Method for screening anti-inflammatory cardiac protection active components of Siji decoction
By constructing a metabolomic correlation model of eicosanoids, the anti-inflammatory and cardioprotective active ingredients in Sini Decoction were screened out, which solved the problem of unclear active ingredients in Sini Decoction and confirmed the anti-inflammatory and cardioprotective effects of ingredients such as Songguoling, Neoline, Tarasamine, and Isoliquiritin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-14
AI Technical Summary
The anti-inflammatory and cardioprotective active ingredients of Si Ni Tang are unclear in the existing technology, and there is a lack of effective screening methods.
By constructing an eicosanoid metabolite profile as a pharmacodynamic target, a correlation model was established between the quantitative information of eicosanoid metabolites in rats with heart failure and the intensity of blood-entering components of Sini Tang at different time points. Twelve anti-inflammatory and cardioprotective active ingredients of Sini Tang were screened out, and their effects were verified through correlation analysis and cell models.
The components such as Songguoling, Neolin, Tarasamine, and Isoliquiritin were screened and found to have clear anti-inflammatory and cardioprotective effects, and their effectiveness in in vivo and in vitro models was verified.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine research technology, specifically relating to a method for screening the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang. Background Technology
[0002] Heart failure is a complex clinical syndrome with high morbidity and mortality, seriously endangering human health and causing enormous clinical pressure and economic costs. Currently, the main treatment strategies for heart failure are chemotherapy and surgery, but these methods have drawbacks such as high cost and certain side effects. Therefore, finding active ingredients for treating heart failure has become a hot research topic for many medical professionals in recent years.
[0003] Eicosanoids, also known as arachidonic acids, are a family of lipid mediators with hundreds of different biological activities produced from arachidonic acid through three enzymatic pathways (cyclooxygenases, lipoxygenases, and cytochrome P450) or non-enzymatic auto-oxidation pathways. Eicosanoids are important precursors of inflammatory factors and cardiovascular active substances in the body, participating in immune and inflammatory responses. Studies have shown that eicosanoid metabolism is closely related to heart failure. Many of its metabolites, such as prostaglandins, leukotrienes, hydroxylated metabolites (HETE), and epi-oxidized eicosatetrienoic acid (EET), have been shown to play important regulatory roles in myocardial remodeling and the development of heart failure. Many molecules can serve as potential targets for heart failure treatment. Therefore, targeting the eicosanoid metabolic network to find active ingredients that effectively regulate it is a reasonable approach to discovering anti-inflammatory and cardioprotective active ingredients in traditional Chinese medicine.
[0004] Sini Tang, composed of Aconitum carmichaelii, dried ginger, and licorice, is a classic formula from Zhang Zhongjing's *Treatise on Cold Damage*. It possesses the effects of restoring yang and rescuing from collapse, and exhibits significant anti-heart failure effects. However, the specific anti-inflammatory and cardioprotective active ingredients within it remain unclear. The blood-transferring components of traditional Chinese medicine (TCM) provide methodological support for discovering these active ingredients. Since there is inevitably a correlation between the blood-transferring components of TCM and its efficacy indicators, this study aims to correlate the amounts of blood-transferring components of TCM at different time points with the amounts of eicosanoic acid-like compounds in the blood at different time points. Through correlation analysis, the anti-inflammatory and cardioprotective active ingredients of Sini Tang can be identified. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a method for screening the anti-myocardial ischemia active ingredients of Sini Decoction in view of the deficiencies of the above-mentioned prior art. This method uses the eicosanoid metabolic profile as the pharmacodynamic target, constructs a correlation model between the quantitative information of eicosanoid metabolites in rats with heart failure at different time points and the intensity of the ingredients of Sini Decoction in the blood at different time points, screens out 12 anti-inflammatory and cardioprotective active ingredients of Sini Decoction, and confirms that 4 ingredients, such as songorine, niolean, taraxamine, and isoliquiritigenin, have definite anti-inflammatory and cardioprotective effects, solving the problem that the anti-inflammatory and cardioprotective active ingredients of Sini Decoction are unclear in the prior art.
