction.

[0028] Figure 4 : Regression coefficients of the common peak and the anti-inflammatory efficacy of water-soluble fraction samples.

[0029] Figure 5 : VIP values ​​of anti-inflammatory efficacy of samples from the common peak and water-soluble fraction.

[0030] Figure 6 : Regression coefficient of antibacterial efficacy of common peak and water-soluble fraction samples.

[0031] Figure 7: VIP values ​​of antibacterial efficacy of samples in the common peak and water-soluble fraction. DETAILED DESCRIPTION

[0032] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0033] Example 1

[0034] A method for establishing a UPLC fingerprint of Gancao Xiexin Decoction comprises the following steps:

[0035] (1) Preparation of test solution

[0036] According to the prescription of Gancao Xiexin Decoction, 20g of licorice, 15g of scutellaria, 15g of dried ginger, 12g of jujube, 13g of pinellia, and 5g of coptis were taken, and the sample mother liquor was extracted by semi-bionic extraction method. Specifically, the water was decocted three times. The extraction conditions were as follows: the pH of the first decoction was 4.0, and the decoction was 1.0h. The pH of the second decoction was 7.0, and the decoction was 0.5h. The pH of the third decoction was 8.0, and the decoction was 0.5h. The decoction liquid-to-material ratio was 1:10. The extraction temperature was 95℃ and reflux extraction was carried out. The pH of each water was HCl (0.1mol.L -1 ) and NaOH (0.1 mol.L -1 ) adjustment; take 20 mL of the prepared sample mother solution, add 20 mL of ethyl acetate solvent, shake the extraction bottle for 5 minutes, let it stand for 10 minutes, take the lower layer solution and extract it three times in a row, combine the three extracts, and evaporate the extract to dryness at 55°C. Use 50% methanol solution to make the volume to 20 mL to obtain a water-soluble fraction sample solution, filter it with a 0.45μm filter membrane to obtain a test solution; the chromatogram of the water-soluble fraction sample is as follows Figure 1 shown.

[0037] (2) Preparation of reference solution

[0038] Liquorice root, baicalin, wogonin, coptisine hydrochloride, glycyrrhizin, ammonium glycyrrhizate, and berberine hydrochloride were mixed and dissolved in methanol to a concentration of 1.0 mg / mL as the water-soluble fraction sample mixed reference solution; the corresponding chromatogram is shown in FIG. Figure 2 As shown, each peak corresponds to 1. liquiritin; 2. baicalin; 3. coptisine hydrochloride; 4. wogonin; 5. berberine hydrochloride; 6. glycyrrhizin; 7. ammonium glycyrrhizate;

[0039] (3) UPLC chromatographic conditions

[0040] Chromatographic column: Shim-pack Scepter C18-200 (3 μm, 4.6 × 250 mm), detection wavelength: 277 nm, flow rate: 0.3 mL min -1 , injection volume 3 μL, column temperature 15 °C;

[0041] Gradient elution: mobile phase A is 0.4% formic acid aqueous solution, mobile phase B is acetonitrile, 0-5 min, the volume percentage of mobile phase B increases from 2% to 6%; 5-10 min, the volume percentage of mobile phase B increases from 6% to 8%; 10-15 min, the volume percentage of mobile phase B increases from 8% to 15%; 15-20 min, the volume percentage of mobile phase B increases from 15% to 20%; 20-30 min, the volume percentage of mobile phase B increases from 20% to 23%; 30-40 min, the volume percentage of mobile phase B increases from 23% to 32%; 40- At 60 min, the volume percentage of mobile phase B increased from 32% to 35%; at 60-80 min, the volume percentage of mobile phase B increased from 35% to 50%; at 80-85 min, the volume percentage of mobile phase B increased from 50% to 80%; at 85-90 min, the volume percentage of mobile phase B increased from 80% to 98%; at 90-95 min, the volume percentage of mobile phase B decreased from 98% to 60%; at 95-100 min, the volume percentage of mobile phase B decreased from 60% to 35%; at 100-105 min, the volume percentage of mobile phase B decreased from 35% to 2%;

[0042] (4) Establishment of UPLC fingerprint

[0043] Samples were injected according to the chromatographic conditions, and UPLC data (CDF format data) were recorded. The raw data were imported into the "Similarity Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version) software. Common peak matching was performed using the multi-point correction method to establish fingerprint spectra. S9 was used as the reference spectrum, and the control spectrum was generated using the median method, with a time window width of 0.5 and a spectrum spacing of 400. Fingerprint spectra of 16 batches of samples were obtained.

