3 apos; , 4apos; use of-dihydroxyflavonols in the treatment of hypertension

Abstract

The invention belongs to the technical field of medicines, and mainly relates to application of 3 ', 4'-dihydroxy flavonol in treatment of hypertension. The invention firstly provides application of 3 ', 4'-dihydroxy flavonol or pharmaceutically acceptable salt thereof in preparation of products for preventing and / or treating hypertension. The invention also provides an application of the 3 ', 4'-dihydroxy flavonol or the pharmaceutically acceptable salt thereof in preparation of a product, and the product is used for reducing blood pressure; the blood vessel function reduction is improved; the blood vessel wall thickening is improved; the heart function reduction is improved; the 3 ', 4'-dihydroxy flavonol can improve any one or more of myocardial cell hypertrophy, and the invention further provides a product which comprises an effective dose of 3 ', 4'-dihydroxy flavonol or pharmaceutically acceptable salt thereof. The application finds that the 3 ', 4'-dihydroxy flavonol and the pharmaceutically acceptable salt thereof can be effectively used for preventing or treating hypertension, and a new way is opened up for the treatment of hypertension or hypertension-related diseases.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and mainly relates to the use of 3′,4′-dihydroxyflavonol in the treatment of hypertension. Background Art

[0002] Hypertension is characterized by a sustained increase in the diastolic and (or) compression phases of the systemic arteries. As the most common and most important risk factor for cardiovascular and cerebrovascular diseases, it has brought a heavy health economic burden to society. The incidence of hypertension is positively correlated with the incidence and mortality of cardiovascular diseases, and cardiovascular diseases have become the leading cause of death in the world. At present, there are many studies on its pathogenesis. It is generally believed that the onset of hypertension is mostly related to environmental and genetic factors. Pathological changes in the body such as vascular remodeling, endothelial cell dysfunction, sympathetic hyperactivity, activation of the renin-angiotensin-aldosterone system, and excessive sodium retention in the kidneys are all considered to be related to it. Hypertension disease is a cardiovascular syndrome with coexistence of risk factors, target organ damage, and clinical diseases, which is caused by the coexistence of multiple risk components. At present, the treatment of hypertension follows the principles of giving priority to long-acting preparations, gradually increasing the dose, combining drugs, and individualized treatment. However, most drugs have a ceiling effect, which eventually causes damage to target organs such as the heart, brain, and kidneys. Therefore, we urgently need to find antihypertensive drugs with better therapeutic effects.

[0003] Flavonols are a class of plant polyphenol compounds, known as flavonoids. They have a variety of biological effects, such as anti-thrombotic, anti-inflammatory, antioxidant and vasodilatory effects. 3′,4′-dihydroxyflavonol (Trihydroxyflavonol) is a synthetic flavonol. Studies have shown that the antioxidant capacity of 3′,4′-dihydroxyflavonol is significantly stronger than that of many natural flavonoids and flavonols. It can quickly maintain the activity of nitric oxide (NO) under the condition of elevated reactive oxygen species (ROS). It has been reported that 3′,4′-dihydroxyflavonol improves endothelial function in diabetic patients by reducing the production of Nox2-dependent superoxide and preventing eNOS uncoupling, thereby increasing NO activity. In addition, in rats and sheep and in the aorta of diabetic patients, 3′,4′-dihydroxyflavonol can effectively reduce cardiovascular function damage associated with oxidative stress after ischemia / reperfusion. However, there are no reports on the effects of 3′,4′-dihydroxyflavonol on cardiovascular diseases such as hypertension. Summary of the invention

[0004] In order to solve the defects of the above-mentioned prior art, the present application discovers for the first time that 3′,4′-dihydroxyflavonol or its pharmaceutically acceptable salt can be effectively used to prevent or treat hypertension, and at the same time has a good therapeutic effect on heart and vascular diseases caused by hypertension and has a high degree of safety.

[0005] Therefore, the first aspect of the present application provides the use of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating hypertension.

[0006] Preferably, the product has any one or more of the following effects:

[0007] 1) Lower blood pressure;

[0008] 2) Improve decreased vascular function;

[0009] 3) Improve blood vessel wall thickening;

[0010] 4) Improve reduced heart function;

[0011] 5) Improve cardiomyocyte hypertrophy;

[0012] 6) Prevent and / or treat hypertension.

[0013] The second aspect of the present application provides the use of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in preparing a product, wherein the product is used for any one or more of the following:

[0014] 1) Lower blood pressure;

[0015] 2) Improve decreased vascular function;

[0016] 3) Improve blood vessel wall thickening;

[0017] 4) Improve reduced heart function;

[0018] 5) Improve cardiomyocyte hypertrophy.

[0019] Preferably, the above-mentioned lowering of blood pressure includes lowering systolic blood pressure, and / or lowering diastolic blood pressure, and / or lowering mean arterial pressure.

[0020] Preferably, the above-mentioned improvement of vascular function includes reducing vascular pulse wave velocity, and / or reducing resistance index, and / or reducing pulsatility index.

[0021] Preferably, the above-mentioned improvement of vascular wall thickening includes reducing the thickness of the abdominal aorta vascular wall and / or reducing smooth muscle cell proliferation.

[0022] Preferably, the above-mentioned improvement of decreased cardiac function includes increasing cardiac ejection fraction and / or decreasing cardiac E / e' index.

[0023] Preferably, the improvement of myocardial cell hypertrophy includes aligning myocardial cells, and / or restoring the health of myocardial cells, and / or reducing the number of enlarged myocardial cells, and / or narrowing the intercellular space.

[0024] Preferably, the above-mentioned hypertension, and / or decreased vascular function, and / or thickening of vascular walls, and / or decreased cardiac function, and / or myocardial cell hypertrophy are caused by angiotensin II.

[0025] Preferably, the above product contains an effective dose of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof.

[0026] Preferably, the effective dosage of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the above product is greater than or equal to 10 mg / kg.

[0027] The third aspect of the present application provides a product comprising an effective dose of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof.

[0028] Preferably, the effective dosage of the 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg.

[0029] The beneficial effects of this application are:

[0030] The present application first discovered that 3′,4′-dihydroxyflavonol and its pharmaceutically acceptable salt exhibited excellent medical value. Only 10 mg / kg / d can be effectively used to prevent or treat hypertension. At the same time, it has a good therapeutic effect on heart and vascular diseases caused by hypertension, can improve reduced vascular function, improve blood vessel thickness, improve reduced heart function, improve cardiomyocyte hypertrophy, has a high degree of safety, and its therapeutic effect is better than the existing hypertension drug Valsartan, which opens up a new way for the treatment of hypertension or hypertension-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A statistical analysis chart showing the effect of 3′,4′-dihydroxyflavonol on systolic blood pressure (SBP) of mice after AngII modeling in this application.

[0032] Figure 2 A statistical analysis chart showing the effect of 3′,4′-dihydroxyflavonol on diastolic blood pressure (DBP) of mice after AngII modeling in this application.