[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a method for screening the anti-inflammatory and cardioprotective active ingredients of Sini Decoction, which is characterized in that the method comprises the following steps: Step 1: Discovery and content determination of eicosanoids related to the pharmacodynamics of Sini Decoction Step 101: Preparation of Sini Decoction: Weigh 300 g of aconite root, 200 g of dried ginger, and 300 g of licorice root respectively, add 10 times the volume of purified water, soak for 1 h, then decoct for 2 h, filter while it is hot with four layers of gauze, decoct the obtained residue with 8 times the volume of purified water for 1 h, filter in the same way, combine the two filtrates, and prepare Sini Decoction with a concentration of 1 g / mL and 3 g / mL after concentration under reduced pressure; Step 102: Preparation of samples: Take clean-grade male SD rats, and establish a heart failure model group, a low-dose Sini Decoction treatment group, a high-dose Sini Decoction treatment group, and a sham operation group. The low-dose Sini Decoction treatment group and the high-dose Sini Decoction treatment group are respectively given 10 g (equivalent to the crude drug) / kg / BW and 30 g (equivalent to the crude drug) / kg / BW of Sini Decoction by gavage every day. The heart failure model group and the sham operation group are given the same amount of normal saline by gavage, and the drug is administered 24 h after modeling, once a day, for 28 consecutive days; Step 103: Analysis of eicosanoic acid-like substances in the samples: Take plasma samples from each group of rats in Step 102, add a methanol / acetonitrile mixed solution (volume ratio 1:2, containing deuterated eicosanoic acid-like internal standard), vortex, incubate at -20℃, centrifuge, and perform solid-phase extraction on the supernatant. Collect the solid-phase extraction eluent, dry under reduced pressure in a vacuum concentrator, redissolve in methanol-water solution, vortex, centrifuge, and perform ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis. Data from each group of plasma samples were obtained and processed using MultiQuant software (version 3.0.3, SCIEX). A multiple reaction monitoring (MRM) mode was employed, and quantification was performed by monitoring the specific precursor-daughter ion pairs of eicosanoids. The quantitative range of the method was 0.2 nmol / L to 40000 nmol / L. A standard curve equation was constructed using weighted least squares linear regression (weight coefficient 1 / x). After calibration, the concentration of eicosanoids in each group of plasma samples was calculated. Step 104: Determination of eicosanoid metabolites related to the efficacy of Si Ni Tang: Volcano plot analysis was performed on the eicosanoid metabolite content in the heart failure model group and sham-operated group rats from Step 102 to identify the eicosanoid metabolites that caused abnormal changes in heart failure rats induced by myocardial infarction, and to determine the eicosanoid metabolites related to heart failure. Then, analysis of variance was used to compare the changes of these eicosanoid metabolites related to heart failure under the administration of the heart failure model group, the low-dose Si Ni Tang treatment group, and the high-dose Si Ni Tang treatment group. The eicosanoids that showed significant reversal after Si Ni Tang intervention were identified as the anti-inflammatory and cardioprotective efficacy indicators of Si Ni Tang. Finally, the eicosanoid metabolites and their content that were significantly reversed by Si Ni Tang were determined. Step 2: Identification of the components absorbed into the blood by Si Ni Tang and determination of their relative peak intensities. Step 201, Sample Preparation and Analysis: Take plasma samples from rats in the high-dose treatment group of Si Ni Tang in Step 102 after administration of the drug, add acetonitrile solution containing pentoxifylline as an internal standard, vortex, centrifuge, and take the supernatant for solid-phase extraction to enrich the Si Ni Tang components in the plasma samples. Collect the solid-phase extraction eluent and concentrate it under reduced pressure, then redissolve it with methanol aqueous solution, vortex mix, centrifuge, and take the supernatant for analysis by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS). Step 202: Identification of components absorbed into the blood by Si Ni Tang: The chemical components in Si Ni Tang were identified using Agilent MassHunter software combined with spectral library database matching. Then, based on the retention time and mass-to-charge ratio information of the extracted ion chromatogram, the components absorbed into the blood by Si Ni Tang were identified. The original UHPLC-Q-TOFMS data were converted into mzML format using ProteoWizard software, and peak extraction, correction and peak area integration were performed using XCMS software to obtain a three-dimensional data matrix of retention time, mass-to-charge ratio and peak intensity. Step 203: Determination of the relative peak intensity of the components absorbed into the blood by Si Ni Tang: From the three-dimensional data matrix of retention time, mass-to-charge ratio and peak intensity in step 302, the information of the components absorbed into the blood by Si Ni Tang is found by retention time and mass-to-charge ratio, and the corresponding peak intensity information is obtained. The mass spectrometry response is corrected by using the internal standard peak intensity to obtain the relative peak intensity information of the components absorbed into the blood by Si Ni Tang after high-dose administration of Si Ni Tang. Step 3: Correlation analysis of the content of eicosanoic acid metabolites related to the efficacy of Sini Decoction and the relative peak intensity of components absorbed into the blood by Sini Decoction. The levels of eicosanoid metabolites related to the efficacy of Sinitang in rats in the high-dose Sinitang group on days 7, 14, and 28, as determined in step 104, were compared with the relative peak intensity information of the Sinitang absorbed into the blood after high-dose administration in step 203 using Pearson correlation analysis. The correlation coefficient r was used to represent the degree of effect of the Sinitang absorbed into the blood on the eicosanoid metabolites related to the efficacy of Sinitang. A correlation of |r|≥0.6 (p<0.05) was considered significant, thus identifying the anti-inflammatory and cardioprotective active ingredients of Sinitang.