[0044] The UPLC fingerprint established is as follows Figure 4As shown, the samples have a total of 46 characteristic fingerprint chromatographic peaks. S1-S16 are the water-soluble fraction samples from batches 1 to 16, respectively, and R is the reference spectrum generated by the software. Using S9 as the reference spectrum and a spectrum spacing of 400, the fingerprint of the water-soluble fraction samples was obtained. The chromatographic results show that the similarities between the 16 batches of samples and the reference fingerprint (R) are 0.999, 1.000, 0.999, 0.999, 0.999, 0.999, 0.999, 0.999, 0.998, 0.998, 0.998, 0.998, 0.998, 0.998, 0.998, 0.998, 0.998, and 0.998, respectively. The RSD value of the similarity is 0.04%, indicating that the 16 batches of samples are not significantly different. The RSD values ​​of the common characteristic fingerprint peak chromatographic peak elution time are in the range of 0.01-0.35%, which is less than 1%, indicating that the common peak elution time is highly consistent. The RSD values ​​of its chromatographic peak area are in the range of 1.47-29.69%, indicating that there are certain differences in the peak areas of individual components of samples between different batches.

[0045] The mass spectrometry conditions were as follows: electrospray ion source (ESI ion source), TOF-MS (+) positive ion scan mode, capillary voltage 5.5 kV, sampling cone voltage 80 V; desolvation gas temperature 550°C; desolvation gas volume flow rate 800 L / h; cone gas volume flow rate 50 L / h; ion source temperature 150°C; collision gas: nitrogen; scan range: m / z 100-2000, and total ion current was collected.

[0046] Mass spectrometry analysis of the water-soluble fraction revealed 20 compounds from 46 common peaks. These 20 compounds were then classified, primarily as ketones, acids, glycosides, flavonoids, and alkaloids. The 46 components were then assigned to medicinal materials using a control chromatogram. Combined with preliminary assumptions about the attribution of these components, 23 common peaks were identified, including peaks 11, 12, 17, 18, 34, 36, 38, and 40, assigned to Glycyrrhiza uralensis, 19, 23, 24, 28, 29, 32, 36, and 39 to Scutellaria baicalensis, 22, 30, and 35 to Coptis chinensis, 15 to Pinellia ternata, and 5, 21, and 31 to Jujube.

[0047] Example 2

[0048] Methodological investigation of water-soluble fraction samples:

[0049] The feasibility and scientificity of the established method were evaluated from three perspectives: precision, repeatability, and stability. Precision was assessed by precisely aspirating a water-soluble fraction sample solution and injecting it six times in a row under optimized chromatographic conditions. The peak elution time and peak area were recorded, and the relative standard deviation (RSD) of these values ​​was calculated. Repeatability was assessed by preparing six parallel aliquots of the water-soluble fraction of Gancao Xiexin Decoction and sequentially injecting and analyzing them under UPLC conditions. The peak elution time and peak area were recorded, and the relative standard deviation (RSD) of these values ​​was calculated. Stability was assessed by precisely aspirating a water-soluble fraction sample solution from the same batch and injecting it at 0, 2, 4, 8, 12, 18, and 24 hours. The optimized chromatographic conditions were followed for injection and analysis. The peak elution time and peak area were recorded, and the relative standard deviation (RSD) of these values ​​was calculated. The specific results are shown in Table 1.

[0050] Table 1: Precision, repeatability and stability of common peaks in water-soluble fraction samples

[0051]

[0052]

[0053] As shown in Table 1, the RSD values ​​of precision retention time and peak area are in the range of 0.01-0.30% and 0.13-8.91%, respectively, both less than 10%; the RSD values ​​of repeatability retention time and peak area are in the range of 0.01-0.32% and 0.73-9.38%, respectively, both less than 10%; the RSD values ​​of stability retention time and peak area are in the range of 0.01-1.32% and 0.47-9.44%, respectively, both less than 10%, which meets the requirements.

[0054] Example 3

[0055] Establishment of mathematical model for sample data

[0056] First, anti-inflammatory and antibacterial tests were carried out on samples of water-soluble parts. The obtained anti-inflammatory and antibacterial efficacy results were analyzed using SPSS.25 statistical analysis software, and PCA analysis was performed by dimensionality reduction; OPLS analysis was performed using SIMCA-P14.1 software to establish a mathematical model; and the GRA correlation value was calculated using EXCEL to obtain the specific pharmacological components related to anti-inflammatory and antibacterial effects.