[0033] Figure 3 The statistical analysis graph showing the effect of 3′,4′-dihydroxyflavonol on mean arterial pressure (MAP) of mice after AngII modeling in the present application is shown.

[0034] Figure 4 The statistical analysis graph showing the effect of 3′,4′-dihydroxyflavonol on the body weight of mice after AngII modeling in the present application.

[0035] Figure 5 Ultrasound diagram showing the effect of 3′,4′-dihydroxyflavonol on the blood flow in the abdominal aorta of mice after AngII modeling.

[0036] Figure 6 The statistical analysis graph of the effect of 3′,4′-dihydroxyflavonol on the pulse wave velocity of the abdominal aorta of mice after AngII modeling in this application is shown.

[0037] Figure 7 Ultrasound graph showing the effect of 3′,4′-dihydroxyflavonol on the resistance index and pulsatility index of the superior mesenteric artery of mice after AngII modeling.

[0038] Figure 8 The statistical analysis graph shows the effect of 3′,4′-dihydroxyflavonol on the superior mesenteric artery resistance index of mice after AngII modeling in the present application.

[0039] Fig. 9 The statistical analysis graph shows the effect of 3′,4′-dihydroxyflavonol on the pulsatility index of the superior mesenteric artery of mice after AngII modeling in the present application.

[0040] Fig.10 The image shows the pathological morphology of the abdominal aorta of mice after AngII modeling with 3′,4′-dihydroxyflavonol in this application.

[0041] Fig.11 Ultrasound images showing the effect of 3′,4′-dihydroxyflavonol on the cardiac function of mice after AngII modeling in the present application.

[0042] Fig.12 The statistical analysis graph shows the effect of 3′,4′-dihydroxyflavonol on the cardiac ejection fraction (EF) of mice after AngII modeling in the present application.

[0043] Fig.13 Ultrasound graph showing the effect of 3′,4′-dihydroxyflavonol in the present application on the early diastolic blood flow velocity (E) at the mitral valve tip and the early diastolic myocardial motion velocity (e′) at the mitral valve ring of the mouse heart after AngII modeling.

[0044] Fig.14 The statistical analysis graph shows the effect of 3′,4′-dihydroxyflavonol on the cardiac contraction and relaxation function (E / e′) of mice after AngII modeling in the present application.

[0045] Fig.15 A diagram showing the effect of 3′,4′-dihydroxyflavonol on the pathological morphology of cardiac tissue in mice after AngII modeling in this application. DETAILED DESCRIPTION

[0046] The following describes the implementation of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. The present application can also be implemented or applied through other different specific implementations, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application.

[0047] Before further describing the specific implementation methods of the present application, it should be understood that the scope of protection of the present application is not limited to the specific specific implementation methods described below; it should also be understood that the terms used in the examples of the present application are for describing specific specific implementation methods rather than for limiting the scope of protection of the present application; in the specification and claims of the present application, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0048] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present application, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present application, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present application can also be used to realize the present application.

[0049] The present application firstly discovered that 3′,4′-dihydroxyflavonol and its pharmaceutically acceptable salts can be used to prepare drugs for preventing and / or treating hypertension, as well as heart and vascular diseases caused by hypertension. The drugs have good therapeutic effects and are safe, thus opening up a new approach for the treatment of hypertension or hypertension-related diseases.

[0050] The 3′,4′-dihydroxyflavonol (Trihydroxyflavonol, DiOHF) is a flavonoid compound with three hydroxyl (-OH) groups, a flavonoid compound with specific biological activity, and has antiviral activity, such as inhibition of influenza A virus. It is a yellow crystal or powder, soluble in organic solvents such as ethanol and dimethyl sulfoxide (DMSO), but insoluble in water. The melting point of 3,4′-dihydroxyflavonol is 275-276°C, the boiling point is about 452.5±45.0°C, the density is about 1.472±0.06g / cm3, and the acidity coefficient (pKa) is about 8.94±0.20.

[0051] The molecular formula of 3′,4′-dihydroxyflavonol is C 15 H 10O5, molecular weight is 270.24, CAS number is 6068-78-6, and its structural formula is as follows:

[0052]

[0053] In the present application, the pharmaceutically acceptable salt refers to the salt of 3′,4′-dihydroxyflavonol, which is prepared from the 3′,4′-dihydroxyflavonol of the present application and a relatively non-toxic acid or base. When the 3′,4′-dihydroxyflavonol of the present application contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of the compound of the present application with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases, and the salts of the inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. The salts of the organic non-toxic bases include salts of primary amines, secondary amines and tertiary amines, including substituted amines and cyclic amines. For example: N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and the like.

[0054] The present application first provides the use of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in preparing a product for preventing and / or treating hypertension.

[0055] In this application, prevention refers to taking measures to prevent the occurrence of a disease or reduce the risk of a disease, which can be divided into primary prevention, which is taking measures before the disease occurs, with the purpose of preventing the occurrence of the disease; secondary prevention: the purpose is to detect and treat the disease in its early stages to prevent the development and deterioration of the disease; tertiary prevention is taking measures after the disease has occurred to prevent further deterioration of the disease or complications. Specifically refers to all actions to suppress symptoms or delay specific symptoms by administering the products described in this application.

[0056] In this application, the treatment refers to a series of positive effects after the disease has begun to develop. Specifically, it can slow down the progression of the disease so that the originally rapid development trend can be controlled; it can interrupt the process of continuous deterioration of the disease and prevent the disease from evolving further in a serious direction; effectively control the severity of the disease to prevent it from exceeding the range that the body can bear; stop the adverse development trend of the disease so that it no longer continues to deteriorate; alleviate the various uncomfortable symptoms caused by the disease and relieve the patient's pain; and even reverse a specific sign, symptom, disorder, disease or the direction or severity of the progression of the disease to a certain extent. However, it should be made clear that this treatment does not necessarily mean that it can involve the complete elimination of all signs, symptoms, diseases or disorders related to the disease, but improves the state of the disease and slows down the signs, symptoms, diseases or disorders related to the disease.

[0057] In a specific embodiment of the present application, the product has any one or more of the following effects:

[0058] 1) Lower blood pressure;

[0059] 2) Improve decreased vascular function;

[0060] 3) Improve blood vessel wall thickening;

[0061] 4) Improve reduced heart function;

[0062] 5) Improve cardiomyocyte hypertrophy;

[0063] 6) Prevent and / or treat hypertension.

[0064] The efficacy refers to the positive effect of the product drug in the organism.

[0065] The present application also provides the use of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the preparation of a product, wherein the product is used for any one or more of the following:

[0066] 1) Lower blood pressure;

[0067] 2) Improve decreased vascular function;

[0068] 3) Improve blood vessel wall thickening;

[0069] 4) Improve reduced heart function;

[0070] 5) Improve cardiomyocyte hypertrophy.

