Application of nicotiflorin in preparation of medicine for preventing and treating heart failure

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of nicotiflorin in preparation of a medicine for preventing and treating heart failure. It is verified that the fireworks glycoside can reduce the pericardial area, the venous extravasated blood area and the venous sinus-arterial ball distance of a heart failure animal model, increase the heart rate, the short-axis shortening rate and the ejection fraction, improve heart tissue pathological changes caused by the heart failure and can be used for preparing the medicine for preventing and treating the heart failure.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of nicotiflorin in the preparation of drugs for preventing and treating heart failure. Background Art

[0002] Heart failure is the end-stage manifestation of various cardiovascular diseases, characterized by fatigue and dyspnea caused by left (or systemic) ventricular dysfunction, usually accompanied by signs of congestion. Heart failure is a progressive disease, and the prognosis deteriorates significantly as the disease progresses, with repeated exacerbations. A multi-faceted approach from prevention to post-onset management is crucial for controlling the heart failure pandemic. Heart failure is common in the middle-aged and elderly populations, and the incidence increases with the rising degree of population aging. According to treatment guidelines, the conventional drug treatment for HF includes angiotensin-converting enzyme inhibitors, β-blockers, diuretics, corticosteroid receptor antagonists, sodium-glucose cotransporter Ⅱ inhibitors, etc.

[0003] Nicotiflorin, also known as kaempferol-3-O-rutinoside, is a plant secondary metabolite and exists in various medicinal plants. Currently, studies have shown that nicotiflorin has anti-inflammatory, antioxidant, antibacterial, antiviral, analgesic, neuroprotective, and anti-myocardial ischemia effects, but there has been no report on its use in preventing and treating heart failure so far. Summary of the Invention

[0004] The purpose of the present invention is to expand the application of nicotiflorin in the preparation of drugs for preventing and treating heart failure, and in particular, to clarify the effect of nicotiflorin in preventing and treating heart failure, especially acute heart failure.

[0005] On the one hand, the present invention relates to the application of nicotiflorin in the preparation of drugs for preventing and / or treating heart failure-related diseases.

[0006] As is well known to those skilled in the art, heart failure drugs focus on improving cardiac function, inhibiting excessive activation of neurohormones, and require long-term management; while conventional tissue injury drugs focus on anti-inflammatory, anti-infective, and promoting repair, and are mostly used short-term or locally. There are significant differences in mechanisms, objectives, risks, and drug use strategies between the two, and they cannot be used interchangeably. Heart failure drugs are mainly used to regulate cardiac function, enhance myocardial contractility, reduce cardiac load; inhibit excessive activation of neurohormones; improve metabolism and energy supply. Tissue injury drugs are mainly used for anti-inflammatory and analgesic effects; promoting repair; anti-infection. Heart failure drugs mainly improve cardiac pumping function, relieve symptoms; delay disease progression, reduce mortality, and prevent complications. Tissue injury drugs mainly reduce inflammatory reactions and pain; promote tissue regeneration and repair; prevent or treat infections.

[0007] Further, in the application provided by the present invention, the symptoms of the heart failure-related diseases include an increase in the pericardial area and the venous congestion area caused by pericardial edema and venous congestion.

[0008] Further, in the application provided by the present invention, the symptoms of the heart failure-related diseases include a decrease in the fractional shortening and ejection fraction.

[0009] Further, in the application provided by the present invention, the symptoms of the heart failure-related diseases include a slowdown in blood flow velocity caused by a weakened cardiac pumping ability.

[0010] Further, in the application provided by the present invention, the symptoms of the heart failure-related diseases include a large number of apoptotic cardiomyocytes and ventricular remodeling with dilation leading to thinning of the ventricular wall.

[0011] Further, in the application provided by the present invention, the heart failure-related diseases include acute heart failure.

[0012] On the other hand, the present invention relates to a drug for preventing and treating heart failure, and the only active ingredient of the drug for preventing and treating heart failure is quercetin-3-O-neohesperidoside.

