Application of cinnamaldehyde to preparation of aldosterone synthase inhibitor

Abstract

The invention discloses application of cinnamaldehyde to preparation of an aldosterone synthase inhibitor, and relates to the field of natural medicine.Firstly, an effective target of cinnamaldehyde playing a role in inhibiting aldosterone synthesis is screened through a network pharmacology analysis tool, and the result indicates that cinnamaldehyde may play a role by acting on aldosterone synthase (CYP11B2); subsequently, the influence of cinnamaldehyde on aldosterone and CYP11B2 is detected through an in-vitro experiment, and cinnamaldehyde is proved to have an inhibiting effect on CYP11B2; finally, human adrenal cortex adenocarcinoma cells (NCI-H295R) and cinnamaldehyde are incubated together, it is found that cinnamaldehyde can inhibit the activity of CYP11B2 of the NCI-H295R cells and reduce the aldosterone level, and it is proved that cinnamaldehyde can inhibit biosynthesis of aldosterone through CYP11B2.

Description

Technical Field

[0001] This invention relates to the field of natural medicines, specifically the use of cinnamaldehyde in the preparation of aldosterone synthase inhibitors. Background Technology

[0002] The renin-angiotensin-aldosterone system (RAAS) is the core system for regulating blood pressure, fluid and electrolyte balance in the human body. It functions through a cascade of renin release, angiotensin production, and aldosterone secretion. It can both temporarily regulate vasoconstriction to raise blood pressure and maintain stable blood volume in the long term through sodium and water retention.

[0003] Aldosterone, as the end-effect hormone of the renin-angiotensin-aldosterone system (RAAS), plays a central role in maintaining electrolyte balance and blood pressure homeostasis. However, excessive aldosterone secretion is an independent risk factor for various cardiovascular metabolic diseases. Its pathological effects extend far beyond sodium retention and potassium excretion, including promoting inflammation, oxidative stress, endothelial dysfunction, myocardial fibrosis, and target organ remodeling. Aldosterone plays a crucial role in the pathophysiological progression of the cardiovascular system. At the vascular level, excessive aldosterone stimulates endothelial dysfunction and the infiltration of inflammatory cells, promoting the formation of atherosclerotic plaques and leading to plaque instability, arterial stiffness, and calcification. In the heart, aldosterone increases cardiac inflammation, fibrosis, and myocardial hypertrophy. From a clinical perspective, high aldosterone levels are associated with an increased risk of cardiovascular events and death. Meanwhile, clinical studies have shown that lowering aldosterone levels can reduce damage to target organs. Current treatments for aldosterone mainly include RAAS inhibitors and aldosterone synthase inhibitors.

[0004] RAAS inhibitors selectively block the binding of angiotensin II (Ang II) to AT1 receptors, inhibiting vasoconstriction, aldosterone secretion, and sodium and water retention, thereby lowering blood pressure and reducing the burden on the heart and kidneys. Classic RAAS inhibitors, such as angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor antagonists (ARBs), are widely used clinically, but they exhibit a significant "aldosterone escape" phenomenon—that is, during treatment, aldosterone levels initially decrease and then rise again, leading to treatment failure or continued progression of target organ damage. Mineralocorticoid receptor antagonists (MRAs) include non-selective and selective MRAs. While non-selective MRAs such as spironolactone and selective MRAs such as eplerenone, as aldosterone receptor antagonists, are used to block the effects of aldosterone (by blocking its binding to its receptor to exert a therapeutic effect), they also have limitations. Because aldosterone receptor antagonists have a structure similar to aldosterone, they bind to specific mineralocorticoid receptors in the cytoplasm, preventing the nuclear translocation of the aldosterone-receptor complex and thus antagonizing aldosterone. However, they do not reduce aldosterone secretion or concentration, and aldosterone can still damage target organs such as the cardiovascular system. Furthermore, these drugs are prone to adverse reactions such as hyperkalemia, gynecomastia in men, and menstrual disorders in women. Therefore, the medical community has long focused on upstream aldosterone synthase (CYP11B2). Developing highly selective aldosterone synthase inhibitors (ASIs) is considered an ideal strategy for precisely controlling aldosterone levels at the source and avoiding the shortcomings of MRAs, and is one of the urgent challenges to be overcome in the cardiovascular field.