[0071] In a specific embodiment of the present application, the above-mentioned lowering of blood pressure includes but is not limited to lowering the systolic blood pressure (SBP) of the subject, and / or lowering the diastolic blood pressure (DBP) of the subject, and / or lowering the mean arterial pressure (MAP) of the subject.

[0072] In certain embodiments of the present application, the lowering of the systolic blood pressure of the subject, the lowering of the diastolic blood pressure of the subject, and the lowering of the mean arterial pressure of the subject means that when 3′,4′-dihydroxyflavonol is used to the subject, the SBP, DBP, and MAP of the subject are significantly decreased compared to the unused control group (#P<0.05).

[0073] In a specific embodiment of the present application, the above-mentioned improvement of reduced vascular function includes reducing vascular pulse wave velocity, and / or reducing resistance index, and / or reducing pulsatility index. Specifically, the reduced vascular function refers to an increase in vascular pulse wave velocity, resistance index or pulsatility index, and the improvement of vascular function refers to reducing vascular pulse wave velocity, resistance index or pulsatility index. For example, based on healthy blood vessels, the product can restore the pulse wave velocity, resistance index or pulsatility index of the blood vessels of the subject to at least 70%, 80%, 90% or 100% of that of healthy blood vessels. The pulse wave velocity of the abdominal aorta reflects the stiffness of the abdominal aorta. An increase in the pulse wave velocity of the abdominal aorta means an increase in vascular stiffness, which may be caused by diseases such as atherosclerosis, hypertension, diabetes, and is also related to aging of blood vessels. The resistance index and pulsatility index of the superior mesenteric artery are important indicators for evaluating the hemodynamic state of the superior mesenteric artery. The resistance index reflects the degree of resistance to blood flow in the blood vessel. A higher resistance index may indicate lesions such as vascular stenosis and arteriosclerosis, resulting in greater resistance when blood flow passes through. On the contrary, a lower resistance index may indicate better vascular elasticity and less blood flow resistance. The pulsatility index reflects the pulsation characteristics of blood vessels. It is closely related to factors such as the contraction and relaxation function of the heart and the elasticity of blood vessels. An abnormal pulsatility index may indicate abnormal heart function or vascular disease, which affects the normal pulsation of the superior mesenteric artery.

[0074] In certain embodiments of the present application, the reduction of vascular pulse wave velocity means that when 3′,4′-dihydroxyflavonol is used on the subject, the pulse wave velocity of the abdominal aorta of the subject is significantly reduced compared with the unused control group (#P<0.05), and is superior to the existing drug Valsartan for treating hypertension.

[0075] In certain embodiments of the present application, the lowering of the resistance index means that when 3′,4′-dihydroxyflavonol is administered to a subject, the superior mesenteric artery resistance index of the subject is significantly lowered compared to a control group that does not administer 3′,4′-dihydroxyflavonol (#P<0.05).

[0076] In certain embodiments of the present application, the reduction of the pulsatility index means that when 3′,4′-dihydroxyflavonol is used for the subject, the superior mesenteric artery pulsatility index of the subject is significantly reduced compared with the unused control group (#P<0.05).

[0077] In a specific embodiment of the present application, the above-mentioned improvement of vascular wall thickening includes but is not limited to reducing the thickness of the abdominal aorta vascular wall of the subject, and / or reducing the proliferation of smooth muscle cells of the subject; specifically, the vascular wall thickening refers to the thickening of each layer of the abdominal aorta vascular wall, the proliferation of smooth muscle cells, etc., and the improvement of vascular thickening refers to reducing the thickness of the abdominal aorta vascular wall, reducing the proliferation of smooth muscle cells, etc. The reduction of the thickness of the vascular wall refers to bringing the vascular wall of the subject close to or reaching the thickness of a healthy blood vessel. For example, based on a healthy blood vessel, the product can make the thickness of the vascular wall of the subject's blood vessel not exceed 130%, 120%, 110% or 100% of the thickness of a healthy blood vessel. The main reasons for the thickening of the vascular wall caused by hypertension are as follows. One is hemodynamic changes: long-term hypertension increases the pressure load in the blood vessels, promotes the proliferation of smooth muscle cells, and thus leads to an increase in the thickness of the vascular wall. The second is growth factor activation: hypertension can activate growth factors such as transforming growth factor β (TGF-β) to promote the proliferation and fibrosis of vascular smooth muscle cells. The third is endothelial dysfunction: hypertension causes endothelial cell damage, affecting the normal regulatory function of blood vessels and leading to reactive thickening. The fourth is inflammatory response: hypertension is accompanied by systemic inflammation, and inflammatory cell infiltration can cause structural changes in the blood vessel wall. The fifth is metabolic abnormalities: hypertension is often associated with metabolic syndromes such as high cholesterol and obesity, which also promote the thickening of blood vessel walls.

[0078] In certain embodiments of the present application, the reduction of the thickness of the abdominal aorta wall and the reduction of smooth muscle cell proliferation of the subject refer to that when 3′,4′-dihydroxyflavonol is used on the subject, the following symptoms of the subject are improved compared with the unused control group: thickening of the abdominal aorta wall, proliferation and migration of subintimal smooth muscle cells, deposition of extracellular matrix, hypertrophy and hyperplasia of media smooth muscle cells, structural disorder, blurred boundaries between the intima and the media, smooth muscle cells are no longer neatly arranged, inflammatory cells infiltrate the vessel wall, monocytes, lymphocytes, etc. are scattered or aggregated, and vessel wall damage is improved.

[0079] In a specific embodiment of the present application, the above-mentioned improvement of reduced cardiac function includes but is not limited to increasing the cardiac ejection fraction of the subject, and / or reducing the cardiac E / e' index of the subject; specifically, the reduced cardiac function refers to a reduced cardiac ejection fraction or a reduced cardiac diastolic function, the improved reduced cardiac function refers to increasing the cardiac ejection fraction or reducing the cardiac E / e' index, and the increased cardiac ejection fraction refers to making the cardiac ejection fraction of the subject approach or reach the score of a healthy heart. For example, based on a healthy heart, the product can restore the cardiac ejection fraction of the subject's heart to at least 70%, 80%, 90% or 100% of that of a healthy heart. A reduced cardiac ejection fraction or a reduced left ventricular shortening fraction is of great significance. This usually means that the heart's ability to pump blood is weakened. Myocardial fibers are often damaged due to underlying diseases such as heart disease, valvular disease, and hypertension, thereby affecting their contractile function. The cardiac E / e' index is an echocardiographic index used to evaluate cardiac diastolic function. It is calculated by the ratio of the early diastolic blood flow velocity of the mitral valve (E peak) to the early diastolic peak velocity of the mitral valve ring (e'). The E / e′ index reflects the left ventricular filling pressure and can be used to assess left ventricular diastolic function.

[0080] In certain embodiments of the present application, the improvement of the cardiac ejection fraction of the subject means that when 3′,4′-dihydroxyflavonol is used for the subject, the left ventricular ejection fraction of the subject is significantly improved compared with the unused control group (#P<0.05), and is superior to the existing drug Valsartan for treating hypertension.

