Establishment of UHPLC (Ultra High Performance Liquid Chromatography) fingerprint spectrum of salvia miltiorrhiza-hawthorn drug pair extract and methodology for determining content of index components

Abstract

The invention relates to the field of quality control of traditional Chinese medicines, in particular to establishment of a UHPLC (Ultra High Performance Liquid Chromatography) fingerprint spectrum of a salvia miltiorrhiza-hawthorn medicine pair extract and methodology for determining the content of index components. The establishment method comprises the following steps: preparing a reference substance solution, namely respectively weighing different reference substances, fixing the volume by using methanol, filtering by using a filter membrane, and taking a subsequent filtrate; preparing a test solution, namely weighing various batches of salvia miltiorrhiza-hawthorn pair extracts in a container, carrying out ultrasonic extraction by using 70% methanol, cooling, fixing the volume, filtering by using a filter membrane, and taking a subsequent filtrate; performing UHPLC determination: respectively sucking the reference substance solution and the test solution, and injecting the reference substance solution and the test solution into a UHPLC chromatographic instrument for determination to obtain a UHPLC chromatogram; and generating the UHPLC fingerprint spectrum. The method can provide a certain reference basis for quality control and evaluation of the salvia miltiorrhiza-hawthorn drug pair.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and in particular to the establishment of a UHPLC fingerprint spectrum of a Danshen-Hawthorn medicinal pair extract and a methodology for determining the content of index components. Background Art

[0002] The Danshen-Shanzha Herb Pair (DSHP) consists of two Chinese herbs: Danshen and Crataegus pinnatifida. Danshen is bitter and slightly cold in nature, entering the heart and liver meridians, and has the effects of promoting blood circulation, removing blood stasis, and relieving pain. Crataegus pinnatifida is sour, sweet, and slightly warm in nature, entering the spleen, stomach, and liver meridians, and is effective in clearing turbidity, lowering lipids, and promoting qi and dispersing blood stasis. Existing quality control research on DSHP and its related preparations is primarily limited to the quantitative analysis of individual characteristic components (such as tanshinones and organic acids) within a single herbal ingredient. This single-index component detection method is unable to fully reflect the overall quality characteristics of the DSHP and its related preparations, and cannot meet the "holistic" quality control requirements of traditional Chinese medicine compound formulas.

[0003] The basis for the effectiveness of traditional Chinese medicine (TCM) lies in the chemical constituents it contains. The content and relative proportions of each constituent directly affect the stability and reproducibility of clinical efficacy. In the field of TCM quality control research, fingerprint technology has become an important technical means for quality control of TCM materials and their preparations due to its holistic, characteristic, and quantifiable characteristics. Ultra-high performance liquid chromatography (UHPLC) technology, with its advantages of high resolution, high sensitivity, and high analysis speed, is particularly suitable for the simultaneous detection and analysis of multiple components in complex TCM systems. Currently, combining UHPLC fingerprint technology with multi-index component content determination has become the mainstream direction of TCM quality control methodology research. A literature search shows that there are few studies on the establishment of UHPLC fingerprints for DSHP extracts and the simultaneous quantitative analysis of multiple index components. Summary of the Invention

[0004] To address the gaps in existing technology, this study established a fingerprint analysis method for 14 batches of DSHP extracts based on UHPLC technology. In addition, an analytical method for the simultaneous quantitative detection of 17 indicator components in the extracts was developed. This study aims to provide technical support for the construction of a quality control system for this classic drug pair and its preparations, while laying a scientific foundation for its clinical application.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for establishing a UHPLC fingerprint of a DSHP extract, the method comprising:

[0007] Preparation of reference solution: Weigh 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2, and epicatechin into a volumetric flask, dissolve and dilute to volume with 10% methanol; weigh isoquercetin, hyperoside, dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA into a volumetric flask; weigh oleanolic acid, ursolic acid, rosmarinic acid, lithospermic acid, and salvianolic acid A into a volumetric flask; weigh salvianolic acid B into a volumetric flask, dissolve and dilute to volume with methanol; the reference solution is obtained.

[0008] Preparation of test solution: Weigh each batch of DSHP extract into a container, add 70% methanol and ultrasonically extract for 1 hour. After cooling, dilute to volume with 70% methanol, and then filter with a 0.22 μm filter membrane. Take the filtrate to obtain the test solution;

[0009] UHPLC determination: Aspirate the reference solution and the test solution separately, inject them into the UHPLC chromatograph for determination, and obtain the UHPLC chromatogram.

[0010] It should be noted that the UHPLC determination conditions are as follows: flow rate, 0.2 mL / min; column temperature, 40°C; injection volume, 2 μL; detection wavelength, 210 nm; mobile phase A, acetonitrile; mobile phase B, 0.1% phosphoric acid water; gradient elution program: 0-5 min, 3% mobile phase A and 97% mobile phase B; 5-15 min, 15% mobile phase A and 85% mobile phase B; 15-17 min, 30% mobile phase A and 70% mobile phase B; 17-22 min, 80% mobile phase A and 20% mobile phase B; 22-22.5 min, 90% mobile phase A and 10% mobile phase B; 22.5-30 min, 3% mobile phase A and 97% mobile phase B; greater than 30 min, 3% mobile phase A and 97% mobile phase B.