[0057] 1. Anti-inflammatory and antibacterial efficacy testing of Gancao Xiexin Decoction:

[0058] A mouse RAW264.7 macrophage inflammation model was constructed to conduct anti-inflammatory tests, and in vitro inhibition tests on Escherichia coli, Salmonella choleraesuis and Bacillus subtilis were conducted to evaluate the anti-inflammatory and antibacterial activities.

[0059] (1) Anti-inflammatory activity detection of water-soluble fraction samples

[0060] 16 batches of water-soluble fraction samples were taken and the concentration was 50 mg mL -1 As the drug concentration, take 6-well plates and adjust the cell concentration per plate to 1×10 5 mL -1 10 μL of 15 μg·mL was added to each well. -1 Cells were stimulated with LPS for 12 hours, then 10 μL of drug was added and incubated for 12 hours in a 37°C, 5% CO2 incubator. Cell supernatants were collected and assayed for NO using a NO detection kit. Six replicates were performed for each drug concentration, along with a model control and a blank control. The results are shown in Table 2.

[0061] Table 2: NO content in 16 batches of water-soluble samples

[0062]

[0063] As shown in Table 2, compared with the blank group, the NO content in the model group increased significantly (P < 0.01), indicating that the inflammatory model was successfully established; compared with the model group, all 16 batches of samples in the water-soluble part had a significant inhibitory effect on NO (P < 0.01), and there was no significant difference between the batches, indicating that the inhibitory effect of the 16 batches of samples was significant and the effect difference was not significant.

[0064] (2) Antibacterial activity test of water-soluble fraction samples

[0065] (1) Escherichia coli inhibition test

[0066] 16 batches of water-soluble fraction samples were taken and Escherichia coli was cultured in LB medium at 37°C for 8 h. The cultured bacteria were diluted 50 times with the medium and 200 μL was inoculated on LB agar plates. After the plates were dry and no liquid flowed, an Oxford cup was slightly heated over a flame and placed on the plates. The Oxford cup was gently pressed to fix it. 16 batches of samples (mass concentration 50 mg·L) were added to the Oxford cups. -1 ), add 180 μL to each sample, incubate at 37°C for 8 hours, and observe the antibacterial effect. An inhibition zone diameter D ≥ 20 mm is considered extremely sensitive, 15-19 mm is highly sensitive, 10-14 mm is moderately sensitive, and less than 10 mm is considered low-sensitive.

[0067] (2) Inhibition of Salmonella choleraesuis test

[0068] 16 batches of water-soluble fraction samples were taken and cultured with LB medium at 37°C for 8 h. The cultured bacteria were diluted 50 times with the medium and 200 μL was inoculated on LB agar plates. After the plates were dry and no liquid flowed, an Oxford cup was slightly heated over a flame and placed on the plates. The Oxford cup was gently pressed to fix it. 16 batches of samples (mass concentration 50 mg·L) were added to the Oxford cups.-1 ), add 180 μL to each sample, incubate at 37°C for 6 hours, and observe the antibacterial effect. An inhibition zone diameter D ≥ 20 mm is considered extremely sensitive, 15-19 mm is highly sensitive, 10-14 mm is moderately sensitive, and less than 10 mm is considered low-sensitive.

[0069] (3) Inhibition of Bacillus subtilis test

[0070] The miraculous bacteria were cultured in LB medium at 37℃ for 8h. The cultured bacteria were diluted 50 times with the medium and 200μL was inoculated on the LB agar plate. After the plate was dry and no liquid flowed, the Oxford cup was slightly heated over a flame and placed on the plate. The Oxford cup was gently pressed to fix it. 16 batches of water-soluble part samples (mass concentration 50mg·L) were added to the Oxford cup. -1 ), add 180 μL to each sample, incubate at 37°C for 6 hours, and observe the antibacterial effect. An inhibition zone diameter D ≥ 20 mm is considered extremely sensitive, 15-19 mm is highly sensitive, 10-14 mm is moderately sensitive, and less than 10 mm is considered low-sensitive.

[0071] The antibacterial effects of the water-soluble fraction samples on Escherichia coli, Salmonella choleraesuis and Bacillus mirabilis are shown in Table 3.

[0072] Table 3

[0073]

[0074] Note: The diameter of the inhibition zone D ≥ 20 mm is extremely sensitive, 15-19 mm is highly sensitive, 10-14 mm is moderately sensitive, and less than 10 mm is low sensitive.

