Trends Sci. 2025; 22(9): 10496

Hypertension remains one of the leading global health concerns, contributing significantly to cardiovascular morbidity and mortality worldwide [1,2]. Despite the availability of a wide range of antihypertensive medications, limitations such as side effects, resistance, and suboptimal efficacy in certain populations underscore the need for novel therapeutic agents with improved safety and multi-target profiles. In this context, natural products have gained increasing attention as alternative or complementary sources of cardiovascular therapy. Their diverse bioactive compounds—especially polyphenols, flavonoids, and tannins-exhibit antioxidant, anti-inflammatory, and vasomodulatory properties, which are beneficial in the management of hypertension. Notably, these compounds have been shown to modulate critical vascular mechanisms, including endothelial function and ion channel activity, particularly voltage-gated Ca²⁺ channels. Since these channels play a pivotal role in vascular smooth muscle contraction and blood pressure regulation, targeting them presents a promising strategy for antihypertensive intervention [3]. However, its direct vascular effects and interaction with key ion transport mechanisms remain insufficiently explored. Therefore, the present study aims to investigate the antihypertensive effects of A41 through a multidisciplinary approach combining in vitro, in vivo, and in silico methodologies. We examined its influence on calcium handling in vascular smooth muscle, its systemic blood pressure-lowering capacity, and its molecular interactions with calcium transport proteins. This integrated strategy is designed to provide mechanistic insight into A41’s mode of action and support its potential development as a novel, nature-derived antihypertensive agent. This study aims to evaluate the antihypertensive potential of A41, a natural bioactive compound, by examining its effects on calcium ion channels through in vitro, in vivo, and in silico approaches.

Materials and methods

Synthesis of propyl ester of gallic acid - A41.

2 g of gallic acid and 50 mL of propyl alcohol and 1 mL of sulfuric acid for the catalyst were placed in a round-bottom flask with a reverse cooler and heated in a nitrogen atmosphere at 98 - 100 C for 8 - 10 h [6,7]. Every half hour, the reaction was checked using thin-layer chromatography. After the reaction, the excess alcohol was distilled under pressure and diluted in 10 mL of ethyl acetate. The organic part was washed 1^st with 15 mL of NaCl water and then with 15 mL of a 5 % NaHCO₃ solution. The separated precipitate was extracted 3 times with ethyl acetate and evaporated under low pressure (Figure 1). Propyl ester of gallic acid is a white-yellow solid substance. The reaction yield is 75 %. The product was checked using thin-layer chromatography [8]. (1:1 hexane/EtOAc), Rf = 0.87. Melts at 145 - 147 C.

Chemicals

Phenylephrine, phentolamine, and verapamil (≥98 % purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animal ethics

All preoperative care procedures and experimental protocols were examined and approved by the Institutional Animal Use Committee. The animals were kept in standard vivarium conditions, including 55 - 65 % humidity, 22 ± 2 C temperature, and ad libitum access to water and laboratory chow. All animal handling was in accordance with the European Directive 2016/63/EU on the protection of animals used for scientific purposes. Ethical clearance was given by the Animal Ethical Committee of the Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan (Protocol No. 133/1a/h, 4 August 2016).

Tissue preparation

Surgery was performed under sodium pentobarbital anesthesia to reduce distress. Experiments were performed on aortic tissue from healthy white male rats (200 - 250 g). Animals were killed by cervical dislocation. Thoracic cavity was opened, and aorta was removed, cleaned of adhering fat and connective tissue, and cut into 3 - 4 mm segments. The aortic rings were put in a 5 mL organ bath filled with Krebs-Henseleit physiological solution with (in mM): NaCl 120.4, KCl 5, NaHCO₃ 15.5, NaH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 2.5, glucose 11.5 and HEPES-buffered to pH 7.4. For certain experiments, a Krebs solution free of Ca²⁺ and with 1 mM EGTA was used. The bath solution was continuously gassed with carbogen (95 % O₂ and 5 % CO₂) and maintained at 37 C using a DAIHAN WATER BATH ultrathermostat [9].

Aortic-ring contraction studies

The aortic rings were suspended in a Radnoti isometric transducer system (USA) using platinum wire hooks and equilibrated for 60 min under normal conditions. The preparations were all stretched with an initial load of 1 g (10 mN). Contractile responses were transferred from the transducer to a signal amplifier and were digitally recorded by a Go-link automatic converter interfaced with a computer. Data were analyzed with Origin Pro v.8.5 SR1 (EULA, Northampton, MA, USA). The isometric contraction force (mN) of the rat aortic tissue in vitro was calculated as a percentage (%) for statistical analysis. The preparations of vascular smooth muscle were studied on an apparatus. The organ bath (5 mL) was connected via a special reservoir circulating the Krebs-Henseleit physiological solution (Figure 2). The physiological temperature was constantly maintained with the help of a thermostat, and the solution was continuously gassed with a mixture of 95 % O₂ and 5 % CO₂. The contractile activity of the aortic vascular segment, mounted in the experimental chamber, was recorded with the help of an ISOMETRIC TRANSDUCER (Grass Instrument, USA) and displayed on the GoLink signal amplifier and support system [10].

Figure 1 Synthesis scheme of the propyl ester derivative of gallic acid (A41).

Figure 2 Diagram of the experimental setup used to monitor isometric contractions in isolated rat aortic vascular tissue. (1) The organ bath (5 mL) is connected to a dedicated reservoir for solution circulation. (2) Krebs-Henseleit solution is used to maintain physiological conditions. (3) A thermostat ensures stable temperature regulation. (4) The system is continuously aerated with a gas mixture containing 95 % oxygen and 5 % carbon dioxide. The aortic tissue is mounted within the experimental chamber for contraction assessment. (5) An isometric transducer (Grass Instrument, USA) captures mechanical responses, while (6) GoLink devices handle signal amplification and system integration.