[0011] Generation of UHPLC fingerprints: The UHPLC chromatograms were processed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. The fingerprints were constructed using the median method. The time window width was set. After multi-point calibration and Mark peak matching, a UHPLC overlay spectrum was generated, which was the UHPLC fingerprint.

[0012] Preferably, the establishment method further comprises performing UHPLC content determination, wherein the UHPLC content determination comprises preparing a reference solution, preparing a test solution, and determining chromatographic conditions.

[0013] Preferably, the establishment method further comprises performing cluster analysis and orthogonal partial least squares discriminant analysis using SIMCA14.1 software, wherein:

[0014] The cluster analysis classified DSHP extracts into two categories, and the classification results were related to the production areas;

[0015] The orthogonal partial least squares discriminant analysis screens out potential markers that affect the quality differences of different batches of DSHP extracts.

[0016] Preferably, the potential markers are dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, salvianolic acid A, 5-hydroxymethylfurfural and proanthocyanidin B2.

[0017] Preferably, the preparation of the reference substance solution includes the preparation of a linear relationship reference substance solution and the preparation of a precision test reference substance solution, and the preparation of the linear relationship reference substance solution includes the preparation of a mixed reference substance I and a mixed reference substance II.

[0018] Preferably, the preparation of the mixed reference substance I comprises: weighing 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2 and epicatechin into a volumetric flask, dissolving and adjusting the volume with 10% methanol; weighing isoquercetin, rosmarinic acid and lithospermic acid into a volumetric flask; weighing hyperoside into a volumetric flask, dissolving and adjusting the volume with methanol; then drawing 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid and lithospermic acid into the same volumetric flask, and adjusting the volume with 10% methanol to obtain the mixed reference substance I.

[0019] Preferably, the preparation of the mixed reference substance II comprises: weighing salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA into a volumetric flask, dissolving and adjusting the volume with methanol; weighing oleanolic acid and ursolic acid into a volumetric flask, dissolving and adjusting the volume with methanol; weighing salvianolic acid B into a volumetric flask, dissolving and adjusting the volume with methanol; then drawing salvianolic acid A, salvianolic acid B, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid and ursolic acid into the same volumetric flask, and adjusting the volume with methanol to obtain the mixed reference substance II;

[0020] Preferably, the preparation of the precision test reference solution comprises: weighing 5-hydroxymethylfurfural, chlorogenic acid, proanthocyanidin B2 and epicatechin into a volumetric flask, dissolving and adjusting the volume with 10% methanol; weighing danshensu into a volumetric flask, dissolving and adjusting the volume with 10% methanol; weighing isoquercetin, tanshinone ⅡA and oleanolic acid into a volumetric flask, dissolving and adjusting the volume with 10% methanol; weighing salvianolic acid B, ursolic acid, rosmarinic acid, lithospermic acid and hyperoside into a volumetric flask, dissolving and adjusting the volume with methanol. ; Weigh salvianolic acid A, dihydrotanshinone I and tanshinone I into a volumetric flask, dissolve them with methanol and make up to volume; then draw 5-hydroxymethylfurfural, tanshinone, chlorogenic acid, proanthocyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid and ursolic acid into the same volumetric flask, make up to volume with 70% methanol to obtain the precision test reference solution.

[0021] Preferably, the preparation of the test solution includes: weighing each batch of DSHP extract into a container, adding 70% methanol for ultrasonic extraction, cooling and then diluting with 70% methanol, then filtering with a 0.1-0.3 μm filter membrane, and taking the filtrate to obtain the test solution.

[0022] In the present invention, the DSHP extract is a freeze-dried extract of Danshen: Crataegus pinnatifida = 1:3 (mass ratio), and its preparation method includes:

[0023] Take Danshen and Crataegus root medicinal materials and dry them in an oven at 50℃;

[0024] Cut the dried Danshen and Hawthorn into small pieces and put them into a 2000 mL round-bottom flask in batches of 15 g Danshen and 45 g Hawthorn. Add 15 times the amount of 70% ethanol and soak for 30 min. Then reflux and extract twice, each time for 40 min.

[0025] The two filtrates were combined, ethanol was recovered, concentrated to a thick paste, and dried in a freeze dryer for 48 h;

[0026] Collect the dry powder, seal it and store it in a desiccator for later use.