[0005] CYP11B2 shares up to 93% homology with 11β-hydroxylase (CYP11B1), the enzyme responsible for cortisol synthesis, and they also share the same substrate. This high degree of structural and functional similarity makes the development of inhibitors with absolute selectivity for CYP11B2 exceptionally difficult. Early-developed ASIs, such as FAD286A (derived from CGP 34,648), showed good antihypertensive and antifibrotic effects in animal models, but they still exhibited some inhibitory activity against CYP11B1, and long-term use may lead to adrenal insufficiency, limiting their clinical translation. In recent years, significant breakthroughs have been made in the development of novel ASIs. Baxdrostat, as a representative of highly selective ASIs, demonstrated in a phase II clinical trial (BrigHTN) that it could produce a dose-dependent decrease in systolic blood pressure (up to 11 mmHg) in patients with refractory hypertension, with an extremely low incidence of hyperkalemia. This finding was published in the *New England Journal of Medicine* in 2022, strongly validating the clinical feasibility of ASI targets. However, finding ASI molecules with novel structures, diverse sources, and better safety remains an important direction for drug development.

[0006] Cinnamaldehyde, chemically known as 3-phenyl-2-propenal, is the main bioactive component of cinnamon (Cinnamomum cassia), a traditional Chinese medicine, and the source of its characteristic flavor. Since ancient times, cinnamon has been used in traditional Chinese medicine to "warm and unblock the meridians, dispel cold and relieve pain," often treating circulatory disorders related to "cold" syndromes, which to some extent relates to certain clinical manifestations of modern cardiovascular and cerebrovascular diseases. Modern pharmacological research has preliminarily revealed the broad bioactivity of cinnamaldehyde. Numerous in vitro and animal model studies have shown that cinnamaldehyde exhibits multiple pharmacological effects, including lowering blood pressure, anti-inflammation, anti-oxidation, improving insulin resistance, and vasodilation. This suggests potential value of cinnamaldehyde in treating metabolic syndrome and related cardiovascular diseases. However, despite these advances, the underlying molecular mechanisms by which cinnamaldehyde exerts its core pharmacological effects, especially whether it acts on a key upstream pathophysiological target, remains unclear. Existing research largely focuses on its downstream effects or relatively generalized signaling pathways, lacking precise identification of a key molecular target that can integrate its pleiotropic effects. This mechanistic ambiguity significantly limits the progress of developing cinnamaldehyde into a modern precision medicine. It is noteworthy that among the various known pharmacological effects of cinnamaldehyde, its benefits partially overlap with those of an important class of clinical drugs—mineralocorticoid receptor antagonists (such as spironolactone), including antihypertensive, anti-fibrotic, and endothelial function improvement. This suggests that the effects of cinnamaldehyde may also be related to key aspects of the renin-angiotensin-aldosterone system. Aldosterone synthase is the final regulatory node of the RAAS system and the only rate-limiting enzyme in aldosterone biosynthesis, making it a cutting-edge and high-value target in current cardiovascular drug development. However, to date, no existing technology or literature has publicly disclosed or suggested that cinnamaldehyde can directly act on aldosterone synthase as its inhibitor. Summary of the Invention

[0007] This invention is the first to propose and confirm that cinnamaldehyde is a natural aldosterone synthase inhibitor, and proposes a new use for "cinnamaldehyde in the preparation of aldosterone synthase inhibitors." This not only provides a completely new, upstream, and precise explanation of the mechanism of action for elucidating the traditional efficacy of cinnamaldehyde and cinnamon—its cardiovascular protective effect is likely partly due to the source inhibition of aldosterone synthesis—but also lays a solid foundation for its development into an innovative drug for treating aldosterone-driven diseases such as primary aldosteronism, refractory hypertension, and heart failure, and has significant scientific significance and clinical application prospects. Furthermore, it provides modern scientific evidence for elucidating the traditional efficacy of cinnamon, possessing significant originality and broad development potential.

