Method for establishing fingerprint spectrum of heart-strengthening granules and determining contents of 15 components

Abstract

According to the method for establishing the fingerprint spectrum of the heart-strengthening granules and determining the contents of the 15 components, under the cooperation of the specific sample solution and the HPLC condition, the fingerprint spectrum capable of reflecting the 39 chemical component characteristic peaks of the medicinal materials of the heart-strengthening granules is obtained, and the content of the 15 components of the heart-strengthening granules is determined based on the single wavelength (260 nm) by taking representative components of the heart-strengthening granules as contrasts. According to the method, the content of 15 symbolic components including tanshinol, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-glucoside, typhin, isorhamnetin 3-O-neohesperidin, astragalus smicus glycoside, formononin, alkannic acid, salvianolic acid B, cinnamaldehyde, calycosin-7-one, salvianolic acid A and formononetin in the heart-strengthening granules is determined, and the quality of the heart-strengthening granule preparation is comprehensively and effectively controlled. The method provided by the invention has the characteristics of total time consumption of 65 minutes, high analysis efficiency, simplicity, convenience, feasibility, strong specificity and the like, and can provide a basis for improving the quality control level of the existing heart-strengthening granules.

Description

Technical Field

[0001] The present invention belongs to the field of quality control of traditional Chinese medicine preparations, and particularly relates to a method for establishing a fingerprint of Jianxin Granules and determining the contents of 15 components. Background Art

[0002] The main components of Jianxin Granules are eight Chinese medicinal materials, namely red ginseng, astragalus root, cattail pollen, salvia miltiorrhiza, polyporus umbellatus, atractylodes macrocephala, cassia twig, and semen lepidii. Jianxin Granules is a compound preparation composed of 8 traditional Chinese medicines, and the types of its chemical components are numerous and complex. The existing technology can only detect the contents of one or two target components, and cannot effectively control the overall quality of Jianxin Granules. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for establishing a fingerprint of Jianxin Granules and determining the contents of 15 components.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A method for establishing a fingerprint of Jianxin Granules, comprising the following steps:

[0006] (1) Weigh Jianxin Granules precisely to prepare a test solution of Jianxin Granules.

[0007] (2) Detect the test solution by high performance liquid chromatography to obtain a chromatogram of Jianxin Granules.

[0008] The chromatographic conditions include: using a C 18 chromatographic column, 4.6 mm × 250 mm, 4 μm; mobile phase: phase A is acetonitrile, and phase B is an aqueous solution of 0.2% phosphoric acid by volume; the gradient elution conditions are: 0 - 5 min, 3% - 3% phase A; 5 - 10 min, 3% - 5% phase A; 10 - 20 min, 5% - 10% phase A; 20 - 45 min, 10% - 20% phase A; 45 - 55 min, 20% - 25% phase A; 55 - 60 min, 25% - 50% phase A; 60 - 65 min, 50% - 80% phase A; the column temperature is 30°C, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the injection volume is 10 μL;

[0009] (3) Analyze the chromatogram of Jianxin Granules by using the similarity evaluation software for traditional Chinese medicine chromatographic fingerprints to obtain the fingerprint of Jianxin Granules.

[0010] Further, the preparation method of the test sample solution includes: grinding the Jianxin Granules into powder, taking 2.0 g of the powder in a stoppered bottle, accurately weighing, transferring it into 25 mL of a 60% (v / v) methanol aqueous solution, weighing, ultrasonicating for 30 min at a power of 250 W and a frequency of 50 kHz, weighing again, making up the lost weight with a 60% (v / v) methanol aqueous solution, shaking well, filtering, taking 20 mL of the filtrate, heating it in a water bath to evaporate the solvent, redissolving it with 2 mL of a 60% (v / v) methanol aqueous solution, passing it through a 0.22 μm microporous membrane, and taking the subsequent filtrate to obtain the solution.

[0011] Further, the attribution of 39 common peaks in the fingerprint of Jianxin Granules to 8 Chinese medicinal materials is shown as follows: peaks 1, 2, 4, 12, and 13 are attributed to Red Ginseng; peaks 6, 7, 14, 23, 27, 29, 33, and 39 are attributed to Astragalus membranaceus; peaks 3, 11, 26, 28, 31, 35, 36, 37, and 38 are attributed to Salvia miltiorrhiza; peaks 18 and 19 are attributed to Typha angustifolia; peaks 24 and 32 are attributed to Cinnamomum cassia; peaks 21 and 30 are attributed to Lepidium apetalum; peaks 5, 8, and 22 are attributed to Polyporus umbellatus; peak 17 is common to Astragalus membranaceus and Typha angustifolia; peak 9 is common to Red Ginseng, Salvia miltiorrhiza, and Atractylodes macrocephala; peak 16 is common to Salvia miltiorrhiza and Typha angustifolia; peaks 25 and 34 are common to Salvia miltiorrhiza and Lepidium apetalum; peak 20 is common to Astragalus membranaceus, Salvia miltiorrhiza, Typha angustifolia, and Cinnamomum cassia.