[0075] As shown in Table 3, all 16 batches of water-soluble samples exhibited inhibitory effects against Escherichia coli, Salmonella choleraesuis, and Bacillus mirabilis. The diameters of the inhibition zones for E. coli and Bacillus mirabilis were greater than 20 nm, indicating extreme sensitivity. The diameters of the inhibition zones for Salmonella were mostly between 15 and 19 mm, indicating high sensitivity. Therefore, it is possible to further identify components with antibacterial efficacy from the water-soluble components of Gancao Xiexin Decoction.

[0076] 2. Software Analysis

[0077] SPSS.25 statistical analysis software was used to perform PCA analysis by dimensionality reduction; SIMCA-P14.1 software was used for OPLS analysis; and EXCEL was used to calculate the GRA correlation value.

[0078] (1) Data analysis principles

[0079] Principal component analysis (PCA) transforms multiple indicators into a few representative composite indices, simplifying the complex interrelationships between variables. Specifically, PCA simplifies multiple measured indicators, reducing their mutual influence, extracting key factors, and then calculating a composite score to obtain a composite index value. The analysis requires multiple variables with a certain degree of correlation between them. Furthermore, the number of principal components is determined based on eigenvalues ​​greater than 0.7 and cumulative variance contributions exceeding 85%. The scores for each principal component are then calculated, and the composite standard value F is finally calculated. The specific formulas are shown in Equations 1, 2, and 3.

[0080] F i =w i1 *X1+w i2 *X2+...+w in *X n (Formula 1)

[0081]

[0082] F=a1F1+a2F2+...+a n F n (Formula 3)

[0083] In formula 1: w in Indicates the weight of each variable of the i-th principal component, X1....X n Indicates the corresponding X value after the data is dimensionless; in formula 2: θ n represents the coefficient corresponding to each variable in the component matrix, Indicates the root value of the eigenvalue corresponding to the i-th principal component; in formula 3: a n represents the variance percentage of the nth principal component, F represents the comprehensive standard value, and F n Represents the standardized value of the nth principal component.

[0084] Orthogonal projection partial least squares (OPLS) is a multivariate statistical analysis method for multiple independent variables using multiple dependent variables. The regression coefficients used here can not only reveal the degree of correlation between variables, positive and negative correlations, and regression equations, but also reveal the comprehensive effects of components on efficacy, enabling validation of different methods. Using the common peak area as the independent variable (X) and the efficacy index as the dependent variable (Y), spectrum-effect relationships are studied, and the regression coefficients for each X corresponding to Y are obtained to establish a regression equation. Variable projection importance (VIP) is an important indicator reflecting the ability of an independent variable to explain the dependent variable. A higher VIP value indicates a stronger explanatory power for the independent variable. It is generally believed that when VIP > 0.7, the independent variable is significantly important in explaining the dependent variable, indicating that the pharmacological component is significantly important.

[0085] Grey relational analysis (GRA) is a statistical analysis technique that is mainly used to analyze the closeness of the relationship between the parent factor and the child factor in the system, so as to determine the main and secondary factors that cause the development and change of the system. It is a quantitative comparative analysis method for the dynamic development trend of the system. The steps of the grey relational analysis method are as follows:

[0086] ①Select reference sequence (parent sequence) Y j , where j = 1, 2, 3…, m; comparison sequence (subsequence) X i , where i = 1, 2, 3 ..., n. The present invention uses the efficacy indicators of anti-inflammatory and antibacterial as the parent sequence (Y), then Y j where j=1, 2; the common peak area of ​​each characteristic fingerprint in the chromatogram is taken as the subsequence (X), and i=1, 2, ..., 46 in the water-soluble part sample.

[0087] ② Perform dimensionless processing on the variables. Common methods include initial value method, mean method, etc. This invention uses mean method to perform data normalization processing.

[0088] ③ Find the difference sequence, maximum difference and minimum difference. Reference sequence Y j Compare with sequence X i The absolute value of the difference on the kth index is the difference sequence value △ 0i (k)=|Y j (k)-X i (k)|, k=1,2,3...n;j=1,2;i=1,2,3...,n;the maximum difference is:△ max =max|Y j (k)-X i (k)|; Minimum difference △ min =min|Y j (k)-X i (k)|.

[0089] ④ Calculate the correlation coefficient ξ, see formula 4.

