Application of fisetin in preparation of products for preventing or treating hypertension

Abstract

The invention discloses a new application of fisetin and an application of a composition containing the fisetin. The new application comprises preparation of a medicine for treating hypertension, preparation of functional food or health care products for improving the blood pressure level, preparation of a medicine for reducing blood pressure, preparation of a medicine for inducing diuresis and discharging sodium and preparation of a preparation for reducing the Sik1 level. Experiments find that fisetin intervention can reduce the blood pressure of spontaneously hypertensive rats for the first time. The fisetin can increase the GSH-Px activity of the kidney, reduce the ROS level, improve the urinary sodium retention state by reducing the expression level of kidney Sik1 and further reduce the hypertension blood pressure level, and it is indicated that fisetin has the effect of preventing and / or treating primary hypertension and / or spontaneous hypertension. The invention provides application of fisetin in reducing hypertension, and provides a new thought for preventing and treating primary hypertension and / or spontaneous hypertension by reducing blood pressure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the use of fisetin in the preparation of products for preventing or treating hypertension. Background Art

[0002] Cardiovascular diseases cause more than 18 million deaths every year, accounting for about one-third of the global death toll. Hypertension is a major risk factor for cardiovascular diseases such as coronary heart disease, stroke, and heart failure. The total hospitalization cost mainly diagnosed with hypertension in China in 2022 was approximately 9.3 billion yuan. Controlling hypertension is crucial for improving the quality of life of residents and reducing the economic burden. Therefore, it is of great significance to deeply explore new strategies for safe and targeted nutritional prevention and treatment of hypertension. The pathological mechanism of hypertension involves multiple links such as abnormal activation of the sympathetic nervous system, oxidative stress, vascular endothelial dysfunction, and chronic inflammatory response. In this context, nutritional therapy has gradually become a research hotspot in hypertension management due to its high safety and characteristics of targeted regulation of metabolic disorders. Research shows that flavonoids, as natural plant active ingredients, can play antihypertensive and organ-protective roles through the following mechanisms: antioxidant and anti-inflammatory effects, improvement of endothelial function, regulation of the renin-angiotensin system (RAS), and inhibition of vascular remodeling. In addition, flavonoids can also achieve multiple protection of hypertension target organs by reducing renal oxidative damage and inhibiting myocardial fibrosis.

[0003] Fisetin (3,3',4',7-tetrahydroxyflavone) is a natural flavonol present in various fruits and vegetables such as strawberries and apples. In recent years, studies have found that fisetin shows significant potential in delaying the progression of age-related chronic diseases (including cardiovascular diseases). As an effective senolytic, fisetin can selectively eliminate senescent cells compared with other flavonoids such as curcumin and quercetin, and has a stronger anti-aging effect. In addition, fisetin is also a high vitamin E equivalent (Trolox) antioxidant, and its antioxidant ability is superior to other subclasses such as quercetin. In the prevention and treatment of cardiovascular diseases, fisetin shows a positive effect in delaying diseases such as atherosclerosis. Fisetin can promote intestinal cholesterol excretion and reduce the incidence risk of atherosclerosis by activating PPARδ to up-regulate the expression of cholesterol efflux transporters (ABCG5 / G8 and ABCB1). In addition, supplementing fisetin can significantly improve the arterial elasticity (pulse wave velocity decreased by 20%) and endothelial function (endothelium-dependent dilation increased by 16%) of elderly mice, and reduce the high DNA methylation in myocardial ischemia-reperfusion injury to maintain normal mitochondrial function to provide cardiac protection.

[0004] Although existing studies have confirmed the important role of fisetin in the prevention and treatment of cardiovascular diseases, whether it has a protective effect on hypertension and its underlying mechanisms are still unclear. Therefore, exploring the role of fisetin in the prevention and treatment of hypertension has important scientific significance and clinical application value. Summary of the Invention

[0005] Based on the above analysis, the present invention discovers that the flavonol compound fisetin has a certain effect on blood pressure control and has certain potential or broad application prospects in the development of functional food active ingredients or drugs for the prevention and / or treatment of hypertension. The present invention is achieved by the following technical means:

[0006] The present invention first discloses new applications of fisetin, including:

[0007] (1) Preparing a drug for treating hypertension;

[0008] (2) Preparing a functional food or health product for improving blood pressure level;

[0009] (3) Preparing a drug for reducing blood pressure;

[0010] (4) Preparing a drug for diuresis and sodium excretion;

[0011] (5) Preparing a preparation for reducing the level of Sik1.

