Modulatory Effects of Bioactive Phytoconstituents on the Amplitude and Gating Properties of Membrane Ion Channels

1. Introduction

Plant-derived natural products, often referred to as phytochemicals, are secondary metabolites that plants produce to protect themselves from environmental stress, pests, and diseases. These compounds provide humans with a vast repertoire of bioactive properties that may contribute to the prevention or treatment of a wide range of diseases and disorders [1,2,3,4]. Among these bioactive properties, their regulatory effects on ion channels in the cell membranes of different cell types have long been an important subject of academic research. This review paper examines 24 phytoconstituents derived from plant natural products. Based on their major structural features, these compounds are broadly classified into five main categories: alkaloids, terpenoids, lignans and acetogenins, polyphenols, and other aromatic and conjugated systems (Figure 1). We present a comprehensive overview of the currently recognized modulatory effects of these compounds on membrane ion channels across a broad range of mammalian cell types.

2. Alkaloids

2.1. Aconitine

Aconitine is an intensely poisonous alkaloid derived from plants of the genus Aconitum (Ranunculaceae) [5,6]. This compound is a proarrhythmic agent known to open tetrodotoxin-sensitive Na⁺ channels in electrically excitable cells [7]. Aconitine-containing herbal extracts have also been reported to exert inhibitory effects on the proliferation of various neoplastic cells [6,8]. Studies have reported that this compound suppresses the delayed-rectifier K⁺ current (I_K(DR)) in neurons, cardiomyocytes, and immune cells, leading to current inactivation upon membrane depolarization [9,10,11,12,13]. Recent findings indicate that aconitine can induce both early and delayed afterdepolarizations in neonatal cardiac cells and can trigger ventricular polymorphic tachycardia in rats [10,13]. These findings underscore the importance of understanding the electrophysiological impact of aconitine and similar compounds, considering their potential therapeutic applications and associated toxicities. Prior to evaluating the therapeutic potential of Aconitum extracts, such as their antitumor effects, it is essential to thoroughly assess the possible toxicities associated with these reagents.

2.2. Arecoline

Areca nut (Areca catechu, family Arecaceae) has long been used as a medical and psychoactive stimulant in China [14] and a leading cause of oral cancer. Arecoline (1,2,5,6-tetrahydro-1-methyl-3-pyridinecarboxylic acid methyl ester) is an alkaloid extracted from areca nut and is a non-selective agonist of muscarinic receptors and that has been shown to have carcinogenicity, cytotoxicity and immunotoxicity [15,16].

The neuroprotective effects of arecoline were found in Alzheimer’s disease [17]. It stimulates production of connective tissue growth factors in human buccal mucosal fibroblasts and induces the apoptosis of HaCaT keratinocytes [15,16]. An earlier report has demonstrated that in U373 and U87MG glioma cells, arecoline produces an inhibitory effect on intermediate-conductance Ca²⁺-activated K⁺ (K_Ca3.1-encoded, or KCNN4-encoded) channels in a concentration-, voltage- and state-dependent fashion [18]. The dissociation constant for arecoline-induced suppression of these channels was calculated to be 11.2 M [18]. Arecoline-mediated membrane depolarization can arise primarily from its inhibition of intermediate-conductance Ca²⁺-activated K⁺ channels in these cells. Moreover, the major effect of arecoline on these channels is thought to be independent of binding to muscarinic receptor(s) [18]. Compounds like arecoline aimed block K_Ca3.1 channels could be tailored to become intriguing agents that potentiate the efficacy of other cytotoxic drugs for management of malignant gliomas or other types of neoplastic diseases [19].

2.3. Berberine

Berberine is an isoquinoline alkaloid found in many medicinal plants of the genera Berberis (Berberudaceae) and Coptis (Ranunculaceae). Preparations from these medicinal plants have been used as folk medicine in the treatment of jaundice, dysentery, hypertension, and other diseases [20]. Berberine alone was reported to possess anti-proliferative effects on human teratocarcinoma and hepatoma cells [21].

An earlier study reported that in human myeloma cells, berberine directly and differentially inhibits delayed-rectifier K⁺ current (I_K(DR)) and Ca²⁺-activated K⁺ current (I_K(Ca)) in a concentration-dependent manner [22]. Berberine also effectively suppresses the proliferation of these cells. Because of the similarity between the potency and IC₅₀ for blocking K⁺ currents and inhibiting cell proliferation by berberine, the results suggest that inhibition of I_K(DR) and I_K(Ca) could be one of the mechanisms underlying berberine-induced anti-neoplastic actions [22]. In addition to its suppression of ion channels, berberine was reported to improve mitochondrial function and to increase apoptotic proteins, including cytochrome c, Bax, and caspase [23]. Therefore, berberine is a bioactive compound with potential for the treatment of various diseases, including glioma and Alzheimer’s disease [24,25,26].

3. Terpenoids

3.1. Cannabidiol

Cannabidiol, which is considered a terpenophenol, is a non-psychoactive cannabinoid derived from the Cannabis plant (Cannabaceae), known for its potential therapeutic effects. It is among over 100 cannabinoids present in the plant and has been shown to be effective in treating various medical conditions, such as epilepsy, bipolar disorder, inflammation, and cancer [27]. Recent studies have demonstrated that cannabidiol influences the activity in the hypothalamic-pituitary-adrenal axis and modulates several ionic currents in excitable cells, including voltage-gated Na⁺ currents (I_Na), M-type K⁺ currents (I_K(M)), and hyperpolarization-activated cation currents (I_h) [28,29,30].

Cannabidiol exposure was noted to result in a concentration-dependent suppression of I_K(M) in pituitary GH₃ lactotrophs, with an IC₅₀ of 3.6 M [30]. This compound also results in the suppression of I_h in the same cell type with an IC₅₀ of 3.3 M [30]. These findings suggest that the responsiveness of these ionic currents is complex and influenced by various factors, including the resting membrane potential, cannabidiol concentration, patterns of action potential firing, or combinations of these variables [30].

3.2. Ent-Kaurane-Type Diterpenoids

The genus Croton (Euphorbiaceae) includes about 1300 species that are widely distributed throughout tropical regions. C. tonkinensis Gagnep., known as Kho sam cho la in Vietnamese, is a tropical shrub native to northern Vietnam and has been used commonly in Vietnam to treat different disorders [31]. C. tonkinensis is a rich source of diterpenoids [31], and its extracts were reported to exert anti-inflammatory and cancer chemopreventive activities [31,32].

