Application of tin-added ferruginine in clerodendrum trichotomum to preparation of medicine for reducing blood pressure and protecting blood vessels

Abstract

The invention discloses application of tin-added ferruginine in clerodendrum trichotomum to preparation of a medicine for reducing blood pressure and protecting blood vessels. The tin-added ferruginine has a good antihypertensive effect and a target organ protection effect on L-NNA induced hypertension rats, has high efficiency and safety, has a multi-target regulation characteristic, is derived from natural products, has relatively low toxic and side effects, and has a unique advantage in treatment of hypertension.

Description

TECHNICAL FIELD

[0001] The application relates to an application of edulinine in an Orixa japonica Thunb., and in particular to an application of edulinine in the Orixa japonica Thunb. in preparation of a blood pressure reducing and blood vessel protecting medicine. BACKGROUND

[0002] As one of the most common chronic non-communicable diseases in the world, hypertension is characterized by an increase in systemic arterial pressure and peripheral arteriolar resistance, accompanied by functional or organic damage to the heart, brain, kidneys and other organs, and is a major risk factor for the occurrence and development of cardiovascular diseases (CVD). Although modern medicine has been researching hypertension for more than a century, hypertension is still difficult to cure, and its mechanism is complex and involves multiple interacting factors, which makes the pathogenesis of hypertension not fully elucidated and the prevention and treatment greatly limited.

[0003] Commonly used drugs for treating hypertension mainly include diuretics, angiotensin II receptor antagonists, calcium antagonists, angiotensin converting enzyme inhibitors, beta receptor blockers, alpha receptor blockers and several other types of drugs, which have certain curative effects, but still have obvious side effects of long-term drug taking, liver and kidney function damage, and increased drug resistance.

[0004] Edulinine (Edu) is an indole alkaloid extracted from the Orixa japonica Thunb. of the Rutaceae Orixa genus, and studies have shown that it has multiple biological activities such as sedation, analgesic anti-inflammatory, partial central inhibition and anticonvulsant. However, no literature and research has disclosed the effect of edulinine on blood pressure reduction and blood vessel protection. SUMMARY

[0005] The purpose of the present application is to provide an application of edulinine in the Orixa japonica Thunb. in preparation of a blood pressure reducing and blood vessel protecting medicine.

[0006] The technical scheme of the present application is that the edulinine in the Orixa japonica Thunb. is applied in preparation of a blood pressure reducing and / or blood vessel protecting medicine.

[0007] In the foregoing application, the extraction method of the edulinine comprises the following steps: S1, crushing the aboveground part of the Orixa japonica Thunb. and extracting with methanol under heating, filtering the obtained extract liquid to remove the filter residue, combining the filtrate and concentrating under reduced pressure to obtain an extract; S2, dissolving the extract with methanol, and then performing silica gel column chromatography, first eluting with petroleum ether / dichloromethane at a volume ratio of 30:70, and then gradient eluting with chloroform / methanol at a volume ratio of 100:0~0:100 to obtain a fraction Fr.3; S3, fraction Fr.3C was subjected to silica gel column chromatography, eluted with petroleum ether / ethyl acetate (20:80 by volume) to obtain fraction Fr.3C; S4, fraction Fr.3C was subjected to normal-phase silica gel column chromatography, eluted with petroleum ether / dichloromethane (30:70 by volume) to obtain tin-miraxanthin.

[0008] In the foregoing application, the temperature of the methanol heated extraction in step S1 is 60-80℃.

[0009] In the foregoing application, the methanol heated extraction in step S1 is performed 2-4 times, each time for 1-3 hours.

[0010] In the foregoing application, the silica gel column in step S2 is 100-200 mesh; and the silica gel column in step S3 is 300-400 mesh.

[0011] A medicine with blood pressure lowering effect, containing tin-miraxanthin extracted from Kopsia fruticosa.

[0012] A medicine with vascular protection effect, containing tin-miraxanthin extracted from Kopsia fruticosa.

[0013] Compared with the prior art, the present application has the following beneficial effects: The research results of the present application show that tin-miraxanthin exhibits better blood pressure lowering effect than sodium nitroprusside in L-NNA-induced hypertensive rats, and can effectively improve the left ventricular systolic dysfunction caused by hypertension, and has obvious therapeutic and protective effects on the left ventricular systolic dysfunction caused by hypertension.

[0014] In addition, tin-miraxanthin can also significantly improve the abnormal biochemical indicators, histopathological changes and fibrosis damage of hypertensive rats. Physiological and biochemical indicators and pathological morphological examination methods show that tin-miraxanthin has a protective effect on the vascular endothelial damage of L-NNA-induced hypertensive rats; by detecting the changes of blood lipids (such as total cholesterol, triglyceride, low-density lipoprotein cholesterol, etc.) and blood sugar (such as fasting blood glucose, glycosylated serum protein, etc.), it is shown that tin-miraxanthin has a selective regulation on LDL level, maintains the stability of HDL, TG and CHO, and does not interfere with the metabolic regulation of blood sugar, has no adverse effects on sugar metabolism, and has good safety of sugar metabolism.

