Plant and fungi derived analgesic natural products targeting voltage-gated sodium and calcium channels

Plants have played an important role in nutrition, recreational usage, and religious practices for as long as we have existed. The first medicinal drugs came from natural sources and existed in the form of herbs, plants, roots, vines, and fungi [ 1 ]. Traditional medicine has been used by cultures worldwide, resulting in a generational legacy that helps keep and improve human health [ 2–7 ]. Throughout the last two centuries and upon the discovery of the active ingredients morphine (from opium, poppy), salicylic acid (from the bark of the white willow tree), and THC (from cannabis), new chemical analogs have been synthesized to improve their pharmacokinetic properties, avoid toxicity, and decrease side effects [ 1 , 8 ]. The Incas, for example, discovered the effectiveness of local anesthetics (LAs) by using coca-plant leaves to alleviate pain [ 9 ]; however, the high frequency of toxicity of cocaine affecting the central nervous and cardiovascular systems prompted the search for more effective and safer LAs. Nowadays, herbal medicines are widely used on up to 80% of the population, consuming them alone or in combination for all kinds of sicknesses [ 10 ]. Of the drugs approved by the US Food and Drug Administration (FDA) between 1980 and 2014, 49% were either natural products or derivatives. Natural products represent an important source for the identification of novel drugs [ 11 , 12 ], including those with anticancer and antimicrobial properties, which have been reported to have an action on intracellular and membrane proteins [ 13–15 ]. In this review, we present a synthesis of the evidence of action of natural products on two families of transmembrane proteins that play important roles in the electrical signaling of cells – the voltage-gated sodium and calcium channels (VGSCs and VGCCs). VGSCs and VGCCs are evolutionarily related ion channels that have 25% sequence similarity [ 16 ]. Both channel families originated from a common ancestor, the bacterial sodium channel NaChBac [ 17 ]. These membrane proteins allow sodium or calcium ions to enter the cell as a result of membrane depolarization [ 18 , 19 ]. They play important roles in action potential generation and propagation, neurotransmitter release, excitation-contraction coupling, and other physiological processes including hormone secretion [ 18 , 19 ]. Although a large number of small molecules have been reported to alter the functional activity of VGSCs [ 17 , 20 , 21 ] and VGCCs [ 22 , 23 ], the effect(s) and mechanism(s) of action of natural products on these channels are still a matter of investigation. In this review, we begin by presenting a brief overview of the structure and function of VGSCs and VGCCs, and then describe recent contributions (from 2000 until 2021) made in the studies of the effects of plant- and fungi-derived natural products on VGSCs and VGCCs.

One of the most serious side effects of chemoradiotherapy for head and neck cancer patients is oral ulcerative mucositis (OUM) [ 125 ]. [6]-gingerol and [6]-shogaol are two ingredients of Hangeshashinto, a Japanese pharmaceutical-grade drug composed of seven herbal extracts. In an OUM-induced pain model, [6]-gingerol and [6]-shogaol produced lower NaV1.8 currents on cultured rat sensory neurons and in oral mucosal sensation. Preincubation of sensory neurons with [6]-gingerol and [6]-shogaol inhibited veratridine-induced substance P release and action potential generation in a dose-dependent manner. Moreover, [6]-shogaol inhibited currents via NaV1.1, NaV1.3, NaV1.6, NaV1.7, and CaV2.2 [ 125 ]. In an oral ulcerative mucositis model [ 126 ], [6]-gingerol plus [6]-shogaol in combination with Ginseng extract increased mechanical threshold and alleviated spontaneous pain. These findings suggest that targeting both VGSCs and VGCCs by [ 6 ]-gingerol and [6]-shogaol is effective for the treatment of OUM-induced pain. Changrolin (2,6-bis[pyrrolidin-1-ylmethyl]-4-[quinazolin-4-ylamino] phenol) is an anti-arrhythmic drug derived from β-dichroine, an active component of the Chinese medicinal herb, Dichroa febrifuga Lour. The molecular basis for its antiarrhythmic effects has been shown to be throughout the inhibition of delayed rectified potassium channels, transient outward potassium channels, VGSC, and VGCC. This multi-current blocking profile observed in rat ventricular preparations seems to lead to a modification of electromechanical function of cardiac myocytes and likely contribute to the termination of arrhythmia [ 127 ]. Curcumin is a polyphenolic compound found in the curry spice turmeric, obtained from the roots of the Curcuma longa plant. This phytochemical has been used for centuries in traditional Indian medicine to treat a variety of diseases and conditions ranging from allergies and arthritis to Alzheimer’s disease, diabetes, congestive heart failure, and