[0012] Further, in the fingerprint of Jianxin Granules, peak 3 is Danshensu, peak 8 is Protocatechuic aldehyde, peak 11 is Caffeic acid, peak 16 is Ferulic acid, peak 17 is Calycosin-7-O-β-D-glucoside, peak 18 is Typhaneoside, peak 19 is Isorhamnetin 3-O-neohesperidoside, peak 20 is Astragalin, peak 27 is Ononin, peak 28 is Lithospermic acid B, peak 31 is Salvianolic acid B, peak 32 is Cinnamaldehyde, peak 33 is Calycosin, peak 36 is Salvianolic acid A, and peak 39 is Formononetin.

[0013] A method for determining the contents of 15 components in Jianxin Granules, comprising the following steps:

[0014] 1) Preparation of the test sample solution

[0015] Grind the Jianxin Granules into powder, take 2.0 g of the powder in a stoppered bottle, accurately weigh, transfer it into 25 mL of a 60% (v / v) methanol aqueous solution, weigh, ultrasonicate for 30 min at a power of 250 W and a frequency of 50 kHz, weigh again, make up the lost weight with a 60% (v / v) methanol aqueous solution, shake well, filter, take 20 mL of the filtrate, heat it in a water bath to evaporate the solvent, redissolve it with 2 mL of a 60% (v / v) methanol aqueous solution, pass it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution;

[0016] 2) Preparation of the reference substance solution

[0017] Weigh appropriate amounts of reference substance powders of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin 3-O-neohesperidoside, astragalin, ononin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, and formononetin separately, dissolve them with methanol, and prepare single reference substance stock solutions with mass concentrations of 1.125, 1.280, 1.850, 1.150, 1.580, 3.050, 1.990, 3.340, 1.990, 0.980, 12.050, 2.300, 1.360, 1.550, and 1.710 mg / mL respectively. Precisely measure appropriate amounts of each reference substance and place them in the same volumetric flask, add 60% (v / v) methanol aqueous solution to make up the volume and dilute, shake well, and obtain a mixed reference substance mother liquor with concentrations of 22.50, 51.20, 37.00, 46.00, 63.20, 76.25, 39.80, 83.50, 39.80, 196.00, 2410.00, 92.00, 54.40, 310.00, and 34.20 μg / mL in sequence;

[0018] 3) Preparation of standard curve

[0019] Take the mixed reference substance mother liquor, dilute it with 60% (v / v) methanol aqueous solution to prepare a series of solutions with gradient concentrations, inject them into a high-performance liquid chromatograph for detection. Use the mass concentration as the abscissa and the corresponding chromatographic peak area as the ordinate, and plot the standard curves of 15 components to obtain linear equations;

[0020] 4) Content determination

[0021] Inject the test solution into a high-performance liquid chromatograph for detection, record the peak area, and substitute the peak area into the linear equation to calculate the percentage content of 15 components;

[0022] Among them, the chromatographic conditions in step 3) and step 4) include: using a C 18 chromatographic column, 4.6 mm × 250 mm, 4 μm; mobile phase: phase A is acetonitrile, phase B is 0.2% (v / v) phosphoric acid aqueous solution; gradient elution conditions are: 0 - 5 min, 3% - 3% phase A; 5 - 10 min, 3% - 5% phase A; 10 - 20 min, 5% - 10% phase A; 20 - 45 min, 10% - 20% phase A; 45 - 55 min, 20% - 25% phase A; 55 - 60 min, 25% - 50% phase A; 60 - 65 min, 50% - 80% phase A; column temperature is 30 °C, flow rate is 1.0 mL / min, detection wavelength is 260 nm, and injection volume is 10 μL.

[0023] The advantages of the present invention are as follows:

[0024] The quality control method proposed in this application is established for the first time. The fingerprint spectrum method of Jianxin Granules with an analysis time of 65 minutes has calibrated 39 common characteristic peaks, determined the attribution of the 39 common characteristic peaks in the formulated medicinal materials, and at the same time, based on a single wavelength (260 nm) with the representative components of Jianxin Granules as the control, the contents of 15 components (salvianic acid A, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin 3-O-neohesperidoside, astragalin, ononin, lithospermic acid B, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, formononetin) are determined, so that the contents of common components are quantified, with high analysis efficiency, simple and easy to operate, which can better reflect the internal quality of the preparation and achieve the overall and effective control of the quality of Jianxin Granules preparation.

[0025] I. The specific parameters for the establishment of the HPLC fingerprint spectrum and the determination of the contents of 15 components in Jianxin Granules, including the detection wavelength and the mobile phase gradient elution program.

[0026] II. The method for preparing the sample solution for the HPLC fingerprint spectrum and the determination of the contents of 15 components in Jianxin Granules, including the extraction method, extraction solvent, and extraction time.

