Use of acacetin or 6-hydroxyflavone in the preparation of anti-angina pectoris drugs

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of acacetin or 6-hydroxyflavone in preparation of anti-angina pectoris drugs, and aims to solve the problems of adverse reactions such as blood pressure drop, reflex tachycardia and the like caused by non-selective vasodilation of existing anti-angina pectoris drugs, wherein the drug improves myocardial blood supply by selectively dilating coronary vessels of the heart, wherein acacetin dilates coronary vessels of the heart by inhibiting cardiac coronary vessel myosin light chain kinase and activating myosin light chain phosphatase, and 6-hydroxyflavone dilates coronary vessels of the heart by inhibiting protein kinase C and enhancing myosin light chain phosphatase activity; and the acacetin or 6-hydroxyflavone significantly reduces the symptoms of myocardial ischemia of rats caused by isopropyl adrenaline, improves characteristic ECG abnormal changes of angina pectoris, does not affect heart rate and systemic blood pressure, and is safer, thereby providing a potential drug selection for anti-angina pectoris treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of farnesin or 6-hydroxyflavone in the preparation of antianginal drugs. Background Technology

[0002] Angina pectoris is a common cardiovascular disease caused by temporary myocardial ischemia and hypoxia due to insufficient blood supply to the coronary arteries. It is mainly manifested by symptoms such as chest pain, chest tightness, and palpitations. More than 112 million people worldwide are affected by it. Some patients may suffer myocardial infarction due to coronary artery blockage, while most non-obstructive angina is caused by coronary artery spasm or microvascular vasodilatory dysfunction, and there is an urgent need for effective drugs to improve ischemic symptoms. Currently, the main drugs used clinically to treat angina pectoris include nitrates, calcium channel blockers, beta-blockers, ranolazine, antiplatelet drugs, and statins. While these drugs can exert some therapeutic effect by dilating coronary arteries or reducing myocardial oxygen consumption, they all lack selective dilation of the coronary arteries and are prone to various side effects during treatment. These include facial flushing and orthostatic hypotension caused by nitrates, cardiac depression caused by calcium channel blockers, negative inotropic and conduction problems caused by beta-blockers, and adverse reactions such as dizziness, bleeding, and elevated transaminase levels caused by other drugs, severely limiting their clinical application. Farnesin and 6-hydroxyflavone, as known flavonoids, have previously been reported to have anticancer and anti-inflammatory pharmacological activities, but no studies have yet revealed their selective dilation of coronary arteries and their anti-anginal effects. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide the application of farnesin or 6-hydroxyflavone in the preparation of antianginal drugs. These drugs improve myocardial blood supply by selectively dilating coronary arteries. Farnesin dilates coronary arteries by inhibiting myosin light chain kinase and activating myosin light chain phosphatase, while 6-hydroxyflavone achieves coronary artery dilation by inhibiting protein kinase C and enhancing myosin light chain phosphatase activity. Furthermore, farnesin or 6-hydroxyflavone can significantly alleviate isoproterenol-induced angina in rats, relieve myocardial ischemia symptoms, without affecting heart rate or systemic blood pressure, exhibiting superior safety and providing a promising drug option for the treatment of angina.

[0004] To achieve the above objectives, the present invention employs the following technical solution:

[0005] This invention provides the use of farnesin or 6-hydroxyflavone in the preparation of antianginal drugs.

[0006] Furthermore, the angina pectoris treated by the antianginal drug is non-obstructive coronary angina.

[0007] The core pathological feature of non-obstructive coronary angina is insufficient myocardial blood supply due to abnormal vascular tension in the coronary arteries without obvious obstruction. Acacia extract or 6-hydroxyflavone can specifically dilate the coronary arteries of the heart and relieve ischemic symptoms by improving myocardial blood flow perfusion, which is especially suitable for the treatment needs of this type of angina.

[0008] Furthermore, the antianginal drug is used to selectively dilate coronary arteries.

[0009] At therapeutic doses, acacia extract or 6-hydroxyflavone have a significantly stronger vasodilatory effect on coronary arteries than on peripheral resistance arteries. Since the tension of peripheral resistance arteries directly affects systemic blood pressure, this selectivity can avoid a drop in blood pressure caused by excessive peripheral vasodilation, thus solving the safety issues caused by the non-selective vasodilatory effect of traditional antianginal drugs.

[0010] Furthermore, the farnesin is administered in the form of a farnesin prodrug, wherein the farnesin prodrug is a water-soluble farnesin prodrug.

[0011] Furthermore, the water solubility of the water-soluble farnesin prodrug is ≥100 mg / mL.

[0012] Acacia extract injection is administered in the form of a water-soluble prodrug. Water-soluble prodrugs can be more conveniently administered via commonly used clinical routes of administration such as injection and nebulized inhalation, and can remain stable in the body environment and be effectively absorbed.

[0013] Furthermore, the active ingredient of the drug is farnesin or 6-hydroxyflavone; the daily dosage of the drug is 1-100 mg / kg.

[0014] Furthermore, when the active ingredient is farnesin, the daily dosage is 5-15 mg / kg.

[0015] Furthermore, when the active ingredient is 6-hydroxyflavone, the daily dosage is 5-15 mg / kg.