Studies have shown that the fractions isolated from C. tonkinensis—specifically croton-01 (ent-18-acetoxy-7-hydroxykaur-16-en-15-one), croton-02 (ent-7,14-dihydroxykaur-16-en-15-one), and croton-03 (ent-1-acetoxy-7,14-dihydroxykaur-16-en-15-one)—are capable of modulating ionic currents in microglial SM826 cells, including the I_K(DR) (encoded by K_V1.3) and the inwardly rectifying K⁺ current (encoded by Kir2.1) [33]. The notable findings showed that in these microglial cells, croton-03 differentially suppresses K_V1.3- and Kir2.1-encoded currents in a concentration-, time-, and state-dependent manner. Voltage-dependent blocking by croton-03 of K_V1.3-encoded current with K_d (apparent affinity constant) and  (fractional electrical distance) values of 5.17 M and 0.091, respectively, was also estimated [33]. Therefore, the presence of these ent-kaurane diterpenoids is likely to disrupt cellular function by modulating the activity of these ionic currents.

Another report further demonstrated that the presence of croton-01, croton-02, and croton-03 produces concentration-dependent inhibition of I_h in pituitary tumor (GH₃) cells with effective IC₅₀ values of 2.89, 6.25, and 2.84 M, respectively [34]. Cell exposure to croton-03 was also noted to decrease the voltage-dependent hysteresis of this current in response to long-lasting isosceles triangular ramp pulse [34]. Therefore, croton derivatives and other structurally related ent-kaurane-type diterpenoids could represent intriguing compounds that use the open/activated state of the HCN channels as a substrate.

3.3. Lutein

Lutein (xanthophyll, ,-carotene-3,3’-diol), derived from a hydride of a (6R)-,-carotene, is one of the few xanthophyll carotenoids that is recognized to exist not only in vegetables and fruits, but also in high concentrations present in the macula of the human retina, where it is thought to act as a yellow filter [35,36]. A previous study showed that in pituitary GH₃ cells, the exposure to lutein is capable of suppressing hyperpolarization-activated cation current in a concentration-, state-, voltage-, and hysteresis-dependent manner [37]. A hyperpolarizing shift of the steady-state activation curve of hyperpolarization-activated cation current was also observed in the presence of lutein.

Furthermore, the subsequent addition of oxaliplatin or ivabradine, but still in the continued presence of 3 M lutein, was capable of adjusting lutein-perturbed inhibition of I_h in GH₃ cells, as shown in Figure 2. Oxaliplatin or ivabradine has been shown to activate or suppress I_h, respectively [38,39,40]. Cyclic nucleotide-gated (CNG) channels (e.g., CNGA2 ion channels) were previously shown to exhibit hysteretic behavior of ion currents [41]. As shown in Figure 3, the experimental results disclosed the capability of I_h strength evoked by double ramp pulse to modify the varying patterns of bursting firing in different excitable cells, including central neurons [37,42].

In these current measurements, the tested cell was held at −40 mV and a long-lasting inverted double ramp pulse was applied [37]. The ramp pulse protocol comprises the downsloping (forward) ramp from −40 to −150 mV followed by the upsloping (backward) limb back to −40 mV, with a total duration of 3.2 sec (i.e., a ramp speed of ±69 mV/sec) as indicated in the upper part of Figure 3A. As demonstrated in Figure 3, the voltage-dependent hysteresis of I_h (i.e., the relationship of forward or backward I_h versus membrane potential) was observed upon activation by triangular double ramp pulse [43]. Notably, the I_h amplitude evoked by the downsloping phase of the inverted triangular ramp pulse was smaller (in absolute value) than that elicited by the upsloping phase, as indicated in the dashed arrows along the current trajectory (Figure 3A). For example, in the control (i.e., lutein was not present), the I_h amplitude at −120 mV taken during the downsloping or upsloping phase of double ramp pulse was markedly different (p < 0.05) (i.e., 48 ± 8 pA [downsloping] versus 249 ± 11 pA [upsloping], n = 7, p < 0.05). Under cell exposure to lutein (3 M), I_h evoked in the downsloping limb of the inverted double ramp pulse was found to decline to a lesser extent than that measured from the upsloping end of the triangular ramp pulse. Moreover, the strength of lutein-induced current inhibition at the downsloping (forward) and upsloping (reverse) limbs of triangular ramp pulse differed significantly. As demonstrated in Figure 3B, the degree of I_h’s hysteresis residing in GH₃ cells with or without the presence of lutein was further quantified. The results showed the effects of lutein and lutein plus ivabradine on the ∆area under the curve (i.e., the shaded region in A) [37]. For example, in addition to its depressive action on I_h magnitude, the addition of 3 μM lutein resulted in a reduction in the area responding to inverted triangular ramp pulse, as demonstrated by a reduction of ∆area from 17.7 ± 2.4 to 8.8 ± 1.6 mV·nA (n = 7, p < 0.05). After the lutein was removed, the hysteretic area returned to 17.2 ± 2.3 mV·nA. As cells were continually exposed to 3 μM lutein, subsequent application of 3 μM ivabradine measurably decreased the ∆area of voltage-dependent hysteresis further.

The investigators further switched to current-clamp potential recordings in order to examine effects of lutein on sag potential in GH₃ cells [37]. Sag potential evoked in response to hyperpolarizing current stimulus has been shown to be closely linked to the presence of I_h in different types of excitable cells [43,44,45,46]. As shown in Figure 4, when the whole-cell potential recordings were achieved, a long-step 2-sec hyperpolarizing current injection with the amplitude of around 25 pA was noticed to induce the occurrence of sag potential (i.e., an abrupt drop-down to a lower level in the membrane potential upon hyperpolarizing current injection) [45]. Cell exposure to ivabradine (3 μM) was effective at suppressing the amplitude of sag potential. Furthermore, the application of 1 or 3 μM lutein resulted in a considerable depression of sag potential evoked in response to hyperpolarizing current stimulus [37]. Together, the findings can be interpreted to mean that, besides its antioxidative or anti-inflammatory properties [47], the existence of lutein can inhibit the amplitude as well as alter gating and hysteretic behavior of I_h, and that lutein’s actions on ionic currents would engage in the modifications of spontaneous action potentials present in electrically excitable cells, presuming that similar in vivo observations occur [37].