[0008] This invention develops a drug using cinnamaldehyde as an aldosterone synthase inhibitor, which is indicated for the treatment of the following aldosterone-related diseases:

[0009] Refractory hypertension

[0010] Primary aldosteronism

[0011] - Chronic heart failure

[0012] Chronic kidney disease

[0013] Refractory hypertension refers to a condition in which a patient's blood pressure remains above the target value (usually ≥140 / 90 mmHg) even after correctly using ≥3 different types of antihypertensive drugs (including diuretics) at the maximum or optimal dose.

[0014] Primary aldosteronism refers to the excessive secretion of aldosterone by the adrenal cortex, which leads to sodium retention, potassium excretion, increased blood volume, and inhibition of the renin-angiotensin system. The main clinical manifestations are hypertension accompanied by hypokalemia.

[0015] Heart failure refers to a pathological state in which the heart's pumping function gradually decreases on the basis of pre-existing chronic heart disease. Even with sufficient venous return, the heart's stroke volume is still insufficient to meet the body's metabolic needs, or it relies on increased filling pressure to compensate. Chronic heart failure is more common in middle-aged and elderly people, as well as patients with hypertension and coronary heart disease.

[0016] Chronic kidney disease is a disease that continuously impairs kidney function. It is usually caused by a variety of factors, most commonly diabetes and hypertension.

[0017] The diagnostic criteria for hypertension are: if blood pressure is measured three times on different days without the use of antihypertensive drugs, and the systolic blood pressure is ≥140 mmHg and / or the diastolic blood pressure is ≥90 mmHg, then hypertension can be diagnosed.

[0018] Patients with hypertension, primary aldosteronism, chronic heart failure, and chronic kidney disease all experience excessive aldosterone levels (over-secretion), leading to corresponding clinical manifestations. Therefore, lowering aldosterone levels is necessary in treatment. Cinnamaldehyde, as an aldosterone synthase inhibitor, can be developed into a drug for precise treatment of the above diseases, that is, it can precisely lower aldosterone levels.

[0019] Cinnamaldehyde can be used as an active ingredient in drugs for treating any of the following: hypertension, primary aldosteronism, chronic heart failure, and chronic kidney disease. It is also applicable when hypertension is refractory. When used as an active ingredient, the proportion of cinnamaldehyde in the drug can be adjusted within the range of 1%-100% (by mass). This invention demonstrates the inhibitory effect of cinnamaldehyde on aldosterone synthase, thus allowing the mass content of cinnamaldehyde in the active ingredient to exceed 50%, or to be 100% by mass. The drug can be dissolved in a solvent, and the concentration of cinnamaldehyde in the resulting solution is preferably 5-20 nmol / L.

[0020] Cinnamaldehyde's advantages as an aldosterone synthase inhibitor are:

[0021] First, source regulation: directly inhibiting the production of aldosterone, which theoretically can prevent "aldosterone escape" and produce synergistic or substitution effects with MRA, unlike traditional receptor antagonists.

[0022] Secondly, the potential for multiple effects: the anti-inflammatory and antioxidant effects of cinnamaldehyde itself can synergize with the aldosterone-inhibiting effect to more comprehensively combat aldosterone-mediated target organ damage and achieve "multi-target" intervention for cardiovascular metabolic diseases.

[0023] Through dual verification using in vitro cell experiments and in vivo animal experiments, cinnamaldehyde has been demonstrated to achieve the following therapeutic effects as an aldosterone synthase inhibitor:

[0024] 1. Significantly reduces aldosterone: Experiments in SHR rats and NCI-H295R cells showed that cinnamaldehyde significantly inhibited aldosterone biosynthesis.