[0013] On the other hand, the present invention relates to a drug for preventing and treating acute heart failure, and the only active ingredient of the drug for preventing and treating acute heart failure is quercetin-3-O-neohesperidoside.

[0014] Further, in the embodiment provided by the present invention, the preparation of the drug contains quercetin-3-O-neohesperidoside, and the rest are pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers and / or excipients for humans and animals. The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants.

[0015] Further, the preparation is an oral preparation or an injection. The oral preparation is tablets, sustained-release tablets, controlled-release tablets, capsules, dripping pills, pellets, suspensions, emulsions, powders or granules, oral liquids, etc.; the injection is a sterilized aqueous or oily solution, a sterile powder injection, a liposome or an emulsion, etc.

[0016] According to actual application needs, for the drug provided by the present invention, according to the methods well-known to those skilled in the art, quercetin-3-O-neohesperidoside can be prepared into a pharmaceutically acceptable salt of quercetin-3-O-neohesperidoside as the active ingredient.

[0017] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of nicotiflorin and has no adverse effects biologically or otherwise. Pharmaceutically acceptable salts refer to converting the base group in the parent compound into a salt form, such as inorganic or organic acid salts of the base group (such as amino group). Generally, the parent compound is reacted with conventional types of acids in a solvent system for preparation. Inorganic acids generally include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids generally include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0019] Through the zebrafish animal model of Ver-induced heart failure, the present invention verifies and discloses that nicotiflorin can reduce the pericardial area, venous congestion area, sinus venosus - bulbus arteriosus distance, increase heart rate, fractional shortening, ejection fraction in the heart failure animal model, and improve the pathological changes of heart tissue caused by heart failure, and can be used for preparing drugs for preventing and treating heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is a schematic diagram of the hearts of zebrafish with Ver-induced heart failure at different concentrations of nicotiflorin. Among them, the black dashed part represents the pericardial area, and the red dashed part represents the venous congestion area.

[0022] Figure 2 It is a bar chart of the pericardial area, venous congestion area, and sinus venosus - bulbus arteriosus (SV - BA) of zebrafish with Ver-induced heart failure at different concentrations of nicotiflorin. Among them, # indicates comparison between the model group and the blank group, * indicates comparison between the positive drug group, different concentration sample groups and the model group, *p < 0.05, ** / ##p < 0.01, ***p < 0.001, **** / #p < 0.0001.

[0023] Figure 3 It is a schematic diagram of the systolic and diastolic ends of the hearts of zebrafish with Ver-induced heart failure at different concentrations of nicotiflorin. Among them, A is the atrium and V is the ventricle.

[0024] Figure 4 Bar chart of the effects of different concentrations of quercetin-3-O-rutinoside on the average heart rate, fractional shortening, and ejection fraction of zebrafish with Verapamil (Ver)-induced heart failure. Among them, bpm represents the number of heart beats per minute, that is, the heart rate. # indicates comparison between the model group and the blank group, * indicates comparison between the positive drug group, different concentration sample groups and the model group, * / #p < 0.05, **p < 0.01, **** / #p < 0.0001.

[0025] Figure 5 Schematic diagram of the effects of different concentrations of quercetin-3-O-rutinoside on the average blood flow velocity of zebrafish with Verapamil (Ver)-induced heart failure.

[0026] Figure 6 Bar chart of the effects of different concentrations of quercetin-3-O-rutinoside on the average blood flow velocity of zebrafish with Verapamil (Ver)-induced heart failure; among them, # indicates comparison between the model group and the blank group, * indicates comparison between the positive drug group, different concentration sample groups and the model group, *** / p < 0.001, #p < 0.0001.

[0027] Figure 7 Schematic diagram of the pathological staining of the heart tissue of zebrafish with Verapamil (Ver)-induced heart failure treated with different concentrations of quercetin-3-O-rutinoside. Detailed implementation manners

[0028] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. %, as used in the following embodiments, is mass percentage unless otherwise specified. The ratios in the following embodiments are mass ratios unless otherwise specified.