[0015] Through joint analysis of metabolomics and proteomics, it is found that tin-miraxanthin mainly targets the regulation of lipid and energy metabolism related proteins and signal pathways to alleviate the metabolic disorder of thoracic aorta tissue under the condition of hypertension. Tin-miraxanthin can regulate multiple metabolic pathways and protein pathways to exert multidimensional therapeutic effects, showing good application prospect in the treatment of hypertension.

[0016] In summary, tin-adding miraculin has good antihypertensive effect and target organ protection effect on L-NNA-induced hypertensive rats, has high efficiency and safety, has multi-target regulation characteristics, and has relatively low toxicity and side effects due to being derived from natural products, and has unique advantages in the treatment of hypertension. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is a graph showing the effects of tin-adding miraculin on the blood pressure and cardiac function of rats in each group; wherein Figures A-B are graphs showing the body weight changes of rats in each group; Figures C-F are graphs showing the cardiac function indicators of rats in each group detected by ultrasound; Figures G-O are graphs showing the blood pressure changes of rats in each group detected by ultrasound; the results are expressed as mean ± standard deviation (n = 6); * indicates p < 0.05, ** indicates p < 0.01, which is the difference between the control group rats and the model group rats; # indicates p < 0.05, ## indicates p < 0.01, which is the difference between the model group rats and the Edu treatment group or the SNP treatment group.

[0018] Figure 2 Figure 2 is a graph showing the effects of tin-adding miraculin on the biochemical indicators and histopathology of rats in each group; wherein Figure A is a graph showing the pathological observation and oil red O staining of rats in each group; Figures B-G are graphs showing the serum total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose (GLU), and glycated serum protein (GSP) levels of rats in each group; the results are expressed as mean ± standard deviation (n = 6); * indicates p < 0.05, ** indicates p < 0.01, which is the difference between the control group rats and the model group rats; # indicates p < 0.05, which is the difference between the model group rats and the Edu treatment group or the SNP treatment group.

[0019] Figure 3 Figure 3 is a superimposed graph of the total ion flow graph detected by QC sample mass spectrometry.

[0020] Figure 4 Figure 4 is a metabolite classification graph.

[0021] Figures 5-8 Figure 5 is a metabolomics analysis graph; wherein Figure 5 A of Figure 5 is a principal component analysis graph of differential metabolites; Figure 5 B of Figure 5 is a Venn diagram of differences between groups; Figure 6 C of Figure 5 is an OPLS-DA score graph of the model group and the control group; Figure 6 D of Figure 5 is an OPLS-DA score graph of the SNP group and the model group; Figure 6 E of Figure 5 is an OPSS-DA score graph of the Edu group and the model group; Figure 7 F of Figure 5 is a volcano plot of differential metabolites between the model group and the control group; Figure 7 G of Figure 5 is a volcano plot of differential metabolites between the SNP group and the model group;Figure 7 H is a volcano plot of differential metabolites between the Edus group and the model group; Figure 8 I is a differential metabolic pathway enrichment map between the model group and the control group; Figure 8 J is a differential metabolic pathway enrichment map between the SNP group and the model group; Figure 8 K is a differential metabolic pathway enrichment map between the Edu group and the model group.

[0022] Figures 9-15 This is a diagram of proteomics analysis; among which... Figure 9 A is a volcano plot of differentially expressed proteins between the model group and the control group; Figure 9 B is a volcano plot of differentially expressed proteins between the SNP group and the model group; Figure 9 C is a volcano plot of differentially expressed proteins between the Edu group and the model group; Figure 10 D is the gene ontology (GO) analysis diagram between the model group and the control group; Figure 11 E is the gene ontology (GO) analysis diagram between the SNP group and the model group; Figure 12 F is the gene ontology (GO) analysis diagram between the Edu group and the model group; Figure 13 G is a differential metabolic pathway enrichment map between the model group and the control group; Figure 14 H is a differential metabolic pathway enrichment map between the SNP group and the model group; Figure 15 I is a differential metabolic pathway enrichment map between the Edu group and the model group.

[0023] Figures 16-18 This is a molecular model diagram of Edu binding to key enzymes in fatty acid degradation; among which... Figures 16-18 The figures for AG represent the molecular docking of Edu with Acads, Acadm, Acat1, Acadl, Hadh, Hadha, and Hadhb, respectively.

[0024] Figure 19 These are iPath pathway analysis diagrams for proteomics and metabolomics; Figure A shows the iPath pathway diagrams for differentially expressed metabolites and genes between the model group and the control group; Figure B shows the iPath pathway diagrams for differentially expressed metabolites and genes between the Edu group and the model group.