[0027] III. The application of the established method in the quality standard control of Jianxin Granules. Description of the Drawings

[0028] Figure 1-1 and Figure 1-2 are all HPLC (260 nm) spectra of different mobile phase systems;

[0029] Figure 2-1 and Figure 2-2 are all HPLC spectra of different absorption wavelengths;

[0030] Figure 3 is the HPLC (260 nm) spectrum of different extraction methods;

[0031] Figure 4 is the HPLC (260 nm) spectrum of different extraction times;

[0032] Figure 5 is the HPLC (260 nm) spectrum of different extraction solvents;

[0033] Figure 6 is the superposition map of the fingerprint spectra of 15 batches of Jianxin Granule samples;

[0034] Figure 7 is the HPLC spectrum of each medicinal material and the test solution;

[0035] Figure 8It is the HPLC chromatogram of Jianxin Granule sample and mixed reference substance solution. Among them, 3 - Danshensu; 8 - Protocatechuic aldehyde; 11 - Caffeic acid; 16 - Ferulic acid; 17 - Calycosin - 7 - O - β - D - glucopyranoside; 18 - Typhaneoside; 19 - Isorhamnetin 3 - O - neohesperidoside; 20 - Astragalin; 27 - Ononin; 28 - Lithospermic acid; 31 - Salvianolic acid B; 32 - Cinnamaldehyde; 33 - Calycosin; 36 - Salvianolic acid A; 39 - Formononetin;

[0036] Figure 9 It is the HPLC chromatogram of mixed reference substance, sample and negative sample test solution;

[0037] Figure 10 It is the cluster heat map analysis of the contents of 15 components in 15 batches of Jianxin Granule. Detailed implementation manners

[0038] To make the above - mentioned features and advantages of the present invention more obvious and understandable, specific embodiments are hereinafter given for detailed description. Unless otherwise specified, the methods of the present invention are all conventional methods in the art.

[0039] Example 1

[0040] 1 Materials

[0041] 1.1 Instruments

[0042] DIONEX U3000 high - performance liquid chromatograph (Thermo Company, USA); KQ - 500DE desktop ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); EX125ZH electronic balance [Ohaus Instruments (Changzhou) Co., Ltd.]; BHS - 2 water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ultrapure water machine (Sichuan Zhuoyue Water Treatment Equipment Co., Ltd.); 5810R centrifuge (Eppendorf Company, Germany).

[0043] 1.2 Drugs and reagents

[0044] Caffeic acid (batch number: 110885-201703), ferulic acid (batch number: 110773-201915), calycosin glucoside (batch number: 111920-202308), and salvianolic acid B (batch number: 111562-201917) were purchased from the China Food and Drug Administration; danshensu (batch number: H09N10S102463); protocatechuic aldehyde (batch number: L17A9D59027), typhalloidin (batch number: Y20A11H116659), isorhamnetin 3-O-neohesperidin (batch number: J16HB188829), astragaloside ( Batch number: F18HB175834) and lithospermic acid (Batch number: Y30S7H22269) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; formononetin (Batch number: PS000674) and formononetin (Batch number: PS000674) were purchased from Chengdu Pusi Biotechnology Co., Ltd.; calycosin (Batch number: 1215-RD-0018) and salvianolic acid A (Batch number: 0305-RF-0102) were purchased from Guangzhou Jiatu Technology Co., Ltd.; cinnamaldehyde (Batch number: DOO19OO6) was purchased from Beijing Manhage Biotechnology Co., Ltd.; the purity of the above reference substances was ≥98%. Astragalus (batch number: X22091502C) was purchased from Tianjin Shengshi Pharmaceutical Co., Ltd., red ginseng (batch number: 2303011) was purchased from Anhui Shouxin Technology Pieces Co., Ltd., Danshen (batch number: 2302031) was purchased from Anhui Puren Chinese Medicine Pieces Co., Ltd., cinnamon twig (batch number: 2310011) was purchased from Bozhou Cijitang Chinese Medicine Pieces Co., Ltd., Atractylodes macrocephala (batch number: 2211020011) was purchased from Bozhou Huqiao Pharmaceutical Co., Ltd., Polyporus umbellatus (batch number: 220901) was purchased from Anhui Fuhua Pharmaceutical Co., Ltd., Pollen Typhae (batch number: 20082802) and Semen Lepidii (batch number: 23021904) were purchased from Anhui Xiehecheng Pharmaceutical Pieces Co., Ltd. The above medicinal materials were used to prepare single-flavor medicines and Yin sample solution; 15 batches of Jianxin Granules, batch numbers 202108004, 202111015, 202202010, 202205013, 202208010, 202211005, 202304006, 202307007, 202309002, 2023120050, 20231209, 202403006, 202406003, 202411003, 202412005, numbered S1 to S15, with a specification of 10 g / bag, all provided by the Preparation Center of the Second People's Hospital Affiliated to Fujian University of Chinese Medicine, methanol and acetonitrile were chromatographically pure, water was ultrapure water, and the other reagents were of analytical grade.

[0045] 2. Investigation of chromatographic and extraction conditions

[0046] 2.1 Selection of mobile phase system

[0047] Take about 2.0 g of Jianxin Keli powder in a stoppered flask, accurately weigh it, transfer it into 25 mL of a 60% (v / v) methanol aqueous solution, weigh it, treat it by ultrasonic wave (power 250 W, frequency 50 kHz) for 30 min, weigh it again, make up the lost weight with a 60% (v / v) methanol aqueous solution, shake well, filter, take 20 mL of the filtrate, evaporate the solvent by heating in a water bath, redissolve it with 2 mL of a 60% (v / v) methanol aqueous solution, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, and inject samples into the mobile phase systems of methanol - pure water, methanol - 0.1% phosphoric acid water, methanol - 0.1% phosphoric acid water, acetonitrile - 0.1% formic acid water, acetonitrile - 0.1% phosphoric acid water, acetonitrile - 0.2% phosphoric acid water, and acetonitrile - 0.3% phosphoric acid water for analysis. Take the overall chromatographic peak resolution as the index to determine the optimal mobile phase system. The results are shown in Figure 1.