3.4. Thymol

Thymol, a monoterpenoid phenol derived from thyme (Thymus vulgaris, Lamiaceae) essential oil, has been widely used as an antiseptic and antimicrobial agent [48,49,50,51]. Its aromatic properties make it a common ingredient in mouthwashes and dental preparations for treating oral infections [48]. Beyond oral care applications, thymol serves as a stabilizer in several therapeutic agents, including the anesthetic halothane, where accumulates in vaporizers during prolonged use. Notably, adverse effects following massive mouthwash ingestion have been attributed to thymol [52,53].

Previous studies have revealed that its biological activities may be partly mediated through modulation of ion channels. In particular, previous studies indicate that thymol influences Ca²⁺ signaling in pituitary GH₃ cells by enhancing capacitative Ca²⁺ entry and depleting intracellular Ca²⁺ stores, involving both thapsigargin–sensitive and thapsigargin–insensitive pools [54]. Moreover, thymol was reported to stimulate I_K(Ca) in GH₃ cells [55]. These stimulatory effects on Ca²⁺ dynamics may contribute to the cellular mechanisms underlying the impact of thymol on neuroendocrine or endocrine functions.

3.5. Triptolide

Triptolide is a diterpene triepoxide isolated from traditional Chinese medicinal vine Trypterygium wilfordii Hook. F. (Celastraceae). It possesses multiple biological activities, including antitumor, immunosuppression, and antifertility effects [56]. Triptolide can protect neurons in the CNS and promote axon growth of dopaminergic neurons [57]. It also promotes spinal cord repair through down-regulation of astrogliosis and inflammation in an animal model of spinal cord injury [58]. This compound has been reported to inhibit proliferation and invasion of malignant glioma cells [59] and induce apoptotic cell death in malignant glioma cells [60]. Due to its small size and high lipid solubility, triptolide can cross the blood-brain barrier and produce significant effects on glial and glioma cells [56,60].

Previous studies showed that in human glioma cells (U373 cells), triptolide inhibits the inwardly rectifying K⁺ current in a concentration-dependent manner with an IC₅₀ of 0.72 M [61]. Triptolide suppresses the activity of the inwardly rectifying K⁺ channels in these cells with no change in single-channel conductance. The biophysical properties of the inwardly rectifying K⁺ currents in U373 cells resemble the Kir4.1-encoded current because of positive mRNA detection of KCNJ10 (Kir4.1) and high sensitivity to inhibition by BaCl₂. KCNJ10, the gene that codes for Kir4.1, is recognized as a putative seizure susceptibility gene in mice and humans [62]. Therefore, the pharmacological actions of triptolide as described recently [56,57,58,59,60] could be partly, if not entirely, connected with its inhibition of the inwardly rectifying K⁺ channels in glial or glioma cells. However, it should be noted that mRNA expression for other Kir channels may also occur in U373 cells.

We conducted a detailed analysis of the atomic interaction between the KCNJ10 protein [63] and triptolide using PyRx software, employing its embedded AutoDock function. Figure 5 illustrates the predicted docking sites of the triptolide molecule. Notably, during the docking analysis with the KCNJ10 channel, triptolide was observed to form hydrogen bonds with residues Arg36(A) and Ile179(A), with bond lengths of 3.08 and 3.06 Å, respectively. Moreover, triptolide exhibited hydrophobic interactions with several residues, including Lys33(A), Asp34(A), Glu177(A), Arg180(A), Phe181(A), Gln183(A), and Thr198(A). These findings suggest a strong binding affinity between triptolide and the amino acid residues of the KCNJ10 channel, estimated at −7.8 kcal/mol. This interaction predominantly occurs within the intracellular domain of chain A in the KCNJ10 channel. This predicted interaction therefore raises the possibility that triptolide-mediated alterations in the magnitude and gating kinetics of the inwardly rectifying K⁺ currents may occur independently of its binding to muscarinic receptors [61].

4. Lignans and Acetogenins

4.1. Gomisin A

Gomisin A (wuweizichu B, wǔwèizi chún yĭ), a dietary dibenzocyclooctadiene lignan compound isolated from the hexane fraction obtained from the fruits of Schisandra chinensis (Schisandraceae) [64], has been reported to have anti-inflammatory, anti-oxidative, antihypertensive, neuroprotective, and anti-proliferative properties [65]. For example, this compound was previously reported to induce protective effects either against hepatic and renal injury induced by CCl₄ exposure, or nitropropionic acid-induced striatal toxicity, through differential regulation of the MAPK signal transduction pathway [66,67]. It could inhibit COX-2, iNOS, IL-6, TNF- and NOּ through the down-regulation of RIP2 and NF-B activation in mouse macrophages [68]. Earlier reports have also shown that gomisin A was able to exert antioxidative effects during osteoblast differentiation and in vascular endothelial cells [65,69], and to induce apoptotic changes in colon carcinoma HCT-116 cells [70]. Alternatively, the active components of S. chinensis were recently demonstrated to be detected after the intragastric administration of the lignans in rats [64]. The effectiveness of crude S. chinensis extract in decreasing prolactin production in pituitary GH₃ cells suggest that such extract could be therapeutically beneficial for patients with hyperprolactinemia and prolactinoma [71].

An interesting report demonstrated that the presence of gomisin A was able to produce an inhibitory action on I_Na in GH₃ cells in a concentration-, a time- and state-dependent manner. Cell exposure to gomisin A was also noticed to accentuate the inactivation rate of voltage-gated Na⁺ current (I_Na), particularly at the slow component of current inactivation [72]. The inhibitory action on I_Na tended to be rapidly developing and readily washed out, and it would concurrently correlate in time with a significant rise in the inactivation rate of the current activated by short depolarizing pulse, while the activation kinetics of I_Na remained unchanged in the presence of gomisin A. In pituitary GH₃ cells, gomisin A differentially and effectively inhibited the transient or late components of I_Na with effective IC₅₀ value of 6.2 or 0.73 M, respectively [72]. The observed effect of gomisin A on the strength and gating of I_Na is primarily ascribed to this molecule acting largely on the voltage-gated Na⁺ (Na_V) channel itself or its accessory subunits.