[0090] ξ i (k)=[Δ(min)+ρ×Δ(max)] / [Δ 0i (k)+ρ×Δ(max)] (Equation 4)

[0091] In formula 4, k is the peak number = 1, 2, 3, ..., n; ρ is the resolution coefficient, ranging from 0 to 1, usually selected as 0.5; Δ0i(k) is the absolute difference between the efficacy index (reference sequence) and the peak area of ​​the characteristic peak (comparison sequence) after averaging; Δ(min) is the minimum difference, and Δ(max) is the maximum difference.

[0092] ⑤Calculate the correlation r i , see formula 5. iReflects the reference sequence Y j Compare with sequence X i The overall correlation between the two groups is set in the reference literature as r i When it is greater than 0.6, it is the main contributor to the spectrum-effect relationship.

[0093]

[0094] In Formula 5, n is the number of data contained in the comparison sequence (i=1, 2, 3...n; k=1, 2, 3).

[0095] (3) Software analysis results of sample data

[0096] PCA analysis: The data were first standardized using SPSS 25 software, and the results are shown in Table 4.

[0097] Table 4: Standardization of water-soluble fraction sample data

[0098]

[0099] The antibacterial effects of the water-soluble fraction samples on the three bacteria were analyzed by dimensionality reduction using SPSS 25 software, and the three principal components, initial eigenvalues, and component matrices were extracted (see Tables 5 and 6).

[0100] Table 5: Initial eigenvalues ​​of the total variance explained by the water-soluble fraction samples

[0101]

[0102] Note: The extraction method is principal component analysis.

[0103] Table 6: Water-soluble fraction sample composition matrix

[0104]

[0105] Note: The extraction method is principal component analysis.

[0106] The weight coefficient w can be calculated again from Tables 4, 5, and 6.

[0107] Calculate the comprehensive standard values ​​of each principal component F1 = 0.50977*X1-0.64738*X2+0.56689*X3, F2 = 0.78393*X1+0.07766*X2-0.61602*X3, and finally calculate the comprehensive index F value = 0.4450F1+0.3028F2+0.2523F3.

[0108] OPLS analysis: The peak area and efficacy index data of the characteristic fingerprint of the water-soluble fraction samples were processed using the mean method to establish mathematical models for anti-inflammatory and antibacterial efficacy, respectively. The anti-inflammatory modeling was as follows: OPLS analysis was performed using SIMCA-P14.1 software, with the common peak area of ​​the water-soluble fraction samples as the independent variable X and the anti-inflammatory efficacy index as the dependent variable Y1. The regression equation was automatically fitted: Y1 = a1X1 + a2X2 + ...a 46 X 46 , where Y1 corresponds to the anti-inflammatory efficacy result, X1~X 46 The peak area corresponding to the common peak, a1~a 46 The regression coefficients of the corresponding common peaks and anti-inflammatory efficacy are as follows: a positive regression coefficient indicates a positive correlation with the efficacy, while a negative regression coefficient indicates a negative correlation with the efficacy. Figure 4 VIP is an important indicator that reflects the ability of the independent variable to explain the dependent variable. The larger the value, the stronger the ability of the independent variable to explain the dependent variable. It is generally believed that when VIP>0.7, the independent variable has significant importance in explaining the dependent variable. Figure 5 The anti-inflammatory mathematical model of water-soluble samples was established based on the regression coefficient:

[0109] Y1=-0.04948X1+0.11156X2+0.03650X3-0.02220X4-0.20263X5-0.04098X6-0.010

[0110] 47X7+0.02171X8+0.00568X9+0.02808X 10 -0.00712X 11 +0.00462X 12 +0.01531X 13 -0.00430X 14 -0.00014X 15 -0.02298X 16 +0.00815X 17 +0.04172X 18 -0.02876X 19 -0.03912X 20 -0.03708X 21 -0.00866X 22 +0.01419X 23 +0.10221X 24 +0.01647X 25 -0.05146X 26 -0.09424X 27 +0.08429X 28 +0.01675X 29+0.00824X 30 -0.03241X 31 +0.02193X 32 -0.03117X 33 -0.02253X 34 +0.05572X 35 -0.03793X 36 -0.19747X 37 -0.13831X 38 -0.12111X 39 +0.09264X 40 -0.06455X 41 +0.15164X 42 +0.25815X 43 +0.09535X 44 -0.05168X 45 +0.04917X 46 .