[0025] Figures 20-22 This is a diagram of KEGG pathway enrichment analysis in proteomics and metabolomics. Figure 20 A is a bubble diagram of KEGG pathway enrichment analysis of differentially expressed proteins and metabolites between the model group and the control group. Figure 21 B is a bubble diagram of KEGG pathway enrichment analysis of differentially expressed proteins and metabolites between the Edu group and the model group. Figure 22 C is a visualization of the KEGG pathway between the model group and the control group;Figure 22 D is the KEGG pathway visualization diagram of Edu group and model group. DETAILED DESCRIPTION

[0026] The application will be further described in connection with the following examples, but not as a limitation on the application.

[0027] Example: The extraction method of the active ingredient tin mirabilis bicarbonate in the common common mountain, comprising the following steps: S1, take 20.0 kg of dry common mountain aboveground part, crush, heat extraction with 100 L of methanol at 80 ℃ for three times, 2 hours each time, filter the obtained extract, remove the residue, combine the filtrate, and concentrate under reduced pressure to obtain 2.1 kg of extract.

[0028] S2, dissolve the extract with appropriate amount of methanol solvent, then pass through 100-200 mesh silica gel column chromatography, first elute with petroleum ether / dichloromethane at a volume ratio of 100:0-0:100, then elute with chloroform / methanol at a volume ratio of 100:0-0:100. The eluent is detected by TLC, and the bismuth potassium iodide color developing agent is colored to obtain 5 fractions Fr.1-Fr.5. The fraction Fr.1 is eluted with petroleum ether / dichloromethane at a volume ratio of 80:20, the fraction Fr.2 is eluted with petroleum ether / dichloromethane at a volume ratio of 50:50, the fraction Fr.3 is eluted with petroleum ether / dichloromethane at a volume ratio of 30:70, the fraction Fr.4 is eluted with dichloromethane / methanol at a volume ratio of 70:30, and the fraction Fr.5 is eluted with dichloromethane / methanol at a volume ratio of 30:70.

[0029] S3, fraction Fr.3 (18.5 g) is subjected to 300-400 mesh silica gel column chromatography, and petroleum ether / ethyl acetate at a volume ratio of 100:0-0:100 is subjected to gradient elution to obtain fractions Fr.3A-Fr.3C; the fraction Fr.3A is eluted with petroleum ether / ethyl acetate at a volume ratio of 80:20, the fraction Fr.3B is eluted with petroleum ether / ethyl acetate at a volume ratio of 50:50, and the fraction Fr.3C is eluted with petroleum ether / ethyl acetate at a volume ratio of 20:80.

[0030] S4, fraction Fr.3C (1.1 g) is subjected to normal phase silica gel column chromatography, and petroleum ether / dichloromethane at a volume ratio of 30:70 is subjected to isocratic elution to separate tin mirabilis bicarbonate (820.0 mg).

[0031] The purity of the tin mirabilis bicarbonate obtained by the above method is 95%.

[0032] From the common mountain in the Geshimi Luoguo base, with the effect of lowering blood pressure and blood vessel protection, can be applied in the preparation of antihypertensive and / or vascular protection drugs.

[0033] Experimental example: Materials and methods: Animals: Wistar rats, male, body weight 200±20g, animal production license number: SYXK (Qian) 2018-0001, provided by the animal center of Guizhou Medical University, the use of experimental animals and related purposes were approved by the ethics committee of Guizhou Medical University, the approval number was NO. 1901109. The animal breeding environment temperature was 20~25℃, humidity 50%~60%, free diet and water, 12h light, 12h dark, the animals were adaptively fed for 7d after entering the room, and the experiment was carried out, all operations were in line with the "guide for the use of laboratory animals".

[0034] Reagents and instruments: Geshimi Luoguo base (Edu); sodium nitroprusside (Kangfeng Cano Pharmaceutical Co., Ltd., batch number: 154154); L-nitroarginine (Shanghai Maikelin Biochemical Technology Co., Ltd., C11466030); PPAR-γ (Protientech, batch number: GB112205); GAPDH (Bioworld, batch number: MB001); basic feed (16% protein, 4% fat and 60% carbohydrate, Chongqing Tengxin Biotechnology Co., Ltd., batch number: 2017-1021); rainbow 180 broad-spectrum protein marker (Beijing Solabio Technology Co., Ltd., batch number: PR1910); BCA protein concentration determination kit (Beijing Solabio Technology Co., Ltd., batch number: PC0020); high-efficiency RIPA tissue / cell rapid lysis solution (Beijing Solabio Technology Co., Ltd., batch number: R0010); PMSF (Beijing Solabio Technology Co., Ltd., batch number: P0100-01); SDS-PAGE gel kit (Beijing Solabio Technology Co., Ltd., batch number: P1200); hypersensitive ECL chemiluminescence kit (Xinsaimai Biotechnology Co., Ltd., batch number: P10300).