[0016] Acacia extract exerts its anti-anginal effect through a specific molecular mechanism. Specifically, it inhibits the activity of myosin light chain kinase (MLCK) in the smooth muscle cells of the coronary arteries, while activating the activity of myosin light chain phosphatase (MLCP). By regulating the phosphorylation state of myosin light chains, it reduces the sensitivity of vascular smooth muscle contractile proteins to calcium ions, induces coronary vasodilation, and ultimately improves myocardial blood supply, thus achieving the anti-anginal effect.

[0017] 6-Hydroxyflavonoids exert their anti-anginal effects through a specific pathway. They inhibit the activity of protein kinase C (PKC) in the smooth muscle cells of the coronary arteries, while enhancing the activity of myosin light chain phosphatase (MLCP), blocking the PKC-mediated vasoconstriction signaling pathway, promoting vascular smooth muscle relaxation, thereby dilating the coronary arteries, improving myocardial ischemia, and achieving the therapeutic goal of relieving angina pectoris.

[0018] Furthermore, the route of administration of the antianginal drug includes one of injection, oral administration, sublingual administration, and transdermal administration.

[0019] Furthermore, the antianginal drug is a pharmaceutical composition comprising pharmaceutically acceptable excipients.

[0020] Pharmaceutically acceptable excipients refer to carriers, excipients, or diluents that are routinely used in the pharmaceutical industry, are non-toxic and harmless to humans and animals, and are compatible with the active ingredient.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention utilizes farnesin or 6-hydroxyflavone as anti-anginal drugs to selectively dilate coronary arteries. The mechanism of action is independent of inhibiting smooth muscle calcium channels or activating potassium channels, thus effectively improving myocardial blood supply while avoiding adverse reactions such as decreased blood pressure and reflexive heart rate increase caused by non-selective systemic vasodilation of traditional drugs. Farnesin primarily dilates coronary arteries by inhibiting the RhoA-Rho kinase (RhoA-ROCK) signaling pathway and myosin light chain kinase, and activating myosin light chain phosphatase. 6-hydroxyflavone similarly dilates coronary arteries by inhibiting the protein kinase C pathway and enhancing myosin light chain phosphatase activity. In an isoproterenol-induced angina pectoris model in rats, farnesin or 6-hydroxyflavone significantly alleviated isoproterenol-induced myocardial ischemia symptoms without affecting heart rate or arterial blood pressure at therapeutic doses. Therefore, this invention provides acacia extract and 6-hydroxyflavone that exhibit superior safety characteristics while maintaining selective diastole of the coronary arteries, offering a promising drug option for the clinical treatment of non-obstructive angina pectoris. Attached Figure Description

[0023] Figure 1 The correlation diagram shows the diastolic effects of farnesin on isolated porcine coronary and mesenteric arteries pre-constricted with U46619 (a thromboxane A2 receptor agonist) and high potassium (KCl): Figure 1 A shows the original recorded curves of the vasodilatory effects of 1 μM, 3 μM, and 10 μM farnesin on the coronary artery (left panel) and mesenteric artery (right panel) of isolated porcine hearts pre-constricted by U46619. Figure 1 B is the concentration-dependent vasodilatory curve of farnesin on coronary and mesenteric arteries of the heart pre-constricted by U46619. Figure 1 C represents the concentration-dependent vasodilatory effect curve of farnesin on coronary and mesenteric arteries pre-constricted by high potassium. Figure 1 *P<0.05, **P<0.01 vs. coronary arteries).

[0024] Figure 2 The correlation diagram shows the effects of nifedipine and 6-hydroxyflavone on the vasodilatory effects of U46619 and high potassium on the coronary and mesenteric arteries of porcine heart: Figure 2 A is the vasodilatory effect curve of nifedipine on coronary and mesenteric arteries pre-constricted by U46619; Figure 2 B is the vasodilatory effect curve of nifedipine on coronary and mesenteric arteries of the heart pre-constricted by high potassium; Figure 2 C represents the vasodilatory effect curve of 6-hydroxyflavone on the coronary and mesenteric arteries of the heart pre-constricted by U46619; Figure 2 D represents the vasodilatory effect curve of 6-hydroxyflavone on coronary and mesenteric arteries of the heart pre-constricted by high potassium. Figure 2 *P<0.05, **P<0.01 vs. coronary arteries).

[0025] Figure 3 The figure shows the effect of farnesin and 6-hydroxyflavone on the concentration-dependent systolic response of the coronary arteries induced by calcium chloride (CaCl2). Figure 3 Figure A shows the effect of farnesin on the concentration-dependent contractile response of the coronary arteries induced by calcium chloride (CaCl2). Figure 3 Figure B shows the effect of 6-hydroxyflavone on the concentration-dependent systolic response of the coronary arteries induced by calcium chloride (CaCl2). Figure 3 Medium *P<0.05, **P<0.01 vs. vehicle).

[0026] Figure 4 The figure shows the effects of PKA (protein kinase A) inhibitors and PKC (protein kinase C) agonists on the coronary vasodilatory effects of farnesin and 6-hydroxyflavone. Figure 4 A and 4B represent the effects of the PKA inhibitor H-89 on the coronary vasodilatory effects of farnesin and 6-hydroxyflavone, respectively. Figure 4 C represents the effect of the PKC agonist PMA (12-myristoyl-13-acetylphorbolol) on the coronary vasodilatory effect of farnesin. Figure 4 D represents the effect of the PKC agonist PMA on the coronary vasodilatory effect of 6-hydroxyflavone (*P<0.05, **P<0.01 vs. solvent).