[0027] Preferably, the chromatographic conditions include: a flow rate of 0.2 mL / min; a column temperature of 40°C; an injection volume of 2 μL; detection wavelengths of 210 nm, 280 nm and 355 nm; mobile phase A is acetonitrile; mobile phase B is 0.1% phosphoric acid water; and a gradient elution program of: 0-5 min, 3% mobile phase A and 97% mobile phase B; 5-15 min, 15% mobile phase A and 85% mobile phase B; 15-17 min, 30% mobile phase A and 70% mobile phase B; 17-22 min, 80% mobile phase A and 20% mobile phase B; 22-22.5 min, 90% mobile phase A and 10% mobile phase B; 22.5-30 min, 3% mobile phase A and 97% mobile phase B; and more than 30 min, 3% mobile phase A and 97% mobile phase B.

[0028] In a second aspect, the application of the establishment method of the present invention in DSHP quality control is provided.

[0029] In a third aspect, the application of the establishment method of the present invention in detecting candidate detection indicators as an auxiliary method for identifying the quality of DSHP is provided.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention successfully established the UHPLC fingerprint and content determination method of 14 batches of the best compatible extracts of Danshen-Hawthorn medicine pair. Through methodological verification, the content determination of 17 index components in DSHP [5-hydroxymethylfurfural, danshensu, chlorogenic acid (F4), proanthocyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid and ursolic acid] was achieved. Through multivariate statistical analysis, dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, salvianolic acid A, 5-hydroxymethylfurfural and proanthocyanidin B2 were the main chemical components affecting the quality differences of different batches of DSHP extracts, providing data support for the subsequent research on quality markers.

[0032] (2) Due to the numerous components in Danshen and Crataegus pinnatifida, and the varying solubility and resolution of each component, multiple chromatographic conditions were investigated, including mobile phase, flow rate, injection volume, column temperature, detection wavelength, solvent, and gradient elution procedure, ultimately selecting the chromatographic conditions of the present invention. The present invention successfully established a UHPLC fingerprint of DSHP extract, which, combined with the determination of potential biomarkers, can provide a reference for DSHP quality control.

[0033] (3) The UHPLC fingerprint establishment method of the DSHP extract provided by the present invention has high precision, good repeatability and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The common peaks of the fingerprint superposition of 14 batches of DSHP optimal compatibility extracts (S1~S14) and their reference fingerprint (R) are identified; A is the fingerprint superposition; B is the reference fingerprint.

[0035] Figure 2 Figure 1 is a multivariate statistical analysis of the contents of 14 batches of optimal DSHP extracts (S1 to S14); A is a PCA hierarchical clustering diagram; B is an OPLS-DA discriminant analysis score diagram; and C is an OPLS-DA discriminant analysis VIP diagram. DETAILED DESCRIPTION

[0036] The following examples illustrate the technical solutions of the present invention, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0037] Example 1 Experimental Materials for Establishing UHPLC Fingerprint

[0038] 1.1 Instrument

[0039] See Table 1.

[0040] Table 1 Instruments and equipment list

[0041]

[0042]

[0043] 1.2 Medicinal Materials

[0044] Salvia miltiorrhiza and Crataegus pinnatifida were purchased from different manufacturers and paired based on the same manufacturer. They were identified by Professor Tang Haifeng of the Air Force Medical University as the dried roots and rhizomes of Salvia miltiorrhiza (Bge.), a plant of the Lamiaceae family, and the dried mature fruits of Crataegus pinnatifida (Bge.), a plant of the Rosaceae family. Detailed information is provided in Table 2. The extracts from 14 batches of samples were concentrated to a thick paste and dried in a freeze dryer for 48 hours. The dried powder was collected and stored in a sealed desiccator until ready for use.

[0045] Table 2 Sample information of Salvia miltiorrhiza and Crataegus pinnatifida

[0046]

[0047] 1.3 Reagents

[0048] The reference material information table is shown in Table 3.

[0049] Table 3 Reference Material Information

[0050] name purity factory batch number Tanshinone I HPLC ≥98% Baoji Chenguang Biotechnology Co., Ltd. HR1217S1 Salvianolic acid B HPLC ≥98% Baoji Chenguang Biotechnology Co., Ltd. HS20304B1 Oleanolic acid HPLC ≥98% Baoji Chenguang Biotechnology Co., Ltd. HR3488W3 Ursolic acid HPLC ≥98% Baoji Chenguang Biotechnology Co., Ltd. HR243W6 Danshensu HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBG1911 Chlorogenic acid HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFCB1410 Hyperoside HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFDE1305 Isoquercetin HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AZDI2601 Proanthocyanidin B2 HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFCG0633 5-Hydroxymethylfurfural HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBI2704 Rosmarinic acid HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBH1513 Lithospermic acid HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBL0505 Salvianolic acid A HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBL0702 Dihydrotanshinone Ⅰ HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFCB2701 Cryptotanshinone HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFDI2551 Tanshinone IIA HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBG1388 epicatechin HPLC ≥98% Chengdu Aifa Biotechnology Co., Ltd. AFBL0906 phosphoric acid HPLC grade Adamas 81920E Methanol HPLC grade Adamas 75851AJ Acetonitrile HPLC grade ThermoFisher A996-4F