[0029] The following embodiments use Verapamil (Ver), which has been widely used in the technical field, to prepare a zebrafish heart failure model.

[0030] Quercetin-3-O-rutinoside was purchased from Chengdu Cromar Biotechnology Co., Ltd., Digoxin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Ver was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the culture water for zebrafish had a concentration of 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4.

[0031] Example 1

[0032] This example provides the effects of quercetin-3-O-rutinoside on the heart of zebrafish with Verapamil (Ver)-induced heart failure

[0033] (1) Experimental method

[0034] Under a microscope, normally developed wild-type zebrafish at 2 dpf (days post-fertilization) were selected and placed into 24-well plates, with 10 fish in each well. A blank group, a model group (Verapamil), a positive drug group (Verapamil + 12.8 μM Digoxin), and different concentration sample groups (Verapamil + 10 μM Nicotiflorin, Verapamil + 20 μM Nicotiflorin, Verapamil + 40 μM Nicotiflorin) were set up. The positive drug group and different concentration sample groups were given the corresponding concentration of the drug and placed in a constant temperature incubator at 28°C ± 1°C for pre-protection for 4.5 h, then the drug was washed off and the fish were washed 2 - 3 times with fish-raising water. Subsequently, Ver was added to the model group, the positive drug group, and different concentration sample groups and acted for 30 min. After the treatment, the zebrafish were anesthetized with 0.3% tricaine by mass concentration for 1 min. The cardiac morphology of the zebrafish was observed under a microscope and photographed, and the pericardial area, venous congestion area, and SV-BA distance of the zebrafish in each group were measured using Image J software.

[0035] (2) Statistical method

[0036] GraphPad Prism 8.0.2 was used for statistical analysis of the data. The data were tested for normality and variance analysis. If the data conformed to normality and homoscedasticity, the t-test was selected; otherwise, the rank sum test was selected, and P < 0.05 indicated that the difference was statistically significant.

[0037] (3) Result analysis

[0038] When Ver induced heart failure in zebrafish, the zebrafish showed pericardial edema and venous congestion, resulting in an increase in the pericardial area and venous congestion area; SV is the part where blood enters the atrium, and BA is the part where blood flows out of the ventricle. The positions of the atrium and ventricle of the heart failure zebrafish changed and no longer overlapped, resulting in a corresponding change in the SV-BA distance.

[0039] Figure 2 It is a columnar statistical chart of the pericardial area, venous congestion area, and SV-BA distance of the heart failure zebrafish induced by Ver with different concentrations of the sample. Compared with the blank group, the pericardial area, venous congestion area, and SV-BA distance of the zebrafish in the model group were significantly increased. Compared with the model group, the pericardial area, venous congestion area, and SV-BA distance of the positive drug group and different concentration sample groups were significantly decreased.

[0040] Example 2

[0041] This example provides the effect of nicotiflorin on the cardiac function of heart failure zebrafish induced by Ver.

[0042] (1) Experimental method

[0043] Under a microscope, select normal-developing wild-type zebrafish at 2 dpf (days post-fertilization) and place them in a 24-well plate, with 10 fish per well. Set up a blank group, a model group (Verapamil), a positive drug group (Verapamil + 12.8 μM Digoxin), and different concentration sample groups (Verapamil + 10 μM Nicotiflorin, Verapamil + 20 μM Nicotiflorin, Verapamil + 40 μM Nicotiflorin). The positive drug group and different concentration sample groups are given the corresponding concentration of the drug and placed in a constant temperature incubator at 28°C ± 1°C for pre-protection for 4.5 h, then the drug is washed off and the fish are washed 2 - 3 times with fish-raising water. Subsequently, Ver is added to the model group, positive drug group, and different concentration sample groups and acts for 30 min. After the treatment, the zebrafish are anesthetized with 0.3% tricaine by mass concentration for 1 min, the heart rate is recorded, the heart morphology of the zebrafish is observed and photographed under a fluorescence microscope, and the short-axis and long-axis distances of the zebrafish in each group are measured using Image J software, and the fractional shortening of the short axis and ejection fraction are calculated.