Blood pressure measurements

Tail-cuff plethysmography using the Sistola device (Neurobotics, Russia) was performed after acclimating animals for 3 consecutive days. Blood pressure readings were taken in triplicate per session. The experiments took place at the “BFM Pharmacology and Screening Laboratory” and the “Plant Cytoprotectors” laboratory of the A. Sodikov Institute of Bioorganic Chemistry. Data were analyzed using the specialized software “AcqKnowledge 4.2 for MP150” (Figure 3).

Figure 3 The “Sistola” device (Neurobotics, Russia) used for non-invasive measurement of arterial blood pressure in rats via the tail artery.

Statistics

Statistical evaluation and graphical representations were performed using Origin Pro 8.5 software (USA). Contractile responses were expressed as a percentage of the maximum contraction induced by phenylephrine (10 mM) or KCl (50 mM) and are shown as mean values from 4 to 6 independent trials (n = 4 - 6). Paired t-tests were used for analyzing combined data sets, while unpaired t-tests assessed differences between separate groups. Results were considered statistically significant at the threshold of p < 0.05.

Molecular docking “software and databases”

The software utilized in this research is freely accessible for academic purposes [11]. The Protein Data Bank (PDB) serves as a global repository for 3-dimensional structural information of biological macromolecules [12]. Protein structures associated with calcium signaling and regulation were retrieved from the PDB, including the L-type calcium channel Cav1.2 (PDB ID: 6jp5), R-type calcium channel Cav2.3 (PDB ID: 7xlq), sodium-calcium exchanger NCX1 (PDB ID: 8sgi), ryanodine receptor type 2 RyR2 (PDB ID: 5c33), and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase SERCA (PDB ID: 6rb2). Additionally, the PubChem database-a comprehensive resource integrating pharmacological, pharmaceutical, chemical, and molecular target data (such as sequences, structures, and pathways)-was used to obtain flavonoid compounds and reference molecules for this study [13]. Each DrugCard entry in PubChem contains over 80 data fields, including information on both chemical substances and target proteins (as presented in Table 1). Visualization of the retrieved PDB structures was carried out using PyMOL (version 1.2), a molecular graphics tool based on Python [http://www.pymol.org]. PyMOL was also employed to examine and display molecular docking results. For docking analysis, AutoDock 4.2-developed by The Scripps Research Institute (www.scripps.edu)-was used. Preparation of input files and configuration of docking parameters were managed via AutoDock Tools (ADT), a graphical interface supporting user interaction. AutoDock provides a robust platform for simulating the interaction and optimal binding pose of small molecules, such as drugs or natural ligands, with macromolecular targets of known 3D structure.

Calculation of inhibition constant (Ki) from binding energy
In molecular docking, the strength of interaction between a ligand and its target is usually represented by binding energy (ΔG) in kcal/mol [14]. The inhibition constant (Ki) can be derived from this energy using a thermodynamic equation.

Results and discussion

The study the role of l-type ca²⁺ and ca²⁺r-type ion channels in the relaxant effect of a-41

It is firmly established that contraction of aortic smooth muscle tissues by 50 mM potassium chloride (KCl) is mostly mediated through the opening of voltage-dependent L-type calcium (Ca²⁺) channels. In the presence of higher extracellular concentrations of K⁺, the ensuing depolarization of the cell membrane changes the membrane potential, thereby opening the L-type Ca²⁺ channels, permitting the entry of Ca²⁺ ions into the cell and contraction of vascular smooth muscle cells. Here, in the present study, we investigated the possible vasorelaxant effect of the compound A-41 on KCl-induced contraction of isolated rat aortic rings. The results indicated that A-41 caused concentration-dependent relaxation of pre-contracted aortic tissues. In particular, at concentrations between 5 - 35 μM, A-41 effectively suppressed KCl (50 mM)-induced contraction with relaxation percentage ranging from 5.0 ± 2.2 to 82.2 ± 2.0 % when compared with the control (Figure 4(A)). These results show that A-41 has a strong ability to interfere with the contractile response to membrane depolarization [15]. The ensuing vasorelaxation suggests that A-41 most probably operates by regulating the influx of calcium via voltage-operated L-type Ca²⁺ channels on the plasma membrane of the smooth muscle cells. Through blockade of intracellular influx of extracellular calcium necessary for contraction, A-41 induces vascular smooth muscle relaxation. To examine more closely if the effect of A-41 is exclusively on L-type Ca²⁺ channels, a pharmacological comparison with verapamil, which is an established L-type calcium channel blocker, was conducted [16]. A submaximal concentration of verapamil (0.1 μM) that caused a partial block of KCl (50 mM)-elicited contractions was added to A-41. Under the experimental conditions used, A-41 was noted to cause an additional relaxing effect, inhibiting the contractile response by 13.0 ± 2.7 %. IC₅₀ (half-maximal inhibitory concentration) obtained for A-41 was approximately 22.10 μM (Figure 4(B)). The additive action explains the synergistic or complementary action between A-41 and verapamil in inhibiting Ca²⁺ influx via L-type channels. Together, the results of the experiment are strong evidence that the vasorelaxant effect of A-41 is mediated primarily through its action on voltage-dependent L-type calcium channels. The drug is able to inhibit KCl-induced contraction by inhibiting calcium influx and thereby decreasing intracellular calcium concentration and causing relaxation of the smooth muscle. The mechanistic similarity of verapamil and A-41 also supports the finding that A-41 is active via modulation or blockage of L-type Ca²⁺ channels, indicating its promise as a therapeutic agent in the management of vascular disease linked with increased calcium channel activity.

