Method for rapidly detecting components of heart-tonifying drink based on liquid chromatography-mass spectrometry

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a heart-tonifying drink component rapid detection method based on a liquid chromatography-mass spectrometry technology. According to the method, the UHPLC-Q-TOF / MS analysis technology is adopted, positive and negative ion mode scanning is adopted, and according to information such as compound retention time, accurate molecular weight, secondary fragment ions and the like, the modes such as reference substance comparison, literature report and online database comparison are combined, so that in-vitro component identification of the heart benefiting drink is rapidly and systematically carried out; the time change of plasma and heart migration components of the atrial fibrillation rats is investigated. The invention provides a research direction and an experimental basis for the subsequent in-depth research of the pharmacodynamic material basis and the action mechanism of the heart-tonifying decoction for treating atrial fibrillation.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a method for rapid detection of ingredients in Yixin Drink based on liquid chromatography-mass spectrometry technology. Background Art

[0002] Atrial fibrillation, also known as AF, is a common, persistent cardiac arrhythmia caused by disrupted atrial electrical activity. It is the most common arrhythmia and a high-risk factor for complications such as stroke, pulmonary embolism, and heart failure. Recurrence of AF occurs in approximately 40%-50% of patients, with a high mortality rate and a high level of invisibility, making it a major global public health issue.

[0003] Yixinyin oral liquid (abbreviated as "Yixinyin") is derived from Zhang Zhongjing's famous prescription Zhigancao Decoction. It is made from 15 herbs, including Zhigancao, Danshen, Astragalus, Sichuan cinnamon twig, and ginger. Currently, Yixinyin has been used in the clinic as an in-hospital preparation for over 30 years and has shown significant efficacy in treating arrhythmias, myocarditis, and coronary heart disease (Lin Houwen, Shen Yang, Shen Limin, et al. Thin-layer chromatography identification of Yixinyin oral liquid [J]. Shizhen Traditional Chinese Medicine, 2001(5): 421-422. Zheng Peiyong, Qin Zhifeng, Wei Xiao. Effect of Yixinyin on SOD and MDA activities in mice with viral myocarditis [J]. Tianjin Journal of Traditional Chinese Medicine, 2004(3): 185-187).

[0004] However, a rapid detection method for the in vitro components of Yixin Drink based on liquid chromatography-mass spectrometry has not been reported yet. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for rapid detection of in vitro components of Yixin Drink based on liquid chromatography-mass spectrometry technology.

[0006] The present invention provides a method for rapid detection of Yixin Drink components based on liquid chromatography-mass spectrometry technology. The method adopts UHPLC-Q-TOF / MS analysis technology, adopts positive and negative ion mode scanning, and according to the retention time, precise molecular weight, and secondary fragment ion information of the compound, combined with reference substance comparison, literature reports, and online database comparison, rapidly and systematically carries out identification of the in vitro components of Yixin Drink and identification of the chemical component distribution of Yixin Drink in the plasma and heart of dosed rats.

[0007] Furthermore, in the rapid detection method of Yixin Drink ingredients based on liquid chromatography-mass spectrometry technology, the UHPLC-Q-TOF / MS conditions are:

[0008] Chromatographic conditions: Column: Waters ACQUITY UPLC HSS T3 column (1 mm × 100 mm, 1.8 μm); Mobile phase: 0.1% formic acid in water (A)-acetonitrile (B), Chromatographic elution gradient: 0-3 min, 3%-5% B; 3-7 min, 5%-60% B; 7-18 min, 60%-70% B; 18-20 min, 70%-95% B; 20-23 min, 95%-95% B), Post-run time: 3 min; Flow rate: 0.35 mL / min; Column temperature: 35 °C; Injection volume: 2 μL;

[0009] Mass spectrometry conditions: an electrospray ionization (ESI) source was used to acquire data in both positive and negative ion modes over the m / z range of 100 to 1700. The ion source temperature was 350°C. The capillary voltages were 4.0 kV for positive ions and 3.5 kV for negative ions. The nebulizer gas pressure was 45 psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350°C, the fragmentor voltage was 140 V, and the collision energy was 30 V.

[0010] Furthermore, the rapid detection method of Yixin Drink ingredients based on liquid chromatography-mass spectrometry technology comprises the following steps:

[0011] (A) In vivo and in vitro component analysis

[0012] (A1) Preparation of reference solution

[0013] Accurately weigh 24 reference substances including 4-hydroxycinnamic acid, apigenin isoliquiritigenin, liquiritin, daidzein, calycosin isoflavone, curcumin, isoliquiritigenin, myristyl ether, tanshinone IIB, glycyrrhetinic acid, caffeic acid, vanillin, salvianolic acid B, formononetin, myristic acid, calycosin isoflavone glycosides, gingerol, ligusticolide A, 3-hydroxycoumarin, salvianolic acid A, astragaloside IV, isoliquiritigenin, gingerol, shogaolide A, and ligusticolide, add 80% methanol, sonicate and dissolve, and finally prepare the mixture to a concentration of 1.0 mg / mL reference solution; 4-hydroxycinnamic acid, apigenin isoliquiritigenin, liquiritin, daidzein, calycosin isoflavones, curcumin, isoliquiritigenin, myristyl ether, tanshinone IIB and glycyrrhetinic acid are prepared as mixed reference substance 1; caffeic acid, vanillin, salvianolic acid B, formononetin, myristic acid, calycosin isoflavone glycosides, gingerol and ligusticolide A are prepared as mixed reference substance 2; 3-hydroxycoumarin, salvianolic acid A, astragaloside IV, isoliquiritigenin, zingerenone A and ligusticolide are prepared as mixed reference substance 3, ready for injection;