[0051] Example 2 UHPLC fingerprint establishment method and UHPLC content determination method

[0052] 2.1UHPLC fingerprint establishment

[0053] 2.1.1 Preparation of reference solution

[0054] Accurately weigh 35.56 mg of 5-hydroxymethylfurfural, 5.80 mg of danshensu, 5.45 mg of chlorogenic acid, 26.98 mg of procyanidin B, and 6.88 mg of epicatechin into a 50 mL volumetric flask, dissolve in 10% methanol, and dose to volume. Accurately weigh 4.23 mg of isoquercetin, 5.35 mg of hyperoside, 5.71 mg of dihydrotanshinone I, 11.01 mg of tanshinone I, 6.17 mg of cryptotanshinone, and 5.80 mg of tanshinone IIA into a 50 mL volumetric flask. Accurately weigh 6.53 mg of oleanolic acid, 6.68 mg of ursolic acid, 5.54 mg of rosmarinic acid, 5.40 mg of lithospermic acid, and 4.16 mg of salvianolic acid A into a 25 mL volumetric flask, and 5.82 mg of salvianolic acid B into a 10 mL volumetric flask, dissolve in methanol, and dose to volume. Then dilute salvianolic acid B to 10 times, and dilute the other reference substances to 20 times. The concentrations are shown in Table 4 below.

[0055] Table 4 Concentration of mixed reference substances (μg / mL)

[0056] name Concentration (μg / mL) 5-Hydroxymethylfurfural 34.849 Danshensu 35.280 Chlorogenic acid 22.442 Proanthocyanidin B2 30.164 epicatechin 73.480 Hyperoside 10.535 Isoquercetin 10.898 Rosmarinic acid 21.599 Lithospermic acid 25.612 Salvianolic acid B 660.128 Salvianolic acid A 8.134 Dihydrotanshinone Ⅰ 5.610 Tanshinone I 11.094 Cryptotanshinone 12.142 Tanshinone IIA 17.081 Oleanolic acid 16.258 Ursolic acid 79.686

[0057] 2.1.2 Preparation of test solution

[0058] Accurately weigh 100 mg of each batch of samples and place them in a 10 mL volumetric flask. Prepare two parallel portions, add 70% methanol and extract them ultrasonically (40 kHz) for 1 hour. Cool and dilute to the mark with 70% methanol. Filter the solution with a 0.22 μm filter membrane and take the filtrate to obtain the test solution.

[0059] 2.1.3 Chromatographic conditions

[0060] Chromatograph: Thermo Vanquish Flex UHPLC liquid chromatograph; chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 mm × 150 mm, 1.7 μm); flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 2 μL; detection wavelength: 210 nm; mobile phase: acetonitrile (A)-0.1% phosphoric acid (B); gradient elution: see Table 5.

[0061] Table 5 Elution gradient

[0062] Time (min) Acetonitrile (A,%) 0.1% phosphoric acid water (B,%) 0 3 97 5 15 85 15 30 70 17 80 20 22 90 10 22.5 3 97 30 3 97

[0063] 2.1.4 Methodological Validation

[0064] The relative retention times of the common peaks and the relative peak areas of peaks 3, 6, 7, 9, 10, 11, 14 and 15, which account for more than 5% of the peak area, were investigated with salvianolic acid B (peak 16, S) as a reference.

[0065] 2.1.4.1 Precision test

[0066] The test solution of batch number S13 was injected continuously for 6 times according to the chromatographic conditions under "2.1.3". With salvianolic acid B (peak 16, S) as the reference, the relative retention time RSDs of the common peaks were calculated to be less than 0.317%, and the relative peak areas of peaks 3, 6, 7, 9, 10, 11, 14, and 15, which accounted for more than 5% of the peak area, were RSDs less than 0.751%, indicating that the instrument had good precision (Tables 6 and 7).

[0067] Table 6 Precision investigation-relative retention time

[0068]

[0069]

[0070] Table 7 Precision investigation - relative peak area

[0071]

[0072] 2.1.4.2 Repeatability test

[0073] Six portions of the test solution with batch number S13 were prepared and injected continuously for determination six times according to the chromatographic conditions under "2.1.3". Using salvianolic acid B (peak 16, S) as a reference, the relative retention time RSDs of the common peaks were calculated to be less than 0.399%, and the relative peak areas of peaks 3, 6, 7, 9, 10, 11, 14, and 15, which accounted for more than 5% of the peak area, were RSDs less than 2.088%, indicating that the method had good reproducibility (Tables 8 and 9).