[0007] The above-mentioned method for screening the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang is characterized in that the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang in step three are Song Guo Ling, Niorin, Tarasamine, and Isoliquiritin.
[0008] Compared with the prior art, the present invention has the following advantages: 1. This invention uses the eicosanoic acid (EA) metabolic spectrum, which is closely related to the body's inflammation and immunity, as the pharmacodynamic target. A quantitative analysis method for the broad-coverage EEA metabolic spectrum in rats with heart failure was established. A correlation model was constructed between the quantitative information of EEA metabolites in rats with heart failure at different time points and the intensity of blood-entering components of Si Ni Tang at different time points. Based on the correlation coefficient, 12 anti-inflammatory and cardioprotective active ingredients of Si Ni Tang were screened and obtained as follows: 6-shogaol, 14-benzoyl-10-hydroxyneoaconitine, neoaconitine, aconitine, polyaconitine, haitisan, songguoling, neorin, aconitine, tarasamine, isoliquiritin, and benzoylneoaconitine.
[0009] 2. This invention integrates the H9c2 cell inflammatory injury model and the H9c2 cell oxygen-glucose deprivation / reoxygenation injury model, and confirms that the four components, Songguoling, Neoline, Tarasamine, and Isoliquiritin, have clear anti-inflammatory and cardioprotective effects.
[0010] The technical solution of the present invention will be further described in detail below through embodiments. Attached Figure Description
[0011] Figure 1 The results of volcano plot analysis of eicosanoic acid metabolite content in rats in the sham-operated group and the heart failure model group after administration of Si Ni Tang on day 28 of this invention.
[0012] Figure 2 A bar chart showing the analysis of variance of 31 eicosanoic acid-like metabolites related to heart failure identified in this invention among the sham surgery group, heart failure model group, low-dose Si Ni Tang group, and high-dose Si Ni Tang group.
[0013] Figure 3 The images show the base peak chromatogram and extracted ion chromatogram obtained from the in vitro UHPLC-Q-TOFMS analysis of the Si Ni Tang formula of this invention.
[0014] Figure 4 This is a heatmap showing the correlation coefficients between the content of eicosanoic acid metabolites related to the efficacy of Sinitang and the relative peak intensity of the components absorbed into the blood by Sinitang in rats in the high-dose Sinitang group on days 7, 14, and 28 of this invention.
[0015] Figure 5 This is a flowchart of the H9c2 cardiomyocyte inflammatory injury model induced by the present invention.
[0016] Figure 6 This is a cell viability analysis diagram of the four highly correlated eicosanoic acid components in this invention on CM-induced inflammatory damage to cardiomyocytes.
[0017] Figure 7 This is a graph showing the analysis of the NO release from cardiomyocytes induced by four highly correlated eicosanoic acid components in this invention.