[0014] (A2) Preparation of Yixinyin Extract Test Solution

[0015] Yixinyin extract was placed in an EP tube, and the same volume of 80% methanol (containing 200 ng / mL warfarin) was added. After ultrasonic extraction for 30 min, the extract was heated at 13,000 r·min. -1 Centrifuge for 15 min under the same conditions, and transfer the supernatant to a brown injection vial for analysis;

[0016] (A3) Preparation of plasma test solution

[0017] 0.2 mL of rat plasma was added with 0.8 mL of methanol (containing 200 ng / mL warfarin), vortexed and then rotated at 4 °C and 13,000 r·min. -1 The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL of 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r·min. -1 Centrifuge for 10 min under the same conditions, pipette the supernatant into a brown injection vial, and pipette 10 μL of each supernatant into the injection vial, mix well, and use as QC;

[0018] (A4) Preparation of Heart Test Solution

[0019] About 100 mg of the rat heart was taken, added with 5 times the volume of ice-cold physiological saline, and homogenized using a low-temperature tissue grinder at -20 °C and 50 Hz. 300 μL of the homogenate from each sample was transferred to a new EP tube, 1.2 mL of methanol (containing 200 ng / mL warfarin) was added, vortexed to mix, and then rotated at 4 °C and 13,000 r·min. -1 The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r·min. -1 Centrifuge for 10 min under the same conditions, pipette the supernatant into a vial, and pipette 10 μL of each supernatant into a vial, mix well, and use as QC;

[0020] (B) UHPLC-Q-TOF / MS conditions

[0021] Chromatographic conditions: Column: Waters ACQUITYUPLC HSS T3 column (1 mm × 100 mm, 1.8 μm); Mobile phase: 0.1% formic acid in water (A)-acetonitrile (B), Chromatographic elution gradient: 0-3 min, 3%-5% B; 3-7 min, 5%-60% B; 7-18 min, 60%-70% B; 18-20 min, 70%-95% B; 20-23 min, 95%-95% B, post-run time: 3 min; Flow rate: 0.35 mL / min; Column temperature: 35 °C; Injection volume: 2 μL;

[0022] Mass spectrometry conditions: an electrospray ionization (ESI) source was used to acquire data in both positive and negative ion modes over the m / z range of 100–1700. The ion source temperature was 350°C. The capillary voltages were 4.0 kV for positive ions and 3.5 kV for negative ions. The nebulizer gas pressure was 45 psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350°C, the fragmentor voltage was 140 V, and the collision energy was 30 V.

[0023] (C) Sample determination

[0024] Determine the chemical components of the reference solution, the Yixinyin extract test solution, and the biological sample test solution in step (A); identify the in vitro components of the Yixinyin extract and the distribution of the chemical components of Yixinyin in the plasma and heart of administered rats based on the retention time, molecular separation characteristics, fragmentation pattern, and liquid-mass behavior of the reference solution, combined with chemical component information reported in literature and provided by online databases;

[0025] (D) Data processing

[0026] The chemical component information (including compound name, precise relative molecular mass, molecular formula, structural formula, secondary ion fragmentation, etc.) of each medicinal ingredient in Yixinyin was retrieved and imported into Agilent PCDL Manager B.08 software to construct a chemical component database. Mass spectral data processing such as peak extraction and peak matching was performed using Masshunter and Msdial software. The chemical components were identified by comparing the secondary mass spectral information with the above-mentioned databases and online databases such as HMDB. For compounds with reference substances, the retention time and mass spectral data of the reference substances were used for confirmation.

[0027] The advantages of the present invention are:

[0028] The present invention adopts UHPLC-Q-TOF / MS analysis technology and positive and negative ion mode scanning. According to information such as compound retention time, precise molecular weight, secondary fragment ions, and combined with reference substance comparison, literature reports, and online database comparison, the in vitro component identification of Yixinyin is carried out rapidly and systematically, and the temporal and quantitative changes of plasma and cardiac migration components in rats with atrial fibrillation are examined, providing a certain reference for clarifying the material basis of the efficacy of Yixinyin in treating atrial fibrillation and in-depth research on its mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 . Experimental design of the rat atrial fibrillation model.

[0030] Figure 2 Partial segment of lead II electrocardiogram recording and atrial fibrillation induction, (A) Lead II electrocardiogram recording of sinus rhythm and atrial fibrillation rhythm, (B) atrial fibrillation inducibility, (C) atrial fibrillation susceptibility, (D) atrial fibrillation duration (Note: ***p < 0.001 compared with the normal group).

[0031] Figure 3 Echocardiographic data of rats, (A) B-mode echocardiography at 30 days, (B) LAD at 30 days, (C) LA area at 30 days, (D) B-mode echocardiography at 37 days, (E) LAD at 37 days, (F) LA area at 37 days (Note: compared with the normal group, *p < 0.05, ***p < 0.001).

[0032] Figure 4 Pathological section results, (A) Masson staining and H&E staining sections, (B) atrial fibrosis area (Note: compared with the normal group, ***p < 0.001).

[0033] Figure 5 Transmission electron microscopy and enzyme-linked immunosorbent assay results, (A) Transmission electron microscopy sections, (B) Flameng score, (C) BNP, (D) TNF-α, (E) IL-6 (Note: compared with the normal group, *p < 0.05, **p < 0.01, ***p < 0.001).