[0012] The present invention also discloses applications of a composition containing fisetin, including:

[0013] (1) Preparing a drug for treating hypertension;

[0014] (2) Preparing a functional food or health product for improving blood pressure level;

[0015] (3) Preparing a drug for reducing blood pressure;

[0016] (4) Preparing a drug for diuresis and sodium excretion;

[0017] (5) Preparing a preparation for reducing the level of Sik1.

[0018] Furthermore, in the above applications, the composition containing fisetin includes: a functional food composition, a health product composition, and a drug composition.

[0019] Furthermore, the functional food composition, the health product composition, and the drug composition include pharmaceutically or foodologically acceptable excipients / adjuvant components or carriers / excipients acceptable in the process of drug preparation.

[0020] Furthermore, in the above applications, the hypertension includes: spontaneous hypertension and essential hypertension.

[0021] Furthermore, in the above application, the drug is a pharmaceutical product, and its dosage forms include: tablets, capsules, granules, pills, ointments, solutions, suspensions or tube feeding preparations.

[0022] Furthermore, in the above application, the health care product is a health food.

[0023] Furthermore, in any of the above applications, the drug, functional food or health care product contains an effective amount of fisetin.

[0024] Furthermore, in any of the above applications, the test subject model of the drug, functional food or health care product is a spontaneously hypertensive rat.

[0025] Furthermore, the administration route of the test subject model is gavage, with a frequency of once a day, continuous gavage for 4 weeks, and the gavage dose is 50 mg / kg.

[0026] Furthermore, in any of the above applications, the blood pressure reduction is to reduce the systolic blood pressure and / or diastolic blood pressure.

[0027] Furthermore, in any of the above applications, the diuretic and natriuretic preparation is to increase the 24-hour urine volume, urinary sodium excretion and fractional sodium excretion.

[0028] Furthermore, in any of the above applications, the reduction of Sik1 level includes: reducing the Sik1 gene transcription level and reducing the Sik1 protein level.

[0029] The beneficial effects of the present invention are as follows:

[0030] 1. The present invention clearly demonstrates that fisetin has a preventive and / or therapeutic effect on hypertension, can reduce the blood pressure level of primary hypertension, including systolic blood pressure and diastolic blood pressure, and has a significant control effect on hypertension.

[0031] 2. Fisetin is applied in functional foods or drugs for the prevention and / or treatment of primary hypertension. It can improve the activity of antioxidant enzymes to reduce the content of reactive oxygen species (ROS) in the kidney. By reducing the expression level of Sik1 in the kidney, it further promotes the excretion of urinary sodium, and this mechanism plays a key role in reducing blood pressure, protecting against hypertension and improving the renal urinary sodium excretion function.

[0032] 3. The present invention for the first time discovers through experiments that fisetin intervention can reduce the blood pressure of spontaneously hypertensive rats. It can increase the activity of GSH-Px in the kidneys, reduce the ROS level, improve the state of urinary sodium retention by reducing the expression level of Sik1 in the kidneys, and further reduce the blood pressure level of hypertension, indicating that fisetin has the effect of preventing and / or treating essential hypertension. The present invention provides the application of fisetin in reducing hypertension, providing a new idea for the prevention and treatment of essential hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Systolic and diastolic blood pressure levels of spontaneously hypertensive rats in the control group, the 25 mg / kg fisetin treatment group, and the 50 mg / kg fisetin treatment group;

[0034] Figure 2 : Effects of 50 mg / kg fisetin treatment on 24-hour urine volume, urinary sodium excretion, and fractional sodium excretion in spontaneously hypertensive rats;

[0035] Figure 3 : Heat map and volcano plot of renal cortex transcriptome of spontaneously hypertensive rats in the control group and treated with 50 mg / kg fisetin;

[0036] Figure 4 : Effects of 50 mg / kg fisetin treatment on the mRNA and protein expression of Sik1 in the renal cortex of spontaneously hypertensive rats;

[0037] Figure 5 : Effects of 50 mg / kg fisetin treatment on the activity of GSH-Px in the renal cortex of spontaneously hypertensive rats;

[0038] Figure 6 : Effects of 50 mg / kg fisetin treatment on the ROS content in the kidneys of spontaneously hypertensive rats. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides a drug or functional food additive ingredient for preventing and treating hypertension. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. In the following examples, for the experimental operations where specific conditions are not particularly specified, generally follow the conventional conditions commonly used in the art, or carry out according to the conditions recommended by relevant product manufacturers. The raw materials, reagents, etc. used in the experiments, unless otherwise specified, are all common products that can be obtained through conventional markets or other commercial channels. Any non-substantial modifications or substitutions made by those skilled in the art based on the present invention shall be considered within the protection scope of the present invention.