[0048] 2.2 Determination of Absorption Wavelength

[0049] Take about 2.0 g of Jianxin Keli powder in a stoppered flask, accurately weigh it, transfer it into 25 mL of a 60% (v / v) methanol aqueous solution, weigh it, treat it by ultrasonic wave (power 250 W, frequency 50 kHz) for 30 min, weigh it again, make up the lost weight with a 60% (v / v) methanol aqueous solution, shake well, filter, take 20 mL of the filtrate, evaporate the solvent by heating in a water bath, redissolve it with 2 mL of a 60% (v / v) methanol aqueous solution, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject samples, compare the chromatograms at wavelengths of 220, 240, 260, 280, 300, and 320 nm, take the total number of chromatographic peaks and resolution as the index to determine the optimal absorption wavelength. The results are shown in Figure 2.

[0050] 2.3 Selection of Extraction Method

[0051] Take 3 portions of the same batch of Jianxin Keli powder, accurately weigh them, transfer them into 25 mL of a 60% (v / v) methanol aqueous solution respectively, and extract them by three extraction methods: standing overnight, ultrasonic extraction for 30 min, and heating under reflux for 30 min. After cooling to room temperature, make up the weight with a 60% (v / v) methanol aqueous solution, shake well, filter, take 20 mL of the filtrate, evaporate the solvent by heating in a water bath, redissolve it with 2 mL of a 60% (v / v) methanol aqueous solution, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject samples, and take the total number of chromatographic peaks and peak area as the index to determine the optimal extraction method. The results are shown in Figure 3 .

[0052] 2.4 Selection of Extraction Time

[0053] Take 4 portions of the same batch of Jianxin Keli powder, each about 2.0 g, in stoppered flasks, accurately weigh, transfer into 25 mL of 60% (v / v) methanol aqueous solution, weigh and record. Ultrasonically extract for 15 min, 30 min, 45 min, and 60 min respectively. After cooling to room temperature, make up the weight with 60% (v / v) methanol aqueous solution, shake well, filter. Take 20 mL of the filtrate, evaporate the solvent by heating in a water bath, redissolve with 2 mL of 60% (v / v) methanol aqueous solution, filter through a 0.22 μm microporous membrane, take the subsequent filtrate, inject the sample. Using the total number of chromatographic peaks and peak area as indicators, determine the optimal extraction time. The results are shown in Figure 4 。

[0054] 2.5 Selection of extraction solvent

[0055] Take 4 portions of the same batch of Jianxin Keli powder, each about 2.0 g, in stoppered flasks. Accurately transfer into 25 mL of 20% (v / v), 40% (v / v), 60% (v / v), and 80% (v / v) methanol aqueous solutions respectively, weigh and record. Ultrasonically treat (power 250 W, frequency 50 kHz) for 30 min. After cooling to room temperature, make up the weight with 60% (v / v) methanol aqueous solution, shake well, filter. Take 20 mL of the filtrate, evaporate the solvent by heating in a water bath, redissolve with 2 mL of 60% (v / v) methanol aqueous solution, filter through a 0.22 μm microporous membrane, take the subsequent filtrate, inject the sample. Using the total number of chromatographic peaks and peak area as indicators, determine the optimal extraction solvent. The results are shown in Figure 5 。

[0056] 3 Methods and results

[0057] 3.1 Chromatographic conditions

[0058] Use a Thermo Scientific Accucore TM XL C 18 chromatographic column (4.6 mm × 250 mm, 4 μm), mobile phase acetonitrile (A) - 0.2% phosphoric acid water (B), gradient elution (0 - 5 min, 3% - 3% A; 5 - 10 min, 3% - 5% A; 10 - 20 min, 5% - 10% A; 20 - 45 min, 10% - 20% A; 45 - 55 min, 20% - 25% A; 55 - 60 min, 25% - 50% A; 60 - 65 min, 50% - 80% A; by volume fraction), column temperature 30 °C, flow rate 1.0 mL / min, detection wavelength 260 nm, injection volume 10 μL.

[0059] 3.2 Preparation of reference substance solution

[0060] Precisely weigh appropriate amounts of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin glucoside, typhanoside, isorhamnetin 3-O-neohesperidin, astragaloside, formononetin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin glucoside, salvianolic acid A, and formononetin reference substance powders, dissolve them in methanol, and prepare solutions with mass concentrations of 1.125, 1.280, 1.850, 1.150, 1.580, 3.050, 1.990, 3.340, 1.990, 0.980, 12.050, 2.300, and 1 .360, 1.550, and 1.710 mg / mL single reference substance stock solutions, accurately measure appropriate amounts of each reference substance and place them in the same volumetric flask, add 60% methanol aqueous solution to dilute to volume, shake well, and obtain mixed reference substance mother solutions with concentrations of 22.50, 51.20, 37.00, 46.00, 63.20, 76.25, 39.80, 83.50, 39.80, 196.00, 2410.00, 92.00, 54.40, 310.00, and 34.20 μg / mL, respectively.