[0081] In certain embodiments of the present application, the reduction of the cardiac E / e' index of the subject means that when 3',4'-dihydroxyflavonol is used for the subject, the E / e' ratio of the subject is significantly reduced compared with the unused control group (#P<0.05).

[0082] In a specific embodiment of the present application, the above-mentioned improvement of myocardial cell hypertrophy includes but is not limited to arranging the myocardial cells of the target, and / or restoring the health of the myocardial cells of the target, and / or reducing the number of enlarged myocardial cells of the target, and / or narrowing the gap between the cells of the target; specifically, the myocardial cell hypertrophy refers to myocardial cell hypertrophy and disordered arrangement of myocardial cells and myofilament bundles and surrounding proliferating loose connective tissue, and widening of the intercellular gap. The improvement of myocardial cell hypertrophy refers to arranging myocardial cells in a neat manner, restoring the health of myocardial cells or reducing the number of enlarged myocardial cells, and narrowing the intercellular gap. The improvement of myocardial cell hypertrophy refers to bringing the myocardial cells of the target close to or reaching a healthy size. When the myocardium is hypertrophic, the number of collagen fibers in the interstitium increases significantly. Under HE staining, the collagen fibers appear pink, which deepens the color of the interstitium. The increase in collagen fibers leads to an increase in the hardness of the myocardium, affecting the diastolic function of the heart. The blood vessels in the interstitium may show dilation, congestion and other manifestations. The thickness of the vascular wall may increase to meet the demand for blood supply during myocardial hypertrophy. In some pathological conditions, myocardial hypertrophy may be accompanied by an inflammatory response, and a small amount of inflammatory cell infiltration, such as lymphocytes, monocytes, etc., may be seen in the interstitium. For example, based on healthy myocardial cells, the product can make the size of the target myocardial cells no more than 130%, 120%, 110% or 100% of the healthy myocardial cells.

[0083] In certain embodiments of the present application, when 3′,4′-dihydroxyflavonol is used as the subject, the following symptoms of hypertensive mice are improved compared to the untreated control group: myocardial cell hypertrophy, enlarged and darkly stained nuclei, widened intercellular spaces, and disordered arrangement of myocardial cells, and the thickening and hardening of the abdominal aorta can be alleviated.

[0084] In a specific embodiment of the present application, the above-mentioned decreased vascular function, and / or thickening of vascular walls, and / or decreased cardiac function, and / or cardiomyocyte hypertrophy are caused by hypertension, and are specifically related to angiotensin II (AngⅡ).

[0085] Blood vessels play an important role in regulating changes in blood pressure. They can secrete a variety of vasoactive substances to regulate the structure and function of blood vessels, mainly involving the renin-angiotensin-aldosterone system (RAAS) and other growth factors and cytokines. RAAS is an endocrine system that plays an important role in cardiovascular physiological regulation. Its overactivation can cause abnormal increases in AngⅡ and its downstream aldosterone, promote the remodeling of vascular smooth muscle and myocardium, lead to vascular and ventricular stiffness, and ultimately lead to the occurrence and development of hypertension, atherosclerosis, myocardial hypertrophy, heart failure and ischemic heart disease. AngⅡ, as an important bioactive peptide in RAAS, can produce vasoconstriction and thus cause an increase in blood pressure.

[0086] In a specific embodiment of the present application, the above product comprises an effective dose of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof.

[0087] In a specific embodiment of the present application, the above-mentioned product must include various forms of 3′,4′-dihydroxyflavonol or its pharmaceutically acceptable salts, and 3′,4′-dihydroxyflavonol or its pharmaceutically acceptable salts is used as the only active ingredient or one of the active ingredients.

[0088] In this application, an effective dose refers to a dose of a drug, treatment or other intervention measure that achieves the desired therapeutic effect without causing unacceptable side effects or toxicity. The effective dose is expressed as the weight of the drug administered per kilogram of body weight to each individual and is administered to the subject every day during the use period.

[0089] In a specific embodiment of the present application, the effective dose of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg. Using 10 mg / kg / d on the subject can be effectively used to prevent or treat hypertension. At the same time, it has a good therapeutic effect on heart and vascular diseases caused by hypertension, can improve reduced vascular function, improve increased vascular thickness, improve reduced cardiac function, improve cardiomyocyte hypertrophy, and has a high degree of safety.

[0090] The present application also provides a product comprising an effective dose of 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof.

[0091] In a specific embodiment of the present application, the product must include various forms of 3′,4′-dihydroxyflavonol or its pharmaceutically acceptable salts, and 3′,4′-dihydroxyflavonol or its pharmaceutically acceptable salts is used as the only active ingredient or one of the active ingredients.

[0092] In a specific embodiment of the present application, the effective dosage of the 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg.

[0093] The product can or can be used to lower blood pressure; improve reduced vascular function; improve vascular wall thickening; improve reduced heart function; improve cardiomyocyte hypertrophy, and the symptoms of reduced vascular function, vascular wall thickening, reduced heart function or cardiomyocyte hypertrophy can be caused by hypertension.

[0094] The product is capable of or can be used for preventing and / or treating hypertension.

[0095] The products in this application include but are not limited to drugs, pharmaceutical compositions, combinations of drugs, reagents, kits, foods, and health products.

[0096] The dosage form or state of the product in the present application is not limited. For example, when the product is a medicine, or a pharmaceutical composition, or a combination of medicines, the dosage form can be one or more of a solution, injection, spray, nasal drops, aerosol, powder spray, tablet, capsule and granule; the medicines in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field; preferably, it is a solution; for another example, when the product is a food or a health product, the state can be liquid or solid.

[0097] The product in the present application may include not only 3′,4′-dihydroxyflavonol or a pharmaceutically acceptable salt thereof, but also a pharmaceutically acceptable auxiliary material or carrier.

[0098] In pharmaceutical preparations, pharmaceutically acceptable excipients refer to those auxiliary materials that do not affect the efficacy and safety of the main ingredients of the drug, are non-toxic in themselves, do not produce adverse, allergic or other adverse reactions, and should be compatible with the 3′,4′-dihydroxyflavonol, that is, they can be mixed with it without significantly reducing the effect of 3′,4′-dihydroxyflavonol under normal circumstances. Examples of these excipients include: sterile water or saline, as a solvent or diluent for the drug; stabilizers, used to maintain the stability of the drug and prevent decomposition; excipients, which help form a specific form of the drug, such as tablets, capsules, etc.; antioxidants, such as ascorbic acid, to prevent drug oxidation; buffers: such as phosphates, citric acid, etc., used to maintain the pH value of the drug; preservatives, to prevent the drug from being contaminated by microorganisms during storage; surfactants, such as polyethylene glycol (PEG), Tween, etc., used to increase the solubility or stability of the drug; chelating agents, such as EDTA, used to bind to metal ions to prevent drug degradation; adhesives, used to fix the drug components together. In addition, the pharmaceutical preparation may also contain: low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; amino acids, such as glycine, glutamic acid, asparagine, arginine and lysine; sugars or carbohydrates, such as polysaccharides and monosaccharides; sugar alcohols, such as mannitol or sorbitol. When preparing aqueous solutions for injection, the following may also be used: physiological saline; isotonic solutions containing glucose or other auxiliary drugs; solubilizers, such as alcohols (ethanol), polyols (such as propylene glycol, PEG) and non-ionic surfactants (such as Tween 80, HCO-50). These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration. The types and proportions of these excipients or carriers can be adjusted according to the type and requirements of the final pharmaceutical preparation.