[0018] Figure 8 This is a cell viability analysis diagram of the oxygen-glucose deprivation / reoxygenation damage model of four highly correlated eicosanoic acid components in this invention. Detailed Implementation
[0019] Example 1 The method for screening the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang in this embodiment includes the following steps: Step 1: Discovery and content determination of eicosanoic acid-like substances related to the efficacy of Si Ni Tang. Step 101: Preparation of Si Ni Tang: Weigh 300g of Aconitum carmichaelii, 200g of dried ginger and 300g of licorice, add 10 times the volume of purified water and soak for 1 hour, then decoct for 2 hours. Filter while hot through four layers of gauze. The residue is then decocted with 8 times the volume of purified water for 1 hour and filtered in the same way. Combine the two filtrates and concentrate under reduced pressure to prepare Si Ni Tang with concentrations of 1g / mL and 3g / mL. Step 102, Sample Preparation: Clean-grade male SD rats (200g±10g) were anesthetized with ether. The left atrial appendage was gently lifted with ophthalmic curved forceps, and the left anterior descending coronary artery was ligated with 5-0 surgical suture about 1mm below the conus aorta and the left atrial appendage to create a myocardial infarction model. The surgical procedure for the sham-operated group was similar to that for the myocardial infarction model, but only one loop of suture was made, and the left coronary artery was not ligated. The surviving rats after the myocardial infarction model were randomly divided into a heart failure model group (n=8), a low-dose Si Ni Tang treatment group (SND-L, n=8), and a high-dose Si Ni Tang treatment group (SND-H, n=8), along with the sham-operated group ( Eight animals were included in the study. The low-dose treatment group (SND-L) and the high-dose treatment group (SND-H) were given 10g (equivalent to raw medicinal material) / kg / BW and 30g (equivalent to raw medicinal material) / kg / BW of Sini Decoction by gavage, respectively. The heart failure model group (Model) and the sham operation group (Sham) were given an equal volume of normal saline by gavage. The drugs were administered 24 hours after modeling, once a day for 28 consecutive days. Blood was collected from the orbital cavity 1 hour after the administration of Sini Decoction on the 7th, 14th and 28th days. The plasma was centrifuged at 3000×g for 10 min at 4℃ and the upper plasma layer was separated as plasma samples and stored at -70℃. Step 103: Analysis of eicosanoic acid-like substances in the samples: Take 200 μL of plasma samples from each group of rats in Step 102, add 200 μL of methanol / acetonitrile mixed solution (volume ratio 1:2, containing deuterated eicosanoic acid-like internal standard), vortex for 5 min, incubate at -20℃ for 30 min, centrifuge at 12000×g for 10 min at 4℃, collect the supernatant for solid-phase extraction (SPE), collect the solid-phase extraction eluent, dry under reduced pressure in a vacuum concentrator at 30℃ and 1500 rpm, then redissolve in 200 μL of 50% methanol aqueous solution, vortex mix for 30 s, centrifuge at 15000×g for 20 min, collect the supernatant for ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis, wherein the ultra-high performance liquid chromatography-tandem mass spectrometry analysis is performed using the ExionLC AD ultra-high performance liquid chromatography system (SCIEX)-QTRAP. The 6500 liquid chromatography-mass spectrometry system was used with an ACQUITY UPLC HSST3 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters). The mobile phase consisted of a 60% (v / v) acetonitrile aqueous solution in phase A and a 50% (v / v) acetonitrile-isopropanol mixture in phase B. The gradient elution program was as follows: (1) 0-2 min, 99.9% A – 70% A; (2) 2 min-4 min, 70% A – 50% A; (2) 4 min-5.5 min, 50% A – 1% A; (2) 5.5 min-7 min, 1% A – 99.9% A. The flow rate was 0.4 mL / min, the column temperature was 40℃, and the injection volume was 10 mL / min. μL was collected and detected using negative ion mode. The parameters were set as follows: ion spray needle voltage -4500V, turbine gas temperature 550℃, collision activation pyrolysis gas at medium intensity, and curtain gas pressure set to 35psi. Data for each group of plasma samples were obtained and processed using MultiQuant software (version 3.0.3, SCIEX). Multiple reaction monitoring (MRM) was employed, and quantification was performed on 73 eicosanoic acid-specific precursor-daughter ion pairs. The quantitative range of the method was 0.2 nmol / L to 40000 nmol / L. A standard curve equation was constructed using weighted least squares linear regression (weight coefficient 1 / x). Detailed method information is shown in Table 1. The t-values in Table 1... R The retention time of eicosanoic acid metabolites is represented by Q1, the mass-to-charge ratio of the parent ion of eicosanoic acid metabolites as determined by multiple reaction monitoring mode is represented by Q3, and the mass-to-charge ratio of the daughter ion of eicosanoic acid metabolites as determined by multiple reaction monitoring mode is represented by Q3. The concentration of eicosanoic acid in each group of plasma samples is obtained by calculation after calibrating the standard curve equation. Table 1. Information on eicosanoid metabolism detection based on UHPLC–MS / MS multiple reaction monitoring mode.