[0061] 3.3 Preparation of test solution

[0062] Grind Jianxin Granules into powder, take about 2.0g of the powder into a stoppered bottle, weigh accurately, transfer into 25mL of 60% methanol aqueous solution, weigh, treat with ultrasound (power 250W, frequency 50kHz) for 30min, weigh again, make up the lost weight with 60% methanol aqueous solution, shake well, filter, take 20mL of the filtrate and heat in a water bath to evaporate the solvent, add 2mL of 60% methanol aqueous solution to re-dissolve, filter through 0.22μm microporous filter membrane, take the filtrate.

[0063] 3.4 Preparation of single herbal medicine and negative sample solutions

[0064] According to the prescription ratio of Jianxin Granule, weigh each medicinal ingredient and the negative sample respectively, and prepare them according to the method under "3.3".

[0065] 3.5 Fingerprint research

[0066] 3.5.1 Precision test

[0067] Take Jianxin granule powder (S3), prepare the test solution according to the method under "3.3", inject the sample 6 times continuously under the conditions under "3.1", record the chromatogram, take peak 17 (calycosin isoflavone glucoside) as the reference peak, calculate the RSD values of the relative peak area and relative retention time of the 39 common peaks of the sample, which are less than 2.64% and 1.94%, respectively, indicating that the instrument has good precision.

[0068] 3.5.2 Repeatability test

[0069] Take the powder of Jianxin Granules (S3), prepare 6 portions of test solution in parallel according to the method under "3.3", inject and determine under the conditions of "3.1", record the chromatogram, take the 17th peak (calycosin-7-O-β-D-glucoside) as the reference peak, calculate the RSDs of the relative peak areas and relative retention times of 39 common peaks in the sample, which are less than 1.75% and 2.24% respectively, indicating that this method has good repeatability.

[0070] 3.5.3 Stability test

[0071] Take the powder of Jianxin Granules (S3), prepare the test solution according to the method under "3.3", inject and determine under the conditions of "3.1" at 0, 2, 4, 8, 12, 16, 24 h at room temperature, record the chromatogram, take the 17th peak (calycosin-7-O-β-D-glucoside) as the reference peak, calculate the RSDs of the relative peak areas and relative retention times of 39 common peaks in the sample, which are less than 1.92% and 2.28% respectively, indicating that the solution has good stability within 24 h.

[0072] 3.5.4 Establishment of fingerprint and similarity analysis

[0073] Take 15 batches of Jianxin Granules samples (S1 - S15), prepare the test solution according to the method under "3.3", inject and determine under the conditions of "3.1", record the chromatogram, import the data into the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" for processing and analysis. Take the S3 batch as the reference chromatogram, adopt the median method, with a time window of 0.1 min, perform multi-point calibration and Mark peak matching to generate the reference chromatogram (R), see Figure 6 , and the results show that 39 common peaks (at 260 nm) are calibrated in 15 batches of samples. The similarities of S1 - S15 batches of samples to the reference chromatogram are 0.998, 0.996, 0.997, 0.995, 0.996, 0.996, 0.992, 0.996, 0.996, 0.998, 0.995, 0.996, 0.996, 0.992, 0.998 respectively, all greater than 0.99, indicating that the composition of the material basis of each batch of Jianxin Granules preparations produced is relatively stable.

[0074] 3.5.5 Attribution and identification of common peaks

[0075] Respectively take the mixed reference substance solution, sample solution, and single-herb solution under "3.2", "3.3", and "3.4", inject and determine under the conditions of "3.1", record the chromatogram, compare the retention times and ultraviolet absorption spectra of each chromatographic peak in the sample solution with the single-herb solution, and determine the attribution of 39 common peaks in Jianxin Granules to 8 herbs. The results are shown in Figure 7, it was shown that peaks 1, 2, 4, 12, and 13 belonged to red ginseng, peaks 6, 7, 14, 23, 27, 29, 33, and 39 belonged to astragalus root, peaks 3, 11, 26, 28, 31, 35, 36, 37, and 38 belonged to salvia miltiorrhiza, peaks 18 and 19 belonged to cattail pollen, peaks 24 and 32 belonged to cassia twig, peaks 21 and 30 belonged to semen lepidii, peaks 5, 8, and 22 belonged to polyporus umbellatus, peak 17 was common to astragalus root and cattail pollen, peak 9 was common to red ginseng, salvia miltiorrhiza, and atractylodes macrocephala, peak 16 was common to salvia miltiorrhiza and cattail pollen, peaks 25 and 34 were common to salvia miltiorrhiza and semen lepidii, and peak 20 was common to astragalus root, salvia miltiorrhiza, cattail pollen, and cassia twig. After consulting the literature and comparing with reference substances, 15 characteristic peaks were identified, namely peak 3 (danshensu), peak 8 (protocatechuic aldehyde), peak 11 (caffeic acid), peak 16 (ferulic acid), peak 17 (calycosin-7-O-β-D-glucoside), peak 18 (typhaneoside), peak 19 (isorhamnetin-3-O-neohesperidoside), peak 20 (astragalin), peak 27 (ononin), peak 28 (lithospermic acid), peak 31 (salvianolic acid B), peak 32 (cinnamaldehyde), peak 33 (calycosin), peak 36 (salvianolic acid A), and peak 39 (formononetin). The results are shown in Figure 8 .