[0035] Thermo Scientific QE-Oribitrap high-resolution mass spectrometry (Thermo Fisher Corporation, USA); Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Corporation, USA).

[0036] Model preparation and drug administration: The concentration of L-NNA physiological saline solution is 1.5 mg / mL, and the dose is 2.25 mg / 100 g·d according to the weight of the rats. The rats are given intraperitoneal injection once a day, and the tail artery blood pressure of the rats is measured by using a small animal non-invasive blood pressure instrument Bp6. The systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) are recorded once a week for 4 weeks, and the vascular endothelial injury type hypertension rat disease model is replicated. After 4 weeks, the rats with SBP higher than 140 mmHg and DBP higher than 90 mmHg are randomly divided into a model group, a positive control group (1 mg / kg / d) and a group (6 mg / kg / d), each group having 6 rats. The rats in the nitroprusside and timiruo fruit alkaloid groups are given intraperitoneal injection of the modeling drugs 1 hour before the administration, and the rats in the model group and the control group are given the same volume of normal saline. The tail artery blood pressure of the rats is measured 15, 30, 45, 60 and 120 minutes after administration once, and the tail artery blood pressure of the rats is measured once a week for 2 weeks.

[0037] Rat weight detection: the weight of the rats is measured once every 7 days.

[0038] Heart index detection: the heart index of the rats in each group before modeling, four weeks after modeling (before administration) and two weeks after administration is investigated by using a small animal B-ultrasound instrument, and the effect of Edu on the heart index of the L-NNA-induced hypertensive rats is investigated.

[0039] Serum factor detection: the rats in each group are given abdominal aortic blood sampling, centrifuged at 3000 rpm for 10 minutes, and the upper serum sample is separated. The content of triglyceride (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), glucose (GLU) and serum protein (GSP) is measured by using a full-automatic biochemical analyzer.

[0040] HE staining method: the thoracic aorta of the rats in each group is fixed in 4% paraformaldehyde fixing solution, routinely embedded in paraffin, sliced, deparaffinated, rehydrated, dyed with hematoxylin-eosin (HE) according to the steps, sealed with neutral gum, observed under a light microscope and images are collected.

[0041] Masson staining method: the thoracic aorta paraffin sections of the rats are deparaffinated, Masson stained and permeabilized, and the tissue lesion condition is observed under a microscope and images are collected.

[0042] Oil red O staining method: the thoracic aorta of rats in each group was fixed in 4% paraformaldehyde fixing solution, and was infiltrated and dehydrated in isopropanol; the sample was placed horizontally in an embedding box and dried in a 60°C constant temperature box for 2 hours; preparation, the dried blood vessels were immersed in 0.5% oil red O staining solution for 30 minutes; after staining, 60% isopropanol was used for differentiation for several seconds until the background was clear, and PBS was used for washing to stop the reaction; finally, the cell nucleus was stained with hematoxylin, and after PBS was returned to blue, gradient ethanol was used for dehydration (70%, 80%, 95%, 100% for 2 minutes each), xylene was used for transparency, neutral gum was used for sealing, and the degree of lipid deposition in the blood vessel wall was observed under an optical microscope and collected for image, and the degree of lipid deposition in the blood vessel wall was evaluated.

[0043] Molecular docking: molecular docking was performed using Maestro software of Schrödinger, and the binding capacity of Edu to Acads, Acadm, Acat1, Acadl, Hadh, Hadha and Hadhb was analyzed. The target protein crystal structure was downloaded from the PDB database, and after being imported into Maestro, the Protein Preparation Wizard module was used for structure modification, water removal, hydrogen bond optimization and energy minimization pretreatment; if the structure has no ligand, the SiteMap module is used to predict the active site. With the ligand or predicted site as the center, the Receptor Grid Generation module is used to generate the docking box. The 2D structure of L-NNA was downloaded from PubChem, and after being imported, the LigPrep module was used to generate the optimized 3D ligand structure. The pretreated protein and ligand were imported into the Ligand Docking module, and the ExtraPrecision precision was set for molecular docking. The lower the docking score, the more stable the binding. Finally, the online analysis of the action mode of the complex was performed by using PLIP v2.3.0 to export the pse file, and further analysis was performed by using PyMOL and DiscoveryStudio software.