[0034] Figure 6In vitro identification results of Yixinyin ingredients, (A) TIC plot in positive ion mode, (B) TIC plot in negative ion mode. Red font indicates comparison with reference substances: 23. caffeic acid; 26. 4-hydroxycinnamic acid; 33. vanillin; 39. 3-hydroxycoumarin; 43. salvianolic acid B; 44. salvianolic acid A; 47. neolicuroside; 50. ononin; 53. daidzein; 56. calycosin; 58. myristic acid; 60. curcumin; 62. calycosin 7-galactoside; 63. astragaloside; 66. isoliquiritigenin; 72. myristicin; 77. gingerol; 82. tanshinone IIb; 84. senkyunolide A; 96. ligustilide; 107. glycyrrhetnic acid.

[0035] Figure 7 TIC diagrams for the identification of Yixinyin components in vivo. TIC diagrams of the components entering blood (A) and exposed to the heart (C) in positive ion mode, and TIC diagrams of the components entering blood (B) and exposed to the heart (D) in negative ion mode. Red fonts indicate comparison with reference substances: 3. caffeic acid; 4. 4-hydroxycinnamic acid; 6. vanillin; 7. liquiritin; 8. salvianolic acid B; 10. neolicuroside; 11. ononin; 12. daidzein; 13. calycosin; 14. myristic acid; 15. curcumin; 16. calycosin 7-galactoside; 17. isoliquiritigenin; 19. myristicin; 21. gingerol; 22. tanshinone IIB; 23. senkyunolide A; 26. glycyrrhetnic acid.

[0036] Figure 8 . Relative peak area-time trend diagram of Yixinyin blood components in the normal and model groups (Note: compared with the normal group, *p<0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0037] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0038] Example:

[0039] 1. Instruments and Materials

[0040] 1.1 Instrument

[0041] An ultra-high-performance liquid chromatography-quadrupole-time of flight / mass spectrometer (UHPLC-Q-TOF / MS) was purchased from Agilent (USA), including a 1290 Infinity UHPLC and 6530 Q-TOF / MS systems. A Centrifuge 5810R high-speed benchtop refrigerated centrifuge was purchased from Eppendorf (Germany). A Waters ACQUITY UPLC HSS T3 column (1 mm × 100 mm, 1.8 μm) was purchased from Sartorius (Germany). A 1 / 100,000 electronic balance was purchased from Sartorius (Germany). A Milli-Q purified water system was purchased from Millipore (USA). A Lyovapor L-200 freeze dryer was purchased from Buchi (Switzerland). An SK7200H ultrasonic cleaner was purchased from Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0042] 1.2 Drugs and reagents

[0043] Yixinyin fluid extract (Codonopsis pilosula: Astragalus membranaceus: Rehmannia root: Ophiopogon japonicus: Angelica sinensis: Salvia miltiorrhiza: Donkey-hide gelatin: Roasted Licorice root: Asparagus cochinchinensis: Jujube: Ginger: Schisandra chinensis: Cannabis sativa seed: Coptis chinensis: Cinnamomum cassia twig, weight ratio 10:20:10:3:3:10:1:3:2:2:1:3.3:1:1:5) was decocted twice with 8 times the volume of water, the first time for 2 hours and the second time for 1 hour. The filtrate was concentrated to a volume of 95% ethanol, and then precipitated. After standing, the supernatant was ethanol recovered to obtain the fluid extract. Acetylcholine chloride (Batch No. BCCH5244) was purchased from MCE, anhydrous calcium chloride (Batch No. WXBD9929V) was purchased from Sigma, and alcohol liquid feed (Batch No. 20231101) was purchased from Jiangsu Xietong. Curcumin (lot number: DST211230-014), myristyl ether (lot number: DAT240904-054), apiosyl isoliquiritigenin (lot number: DST241028-166), 4-hydroxycinnamic acid (lot number: DSTDD028101), gingerol (lot number: DSTDJ002702), and vanillin (lot number: DSTDX009001) were purchased from Lemitian, while myristic acid (lot number: 13390), isoliquiritigenin (lot number: 16690), and tanshinone IIB (lot number: 18978-G240701) were purchased from Shidan. Chuanxiong lactone A (batch number: J09GB153719), liquiritin (batch number: Z13J11X108109), calycosin (batch number: Y16O11H127829), and daidzein (batch number: B20227) were all purchased from Yuanye Biotechnology. Glycyrrhetinic acid (batch number: N1110AS), salvianolic acid B (batch number: 01011BS), formononetin (batch number: MB6579), caffeic acid (batch number: D1220AS), and calycosin (batch number: D0806AS) were all purchased from Meilun Biotechnology. The purity of the reference substances was greater than 98%. The BNP enzyme-linked immunosorbent assay kit (batch number: G20241009) was purchased from Langton Biotechnology Co., Ltd.; the TNF-α methanol enzyme-linked immunosorbent assay kit (batch number: 202409) was purchased from Jingmei Biotechnology Co., Ltd.; and the IL-6 enzyme-linked immunosorbent assay kit (batch number: A30640615) was purchased from Lianke Biotechnology Co., Ltd. Acetonitrile was of mass spectrometry grade (Merck, Germany); formic acid was of mass spectrometry grade (Thermo Fisher, USA); ultrapure water was prepared using a MILI-Q water purification system (Millipore, USA); all other reagents were of analytical grade.

[0044] 1.3 Animals

[0045] Twenty-eight SPF male Sprague-Dawley rats, weighing 200 ± 20 g, were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., Animal Production License No. SCXK (Beijing) 2021-0006, and Laboratory Animal Use License No. 110011231108698637. The rats were housed in a controlled 12-h light-dark cycle with free access to food and water.