[0027] Figure 5 Figure showing the effects of inhibitors of RhoA-Rho kinase (RhoA-ROCK) signaling pathway and myosin signaling pathway-related molecules on the coronary vasodilatory effects of farnesin and 6-hydroxyflavone: Figure 5 A represents the effect of the ROCK inhibitor Y-27632 on the coronary vasodilatory effect of farnesin. Figure 5 B represents the effect of Y-27632 on the coronary vasodilatory effect of 6-hydroxyflavone; Figure 5 C and 5D represent the effects of the myosin light chain phosphatase inhibitor Calyculin-A (calyx spongymycin A) on the coronary vasodilatory effects of farnesin and 6-hydroxyflavone, respectively. Figure 5 E represents the effect of the myosin light chain kinase inhibitor ML-7 on the coronary vasodilatory effect of farnesin. Figure 5 F represents the effect of ML-7 on the coronary vasodilatory effect of 6-hydroxyflavone. Figure 5 Medium *P<0.05, **P<0.01 vs. vehicle).

[0028] Figure 6 The figure shows the effect of farnesin on the expression levels of molecules related to the coronary myosin signaling pathway: Figure 6 A represents the expression level of RhoA protein. Figure 6 B represents the expression level of ROCK2 (Rho kinase 2) protein. Figure 6 C represents the expression level of pMYPT1 (phosphorylated myosin light chain phosphatase targeting subunit 1). Figure 6 D represents the expression level of pCPI-17 (phosphorylated protein phosphatase 1 inhibitor 17) protein. Figure 6 E represents the MLCK protein expression level (n=5, **P<0.01 vs. solvent; #P<0.05, ##P<0.01 vs. U46619).

[0029] Figure 7 The figure shows the effect of 6-hydroxyflavone on the expression levels of coronary PKC and myosin signaling pathway-related molecules: Figure 7 A and 7B represent the expression levels of PKC and PKCδ (phosphorylated protein kinase C), respectively. Figure 7 C represents the expression level of pCPI-17 protein. Figure 7 D represents the expression level of pMYPT1 protein (n=5, **P<0.01 vs. solvent group; #P<0.05, ##P<0.01 vs. U46619).

[0030] Figure 8 The figure shows the effect of farnesin on coronary blood flow in isolated hearts and blood pressure in anesthetized rats: Figure 8A showed that farnesin can increase coronary blood flow in isolated rat hearts in a concentration-dependent manner; Figure 8 B shows the plasma abacus concentration changes at different time points after intravenous infusion of abacus prodrug in rats. Figure 8 C showed that, at concentrations that effectively increased coronary blood flow, farnesin did not affect the heart rate, systolic blood pressure, or diastolic blood pressure in anesthetized rats.

[0031] Figure 9 The related figure shows the antagonistic effect of farnesin and 6-hydroxyflavone on isoproterenol-induced angina in rats: Figure 9 A is a representative electrocardiogram of rats with isoproterenol-induced angina pectoris, showing downward extension of the S wave and flattening of the T wave; Figure 9 B shows a representative electrocardiogram of a rat with angina pectoris after treatment with acacia extract, which shows a significant improvement in myocardial ischemia-related waveforms. Figure 9 C represents the statistical effect of farnesin, nifedipine, and 6-hydroxyflavone on the amplitude of S-wave depression in the electrocardiogram of rats with angina pectoris (##P<0.01 vs. control; *P<0.05, **P<0.01 vs. saline or DMSO). Detailed Implementation

[0032] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the application will be further described in detail below with reference to embodiments. However, this should not be construed as limiting the scope of this application to the following examples. All other embodiments obtained by those skilled in the art without creative effort without departing from the above-described methodological spirit of this application are within the scope of protection of this application.

[0033] The singular forms “for,” “or,” “a,” “any,” and “described” used in this application are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, the terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0034] The core of this invention lies in providing the application of farnesin and 6-hydroxyflavone in the preparation of antianginal drugs. Its key advantage is its selective vasodilatory effect on the coronary arteries without affecting peripheral resistance arterial tension or systemic blood pressure. This addresses the clinical pain point of existing antianginal drugs (such as nifedipine) causing hypotension due to a lack of vascular selectivity. To comprehensively verify the scientific validity, effectiveness, and safety of this technical solution, a detailed demonstration will be conducted from five dimensions: vascular selective vasodilatory function, molecular mechanism of action, in vitro and in vivo efficacy, in vivo safety, and antianginal pharmacological activity.

[0035] Experimental animals and vascular samples: The porcine coronary arteries (taken from isolated porcine hearts) and mesenteric arteries (taken from the greater omentum of porcine vessels, belonging to peripheral resistance arteries) used for the determination of isolated arterial vascular tension in this study were obtained from fresh porcine hearts and greater omentum from the Sha Tin Slaughterhouse in Hong Kong or the Xinle Meat Processing Centre in Nanjing. After acquisition, they were immediately placed in oxygenated frozen Krebs-Heneseleit solution and rapidly transferred to the laboratory. Subsequently, the target vessels were isolated and prepared into 2-3 mm vascular rings, which were stored in preservation solution at 4°C (maximum storage time 48 hours) for later use. The experimental animals were male SD rats, 6-8 weeks old and weighing 200-300g. All experimental protocols were approved by the Animal Research Ethics Committee of the Faculty of Medicine, University of Hong Kong.