[0074] Table 8 Repeatability test-relative retention time

[0075]

[0076]

[0077] Table 9 Repeatability test - relative peak area 2.1.4.3 Stability test

[0078]

[0079] Prepare the test solution with batch number S13, and place it at room temperature for 0, 1, 2, 4, 8, 12, and 24 hours respectively. Then, inject the sample and measure it according to the chromatographic conditions under "2.1.3". Using salvianolic acid B (peak 16, S) as the reference, the relative retention time RSD of each common peak was calculated to be less than 0.389%, and the relative peak area RSD of peak 3, peak 6, peak 7, peak 9, peak 10, peak 11, peak 14, and peak 15 with a peak area greater than 5% was less than 3.081%, indicating that the sample has good stability and meets the requirements (Table 10 and Table 11).

[0080] Table 10 Stability test - relative retention time

[0081]

[0082]

[0083] Table 11 Stability test - relative peak area

[0084]

[0085]

[0086] 2.1.4.4 Fingerprint Generation and Similarity Evaluation

[0087] Fourteen batches of extract samples containing the optimal DSHP compatibility ratio were selected for the experiment. Test solutions were prepared according to the method in "2.1.2," and analysis was performed according to the chromatographic conditions in "2.1.3." The test results were exported in CDF format and subsequently imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) for data processing. Fingerprints were constructed using the median method, with a time window set to 0.2 minutes. Sample S1 was used as a reference. After multi-point calibration and Mark peak matching, a control fingerprint (R) and a UHPLC characteristic fingerprint were generated, and similarity evaluation and analysis were completed.

[0088] 2.2 UHPLC content determination

[0089] 2.2.1 Preparation of reference solution

[0090] 2.2.1.1 Preparation of linear relationship reference solution

[0091] Since the solubility of highly polar components such as 5-hydroxymethylfurfural and danshensu in methanol is low, 10% methanol was used to prepare the stock solutions of highly polar components.

[0092] Mixed Reference Substance I: Accurately weigh 35.56 mg of 5-hydroxymethylfurfural, 12.00 mg of danshensu, 11.45 mg of chlorogenic acid, 215.39 mg of procyanidin B, and 37.49 mg of epicatechin into a 50 mL volumetric flask and dissolve in 10% methanol until volume is reached. Accurately weigh 11.12 mg of isoquercetin, 11.02 mg of rosmarinic acid, and 10.89 mg of lithospermic acid into a 50 mL volumetric flask, and 10.75 mg of hyperoside into a 100 mL volumetric flask and dissolve in methanol until volume is reached. Then, pipette 0.5 mL of 5-hydroxymethylfurfural, 1.5 mL of danshensu, 1 mL of chlorogenic acid, 21 mL of procyanidin B, 1 mL of epicatechin, 1 mL of hyperoside, 0.5 mL of isoquercetin, 1 mL of rosmarinic acid, and 1.2 mL of lithospermic acid into the same 10 mL volumetric flask and bring to volume with 10% methanol.

[0093] Mixed Reference Substance II: Accurately weigh 16.60 mg of salvianolic acid A, 11.45 mg of dihydrotanshinone I, 11.32 mg of tanshinone I, 12.39 mg of cryptotanshinone, and 11.62 mg of tanshinone IIA into a 100 mL volumetric flask, dissolve in methanol, and bring to volume. Accurately weigh 16.59 mg of oleanolic acid and 16.94 mg of ursolic acid into a 50 mL volumetric flask, dissolve in methanol, and bring to volume. Accurately weigh 33.68 mg of salvianolic acid B into a 10 mL volumetric flask, dissolve in methanol, and bring to volume. Then, pipette 0.5 mL of salvianolic acid A, 2 mL of salvianolic acid B, 0.5 mL of dihydrotanshinone I, 1 mL of tanshinone I, 1 mL of cryptotanshinone, 1.5 mL of tanshinone IIA, 0.5 mL of oleanolic acid, and 2.4 mL of ursolic acid into the same 10 mL volumetric flask with methanol and bring to volume.