[0020] ARA: Arachidonic acid; EPA: Eicosapentaenoic acid; DGLA: Dihydrogamma-linolenic acid Step 104: Determination of eicosanoic acid metabolites related to the efficacy of Si Ni Tang: Volcano plot analysis was performed on the eicosanoic acid metabolite levels in rats in the sham surgery group (Sham) and the heart failure model group (Model) after administration of Si Ni Tang on day 28 in Step 102 to identify the eicosanoic acid metabolites that caused abnormal changes in myocardial infarction-induced heart failure rats. The results are as follows: Figure 1 As shown, 31 eicosanoic acid-like metabolites (red dots ● and blue dots ●) associated with heart failure were identified from 73 eicosanoic acid metabolites: LXB4, 12-HETE, 5-oxoETE, LXA4, TXB2, 16-HETE, 20-HETE, LTB4, PGA2, 17-HETE, 11-keto-TXB2, 11β-PGE2, 11β-PGF2α, 12-OxoETE, PGE2, PGD2, 15-HETE, PGB2, 18-HETE, 20-COOH-LTB4, 6-trans- LTB4, 15-keto-PGF2α, 8-HETE, TXB1, PGD1, PGE1, PGF1α, 12-HEPE, 11-HEPE, 15-HEPE, and TXB3 were analyzed. Analysis of variance was then used to compare the changes in these 31 eicosanoid metabolites associated with heart failure under different treatment groups: a heart failure model group (Model), a low-dose Sini Tang treatment group (SND-L), and a high-dose Sini Tang treatment group (SND-H). The aim was to identify eicosanoids that showed significant reversion after Sini Tang intervention, which were then used as indicators of the anti-inflammatory and cardioprotective efficacy of Sini Tang. The results of the analysis of variance are as follows: Figure 2 As shown (##p<0.01 vs sham surgery group; *p<0.05, **p<0.01 vs heart failure model group), the following 24 eicosanoic acid metabolites and their contents that were significantly reversible by Si Ni Tang were finally identified: LXB4, 12-HETE, 5-oxoETE, LXA4, TXB2, 16-HETE, 20-HETE, LTB4, 11-keto-TXB2, 11β-PGE2, 11β-PGF2α, 12-OxoETE, PGE2, PGD2, 15-HETE, PGB2, 6-trans-LTB4, 15-keto-PGF2α, TXB1, PGF1α, 12-HEPE, 11-HEPE, 15-HEPE, TXB3; Step 2: Identification of the components absorbed into the blood by Si Ni Tang and determination of their relative peak intensities. Step 201, Sample Preparation and Analysis: Take 100 μL of plasma samples from rats in the high-dose Si Ni Tang treatment group on days 7, 14, and 28 after drug administration (1 hour). Add 200 μL of acetonitrile solution containing 500 ng / mL pentoxifylline as an internal standard. Vortex for 1 min, centrifuge at 4℃ and 12000×g for 10 min. Collect the supernatant for solid-phase extraction to enrich the Si Ni Tang components in the plasma samples. Collect the solid-phase extraction eluent and concentrate under reduced pressure. Redissolve in 100 μL of 80% (v / v) methanol aqueous solution, vortex for 1 min, centrifuge at 15000×g for 20 min, and collect the supernatant for ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS). UHPLC-Q-TOFMS analysis was performed using an Agilent 1290 ultra-high performance liquid chromatography system, and mass spectrometry was performed using an Agilent 6545 system. The Q-TOF mass spectrometer was used with an ACQUITY UPLC BEN T3 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters). The mobile phase A was a 0.1% formic acid aqueous solution (containing 5% acetonitrile), and the mobile phase B was acetonitrile (containing 0.1% formic acid). The gradient elution program was as follows: (1) 0-14 min, 2% B – 28% B; (2) 14 min-25 min, 28% B – 51% B; (3) 25 min-28 min, 51% B – 95% B; (4) 28 min-30 min, 95% B – 95% B; (5) 30 min-30.1 min, 95% B – 2% B. The flow rate was 0.3 mL / min, the column temperature was 40℃, the injection volume was 2 μL, and the mass spectrometer was in full scan mode with a scan range of m / z 100–1200. Step 202: Identification of components absorbed into the blood by Si Ni Tang: The chemical components in Si Ni Tang were identified using Agilent MassHunter software combined with spectral library database matching. Then, based on the retention time and mass-to-charge ratio information of the extracted ion chromatogram, the components absorbed into the blood by Si Ni Tang were