Next, the SCN9A (Na_V1.7) protein was docked with gomisin A using PyRx software. The predicted binding sites of gomisin A on this channel protein are illustrated in Figure 6. This compound establishes hydrophobic interactions with several residues, including Ser32(I), Ser820(B), Val821(B), Leu875(A), Phe876(A), Val914(A), Tyr915(A), Pro916(A), and Tyr917(A), which are located in the membrane-embedded segment of the channel. Since the results have shown an inhibitory effect of gomisin A on I_Na and a predicted interaction between SCN9A protein and gomisin A, this compound could potentially be applied to modulate the activity of Na_V1.7 channels in different cell types, thereby influencing their functional activity.

4.2. Honokiol

Honokiol, a hydroxylated biphenyl compound obtained from Magnolia officinalis and from other species (Magnoliaceae), has been used in traditional Asian medicines (Houpo, Hou p’u, or Saiboku-to) [73]. Honokiol is recognized to be a potential natural compound that has been demonstrated to exert multiple effects on different cellular processes in various cancer models [74]. Previous studies have also revealed the effectiveness of this compound in modulating the functional activities of neuroendocrine or endocrine cells. For example, an earlier study has demonstrated that honokiol could induce cell cycle arrest and programmed cell death in vitro and in vivo in human thyroid neoplastic cells [75]. Several investigations have also reported the ability of M. officinalis bark or honokiol to modify the secretion of catecholamines from adrenal chromaffin cells [76]. Moreover, honokiol was noted to exert antidepressant effects by normalizing the hypothalamic-pituitary-adrenal axis [77].

Previous studies have demonstrated that the presence of honokiol produces concentration-dependent inhibition of I_h in GH₃ cells with an IC₅₀ value of 2.1 M (Figure 7) [78]. Honokiol shifts the steady-state activation curve of I_h toward a more negative potential with no modification in the gating charge of the current. This compound was able to attenuate the voltage-dependent hysteresis of I_h elicited by long-lasting triangular ramp pulse. It can also depress I_h amplitude in Rolf B1.T olfactory neurons [78]. The results unveiled the evidence that the honokiol presence is effective at inhibiting I_h in pituitary GH₃ cells and in Rolf B1.T olfactory neurons. The inhibition of these ionic currents was noted to be rapid in onset and is hence likely to be responsible for its modulatory action on functional activities in sensory neurons such as Rolf B1.T cells. Such inhibitory actions could lead to modifications in the firing behavior of electrically excitable cells, hence altering neuronal function [78].

To examine how honokiol interacts with the HCN channel protein, we used the structure of HCN3 protein from PDB. The HCN3 channel protein structure was acquired from PDB (PDB ID: 8IO3), and PyRx software was used to see how the honokiol molecule docks into the channel protein. Figure 8 presents the predicted docking sites of honokiol for interaction with amino-acid residues of the HCN3 channel. Specifically, the honokiol molecule was predicted to form hydrophobic contacts with residuals Phe60(D), Tyr89(D), Phe94(D), Ile237(D), and Phe238(D), as well as hydrogen bonds with residuals Ser90(D) and Asp91(D), with estimated distances of 2.95 and 3.10 Å, respectively. It is therefore anticipated that honokiol can interact with HCN channels to modulate the amplitude and gating kinetics of I_h [78].

Recent reports have demonstrated the ability of honokiol to exert antioxidant, anti-inflammatory, neurotrophic, and anti-apoptotic effects [79]. We also conducted a predicted docking analysis between honokiol and superoxide dismutase (SOD). SOD is a key antioxidant enzyme that plays a central role in controlling oxidative stress and modulating inflammation. We explored how SOD protein could be optimally docked with honokiol molecule by using PyRx software. The protein structure of SOD was obtained PDB (PDB ID: 1DO5). The predicted docking sites of the honokiol molecule with which the amino-acid residues can interact are presented in Figure 9. Notably, the honokiol molecule was predicted to form hydrophobic contacts with certain residues, including Asn142(B), Asn142(C), Ser143(B), Ser143(C), Gly145(C), Asn146(C), Ser233(A), Ala234(A), Gly235(A), and Leu236(A). The honokiol molecule was also predicted to form a hydrogen bond with residue Asp136(B) with a distance of 2.79 Å. These docking results suggest that honokiol can bind to SOD protein with a binding affinity of −7.4 kcal/mol.

4.3. Ganoderma Triterpenoids

Ganoderma mushrooms (Língzhī in Chinese, or Reishi in Japanese) are a traditional Chinese herbal medicine that has been widely accepted as a nutritional supplement around the world. Among many species of the mushrooms, Ganoderma lucidum (Ganodermataceae) is most commonly used and it is commercially cultivated under controlled conditions to obtain mushrooms with more consistent chemical composition [80]. G. lucidum was reported to possess a variety of biological activities, such as antihypertensive, hypoglycemic, and hypocholesterolemic activities among other medicinal benefits [80,81]. The primary bioactive compounds were noted to include triterpenoids [81,82]. G. lucitum was also shown to prevent cardiac damage in animal models by alleviating the oxidative stress associated with myocardial injury [83].

Triterpenes are types of terpenes having a basic skeleton of C₃₀ carbons. Triterpenoids have molecular weights ranging from 400 to 600 Da, and their chemical structure is complex and noted to be highly oxidized [84]. The triterpenoid fraction of Ganoderma, consisting of more than 300 lanostane triterpenoids, has been increasingly demonstrated to be effective at exerting biological actions, including providing effective antioxidant activities for prevention of myocardial injury, and producing neuroprotective actions [81,83,85]. Ganoderma triterpenoids could also suppress inflammatory response by directly scavenging the free radicals or systemically enhancing the antioxidant enzymes, thereby lowering lipid peroxidase in chicken livers or mice [83,85]. The aqueous extract was reported to exert anti-convulsant, anti-depressive, anxiolytic and anti-nociceptive actions [86].