various malignancies. Studies have demonstrated that curcumin exhibits antioxidant, antifungal, and antitumor activities [ 128 ]. Curcumin has been shown to directly modulate the activity of several types of ion channels, such as the cystic fibrosis transmembrane conductance (CFTR) Cl − channel, the inositol 1,4,5-triphosphate receptor calcium channel, and the voltage-gated Kv1.4 potassium channel. It has also been shown that curcumin can inhibit the Adrenocorticotropic-hormone- and Angiotensin-II-stimulated cortisol secretion in adrenal zona fasciculata (AZF) cells potentially via the inhibition of calcium entry through CaV3.2 channels [ 129 ]. Additionally, curcumin was demonstrated to effectively prevent and/or ameliorate diabetic neuropathic pain (DNP). This compound decreases the pain threshold (mechanical and thermal) of DNP rats. It was also reported that the increase in TTX-R sodium currents caused by DNP is ameliorated by curcumin [ 130 ]. These findings suggest that by acting on different targets, curcumin can relieve pain. Eugenol, the main phenolic component in the essential oils extracted from cloves, has been extensively used in dental clinics as analgesic. It has been described that eugenol can influence cloned T-type channel isoforms expressed in HEK293 cells. Treatment of trigeminal ganglion (TG) neurons with eugenol inhibited all three isoforms of T-type calcium channels in a concentration-dependent manner. Interestingly, eugenol had little effect on the current kinetics of CaV3.1 and CaV3.2, but did not affect the inactivation kinetics of CaV3.3 channels [ 131 ]. Reduction of channel availability enhanced eugenol inhibition sensitivity for CaV3.1 and CaV3.2, but not for CaV3.3. Moreover, eugenol inhibition of T-type channel isoforms was found to be use-dependent. Taken together, these findings suggest that T-type calcium channels are molecular targets for the pain-relieving effects of eugenol [ 131 ]. On the other hand, methyl eugenol, a related phenylpropanoid, also used for the treatment of toothache and other types of pain, was shown to preferentially bind to NaV1.7 channels in the inactivated and/or open state [ 132 ]. In whole-cell patch-clamp recordings, methyl eugenol tonically inhibited transiently expressed NaV1.7 channels in a concentration- and voltage-dependent manner, suggesting that the antinociceptive and anesthetic effects of this compound could result in part from its inhibitory action on VGSCs. Linalool is a major component of essential oils such as Jasmine, Rosemary, and Lavender and has been shown to possess various biological effects in the sensory and central nervous systems. Linalool significantly and reversibly suppressed VGSCs in newt olfactory receptor cells (ORCs) as well as membrane voltage-gated currents in newt retinal cells and cerebellar Purkinje cells [ 133 ]. The mechanism for this voltage-gated channel block is still unclear, but given that linalool can interfere with the lipids of somatic membranes, this could affect ionic channels directly as some other odorants have been previously reported [ 134–137 ]. Additionally, some evidence suggests that linalool also suppresses calcium currents in ORCs, by inhibiting the KCl-induced [Ca 2+ ] response of ORCs [ 133 ]. Moreover, linalool along with Rosmarinic acid reduces CaV3.2 conductance and shifts its steady-state inactivation when tested on HEK-293 cells. In line with this, no change in the activation properties was observed, indicating that these natural compounds preferentially bind and stabilize CaV3.2 channels in the inactivated state [ 138 ]. The inhibition of the T-type Calcium channel CaV3.2 by these natural products may contribute to their neuroprotective and anticonvulsant properties. Naringenin is a flavonoid found in citrus fruits, tomatoes, and figs, and has been shown to reduce inflammatory pain in mice by lowering IL-33, TNF-α, IL-1β, and NFκB activation [ 139 , 140 ]. Furthermore, Naringenin inhibits prostate cancer metastasis by decreasing mRNA level expression of SCN9A gene, which encodes NaV1.7 VGSC [ 141 ]. Naringenin inhibited depolarization-evoked calcium influx in rat DRG neurons stimulated by acetylcholine, ATP, and capsaicin. This compound was discovered to bind to the VGCC regulator collapsing response mediator protein 2 (CRMP2), in the polar cavity formed by the amino acids P79, Q81, K254, and H198 [ 142 ]. In electrophysiology recordings, Naringenin inhibited calcium and NaV1.8 currents in DRG neurons, while also lowered the frequency of sEPSC in substantia gelatinosa neurons. In addition, Naringenin alleviated pain caused by spinal nerve injury (SNI) and reduced postsurgical mechanical allodynia but had no effect on heat-induced nociceptive thresholds. Interestingly, the antinociceptive potential of Naringenin is sex-dependent, as it is only found in males. This data provides evidence that by targeting both VGCCs and VCSCs, Naringenin elicits its analgesic effects.