[0044] Serum metabolomics analysis: 50 μL of serum from the thoracic aorta of rats in each group was taken, 300 μL of 20% acetonitrile methanol internal standard extraction solution was added, and vortex mixing was performed for 3 minutes. Centrifugation was performed at 4°C and 12,000 rpm for 10 minutes. After centrifugation, 200 μL of supernatant was taken and placed in a -20°C refrigerator for 30 minutes. Centrifugation was performed again at 4°C and 12,000 rpm for 3 minutes, and 180 μL of supernatant was taken for machine analysis. Chromatographic separation was performed using a Waters ACQUITY Premier HSS T3 column (1.8 μm, 2.1 mm x 100 mm), mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; the column temperature was set to 40°C, the flow rate was 0.4 mL / min, and the injection volume was 4 μL. After the raw mass spectrometry data was converted to mzXML format, XCMS software was used for peak extraction, alignment, retention time correction, filtering, filling and re-correction, etc. Pretreatment was performed, and then compound identification was performed. Substances with a comprehensive score of 0.5 or more and a QC sample coefficient of variation (CV) of less than 0.3 were selected. Subsequently, positive and negative ion mode data were combined (preferably retaining the substance with the highest qualitative level and the smallest CV value), and relevant analysis files were obtained.

[0045] Proteomics analysis: protein quantification analysis was performed on the thoracic aorta tissue of rats in each group, and 3 samples were selected from each group. After protein extraction, the sample concentration was determined by BCA method, and then the sample was subjected to reduction alkylation treatment. Polypeptide samples were prepared by trypsin enzyme digestion, and the obtained polypeptides were desalted and quantified. DIA detection technology was used for analysis by ultra-high performance liquid chromatography tandem mass spectrometry. Qualitative and quantitative analysis was performed by Spectronaut™ software based on the spectrum library, and further bioinformatics analysis was carried out. For the comparison group containing T test P value, a volcano plot was drawn to screen differential proteins, and then the differential proteins obtained by screening were introduced into the KOBSA website for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

[0046] Statistical analysis: statistical analysis was performed using GraphPad Prism 9.5.0 software, data were expressed as mean ± standard deviation (±s), and one-way ANOVA was used for comparison between groups. P<0.05 indicates that the difference is statistically significant.

[0047] Experimental results: 1. Investigation of the antihypertensive effect of stanniferine on L-NNA-induced hypertensive rats: ​Quantitative evaluation of left ventricular systolic function is mainly based on the detection of left ventricular cavity size and volume changes. In clinical practice, indicators such as ejection fraction (EF) and fractional shortening (FS) are commonly used to evaluate left ventricular function. In the L-NNA-induced hypertensive rat model constructed in this study, the experimental results are shown in Figure 1 As can be seen from the C-F of Figure 1 , both EF and FS values are significantly reduced, indicating that this modeling method can effectively induce impaired left ventricular systolic function in rats. After Edu treatment, both EF and FS indicators were significantly improved, indicating that Edu has a significant therapeutic effect on left ventricular systolic dysfunction caused by L-NNA-induced hypertension. As can be seen from the A-B of Figure 1 , there were no significant differences in body weight changes among the groups in the first 4 weeks, but changes began to appear from the 5th week to the 6th week, with the Edu group maintaining relatively stable body weight.

[0048] Hypertension is a clinical syndrome characterized by persistent elevation of systemic arterial blood pressure (including systolic and / or diastolic pressure). It is usually defined as systolic pressure ≥ 130 mmHg and / or diastolic pressure ≥ 80 mmHg. In this study, the experimental results are shown in Figure 1 As can be seen from the G-O graph of Figure 1 , after intraperitoneal injection of L-NNA for 1 week, the blood pressure of rats showed a gradual upward trend; by the 4th week, 90% of rats had systolic blood pressure (SBP) exceeding 140 mmHg and diastolic blood pressure (DBP) exceeding 90 mmHg; in the single-dose experiment, compared with the model group, both the SNP group and the Edu group showed significant decreases in systolic blood pressure; in terms of diastolic blood pressure, the SNP group showed statistically significant decreases at 60 and 120 minutes after administration, while the Edu group showed statistically significant decreases at 15, 30, 60, and 120 minutes after administration.

[0049] Therefore, the above experiments demonstrate that tinactin has a blood pressure-lowering effect.

[0050] 2. Improvement of tinactin on abnormal biochemical indicators and histopathological damage: As can be seen from the C-F of Figure 2As shown in Figure A, pathological section results indicate that in the control group, the aorta showed focal intimal hyperplasia, focal smooth muscle-like degeneration and vacuolar degeneration in the media, and focal fibrinoid degeneration in the adventitia; in the model group, the aorta showed focal intimal hyperplasia, extracellular lipids, focal smooth muscle hyperplasia, hyaline degeneration and vacuolar degeneration in the media, and fibrinoid degeneration in the adventitia; in the sodium nitroprusside (SNP) group, the aorta showed focal intimal hyperplasia, extracellular lipids, and scattered inflammatory cells attached to the intima; and in the tinamylose (Edu) group, the aorta showed focal intimal hyperplasia, extracellular lipids, and scattered inflammatory cells attached to the intima. Masson staining results showed that the aorta of rats in the model group exhibited disordered and damaged elastic fiber structure, with a significant increase in collagen content. The blue staining area of ​​collagen fibers in the SNP and Edu groups was significantly reduced compared to the model group, and the elastic fiber structure of the vessel wall was relatively intact, with only slight breaks visible in local areas; the smooth muscle cells were arranged more neatly than those in the model group, and the cell nuclei were clearly visible with blue-purple staining. In addition, Oil Red O staining showed that the vascular tissue structure of rats in each group was intact and no obvious lipid droplets were observed, suggesting that there was no significant lipid deposition in the vascular wall of rats in each group.