[0094] 2.2.1.2 Preparation of reference solution for precision test

[0095] Accurately weigh 35.56 mg of 5-hydroxymethylfurfural, 11.45 mg of chlorogenic acid, 15.39 mg of proanthocyanidin B2, and 37.49 mg of epicatechin to a 50 mL volumetric flask, dissolve in 10% methanol, and adjust to volume; accurately weigh 12.02 mg of danshensu to a 25 mL volumetric flask, dissolve in 10% methanol, and adjust to volume; accurately weigh 11.12 mg of isoquercetin, 11.63 mg of tanshinone ⅡA, and 16.59 mg of oleanolic acid to a 50 mL volumetric flask, dissolve in methanol, and adjust to volume; accurately weigh salvianolic acid B to a 50 mL volumetric flask, dissolve in 10% methanol, and adjust to volume; accurately weigh 12.02 mg of danshensu to a 25 mL volumetric flask, dissolve in 10% methanol, and adjust to volume; accurately weigh 11.12 mg of isoquercetin, 11.63 mg of tanshinone ⅡA, and 16.59 mg of oleanolic acid to a 50 mL volumetric flask, dissolve in methanol, and adjust to volume; accurately weigh 12.02 mg of danshensu to a 25 mL volumetric flask, dissolve in 10% ... 84.20 mg, ursolic acid 16.98 mg, rosmarinic acid 20.93 mg, lithospermic acid 25.58 mg, and hyperoside 12.55 mg were dissolved in methanol in a 25 mL volumetric flask and the volume was adjusted to the desired volume. 16.60 mg of salvianolic acid A, 11.45 mg of dihydrotanshinone I, and 11.32 mg of tanshinone I were accurately weighed separately and dissolved in methanol in a 100 mL volumetric flask and the volume was adjusted to the desired volume. Pipette 0.04 mL of 5-hydroxymethylfurfural, 0.36 mL of danshensu, 0.54 mL of chlorogenic acid, 21.25 mL of procyanidin B, 0.58 mL of epicatechin, 0.2 mL of hyperoside, 0.25 mL of isoquercetin, 0.2 mL of rosmarinic acid, 0.3 mL of lithospermic acid, 1.6 mL of salvianolic acid B, 0.35 mL of salvianolic acid A, 0.06 mL of dihydrotanshinone I, 0.24 mL of tanshinone I, 0.4 mL of cryptotanshinone, 0.65 mL of tanshinone II A, 0.4 mL of oleanolic acid, and 1.1 mL of ursolic acid into the same 10 mL volumetric flask, and dilute to volume with 70% methanol.

[0096] See Table 12.

[0097] Table 12 Concentration of mixed reference substances (μg / mL)

[0098]

[0099]

[0100] 2.2.2 Preparation of test solution

[0101] Take 100 mg of each batch of samples, accurately weigh them, and place them in a 10 mL volumetric flask. Prepare two parallel portions, add 70% methanol and extract them ultrasonically (40 kHz) for 1 hour. Cool them, make up to the mark with 70% methanol, filter the solution with a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0102] 2.2.3 Chromatographic conditions

[0103] The chromatographic conditions were the same as those in 2.1.3. The detection wavelength for oleanolic acid and ursolic acid was 210 nm, the detection wavelength for chlorogenic acid, hyperoside, isoquercetin, rosmarinic acid, and lithospermic acid was 355 nm, and the detection wavelength for the remaining components was 280 nm.

[0104] 2.2.4 Methodological Validation

[0105] 2.2.4.1 Linear relationship investigation

[0106] Proanthocyanidin B2 and lithospermic acid used the "2.1.1." precision test reference solution as the highest concentration point working solution, and the remaining 15 components to be tested used mixed reference substances I and II as the highest concentration point working solution. Other concentration working solutions were prepared by diluting mixed reference substances I and II to 2.5, 5, 10, 20, and 40 times, respectively. Two portions of each concentration working solution were prepared in parallel, and a standard curve was drawn with concentration (μg / mL) as the horizontal axis and peak area mean (n=2) as the vertical axis. Table 13 shows that the linear relationship of each component to be tested is good, R 2 Greater than 0.9997.

[0107] Table 13 Linear relationship investigation

[0108]

[0109]

[0110] 2.2.4.2 Precision test

[0111] Inject the precision test reference solution in section 2.2.1.2 five times in succession, and calculate the RSD of the peak area of ​​each analyte. The results in Table 14 show that the precision of this method meets the requirements, with the RSD of the peak area of ​​each analyte being less than 1.5%.

[0112] Table 14 System suitability test

[0113]

[0114] 2.2.4.3 Repeatability test

[0115] Six portions of sample batch S10 were accurately weighed and prepared into test solutions, and the content of each component was determined. The results in Table 15 show that the repeatability of this method meets the requirements, with the RSD of the content of each component being determined being less than 1.5%.

[0116] Table 15 Repeatability test

[0117]

[0118] 2.2.4.4 Stability test

[0119] Sample 1 (S1) from the repeatability test was measured at room temperature for 0, 1, 4, 8, 12, 18, and 24 hours, and the RSD of the peak area of ​​each analyte was calculated. The results are shown in Table 16. The sample remained stable at room temperature for 24 hours, with the RSD of the peak area of ​​each analyte less than 3%.

[0120] Table 16 Stability test

[0121]

[0122]

[0123] 2.2.4.5 Sample recovery

[0124] Accurately weigh 50 mg of sample batch S10 into a 10 mL volumetric flask. Repeat for nine replicates. Add 1.4 mL, 2.8 mL, and 4.2 mL of the precision test reference solution described in section 2.2.1.2 to each three-dose sample. Add an appropriate amount of 70% methanol and ultrasonically condition (40 kHz) for 1 hour before derating to volume. Calculate the recovery of each analyte using the formula [Amount measured (μg) / Amount added (μg) × 100%]. Tables 17-33 show that the recovery of each analyte meets the requirements.