[0046] 2. Methods

[0047] 2.1 Atrial Fibrillation Model Construction

[0048] 2.1.1 Animal modeling, grouping, and drug administration

[0049] Twenty-eight healthy SD rats weighing 200±20g were selected and randomly divided into a normal group, a normal + Yixinyin group, a model group, and a model + Yixinyin group (n=7). Rats in the normal and normal + Yixinyin groups were given a liquid control diet, while rats in the model and model + Yixinyin groups were given a 4% alcohol liquid diet for 30 days. On the 30th day, the left atrial volume of the rats was monitored and the dilation was observed to confirm left atrial enlargement. The model and model + Yixinyin groups were injected with acetylcholine-calcium chloride (66 μg / ml-10 mg / ml) via the tail vein for 7 consecutive days. On the last day of injection, the electrocardiogram (ECG) of the rats was monitored and recorded. After confirming atrial fibrillation-like ECG, the normal + Yixinyin and model + Yixinyin groups were gavaged with Yixinyin (31 g / kg / d). The normal and model groups were gavaged with an equal volume of normal saline for 7 consecutive days. The experimental flow chart of induced atrial fibrillation in rats is shown in the following figure. Figure 1 .

[0050] 2.1.2 Biological sample collection

[0051] At 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after the last administration, 1.0 mL of blood sample was collected from the orbital venous plexus of each rat in an EDTA K2 anticoagulant tube, allowed to stand at room temperature for 1 h, and centrifuged at 4 °C and 3,000 rpm for 10 min to obtain plasma, which was then aliquoted and transferred to a -80 °C refrigerator until sample processing.

[0052] After blood sample collection, the rat heart was perfused to drain the blood, and the heart and other biological samples were quickly removed. The samples were rinsed in ice-cold saline to remove surface blood and other contaminants. The surface moisture was absorbed with filter paper, and the samples were quickly frozen in liquid nitrogen and transferred to a -80 °C refrigerator for storage until sample preprocessing.

[0053] 2.1.3 Model Evaluation Indicators and Detection Methods

[0054] Confirmation of Left Atrial Dilatation: On the 30th day of the experiment, left atrial volume was monitored by echocardiography to confirm left atrial dilatation. Confirmation of Atrial Fibrillation: After the final tail vein injection, electrocardiograms were monitored to observe the presence of atrial fibrillation-like electrocardiograms (ECGs). The inducibility rate, susceptibility to atrial fibrillation, and duration of atrial fibrillation were recorded. Myocardial Histopathological Examination: After blood collection, heart tissue was dissected from each group and stained with hematoxylin and eosin (HE) and Masson stains. Pathological changes in heart tissue were observed microscopically. Observation of Mitochondria in Myocardial Tissue: Heart tissue blocks were excised, embedded, stained, and then mitochondrial morphology was observed under a transmission electron microscope. Myocardial Injury Markers and Biochemical Factors Detection: After confirming the onset of atrial fibrillation, blood was collected from the abdominal aorta of each group under anesthesia. Enzyme-linked immunosorbent assays were used to measure the corresponding markers in each group, including rat brain natriuretic peptide (BNP), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6).

[0055] 2.2 Analysis of in vivo and in vitro components

[0056] 2.2.1 Preparation of reference solution

[0057] Accurately weigh appropriate amounts of 24 reference substances, add 1 mL of 80% methanol, and sonicate to dissolve them. Finally, prepare reference substance solutions with a concentration of 1.0 mg / mL. 4-Hydroxycinnamic acid, apigenin isoliquiritigenin, liquiritin, daidzein, calycosin isoflavones, curcumin, isoliquiritigenin, myristyl ether, tanshinone IIB, and glycyrrhetinic acid are prepared as mixed reference substance 1. Caffeic acid, vanillin, salvianolic acid B, formononetin, myristic acid, calycosin isoflavone glycosides, gingerol, and ligusticolide A are prepared as mixed reference substance 2. 3-Hydroxycoumarin, salvianolic acid A, astragaloside IV, isoliquiritigenin, zingiberenone A, and ligusticolide are prepared as mixed reference substance 3 and are ready for injection.

[0058] 2.2.2 Preparation of Yixinyin extract test solution

[0059] Take an appropriate amount of Yixinyin extract in an EP tube, add the same volume of 80% methanol (containing 200 ng / mL warfarin), and extract by ultrasonic for 30 min at 13,000 r·min. -1 Centrifuge for 15 min under the same conditions, and aspirate the supernatant into a brown injection vial for injection analysis.

[0060] 2.2.3 Preparation of plasma test solution

[0061] 0.2 mL of plasma from the rats in the normal group, model group, normal + Yixinyin group, and model + Yixinyin group under item 2.1.2 was added with 0.8 mL of methanol (containing 200 ng / mL warfarin), vortexed, and then rotated at 4 °C and 13,000 r·min. -1The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL of 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r·min. -1 Centrifuge for 10 min under the same conditions, aspirate the supernatant into a brown injection vial, and aspirate 10 μL of each supernatant into the injection vial, mix well, and use as QC.

[0062] 2.2.4 Preparation of Heart Test Solution

[0063] Approximately 100 mg of rat hearts were collected from the normal group, model group, normal group + Yixinyin group, and model + Yixinyin group. Five volumes of ice-cold saline were added and homogenized using a cryogenic tissue grinder at -20°C and 50 Hz. 300 μL of the homogenate from each sample was transferred to a new EP tube, 1.2 mL of methanol (containing 200 ng / mL warfarin) was added, vortexed, and then centrifuged at 4°C and 13,000 rpm. -1 The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL of 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r·min. -1 Centrifuge for 10 min under the same conditions, aspirate the supernatant into a sampling vial, and aspirate 10 μL of each supernatant into a sampling vial, mix well, and use as QC.