It is established that vascular smooth muscle cell contraction is not exclusively controlled by voltage-dependent L-type Ca²⁺ channels. Intracellular calcium transport mechanisms, i.e., those associated with the sarcoplasmic reticulum (SR), also play a crucial role in calcium regulation and contraction. In this regard, the subsequent experiments were conducted to determine compound A-41’s impact on contraction induced by the α-adrenoceptor agonist phenylephrine (1 μM), which stimulates receptor-operated calcium channels [17]. Phenylephrine is known to cause contraction mainly by raising intracellular calcium concentration ([Ca²⁺]ᵢ) due to the mobilization of Ca²⁺ from the SR and also via receptor-operated Ca²⁺ channels on the plasma membrane. In the aforementioned studies, A-41 exhibited potent vasorelaxant activity, markedly inhibiting phenylephrine (1 μM)-induced contractions by 87.0 ± 2.7 % at its highest test concentration (50 μM) in comparison to the untreated control (Figure 4(C)). These results implicate that A-41 might be producing the relaxant effect through blockade of receptor-operated Ca²⁺ channels. To explore this possibility further, the activity of A-41 was compared with that of phentolamine, a selective α-adrenoceptor antagonist, and with those flavonoids which are found to antagonize receptor-mediated communication. In the absence of phentolamine, as noted above, A-41 (50 μM) also significantly suppressed the contraction evoked by phenylephrine. Phentolamine (10 μM) suppressed phenylephrine-induced contraction by 75.0 ± 2.4 % compared with the control. Surprisingly, in the presence of both phentolamine and A-41, the contraction force was further decreased to 66.0 ± 2.7 % (Figure 4(D)). These findings are strong evidence that the vasorelaxant action of A-41, as modeled by compound A-41, is mediated at least in part by inhibition of receptor-operated Ca²⁺ channels [18]. The further observation that contraction was also inhibited by the presence of the α-adrenoceptor antagonist phentolamine is further evidence for this mechanism, suggesting that A-41 disrupts α-adrenoceptor-mediated calcium entry mechanisms [18].

The study the role of endothelium in the relaxant effect of a-41

The endothelium plays a critical role in regulating vascular tone by producing local mediators such as nitric oxide (NO), a key endothelial-derived vasodilator. Endothelial dysfunction (ED), which arises from structural and functional impairments of endothelial cells, is a major contributor to the development of cardiovascular diseases like hypertension and atherosclerosis [19,20].

Figure 4 (A) Dose-dependent, linear vasorelaxant effect of compound A-41 on KCl-induced contraction. (B) Comparison of A-41 with the calcium channel blocker verapamil. (C) Effect of A-41 on receptor-operated Ca²⁺ ion channels. (D) Confirmation of A-41’s mechanism using the α-adrenergic antagonist phentolamine. Data represent mean ± SEM, n = 3 - 4, p < 0.05.

Figure 5 Relaxant effect of A-41 on the contraction induced by Phe 1 μM in the undenuded and denuded of the rat aortic blood vessel endothelial layer. Contraction force elicited by 1 μM Phe was taken as 100 % of control. (in all cases *p < 0.05, **p < 0.01; n = 5).

ED results from an imbalance between vasodilatory and vasoconstrictive mediators, often triggered by oxidative stress and modifiable risk factors such as smoking, poor diet, and metabolic disturbances. NO is synthesized in endothelial cells from L-arginine via endothelial nitric oxide synthase (eNOS), which is regulated by calcium-calmodulin and inhibited when bound to caveolin. Stimuli such as acetylcholine or bradykinin release eNOS from caveolin, enhancing NO production. Statins at low concentrations can also stimulate NO synthesis by disrupting the eNOS-caveolin complex, contributing to their vascular protective effects. Oxidative stress reduces NO availability by accelerating its degradation through reactive oxygen species, diminishing its vasodilatory effect. NO promotes vascular smooth muscle cell (SMC) relaxation via the NO/cGMP/PKG pathway, lowering intracellular Ca²⁺ levels and enhancing vasodilation. To assess the role of the endothelium in the relaxant effect of A-41, experiments were conducted on endothelium-denuded aortic rings. The removal of the endothelial layer relatively reduced A-41’s vasorelaxant effect by 31.0 ± 2.5 %, (Figure 5) indicating that the endothelium-and likely NO signaling-plays a key role in mediating its action [21].

Molecular docking study of a-41 with aortic ion channels

To further elucidate the molecular mechanisms underlying our in vitro findings, molecular docking studies were conducted. These experiments aimed to assess the interaction of the A-41 compound with key ion channels involved in vascular smooth muscle function. Specifically, we examined the binding affinity and inhibitory constants (Ki) of A-41 with the following aortic ion channels. The docking results provided insight into the binding energies and potential inhibitory interactions of A-41 at the molecular level, supporting its vasorelaxant activity observed in vitro. After docking of the A41 ligand with a series of calcium-handling targets - Ca²⁺ L-type, Ca²⁺ R-type, RyR2 receptor, SERCA, Ca²⁺-ATPase, and NCX channels - the binding energies of the resulting complexes were calculated. The lowest binding energy was –6.6 kcal/mol, with Ca²⁺-ATPase and NCX, showing the strongest interaction among all the targets investigated. From these binding energies, the inhibitory potencies of the ligand (Ki values) for the proteins were then determined [22]. In order of the rising Ki values (declining affinity), the targets are as follows:

Ca²⁺ R-type > RyR2 receptor > SERCA > L-type Ca²⁺ > Ca²⁺-ATPase – NCX, a gradient of diminishing interactions among these calcium-regulatory proteins (Table 1).