identified. The original UHPLC-Q-TOFMS data were converted into mzML format using ProteoWizard software, and chromatographic peak extraction, correction, and peak area integration were performed using the XCMS software package (https: / / xcmsonline.scripps.edu / landing_page.php?pgcontent=mainPage). Except for setting fwhm=15, bw=10, and snthresh=5, all other XCMS parameters were set to default values, resulting in a three-dimensional data matrix of retention time, mass-to-charge ratio, and peak intensity. Figure 3Figure A shows the chemical composition spectrum of the original Si Ni Tang decoction, and Figure B shows the blood absorption spectrum of Si Ni Tang in the high-dose treatment group. Comparing Figure A and Figure B, it can be seen that 19 Si Ni Tang components were identified in the blood of rats in the high-dose treatment group. The combined extracted ion chromatogram results are shown in Figure B. The peak numbers of the 19 Si Ni Tang components in Figure B correspond to the following components: Peak 1: Senbusine A, Peak 2: Mesaconine, Peak 3: 16β-Hydroxycardiopetaline, Peak 6: Karakorine, Peak 7: Isotalatizidine, Peak 8: Aconine, Peak 9: Songorine, Peak 10: Hetisine, peak 11: Hypaconine, peak 12: Fuziline, peak 13: Neoline, peak 15: Talatizamine, peak 16: isoliquiritigenin, peak 17: Chasmanine, peak 18: 14-acetyineoline, peak 19: 14-acetyltalatizamine, peak 20: 14-benzoy-10-OH-mesaconine, peak 22: benzoylmesaconine, peak 31: 6-shogaol; Step 203: Determination of the relative peak intensities of the components absorbed into the bloodstream by Si Ni Tang: From the three-dimensional data matrix of retention time, mass-to-charge ratio, and peak intensity obtained in Step 302, information on 19 components absorbed into the bloodstream by Si Ni Tang was found through retention time and mass-to-charge ratio, and the corresponding peak intensity information was obtained. Mass spectrometry response correction was performed using internal standard peak intensity to obtain the relative peak intensity information of the 19 components absorbed into the bloodstream by Si Ni Tang 1 hour after gavage administration on days 7, 14, and 28 (e.g., ...). Figure 3 (as shown in Figure B). Step 3: Correlation analysis of the content of eicosanoic acid metabolites related to the efficacy of Sini Decoction and the relative peak intensity of components absorbed into the blood by Sini Decoction. The levels of eicosanoid metabolites related to the efficacy of Sinitang in rats in the high-dose Sinitang group at days 7, 14, and 28, as determined in step 104, were analyzed using Pearson correlation with the relative peak intensity information of the Sinitang absorbed into the blood after high-dose administration in step 203. The correlation coefficient r represents the effect of the absorbed Sinitang components on the eicosanoid metabolites related to the efficacy of Sinitang. A correlation of |r| ≥ 0.6 (p < 0.05) indicates a significant correlation. This process was used to screen for the anti-inflammatory and cardioprotective active components of Sinitang. The results are as follows: Figure 4 As shown (red dots ● represent high positive correlation (r≥0.6); blue dots ● represent high negative correlation (r≤-0.6); yellow dots ● represent low correlation (|r|<0.6)), from Figure 4 It can be seen that the 12 components showed a broad and significant correlation with eicosanoic acid metabolites: 6-shogaol, 14-benzoy-10-OH-mesaconine, mesaconine, aconine, Karakorine, hetisine, songrine, neoline, fuziline, talatizamine, isoliquiritigenin, and benzoylmesaconine.
[0021] The activity verification of the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang screened in this embodiment. To verify the accuracy and effectiveness of the correlation analysis, four components highly correlated with eicosanoic acid metabolism—songrine, neoline, talatizamine, and isoliquiritigenin—were selected to verify their anti-inflammatory and cardioprotective effects using an H9c2 cell inflammatory injury model and an H9c2 cell oxygen-glucose deprivation / reoxygenation injury model.