[0076] 3.6 Determination of the Contents of 15 Components in Jianxin Granules

[0077] 3.6.1 Specificity Test

[0078] Take the mixed reference substance solution, test solution, and negative solution under items "3.1", "3.2", and "3.3" and inject them for determination under the conditions of item "3.1". The chromatograms are shown in Figure 9 . The results showed that the resolution of each component was good, and the chromatographic peaks of the negative samples had no interference, indicating good method specificity.

[0079] 3.6.2 Investigation of Linear Relationship

[0080] Take the mixed reference substance solution under item "3.2" and dilute it with a 60% (v / v) methanol aqueous solution to prepare a series of solutions with gradient concentrations. Inject them for determination under the conditions of item "3.1", record the peak areas, use the mass concentration as the abscissa (X) and the corresponding chromatographic peak areas as the ordinate (Y) to plot the linear regression equation and calculate the correlation coefficient (r). Gradually dilute the mixed standard, use the mass concentration with a signal-to-noise ratio (S / N) approximately 3:1 as the detection limit (LOD), and use the mass concentration with an S / N approximately 10:1 as the quantification limit (LOQ). The results are shown in Table 1.

[0081] Table 1 Linear Relationships of Each Component

[0082]

[0083] 3.6.3 Precision Investigation

[0084] Take the mixed reference substance solution under item "3.2", and inject samples continuously 6 times within 1 day under the conditions of item "3.1" and repeat the injection for 3 consecutive days (inject 3 times a day) for determination. Record the peak areas of 15 components. The RSD values of the intra-day precision (n = 6) of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin 3-O-neohesperidoside, astragalin, ononin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, formononetin are 0.71%, 1.33%, 0.70%, 1.59%, 1.20%, 1.40%, 1.51%, 1.34%, 1.49%, 1.20%, 1.17%, 1.55%, 1.59%, 0.80%, 1.16% respectively; the RSD values of the inter-day precision (n = 9) are 2.09%, 2.08%, 1.85%, 2.07%, 1.23%, 1.31%, 1.33%, 1.16%, 1.46%, 1.30%, 1.24%, 1.79%, 2.14%, 1.34%, 2.01% respectively, indicating good instrument precision.

[0085] 3.6.4 Stability investigation

[0086] Take about 2 g of the sample (S3), prepare the test solution according to the method under item "3.3", and measure it under the conditions of item "3.1" after standing at room temperature for 0, 2, 4, 8, 12 and 24 h. Record the peak areas of 15 components. The RSD values of the peak areas of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin 3-O-neohesperidoside, astragalin, ononin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, formononetin are 1.68%, 2.41%, 1.99%, 2.70%, 0.41%, 2.55%, 0.44%, 0.97%, 0.49%, 1.31%, 1.27%, 0.49%, 1.03%, 0.86%, 0.91% respectively, indicating good stability of the test solution within 24 h.

[0087] 3.6.5 Repeatability investigation

[0088] Take samples (S3) of the same batch, about 2 g each, and prepare 6 portions in parallel according to the method under "3.3". Inject and measure under the conditions of "3.1", record the peak areas, substitute them into the linear equation under "3.6.2" to calculate the contents of each component and their RSD values. The average contents of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin 3-O-neohesperidoside, astragalin, ononin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, formononetin were 0.172, 0.036, 0.032, 0.017, 0.056, 0.033, 0.014, 0.072, 0.023, 0.209, 3.005, 0.105, 0.046, 0.238, 0.035 mg·g-1 respectively, and the RSD values were 2.40%, 0.91%, 1.97%, 1.27%, 1.41%, 2.38%, 1.07%, 1.73%, 1.99%, 0.80%, 0.42%, 0.54%, 1.38%, 0.78%, 1.68% respectively, indicating that this method has good repeatability.

[0089] 3.6.6 Recovery Test by Standard Addition

[0090] Take 6 portions of the samples (S3) of the same batch with known contents for the repeatability investigation, about 1.0 g each, and weigh accurately. Add each reference substance according to an approximate ratio of 1:1 to the existing content of the sample. Prepare the test solution according to the method under "3.3". Inject and measure under the conditions of "3.1", and calculate the recovery by standard addition. The results are shown in Table 2.