[0099] For example, when the product is a drug, a combination of drugs, or a pharmaceutical composition, it can be administered orally, and dosage forms suitable for oral administration include solid dosage forms and liquid preparations. The solid dosage forms include capsules, tablets, pills, powders and granules. In these solid dosage forms, 3', 4'-dihydroxyflavonol or its pharmaceutically acceptable salt is obtained according to the prior art with at least one conventional inert excipient, filler or solubilizer, adhesive, moisturizer, disintegrant, solubilizer, adsorbent, lubricant. In capsules, tablets and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compound in such a composition can be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and wax substances. If necessary, the active compound can also be formed into a microcapsule form with one or more of the above excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers, emulsifiers, etc.

[0100] For example, when the product is a drug, a combination of drugs, or a pharmaceutical composition, it can be administered enterally or parenterally, such as by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation, such as into the body, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or introduced into the body after being mixed or wrapped with other substances. Preferably, it is administered enterally. Therefore, preferably, the pharmaceutical composition also includes an antioxidant, a buffer, an antibacterial agent, and a solute that makes the preparation isotonic with the subject's blood, as well as an aqueous and non-aqueous sterile suspension, which may contain a suspending agent, a solubilizer, a thickener, a stabilizer and a preservative.

[0101] In the product of the present application, the 3',4'-dihydroxyflavonol or its pharmaceutically acceptable salt can be a single active ingredient, or it can be combined with one or more other active ingredients that have therapeutic effects on hypertension to form a combined preparation, and the efficacy and safety of each component do not conflict. Other active ingredients can be various other drugs that can be used to treat hypertension, such as diuretics (hydrochlorothiazide, indapamide), calcium channel blockers (nifedipine controlled release tablets, amlodipine, levamlodipine), angiotensin converting enzyme inhibitors (benazepril, enalapril, perindopril), angiotensin receptor antagonists (losartan, valsartan, irbesartan), beta-blockers (metoprolol, bisoprolol), alpha-blockers (prazosin, terazosin), renin inhibitors (such as remicycline), angiotensin receptor neprilysin inhibitors (such as sacubitril valsartan). The content of each ingredient in the combined preparation is generally a safe and effective amount, which can be adjusted based on actual usage (eg, patient weight, application type, disease condition, severity).

[0102] When the product in this application is used as a preparation, it is preferably in unit dosage form, which means that the preparation is subdivided into unit doses containing appropriate amounts of active ingredients. The unit dosage form can be a solution, injection, injection, capsule, tablet or any dosage form; in addition, the unit dosage form can also be a packaged preparation, such as a solution, injection, tablet, capsule and powder packaged in a vial or ampoule. The amount of the active ingredient in the unit dosage preparation can be changed or adjusted from 0.1 mg to 1000 mg, depending on the specific application and efficacy of the active ingredient.

[0103] In a specific embodiment of the present application, when the drug is used to treat hypertension in a subject, the product in an effective dose must be accurately administered to the subject, so that the hypertension originally suffered by the subject can be effectively suppressed, reduced or even alleviated, and / or the reduced vascular function caused by hypertension can be successfully curbed, including the adverse trend of vascular wall thickening can be slowed down, the condition of reduced heart function can be improved, and the phenomenon of myocardial cell hypertrophy can be suppressed.

[0104] The product in the present application is directed to an organism suffering from hypertension, or an organism in need of prevention and / or treatment of hypertension, specifically various mammals, including but not limited to rodents, artiodactyls, perissodactyls, lagomorphs, primates, such as humans, monkeys, other primates, migratory ants, cattle, horses, donkeys, pigs, dogs, cats, mice, rabbits, rats, guinea pigs, hamsters, foxes, deer, etc.; in a preferred embodiment of the present application, the target of action is a human or a mouse.

[0105] The product in this application can also be used in combination with other methods for treating hypertension, which can be used simultaneously or in a sequential order. Other methods for treating hypertension include but are not limited to other drug treatments or non-drug treatments, such as dietary adjustment, weight control, regular exercise, smoking cessation and alcohol restriction, stress management, and adequate sleep.

[0106] The present application is further described below by way of examples, but the scope of the present application is not limited thereby. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present application all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. These techniques have been fully described in the existing literature. Unless otherwise specified, the instruments, reagents, and materials used in the examples can be obtained by conventional means.

[0107] In the examples of this application, the material information used is as follows:

[0108] Experimental animals: SPF-grade C57BL / 6 male mice were purchased from Slack Laboratory Animal Co., Ltd., with the experimental animal use license number: SYXK(Min)2020-0002.

[0109] Experimental drugs and main reagents: osmotic micropump (Alzet, 2004D); angiotensin II (AngII; Abcam, ab120183); 3′,4′-dihydroxyflavonol (MCE, Hy-111804); valsartan (Sigma, PHR315); eosin stain (Beijing Solarbio Company, G1100); Cole hematoxylin stain (Beijing Solarbio Company, G1140); isoflurane for animals (Shenzhen Ruiwode Life Science Technology Co., Ltd., 970-00026-00); 4% paraformaldehyde universal tissue fixative (Anhui biosharp Company, 23319084), anhydrous ethanol (Xilong Science Co., Ltd., 1280340101602), xylene (Fuzhou Hongxin Glass Instrument Co., Ltd., X820585) and other chemical reagents.

[0110] The main instruments of the experiment are: manual pipette (Raining Company, USA); AB204-N electronic balance scale (MerrlerToledo Company, Switzerland); non-invasive rat tail sphygmomanometer (Kent Co., Ltd., USA); Vevo 2100 ultra-high resolution small animal ultrasound imaging system (VisualSonics Company, Canada); inhalation small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd.); automatic biological tissue dehydration machine (Hubei Xiaogan Yaguang Medical Electronic Technology Co., Ltd.); biological tissue paraffin embedding machine (Hubei Xiaogan Yaguang Medical Electronic Technology Co., Ltd.); fully automatic paraffin sectioning machine (Leica Company, Germany); intelligent tissue section analysis system (Leica Company, Germany).