[0025] 2. Significant blood pressure lowering effect: Experiments on SHR rats have confirmed that cinnamaldehyde has a certain blood pressure lowering effect.

[0026] 3. Strong targeting: Experiments in SHR rats and NCI-H295R cells showed that cinnamaldehyde can inhibit the expression of CYP11B2 gene and protein.

[0027] At the same time, it has a natural source and high safety profile: As a natural product, cinnamaldehyde has a known history of use in food and medicine, and its safety profile may be superior to that of newly chemically synthesized drug molecules, giving it a unique safety advantage in developing into functional foods or drugs.

[0028] 1. Fewer side effects: Avoids the anti-androgen side effects of receptor antagonists;

[0029] 2. Low toxicity: Cinnamaldehyde is widely used as a food additive and has good safety profile;

[0030] 3. Low risk of hyperkalemia: theoretically safer than receptor antagonists;

[0031] 4. Long-term experience: Cinnamon has been used as a traditional Chinese medicine for thousands of years.

[0032] Finally, the advantages of using cinnamaldehyde as an aldosterone synthase inhibitor: This drug offers the following advantages when used to treat related indications:

[0033] 1. Wide range of sources: Cinnamaldehyde can be extracted from cinnamon or chemically synthesized;

[0034] 2. Low cost: Cinnamaldehyde is easy to extract, and there are mature extraction processes available now. The production cost is much lower than that of synthetic CYP11B2 inhibitors.

[0035] 3. Easy to prepare: It can be prepared into various dosage forms, such as powder, capsules, tablets, liquids, etc.

[0036] 4. Good patient compliance: Natural source, high patient acceptance. Attached Figure Description

[0037] Figure 1 Network pharmacology construction and analysis of cinnamaldehyde in the treatment of primary aldosteronism; A: Veen diagram showing potential targets of cinnamaldehyde in the treatment of primary aldosteronism, Drug: cinnamaldehyde; Disease: primary aldosteronism; B: GO analysis of candidate target genes.

[0038] Figure 2 Effects of cinnamaldehyde on SHR and WKY rats, where WKY and SHR represent the corresponding rats, and WKY+CA and SHR+CA represent rats that underwent cinnamaldehyde intervention; A shows the effect of cinnamaldehyde on blood pressure in SHR and WKY rats; B shows the effect of cinnamaldehyde on serum aldosterone levels in SHR and WKY rats; C shows the effect of cinnamaldehyde on CYP11B2 mRNA levels in SHR and WKY rats; D shows representative Western blotting images of the effect of cinnamaldehyde on CYP11B2 protein levels in SHR and WKY rats, where β-actin was used as the internal control protein; CA "-" indicates no intervention, and CA "+" indicates intervention; E shows the Western blotting quantitative analysis of the effect of cinnamaldehyde on CYP11B2 protein levels in SHR and WKY rats; F shows representative immunofluorescence images of the effect of cinnamaldehyde on CYP11B2 protein levels in SHR and WKY rats; G shows the immunofluorescence quantitative analysis of the effect of cinnamaldehyde on CYP11B2 protein levels in SHR and WKY rats.

[0039] Figure 3The images show the effects of cinnamaldehyde on NCI-H295R cells. A shows the effect of different concentrations of Ang II on aldosterone secretion in NCI-H295R cells; B shows the effect of different concentrations of cinnamaldehyde on aldosterone secretion in NCI-H295R cells; C shows the effect of different concentrations of cinnamaldehyde on CYP11B2 mRNA levels in NCI-H295R cells; D shows representative Western blotting images of the effect of different concentrations of cinnamaldehyde on CYP11B2 levels in NCI-H295R cells; E shows the quantitative Western blotting analysis of the effect of different concentrations of cinnamaldehyde on CYP11B2 levels in NCI-H295R cells; F shows representative immunofluorescence images of the effect of cinnamaldehyde on CYP11B2 levels in NCI-H295R cells; and G shows the quantitative immunofluorescence analysis of the effect of cinnamaldehyde on CYP11B2 levels in NCI-H295R cells. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below in conjunction with network pharmacology analysis and animal experiments.