Previous work has demonstrated that the inhibition by Ganoderma triterpenoids of I_h in GH₃ cells did not simply decrease current magnitude, but also altered the kinetics of the current, thereby indicating that they are able to produce a dose-, time- and state-dependent inhibition of I_h [87]. The steady-state activation curve of I_h in the presence of Ganoderma triterpenoids was shifted along the voltage axis toward the less depolarized potential. The IC₅₀ value for Ganoderma triterpenoids-mediated inhibition of I_h observed in GH₃ cells (11.7 g/mL) is comparable to concentrations reported for their antioxidative and neuroprotective effects [81,87]. Such intriguing actions could therefore be of pharmacological relevance and appear to be upstream of its effects on oxidative stress occurring inside the cell [80,81].

4.4. Sesamin and Sesamolin

Sesame seeds and sesame oil obtained from Sesamum indicum (Pedaliaceae) have been recognized as health foods in Asian countries [88]. In comparison with other edible oils extracted from diverse seeds, sesame oil is extremely stable due possibly to the effective antioxidant activities presumably attributed to its abundance of lipid-soluble furofuran lignans such as sesamin and sesamolin [88].

Emerging research has demonstrated that sesamin and sesamolin, the two major furofuran lignans of sesame oil, exhibit multiple biological activities. These include suppression of lipid peroxidation in erythrocytes [89], inhibition of intestinal absorption of cholesterol and hepatic 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase activity [90], prevention of chemically induced mammary cancer, inhibition of 5-desaturase and chain elongation of C18 fatty acids, protection of hypoxic neuronal and PC12 cells by suppressing ROS generation and MAPK activation [91], as well as antihypertensive and cardioprotective effects [92].

Previous studies have shown that sesamin or sesamolin differentially and effectively inhibited the transient and late components of I_Na in a concentration-dependent manner in pituitary GH₃ cells [93]. The IC₅₀ value of sesamin needed to lower the peak and the sustained I_Na were estimated to be 7.2 and 0.6 M, respectively [93]. The addition of sesamin results in producing a modification of the inactivation kinetics of I_Na in response to brief depolarization. According to a Markovian model designed from the SCN8A channel adopted previously [93,94], sesamin-induced changes in the gating kinetics of Na_V channel could be ascribed to its lowering of the probability of open (O) and open-blocked (OB) states of the channel. The results suggest that the inhibition by sesamin of these ion channels can be one of the ionic mechanisms underlying its remarkable changes in the functional activities of different types of electrically excitable cells, assuming that similar observations can be found in vivo [93]. Sesamin and other structurally similar compounds may serve as modulatory agents of Na_V channels in neoplastic or neuroendocrine tumor cells within the field of cancer neuroscience, thereby suppressing tumor invasiveness and metastasis [95].

4.5. Squamocin

Squamocin is a bis-tetrahydrofuran acetogenin isolated from several genera of the plant family Annonaceae, including Annona squamosa (custard apple), Annona muricata (soursop), and Asimina triloba (pawpaw). Its structure is characterized by a long, alkyl chain bearing a terminal ,-unsaturated -lactone ring, two tetrahydrofuran rings, and some oxygenated substituents along the chain. Squamocin is known to possess insecticidal properties [96] and anti-tumor effects [97]. Squamocin has been shown to inhibit the activity of mitochondrial NADH:ubiquinone oxidoreductase [98]. In addition, the cytotoxicity caused by asimicin, an analogue of squamocin, was thought to be associated with the regulation of membrane conformation [99]. It has recently been reported that squamocin could induce apoptosis in HL-60 leukemia cells and this effect was implicated in the activation of stress-activated protein kinase [100].

Previous studies reveal an interesting finding that squamocin produces a stimulatory effect on I_K(Ca) in human coronary smooth muscle cells [101]. The stimulatory effect on I_K(Ca) caused by squamocin was presumably related to changes in the level of intracellular Ca²⁺ concentrations. The results showing that the removal of extracellular Ca²⁺ inhibited the squamocin-induced increase in I_K(Ca) amplitude, also suggest that both extracellular Ca²⁺ and internal Ca²⁺ release contribute to the increase in the current amplitude in these cells. It is likely that squamocin opens membrane ion channels with high Ca²⁺ permeability, can presumably account for its stimulatory effect on I_K(Ca) [101]. The elucidation of the mechanisms by which the actions of squamocin and other structurally related compounds in different types of cells occur remains the aim of further research.

5. Polyphenols

5.1. Curcumin

Curcumin (diferuloylmethane, 1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6- heptadiene-3,5-dione), a major constituent of the spice turmeric obtained from Curcuma longa (Zingiberaceae), is a bright yellow diarylheptanoid produced by some plants. It is the principal curcuminoid of turmetic, which is a member of the ginger family (Zingiberaceae). This compound is largely used as a herbal supplement, cosmetics ingredient, food flavoring and food coloring [102]. Particularly, this nutraceutical compound has been demonstrated to possess beneficial properties in a variety of diseases ranging from cancer to diabetes mellitus [103]. For example, curcumin has been recently reported to influence insulin release from isolated pancreatic islets [104].

Curcumin has been reported to exert a concentration- and state-dependent depressant action on I_K(DR) in pancreatic -cells, particularly in the rat insulinoma cell line INS-1 [105]. The inhibitory action on this K⁺ current tends to correlate in time with a significant increase in the inactivation rate of the currents in response to membrane depolarization, while the activation kinetics of the current remained unaltered in the presence of curcumin [105].

The estimated peak of plasma curcumin concentration was previously reported to reach 3.14 g/mL (around 8.57 M) [106], a value which is apparently greater than the K_D (1.26 M) and IC₅₀ (3.32 M) required for curcumin-mediated inhibition of I_K(DR) seen in INS-1 cells [105]. Therefore, the inhibitory effects of curcumin and curcuminoids on these K⁺ currents in pancreatic -cells are expected to occur at concentrations achievable in the human organisms, although whether the effects of curcumin and other curcuminoid derivatives on ionic currents in humans still remains to be further delineated.

5.2. Columbianadin

Columbianadin is one of the main bioactive constituents isolated from the underground part of Angelica pubescens Maxim. f. biserrata Shan et Yuan (Apiaceae) (Angelicae Pubescentis Radix, or “Duho [Dú huó]” in China). Columbianandin is a coumarin-type compound (an angular dihydrofuranocoumarin) which is recognized to have various biological activities that include analgesic, anti-inflammatory and anti-neoplastic effects [107,108]. This compound has been reported to induce changes in cellular proliferation or apoptosis [108]. It has also been previously demonstrated that carrageenan and lipopolysaccharide-induced inflammatory reaction are ameliorated by the application of columbianadin [107].