[0040] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0041] Fisetin, whose chemical formula is C 15H 10 O6, a natural flavonol, was purchased from MedChemExpress, product number: HY-N0182.

[0042] Combined with the examples, the implementation process of the present invention will be specifically described below:

[0043] Example 1

[0044] 1.1 Materials:

[0045] 1.1.1 Experimental animals: Male spontaneously hypertensive rats (SHR), SPF level, 8 weeks old, weighing 219.5 ± 6.1 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and adaptively fed in an SPF-level laboratory for one week. Experimental animal production license number: SCXK(Beijing)2021-0006; Experimental animal use license number: SYXK(Chongqing)2022-0016.

[0046] 1.1.2 Main reagents: Sodium determination kit, creatinine determination kit, glutathione peroxidase determination kit, purchased from Nanjing Jiancheng Bioengineering Institute; Sik1 primers were purchased from Shanghai Sangon Biotech Co., Ltd.; Sik1 antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd.; BCA protein content detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd.; Superoxide anion reactive oxygen species detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0047] Instruments and consumables: Non-invasive rat tail blood pressure monitor, Beijing Zhongshi Technology Co., Ltd.; Electronic analytical balance, Shanghai Jingtian Electronic Instrument Co., Ltd.; Low-temperature high-speed centrifuge, Eppendorf, Germany; Microplate reader, Molecular Devices, USA; Vortex oscillator, Scientific Industries, USA; 0.22um filter, Millipore, USA; Disposable sterile syringe (1ml), Shandong Weigao Group Co., Ltd.

[0048] Transcriptome sequencing analysis was completed by Shanghai NeoBiotech Co., Ltd.

[0049] Preparation of fisetin working solution: Weigh fisetin powder and sequentially add 45% PEG300, 5% Tween-80, and 50% normal saline, and dissolve it with ultrasonic assistance to prepare a working solution with a concentration of 10 mg / ml. Use it directly and prepare it freshly.

[0050] 1.2 Experimental methods

[0051] 1.2.1 Intervention and grouping of animal models

[0052] Fifteen male SHR rats were adaptively fed for 1 week. Before modeling, the tail artery blood pressure of rats in each group was measured using the ZS-Z-IV non-invasive blood pressure measurement and analysis system, repeated 3 times, and the average value was taken and recorded. Fisetin or the same volume of solvent was intragastrically administered to various rats at a fixed time period every day for 4 weeks. The specific random grouping information is as follows: control group (intragastric administration of solvent), experimental group 1 (intragastric administration of 25 mg / kg fisetin), experimental group 2 (intragastric administration of 50 mg / kg fisetin). The tail artery blood pressure levels of each rat in the waking state were monitored weekly through the non-invasive rat tail manometry system.

[0053] 1.2.2 Blood pressure measurement

[0054] Before intervention (baseline) and weekly after modeling, the systolic and diastolic blood pressures of the tail artery of rats in the waking state were measured using the non-invasive rat tail blood pressure measurement and analysis system, repeated 5 times, and the average value was taken.

[0055] 1.2.3 Detection of urinary sodium excretion function

[0056] Before intervention (baseline) and at the 4th week of modeling, 24-hour urine volume was collected and recorded using a metabolic cage, and urinary sodium excretion and sodium excretion fraction were measured and calculated using sodium and creatinine assay kits. The 24-hour urine was centrifuged at 4000 r / min for 5 min to obtain the supernatant, which was stored in a refrigerator at 4°C for later use. The blood was centrifuged at 4000 r / min for 5 min to obtain the serum, which was stored in a refrigerator at -20°C for later use.

[0057] (1) The urine sodium content was measured by colorimetry, and the experimental steps were carried out according to the instructions of the sodium detection kit.

[0058] (2) The urine and serum creatinine contents were measured by the sarcosine oxidase method, and the experimental steps were carried out according to the instructions of the creatinine detection kit.