[0041] I. Network Pharmacological Analysis

[0042] (1) Obtain the active pharmaceutical ingredient of cinnamaldehyde and its potential target.

[0043] First, using "cinnamaldehyde" as the keyword, the SDF file of cinnamaldehyde was downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov). Then, the active ingredient targets of cinnamaldehyde were predicted using the SwissTargetPrediction platform (http: / / www.swisstargetprediction.ch), with the screening criterion being NormFit > 0.8. The target names were converted into uniform gene names using the Uniprot database (https: / / www.uniprot.org), and duplicate values ​​were integrated and removed, ultimately yielding 29 drug component targets.

[0044] (2) Construction of targets for primary aldosteronism

[0045] Using "primary hyperaldosteronism" as the keyword, we collected disease targets for primary hyperaldosteronism through the GeneCards (https: / / www.genecards.org / ) database. GeneCards set a score ≥ 2, resulting in 880 potential targets for primary hyperaldosteronism.

[0046] (3) Drug-disease intersection targets

[0047] Based on the 29 active pharmaceutical ingredient targets and 880 potential targets (target genes) for primary aldosteronism screened in steps (1) and (2), the intersection targets of cinnamaldehyde for the treatment of primary aldosteronism were obtained using the Venny diagram online analysis software (https: / / bioinformatics.psb.ugent.be / webtools / Venn / ), which are 7 potential targets for the treatment of primary aldosteronism with cinnamaldehyde.

[0048] (4) GO enrichment analysis and KEGG pathway analysis

[0049] Seven overlapping targets were imported into the DAVID (https: / / david.ncifcrf.gov / home.jsp) database for Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

[0050] Analysis conclusion:

[0051] Twenty-nine drug targets for cinnamaldehyde were predicted using the SwissTargetPrediction platform. A total of 880 target genes for primary aldosteronism were collected from the GeneCards database. Seven overlapping targets for cinnamaldehyde in treating primary aldosteronism were identified using Venny graph online analysis software: F3, ADH1A, TLR4, NOS2, ADH4, CYP11B1, and CYP11B2. Figure 1 A), these were selected as candidate genes. Further analysis of the candidate genes' GO function and KEGG pathway revealed that they were mainly enriched in the biosynthesis of aldosterone and cortisol (A). Figure 1 (B) indicates that cinnamaldehyde has a significant impact on aldosterone synthesis. Literature review shows that both CYP11B1 and CYP11B2 are located on the long arm of chromosome 8, 8q21-22, and their amino acid sequences share 93% homology. However, the CYP11B1 gene is mainly involved in cortisol synthesis, while the CYP11B2 gene primarily encodes aldosterone synthase, possessing 11β-hydroxylase activity and participating in aldosterone synthesis. CYP11B2 is also the most important rate-limiting enzyme in aldosterone synthesis; therefore, cinnamaldehyde may have an inhibitory effect on aldosterone synthase.

[0052] II. Animal Experiments

[0053] 2.1 Reagents and Animal Sources

[0054] Drugs and main reagents involved in the examples: Cinnamaldehyde and angiotensin II were purchased from MedChemExpress (New Jersey, USA); SHR rats (spontaneously hypertensive rats with a 100% incidence of spontaneous hypertension) and WKY rats (normal blood pressure, used as a blood pressure control for SHR rats) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China); human adrenocortical adenocarcinoma cells (NCI-H295R) were purchased from Fenghui Biotechnology Co., Ltd. (Hunan, China); rat plasma aldosterone ELISA kit was purchased from Wuhan Elabscience Biotechnology Co., Ltd. (Hubei, China); CYP11B2 antibody and TRIzol Reagent kit were purchased from Thermo Fisher Scientific (Massachusetts, USA). All reagents or instruments without specified manufacturers are conventional products that can be obtained through commercial purchase.