Voltage-gated L-type Ca²⁺ ion channel

Calcium ions (Ca²⁺) are significant secondary messengers in eukaryotic cells and are crucial in controlling various physiological and biochemical processes. Of special interest is the influx of Ca²⁺ in mediating cellular response in cardiac, smooth muscle, and neural tissues. One of the major routes for the entrance of calcium into cells is through Voltage-Dependent Calcium Channels (VDCCs). Of these, L-type calcium channels (Cav1 family) stand out due to their wide distribution and the key role that they play in cellular communication. L-type channels are particularly very significant in cardiac and vascular smooth muscle cells, mediating calcium influx required in processes like muscle contraction, secretion of hormones, gene expression, and signal transduction. Control and function of L-type channels are of high significance in a wide range of pathophysiological conditions, e.g., cardiovascular diseases, hypertension, neurological disease, and metabolic syndrome. In view of such situations, L-type channels are of high value for physiological and pharmacological/clinical study alike [23,24]. Over the last few years, computational modeling and experimental techniques have been heavily utilized to analyze the structure, functional modulation, and interaction of L-type calcium channels with natural products-particularly flavonoids-with emphasis on their increasing therapeutic significance. In this research, the biological significance of L-type calcium channels, modulators of their activity, and flavonoid-derived ligand inhibitory activity are investigated. When the A41 ligand was docked onto the Ca²⁺ L-type calcium channel, its binding energy was –6.3 kcal/mol, indicating a stable interaction. The inhibitory constant (Ki) was determined from this binding energy as 24.152 µM, which categorizes the ligand as a weak inhibitor for the L-type channel (Table 1(A)). To induce functional inhibition, the ligand will have to contact essential amino acid residues necessary for channel function. According to docking results, A41 contacts several such essential residues, which include:

LEU F:269, SER F:265, TYR:396, ASP F:598, PRO A:75, ASP A:586, ARG A:593

Nature of these contacts are: Normal hydrogen bonds, Carbon-hydrogen bonds Unfavorable acceptor–acceptor interactions Alkyl and π-alkyl interactions. These results demonstrate that A41 interacts with structural and potentially functional areas of the L-type calcium channel and reveal its mechanism of action and potential pharmacological relevance.

Receptor mediated Ca²⁺R-type calcium channels

R-type calcium channels (Cav2.3) are members of the voltage-dependent calcium channel family (VDCCs), or more specifically the Cav2 subfamily. They are “R-type” by designation because they are resistant to most conventional calcium channel blockers and have intermediate electrophysiological properties between T- and L-type channels [25]. Upon docking with R-type calcium channels, the binding energy obtained was calculated to be −5.5 kcal/mol and is a very weak binding. From the binding energy, Ki value or inhibition constant was estimated to be 93.165 µM and A41 was found to be a weak inhibitor of R-type calcium channels (Table 1(B)). The docking analysis showed that A41 molecule binds to specific amino acid residues of the R-type calcium channel, i.e., PRO A:1479, GLN A:1174, TYR A:1469, ASN A:1466, and LYS A:1345. These bindings are complemented by a range of non-covalent interactions including normal hydrogen bonds, carbon hydrogen bonds, unfavorable donor–donor interactions, pi-cation, pi–sigma, pi–pi T-shaped, alkyl, and pi-alkyl interactions.

SERCA (sarcoplasmic reticulum Ca²⁺ion channel)

SERCA is a significant enzyme that transfers calcium ions (Ca²⁺) from cytoplasm into the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) in muscle cells utilizing the hydrolysis energy of ATP. The process has a central function in intracellular calcium management, muscle contraction and relaxation, and signaling transduction. SERCA is P-type ATPase as it goes through phosphorylation/ dephosphorylation during the transfer of calcium. It exists as a solitary polypeptide chain with the transmembrane domain that crosses the SR or ER membrane and the cytoplasmic domain that can bind ATP [26]. It uses the energy gained from hydrolysis of ATP to translocate 2 calcium ions per ATP molecule, contrary to the direction of the concentration gradient, to the lumen of the ER or SR. SERCA also plays a critical role in the relaxation of muscles because it actually pumps calcium ions out of the cytoplasm following a contraction, resulting in reduced cytosolic calcium, and thus induces relaxation of muscles. Besides muscle tissue, SERCA is present in many other cell types and performs significant roles in signal transduction, protein folding, and regulation of many calcium-dependent enzymes. Recent studies imply that increasing SERCA activity by pharmacological agents or gene therapy might have therapeutic effects for the treatment of cardiac and skeletal muscle diseases, as well as enhancing calcium homeostasis in neurons. Molecular docking of the A41 ligand into the SERCA (Sarco/Endoplasmic Reticulum Ca²⁺-ATPase) was −5.9 kcal/mol binding energy, indicating a moderate degree of interaction strength (Table 1(C)). From this binding energy, the inhibition constant (Ki value) was estimated to be 40.070 µM, suggesting that A41 is a moderate inhibitor of SERCA. The binding study showed a number of significant interactions between the A41 molecule and specific amino acid residues of SERCA, such as LYS A:158, PRO A:160, ASN A:39, THR A:230, LEU A:41 and GLY A:227. Such interactions were formed by a series of non-covalent interactions, such as classical hydrogen bonds, carbon–hydrogen bonding, unfavorable acceptor acceptor, alkyl relationships, and pi-pi stacking. These findings highlight the molecular mechanism underlying the inhibitory action of ligand to SERCA, and specific amino acid residues are involved in the binding event.