[0091] Table 2 Results of Recovery Test by Standard Addition for 15 Components

[0092]

[0093]

[0094]

[0095] 3.6.7 Determination of Sample Content

[0096] Take 15 batches of Jianxin Granule samples (S1 - S15), inject and measure according to the corresponding solution preparation method and chromatographic conditions, record the peak areas, substitute them into the linear equation under "3.6.2" to calculate the contents of each component. The results are shown in Table 3; Import the 15 content data of the 15 batches of samples into Origin 2024 software, and perform heat map clustering analysis on them using the Heat Map Dendrogram.opx plug-in, as shown in Figure 10It shows that it is consistent with the HCA clustering results. The 15 batches of samples are mainly clustered into two categories, that is, S3, S10 and S15 belong to the first category, and the remaining batches belong to the second category, indicating that the contents of the 15 determined components can reflect the quality differences of Jianxin Granules in different batches. And according to the color difference of the heat map squares, it can be intuitively found that the overall component content of the first category of samples is significantly higher than that of the second category, which may be related to factors such as raw medicinal materials and preparation processes.

[0097] Table 3 Determination results of the contents of 15 components in 15 batches of Jianxin Granules (n = 3, mg / g)

[0098] Component S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 Mean Danshensu 0.076 0.070 0.172 0.073 0.082 0.095 0.125 0.056 0.097 0.204 0.078 0.074 0.074 0.134 0.160 0.104 Protocatechuic Aldehyde 0.056 0.058 0.036 0.043 0.022 0.031 0.043 0.061 0.057 0.052 0.024 0.023 0.060 0.056 0.037 0.044 Caffeic Acid 0.012 0.015 0.032 0.014 0.011 0.016 0.011 0.026 0.016 0.018 0.013 0.009 0.015 0.011 0.019 0.016 Ferulic Acid 0.010 0.012 0.017 0.013 0.011 0.015 0.020 0.009 0.019 0.013 0.009 0.011 0.011 0.023 0.011 0.014 Calycosin-7-O-β-D-glucoside 0.014 0.044 0.056 0.018 0.038 0.048 0.030 0.017 0.045 0.056 0.017 0.036 0.042 0.031 0.054 0.036 Typhaneoside 0.035 0.058 0.033 0.057 0.009 0.009 0.025 0.046 0.058 0.035 0.021 0.006 0.053 0.027 0.032 0.034 Isorhamnetin 3-O-neohesperidoside 0.018 0.015 0.014 0.015 0.013 0.011 0.017 0.016 0.015 0.017 0.013 0.018 0.014 0.016 0.002 0.014 Astragalin 0.050 0.077 0.072 0.061 0.059 0.080 0.073 0.051 .068 0.075 0.051 0.056 0.064 0.076 0.057 0.065 Ononin 0.013 0.021 0.023 0.007 0.015 0.018 0.012 0.008 0.021 0.023 0.007 0.014 0.019 0.013 0.021 0.016 Lithospermic Acid 0.144 0.201 0.209 0.161 0.110 0.144 0.175 0.145 0.203 0.198 0.154 0.106 0.185 0.181 0.235 0.170 Salvianolic Acid B 1.758 2.499 3.005 2.448 1.260 1.620 1.857 1.766 .533 2.749 2.324 1.192 2.313 1.916 2.866 2.140 Cinnamaldehyde 0.045 0.098 0.105 0.035 0.050 0.064 0.055 0.045 0.107 0.106 0.032 0.049 0.098 0.057 0.100 0.070 Calycosin 0.035 0.023 0.046 0.035 0.024 0.029 0.023 0.035 0.026 0.045 0.034 0.023 0.024 0.024 0.042 0.031 Salvianolic Acid A 0.121 0.190 0.238 0.116 0.114 0.142 0.121 0.118 0.192 0.239 0.110 0.109 0.175 0.128 0.232 0.157 Formononetin 0.035 0.022 0.035 0.022 0.022 0.025 0.022 0.036 0.022 0.035 0.021 0.021 0.020 0.022 0.033 0.026

[0099] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for establishing the fingerprint of Jianxin Granules, characterized in that, It includes the following steps: (1) Weigh the Jianxin Granules precisely and prepare the test solution of Jianxin Granules. (2) Detect the test solution by high performance liquid chromatography to obtain the chromatogram of Jianxin Granules. The chromatographic conditions include: using a C 18 chromatographic column, 4.6 mm × 250 mm, 4 μm; Mobile phase: Phase A is acetonitrile, and Phase B is an aqueous solution of 0.2% phosphoric acid by volume; the gradient elution conditions are as follows: 0 - 5 min, 3% - 3% Phase A; 5 - 10 min, 3% - 5% Phase A; 10 - 20 min, 5% - 10% Phase A; 20 - 45 min, 10% - 20% Phase A; 45 - 55 min, 20% - 25% Phase A; 55 - 60 min, 25% - 50% Phase A; 60 - 65 min, 50% - 80% Phase A; the column temperature is 30 °C, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the injection volume is 10 μL. (3) Analyze the chromatogram of the Jianxin Granules with the similarity evaluation software for traditional Chinese medicine chromatographic fingerprints to obtain the fingerprint of Jianxin Granules.