[0125] Table 175-Hydroxymethylfurfural sample recovery

[0126]

[0127]

[0128] Table 18 Danshensu sample recovery

[0129]

[0130] Table 19 Chlorogenic acid sample recovery

[0131]

[0132] Table 20 Proanthocyanidin B2 recovery

[0133]

[0134]

[0135] Table 21 epicatechin sample recovery

[0136]

[0137] Table 22 Hyperoside sample recovery

[0138]

[0139]

[0140] Table 23 Isoquercetin sample recovery

[0141]

[0142] Table 24 Rosmarinic acid sample recovery

[0143]

[0144] Table 25 lithospermic acid recovery

[0145]

[0146]

[0147] Table 26 Salvianolic acid B recovery

[0148]

[0149] Table 27 Salvianolic acid A sample recovery

[0150]

[0151] Table 28 Dihydrotanshinone Ⅰ sample recovery

[0152]

[0153]

[0154] Table 29 Tanshinone I sample recovery

[0155]

[0156] Table 30 Cryptotanshinone sample recovery

[0157]

[0158]

[0159] Table 31 Tanshinone Ⅱ A sample recovery

[0160]

[0161] Table 32 Oleanolic acid recovery

[0162]

[0163] Table 33 Ursolic acid recovery

[0164]

[0165]

[0166] 2.2.4.6 Content determination

[0167] Take 14 batches of samples and prepare two test solutions in parallel for each batch of samples according to the method in "2.2.2". Inject and measure the samples under the chromatographic conditions in "2.2.3" and calculate the contents of the above 17 components.

[0168] Example 3 Results

[0169] 3.1 Establishment and similarity evaluation of the fingerprint of the best compatible DSHP extract

[0170] The results of multiple batches of samples were sequentially imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software in CDF format to obtain the superimposed spectra of 14 batches of samples. Figure 1 .Depend on Figure 1 It can be seen that a total of 26 common peaks were calibrated and 17 chemical components were identified, namely 5-hydroxymethylfurfural (peak 1), tanshinone (peak 2), chlorogenic acid (peak 4), proanthocyanidin B2 (peak 6), epicatechin (peak 7), hyperoside (peak 11), isoquercetin (peak 12), rosmarinic acid (peak 14), lithospermic acid (peak 15), salvianolic acid B (peak 16, S), salvianolic acid A (peak 18), dihydrotanshinone I (peak 19), tanshinone I (peak 20), cryptotanshinone (peak 21), tanshinone IIA (peak 23), oleanolic acid (peak 25), and ursolic acid (peak 26).

[0171] Taking the control fingerprint (R) as a reference, the similarity of the fingerprints of 14 batches of DSHP is shown in Table 34. The results in Table 34 show that the fingerprint similarity of the best compatible DSHP extracts is greater than 0.9, and the material basis composition of different batches of DSHP samples is relatively stable.

[0172] Table 34 Similarity results

[0173]

[0174]

[0175] 3.2 DSHP index component content determination results

[0176] The results of the content determination of 14 batches of DSHP are shown in Table 35. The top five components with the highest average content were salvianolic acid B, ursolic acid, epicatechin, lithospermic acid, and proanthocyanidin B2, which were 1.781%, 0.419%, 0.096%, 0.089%, and 0.081%, respectively. The contents of the 17 components were imported into SIMCA14.1 software for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to evaluate the comprehensive quality of the 14 batches of DSHP extracts. The hierarchical cluster analysis in the PCA model divided the 14 batches of extracts into two categories, with S8, S1, S5, S9, S10, S11, and S12 as one group and S3, S4, S13, S2, S14, S6, and S7 as another group, showing certain differences in production areas ( Figure 2 (A) OPLS-DA is a multivariate statistical analysis method primarily used for classification and feature selection, screening biomarkers of drug action, and evaluating drug effects. Figure 2 Figure B shows that the results of OPLS-DA and PCA cluster analysis are consistent, and the overall quality of S14 is good. The VIP diagram of the 17 variables in this model was extracted ( Figure 2 In Figure C, variables with VIP values ​​greater than 1 were used as characteristic variables contributing to significant differences between groups. Peaks with VIP values ​​greater than 1 were identified as dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, salvianolic acid A, 5-hydroxymethylfurfural, and procyanidin B2. Therefore, these seven components may be the primary potential markers for quality differences between batches of DSHP extracts.

[0177] Table 35 Content determination results (%)

[0178]

[0179]

[0180] As can be seen, the present invention successfully established UHPLC fingerprints and content determination methods for 14 batches of optimally compatible DSHP extracts. Through methodological validation, the content of 17 indicator components in DSHP [5-hydroxymethylfurfural, tanshinone, chlorogenic acid (F4), procyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid, and ursolic acid] was determined. Through multivariate statistical analysis, dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, salvianolic acid A, 5-hydroxymethylfurfural, and procyanidin B2 were the main chemical components affecting the quality differences between different batches of DSHP extracts, providing data support for subsequent research on quality markers.