It is important to note that the presence of columbianadin could depress I_Na in a concentration-, time-, and state-dependent manner in GH₃ cells [109]. This compound was found to differentially inhibit the peak and late amplitudes of I_Na activated by rapid membrane depolarization with effective IC₅₀ values of 14.7 and 2.8 M, respectively [109]. It can shift the midpoint of the I_Na inactivation curve along the voltage axis to a more negative potential, despite its inability to alter the activation curve of the current. Subsequent addition of columbianadin was capable of depressing the tefluthrin-induced increase in the amplitude of persistent I_Na activated by the upright isosceles-triangular ramp at either upsloping or downsloping phase. Tefluthrin, a pyrethroid insecticide, can simulate I_Na [110,111,112]. In HL-1 cardiomyocytes, columbianadin was effective at depressing I_Na as well as at decreasing the slow component of the inactivation time constant of the current [109]. The experimental results suggest that columbianadin-mediated changes in the amplitude and gating kinetics of ionic currents tend to be upstream of its action either on cytosolic NOD1/NF-B activation [113] or on the activity of antioxidant enzymes [114], and that they could conceivably participate in the adjustments of different functional activities in electrically excitable cells (e.g., GH₃ cells or HL-1 cardiomyocytes) occurring in vivo.

5.3. Eugenol

Eugenol (4-allyl-2-methoxyphenol) is an aromatic molecule found in several plants including clove (Syzygium aromaticum, Myrtaceae), bay leaves (Laurus nobilis, Lauraceae), and allspice (Pimenta dioica, Myrtaceae), and has been used in dental practice to relieve pain arising from a variety of sources, such as pulpal inflammations and dentin hypersensitivity [115]. Furthermore, eugenol is neuroprotective against excitotoxicity, cerebral ischemia and the toxic effects of amyloid- peptides [116,117]. This compound has been demonstrated to suppress epileptiform field potentials and spreading depression in hippocampus and neocortex. These results suggest that the anti-convulsive properties of eugenol are mediated through the effects on neuronal ion fluxes by blocking Na_V channels [118,119,120].

The evidence has emerged that the effects on ion channels may be an important mechanism underlying eugenol-induced actions in neurons. Previous studies demonstrated the ability of eugenol to bind to vanilloid receptors and, in turn, to activate non-selective cation channels [121]. A recent work has also reported that it produced an inhibition of voltage-gated Ca²⁺ current through Ca_V2.3 channels in the E52 cell line [122]. Such inhibition is thought to be direct and independent of the binding to vanilloid receptors or to -adrenergic receptors [121,122].

Previous reports have demonstrated that the exposure to eugenol differentially inhibited the transient and late components of I_Na in differentiated NG108-15 neuronal cells in a concentration-dependent manner [123]. The IC₅₀ values of eugenol required for the inhibition of the transient and late I_Na were 8.9 and 1.6 M, respectively [123]. Eugenol can also diminish the amplitude of persistent I_Na evoked by long-lasting ramp pulse, and tefluthrin reversed the eugenol-induced inhibition of this current. This compound could decrease the frequency of spontaneous action potentials. It is therefore anticipated that the observed effects of this agent on ionic currents could be an important mechanism underlying its actions in neurons occurring in vivo [117,120,123].

5.4. Resveratrol

Resveratrol (trans-3,4’,5-trihydroxystilbene), a polyphenolic phytoalexin, is derived from various plants, including grape (Vitis vinifera, Vitaceae), peanuts (Arachis hypogaea, Fabaceae), and various berries. It has been demonstrated that resveratrol displays several pharmacological activities, including anti-platelet, anti-carcinogenic, anti-viral, and cardioprotective effects [124,125,126]. Moreover, there is accumulating evidence indicating that resveratrol exhibits neuroprotective effects [3,125,127]. In fact, resveratrol attenuates kainic acid-mediated convulsion and the associated neurotoxicity and also protects against pentylenetetrazol-induced seizure [128,129].

An important finding has reported that the suppressing effect of resveratrol in rat cortical neurons on action potential firing rate may be mediated by both stimulation of I_K(Ca) and suppression of I_Na [130]. The increased amplitude of I_K(Ca) is attributed to the stimulation of large-conductance Ca²⁺-activated K⁺ channels [124,130]. Inhibition of I_Na by resveratrol was reported to account for its analgesic effects [131]. With dual effects on I_K(Ca) and I_Na, resveratrol might have the potential as a broad-spectrum anti-seizure medication.

5.5. Gastrodigenin and Gastrodin

Gastrodigenin (p-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol) is a phenolic compound found in the traditional Chinese herbal medicine Gastrodia elata Blume (Orchidaceae), which is commonly known as Tian Ma in Chinese or Chunma in Korean [132]. Gastrodin, the glucoside of gastrodigenin, is the major bioactive ingredient which can be isolated from G. elata. In East Asian countries, the dried rhizomes have been widely used for centuries to treat many neurological and psychiatric disorders, including convulsive disorders, dizziness, dementia, depression and migraines [133,134,135,136]. Increasing research is being conducted to determine the underlying molecular mechanisms through which the active constituents of G. elata may exert their pharmacological effects.

Previous studies have evaluated two structurally related compounds, gastrodigenin and gastrodin, for their actions on different types of ionic currents in pituitary cells and hippocampal neurons. In pituitary GH₃ lactotrophs, gastrodigenin and gastrodin suppressed the amplitude of I_K(M) in a time- and concentration-dependent manner [137]. The IC₅₀ value of gastrodigenin and gastrodin needed to inhibit I_K(M) in GH₃ cells were estimated to be 12.1 and 19.4 M, respectively [137]. Gastrodigenin-induced inhibition of I_K(M) was associated with a slowing of the activation time course of I_K(M), while gastrodin-mediated inhibition was associated with an increase in the activation time course of the current. Gastrodigenin shifted the steady-state activation curve of I_K(M) toward less depolarized potential with no change in the gating charge of the current [137]. The addition of gastrodigenin or gastrodin also effectively inhibited I_K(M) amplitude in hippocampal mHippoE-14 neurons. Therefore, because gastrodin can penetrate through the blood-brain barrier, different types of ionic currents, particularly low-threshold I_K(M) with a slowly activating and deactivating properties, may be a relevant target for the regulatory actions of these agents in endocrine or neuroendocrine cells, or central neurons, if similar in vivo findings occur [134,135,136,137].