[0111] Antibacterial modeling: The method is the same as above, with the common peak area of ​​the water-soluble fraction samples as the independent variable X, and the anti-inflammatory efficacy index as the dependent variable Y2. The regression coefficient is shown in Figure 6 VIP value see Figure 7 The antibacterial mathematical model of the ester layer sample was established based on the regression coefficient:

[0112] Y2=0.03208X1-0.57574X2-0.14924X3-0.30080X4-0.07895X5-0.01477X6-0.06597X7-0.07526X8+0.04278X9+0.00187X 10 +0.01920X 11 -0.02174X 12 -0.04240X 13 -0.01153X 14 +0.01936X 15 +0.02612X 16 +0.11386X 17 -0.14276X 18 +0.21253X 19 +0.01227X 20 +0.19099X 21 +0.12653X 22 +0.15968X 23 +0.01098X 24 +0.18724X 25 +0.27711X 26 -0.21121X27 -0.05584X 28 +0.04803X 29 +0.01434X 30 -0.02906X 31 +0.01967X 32 +0.23439X 33 +0.00493X 34 -0.26261X 35 +0.53564X 36 -0.34400X 37 +0.55724X 38 -0.08397X 39 -0.07313X 40 +0.15903X 41 -0.23409X 42 +0.17930X 43 +0.20873X 44 -0.09328X 45 +0.24996X 46 .

[0113] GRA analysis: The correlation between the common peak area of ​​the water-soluble fraction samples and the anti-inflammatory and antibacterial efficacy was calculated by formula 4 and 5. When the correlation is greater than 0.6, it can be regarded as an effective anti-inflammatory and antibacterial chromatographic peak. Therefore, the correlation between the common peak of the water-soluble fraction samples and the anti-inflammatory efficacy is r 45 >r 27 >r 42 >r4>r 46 >r1>r 30 >r 24 >r 17 >r6>r 33 >r 12 >r3>r 15 >r 14 >r 26 >r9>r 16 >r8>r 32 >r 29 >r7>r 25 >r 11 >r 31 >r 28 >r 13 >r5>r 20 >r 21 >r 40 >r 10 >r 18 >r 22 >r 43 >r 37 >r23 >r 19 >r 39 >r 36 >r 41 >r 38 >r 44 >r2>r 34 >r 35 , and the correlation with the antibacterial efficacy is r 45 >r 27 >r 46 >r4>r 42 >r1>r 33 >r 26 >r 30 >r3>r 12 >r 17 >r 14 >r 15 >r 25 >r6>r9>r 16 >r7>r 24 >r8>r 13 >r 29 >r 28 >r 31 >r 32 >r5>r 11 >r 20 >r 21 >r 18 >r 40 >r 22 >r 10 >r 23 >r 19 >r 39 >r 36 >r 41 >r 43 >r 37 >r 35 >r 34 >r2>r 38 >r 44 .

[0114] Based on the data of GRA analysis, OPLS and PCA analysis, combined with spectrum-effect relationship research, the key component groups of the water-soluble part samples of Gancao Xiexin Decoction that produce anti-inflammatory and antibacterial effects were screened with the criteria of grey correlation degree > 0.6, positive OPLS analysis regression coefficient (positive correlation) and VIP value > 0.7. The chromatographic peaks related to anti-inflammatory effects of the water-soluble part samples of Gancao Xiexin Decoction were 2, 3, 8, 9, 10, 12, 13, 17, 18, 23, 24, 25, 28, 29, 30, 32, 35, 40, 42, and 44, a total of 20. Combined with previous research and reference maps, 13 anti-inflammatory active ingredients were analyzed from the water-soluble part samples, namely 5,7-dihydroxy-6-methoxyflavonoid-7-O-glucuronide ( 8), sucrose (10), glycyrrhizin-4'-O-β-D-glucose-(1→6)-O-β-D-glucoside (12), glycyrrhizin (17), nor-wogonin-7-O-β-D-glucuronide (23), baicalin (24), magnolamine (25), 5,7,8-trihydroxyflavone-7-O-glucoside (28), baicalin (29), coptisine (30), wogonin ( 32), berberine (35), ammonium glycyrrhizate (40), these components are closely related to anti-inflammatory effects; the chromatographic peaks related to antibacterial activity of the water-soluble fraction of Gancao Xiexin Decoction were 1, 9, 10, 11, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 29, 30, 32, 33, 34, 36, 38, 41, 43, 44, a total of 25, 14 of which were resolved, namely Sucrose (10), formononetin (11), glycyrrhizin (17), cyclic adenosine monophosphate (21), palmatine hydrochloride (22), baicalin (24), magnolamine (25), 5,7,8-trihydroxyflavone-7-O-glucoside (28), scutellarin (29), coptisine (30), wogonin (32), glycyrrhizin (36), and ural glycyrrhizin P (38) are related to antibacterial effects. Among them, 8 active ingredient groups with both anti-inflammatory and antibacterial effects were analyzed, namely sucrose, glycyrrhizin, 5,7,8-trihydroxyflavone-7O-glucoside, baicalin, magnolamine, scutellarin, coptisine, and wogonin. These ingredients mainly belong to sugars, glycosides, flavonoids, and alkaloids.