[0051] like Figure 2 As shown in the BE diagram, the blood lipid and blood glucose test results at the end of week 6 showed that, in terms of blood lipids, compared with the control group, the levels of triglycerides (TG), total cholesterol (CHO), and low-density lipoprotein cholesterol (LDL) in the model group showed an increasing trend, but the differences between the groups were not statistically significant (P>0.05); while the level of high-density lipoprotein cholesterol (HDL) was significantly reduced (P<0.05), indicating that the model group had already shown early changes in abnormal blood lipid profiles. The levels of TG, CHO, and HDL in the Edu intervention group were not statistically different from those in the control group (P>0.05), indicating that Edu did not have a significant effect on normal blood lipid profiles. Compared with the model group, the LDL levels in both the SNP group and the Edu group were significantly reduced (P<0.05), but the LDL level in the SNP group was also significantly lower than that in the control group (P<0.05), suggesting that SNP may have an additional effect on normal lipid metabolism, while the effect of Edu on reducing LDL is relatively selective. Therefore, succinic acid has selective, safe and potentially physiologically adaptive advantages in improving hypertension-related metabolic disorders, and its effect on regulating LDL is closer to the normal physiological state than that of SNP.

[0052] This study measured glucose (GLU) to determine whether model construction or drug intervention affected metabolic stability. Results are as follows: Figure 2As shown in the FG plot, compared with the Control group, the GLU level in the Model group was significantly lower (P<0.05), suggesting that the modeling process may be accompanied by abnormal blood glucose regulation; while the GLU level in the Edu intervention group was not statistically different from that in the Model group and the Control group (P>0.05). Combined with the fact that there was no significant change in the glycated serum protein (GSP) level in each group, it is further confirmed that Edu did not cause an increase or decrease in blood glucose at the current dose and during the experimental period, had no adverse effects on glucose metabolism, and had good metabolic safety.

[0053] 3. Serum metabolomics investigation of the regulatory mechanism of tin-based metaboloside in improving metabolic disorders in hypertensive rats: To investigate the absorption of compounds from serum by Edu, this study employed ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-Q-Exactive Orbitrap-HRMS) to analyze changes in serum components in hypertensive rats after intraperitoneal injection of Edu. See [link to study]. Figure 3 The overlay plot of the total ion current (TIC) chromatograms of the AB and QC samples shows high overlap in the total ion current curves for metabolite detection, indicating consistent retention times and peak intensities. This suggests good signal stability for mass spectrometry when detecting the same sample at different times. The results showed the detection of 3302 compounds and the resolution of 3137 compounds. Metabolite numbers, integral values, and corresponding metabolite names for some metabolites are shown in the metabolite classification chart (see [link to metabolite classification chart]). Figure 4 In the CD mode, benzene and its derivatives (18.33%), organic acids (13.5%), and heterocyclic compounds (12.74%) are the three main components, accounting for a total of 44.57%. In the negative ion mode, amino acids and their metabolites (18.74%) are the most dominant components, organic acids (8.85%) and benzene derivatives (8.85%) are tied for second place, and heterocyclic compounds (9.68%) are ranked third.