5.6. Pterostilbene

Pterostilbene (trans-3,5-dimethoxy-4’-hydroxystilbene) is a natural dimethylated analog of resveratrol isolated from Pterocarpus marsupium Roxb. (Fabaceae), a tree native to India, Nepal, and Sri Lanka. The extracts of P. marsupium containing pterostilbene have been traditionally used in Ayurvedic medicine for treating various disorders [138]. This compound has been reported to have benefits for the prevention or treatment of different kinds of cancers, as mounting evidence has demonstrated its inhibitory effects on almost every cellular event that promotes tumor progression toward metastasis in both apoptosis-dependent and apoptosis-independent manners [139,140,141].

In earlier studies, the addition of pterostilbene inhibited I_h effectively in a concentration- and time-dependent manner in pituitary GH₃ cells. The I-V relationships of I_h established at various levels of hyperpolarizing steps were also established and depicted in Figure 10. It was noted that the presence of pterostilbene (1 M) significantly reduced the slope of the linear fit of I_h amplitudes to the voltages between −130 and −100 mV from 27.2 ± 1.3 to 13.1 ± 1.1 nS (n = 8, p < 0.05). The steady state activation curve of I_h was distinctly shifted to more hyperpolarizing potentials by 11 mV, producing channel opening at more negative voltages. The results thus demonstrate that pterostilbene has a conceivable depressant action on I_h functionally expressed in GH₃ cells [142].

Furthermore, as shown in Figure 11, the presence of pterostilbene raised the amplitude of macroscopic I_K(Ca) in GH₃ cells. In this set of whole-cell current recordings, GH₃ cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl₂, and the recording pipette was filled with a K⁺-enriched solution. In an attempt to inactivate most of the voltage-gated K⁺ currents [143], we then maintained the examined cells at the level of 0 mV, and then applied a series of voltage pulses between 0 and +50 mV with 10-mV steps. Within 1 min of exposing GH₃ cells to pterostilbene (3 M), the amplitude of I_K(Ca) elicited by this voltage profile evidently rose (Figure 11). For example, the addition of 3 M pterostilbene increased I_K(Ca) amplitude elicited by depolarizing pulse from 0 to +50 mV from 568 ± 35 to 1005 ± 89 pA (n = 8, p < 0.05). As the compound was washed out, current amplitude returned to 621 ± 38 pA (n = 8). The averaged I-V relationships of I_K(Ca) amplitude in the control, during the exposure to 3 M pterostilbene and after washout of the compound were depicted in Figure 11B. The addition of 3 M pterostilbene substantially increased the whole-cell conductance of I_K(Ca) measured at the voltages between +30 and +50 mV to 19.6 ± 0.8 nS (n = 8, p < 0.05) from control value of 12.6 ± 0.5 nS (n = 8). Therefore, pterostilbene can increase the amplitude of I_K(Ca) observed in these cells and its stimulation of I_K(Ca) was largely attributable to the activation of large-conductance Ca²⁺-activated K⁺ channels [142].

6. Other Aromatic or Conjugated Systems

6.1. Isoplumbagin and Plumbagin

Isoplumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone) is a naturally occurring quinone from Lawsonia inermis (Lythraceae) or Plumbago europarea (Plumbaginaceae) [144] . Similar to isoplumbagin, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), another hydroxyl-1,4-napththoquinone, is another alkaloid obtained from the roots of the plants of Plumbago genus. Isoplumbagin and plumbagin have been demonstrated to exert antineoplastic activity against an array of cancer cells, including oral and tongue squamous cell carcinoma, glioblastoma, non-small cell lung carcinoma, and breast, cervical, endometrial, pancreatic and prostate cancers [145,146]. Alternatively, plumbagin was shown to induce apoptotic changes in lung cancer and neuronal cells via caspase-9 activation and targeting mitochondrial-mediated ROS induction [147,148]. Plumbagin has been reported to modify release of pituitary gonadotropin [149]. Previous studies have shown the ability of 2-mercaptophenyl-1,4-naphthoquinone, a naphthoquinone derivative, to overcome the elevation of intracellular Ca²⁺ in platelets caused by ADP and collagen [150], as well as to suppress the amplitude of I_K(Ca) and I_K(DR) in GH₃ cells [151].

Emerging evidence has shown that isoplumbagin modulates the magnitude, gating kinetics, and voltage-dependent hysteresis of erg (ether-à-go-go-related gene)-mediated K⁺ currents (I_K(erg)) obtained in GH₃ cells [152]. In addition, these K⁺ currents in MA-10 Leydig tumor cells were also found to be blocked by isoplumbagin [152]. The experimental observations suggest that the reduction by isoplumbagin or plumbagin of I_K(erg) could potentially contribute to its anti-neoplastic actions, presuming that similar in vivo findings occur.

The IC₅₀ values required for isoplumbagin-mediated block of peak or sustained I_K(erg) were 18.3 or 2.4 M, respectively [152]. From the first-order reaction scheme, the K_D value was quantitatively calculated to be 2.58 M, a value that was noted to dovetail with the IC₅₀ (i.e., 2.4 M) for isoplumbagin-induced inhibition of sustained I_K(erg) evoked by long-lasting membrane hyperpolarization. An earlier study has shown that the isoplumbagin at a concentration of 2.5 M could suppress the activity of mitochondrial respiration through a mechanism of its inhibitory action on complex IV activation [153]. Moreover, any modifications by isoplumbagin or plumbagin of I_K(erg) depend not only on the concentration of these compounds given but also on different confounding variables, such as the pre-existing level of the resting potential and various discharge patterns of action potentials or their combinations, presuming that the magnitude of I_K(erg) is adequately expressed in the cells examined. Therefore, whatever the detailed mechanism of isoplumbagin-inhibited actions on I_K(erg) remains unresolved, the block by isoplumbagin or plumbagin of I_K(erg) would be of pharmacological relevance [154]. It is likely that the presence of isoplumbagin or plumbagin may induce prolactin secretion in vivo directly through the inhibition of I_K(erg) [155,156]. However, to what extent isoplumbagin-mediated block of I_K(erg) affects cardiac function [156,157] remains to be delineated.