[0115] Comparison between the calculated values ​​of the mathematical model and the measured values:

[0116] Three batches of water-soluble samples of Licorice Decoction for the Purgation of Heart were prepared in the same manner as the research subjects. The UPLC was injected under the same chromatographic conditions to obtain the corresponding common chromatographic fingerprint peak area (X). The corresponding pharmacodynamic value (Y) was calculated using the mathematical model established in the early stage as the calculated value. At the same time, cell tests and antibacterial tests were carried out under the same conditions as in the early stage to obtain the anti-inflammatory and antibacterial data values ​​of the water-soluble samples as the measured values. The two groups of values ​​were compared and the RSD value was calculated to verify the accuracy of the mathematical model. When the RSD value was less than 10%, the established model was proven to be feasible. The results are shown in Table 7.

[0117] Table 7: Verification results of water-soluble fraction samples

[0118]

[0119] As shown in Table 7, the RSDs between the calculated values ​​from the established mathematical formula and the measured values ​​from actual testing were both less than 10%, validating the feasibility of the model. When testing the anti-inflammatory and antibacterial efficacy of Gancao Xiexin Decoction, the water-soluble fingerprint and mathematical model were used to rapidly calculate its efficacy.

Claims

1. A method for establishing a UPLC fingerprint of a traditional Chinese medicine compound, characterized in that: The steps include: (1) Preparation of test solution According to the prescription of Gancao Xiexin Decoction, 20 g of licorice, 15 g of scutellaria, 15 g of dried ginger, 12 g of jujube, 13 g of pinellia, and 5 g of coptis were taken, and 16 batches were weighed. The sample mother liquor was extracted by semi-bionic extraction method. 20 mL of the sample mother liquor was added to 20 mL of ethyl acetate solvent, and the solution was shaken in the extraction bottle for 5 minutes and allowed to stand for 10 minutes. The lower layer solution was extracted three times in succession, and the three extracts were combined. The extract was rotary evaporated to dryness at 55 ° C and diluted to 20 mL with 50% methanol solution to obtain the water-soluble sample solution. The sample solution was filtered with a 0.45 μm filter membrane to obtain the test solution; (2) Preparation of reference solution Liquorice inoside, baicalin, wogonin, coptisine hydrochloride, glycyrrhizin, ammonium glycyrrhizate, and berberine hydrochloride were mixed and dissolved in methanol to a concentration of 1.0 mg / mL as the reference solution; (3) UPLC chromatographic conditions Chromatographic column Shim-pack Scepter C18-200, 3μm, 4.6×250mm, gradient elution: mobile phase A is 0.4% formic acid aqueous solution, mobile phase B is acetonitrile, 0~5min, the volume percentage of mobile phase B increases from 2% to 6%; 5~10min, the volume percentage of mobile phase B increases from 6% to 8%; 10~15min, the volume percentage of mobile phase B increases from 8% to 15%; 15~20min, the volume percentage of mobile phase B increases from 15% to 20%; 20~30min, the volume percentage of mobile phase B increases from 20% to 23%; 30~40min, the volume percentage of mobile phase B increases from 23% to 32%; 40~60min, the volume percentage of mobile phase B increases from 10% to 20%. The volume percentage of mobile phase B increased from 32% to 35%; from 60 to 80 min, the volume percentage of mobile phase B increased from 35% to 50%; from 80 to 85 min, the volume percentage of mobile phase B increased from 50% to 80%; from 85 to 90 min, the volume percentage of mobile phase B increased from 80% to 98%; from 90 to 95 min, the volume percentage of mobile phase B decreased from 98% to 60%; from 95 to 100 min, the volume percentage of mobile phase B decreased from 60% to 35%; from 100 to 105 min, the volume percentage of mobile phase B decreased from 35% to 2%; in the UPLC chromatographic conditions of step (3), the detection wavelength was 277 nm and the flow rate was 0.3 mL min -1 , injection volume 3 μL, column temperature 15 °C; (4) Establishment of UPLC fingerprint Samples were injected according to the chromatographic conditions, UPLC data were recorded, and the raw data were imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" software. Common peak matching was performed using the multi-point correction method to establish the fingerprint. The reference spectrum was generated using the median method, a time window width of 0.5, and a spectrum spacing of 400, and 16 batches of fingerprints of water-soluble fraction samples were obtained.