RyR2 (Ryanodine) receptor

Ryanodine receptor 2 (RyR2) is a huge calcium (Ca²⁺) channel found on the sarcoplasmic reticulum (SR) membrane of cardiac muscle cells. It is very important for the contraction of cardiac muscle. When there is electrical stimulation of the cardiac cell, the L-type calcium channels (Cav1.2) present on the cell membrane open and permit the ingress of some Ca²⁺ into the cell. This calcium influx triggers RyR2 receptors to release a massive amount of Ca²⁺ from the SR calcium reservoir [27]. This process is referred to as calcium-induced calcium release (CICR). The RyR2 receptor is not only required for normal heart function, but it is also required for the maintenance of blood circulation, regulation of blood pressure, and adequate supply of oxygen to the whole body. Its normal functioning stabilizes heart rhythm and is one of the critical determinants in the prevention of heart failure, arrhythmias, and sudden cardiac death. RyR2 function is controlled by inherent mechanisms (phosphorylation and oxidation) and external inputs (drugs and genetic mutations). The receptor's mutation or dysregulation has been linked to catastrophic cardiac diseases like catecholaminergic polymorphic ventricular tachycardia (CPVT), heart failure, and sudden cardiac death. Because of its central role in cardiac function, the RyR2 receptor continues to be at the center of numerous scientific studies and is a potential pharmacological target for drug therapy [28,29].

From our research, we noted that A41 ligand binds to RyR2 receptor with the binding energy of –5.9 kcal/mol, which is equivalent to the calculated inhibitory constant (Ki) of 24.152 µM (Table 2(A)). The fact that A41 qualifies as a weak inhibitor of the receptor implies that the compound binds to some amino acids that are crucial for the functioning of RyR2 structure. As per our docking results, A41 ligand binds with amino acid residues ALA A:698, PHE A:723, ASP A:721, and ASN A:716 via pi-alkyl, pi-anion, and classical hydrogen bonds. These bindings keep the lipophilic contacts, electrostatic interactions, and binding of the ligand overall in the active site of the receptor stable.

Ca²⁺-ATPase

Ca²⁺-ATPase is a membrane-bound enzyme responsible for regulating intracellular concentration of calcium ion (Ca²⁺). It harnesses the energy derived from the hydrolysis of ATP molecules to transport Ca²⁺ ions from the cytoplasm through to the plasma membrane or into organelles, such as sarcoplasmic and endoplasmic reticulum. The activity aids in calcium homeostasis within the cell. Ca²⁺-ATPase regulates the transport of calcium ions to prevent excess calcium accumulation in the cytoplasm [30]. By moving Ca²⁺ away from the cytoplasm or into storage spaces such as the sarcoplasmic or endoplasmic reticulum, it prevents internal imbalance (homeostasis) in the cell. This is particularly necessary in muscle and heart cells, where the enzyme prevents muscle relaxation by pumping calcium out following contraction. Aside from its structural role, calcium takes part in activities like nerve impulse conduction, hormone release, signaling, and gene expression, all of which are Ca²⁺-ATPase-regulated. When Ca²⁺-ATPase is impaired, calcium will pile up inside the cell, causing ailments like heart failure, muscle disease, and nervous diseases. Ca²⁺-ATPase can thus be referred to as an important pharmacological target. Most drugs affecting the heart function by promoting the action of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA). In our research, while Ca²⁺-ATPase was docked in the presence of the ligand A41, binding energy was -6.6 kcal/mol, and it was observed that there is a good interaction between Ca²⁺-ATPase and A41 molecule. From this binding energy, inhibition constant (Ki value) was observed to be 14.558 µM, which reflects weak inhibitory activity (Table 2(B)). The ligand A41 binds to the following amino acids in Ca²⁺-ATPase: ALA A:516, CYS A:560, LEU A:561, PHE A:487, LYS A:514, ARG A:559, and ARG A:677. These interactions involve traditional hydrogen bonds, pi-sigma bonds, alkyl and pi-alkyl, pi-cation bonds, and donor-donor hydrogen bonds. These non-covalent interactions bind and stabilize the ligand to the enzyme, although the interaction is not strong enough for effective inhibition.

Table 1 Molecular docking results of A-41 with various calcium ion channels. (A) L-type Ca²⁺ channel: 2D and 3D structures, binding energy, and inhibition constant. (B) R-type Ca²⁺ channel: Structural views along with docking parameters. (C) SERCA Ca²⁺ pump: visual representation and corresponding interaction metrics.

Table 2 Molecular docking analysis of the A-41 compound with selected calcium ion channels. (A) RyR2 Ca²⁺ release channel: 2D/3D structural representations, binding affinity, and inhibitory constant (Ki). (B) Ca²⁺-ATPase: Docking visualization and key interaction parameters. (C) NCX (Na⁺/Ca²⁺ exchanger): Structural models and associated binding energies and Ki values.

NCX (sodium and calcium exchanger)

Na⁺/Ca²⁺ exchanger (NCX) is an essential membrane-bound ion transport protein that plays a role in intracellular calcium homeostasis by bidirectional exchange of sodium (Na⁺) and calcium (Ca²⁺) ions across the plasma membrane. Having a tendency to work in its forward mode, NCX extrudes one Ca²⁺ ion from the cytoplasm in return for the inward entry of 3 Na⁺ ions powered by the electrochemical gradient of sodium. This electrogenic, ATP-independent antiporter plays a crucial role in excitable cells like cardiac myocytes and neurons to regenerate low cytosolic levels of calcium after the stimulation of a cell. NCX is critical in the egress of intracellular Ca²⁺, complementing other mechanisms of Ca²⁺ egress to avoid Ca²⁺ overload, which might result in cytotoxicity, mitochondrial impairment, or cell death [31,32]. It is also especially crucial in neuroprotection, synaptic transmission, and cardiac relaxation, through calcium regulation under ischemia, oxidative stress, or excitotoxicity. Changes in NCX function have been shown to be involved in the etiology of a range of diseases, such as cardiac arrhythmias, congestive heart failure, epilepsy, and neurodegenerative diseases, making it a valuable drug target. Current research aims to develop selective NCX modulators to activate or inhibit its function, based on the disease condition. In our molecular docking experiments, the A41 ligand exhibited a binding energy of –6.6 kcal/mol against NCX, a strong interaction and moderate inhibition activity, with a Ki of 14.558 µM. A41 molecule established contacts with the important residues of the NCX protein, including ILE A:163, ILE A:832, PHE A:169, VAL A:99, and THR A:103, by alkyl contacts and normal hydrogen bonds (Table 2(A)). Such interactions confirmed the close and stable ligand binding, which defined its inhibitory function. These findings suggest A41 as a candidate compound for NCX targeting to develop drugs for therapy of calcium-pathology-related diseases in cardiovascular and neurologic disease.