[0055] Animal source: The experimental animals were clean-grade SHR and WKY rats, 8 weeks old, male, with a body weight of 180±10 g. Requirements for the feeding environment: Good ventilation and air filtration system, quiet environment, room temperature maintained at 20°C, humidity controlled at 40-50%, bedding and drinking water replaced daily, and free access to water and food. All rats were purchased from Huafukang Biotechnology Co., Ltd. (Beijing) (license number scxk(Beijing)2019-0008).

[0056] Intervention (CA) period: Dissolve cinnamaldehyde in corn oil and intragastrically administer it to rats once every morning at a dose of 100 mg / kg for one month. Observe the mental state, diet, water intake, and activity status of rats daily, and record the basic physiological parameters of rats weekly, including blood pressure, heart rate, body weight, etc.

[0057] Cell culture: Use DMEM / F12 (1:1) basal medium supplemented with 2.5% Nu-Serum, 1% ITS+ premix (insulin-transferrin-sodium selenite), and 1% penicillin-streptomycin double antibody. Routinely culture human adrenocortical adenocarcinoma cells (NCI-H295R) in a constant temperature incubator with saturated humidity at 37°C and 5% CO2, and passage them at a ratio of 1:3 every 2-3 days. Take cells in the logarithmic growth phase for experiments, and treat them with induction media containing different concentrations (20-500 nmol / L) of Ang II (angiotensin II) for 24 hours, and screen out the optimal intervention concentration of Ang II. All operations are carried out in a laminar flow hood, and mycoplasma detection is regularly performed to ensure that the cells are contamination-free.

[0058] 2.2 Test methods and procedures

[0059] Non-invasive tail blood pressure measurement, using a non-invasive tail artery manometer (BP-2010A): First, the rats were placed in a room at 22°C±2°C for acclimatization for 30 minutes. Then, the rats were placed on a temperature-controlled platform, and the tail bladder of the non-invasive tail artery manometer was inserted into the rat's tail. The tail artery was slowly heated to 39°C to fully dilate it. A four-channel signal acquisition device was connected, and after the computer-acquired signal stabilized, the device was inflated until the pulse signal became a straight line. Then, the pressure was gradually reduced, and blood pressure information was automatically acquired when a regular waveform signal appeared. Measurements were taken every 3 minutes for each rat, repeated 3 times, and the average value was taken. Blood pressure measurements for each group were taken at the same time each day.

[0060] Enzyme-linked immunosorbent assay (ELISA): After the intervention period, rats were fasted for 12 hours and anesthetized with isoflurane. Whole blood was collected via the abdominal aorta and centrifuged at 3000 rpm for 15 minutes at 4°C in pre-chilled EDTA anticoagulant tubes. The supernatant was separated to obtain plasma, which was stored at -80°C for testing. The rat aldosterone ELISA kit was strictly followed according to the instructions. Plasma samples and standards were added to pre-coated antibody-rich microwells. After incubation and washing, biotinylated detection antibodies were added, followed by horseradish peroxidase-labeled streptavidin. Finally, TMB substrate was added for color development, and the reaction was terminated with stop solution. The absorbance of each well was measured at 450 nm. The aldosterone concentration (pg / mL) of each sample was calculated using a standard curve. All samples were replicated to ensure accuracy.

[0061] Real-time quantitative PCR: Adrenal gland tissue was rapidly isolated from euthanized rats, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. Total RNA was extracted from the tissue using the TRIzol method, and RNA purity was detected using a NanoDrop micro-spectrophotometer (A260 / A280 ratios were all within the range of 1.8-2.0). 1 μg of total RNA was used for reverse transcription to synthesize cDNA according to the TRIzol Reagent kit instructions. Using the cDNA as a template, amplification was performed on an Applied Biosystems 7500 real-time quantitative PCR instrument using the SYBR Premix Ex Taq II kit. The reaction program included: 95°C pre-denaturation for 30 seconds; 95°C for 5 seconds, 60°C for 34 seconds, for a total of 40 cycles; finally, melting curve analysis was performed. The relative expression level of CYP11B2 mRNA was calculated using the 2^(-ΔΔCt) method. All samples were tested in triplicate to ensure the reliability of the experimental results.