[0059] 1.2.4 Renal cortex transcriptome sequencing analysis

[0060] The renal cortex of rats in the control group and experimental group 2 (50 mg / kg fisetin) at the 4th week of modeling was taken, and eukaryotic reference transcriptome sequencing (T7, 6G) was performed by Shanghai Zhongke New Life Company, and the results were statistically analyzed through the company's relevant platform. The results were further analyzed by bioinformatics using R language 4.2.1 to draw heat maps and volcano plots.

[0061] 1.2.5 Fluorescent quantitative PCR (qPCR)

[0062] First, the renal cortex was ground at 4°C, and then RNA was extracted using the Trizol method and its concentration was measured. Subsequently, the RNA was converted into a cDNA template using the TAKARA reverse transcription kit according to the operating steps. Finally, a fluorescence quantitative PCR experiment was performed using the TAKARA qPCR kit to obtain the CT value of the target gene. The relative expression fold of the target gene was calculated by the ΔΔCT method.

[0063] 1.2.6 Immunoblotting (Western blotting)

[0064] After grinding the renal cortex at 4°C, the tissue protein was extracted after lysing with RIPA lysis buffer. After detecting the protein concentration, 5×loading buffer was added at a ratio of 4:1, and it was denatured at 100°C for a high temperature. Then, protein gel electrophoresis was performed using 10% SDS-PAGE and transferred to an NC membrane. After blocking with 5% skim milk, it was incubated with the primary antibody at 4°C overnight, and then incubated with the secondary antibody again. Finally, detection was carried out using a chemiluminescence instrument.

[0065] 1.2.7 Detection of glutathione peroxidase (GSH-Px) in the renal cortex

[0066] The activity of GSH-Px in fresh renal cortex was determined by colorimetry, and the experimental steps were carried out according to the instructions of the glutathione peroxidase detection kit.

[0067] 1.2.8 Detection of superoxide anion reactive oxygen species in the renal cortex

[0068] Four weeks after rat model establishment, the renal tissue was fixed and dehydrated overnight in sucrose and 4% paraformaldehyde. After embedding with OCT, frozen sections with a thickness of 4 μm were made. The sections were incubated with a 10 μM concentration of ROS fluorescent probe - dihydroethidium (DHE) for 30 minutes at 37°C in the dark, and after washing 3 times with PBS, fluorescence microscopy was performed.

[0069] 1.3 Experimental results

[0070] 1.3.1 The results are as Figure 1 shown: At the baseline state, the blood pressure values of the control group, experimental group 1, and experimental group 2 were similar and all higher than the normal blood pressure value. When fisetin was intervened for 4 weeks, the systolic and diastolic blood pressure levels of the rats in experimental group 2 (50 mg / kg fisetin) were significantly decreased (P < 0.01). The experimental results show that continuous intervention with 50 mg / kg fisetin for 4 weeks can reduce the systolic and diastolic blood pressure levels of spontaneously hypertensive rats and has a good blood pressure-lowering effect on essential hypertension.

[0071] 1.3.2 The results are as Figure 2Shown as follows: At the baseline state, the 24-hour urine volume, urinary sodium excretion, and fractional sodium excretion in the control group, experimental group 1, and experimental group 2 were similar. When fisetin was intervened for 4 weeks, the 24-hour urine volume, urinary sodium excretion, and fractional sodium excretion in the rats of experimental group 2 (50 mg / kg fisetin) increased significantly (P < 0.01). The experimental results showed that the 50 mg / kg fisetin intervention for 4 weeks could improve the state of urinary sodium retention in spontaneous rats and had a natriuretic effect on essential hypertension.

[0072] 1.3.3 Results are as Figure 3 Shown as follows: After 4 weeks of fisetin intervention in the control group and experimental group 2, there were differential changes in 360 transcriptional genes in the renal cortex. Among the down-regulated differential genes, the gene Sik1 related to the regulation of urinary sodium excretion ranked tenth and was among the top of the differential genes. The experimental results showed that the improvement of urinary sodium excretion function in spontaneous rats by fisetin intervention might be related to the down-regulation of renal Sik1 expression.

[0073] 1.3.4 Results are as Figure 4 Shown as follows: After 4 weeks of fisetin intervention in the control group and experimental group 2, the expression of Sik1 mRNA in the renal cortex of the rats in experimental group 2 was significantly lower than that in the control group (P < 0.01). Results are as Figure 4 Shown as follows: After 4 weeks of fisetin intervention in the control group and experimental group 2, the expression of Sik1 protein in the renal cortex of the rats in experimental group 2 was significantly lower than that in the control group.