Effect of A41 compound on blood pressure in vivo studies

The effect of the A41 compound on blood pressure was evaluated in vivo using the tail cuff method. Rats were divided into 4 groups, each receiving different doses of the A41 compound: 10 mg/kg, 20 mg/kg, and 30 mg/kg. Prior to the experiment, the baseline blood pressure (0 h) of all animals was measured. Afterward, the A41 compound was administered intravenously, and its effect on the cardiovascular system was monitored for 3 h, with measurements taken every hour [33].

Effect of A41 Compound at 10 mg/kg dose

In the control group, the baseline systolic blood pressure was 133.0 ± 13.1 mmHg, and the diastolic blood pressure was 98.5 ± 9.7 mmHg. In the group receiving 10 mg/kg of A41, the blood pressure changes were as follows:

1 h: Systolic blood pressure decreased to 132.3 ± 13.1 mmHg, and diastolic blood pressure decreased to 104.5 ± 10.3 mmHg.

2 h: Systolic blood pressure significantly decreased to 93.0 ± 9.2 mmHg, and diastolic blood pressure dropped to 73.0 ± 7.1 mmHg.

3 h: Blood pressure partially recovered, with systolic blood pressure reaching 84.8 ± 8.5 mmHg, and diastolic blood pressure returning to 56.3 ± 5.6 mmHg.

The 10 mg/kg dose showed the most significant decrease in blood pressure, followed by partial recovery. This dose was identified as the optimal dose.

Effect of A41 compound at 20 mg/kg dose

In the control group, systolic blood pressure was 118.0 ± 11.5 mmHg, and diastolic blood pressure was 92.8 ± 9.1 mmHg. In the group receiving 20 mg/kg of A41, the blood pressure changes were as follows:

1 h: Systolic blood pressure decreased to 112.8 ± 11.1 mmHg, and diastolic blood pressure decreased to 79.8 ± 7.8 mmHg.

2 h: Systolic blood pressure decreased significantly to 76.3 ± 7.5 mmHg, and diastolic blood pressure decreased to 55.8 ± 5.4 mmHg.

3 h: Blood pressure partially recovered, with systolic blood pressure reaching 104.5 ± 9.9 mmHg, and diastolic blood pressure returning to 68.3 ± 6.7 mmHg.

The 20 mg/kg dose also resulted in a significant decrease in blood pressure, but compared to the 10 mg/kg dose, the duration of the reduction was shorter. This dose also demonstrated a significant effect.

Effect of A41 compound at 30 mg/kg dose

The baseline systolic blood pressure was 104.3 ± 10.2 mmHg, and diastolic blood pressure was 76.3 ± 7.5 mmHg. In the group receiving 30 mg/kg of A41, the blood pressure changes were as follows:

1 h: Systolic blood pressure was 101.8 ± 10.0 mmHg, and diastolic blood pressure was 78.5 ± 7.7 mmHg.

2 h: Systolic blood pressure increased to 106.3 ± 10.5 mmHg, and diastolic blood pressure increased to 76.0 ± 7.4 mmHg.

3 h: Systolic blood pressure was 103.8 ± 10.4 mmHg, and diastolic blood pressure was 84.8 ± 8.5 mmHg (Figures 6(A) and 6(B)).

The 30 mg/kg dose did not result in a significant effect on blood pressure, and its efficacy was lower compared to the 20 mg/kg dose. This suggests a lower impact at higher doses. The effect of the A41 compound on blood pressure was evaluated at various doses. At both 10 mg/kg and 20 mg/kg doses, a significant reduction in blood pressure was observed, while at the 30 mg/kg dose, minimal changes occurred. The most effective dose was 10 mg/kg, which showed a significant decrease in blood pressure followed by partial recovery. Further studies are required to explore the mechanism of action of the compound in greater detail (Figure7).

Figure 7 Original systolic recordings obtained using the Neurobotics software. (These results were used as sample data for statistical analysis.) (Control Group).

Evaluation of the effect of A41 compound in the adrenaline-induced hypertension model using the tail-cuff method

Before starting the experiments, the most effective dose of A41 compound was determined, and subsequent studies were conducted based on this dose. During the experiment, the rats were divided into the following groups: healthy control group, adrenaline-induced hypertension control group, and experimental group where A41 compound was administered on an adrenaline background. Each group consisted of healthy male rats (n = 4) weighing 300 - 350 g. Initially, systolic (SBP) and diastolic (DBP) blood pressures of all rats were measured using the Tail-Cuff method. The baseline measurements were as follows: in the control group, SBP was 93.3 ± 8.3 mmHg, DBP was 68.3 ± 5.7 mmHg; in rats receiving 10 mg/kg of A41, SBP was 85.0 ± 6.3 mmHg, DBP was 61.0 ± 5.8 mmHg.