[0064] 2.3 UHPLC-Q-TOF / MS conditions

[0065] 2.3.1 Chromatographic conditions

[0066] Chromatographic column: Waters ACQUITY UPLC HSS T3 column (1 mm × 100 mm, 1.8 μm); mobile phase: 0.1% formic acid in water (A)-acetonitrile (B), chromatographic elution gradient: 0-3 min, 3%-5% B; 3-7 min, 5%-60% B; 7-18 min, 60%-70% B; 18-20 min, 70%-95% B; 20-23 min, 95%-95% B, post-run time: 3 min; flow rate: 0.35 mL / min; column temperature: 35 °C; injection volume: 2 μL.

[0067] 2.3.2 Mass spectrometry conditions

[0068] An electrospray ionization (ESI) source was used to collect data in both positive and negative ion modes over the m / z range of 100 to 1700. The ion source temperature was 350°C, the capillary voltages were 4.0 kV for positive ions and 3.5 kV for negative ions, the nebulizer gas pressure was 45 Psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350°C, the fragmentor voltage was 140 V, and the collision energy was 30 V.

[0069] 2.4 Sample determination

[0070] Determine the chemical composition of the reference substance solution, Yixinyin extract test solution, and biological sample test solution under 2.2. Based on the retention time, molecular separation characteristics, and cleavage behavior of the reference substance, combined with chemical composition information from literature reports and online databases, identify the in vitro components of Yixinyin extract and the distribution of the chemical components of Yixinyin in the plasma and heart of administered rats.

[0071] 2.5 Data Processing

[0072] Chemical component information (including compound name, exact relative molecular mass, molecular formula, structural formula, and secondary ion fragmentation) for each of the medicinal ingredients in Yixinyin was retrieved from online Chinese and English databases such as CNKI, TCMSP, Pubchem, and Pubmed. This information was then imported into Agilent PCDL Manager B.08 software to construct a chemical component database. Mass spectrometry data processing, including peak extraction and peak matching, was performed using Masshunter and Msdial software. Chemical component identification was performed by comparing secondary mass spectrometry data with these databases and online databases such as HMDB. For compounds for which reference standards were available, confirmation was performed using the reference standards' retention times and mass spectrometry data.

[0073] 3. Results

[0074] 3.1 Atrial Fibrillation Model Evaluation

[0075] 3.1.1 Atrial fibrillation inducibility, susceptibility, and duration in model rats

[0076] On the 37th day of the experiment, after the tail vein injection of acetylcholine-calcium chloride, the rats' electrocardiograms were monitored. The absence of p waves and the presence of f waves on the electrocardiogram indicated that atrial fibrillation was successfully induced ( Figure 2 A). The induction rate of rats in the model group was 85% ( Figure 2 B). Figure 2 C and Figure 2 As shown in Figure D, the induction time of atrial fibrillation in the model group was approximately 13.11 s, and the duration of atrial fibrillation was 20.64 s.

[0077] 3.1.2 Changes in LAD and LA areas in model rats

[0078] On the 30th day of the experiment, the rats were monitored by echocardiography and the echocardiographic images of the atrial end-diastole were obtained ( Figure 3 A) The left atrial diameter (LAD) and left atrial area (LA area) of the rats in the model group increased, and the left atrium was dilated ( Figure 3 B, 3C). On the 37th day of the experiment, the left atrium of the rats was examined again by ultrasound ( Figure 3 D), compared with the normal group, the LAD and LA area of ​​the rats in the model group were enlarged ( Figure 3 E, 3F), the left atrium of rats in the model group showed obvious dilation.

[0079] 3.1.3 Atrial fibrosis in rat models

[0080] The rat heart was stained with Masson staining and H&E staining, and the staining results are shown as follows Figure 4 As shown in A. Since atrial fibrosis is a prominent feature of atrial remodeling, the effect of Yixinyin on fibrosis and collagen deposition was evaluated. Masson staining results showed that the atrial tissue fibers of the model group rats were arranged in disorder, with a large number of blue collagen fibers produced, and obvious atrial tissue fibrosis ( Figure 4 B). H&E staining results showed that the cardiomyocytes of the rats in the normal group were neatly arranged and had no abnormal cell morphology. Compared with the normal group, the cardiomyocytes of the rats in the model group were disordered, with a small amount of cell aggregation.

[0081] 3.1.4 Changes in mitochondria, myocardial injury indicators, and inflammatory factors in model rat myocardial tissue

[0082] Figure 5 A shows representative electron microscopy images of mitochondria beneath the sarcolemmal membrane and between myofibrils. Figure 5 B shows the Flameng score for mitochondrial morphology evaluation. As shown in the figure, the mitochondrial structure of the normal group is intact, the mitochondrial cristae are not broken, and there is no obvious swelling; the mitochondrial results of the model group are basically intact, the mitochondrial cristae are broken, and the mitochondria are moderately swollen. Figure 5 As shown in C, compared with the normal group, the level of brain natriuretic peptide (BNP), an indicator of myocardial injury, in the model group was significantly increased. By comparing the levels of tumor necrosis factor (TNF-α) and interleukin 6 (IL-6) in the rats of each group, it was found that ( Figure 5 D, 5E), compared with the normal group, the levels of TNF-α and IL-6 in the rats in the model group were significantly increased.