[0036] Example 1

[0037] Measurement of isolated arterial vessel tension and verification of selective vasodilation:

[0038] 1) Vascular ring balancing: The prepared coronary artery or mesenteric artery vascular rings were connected and fixed to a support using stainless steel triangular rings. The rings were placed in a McLaurel bath containing 15 mL of Krebs-Hensseleit balanced salt solution. A mixture of 95% oxygen and 5% carbon dioxide was continuously circulated through the bath, and the temperature was maintained at 37°C. The vascular rings were then connected to a tension sensor, which was then connected to an RM5240 multi-channel physiological signal acquisition and analysis system manufactured by Chengdu Instrument Factory to record changes in vascular tension. During the experiment, a preload of 1.5 g was continuously applied to the sample, and balancing was maintained for 1 hour until the tension stabilized, thus ensuring the stable function of the vascular rings.

[0039] 2) Preliminary detection of vasomotor function and endothelial integrity: Vascular function was detected by stimulating blood vessels with 60mM KCl, followed by vasodilation with normal Krebs-Heneseleit solution. This process was repeated until the difference in vasoconstriction amplitude induced by two consecutive 60mM KCl tests was less than 10%, indicating normal vasomotor function. Only then could the specimen be used for formal experiments. For endothelial integrity detection, prostaglandin H2 / thromboxane A2 analog U46619 was used. Blood vessels were stimulated at a concentration of 20nM until the vasomotor contraction reached a plateau. Then, 10μM bradykinin was added. If significant vasodilation occurred, it indicated that the endothelium was intact.

[0040] 3) Drug administration: In the formal experiment, 20 nM U46619 or 60 mM KCl (high potassium) was used to stimulate the arterial vessels to pre-constrict to a steady-state level, and then the cumulative concentration of the experimental drug on the vasodilatory effect was observed. The results are as follows: Figure 1 and Figure 2 As shown.

[0041] Results and Analysis:

[0042] Verification of the selective vasodilatory effect of farnesin: The differences in the vasodilatory effects of farnesin on coronary and mesenteric artery vascular rings were compared in isolated coronary artery and mesenteric artery vascular ring specimens. Figure 1 As shown in Figure A, based on the coronary and mesenteric artery contraction induced by 20nMU46619, the administration of farnesin at concentrations of 1 μM, 3 μM, and 10 μM showed that, at the same concentration, farnesin had a significantly stronger effect on dilating the coronary arteries than on the mesenteric arteries.

[0043] Furthermore, the differences in the vasodilatory effects of farnesin at concentrations of 1 μM, 3 μM, 10 μM, and 30 μM were investigated in coronary and mesenteric arteries pre-constricted with 20 nM U46619 or 60 mM KCl. Figure 1 B represents the concentration-dependent vasodilatory curve of farnesin on the pre-constrictive vasoconstriction effect of U46619. Figure 1 C represents the concentration-dependent vasodilatory curve of acaciain in response to high potassium pre-constriction. The vasodilatory curve was fitted using the Hill equation to obtain the 50% effective concentration (EC50) of acaciain in the coronary and mesenteric arteries. 50 ): Acaciain-induced vasoconstriction of coronary and mesenteric arteries by U46619 50 ECGs of 5.4 μM and 10.6 μM, respectively, induced vasoconstriction in the coronary and mesenteric arteries by high potassium levels. 50 The concentrations were 9.2 μM and 18.5 μM, respectively. The results confirmed that, regardless of whether it was under U46619 or high-potassium pre-constriction conditions, the vasodilatory effect of farnesin on the coronary arteries was significantly stronger than its vasodilatory effect on the mesenteric arteries, indicating that farnesin has a selective vasodilatory effect on the coronary arteries.

[0044] Verification of the selective vasodilatory effect of 6-hydroxyflavone: The difference in the vasodilatory effect of 6-hydroxyflavone on the coronary and mesenteric arteries pre-constricted with 20 nM U46619 or 60 mM KCl was investigated. Figure 2 C represents the concentration curve of the diastolic effect of 6-hydroxyflavone on the coronary and mesenteric arteries pre-constricted by U46619. Figure 2 D represents the concentration curve of 6-hydroxyflavone on the vasodilatory effect of high-potassium preconstricted coronary and mesenteric arteries. The EC50 of 6-hydroxyflavone on the vasodilatory effect of U46619 on preconstricted coronary and mesenteric arteries is shown. 50 EC50 showed that 7.4 μM and 17.5 μM, respectively, had vasodilatory effects on coronary and mesenteric arteries pre-constricted by high potassium. 50 The concentrations were 12.2 μM and 30.1 μM, respectively. The results showed that the effects of 6-hydroxyflavone were similar to those of farnesin, exhibiting a selective vasodilatory effect on the coronary arteries of the heart.

[0045] Verification of Selective Vasodilation Effect of Control Drug: Nifedipine is a commonly used drug in clinical practice for the treatment of angina pectoris. To clarify the selective advantage of the drugs acacia extract and 6-hydroxyflavone in this invention, nifedipine was used as a control drug. Its vasodilatory effect on preconstricted vessels (20 nM U46619 or 60 mM KCl) was studied in coronary and mesenteric artery specimens. Results are as follows... Figure 2 As shown in A and 2B, nifedipine has almost equal vasodilatory effects on coronary and mesenteric artery vasoconstriction caused by U46619 and high potassium pre-constriction: it dries the EC50 of coronary and mesenteric arteries pre-constricted by U46619. 50 EC50 levels were 59.6 nM and 46.9 nM, respectively, for diastolic high-potassium presystolic coronary and mesenteric arteries. 50 The concentrations were 4.6 nM and 3.5 nM, respectively, indicating that the control substance nifedipine does not have a selective diastolic effect on the coronary arteries.