[0111] Statistical analysis IBM SPSS Statistics (version 27.0) software was used for statistical analysis, and the results were expressed as mean ± standard deviation. Normality test and variance homogeneity test were performed first. If the data conformed to normal distribution, the difference between the two groups was analyzed by Student-t test, i.e., t test. The data of two or more groups were analyzed by one-way analysis of variance and two-way analysis of variance; if the data did not conform to normal distribution, the rank sum test was used. P < 0.05 indicated that there was a significant difference between the groups.

[0112] Example 1 Drug Preparation

[0113] Weigh an appropriate amount of 3′,4′-dihydroxyflavonol (Trihydroxyflavonol) powder, dissolve it in a corresponding volume of high-pressure ultrapure water at a dose of 10 mg / kg / d according to the average weight of mice, place it in an ultrasound machine to maintain low-temperature ultrasound for about 1 hour after preparation, and store it in a 4°C refrigerator for use to obtain a 3′,4′-dihydroxyflavonol solution, which is prepared and used immediately.

[0114] AngⅡ dry powder was stored at -20℃. Before the experiment, an appropriate amount of AngⅡ powder was weighed and dissolved in high-pressure ultrapure water to prepare a solution with a mother liquor concentration of 1mM. After preparation, the solution was divided and stored in a -80℃ refrigerator for future use and prepared before use.

[0115] Weigh an appropriate amount of valsartan powder and dissolve it in high-pressure water. Dissolve it in a corresponding volume of high-pressure ultrapure water at a dose of 10 mg / kg / d according to the weight of the mouse. After preparation, store it in a 4°C refrigerator for later use to obtain the valsartan solution, which is prepared before use.

[0116] Example 2 Animal Grouping and Model

[0117] Animals: 12 male C57BL / 6 mice (8-10 weeks old, weighing 29±2g) were purchased from Shanghai Slake Experimental Animal Co., Ltd. and kept in the SPF-level laboratory of the Experimental Animal Center of Fujian University of Traditional Chinese Medicine, with a temperature of 23±1℃, humidity controlled at 50%-60%, 12h light / dark cycle, and adequate diet and water. All animal experimental operations were in accordance with the regulations of the Laboratory Animal Ethics and the Animal Management Association of Fujian University of Traditional Chinese Medicine, and the ethical review number is 2W2024005.

[0118] Animal grouping, model construction and drug treatment:

[0119] Animal grouping: Twelve male C57BL / 6 mice were randomly divided into four groups according to basal blood pressure: Control group, AngII group, AngII+Trihydroxyflavonol (10 mg / kg / d) group, and AngII+Valsartan (10 mg / kg / d) group, with 3 mice in each group.

[0120] Preparation of AngII mini-osmotic pumps: The day before surgery, the mini-osmotic pumps were taken out in the clean bench, and 200 μL of AngII solution was injected into the mini-osmotic pumps of the AngII group, AngII+Trihydroxyflavonol (10 mg / kg / d) group, and AngII+Valsartan (10 mg / kg / d) group, respectively, to obtain preset osmotic mini-pumps containing AngII.

[0121] The osmotic minipumps in the Control group were injected with 200 μL of saline solution to obtain osmotic minipumps pre-loaded with saline.

[0122] The osmotic mini-pumps of all groups were immersed in physiological saline in a 37°C cell culture incubator overnight and used for pump implantation surgery the next day.

[0123] Implantation surgery: One day before the surgery, the hair on the back of the mice's neck was removed, and the operating table was routinely disinfected. The mice were anesthetized with isoflurane, placed in a prone position, and disinfected with iodine. The skin was cut with scissors, and the subcutaneous tissue was bluntly separated. The pre-placed osmotic micropumps containing AngII were implanted subcutaneously in the mice of the AngII group, AngII+Trihydroxyflavonol (10 mg / kg / d) group, and AngII+Valsartan (10 mg / kg / d) group; at the same time, the pre-placed osmotic micropumps containing saline were implanted subcutaneously in the mice of the Control group. After the corresponding osmotic micropumps were implanted in each group, the skin was sutured and disinfected with iodine.

[0124] From the second day after implantation, the patients were gavaged; the AngII+Trihydroxyflavonol (10 mg / kg / d) group was gavaged with Trihydroxyflavonol solution; the AngII+Valsartan (10 mg / kg / d) group was gavaged with Valsartan solution. The control group was implanted with a miniature osmotic pump containing 200 μL of saline solution, and distilled water was gavaged later. The intervention was once a day for a total of 4 weeks.

[0125] Example 3 Effect of 3′,4′-dihydroxyflavonol on blood pressure and body weight of mice

[0126] Blood pressure measurement: The systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) of the tail artery of mice were monitored by the Kent non-invasive rat tail artery blood pressure monitor from the United States. The measurement was performed once a week, and the data were statistically analyzed. The specific operation is as follows:

[0127] (1) Install the rat-tail cannula non-invasive sphygmomanometer and check whether the instrument has any abnormal conditions such as air leakage.

[0128] (2) Ensure that the experimental animals are in a calm state. Fix the mouse in a restraint, leaving its tail exposed, and ensure that the mouse's head can be extended out of the restraint to allow normal breathing.

[0129] (3) The mice were then placed on a heating plate, and an occlusive ring and a volume pressure sensing sleeve were installed at the base of the tail. The appropriate heating level was selected according to the room temperature to heat the tail. The restraint was covered with a black towel to keep the mice in a dark environment. The experimental environment was kept quiet. The mice were allowed to rest for 5 minutes before measurement began. Each mouse was measured for at least 10 cycles, each cycle lasting 30 seconds. The SBP, DBP, and MAP of qualified mice were recorded and their average values ​​were taken.

[0130] (4) Once a week, record the systolic blood pressure, diastolic blood pressure, mean arterial pressure and body weight of each animal, and calculate the mean and standard deviation of each group.

[0131] Experimental results: The effects of 3′,4′-dihydroxyflavonol on blood pressure are shown in Figure 1 , 2 and 3.

[0132] like Figure 1As shown in the figure, compared with the Control group, the SBP of AngII mice in the AngII group increased significantly after intervention (*P<0.05). Compared with the AngII group, after the intervention of 3′,4′-dihydroxyflavonol, the SBP of the AngII+Trihydroxyflavonol group decreased significantly (#P<0.05). And the effect was better than valsartan. The results showed that 3′,4′-dihydroxyflavonol could significantly improve the increase of SBP.

[0133] like Figure 2 As shown in the figure, compared with the Control group, the DBP of the AngII group mice after AngII intervention was significantly increased (*P<0.05). Compared with the AngII group, the DBP of the AngII+Trihydroxyflavonol group mice after Trihydroxyflavonol intervention was significantly decreased (#P<0.05), which was comparable to the effect of valsartan treatment.

[0134] like Figure 3 As shown in the figure, the MAP of mice increased significantly after AngII intervention, and the statistical difference was significant compared with the Control group (*P<0.05). After 3′,4′-dihydroxyflavonol intervention, the MAP of the AngII+Trihydroxyflavonol group decreased significantly compared with the AngII group (#P<0.05). The results showed that 3′,4′-dihydroxyflavonol intervention could significantly inhibit the increase of MAP in the AngII group.