[0083] 3.2 Analysis of Yixinyin components in rats with atrial fibrillation

[0084] 3.2.1 Analysis of chemical components of Yixin Drink in vitro

[0085] The chemical components in the Yixin Drink test sample and mixed reference solution were qualitatively analyzed based on UHPLC-Q-TOF / MS technology. Figure 6 As shown in the figure, a total of 111 components were identified using this method, with good responsiveness and good separation. Among them, there are 18 flavonoids, 15 organic acids, 11 terpenes, 8 phenolic acids, 7 ketones, 6 phenols, 5 esters, 4 saponins, 4 aldehydes, 4 lignins, 4 amino acids, and 25 other compounds.

[0086] 3.2.3 Analysis of chemical components in Yixin Drink

[0087] Mass spectrometry data were collected and analyzed from the plasma, heart, liver and other biological samples of normal rats and atrial fibrillation rats after oral administration of Yixinyin, including comparison of their retention time (Rt), molecular formula, accurate molecular weight (m / z) and secondary ion fragmentation data with the in vitro chemical component database of Yixinyin and the information in the mixed standard solution. Figure 7 As shown, a total of 27 chemical components were identified in rats, with 27 in plasma and 8 in heart, respectively. These included 8 flavonoids, 5 organic acids, 4 phenols, 4 terpenes, 2 alkaloids, and 4 other compounds. Comparison with the spectroscopic data of individual herbs revealed that 4 of these compounds were derived from Glycyrrhiza uralensis, 5 from Astragalus membranaceus, 3 from Angelica sinensis, 4 from Salvia miltiorrhiza, 3 from Zingiber officinale, 2 from Schisandra chinensis, 1 from Codonopsis pilosula, and 1 from Ophiopogon japonicus. Furthermore, caffeic acid was derived from Salvia miltiorrhiza and Astragalus membranaceus, 4-hydroxycinnamic acid from Angelica sinensis and jujube, myristic acid from Codonopsis pilosula and Astragalus membranaceus, and myristicin from Salvia miltiorrhiza and jujube.

[0088] Table 1. Identification results and tissue distribution of Yixinyin components

[0089]

[0090] Note: WWZ (Schisandra chinensis), DG (Angelica sinensis), DanS (Danshen miltiorrhiza), HQ (Astragalus membranaceus), GZ (Cinnamomum cassia), DZ (Zidojujube), GC (Glycyrrhiza uralensis), DS (Codonopsis pilosula), SJ (Zingiber officinale), MD (Ophiopogon japonicus).

[0091] 3.1.7 Screening of different components between Yixinyin groups

[0092] The internal standard normalization of each sample in the two Yixinyin treatment groups was performed using the built-in software of UHPLC-Q-TOF / MS, and the relative concentrations of 18 blood-entering components and 7 heart-exposed components were obtained (Table 2), as well as the relative peak area-time trend diagram of the blood-entering components ( Figure 8 ). The results of time-quantity characteristics showed that the component basis in the normal + Yixinyin group and the model + Yixinyin group was basically the same, but there were differences in exposure. Compared with the normal medication group, the relative concentrations of 4-hydroxycinnamic acid, vanillin, salvianolic acid B, formononetin, daidzein, calycosin isoflavones, isoliquiritigenin, myristyl ether, lignanolide A, and glycyrrhetinic acid in the plasma samples of the model medication group were increased, indicating that the plasma exposure of the above compounds in the model group increased. Among them, the exposure of glycyrrhetinic acid, daidzein, calycosin isoflavones, lignanolide A, and myristyl ether in the blood at each time point were higher than those in the normal medication group ( Figure 8 Compared with the normal administration group, the relative concentrations of caffeic acid, calycosin, and myristicin in the heart samples of the model administration group were slightly increased, indicating that the cardiac exposure of the above compounds in the model group was increased.

[0093] Table 2. Relative concentration analysis of Yixinyin components in rat plasma and heart tissue

[0094]

[0095] 4. Discussion

[0096] Clinically, the diagnosis of atrial fibrillation is primarily based on observing the presence of atrial fibrillation-like waves on the electrocardiogram (ECG), while also monitoring the patient's left atrial diameter and myocardial BNP levels. Excessive alcohol consumption has long been considered a risk factor for cardiovascular disease and adverse atrial remodeling. Long-term drinking can increase susceptibility to atrial fibrillation and promote its occurrence and progression. This study was the first to use a rat atrial fibrillation model induced by alcohol-induced left atrial dilation combined with acetylcholine and calcium chloride (ACh-CaCl2) (Yu LM, Dong X, Xu YL, et al. Icariin attenuates excessive alcohol consumption-induced susceptibility to atrial fibrillation through SIRT3 signaling[J]. BiochimicaEt Biophysica Acta. Molecular Basis of Disease, 2022, 1868(10): 166483. YueH, Liang W, Zhan Y, et al. Colchicine: Emerging therapeutic effects on atrial fibrillation by alleviating myocardial fibrosis in a rat model[J]. Biomedicine & Pharmacotherapy, 2022, 154: 113573. Zhou Q, Chen B, Chen X, etal. Arnebiae Radix prevents atrial fibrillation in rats by amelioratingatrial remodeling and cardiac function[J]. Journal of Ethnopharmacology, 2020, 248: 112317. This model simulates the condition of atrial fibrillation (AF) in people with chronic alcohol consumption. The study showed that compared with the normal control group, the model group showed an increased incidence of AF, a shortened duration of AF, a prolonged duration of AF, increased myocardial fibrosis, atrial dilatation, abnormal mitochondrial morphology, myocardial damage, and elevated levels of inflammatory factors. These indicators were consistent with clinical findings, and the trends of the model parameters were also consistent with clinical findings.