Figure 8 (A) Administration of the A41 compound at doses of 10, 20 and 30 mg/kg resulted in a dose-dependent reduction in systolic and diastolic blood pressure. (B) These findings illustrate the antihypertensive effect of A41 against adrenaline-induced hypertension (n = 4, p < 0.05).

Subsequently, adrenaline hydrochloride was intravenously injected into all groups (except the healthy control group), and changes in blood pressure were monitored [34]. After adrenaline administration, blood pressure in the hypertensive model rats increased as follows: SBP 138.3 ± 13.6 mmHg, DBP 102.8 ± 10.1 mmHg. In the experimental group where A41 compound was administered along with adrenaline, the changes in blood pressure were relatively lower: A41 (10 mg/kg) group - SBP 124.0 ± 11.2 mmHg, DBP 94.5 ± 9.3 mmHg. In the next phase, after the intravenous administration of A41 compound, blood pressure was measured hourly in each group. The control group was used solely to show the effect of adrenaline and did not receive any other preparations. In this group, the blood pressure readings were as follows: 1^st hour - SBP 133.3 ± 14.9 mmHg, DBP 95.5 ± 9.3 mmHg; 2^nd hour - SBP 118.8 ± 10.0 mmHg, DBP 90.8 ± 9.1 mmHg; 3^rd hour - SBP 114.8 ± 6.6 mmHg, DBP 83.8 ± 6.4 mmHg (Figure 8).

The antihypertensive effects of A41 compound were observed as follows:

The 10 mg/kg dose of A41 compound showed the most effective results:

1 h: SBP 107.3 ± 10.7 mmHg, DBP 71.8 ± 7.2 mmHg

2 h: SBP 92.8 ± 5.8 mmHg, DBP 67.0 ± 5.6 mmHg

3 h: SBP 82.8 ± 8.3 mmHg, DBP 61.3 ± 5.9 mmHg

These results show that the A41 compound was effective in lowering blood pressure in adrenaline-induced hypertension. The 10 mg/kg dose of A41 compound demonstrated a pronounced antihypertensive effect. This finding is significant in determining the potential therapeutic possibilities of A41.

Discussions

The current research explores compound A-41’s vasorelaxant effect and probable antihypertensive action and investigates the mechanisms of its action using a battery of in vitro and in vivo experimental models, supported by molecular docking simulations. Together, the results validate that A-41 exhibits remarkable vasorelaxant effect via several mechanisms, mainly through the regulation of calcium homeostasis in vascular smooth muscle cells. Our results with KCl-induced contractions of isolated rat aortic rings revealed that A-41 elicits concentration-dependent relaxation from moderate (5.0 ± 2.2 %) at 5 μM to significant (82.2 ± 2.0 %) at 35 μM. The calculated IC₅₀ value of around 22.10 μM reflects moderate potency. A-41’s potent blockade of KCl-induced contraction is highly indicative of interference with voltage-dependent L-type calcium channels, which play a significant role in membrane depolarization-stimulated contraction of vascular smooth muscle. The mechanism was also pharmacologically verified by comparison with verapamil, an established L-type calcium channel blocker. Of note, A-41 showed additive effects with verapamil, suggesting complementary action at different sites on L-type channels or activation of other mechanisms for calcium regulation.

In addition to voltage-dependent calcium channels, our results show that A-41 also acts on receptor-operated calcium channels. This was demonstrated by its strong inhibition (87.0 ± 2.7 % at 50 μM) of phenylephrine-induced contraction, which is mostly mediated by the stimulation of α-adrenoceptors and the subsequent mobilization of calcium from intracellular stores and also from receptor-operated channels. The additive inhibitory effect of co-administration of A-41 with phentolamine (α-adrenoceptor antagonist) is also a pointer to the multi-target mechanism of action because this would imply that A-41 interferes with α-adrenoceptor-mediated calcium entry processes in addition to intracellular calcium stores.

The endothelium dependency experiments provided a glimpse into A-41’s mechanism of action. The marked attenuation (31.0 ± 2.5 %) of vasorelaxant activity following endothelial denudation indicates that endothelium-derived factors, and specifically nitric oxide (NO), are crucial in A-41’s vasodilatory effect. This finding is particularly significant considering that endothelial dysfunction is a major contributor to the pathophysiology of cardiovascular diseases like hypertension and atherosclerosis.

The ability of A-41 to potentially cause NO production or enhance its bioavailability presents an additional therapeutic advantage as it can reverse endothelial dysfunction in pathologic states with oxidative stress and reduced availability of NO.

Molecular docking research has offered additional mechanistic insights by elucidating the interactions of A-41 with other calcium-handling proteins. Binding energy analysis revealed various affinities towards targets, with optimal interactions being with NCX and Ca²⁺-ATPase (both –6.6 kcal/mol), followed by L-type Ca²⁺ channels (–6.3 kcal/mol), RyR2 receptors and SERCA (both –5.9 kcal/mol), and Ca²⁺ R-type channels (–5.5 kcal/mol).

The inhibitory constants (Ki values) calculated were between 14.558 μM against NCX and Ca²⁺-ATPase and 93.165 μM against R-type calcium channels, and A-41 is thus a weak to moderate inhibitor against these targets. The determination of the exact amino acid residues participating in these interactions offers useful structural information for the further optimization of A-41 derivatives.

This multi-targeting profile of A-41 uncovered by these docking studies is also consistent with our in vitro results and predicts a global mechanism for the regulation of calcium homeostasis. By regulating several calcium transport mechanisms simultaneously-voltage-gated channels (L-type and R-type), intracellular calcium release channels (RyR2), calcium pumps (SERCA and Ca²⁺-ATPase), and exchangers (NCX)-A-41 would theoretically be able to regulate intracellular calcium concentrations effectively through complementary pathways, which could be an advantage over single-target calcium channel blockers.