[0046] This result precisely explains why nifedipine treatment for angina causes a decrease in systemic blood pressure: the mesenteric artery is a peripheral resistance artery, and its vascular tone directly affects the peripheral vascular resistance of the systemic circulation, which is closely related to the maintenance of systemic blood pressure. Nifedipine lacks vascular selectivity; while dilating coronary arteries to increase coronary blood supply and relieve myocardial ischemia, it also dilates peripheral resistance arteries such as the mesenteric artery to an equal extent, leading to a significant decrease in peripheral vascular resistance. According to the physiological mechanism of blood pressure formation, a decrease in peripheral vascular resistance directly leads to a reduction in blood pressure against the vessel walls, ultimately causing a decrease in systemic blood pressure. This is the core reason why hypotension may occur as a side effect of nifedipine in clinical use.

[0047] Example 2

[0048] Investigation into the molecular mechanism by which farnesin and 6-hydroxyflavone dilate coronary arteries:

[0049] 1) Calcium Sensitization Investigation Experiment: Vasodilatory-constrictive states are related to the sensitivity of intracellular myosin to calcium ions, which is related to the phosphorylation level of myosin. To investigate whether the dilation of coronary arteries by farnesin and 6-hydroxyflavone is related to the sensitivity of intracellular contractile proteins in smooth muscle cells to calcium ions, the following experiment was conducted:

[0050] After the coronary arteries were equilibrated in the specimen bath, they were pretreated for 50 min by incubation in calcium-free physiological solution with DMSO, 10 µM farnesin, or 10 µM 6-hydroxyflavone, respectively. Then, CaCl2 at concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 mM was added to the tissue bath every 20 min to observe the response of the coronary arteries to different concentrations of CaCl2. 2+ Changes in contraction tension, results as follows Figure 3 As shown.

[0051] 2) PKA / PKC Pathway Investigation Experiment: Protein kinases PKA and PKC signaling pathways regulate vasodilation and vasoconstriction. cAMP (cyclic adenosine monophosphate) and the cAMP-mediated PKA signaling pathway promote phosphorylation of the MYPT1 subunit of MLCP (myosin light chain phosphatase) in a cAMP-dependent manner, block Rho-related kinase ROCK phosphorylation, and induce MLC (myosin light chain) dephosphorylation, thus causing vasodilation. PKC inhibits myosin light chain phosphatase by promoting the phosphorylation of CPI-17, thereby inducing vasoconstriction. It can also directly cause vasoconstriction by activating myosin light chain kinase activity, leading to myosin light chain phosphorylation. To investigate whether the dilation of coronary arteries by farnesin and 6-hydroxyflavone is related to the activation of the PKA signaling pathway and the inhibition of the PKC pathway, the following experiment was conducted:

[0052] The coronary arteries were incubated with 100 nM PKA inhibitor H-89 for 30 min, and the vasodilatory effects of farnesin and 6-hydroxyflavone on U46619-preconstricted coronary arteries were detected. The coronary artery annulus was pre-incubated with 100 nM PKC agonist PMA (Phorbol 12-myristate 13-acetate) for 30 min, and the vasodilatory effects of farnesin and 6-hydroxyflavone on 20 nM U46619-preconstricted vessels were detected. The results are as follows: Figure 4 As shown.

[0053] 3) Rho-ROCK Pathway Investigation Experiment: Rho-related kinases (Rho kinase / ROCK) have pleiotropic functions, including regulating smooth muscle cell contraction. The Rho kinase / ROCK signaling pathway is an important pathway for smooth muscle cell calcium sensitization, phosphorylating myosin light chain phosphatase target subunit 1 and regulating vascular function. The ROCK signaling pathway can also promote myosin light chain phosphorylation through a similar effect to myosin light chain kinase, resulting in vasoconstriction. To investigate whether the dilation of coronary arteries by farnesin and 6-hydroxyflavone is related to the inhibition of the Rho-ROCK pathway, the following experiment was conducted:

[0054] Coronary artery vascular rings were incubated for 30 min with 1 µM ROCK inhibitor Y-27632, 100 nM myosin light chain phosphatase inhibitor Calyculin-A, 10 µM myosin light chain kinase inhibitor ML-7, or solvent DMSO, respectively, to detect whether these inhibitors affected the dilation of coronary artery vascular rings pre-contracted by farnesin and 6-hydroxyflavone.

[0055] Results and Analysis:

[0056] The effect of vasodilation is related to calcium sensitization: according to Figure 3The results showed that coronary arteries pretreated with farnesin or 6-hydroxyflavone exhibited significantly reduced contractile tension induced by the same CaCl2 concentration gradient. This indicates that both substances reduce the sensitivity of intracellular contractile proteins to calcium ions, thereby inducing myosin dephosphorylation and ultimately achieving coronary artery dilation.