[0097] Compounds exhibit differences in absorption, metabolism, and excretion between normal and pathological conditions. Plasma and target organ exposure, to some extent, reflects the selective effects of traditional Chinese medicines on specific physiological organs. This study first validated the therapeutic effects of Yixinyin on rats with atrial fibrillation. Furthermore, a control group plus Yixinyin and an atrial fibrillation plus Yixinyin group were designed to investigate the differences in the amount and content of Yixinyin's plasma and cardiac transit components between normal and atrial fibrillation rats. This study, in turn, explored the characteristics of the compound's therapeutic effects on atrial fibrillation. Furthermore, a temporal and quantitative analysis was conducted to understand the dynamic behavior of Yixinyin's blood-entering components in vivo. First, a rapid detection method for Yixinyin components was established using UHPLC-Q-TOF / MS. 111 chemical components were identified and used as a database for in vivo component identification. Based on this database, 27 components were detected in the plasma of normal and atrial fibrillation rats, and 8 components were detected in cardiac tissue. Results showed that all of these components were detectable in both normal and atrial fibrillation rats, but differences in their content did exist. Among them, salvianolic acid B from Danshen (Salvia miltiorrhiza), glycyrrhetinic acid and isoliquiritigenin from Glycyrrhiza uralensis (Glycyrrhiza uralensis), formononetin, calycosin, and daidzein from Astragalus membranaceus (Astragalus membranaceus), and ligusticholide A from Angelica sinensis (Angelica sinensis) showed higher plasma exposure in the model rats than in normal rats. This may be due to alcohol altering the permeability of vascular endothelial cells, leading to increased plasma levels of these components; or it may be due to decreased metabolic excretion of these compounds under the model, resulting in increased retention in the body. Furthermore, liquiritin, calycosin, gingerol, caffeic acid, 4-hydroxycinnamic acid, myristic acid, and myristic ether were detected in cardiac tissue, suggesting that these compounds may exert therapeutic effects in the heart.

[0098] Research has shown that liquiritin, calycosin, and gingerol, which are distributed in cardiac tissue, are representative components and have been shown in modern pharmacological studies to be useful in treating patients with arrhythmias. For example, liquiritin, the main active ingredient in licorice, can regulate inflammation, oxidative stress, and apoptosis. It is one of the active components of licorice that counteracts aconitine-induced cardiotoxicity, significantly reducing heart rate in model animals and demonstrating a protective effect against aconitine-induced myocardial damage. Calycosin exhibits multiple antioxidant, anti-inflammatory, and anti-injury properties. It can improve myocardial function by activating the PI3K-Akt signaling pathway, controlling infarct size, and reducing myocardial damage. Gingerols exhibit anti-inflammatory and oxidative stress-inhibiting activities. Gingerol can effectively inhibit the production of inflammatory factors such as NO and IL-1β and block the activation of NK-kB, resulting in anti-inflammatory effects.

[0099] In summary, combined with the research results of the in vivo exposure components of Yixinyin, it is speculated that the effect of Yixinyin in improving atrial remodeling in rats with atrial fibrillation may be due to the fact that the components liquiritin, calycosin and gingerol enter the heart tissue and exert their effect.

[0100] 5. Conclusion

[0101] The present invention uses UHPLC-Q-TOF / MS technology to clarify the temporal and quantitative characteristics of the in vitro chemical components and cardiac migration components of Yixin Drink. By comparing the changes in the components of plasma and heart tissue of normal rats and rats with atrial fibrillation after oral administration of Yixin Drink, 27 major blood-entering components and 8 targeted heart-entering components were identified as the effective component group of Yixin Drink for treating atrial fibrillation. Combined with the research results on the in vivo exposure of glycyrrhizin, calycosin and gingerol, it is speculated that these three components may be the key substances for Yixin Drink to enter the heart tissue and exert its effect. The present invention provides a research direction and experimental basis for the subsequent in-depth exploration of the pharmacological material basis and mechanism of action of Yixin Drink in treating atrial fibrillation.

[0102] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A rapid detection method for Yixin Drink ingredients based on liquid chromatography-mass spectrometry technology, characterized in that: The method uses UHPLC-Q-TOF / MS analysis technology, scanning in positive and negative ion modes, and based on the compound retention time, precise molecular weight, and secondary fragment ion information, combined with reference sample comparison, literature reports, and online database comparison, to quickly and systematically carry out the in vitro identification of Yixinyin's components and the chemical composition distribution of Yixinyin in the plasma and heart of dosed rats.

2. The rapid detection method of Yixinyin ingredients based on liquid chromatography-mass spectrometry technology according to claim 1 is characterized in that: The UHPLC-Q-TOF / MS conditions are: Chromatographic conditions: Column: Waters ACQUITYUPLC HSS T3 column, 1 mm × 100 mm, 1.8 μm; Mobile phase: 0.1% formic acid in water (A)-acetonitrile (B), chromatographic elution gradient: 0-3 min, 3%-5% B; 3-7 min, 5%-60% B; 7-18 min, 60%-70% B; 18-20 min, 70%-95% B; 20-23 min, 95%-95% B, post-run time: 3 min; flow rate: 0.35 mL / min; column temperature: 35 °C; injection volume: 2 μL; Mass spectrometry conditions: an electrospray ionization (ESI) source was used to acquire data in both positive and negative ion modes over the m / z range of 100 to 1700. The ion source temperature was 350°C. The capillary voltages were 4.0 kV for positive ions and 3.5 kV for negative ions. The nebulizer gas pressure was 45 psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350°C, the fragmentor voltage was 140 V, and the collision energy was 30 V.