[0135] At the same time, body weight measurement showed that 3′,4′-dihydroxyflavonol intervention had no significant effect on the body weight of mice, indicating that 3′,4′-dihydroxyflavonol has good safety ( Figure 4 ).

[0136] Example 4 Study on the Effect of 3′,4′-dihydroxyflavonol on Pulse Wave Velocity of Abdominal Aorta, Resistance Index and Pulsatility Index of Superior Mesenteric Artery in Mice

[0137] Abdominal aorta pulse wave velocity and thickness measurements:

[0138] (1) Echocardiography: Four weeks after intragastric administration, the abdominal aorta vascular function of mice in each group was detected using a Vevo 2100 small animal ultrasound instrument.

[0139] (2) Abdominal hair removal and animal anesthesia: Before testing, the abdominal hair of the experimental animals was removed, and each animal was anesthetized with 2% isoflurane inhalation. The animals were placed in a supine position on a 37°C constant temperature heating plate.

[0140] (3) Obtaining abdominal aorta section: Apply an appropriate amount of coupling agent to the abdomen of the mouse, place the probe (46MHz) parallel to the middle of the abdomen, and use B-mode ultrasound to record the abdominal aorta. Place the probe perpendicular to the middle of the abdomen, and use M-mode ultrasound to record the abdominal aorta section and the conduction conditions at the proximal and distal ends. Measure the abdominal aorta wall thickness at three different locations, and calculate the abdominal aorta pulse wave velocity (PWV) of the mouse by the abdominal aorta vessel length and terminal delay time. Then slide the ultrasound probe to the right superior mesenteric artery to obtain the superior mesenteric artery blood flow image, and randomly select three blood flow images to measure the superior mesenteric artery resistance index and pulsatility index.

[0141] (4) Analysis and calculation of ultrasound results: After the operation, Vevo Strain Software (VevoLAB 5.8.0) was used to calculate the superior mesenteric artery resistance index (RI) and pulsatility index (PI) of each group of animals, and the average value was calculated three times. The pulse wave velocity (PWV) of the abdominal aorta was calculated according to the following formula: Pulse wave velocity: PWV (m / s) = vessel length / (distal-proximal time difference).

[0142] After AngII induction, mice were treated with 3′,4′-dihydroxyflavonol for 4 weeks. The pulse wave velocity of the abdominal aorta, the resistance index of the superior mesenteric artery, and the pulsatility index of each group of mice were detected and analyzed by small animal ultrasound. Figure 5-9 .

[0143] Studies have shown that an increase in abdominal aorta PWV is positively correlated with the severity of atherosclerotic lesions and can be used as an effective indicator for early detection and evaluation of atherosclerosis. Figure 5 and Figure 6 It can be seen that compared with the Control group, the abdominal aortic pulse wave velocity of the mice in the AngII group was significantly increased (*P<0.05), while Trihydroxyflavonol intervention could significantly reduce the abdominal aortic pulse wave velocity of the mice (#P<0.05). Moreover, the effect of Trihydroxyflavonol in improving the abdominal aortic pulse wave velocity was better than that of valsartan (&P<0.05).

[0144] Long-term hypertension can accelerate the process of arteriosclerosis, leading to thickening and hardening of the wall of the superior mesenteric artery and narrowing of the lumen. This makes blood flow encounter greater resistance when passing through the blood vessels, thus causing an increase in the resistance index. Figure 7 and Figure 8It can be seen that compared with the Control group, the superior mesenteric artery resistance index of mice in the AngII group was significantly increased (*P<0.05), while the superior mesenteric artery resistance index of mice was significantly decreased after Trihydroxyflavonol intervention (#P<0.05).

[0145] Pulsatility index is an important indicator to measure the blood flow resistance of the superior mesenteric artery. Figure 7 and 9 It can be seen that compared with the Control group, the pulsatility index of the superior mesenteric artery of mice in the AngII group was significantly increased (*P<0.05), while the pulsatility index of the superior mesenteric artery of mice was significantly decreased after Trihydroxyflavonol intervention (#P<0.05).

[0146] Example 5 Investigation of the effect of 3′,4′-dihydroxyflavonol on the pathological morphology of the abdominal aorta in mice

[0147] HE staining

[0148] (1)Fixed:

[0149] After sampling, the mouse heart tissue and abdominal aorta were fixed in 4% paraformaldehyde for 24 hours, taken out, placed in an embedding box, and the heart number was clearly marked. The embedding box was then immersed in 70% ethanol overnight.

[0150] (2) Dehydration and wax dipping:

[0151] The samples were immersed in the following reagents for dehydration and wax in turn: the heart tissue was dehydrated and waxed in the following order: 80% ethanol (30 min) → 90% ethanol (30 min) → 95% ethanol (30 min) → 100% ethanol I (10 min) → 100% ethanol II (10 min) → 100% ethanol III (30 min) → xylene I (5 min) → xylene II (10-15 min) → paraffin I (10 min) → paraffin II (10 min) → paraffin III, 25 min.

[0152] (3) Embedding:

[0153] Preheat the embedding mold and wax block in advance, place the tissue in an upright position in the center of the bottom of the mold, gently place the embedding box on top of the mold, fill it with paraffin from the top, transfer it to the cold table and wait for the paraffin to solidify, then put it in a -20℃ refrigerator for 30 minutes, pry out the wax block from the mold, and store it at room temperature for subsequent experiments.

[0154] (4) Slicing:

[0155] The tissue wax block was precooled and fixed on a microtome. The initial setting was 10 μm for coarse trimming. When the tissue structure was complete, it was adjusted to 4 μm for fine trimming and slicing. The slices were flattened in 37°C pure water, fished out and placed on an adhesive slide. The slices were baked at 60°C for 1 hour, and then stored at room temperature for use after baking for 3 hours.

[0156] (5) Dewaxing:

[0157] Immerse in the reagents in the following order: xylene I (25 min) → xylene II (25 min) → xylene III (15 min) → 100% anhydrous ethanol I (5 min) → 100% anhydrous ethanol II (5 min) → 95% anhydrous ethanol (5 min) → 80% anhydrous ethanol (5 min) → 70% anhydrous ethanol (5 min) → ultrapure water I (5 min) → ultrapure water II (5 min).

[0158] (6) Hematoxylin staining:

[0159] The slides were stained in hematoxylin solution for 1 min and then washed with pure water.

[0160] (7) Eosin staining:

[0161] Immerse the slides in eosin solution for 10 seconds and then wash with pure water.

[0162] (8) Sealing:

[0163] After staining, blow dry the slides immediately, add an appropriate amount of neutral gum to seal the slides, and dry them in a ventilated place.

[0164] (9) Microscope observation:

[0165] An intelligent tissue section analysis system was used to observe and collect images at a 400× field of view. Images of 6 different areas were collected for each mouse to observe the pathological changes in the heart and abdominal aorta of each group of mice.