[0062] Immunofluorescence: Select the desired sections by observation under a light microscope, place them in a 37°C oven for 1 hour, and rinse three times with PBS for 10 minutes each time. Adjust the water bath temperature to 95°C, place the immunohistochemistry rinsing box containing 1× sodium citrate antigen retrieval solution in the water bath, and place the rinsed sections in the heat retrieval solution. After 20 minutes, remove them and cool them at room temperature for about 1 hour. Rinse three more times with PBS for 10 minutes each time. Wipe off the surface moisture of the sections, draw circles around the tissue sample with an immunohistochemistry pen, and add 20 µL of permeabilizing blocking solution with a pipette. Block at room temperature for 1 hour. Aspirate the blocking solution around the sample, add primary antibody, and incubate overnight at 4°C. Rinse three times with PBS for 10 minutes each time; add the corresponding secondary antibody and incubate at room temperature in the dark for 2 hours. Add 30 µL of DAPI (4',6-diamidinyl-2-phenylindole) to the surface of each sample, and mount with a glass slide after 3-5 minutes. Images were observed and acquired under a laser confocal microscope, and the average fluorescence intensity was analyzed using ImageJ software for semi-quantitative comparison.

[0063] NCI-H295R cells were seeded at an appropriate density in confocal culture dishes. When the cell confluence reached 60%-70%, they were treated with 100 nmol / LAng II for 24 hours to induce CYP11B2 expression. The culture medium was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. The cells were then permeabilized with 0.5% Triton X-100 for 20 minutes, followed by blocking with 5% BSA at room temperature for 1 hour. Rabbit anti-human CYP11B2 primary antibody (1:200 dilution) was added, and the cells were incubated overnight at 4°C. After washing with PBS, goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 (1:500 dilution) was added, and the cells were incubated at room temperature in the dark for 1 hour. Finally, the nuclei were stained with DAPI for 10 minutes, mounted with anti-fluorescence quenching mounting medium, and observed and images were acquired under a laser confocal microscope. The average fluorescence intensity was analyzed using ImageJ software for semi-quantitative comparison.

[0064] Protein expression detection: Weigh 0.1g of the corresponding tissue, add cell lysis buffer and homogenize thoroughly. Sonicate for 10s, then lyse on ice for 1h. Centrifuge at 12000rpm / min, 4℃, for 30min. Add loading buffer to the sample, boil for 5min to denature, cool, and store at -80℃ for later use. Prepare an 8% gel according to the protein molecular weight, and perform electrophoresis at 120V for 90min. Then, perform electroporation and blocking sequentially, adding CYP11B2 primary antibody overnight. The next day, wash repeatedly with PBS, add fluorescent secondary antibody, incubate for 60min, wash again with PBS, and finally collect fluorescence signals for band analysis.

[0065] Statistical methods: The results of this experiment are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was performed using SPSS 22.0, and the Turkey t-test was used to compare differences between groups. A p-value < 0.05 was considered statistically significant.

[0066] The above methods were used to conduct experiments on rat and human adrenocortical adenocarcinoma cells (NCI-H295R), respectively, and the results were obtained. Figure 2 and Figure 3 The results are shown.