[0044] Fractional shortening of the ventricular short axis (%) = (end-diastolic width - end-systolic width) / end-diastolic width × 100% Ejection fraction (%) = stroke volume / end-diastolic volume of the ventricle × 100% (stroke volume = end-diastolic volume - end-systolic volume)

[0045] (2) Statistical method

[0046] GraphPad Prism 8.0.2 is used for statistical analysis of the data. The data are tested by normality test and analysis of variance test. If the data meet normality and homoscedasticity, the t-test is selected; otherwise, the rank sum test is selected, and P < 0.05 indicates that the difference is statistically significant.

[0047] (3) Result analysis

[0048] The heart rate of zebrafish refers to the number of heartbeats per minute of zebrafish, and the heart rate decreases when zebrafish develop heart failure. At the end of ventricular ejection in zebrafish, the ventricular volume is the smallest, and this ventricular volume is called the end-systolic volume at this time; the filling volume at the end of ventricular diastole is the largest, and the ventricular volume at this time is called the end-diastolic volume. The fractional shortening of the short axis and ejection fraction are calculated according to the formula using the long-axis and short-axis distances at the systolic and diastolic ends of zebrafish. When zebrafish develop heart failure, their fractional shortening of the short axis and ejection fraction decrease.

[0049] Figure 4Bar chart of the average heart rate, fractional shortening, and ejection fraction of zebrafish with Verapamil-induced heart failure treated with samples of different concentrations. Compared with the blank group, the heart rate, fractional shortening, and ejection fraction of zebrafish in the model group were significantly decreased. Compared with the model group, the heart rate, fractional shortening, and ejection fraction of the positive drug group and the medium-concentration sample group were significantly increased. Although there was no significant difference, the heart rate, fractional shortening, and ejection fraction of the low- and high-concentration sample groups showed an increasing trend.

[0050] Example 3

[0051] This example provides the effect of nicotiflorin on the heart tissue pathology of zebrafish with Verapamil-induced heart failure.

[0052] (1) Experimental method

[0053] Under a microscope, select 2-day post-fertilization (dpf) wild-type zebrafish larvae with normal development and place them in a 24-well plate, 10 tails per well. Set up a blank group, a model group (Verapamil), a positive drug group (Verapamil + 12.8 μM Digoxin), and different concentration sample groups (Verapamil + 10 μM Nicotiflorin, Verapamil + 20 μM Nicotiflorin, Verapamil + 40 μM Nicotiflorin). The positive drug group and different concentration sample groups were given the corresponding concentration of the drug and placed in a 28 °C constant temperature incubator for pre-protection for 4.5 h, then the drug was washed off and the fish were washed 2 - 3 times with fish water. Subsequently, Verapamil was added to the model group, the positive drug group, and different concentration sample groups and allowed to act for 30 min. After the treatment, the fish were washed 3 times with fish water. The blood flow velocity was recorded and analyzed using a microscope and Blood Flow v5.18.0.0 and MicroZebraLab BloodFlow v 3.4.6 software.

[0054] (2) Statistical method

[0055] GraphPad Prism 8.0.2 was used for statistical analysis of the data. The data were tested for normality and analysis of variance. If the data met the normality and homogeneity of variance, a t-test was selected; otherwise, a rank sum test was selected, and P < 0.05 indicated that the difference was statistically significant.

[0056] (3) Result analysis

[0057] When zebrafish develop heart failure, the heart's ability to pump blood weakens, resulting in a significant slowdown in blood flow velocity and even the appearance of no blood flow.

[0058] Figure 6Bar chart of the average blood flow velocity of zebrafish with Ver-induced heart failure treated with samples at different concentrations. Compared with the blank group, the blood flow velocity in the model group was significantly decreased. Compared with the model group, the blood flow velocity in the sample groups at different concentrations was significantly increased.