[0054] Principal component analysis (PCA) and orthogonal principal component analysis (OPLS-DA) were used to elucidate the differences between different groups. The results are as follows: Figure 3 As shown in the PCA scatter plot, the variability between and within groups meets the requirements. Figure 5 A). Furthermore, OPLS-DA analysis showed that the metabolic profiles of the model group and the Edu group exhibited their own clusters, each located in one of the four independent regions of the scatter plot. This result suggests that Edu has the potential to improve metabolic abnormalities or serve as a distinguishing indicator at the metabolic level. Figure 6 CE). Differential metabolites were identified based on criteria including fold change (FC>1 or FC<−1), p-value <0.05, and VIP>1, and volcano plots of the differential metabolites were drawn, such as... Figure 7F-H. Compared with the normal control group, 379 metabolites in the model group showed significant changes, of which 165 metabolites were up-regulated and 214 were down-regulated; compared with the model group, 505 metabolites in the Edu treatment group showed significant changes, of which 150 metabolites were up-regulated and 355 were down-regulated; and 505 metabolites in the SNP treatment group showed significant changes, including 287 up-regulated and 218 down-regulated. The difference in the treatment of Edu and SNP metabolites was analyzed by Venn diagram, and the results are shown in Figure 5 B of FIG. 6, showing that there are 3 common differential metabolites in the treatment of Edu and SNP. The differential metabolites were introduced into the KEGG database for pathway analysis, and the metabolic pathways with p value < 0.05 and impact value > 0.1 were selected as potential target pathways, and the 20 pathways with the smallest p value were selected to construct the KEGG bubble chart, as shown in Figure 8 I-K of FIG. 6. Compared with the normal control group, the glycerophospholipid metabolism, linoleic acid metabolism, retrograde endocannabinoid signaling, linolenic acid metabolism, arachidonic acid metabolism, and metabolic pathways of hypertensive rats were significantly changed. After Edu treatment, arachidonic acid metabolism, retrograde endocannabinoid signaling, and metabolic pathways were significantly changed, of which the change in metabolic pathways was the most significant.

[0055] 4. Proteomics analysis: In order to clarify the differences between each group, PCA method was used, and it was found that the cumulative relative explanation variance of PCA1 and PCA2 was 61.5%, indicating that there was a significant difference between the protein samples of the model group and the Edu treatment group. In order to check the changes of differential proteins, we set the standard of p value < 0.05, FC≥1.5 or FC≤1.5. The results showed that compared with the normal control group, 292 proteins in the model group were significantly changed, of which 245 proteins were increased and 47 proteins were decreased; compared with the model group, 320 proteins in the Edu treatment group were significantly changed, of which 285 proteins were increased and 35 proteins were decreased; and 188 proteins in the SNP positive control group were significantly changed, of which 86 proteins were increased and 102 proteins were decreased (see Figure 9 A-C).

[0056] GO annotation is divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF), which elucidates the biological function of proteins from multiple angles. GO results show that BP is mainly associated with metabolic process, CC is substantially associated with mitochondrial protein complex in cells, and MF is associated with oxidoreductase activity (see Figures 10-12). KEGG pathway analysis of differentially expressed proteins showed that the bubble chart of common regulation between model and control groups showed that proteins were enriched in fatty acid degradation, TCA cycle, pyruvate metabolism, oxidative phosphorylation, etc., which had significant significance for lipid metabolism and energy metabolism, and were closely related to fatty acid elongation and myocardial contraction; Edu and model groups showed significant intersection in TCA cycle, fatty acid degradation, oxidative phosphorylation, and fatty acid elongation pathways; the single nucleotide polymorphism group only showed limited performance in TCA cycle and fatty acid degradation pathways. In the fatty acid degradation pathway, the up-regulated proteins in the Edu group were basically completely corresponding to the down-regulated proteins in the control group, significantly more than the SNP group (see Figures 13-15 ), indicating that tinlofoline targeted regulation of lipid and energy metabolism.

[0057] 5. Molecular docking: Molecular docking technology was used to simulate the binding ability of Edu to key enzymes of fatty acid degradation (Acads, Acadm, Acatl, Acadl, Hadh, Hadha, and Hadhb), and to further explore the effect of Edu on key enzymes of fatty acid degradation. The results, as shown in Figures 16-18 , showed that the binding energy of Acads, Acadm, Acatl, Acadl, Hadh, Hadha, and Hadhb to Edu was -8.4, -8.1, -7.3, -6.3, -7.1, -6.9, and -6.6 kcal / mol, respectively, indicating that Edu had good binding ability to key enzymes of fatty acid degradation, which suggested that Edu could delay the progression of hypertension by interfering with the fatty acid degradation pathway.

[0058] 6. Comprehensive analysis of metabolomics and proteomics: 6.1 iPath pathway analysis There is a close interaction and regulation relationship between proteins and metabolites. First, for proteomics, FC>1.3 or FC<1 / 1.3, p<0.05 were selected as the screening criteria for differentially expressed proteins. In metabolomics analysis, |log2FC|>0, p<0.05 was the basic criterion, and VIP value>1 was introduced as an additional screening condition when data was available. Differentially expressed proteins and differential metabolites were visualized by bar charts. To further explore the co-regulation relationship of differential proteins and differential metabolites in biological pathways and their change characteristics under the pathological state of hypertension and Edu intervention, iPath software was used to systematically visualize the biochemical pathways co-enriched by differential metabolites and differential proteins. In this analysis, nodes represent various biochemical molecules (such as metabolic intermediates, enzymes, etc.), and connecting lines represent their participation in biochemical reactions or metabolic flow, thereby directly showing the network association between different metabolic and signaling pathways. The iPath pathway visualization results are as follows: Figure 19As shown, compared with the control group, the model group showed significant differences in multiple core metabolic and biosynthetic pathways such as lipid metabolism, carbohydrate metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism, and secondary metabolite biosynthesis Figure 19 A), suggesting that the pathological state of hypertension is accompanied by multi-pathway metabolic disorders. On the other hand, comparison between the Edu intervention group and the model group showed that Edu mainly showed significant differences in lipid metabolism, carbohydrate metabolism, amino acid metabolism, and energy metabolism Figure 19 B), suggesting that Edu may exert its antihypertensive and metabolic protective effects by regulating these key metabolic networks.