Claims

1. A method for establishing a UHPLC fingerprint of a Salvia miltiorrhiza-crataegi extract, characterized in that: The establishment method comprises: Preparation of reference solution: Weigh 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2, and epicatechin into a volumetric flask, dissolve and dilute to volume with 10% methanol; weigh isoquercetin, hyperoside, dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA into a volumetric flask; weigh oleanolic acid, ursolic acid, rosmarinic acid, lithospermic acid, and salvianolic acid A into a volumetric flask; weigh salvianolic acid B into a volumetric flask, dissolve and dilute to volume with methanol; the reference solution is obtained. Preparation of test solution: Weigh each batch of Danshen-Hawthorn extract into a container, add 70% methanol and ultrasonically extract for 1 hour. After cooling, dilute to volume with 70% methanol, and filter through a 0.22μm filter membrane. Take the filtrate to obtain the test solution; UHPLC determination: Aspirate the reference solution and the test solution separately, inject them into the UHPLC chromatograph for determination, and obtain the UHPLC chromatogram; Generation of UHPLC fingerprints: The UHPLC chromatograms were processed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. The fingerprints were constructed using the median method. The time window width was set. After multi-point calibration and Mark peak matching, a UHPLC overlay spectrum was generated, which was the UHPLC fingerprint.

2. The establishment method according to claim 1, characterized in that The established method also includes performing UHPLC content determination, which includes preparing a reference solution, preparing a test solution, and determining chromatographic conditions.

3. The establishment method according to claim 1, characterized in that The establishment method also includes using SIMCA14.1 software to perform cluster analysis and orthogonal partial least squares discriminant analysis, wherein, The cluster analysis classified the extracts of the Danshen-Hawthorn pair into two categories, and the classification results were related to the production areas; The orthogonal partial least squares discriminant analysis screens out potential markers that affect the quality differences of different batches of Danshen-Hawthorn medicinal pair extracts.

4. The establishment method according to claim 3, characterized in that: The potential markers are dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, salvianolic acid A, 5-hydroxymethylfurfural and proanthocyanidin B2.

5. The establishment method according to claim 2, characterized in that: The preparation of the reference substance solution includes the preparation of the linear relationship reference substance solution and the preparation of the precision test reference substance solution, and the preparation of the linear relationship reference substance solution includes the preparation of the mixed reference substance I and the preparation of the mixed reference substance II.

6. The establishment method according to claim 5, characterized in that: The preparation of the mixed reference substance I includes: weighing 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2 and epicatechin into a volumetric flask, dissolving and adjusting the volume with 10% methanol; weighing isoquercetin, rosmarinic acid and lithospermic acid into a volumetric flask; weighing hyperoside into a volumetric flask, dissolving and adjusting the volume with methanol; then aspirating 5-hydroxymethylfurfural, danshensu, chlorogenic acid, procyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid and lithospermic acid into a volumetric flask, adjusting the volume with 10% methanol, and obtaining the mixed reference substance I.

7. The establishment method according to claim 5, characterized in that: The preparation of the mixed reference substance II includes: weighing salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA into a volumetric flask, dissolving and adjusting the volume with methanol; weighing oleanolic acid and ursolic acid into a volumetric flask, dissolving and adjusting the volume with methanol; weighing salvianolic acid B into a volumetric flask, dissolving and adjusting the volume with methanol; then aspirating salvianolic acid A, salvianolic acid B, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid and ursolic acid into a volumetric flask, adjusting the volume with methanol, and obtaining the mixed reference substance II.

8. The establishment method according to claim 5, characterized in that: The preparation of the precision test reference solution comprises: weighing 5-hydroxymethylfurfural, chlorogenic acid, proanthocyanidin B2 and epicatechin into a volumetric flask, dissolving them with 10% methanol and adjusting the volume to the desired volume; weighing danshensu into a volumetric flask, dissolving them with 10% methanol and adjusting the volume to the desired volume; weighing isoquercetin, tanshinone ⅡA and oleanolic acid into a volumetric flask, dissolving them with 10% methanol and adjusting the volume to the desired volume; weighing salvianolic acid B, ursolic acid, rosmarinic acid, lithospermic acid and hyperoside into a volumetric flask, dissolving them with methanol and adjusting the volume to the desired volume; weighing Take salvianolic acid A, dihydrotanshinone I and tanshinone I into a volumetric flask, dissolve them with methanol and make up to volume; then draw 5-hydroxymethylfurfural, tanshinone, chlorogenic acid, proanthocyanidin B2, epicatechin, hyperoside, isoquercetin, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA, oleanolic acid and ursolic acid into the same volumetric flask, make up to volume with 70% methanol to obtain the precision test reference solution.

9. Application of the established method according to any one of claims 1 to 8 in the quality control of the Danshen-Hawthorn medicinal pair.

10. Use of the established method according to any one of claims 1 to 8 in detecting candidate detection indicators to assist in identifying the quality of the Danshen-Hawthorn drug pair.

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