[0067] 2.3 Experimental Results

[0068] like Figure 2 As shown, SHR rats treated with cinnamaldehyde exhibited a significant decrease in blood pressure, indicating that cinnamaldehyde can significantly reduce the blood pressure of SHR rats. Conversely, WKY rats treated with cinnamaldehyde showed no significant change in blood pressure, indicating that cinnamaldehyde had no significant effect on the blood pressure of WKY rats. Figure 2 A); Plasma aldosterone levels in SHR rats after administration of cinnamaldehyde ( Figure 2 B) Adrenal CYP11B2 protein expression ( Figure 2 DG) and adrenal CYP11B2 mRNA ( Figure 2 C) The levels of cinnamaldehyde were significantly reduced, with no significant effect on WKY rats, indicating that cinnamaldehyde can reduce aldosterone biosynthesis through CYP11B2. Furthermore, cinnamaldehyde was observed to have no significant effect on heart rate and body weight in SHR and WKY rats, indicating that cinnamaldehyde is safe to use.

[0069] like Figure 3 As shown, the effects of different concentrations of Ang II (20, 50, 100, 200, 500 nmol / L) on aldosterone secretion in NCI-H295R cells were first observed. It was found that 100 nmol / L of Ang II had a better stimulatory effect on aldosterone secretion in NCI-H295R cells. Figure 3 A). After stimulation with 100 nmol / L Ang II, the effects of different concentrations of cinnamaldehyde (0, 1, 2, 5, 10, 20 μmol / L) on aldosterone secretion in NCI-H295R cells were further observed. It was found that cinnamaldehyde could reduce aldosterone secretion in a dose-dependent manner, with 10 nmol / L cinnamaldehyde showing a better inhibitory effect on aldosterone secretion in NCI-H295R cells. Figure 3B). The effects of different concentrations of cinnamaldehyde on the expression of CYP11B2 mRNA and protein in NCI-H295R cells were observed. It was found that cinnamaldehyde could reduce CYP11B2 gene and protein expression in a dose-dependent manner, and 10 nmol / L cinnamaldehyde had a good inhibitory effect on the expression of CYP11B2 gene and protein in NCI-H295R cells. Figure 3 CG).

[0070] The above conclusions indicate that cinnamaldehyde has an inhibitory effect on aldosterone synthase and aldosterone biosynthesis.

[0071] Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that many other modifications and implementations can be devised by those skilled in the art, which will fall within the scope and spirit of the principles disclosed herein.

Claims

1. The use of cinnamaldehyde in the preparation of aldosterone synthase inhibitors, characterized in that, Cinnamaldehyde is an inhibitor of aldosterone synthase.

2. The use of cinnamaldehyde according to claim 1 in the preparation of aldosterone synthase inhibitors, characterized in that, It is used when an excessive secretion of aldosterone causes disease.

3. The use of cinnamaldehyde according to claim 1 in the preparation of aldosterone synthase inhibitors, characterized in that, The aldosterone synthase inhibitor is the active ingredient in a drug for treating any one of hypertension, primary aldosteronism, chronic heart failure, or chronic kidney disease.

4. The use of cinnamaldehyde according to claim 3 in the preparation of aldosterone synthase inhibitors, characterized in that, The hypertension mentioned is refractory hypertension.

5. The use of cinnamaldehyde in the preparation of aldosterone synthase inhibitors, characterized in that, Cinnamaldehyde is an active ingredient in drugs used to treat any one of hypertension, primary aldosteronism, chronic heart failure, and chronic kidney disease, wherein the mass content of cinnamaldehyde in the active ingredient exceeds 50%.

6. The use of cinnamaldehyde according to claim 5 in the preparation of aldosterone synthase inhibitors, characterized in that, The active ingredient contains 100% cinnamaldehyde by mass.

7. The use of cinnamaldehyde according to claim 5 or 6 in the preparation of aldosterone synthase inhibitors, characterized in that, The drug is dissolved in a solvent, and the concentration of cinnamaldehyde in the resulting solution is 5~20 nmol / L.

8. The use of cinnamaldehyde according to claim 5 or 6 in the preparation of aldosterone synthase inhibitors, characterized in that, The drug is dissolved in a solvent, and the concentration of cinnamaldehyde in the resulting solution is 10 nmol / L.

Images