[0074] 1.3.5 Results are as Figure 5 Shown as follows: After 4 weeks of fisetin intervention in the control group and experimental group 2, the activity of GSH-Px in the renal cortex of the rats in experimental group 2 was significantly increased compared with that in the control group (P < 0.05).

[0075] 1.3.6 Results are as Figure 6 Shown as follows: After 4 weeks of fisetin intervention in the control group and experimental group 2, the content of ROS in the kidneys of the rats in experimental group 2 was significantly reduced compared with the control.

[0076] 1.4 Conclusion

[0077] The experimental results of the present invention showed that fisetin had the effect of reducing blood pressure in spontaneous hypertensive rats, could increase the activity of GSH-Px in the kidney tissue, reduce the content of ROS, and reduce the transcriptional and protein expression levels of Sik1 to improve the urinary sodium excretion function and reduce blood pressure, clarifying the beneficial effect of fisetin on essential hypertension blood pressure.

[0078] Spontaneously hypertensive rats (SHR) are the most widely used animal models for studying essential hypertension. Similar to the clinical characteristics of essential hypertension, the development of hypertension in SHR rats begins with a mild increase in blood pressure, followed by a progressive stage of hypertension, and finally a stage of persistent hypertension, simulating the chronic development process of human essential hypertension. The response of SHR rats to commonly used antihypertensive drugs (such as ACE inhibitors) is basically the same as that of human patients, making them the main animal models for antihypertensive drug screening. Therefore, the fisetin protected by the present invention for blood pressure control includes both essential hypertension and spontaneous hypertension.

[0079] The preferred embodiments of the present invention have been elaborated in detail above. However, the protection scope of the present invention is not limited to these specific implementation details. Any equivalent replacement or modification of the technical solutions within the framework of the core technical concept of the present invention should be included in the protection scope of the present invention. In addition, it should be noted that the various specific technical features mentioned in the above embodiments can be combined in any suitable way on the premise that they do not conflict with each other. Given the diversity of combination methods, to avoid redundancy, the present invention does not explain each possible combination case one by one. Further, different embodiments of the present invention can also be combined with each other, as long as such combination does not violate the basic idea of the present invention, it should be regarded as the content covered by the present invention.

Claims

1. A new application of fisetin, including: (1) Preparing a drug for treating hypertension; (2) Preparing a functional food or health product for improving blood pressure level; (3) Preparing a drug for reducing blood pressure; (4) Preparing a drug for diuresis and sodium excretion; (5) Preparing a preparation for reducing the level of Sik1.

2. An application of a composition containing fisetin, including: (1) Preparing a drug for treating hypertension; (2) Preparing a functional food or health product for improving blood pressure level; (3) Preparing a drug for reducing blood pressure; (4) Preparing a drug for diuresis and sodium excretion; (5) Preparing a preparation for reducing the level of Sik1.

3. The application according to claim 2, wherein: The composition containing fisetin includes: a functional food composition, a health product composition, and a drug composition.

4. The application according to claim 1 or 2, wherein: The hypertension includes: spontaneous hypertension and essential hypertension.

5. The application according to claim 1 or 2, wherein: The drug is a medicine, and its dosage forms include: tablets, capsules, granules, pills, ointments, solutions, suspensions, or tube feeding preparations.

6. The application according to claim 1 or 2, wherein: The health product is a health food.

7. The application according to claim 1 or 2, wherein: The drug, functional food, or health product contains an effective amount of fisetin.

8. The application according to claim 1 or 2, wherein: The test animal model of the drug, functional food, or health product is a spontaneously hypertensive rat.

9. The application according to claim 8, wherein: The administration route of the test animal model is gavage, with a frequency of 1 time / d, continuous gavage for 4 weeks, and a gavage dose of 50 mg / kg.

10. The application according to claim 1 or 2, wherein: Reducing blood pressure means reducing systolic blood pressure and / or diastolic blood pressure; The diuresis and sodium excretion preparation means increasing the 24-hour urine volume, urinary sodium excretion, and sodium excretion fraction; Reducing the level of Sik1 includes: reducing the transcriptional level of the Sik1 gene and reducing the protein level of Sik1.

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