[0057] The relationship between vasodilation and the PKA / PKC pathway: According to Figure 4 The results showed that the PKA inhibitor H-89 did not affect the diastolic effect of farnesin and 6-hydroxyflavone on U46619 preconstricted coronary arteries, indicating that their diastolic effect was not related to the activation of the PKA signaling pathway. However, the PKC agonist PMA did not affect the diastolic effect of farnesin, but significantly reduced the diastolic effect of 6-hydroxyflavone, indicating that farnesin was not related to the inhibition of the PKC signaling pathway, while the diastolic effect of 6-hydroxyflavone depended on the inhibition of the PKC signaling pathway.

[0058] The relationship between vasodilation and the Rho-ROCK pathway: According to Figure 5 The results showed that the ROCK inhibitor Y-27632 could enhance the coronary vasodilatory effect of farnesin on U46619 preconstriction. Figure 5 A), but it has no enhancing effect on 6-hydroxyflavone ( Figure 5 B); the myosin light chain phosphatase inhibitor Calyculin-A significantly inhibited the coronary vasodilatory effects of acaciain and 6-hydroxyflavone (B). Figure 5 C Figure 5 D), and its inhibitory effect on acaciatin is stronger; the myosin light chain kinase inhibitor ML-7 significantly enhances the coronary artery dilating effect of acaciatin (D). Figure 5 E), but does not affect 6-hydroxyflavone ( Figure 5 F). The results indicate that farnesin may dilate coronary arteries by inhibiting ROCK and myosin light chain kinase and enhancing myosin light chain phosphatase activity, while 6-hydroxyflavone, in addition to inhibiting PKC, also exerts its effect by enhancing myosin light chain phosphatase activity.

[0059] Example 3

[0060] Coronary artery vascular-related protein expression detection:

[0061] The levels of vascular smooth muscle-related proteins were detected using Western blotting analysis.

[0062] 1) Sample preparation: The coronary artery tissue samples were weighed, cut into small pieces, wrapped in aluminum foil, placed in cryovials, and flash-frozen in liquid nitrogen for later use. The flash-frozen tissue was placed in a mortar, ground with a small amount of RIPA lysis buffer, transferred to 2 mL EP tubes, homogenized using a homogenizer, and then centrifuged at 16,000 rpm for 15 min in a refrigerated high-speed centrifuge. The supernatant was collected for later use.

[0063] 2) Protein concentration determination and denaturation: The protein concentration in the supernatant was determined by the BCA method. Proteins of equal concentration were mixed with SDS sample buffer and denatured at 95°C for 5 min.

[0064] 3) Electrophoresis and membrane transfer: The denatured sample was loaded with 8% SDS-PAGE solution for electrophoresis, and then transferred to a nitrocellulose membrane after electrophoresis.

[0065] 4) Blocking and Incubation: Block the membrane with 5% nonfat milk in 0.1% Tris-buffered saline (TTBS) containing Tween for 2 hours. After blocking, add primary antibodies for detection. The primary antibodies used include anti-ROCK2, MLCK, pMYPT1 (Thr853), PKC, pPKCδ antibody (Affinity Bioscience), and anti-RhoA, pCPI-17, and GAPDH antibody (Santa Cruz Biotech). Incubate overnight at 4°C. Wash three times with TTBS, and then incubate the membrane with secondary antibody m-IgGkBP-HRP (Santa Cruz Biotech) in TTBS at room temperature for 2 hours.

[0066] 5) Development and quantification: The membrane was washed three times with TTBS, and then the protein bands were visualized on X-ray film using a chemiluminescence detection system (ECL, GE Healthcare). The band intensity was measured using the image analysis software Gel-Pro Analyser.

[0067] To investigate the effects of farnesin and 6-hydroxyflavone on the levels of calcium sensitization-related proteins in the coronary arteries, the following experiments were conducted:

[0068] Coronary arteries were treated in a tissue bath for 60 min with dimethyl sulfoxide (DMSO), U46619, and U46619 with 10 µM or 30 µM farnesin or 10 µM or 30 µM 6-hydroxyflavone, respectively. Coronary artery tissue proteins were then extracted and analyzed by Western blotting. The effects of farnesin on the expression levels of RhoA-ROCK pathway-related proteins (RhoA, ROCK2, pCPI-17, pMYPT1) and myosin light chain kinase (MLCK) were investigated, as were the effects of 6-hydroxyflavone on the expression of PKC, PKCδ, pCPI-17, and pMYPT1. The results are as follows: Figure 6 and Figure 7 As shown.

[0069] Results and Analysis:

[0070] like Figure 6 As shown, in solvent-treated coronary arteries, U46619 significantly upregulated RhoA (… Figure 6 A), ROCK2 Figure 6 B), Myosin phosphatase target subunit-1 (pMYPT1Thr853), Figure 6 C), pCPI-17, and myosin light chain kinase (MLCK, Figure 6 The protein expression level of E) indicates that the process of U46619-induced coronary artery vasoconstriction is closely related to the activation of the aforementioned vasoconstriction-related proteins. Furthermore, 10µM and 30µM concentrations of farnesin significantly inhibited the increase in the expression of these proteins induced by U46619, suggesting that farnesin exerts its effect of dilating coronary arteries by inhibiting the activity of these vasoconstriction-related proteins and indirectly activating myosin phosphatase.