The blood pressure in vivo experiments also confirmed the therapeutic action of A-41 as an antihypertensive compound. The dose-response study identified 10 mg/kg as the most effective dose, which caused profound reduction in blood pressure and recovery, albeit partial, within 3 h. Interestingly, the 30 mg/kg dose failed to exert any significant action, indicating a bell-shaped dose-response curve that should be further investigated. In the model of adrenaline-induced hypertension, A-41 (10 mg/kg) exhibited excellent antihypertensive activity in the form of a gradual decrease in systolic and diastolic blood pressure lasting for 3 h. These results are most comforting as they validate the translatability of A-41’s in vitro vasorelaxant activity to in vivo with therapeutic importance. The demonstrated action of A-41 in the model of adrenaline-induced hypertension is of particular interest considering the pathophysiological importance of this model. Adrenaline-induced hypertension combines α-adrenergic receptor-mediated vasoconstriction with cardiac output augmentation, and both of these actions simulate phenomena of sympathetic hyperactivity commonly encountered in essential hypertension. The implication that A-41 was able to block such actions is conducive to its prospective value in hypertension of high sympathetic tone.

Conclusions

In conclusion, our extensive study demonstrates that compound A-41 displays excellent vasorelaxant and antihypertensive activity through multiple mechanisms of action. A-41 primarily modulates calcium homeostasis through inhibition of voltage-dependent and receptor-operated calcium channels, along with a possible enhancement of endothelium-derived nitric oxide signaling. Molecular docking studies revealed interactions with an array of calcium-handling proteins, confirming A-41’s multi-target profile. In vivo studies authenticated these findings, demonstrating dose-dependent reduction of blood pressure in normotensive and adrenaline-induced hypertensive models. A-41’s multi-modal action is a promising strategy for antihypertensive treatment and has the potential to be more effective and have a better side effect profile than single-target molecules. The 10 mg/kg dose needed for full effect established herein gives a basis for additional pharmacokinetic study and chronic toxicity testing. Additional studies should be aimed at maximizing the structure of A-41 for greater potency and selectivity, chronic safety and efficacy trials, and exploration of possible synergistic interactions with traditional antihypertensive drugs. These findings collectively position A-41 as a lead compound of promise for the development of novel antihypertensive drugs targeting calcium channel modulation and endothelial function with therapeutic application in hypertension and cardiovascular disorders related to it.

Declaration of Generative AI in Scientific Writing

Only minimal assistance was used from QuillBot for paraphrasing selected sentences. All scientific content, interpretation, and conclusions were developed independently by the authors.

CRediT author statement

Shodiyakhon Sodiqova performed the in vitro experiment, Shokhida Kadirova was responsible for the extraction of the substances, Anvar Zaynabiddinov acted as the PhD supervisor, Izzatullo Abdullaev prepared the original draft of the manuscript, Lazizbek Makhmudov conducted the in vivo experiments, Ulugbek Gayibov supervised the manuscript drafting process, Mukhabbat Yuldasheva supervised the substance extraction process, Madina Xolmirzayeva served as a scientific supervisor, Rakhmatilla Rakhimov contributed to the provision and handling of the substances, Azizbek Mutalipov and Hayotbek Karimjonov contributed equally to the research and data analysis.

References

[1] MD Cesare, P Perel, S Taylor, C Kabudula, H Bixby, TA Gaziano, DV McGhie, J Mwangi, B Pervan, J Narula, D Pineiro and FJ Pinto. Global Heart 2024; 19(1), 11.

[2] IZOGL Abdullaev, UG Gayibov, SZ Omonturdiev, SF Azamjonovna, SN Gayibova and TF Aripov. Molecular pathways in cardiovascular disease under hypoxia: Mechanisms, biomarkers, and therapeutic targets. The Journal of Biomedical Research 2025; 39(2), 1-16.

[3] CJ Hutchings, P Colussi and TG Clark. Ion channels as therapeutic antibody targets. MAbs 2019;11(2), 265-296.

[4] AA Abdullaev, DR Inamjanov, DS Abduazimova, SZ Omonturdiyev, UG Gayibov, SN Gayibova and TF Aripov. Sílybum mariánum’s impact on physiological alterations and oxidative stress in diabetic rats. Biomedical & Pharmacology Journal 2024; 17(2), 1291-1300.

[5] BK Joseph, KM Thakali, CL Moore and SW Rhee. Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches. Pharmacological Research 2013; 70(1), 126-138.

[6] O Gaibullayeva, A Islomov, D Abdugafurova, B Elmurodov, B Mirsalixov, L Mahmudov, I Adullaev, K Baratov, S Omonturdiev and S Sa’dullayeva. Inula helenium L. root extract in sunflower oil: Determination of its content of water-soluble vitamins and immunity-promoting effect. Biomedical & Pharmacology Journal 2024; 17(4), 2729-2737.

[7] AQQ Azimova, AX Islomov, SA Maulyanov, DG Abdugafurova, LU Mahmudov, IZ o`g`li Abdullaev, AS Ishmuratova, SQQ Siddikova and IR Askarov. Determination of vitamins and pharmacological properties of Vitis vinifera L. plant fruit part (mixed varieties) syrup-honey. Biomedical and Pharmacology Journal 2024; 17(4), 2779-2786.

[8] AV Mahmudov, OS Abduraimov, SB Erdonov, UG Gayibov and LY Izotova. Bioecological features of Nigella sativa L. in different conditions of Uzbekistan. Plant Science Today 2022; 9(2), 421-426.