2. The method for establishing a UPLC fingerprint of a Chinese herbal compound according to claim 1, wherein: The mother liquor of the sample is extracted in step (1) by decocting it three times. The specific extraction conditions are as follows: the pH value of the first decoction is 4.0 and the decoction is 1.0h; the pH value of the second decoction is 7.0 and the decoction is 0.5h; the pH value of the third decoction is 8.0 and the decoction is 0.5h; the decoction liquid-to-material ratio is 1:10; the extraction temperature is 95°C and the reflux extraction is carried out; the pH value of each decoction is 0.1 mol·L -1 HCl and 0.1 mol·L -1 Adjust with NaOH.

3. The method for establishing a UPLC fingerprint of a Chinese herbal compound according to claim 2, wherein: The water-soluble fraction samples were subjected to mass spectrometry analysis. The UPLC fingerprint of the water-soluble fraction of Gancao Xiexin Decoction established in step (4) had 46 common peaks, and 23 compound components were analyzed. These 23 compounds were classified into types and found to be mainly ketones, acids, glycosides, flavonoids, and alkaloids. Among them, 11, 12, 17, 18, 34, 36, 38, and 40 common peaks were attributed to Glycyrrhiza uralensis, 19, 23, 24, 28, 29, 32, 36, and 39 were attributed to Scutellaria baicalensis, 22, 30, and 35 were attributed to Coptis chinensis, 15 was attributed to Pinellia ternata, and 5, 21, and 31 were attributed to Jujube. Among them, peak 4 was phenylalanine, peak 5 was adenosine, peak 6 was glycyrrhizic acid, and peak 8 was 5,7-dihydroxy-6-methoxyflavonoid-7- Peak 10 is sucrose, Peak 11 is formononetin, Peak 12 is liquiritin-4'-O-β-D-glucose-(1→6)-O-β-D-glucoside, Peak 17 is liquiritin, Peak 21 is cyclic adenosine monophosphate, Peak 22 is palmatine hydrochloride, Peak 23 is 5,7,8-trihydroxyflavone-7O-glucoside, Peak 24 is 5,7,8-trihydroxyflavone-7O-glucoside. Peak No. 1 is baicalin, Peak No. 25 is magnolamine, Peak No. 28 is norwogonin-7-O-β-D-glucuronide, Peak No. 29 is scutellarin, Peak No. 30 is coptisine, Peak No. 31 is jujube benzyl glycoside, Peak No. 32 is wogonin, Peak No. 35 is berberine, Peak No. 36 is glycyrrhizin, Peak No. 38 is ural glycyrrhizin P, and Peak No. 40 is ammonium glycyrrhizate.

4. The method for establishing a UPLC fingerprint of a Chinese herbal compound according to claim 3, wherein: The water-soluble part sample had a total of 20 anti-inflammatory active ingredient groups, of which 13 were analyzed, namely 5,7-dihydroxy-6-methoxyflavone-7-O-glucuronide, sucrose, glycyrrhizin-4'-O-β-D-glucose-(1→6)-O-β-D-glucoside, glycyrrhizin, norwogonin-7-O-β-D-glucuronide, baicalin, magnolamine, 5,7,8-trihydroxyflavone-7-O-glucoside, scutellarin, coptisine, baicalin, berberine and ammonium glycyrrhizate; the antibacterial active ingredient group had a total of 25 components, of which 14 were analyzed, namely sucrose, formononetin, glycyrrhizin, cyclic adenosine monophosphate, palmatine hydrochloride, baicalin, magnolamine, 5,7,8-trihydroxyflavone-7-O-glucoside, baicalin, coptisine, baicalin, berberine and ammonium glycyrrhizate. O-glucoside, magnolamine, scutellarin, coptisine, wogonin, liquiritigenin and uralenic saponin P.

5. The application of the fingerprint established by the method for establishing the UPLC fingerprint of a traditional Chinese medicine compound according to any one of claims 1 to 4, characterized in that: The quality inspection of Licorice Decoction for Purging the Heart is specifically about the anti-inflammatory and antibacterial efficacy of Licorice Decoction for Purging the Heart.

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