[0166] HE staining was used to detect the changes of 3′,4′-dihydroxyflavonol on the morphology of the abdominal aorta of mice. Fig.10 . Compared with the Control, the abdominal aorta wall of mice induced by AngII was thickened, the subintimal smooth muscle cells proliferated and migrated, the extracellular matrix was deposited, the media smooth muscle cells were hypertrophic and proliferated, the structure was disordered, the boundary between the intima and the media was blurred, and the smooth muscle cells were no longer neatly arranged. At the same time, there was inflammatory cell infiltration in the vascular wall, and monocytes, lymphocytes, etc. were scattered or aggregated, aggravating the damage to the vascular wall. The Trihydroxyflavonol intervention group can improve the pathological thickening of the abdominal aorta wall of mice after AngⅡ stimulation.

[0167] Example 6 Investigation of the Effect of 3′,4′-Dihydroxyflavonol on Cardiac Function in Mice

[0168] Echocardiography to detect cardiac function:

[0169] The chest hair removal and animal anesthesia steps were the same as those described in Example 4. The ultrasound probe (frequency was 400 MHz) was then placed vertically on the left chest of the mouse, and the probe was adjusted so that its notch was facing the head of the mouse. The probe was slowly fine-tuned in the B-mode to obtain a clear heart image as much as possible, and then the cardiac cycle diagram in the M-mode was collected. At least three cardiac cycles were measured for each mouse, and the average value was taken for calculation.

[0170] Small animal ultrasound was used to assess changes in cardiac function, and a decrease in left ventricular ejection fraction (LVEF) indicated impaired left ventricular systolic function and decreased cardiac pumping capacity. Fig.11 and 12 In the experiment, compared with the control group, the LVEF of the mice in the AngII group was significantly reduced (*P<0.05); while the intervention of Trihydroxyflavonol could significantly improve the left ventricular ejection fraction (#P<0.05). Moreover, the effect of Trihydroxyflavonol in improving LVEF was better than that of valsartan (&P<0.05).

[0171] In cardiac ultrasound, E / e′ is an important indicator for evaluating left ventricular filling pressure. Fig.13 and 14 Compared with the Control group, the E / e' ratio of the mouse heart was significantly increased, indicating an increase in left ventricular filling pressure (*P<0.05), which may be related to cardiac diastolic dysfunction. Compared with the AngII group, after Trihydroxyflavonol intervention, the E / e' ratio was significantly reduced (#P<0.05), effectively improving the diastolic function of the mouse heart.

[0172] Example 7 Study on the Effect of 3′,4′-Dihydroxyflavonol on the Pathological Morphology of Mouse Heart Disease

[0173] The HE staining experiment was the same as in Example 5.

[0174] HE staining was used to detect the effect of Trihydroxyflavonol intervention on the pathological morphology of cardiac tissue in mice with hypertension induced by AngII. Fig.15 The myocardial cells of the mice in the Control group were arranged tightly and neatly, with clear nuclei and uniform cytoplasmic staining without degeneration and necrosis. Compared with the Control group, myocardial cell hypertrophy, enlarged and deeply stained nuclei, widened intercellular spaces, and disordered arrangement of myocardial cells were observed in the heart tissue of the AngII group. Trihydroxyflavonol intervention can effectively improve the changes in the pathological morphology of myocardial tissue.

[0175] In summary, 3′,4′-dihydroxyflavonol can significantly lower the blood pressure of AngII model mice, improve vascular function, relieve abdominal aorta thickening and hardening, and improve the condition of reduced heart function caused by hypertension; it can effectively regulate the pathological change of cardiomyocyte hypertrophy.

[0176] The above embodiments are merely illustrative of the principles and effects of the present application and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed in the present application shall still be covered by the claims of the present application.

Claims

1. Use of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating hypertension.

2. The use according to claim 1, characterized in that The product has any one or more of the following effects: 1) Lower blood pressure; 2) Improve decreased vascular function; 3) Improve blood vessel wall thickening; 4) Improve reduced heart function; 5) Improve cardiomyocyte hypertrophy; 6) Prevent and / or treat hypertension.

3. The use according to claim 2, characterized in that: The product has one or more of the following characteristics: 1) The lowering of blood pressure includes lowering systolic blood pressure, and / or lowering diastolic blood pressure, and / or lowering mean arterial pressure; 2) The improvement of vascular function includes reducing vascular pulse wave velocity, and / or reducing resistance index, and / or reducing pulsatility index; 3) Improving vascular wall thickening includes reducing the thickness of the abdominal aorta vascular wall and / or reducing smooth muscle cell proliferation; 4) The improvement of decreased cardiac function includes increasing cardiac ejection fraction and / or decreasing cardiac E / e' index; 5) The improvement of cardiomyocyte hypertrophy includes aligning cardiomyocytes, and / or restoring the health of cardiomyocytes, and / or reducing the number of enlarged cardiomyocytes, and / or narrowing the intercellular space.

4. The use according to claim 2, characterized in that: The hypertension, and / or decreased vascular function, and / or thickening of vascular walls, and / or decreased cardiac function, and / or cardiomyocyte hypertrophy are caused by angiotensin II.

5. The use according to any one of claims 1 to 4, characterized in that: The product comprises an effective dose of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof; Preferably, the effective dosage of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg.

6. Use of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the preparation of a product, wherein the product is used for any one or more of the following: 1) Lower blood pressure; 2) Improve decreased vascular function; 3) Improve blood vessel wall thickening; 4) Improve reduced heart function; 5) Improve cardiomyocyte hypertrophy.

7. The use according to claim 6, characterized in that The product has one or more of the following characteristics: 1) The lowering of blood pressure includes lowering systolic blood pressure, and / or lowering diastolic blood pressure, and / or lowering mean arterial pressure; 2) The improvement of vascular function includes reducing vascular pulse wave velocity, and / or reducing resistance index, and / or reducing pulsatility index; 3) Improving vascular wall thickening includes reducing the thickness of the abdominal aorta vascular wall and / or reducing smooth muscle cell proliferation; 4) The improvement of decreased cardiac function includes increasing cardiac ejection fraction and / or decreasing cardiac E / e' index; 5) The improvement of cardiomyocyte hypertrophy includes aligning cardiomyocytes, and / or restoring the health of cardiomyocytes, and / or reducing the number of enlarged cardiomyocytes, and / or narrowing the intercellular space.

8. The use according to claim 6, characterized in that The decreased vascular function, and / or thickening of vascular walls, and / or decreased cardiac function, and / or cardiomyocyte hypertrophy are caused by angiotensin II.

9. The use according to any one of claims 6 to 8, characterized in that: The product comprises an effective dose of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof; Preferably, the effective dosage of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg.

10. A product comprising an effective dose of 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof; preferably, the effective dose of the 3',4'-dihydroxyflavonol or a pharmaceutically acceptable salt thereof in the product is greater than or equal to 10 mg / kg.

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