[0059] 6.2 KEGG pathway enrichment analysis Fisher's exact test was used to perform KEGG pathway enrichment analysis on the differential proteins and metabolites, respectively. When P<0.05, it was considered to be a significantly enriched pathway. The enrichment results are shown in Figure 7 As shown, the horizontal axis represents the log2 conversion of the enrichment fold, and the vertical axis is the KEGG pathway description. Triangles are metabolites, and circles are proteins. The color of the point represents the enrichment significance, and red represents strong enrichment significance. The size of the point represents the number of metabolites or proteins in the pathway. According to the results of Figures 20-21 Based on the results of KEGG pathway enrichment analysis, in the comparison between the model group and the control group, fatty acid metabolism-related pathways (such as fatty acid elongation, beta-oxidation, fatty acid biosynthesis and degradation) were significantly enriched, and occupied multiple metabolic-related entries in the top five significant pathways in the metabolic network; in the comparison between Edu and the model group, fatty acid metabolism showed a higher enrichment fold. These results indicate that fatty acid metabolism is one of the main metabolic pathways in the response of hypertension pathological state and Edu intervention to reduce blood pressure and vascular protection effect. Edu regulates key enzymes or metabolic nodes in the fatty acid metabolism network, affects the balance of fatty acid decomposition and synthesis, and further regulates lipid metabolism homeostasis and energy metabolism pathways, which plays a role in improving vascular endothelial function, reducing oxidative stress and inflammatory response, regulating vascular tone, and protecting target organs in hypertension.

[0060] According to the results of KEGG pathway analysis, combined with Figure 22The visualization analysis of differential proteins and metabolites (circles represent metabolites, and boxes represent proteins; red represents up-regulation, green represents down-regulation, and yellow indicates multiple expression patterns in the same box) showed that, in the comparison between the model group and the control group, the fatty acid biosynthesis pathway was one of the metabolic pathways that were significantly enriched in differential expression analysis, and a total of 28 proteins in the pathway were significantly up-regulated, and no protein was down-regulated; and in the comparison between Edu and the model group, 66 proteins in the pathway were significantly changed, including 8 up-regulated and 58 down-regulated. These results showed that the fatty acid biosynthesis pathway was one of the most significantly regulated metabolic pathways in this study, and may serve as a key metabolic pathway in the pathology of hypertension and the response to Edu intervention.

[0061] It should be understood that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them. For those skilled in the art, the technical solutions described in the above examples can be modified, or some of the technical features can be replaced by equivalents; and all these modifications and replacements shall fall within the protection scope of the appended claims of the present application.

Claims

1. The application of styromycin from *Changshania spp.* in the preparation of antihypertensive and / or vascular protective drugs.

2. The application according to claim 1, characterized in that: The extraction method of the tin-containing methyl methoxylate includes the following steps: S1. The above-ground parts of *Changshan* were crushed and extracted with methanol by heating. The resulting extract was filtered to remove the residue. The filtrates were combined and concentrated under reduced pressure to obtain the extract. S2. Dissolve the extract in methanol, then perform silica gel column chromatography. First, elute isocratically with petroleum ether / dichloromethane at a volume ratio of 30:70, then elute with chloroform / methanol at a volume ratio of 100:0~0:100 to obtain fraction Fr.

3. S3. Fraction Fr.3 was subjected to silica gel column chromatography and eluted with petroleum ether / ethyl acetate at a volume ratio of 20:80 to obtain fraction Fr.3C. S4. The fraction Fr.3C was subjected to normal-phase silica gel column chromatography and isocratic elution with petroleum ether / dichloromethane at a volume ratio of 30:70 to obtain tin-based mirocholic alkaloid.

3. The application according to claim 2, characterized in that: In step S1, the temperature for methanol extraction by heating is 60~80℃.

4. The application according to claim 2, characterized in that: In step S1, methanol is extracted by heating 2 to 4 times, each time for 1 to 3 hours.

5. The application according to claim 2, characterized in that: In step S2, the silicone column is 100-200 mesh; in step S3, the silicone column is 300-400 mesh.

6. A drug for lowering blood pressure, characterized in that: It contains succinyl alkaloid extracted from *Changshan*.

7. A drug with vascular protection, characterized in that: It contains succinyl alkaloid extracted from *Changshan*.

Images