3. The rapid detection method of Yixinyin ingredients based on liquid chromatography-mass spectrometry technology according to claim 1 is characterized in that: The following steps are involved: (A) In vivo and in vitro component analysis (A1) Preparation of reference solution Accurately weigh 24 reference substances including 4-hydroxycinnamic acid, apigenin isoliquiritigenin, liquiritin, daidzein, calycosin isoflavone, curcumin, isoliquiritigenin, myristyl ether, tanshinone IIB, glycyrrhetinic acid, caffeic acid, vanillin, salvianolic acid B, formononetin, myristic acid, calycosin isoflavone glycosides, gingerol, ligusticolide A, 3-hydroxycoumarin, salvianolic acid A, astragaloside IV, isoliquiritigenin, gingerol, shogaolide A, and ligusticolide, add 80% methanol, sonicate and dissolve, and finally prepare the mixture to a concentration of 1.0 mg / mL reference solution; 4-hydroxycinnamic acid, apigenin isoliquiritigenin, liquiritin, daidzein, calycosin isoflavones, curcumin, isoliquiritigenin, myristyl ether, tanshinone IIB and glycyrrhetinic acid are prepared as mixed reference substance 1; caffeic acid, vanillin, salvianolic acid B, formononetin, myristic acid, calycosin isoflavone glycosides, gingerol and ligusticolide A are prepared as mixed reference substance 2; 3-hydroxycoumarin, salvianolic acid A, astragaloside IV, isoliquiritigenin, zingerenone A and ligusticolide are prepared as mixed reference substance 3, ready for injection; (A2) Preparation of Yixinyin Extract Test Solution Yixinyin extract was placed in an EP tube, and the same volume of 80% methanol containing 200 ng / mL warfarin was added. After ultrasonic extraction for 30 min, the tube was rotated at 13,000 r·min. -1 Centrifuge for 15 min under the same conditions, and transfer the supernatant to a brown injection vial for analysis; (A3) Preparation of plasma test solution To 0.2 mL of rat plasma, 0.8 mL of methanol containing 200 ng / mL warfarin was added, vortexed and then rotated at 4 °C and 13,000 r·min. -1 The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL of 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r·min. -1 Centrifuge for 10 min under the same conditions, pipette the supernatant into a brown injection vial, and pipette 10 μL of each supernatant into the injection vial, mix well, and use as QC; (A4) Preparation of Heart Test Solution About 100 mg of the rat heart was taken, added with 5 times the volume of ice-cold physiological saline, and homogenized using a low-temperature tissue grinder at -20 °C and 50 Hz. 300 μL of the homogenate from each sample was transferred to a new EP tube, and 1.2 mL of methanol containing 200 ng / mL warfarin was added. The tube was vortexed and then rotated at 4 °C and 13,000 r·min. -1 The supernatant was aspirated and dried under nitrogen at room temperature. The residue was dissolved in 200 μL 80% methanol, vortexed and mixed, and centrifuged again at 4 °C and 13,000 r min. -1 Centrifuge for 10 min under the same conditions, pipette the supernatant into a vial, and pipette 10 μL of each supernatant into a vial, mix well, and use as QC; (B) UHPLC-Q-TOF / MS conditions Chromatographic conditions: Column: Waters ACQUITYUPLC HSS T3 column 1 mm × 100 mm, 1.8 μm; Mobile phase: 0.1% formic acid in water (A)-acetonitrile (B), chromatographic elution gradient: 0-3 min, 3%-5% B; 3-7 min, 5%-60% B; 7-18 min, 60%-70% B; 18-20 min, 70%-95% B; 20-23 min, 95%-95% B, post-run time: 3 min; flow rate: 0.35 mL / min; column temperature: 35 °C; injection volume: 2 μL; Mass spectrometry conditions: electrospray ionization (ESI) was used to collect data in both positive and negative ion modes, with a data acquisition range of m / z 100–1700. The ion source temperature was 350 °C. The capillary voltage was 4.0 kV for positive ions and 3.5 kV for negative ions. The nebulizer gas pressure was 45 psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350 °C, the fragmentor voltage was 140 V, and the collision energy was 30 V. (C) Sample determination Determine the chemical components of the reference solution, Yixinyin extract test solution, and biological sample test solution in step (A); identify the in vitro components of Yixinyin extract and the distribution of the chemical components of Yixinyin in the plasma and heart of administered rats based on the retention time, molecular separation characteristics, cleavage pattern, and liquid-mass behavior of the reference solution, combined with the chemical component information reported in literature and provided by online databases.

4. The rapid detection method of Yixinyin ingredients based on liquid chromatography-mass spectrometry technology according to claim 3 is characterized in that: The following steps are also included: (D) Data processing: The chemical component information (including compound name, precise relative molecular mass, molecular formula, structural formula, secondary ion fragmentation, etc.) of each medicinal ingredient in Yixinyin was retrieved from online Chinese and English databases such as CNKI, TCMSP, Pubchem, and Pubmed. The chemical component database of the prescription was constructed using Agilent PCDL Manager B.08 software. Mass spectrometry data processing, such as peak extraction and peak matching, was performed using Masshunter and Msdial software. The chemical components were identified by comparing the secondary mass spectrometry information with the above-mentioned databases and online databases such as HMDB. For compounds with reference substances, the retention time and mass spectrometry data of the reference substances were used for confirmation.

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