[0022] 1. Activity validation based on an H9c2 cell inflammatory injury model The supernatant produced by macrophages (RAW264.7) induced with LPS at a concentration of 1 μg / mL was used as the conditioned medium (CM) for inducing H9c2 cardiomyocyte inflammatory injury. The procedure for inducing the H9c2 cardiomyocyte inflammatory injury model is as follows: Figure 5As shown. Well-grown H9c2 cardiomyocytes were seeded into 96-well plates and divided into a control group, a CM model group (i.e., an inflammatory injury model group), and treatment groups with different concentrations of strongly correlated components (songrine, neoline, and talatizamine concentrations were 10 μM, 20 μM, 50 μM, 75 μM, and 100 μM, respectively; isoliquiritigenin concentrations were 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM, respectively). Each component and each concentration was set at 6 wells. Cells were incubated in replicates for 12 hours in a cell culture incubator. The original culture medium was discarded, and serum-free medium was added for starvation culture for 4 hours. Subsequently, different concentrations of strongly correlated components (songrine, neoline, talatizamine, isoliquiritigenin) were added and incubated for 6 hours, followed by conditioned medium culture for 24 hours. After the culture time was reached, the supernatant was aspirated for use (this supernatant was used for nitric oxide assay). 100 μL of CCK-8 solution was added to each well, and the absorbance was measured at 450 nm. Cell viability was calculated. The results are shown below. Figure 6 As shown (##p<0.01 vs control group; *p<0.05, **p<0.01 vs CM model group), from... Figure 6 It can be seen that the cell viability of each component treatment group was significantly improved compared with the CM model group. Meanwhile, the analysis results of the four eicosanoic acid-related components (songrine, neoline, talatizamine, and isoliquiritigenin) on the NO release induced by cardiomyocytes in the CM model group are as follows: Figure 7 As shown (##p<0.01 vs control group; *p<0.05, **p<0.01 vs CM model group), from... Figure 7 It can be seen that succinate, niorin, tarassamine, and isoliquiritin can reduce the release of nitric oxide (NO) in a concentration-dependent manner in the H9c2 cell inflammatory injury model. Nitric oxide (NO) is widely recognized as a mediator and regulator of inflammatory responses. Therefore, the above results confirm that the components in Sini Decoction that are highly related to eicosanoic acid metabolism have anti-inflammatory and cardioprotective effects.
[0023] 2. Activity validation based on the H9c2 oxygen-glucose deprivation / reoxygenation injury model Given the multifactorial pathogenesis of heart failure induced by myocardial infarction, in order to verify the accuracy of the screening method, a model of oxygen-glucose deprivation / reoxygenation injury in H9c2 cells induced by sodium dithionite combined with glucose deprivation was further used to evaluate the cardioprotective activity of sine sulfadiazine, neorin, tarassamine, and isoliquiritigenin.
[0024] H9c2 cardiomyocytes were used at 5×10 3Cells were seeded at a density of 1 cell / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell density reached approximately 40%. The culture medium was then discarded, and glucose-free and serum-free medium containing different concentrations of components strongly associated with Si Ni Tang (songrine, neoline, and talatizamine at concentrations of 10 μM, 20 μM, 50 μM, 75 μM, and 100 μM; isoliquiritigenin at concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM) was added. The cells were incubated for 6 hours, followed by co-incubation with 3 mM sodium hyposulfite (Na2S2O4) to induce chemical ischemia for 8 hours, followed by reoxygenation for 2 hours. The culture medium was then discarded, and 100 μL of CCK-8 solution was added to each well. The absorbance was measured at 450 nm, and cell viability was calculated. A control group and a CM model group (oxygen-glucose deprivation / reoxygenation injury model group) were also established. The results are as follows: Figure 8 As shown, from Figure 8 It can be seen that pretreatment with four components—Songguoling, Neolin, Tarasamine, and Isoliquiritin—increased cell viability. Specifically, Neolin at its optimal concentration increased cell viability by 16.44%, Songguoling by 10.05%, Isoliquiritin by 15.60%, and Tarasamine by 11.23%. These results further validate the effectiveness of the anti-inflammatory cardioprotective active ingredients screened through correlation analysis between plasma chemical components and eicosanoic acid metabolomics.
[0025] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. A method for screening the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang, characterized in that, The method includes the following steps: Step 1: Discovery and content determination of eicosanoic acid-like substances related to the efficacy of Si Ni Tang. Step 101: Preparation of Si Ni Tang: Weigh 300g of Aconitum carmichaelii, 200g of dried ginger and 300g of licorice, add 10 times the volume of purified water and soak for 1 hour, then decoct for 2 hours. Filter while hot through four layers of gauze. The residue is then decocted with 8 times the volume of purified water for 1 hour and filtered in the same way. Combine the two filtrates and concentrate under reduced pressure to prepare Si Ni Tang with concentrations of 1g / mL and 3g / mL. Step 102, Sample Preparation: Clean-grade male SD rats were used to establish a heart failure model group, a low-dose Si Ni Tang treatment group, a high-dose Si Ni Tang treatment group, and a sham operation group. The low-dose Si Ni Tang treatment group and the high-dose Si Ni Tang treatment group were administered 