6.2. Verteporfin

Verteporfin (Visudyne^®), a benzoporphyrin derivative, is a compound therapeutically tailored as a photosensitizer for photodynamic therapy, because this agent can effectively eliminate aberrant blood vessels in the eye associated with conditions such as either the wet form of macular degeneration or abnormal choroidal neovascularization [158,159,160].

The photodynamic therapy with different photosensitizers such as hypericin or verteporfin has been previously demonstrated to be effective in the treatment of different types of hyperplastic or neoplastic tissues, including the residual small tumor in the pituitary gland [161,162]. Previous work indeed reported the capability of photosensitizers (e.g., rose Bengal, a fluorescein-derivative photosensitizer) to modify membrane ionic current in GH₃ cells and heart cells [163]. Alternatively, earlier reports have also revealed that verteporfin could induce anterior ischemic optic neuropathy in rodents [158,164].

A previous report discloses important findings showing that verteporfin produced a stimulatory effect on Ca²⁺-activated K⁺ current (I_K(Ca)) in pituitary GH₃ cells; however, it mildly depressed I_K(DR) amplitude with no modification of I_Na or I_K(M) [165]. The stimulatory effect on I_K(Ca) caused by verteporfin was presumably related to changes in the level of intracellular Ca²⁺ concentrations. The results showing that removal of extracellular Ca²⁺ suppressed verteporfin-induced increase in I_K(Ca) also suggest that extracellular Ca²⁺ and internal Ca²⁺ release both contribute to the increase in the amplitude of I_K(Ca) in these cells.

The EC₅₀ value for either verteporfin-induced stimulation of I_K(Ca) observed in pituitary GH₃ cells or increase in the activity of large-conductance Ca²⁺-activated K⁺ channels in 13-06-MG glioma cells (a human glioblastoma multiforme cell line) was estimated to be 2.4 or 1.9 M, respectively [165]. These values are quite close to those that are either therapeutically achievable or required for the inhibitory actions on YAP-TEAD (YAP-TEA domain transcription factor) complex in different types of anaplastic or neoplastic cells [166,167]. Because verteporfin increased I_K(Ca) amplitude within minutes in GH₃ cells, it is plausible to suggest a mechanistic association between its anti-neoplastic effects and its stimulatory actions on I_K(Ca), possibly occurring before the compound passes through the cell membrane [1,165,168].

7. Conclusions

This review illustrates that five major chemical classes of phytoconstituents exert distinct and specific modulatory effects on membrane ionic currents, providing a mechanistic basis for their potential pharmacological activities. Alkaloids predominantly exert inhibitory effects on voltage-gated K⁺ currents; however, aconitine specifically affects the inactivation of I_Na and I_K(DR). Terpenoids modulate I_K(M), I_h, and I_K(DR). Compounds within the lignan and acetogenin groups exhibit inhibitory actions on I_Na and I_h. Polyphenolic compounds influence, to varying extents, I_K(DR), I_Na, I_h, I_K(erg), I_K(M), and I_K(Ca). In contrast, other aromatic or conjugated compounds can regulate I_K(erg) and I_K(Ca). Moreover, individual phytoconstituents also display distinct specificities with respect to both the amplitude and gating kinetics of different ionic currents across various cell types.

Ion channel gating kinetics refers to the dynamic rates governing transitions among the functional states of a channel—closed, open, and inactivated—and encompasses activation, inactivation, deactivation, and recovery kinetics [111,169]. By utilizing electrophysiological techniques—particularly voltage-clamp methods—and incorporating digital-to-analog conversion to generate a variety of voltage-clamp profiles, this approach remains the principal method currently used to investigate voltage-clamp ionic currents [169,170]. The docking predictions also indicate that several amino acid residues within many ion channel structures are capable of forming molecular interactions with various phytoconstituents, including hydrogen bonds and hydrophobic contacts. Collectively, these findings have important pharmacological and toxicological implications for the various bioactivities of individual phytoconstituents [1,2,3,4,171].

In addition, phytoconstituents often modulate ion channel proteins situated on the cell surface or interact with nearby membrane phospholipids. Because these actions occur at or within the plasma membrane, the compounds do not necessarily need to enter the cell. As a result, their effects typically appear rapidly and extremely low concentrations are not required for efficacy [171]. In contrast, when their targets reside within the cytoplasm—such as intracellular signaling pathways—or within the nucleus, where they influence transcriptional or epigenetic processes, the onset of action is generally slower. Access to these intracellular targets requires the compounds to cross the plasma membrane, a process largely determined by their lipid solubility (e.g., reflected by the partition coefficient). Consequently, although some bioactive phytoconstituents appear to demonstrate biological effects at relatively low concentrations in cell-free assays, such as western blotting, immunofluorescent assay, and co-immunoprecipitation [2,3,16,23,24,59,79,100,139], substantially higher concentrations are often required to elicit measurable intracellular responses [171].

It is also important to recognize that, in addition to the phytoconstituents described in this article, numerous other compounds with significant modulatory effects on ion channels are likely to be discovered as electrophysiological methodologies—such as automated patch-clamp recording—continue to advance and gain wider application. Moreover, with the exception of certain ligand-gated channels and transient receptor potential (TRP) channels, voltage-gated ion currents are typically activated either rapidly—within a few milliseconds (e.g., I_Na)—or more slowly, over seconds (e.g., I_K(erg) and I_h) [1,43,169,170]. Moreover, the magnitude, gating kinetics, and voltage-dependent hysteresis of voltage-gated ionic currents vary significantly across different cell types [43,152,154,169,170]. In contrast, antineoplastic or antioxidative effects generally require several hours to become apparent. Therefore, clarifying how rapid modulation of ion channels can ultimately translate into these slower antineoplastic, antioxidative, or immunomodulatory outcomes represents an important direction for future research [1]. Addressing this temporal gap is an important objective for future research and may uncover ion channel activity as an early initiator of subsequent intracellular (cytoplasmic or nuclear) responses [1,171].