[0059] Example 4

[0060] This example provides the pathological effects of nicotiflorin on the heart tissue of zebrafish with Ver-induced heart failure.

[0061] (1) Experimental method

[0062] Under a microscope, select 2-day post-fertilization (dpf) wild-type zebrafish larvae with normal development and place them in a 24-well plate, 10 tails per well. Set up a blank group, a model group (Verapamil), a positive drug group (Verapamil + 12.8 μM Digoxin), and sample groups at different concentrations (Verapamil + 10 μM Nicotiflorin, Verapamil + 20 μM Nicotiflorin, Verapamil + 40 μM Nicotiflorin). The positive drug group and the sample groups at different concentrations are given the corresponding concentration of the drug and placed in a constant temperature incubator at 28 °C for pre-protection for 4.5 h, then the drug is washed off and the fish are washed 2 - 3 times with fish-raising water. Subsequently, Ver is added to the model group, the positive drug group, and the sample groups at different concentrations and acts for 30 min. After the treatment is completed, the fish are washed 2 - 3 times with fish-raising water, 5 - 10 fish are taken and anesthetized in an appropriate amount of tricaine, and then transferred to 4% paraformaldehyde to fix the zebrafish for 96 h, dehydrated with gradient ethanol, ethanol-benzene, and xylene, embedded in paraffin in an embedding machine, sectioned at 4 μm, dewaxed to water, rinsed with pure water, and then stained with hematoxylin-eosin (HE), the stained sections are washed and placed in gradient ethanol; dehydrated to transparency in xylene, air-dried, sealed with neutral gum, and observed and photographed under a microscope.

[0063] (2) Result analysis

[0064] When heart failure occurs, a large number of cardiomyocytes undergo apoptosis, and ventricular remodeling occurs with dilation, resulting in a thinning of the ventricular wall.

[0065] Figure 7 Shows the effects of samples at different concentrations on the pathology of zebrafish with Ver-induced heart failure. Compared with the blank group, the ventricular wall thickness in the model group was thinner. Compared with the model group, the heart wall thickness in the sample groups at different concentrations increased.

[0066] From the above experimental results, it can be seen that nicotiflorin can significantly improve the symptoms of Ver-induced heart failure, including reducing the pericardial area, venous congestion area, SV-BA distance, increasing the heart rate, fractional shortening, ejection fraction, increasing the blood flow velocity, and improving the heart tissue pathology.

[0067] The present invention for the first time discovers that quercetin-3-O-rutinoside can be used for treating heart failure, adding a new use for quercetin-3-O-rutinoside, providing a new treatment means for treating heart failure, and also providing a reference for the application development of quercetin-3-O-rutinoside.

[0068] The above-described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents the preferred embodiments of the present invention. All other embodiments obtained by relevant deductions and substitutions made by those of ordinary skill in the art under the condition of the conception of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. Use of nicotinic acid in the preparation of drugs for preventing and / or treating heart failure related diseases.

2. The use according to claim 1, characterized in that: Symptoms of the heart failure-related disease include pericardial edema and venous congestion leading to increased pericardial area and venous congestion area.

3. The use according to claim 1, characterized in that: Symptoms of the heart failure-related diseases include fractional shortening and decreased ejection fraction.

4. The use according to claim 1, characterized in that: Symptoms of heart failure-related diseases include a decrease in the heart's ability to pump blood, resulting in a decrease in blood flow.

5. The use according to claim 1, characterized in that: Symptoms of the heart failure-related diseases include massive apoptosis of cardiomyocytes and dilation of ventricular remodeling leading to thinning of the ventricular wall.

6. The use according to claim 1, characterized in that: The heart failure-related diseases include acute heart failure.

7. A drug for preventing and treating heart failure, characterized in that: The only effective component of the drug for preventing and treating heart failure is nicotinic acid glycoside.

8. A drug for preventing and treating acute heart failure, characterized in that: The only effective component of the drug for preventing and treating acute heart failure is nicotinic acid glycoside.

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