10g (equivalent to the original medicinal material) / kg / BW and 30g (equivalent to the original medicinal material) / kg / BW of Si Ni Tang by gavage, respectively, every day. The heart failure model group and the sham operation group were administered an equal volume of physiological saline by gavage. The drugs were administered once a day for 28 consecutive days, starting 24 hours after model establishment. Step 103: Analysis of eicosanoic acid-like substances in the samples: Take plasma samples from each group of rats in Step 102, add a methanol / acetonitrile mixed solution (volume ratio 1:2, containing deuterated eicosanoic acid-like internal standard), vortex, incubate at -20℃, centrifuge, and perform solid-phase extraction on the supernatant. Collect the solid-phase extraction eluent, dry under reduced pressure in a vacuum concentrator, redissolve in methanol-water solution, vortex, centrifuge, and perform ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis. Data from each group of plasma samples were obtained and processed using MultiQuant software (version 3.0.3, SCIEX). A multiple reaction monitoring (MRM) mode was employed, and quantification was performed by monitoring the specific precursor-daughter ion pairs of eicosanoids. The quantitative range of the method was 0.2 nmol / L to 40000 nmol / L. A standard curve equation was constructed using weighted least squares linear regression (weight coefficient 1 / x). After calibration, the concentration of eicosanoids in each group of plasma samples was calculated. Step 104: Determination of eicosanoid metabolites related to the efficacy of Si Ni Tang: Volcano plot analysis was performed on the eicosanoid metabolite content in the heart failure model group and sham-operated group rats from Step 102 to identify the eicosanoid metabolites that caused abnormal changes in heart failure rats induced by myocardial infarction, and to determine the eicosanoid metabolites related to heart failure. Then, analysis of variance was used to compare the changes of these eicosanoid metabolites related to heart failure under the administration of the heart failure model group, the low-dose Si Ni Tang treatment group, and the high-dose Si Ni Tang treatment group. The eicosanoids that showed significant reversal after Si Ni Tang intervention were identified as the anti-inflammatory and cardioprotective efficacy indicators of Si Ni Tang. Finally, the eicosanoid metabolites and their content that were significantly reversed by Si Ni Tang were determined. Step 2: Identification of the components absorbed into the blood by Si Ni Tang and determination of their relative peak intensities. Step 201, Sample Preparation and Analysis: Take plasma samples from rats in the high-dose treatment group of Si Ni Tang in Step 102 after administration of the drug, add acetonitrile solution containing pentoxifylline as an internal standard, vortex, centrifuge, and take the supernatant for solid-phase extraction to enrich the Si Ni Tang components in the plasma samples. Collect the solid-phase extraction eluent and concentrate it under reduced pressure, then redissolve it with methanol aqueous solution, vortex mix, centrifuge, and take the supernatant for analysis by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS). Step 202: Identification of components absorbed into the blood by Si Ni Tang: The chemical components in Si Ni Tang were identified using Agilent MassHunter software combined with spectral library database matching. Then, based on the retention time and mass-to-charge ratio information of the extracted ion chromatogram, the components absorbed into the blood by Si Ni Tang were identified. The original UHPLC-Q-TOFMS data were converted into mzML format using ProteoWizard software, and peak extraction, correction and peak area integration were performed using XCMS software to obtain a three-dimensional data matrix of retention time, mass-to-charge ratio and peak intensity. Step 203: Determination of the relative peak intensity of the components absorbed into the blood by Si Ni Tang: From the three-dimensional data matrix of retention time, mass-to-charge ratio and peak intensity in step 302, the information of the components absorbed into the blood by Si Ni Tang is found by retention time and mass-to-charge ratio, and the corresponding peak intensity information is obtained. The mass spectrometry response is corrected by using the internal standard peak intensity to obtain the relative peak intensity information of the components absorbed into the blood by Si Ni Tang after high-dose administration of Si Ni Tang. Step 3: Correlation analysis of the content of eicosanoic acid metabolites related to the efficacy of Sini Decoction and the relative peak intensity of components absorbed into the blood by Sini Decoction. The levels of eicosanoid metabolites related to the efficacy of Sinitang in rats in the high-dose Sinitang group on days 7, 14, and 28, as determined in step 104, were compared with the relative peak intensity information of the Sinitang absorbed into the blood after high-dose administration in step 203 using Pearson correlation analysis. The correlation coefficient r was used to represent the degree of effect of the Sinitang absorbed into the blood on the eicosanoid metabolites related to the efficacy of Sinitang. A correlation of |r|≥0.6 (p<0.05) was considered significant, thus identifying the anti-inflammatory and cardioprotective active ingredients of Sinitang.
2. The method for screening the anti-inflammatory and cardioprotective active ingredients of Si Ni Tang according to claim 1, characterized in that, The anti-inflammatory and cardioprotective active ingredients of Si Ni Tang mentioned in step three are Song Guo Ling, Niorin, Tarasamine, and Isoliquiritin.