[0071] Figure 7 To analyze the molecular mechanism of 6-hydroxyflavone in dilating coronary arteries using Western blotting, U46619 significantly upregulated PKC in solvent-treated coronary arteries. Figure 7 A), PKCδ ( Figure 7 B), pCPI-17 ( Figure 7 C) and pMYPT1 ( Figure 7 The protein expression levels of D) were significantly reduced; however, after treatment with 10µM and 30µM 6-hydroxyflavone, the elevated protein expression levels induced by U46619 were significantly inhibited. This indicates that 6-hydroxyflavone activates myosin light chain phosphatase (MLCP) by inhibiting the expression of PKC, PKCδ, pCPI-17 and pMYPT1, thereby exerting a dilating effect on coronary arteries.

[0072] Example 4

[0073] Detection of the effects of isolated rat heart perfusion and coronary blood flow:

[0074] 1) Preparation and perfusion of isolated heart: Male SD rats (6-8 weeks old, 200-300g) were anesthetized with sodium phenobarbital, and the heart was isolated after thoracotomy and placed in a culture dish containing Krebs-Hensseleit (KH) solution. The connective tissue and fat around the heart were removed. The aorta of the heart was fixed on a Langendorff isolated perfusion device, and perfusion was performed using Krebs-Hensseleit solution at 37℃ and oxygenated with 95% O2 + 5% CO2. Coronary blood flow was measured. After the coronary blood flow stabilized, the effect of different drug concentrations on the coronary blood flow of isolated rat hearts was determined.

[0075] 2) Coronary blood flow measurement: The experiment was divided into a solvent control group (DMSO) and a farnesin administration group, with farnesin at three concentration gradients of 0.3, 1, and 3 μM. After the perfusion system stabilized, the corresponding concentration of farnesin or an equal volume of solvent was added to the perfusion fluid, and the coronary blood flow was continuously recorded at each time point. The sample size for each group was n=5. Statistical analysis was performed using one-way ANOVA. The results are shown below. Figure 8 As shown.

[0076] Results and Analysis:

[0077] In vitro perfusion experiments showed no significant change in coronary blood flow in the solvent control group (DMSO), while farnesin increased coronary blood flow in isolated rat hearts in a concentration-dependent manner (n=5, P<0.01 vs. solvent group). Figure 8 A) confirmed that farnesin can increase coronary blood flow by dilating coronary arteries.

[0078] Example 5

[0079] Detection of arterial blood pressure and plasma farnesin concentration in rats:

[0080] 1) Animal anesthesia and intubation: Rats were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, with anesthetic supplements administered as needed during the experiment; animals were tracheally intubated, and the room was ventilated with indoor air, with the body temperature maintained at 37°C using a temperature control system. A PE-50 catheter was used to measure blood pressure via the right carotid artery; a jugular vein catheter was used for drug administration, and a femoral vein catheter was used for blood collection.

[0081] 2) Administration: Administer the water-soluble farnesin prodrug (99% purity) via constant-rate intravenous infusion at a rate of 250 μg / kg / min for 40 minutes. Previous studies have confirmed that this water-soluble prodrug can be rapidly converted into farnesin in vivo to exert its pharmacological effects.

[0082] 3) Indicator monitoring and sample collection: Throughout the process, the ECG and arterial blood pressure in lead II were monitored using the RM6240 multi-channel physiological signal acquisition and analysis system. Approximately 0.2 mL of blood samples were collected from the femoral vein before administration (0 min), and 5 min, 10 min, 30 min, and 40 min after administration, and placed in heparinized tubes for later use.

[0083] 4) Plasma sample testing: Plasma samples were processed using methanol liquid-liquid extraction, with pentamethylquercetin as an internal standard. Detection was performed using a Waters HPLC system (equipped with an Alltech C18 column, inner diameter 250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol-water-phosphoric acid (70:30:0.15), with a flow rate of 1.0 mL / min and a UV detection wavelength of 260 nm. This was used to quantitatively analyze the concentration of farnesin in the plasma. The results are as follows: Figure 8 As shown.

[0084] Results and Analysis:

[0085] Plasma concentration measurements showed that the concentration of farnesin gradually increased during drug administration, reaching a steady-state level at 15 minutes, and reaching a plasma concentration of 1245 ng / mL (4.2 μM, n=5) at 30 minutes. Figure 8 B), after drug administration was stopped, plasma acaciain levels gradually decreased; throughout the process, monitoring showed no significant fluctuations in rat heart rate, and no significant decreases in arterial systolic or diastolic blood pressure. Figure 8 C). The results showed that the concentration of farnesin observed in isolated hearts that significantly increased coronary blood flow did not affect heart rate or arterial blood pressure in anesthetized rats, indicating that farnesin has a selective diastolic effect on the coronary arteries.

[0086] Example 6

[0087] Establishment of a rat model of angina pectoris using isoproterenol and verification of the anti-anginal effect of the drug:

[0088] 1) Experimental animals and pretreatment: Rats were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital. The anesthetic was supplemented as needed during the experiment. The body temperature was maintained at 37°C by a temperature control system. Tracheal intubation was performed and room air was introduced to ensure unobstructed breathing.

[0089] 2) Model Construction and Monitoring Preparation: A PE-50 catheter was used to measure blood pressure via the right carotid artery. During the experiment, data acquisition and monitoring of lead II electrocardiogram (RM6240) and arterial blood pressure were conducted. Sublingual vein puncture was performed using a pediatric scalp needle for drug infusion. Isoproterenol (10µg / kg / min) was injected intravenously at a constant rate for 5 minutes to establish an angina pectoris model (based on ST segment changes on electrocardiogram) and observe the therapeutic effect of the drug on angina pectoris.