2. The method according to claim 1, wherein The preparation method of the test solution includes: Grind the Jianxin Granules into powder, take 2.0 g of the powder in a stoppered flask, weigh precisely, transfer it into 25 mL of a 60% methanol aqueous solution by volume, weigh again, ultrasonically treat it for 30 min at a power of 250 W and a frequency of 50 kHz, weigh again, make up the lost weight with a 60% methanol aqueous solution by volume, shake well, filter, take 20 mL of the filtrate, heat it in a water bath to evaporate the solvent to dryness, dissolve it again with 2 mL of a 60% methanol aqueous solution by volume, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it.

3. The method according to claim 1, wherein Among the fingerprints of Jianxin Granules, the attribution of 39 common peaks to 8 medicinal materials is shown. Peaks 1, 2, 4, 12, and 13 belong to red ginseng, peaks 6, 7, 14, 23, 27, 29, 33, and 39 belong to astragalus membranaceus, peaks 3, 11, 26, 28, 31, 35, 36, 37, and 38 belong to salvia miltiorrhiza, peaks 18 and 19 belong to pollen typhae, peaks 24 and 32 belong to cassia twig, peaks 21 and 30 belong to semen lepidii, peaks 5, 8, and 22 belong to polyporus umbellatus, peak 17 is common to astragalus membranaceus and pollen typhae, peak 9 is common to red ginseng, salvia miltiorrhiza, and atractylodes macrocephala, peak 16 is common to salvia miltiorrhiza and pollen typhae, peaks 25 and 34 are common to salvia miltiorrhiza and semen lepidii, and peak 20 is common to astragalus membranaceus, salvia miltiorrhiza, pollen typhae, and cassia twig.

4. The method according to claim 1, characterized in that, In the fingerprint of Jianxin Granule, peak 3 is danshensu, peak 8 is protocatechuic aldehyde, peak 11 is caffeic acid, peak 16 is ferulic acid, peak 17 is calycosin-7-O-β-D-glucoside, peak 18 is typhaneoside, peak 19 is isorhamnetin 3- O neohesperidoside, peak 20 is astragalin, peak 27 is ononin, peak 28 is lithospermic acid, peak 31 is salvianolic acid B, peak 32 is cinnamaldehyde, peak 33 is calycosin, peak 36 is salvianolic acid A, peak 39 is formononetin.

5. A method for determining the contents of 15 components in Jianxin Granules, characterized in that, It includes the following steps: 1) Preparation of the test solution Grind the Jianxin Granules into powder, take 2.0 g of the powder in a stoppered flask, weigh precisely, transfer it into 25 mL of a 60% methanol aqueous solution by volume, weigh again, ultrasonically treat it for 30 min at a power of 250 W and a frequency of 50 kHz, weigh again, make up the lost weight with a 60% methanol aqueous solution by volume, shake well, filter, take 20 mL of the filtrate, heat it in a water bath to evaporate the solvent to dryness, dissolve it again with 2 mL of a 60% methanol aqueous solution by volume, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it; 2) Preparation of the reference solution Accurately weigh appropriate amounts of reference substance powders of danshensu, protocatechuic aldehyde, caffeic acid, ferulic acid, calycosin-7-O-β-D-glucoside, typhaneoside, isorhamnetin-3- O neohesperidoside, astragalin, ononin, lithospermic acid, salvianolic acid B, cinnamaldehyde, calycosin, salvianolic acid A, formononetin, dissolve them with methanol, and prepare single reference substance stock solutions with mass concentrations of 1.125, 1.280, 1.850, 1.150, 1.580, 3.050, 1.990, 3.340, 1.990, 0.980, 12.050, 2.300, 1.360, 1.550, 1.710 mg / mL respectively. Accurately measure appropriate amounts of each reference substance and place them in the same volumetric flask, add 60% (v / v) methanol aqueous solution to make up the volume and dilute, shake well, and obtain a mixed reference substance mother liquor with concentrations of 22.50, 51.20, 37.00, 46.00, 63.20, 76.25, 39.80, 83.50, 39.80, 196.00, 2410.00, 92.00, 54.40, 310.00, 34.20 μg / mL in sequence; 3) Preparation of the standard curve Take the mother liquor of the mixed reference substance, dilute it with a 60% methanol aqueous solution by volume to prepare a series of solutions with gradient concentrations, inject them into a high performance liquid chromatograph for detection, use the mass concentration as the abscissa and the corresponding chromatographic peak area as the ordinate, and plot the standard curves of 15 components to obtain the linear equations; 4) Content determination Inject the test solution into a high performance liquid chromatograph for detection, record the peak area, substitute the peak area into the linear equation, and calculate the percentage content of 15 components; Among them, the chromatographic conditions for steps 3) and 4) include: using a C 18 chromatographic column, 4.6 mm × 250 mm, 4 μm; mobile phase: phase A is acetonitrile, and phase B is an aqueous solution of 0.2% phosphoric acid by volume; the gradient elution conditions are: 0 - 5 min, 3% - 3% phase A; 5 - 10 min, 3% - 5% phase A; 10 - 20 min, 5% - 10% phase A; 20 - 45 min, 10% - 20% phase A; 45 - 55 min, 20% - 25% phase A; 55 - 60 min, 25% - 50% phase A; 60 - 65 min, 50% - 80% phase A; the column temperature is 30°C, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the injection volume is 10 μL.

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