[0090] 3) Grouping and administration: Rats were randomly divided into 6 groups, with 8-10 rats in each group. The specific grouping and administration methods are as follows:

[0091] Saline group (n=10): 1 mL / kg of normal saline was injected intravenously;

[0092] The solvent control group (DMSO group, n=8): 1 mL / kg DMSO was injected intraperitoneally;

[0093] Acacia prodrug group (n=8 / group): 5 mg / kg, 10 mg / kg, and 15 mg / kg of water-soluble acacia prodrug were administered intravenously;

[0094] 6-Hydroxyflavonoid group (n=8-10 / group): Intraperitoneal injection of 5 mg / kg, 10 mg / kg, and 15 mg / kg of 6-hydroxyflavonoid;

[0095] Positive control group (nifedipine group, n=9): 50 μg / kg nifedipine (a commonly used antianginal drug in clinical practice) was injected intravenously.

[0096] Arterial systolic blood pressure, diastolic blood pressure, and heart rate were recorded before and after drug administration. Simultaneously, ST segment changes on electrocardiograms were dynamically recorded at 1 min, 3 min, and 5 min after isoproterenol infusion. The mean ST segment depression amplitude for each group was statistically analyzed. Results are as follows: Figure 9 As shown.

[0097] Results and Analysis:

[0098] like Figure 9 As shown, after isoproterenol infusion in the saline and DMSO groups, the electrocardiograms exhibited typical features of myocardial ischemia—significant ST segment depression and slightly flattened T waves. Figure 9 A); Both the acacia prodrug group and the 6-hydroxyflavone group showed a dose-dependent reduction in ST segment depression. Figure 9 (B, 9C) Among them, the ST segment depression amplitude of the 5-15 mg / kg acacia extract group and the 5-15 mg / kg 6-hydroxyflavone group was significantly reduced at 5 min after infusion compared with the normal saline group (P<0.05 or P<0.01); the nifedipine group also significantly inhibited ST segment depression (P<0.01). The above results confirm that acacia extract and 6-hydroxyflavone have significant antianginal effects.

[0099] Effects on arterial blood pressure: Table 1 shows the comparison of blood pressure data before and after administration. The results showed that the systolic blood pressure in the nifedipine group decreased significantly by 15.5 mmHg compared to before administration (P<0.01), and the diastolic blood pressure also decreased synchronously. However, there were no significant changes in systolic blood pressure, diastolic blood pressure, and heart rate in the various dose groups of the farnesol prodrug and the 6-hydroxyflavone group (P>0.05). This difference indicates that farnesol and 6-hydroxyflavone, unlike nifedipine, do not cause a decrease in systemic blood pressure when exerting their antianginal effect, demonstrating superior safety.

[0100] Table 1. Statistical analysis of changes in arterial blood pressure (systolic / diastolic) and heart rate before and after drug administration in each group of anesthetized rat models (mean ± standard deviation)

[0101]

[0102] Through the above embodiments, this invention comprehensively verifies the core value of farnesin and 6-hydroxyflavone as antianginal drugs. Not only are they structurally simple, but they also possess unique pharmacological effects, namely significant selective coronary artery dilation. Their mechanism of action differs from traditional antianginal drugs, not relying on inhibition of smooth muscle calcium channels or activation of potassium channels. In vitro and in vivo experiments have confirmed that both can effectively increase coronary blood flow and improve symptoms of myocardial ischemia in angina pectoris. Furthermore, at therapeutic doses, they do not affect systemic blood pressure and heart rate, exhibiting significantly better safety than existing drugs such as nifedipine. These results fully demonstrate that farnesin and 6-hydroxyflavone are highly promising natural small-molecule antianginal drug candidates, which can be used alone or in combination in clinical applications. They provide a new direction for the precise treatment of clinical antianginal diseases, especially non-obstructive coronary angina, and have significant clinical translational value and application prospects.

[0103] The above results demonstrate and describe the basic principles and main features of this application, as well as its advantages.

[0104] The above results and accompanying drawings describe embodiments of the present invention, but the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and these all fall within the protection scope of the present invention.

Claims

1. Application of farnesin or 6-hydroxyflavone in the preparation of antianginal drugs.

2. The application according to claim 1, characterized in that, The angina pectoris treated by the antianginal drugs is non-obstructive coronary angina.

3. The application according to claim 1, characterized in that, The antianginal drug is used to selectively dilate the coronary arteries of the heart.

4. The application according to claim 1, characterized in that, The acacia extract is administered in the form of acacia extract prodrug, and the acacia extract prodrug is a water-soluble acacia extract prodrug.

5. The application according to claim 4, characterized in that, The water solubility of the water-soluble farnesin prodrug is ≥100 mg / mL.

6. The application according to claim 1, characterized in that, The active ingredient of the antianginal drug is farnesin or 6-hydroxyflavone; the daily dosage of the antianginal drug is 1-100 mg / kg.

7. The application according to claim 6, characterized in that, When the active ingredient is farnesin, the daily dosage is 5-15 mg / kg.

8. The application according to claim 6, characterized in that, When the active ingredient is 6-hydroxyflavone, the daily dosage is 5-15 mg / kg.

9. The application according to claim 1, characterized in that, The routes of administration for the antianginal drugs include injection, oral administration, sublingual administration, and transdermal administration.

10. The application according to any one of claims 1-9, characterized in that, The antianginal drug is a pharmaceutical composition containing pharmaceutically acceptable excipients.

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