The VGSC core is the pore-forming α subunit of 250 kDa, topologically structured in 24 segments arranged in four homologous domains, each one constituted by six transmembrane segments [ 19 ]. The symmetric quartet of S1–S4 segments houses the voltage-sensing module. Structurally, this quartet surrounds the pore formed by S5 and S6 [ 19 ]. Even though the expression of the α subunit is sufficient for the channel function, one or two β subunits of 30–40 kDa with single membrane-spanning protein topology can join each α subunit and modify their biophysical properties [ 17 , 19 ]. VGSC’s α-subunits are broadly expressed in central and peripheral nervous systems. The family of VGSCs has nine members (NaV1.1–1.9), which can be further divided according to their sensitivity (S) or resistance (R) to tetrodotoxin (TTX). For instance, TTX-S channels, NaV1.1–NaV1.7, are blocked by 500 nM–1 μM of TTX, whereas NaV1.8 and NaV1.9 are resistant to this toxin [ 19 ]. TTX-S channels have very fast kinetics, exhibiting fast inactivation that typically occurs within 5–10 ms. NaV1.8 and NaV1.9, however, have slow and ultra-slow kinetics, and produce persistent currents for several hundred milliseconds [ 19 ]. In response to membrane depolarizations, activation of VGSC allows current influx through these channels, which contributes to action potential generation and propagation [ 19 ]. The central pore-forming α1 of VGCCs subunit holds a transmembrane arrangement like the α subunit of sodium channels [ 24 ]. According to their voltage sensitivity, VGCCs are divided into high-voltage-activated (HVA) and low-voltage-activated (LVA) calcium channels [ 25 ]. HVA calcium channels (L-, P/Q-, N-, and R-type) are activated at membrane potential more positive than −30 mV. The α1 subunit of HVA channels is associated with up to three distinct classes of auxiliary subunits: the intracellular β subunit; a membrane-associated, disulfide-linked α2δ subunit; and a transmembrane γ subunit [ 24 ]. In contrast, the α1 subunit of LVA calcium channels (T-type) does not require ancillary subunits [ 24 ]. These are activated by small membrane depolarization that allows calcium entry near the resting membrane potential [ 26 ]. VGCCs pharmacological signature profile is unique: dihydropyridines block L-type channels, ω-Conotoxin GVI blocks N-type, ω-Agatoxin blocks P/Q-type, SNX-482 inhibits R-type, while TTA-A2 and Z944 block T-type channels [ 27 , 28 ]. Table 1. Composition and source of natural products, their ion channel targets and effects on heterologous cells and neurons, and analgesic actions in vivo. Natural product Chemical composition Source Target Model and concentration tested Ref. -Not reported- - Allium macrostemon Bunge Water decoction ↓NaV1.7 HEK cells tested with 50 mg/L. Alleviates pain in Formalin-induced and Acetic-acid-induced pain. [ 41 ] Alpha-allocryptopine Alkaloid Corydalis decumbens ↑NaV1.5T353I HEK cells tested with 30 µM. Rescues NaV1.5 T353I plasma membrane expression. [ 44 ] Alpha-bisabolol Terpene Cannibus sativa ↓CaV3.2 ↓CaV3.3 ↓CaV2.1 Inhibited recombinant human CaV3.2 in HEK cells with IC 50 of 4.5 ± 1.1 μM [ 98 ] Astragalin Flavonol glycoside Roots of Astragalus membranaceus ↑L-type current Rat pancreatic islets tested with 100 µM. Hypoglycemic effect in hyperglycemic rats. [ 103 ] Betulinic acid Pentacyclic triterpenoid Hyptis emoryi ↓CaV3.2 ↓CaV3.3 ↓CaV2.2 HEK cells and rat DRGs tested with 20 µM. Alleviates Chemotherapy-induced peripheral neuropathy, HIV-induced sensory neuropathy, and partial sciatic nerve ligation at 2 µg/5 µl. [ 104 ] Bulleyaconitine A Diterpenoid alkaloid Aconitum bulleyanum ↓NaV Pituitary GH3 cells tested with 10 µM. Sensory and motor block of rat sciatic nerve, tested with 0.375 to 0.75 mM. [ 46 ] Camphene Monoterpene Cannibus sativa ↓CaV3.2 ↓CaV3.3 ↓CaV2.1 Inhibited recombinant human CaV3.2 in HEK cells with IC 50 of 7.7 ± 1.8 μM [ 98 ] Cannabidiol, Δ9-tetrahydrocannabinol Benzoic acid Diterpenoid Cannabis sativa ↓CaV3.1–3.3 HEK cells tested with 10–30 μM. Mouse trigeminal ganglion neurons tested with 1 µM. [ 114 ] Changrolin Phenol Dichroa febrifuga Lour ↓NaV Rat ventricular myocytes. IC 50  = 10.19 µM. [ 127 ] ↓CaV (L-type) Rat ventricular myocytes. IC 50  = 74.73 µM. Cordycepin Nucleotide analog Cordyceps spp.(fungus) ↓NaV Rat hippocampal CA1 pyramidal neurons tested with 80 µM. [ 51 ] Curcumin Polyphenol Curcuma longa ↓TTX Nav Rat DRG neurons tested with 100 mg/kg. Analgesic effect in diabetic neuropathic pain model induced with STZ. [ 130 ] ↓CaV3.2 Bovine adrenal cells tested with 5 µM. [ 129 ] Evodiamine Alkaloid Evodia rutaecarpa ↓L-type CaV Rat colon smooth muscle cells tested with 10 and 100 µM. [ 118 ] Eugenol, essential oil Phenolic acid Cloves ↓CaV3 HEK cells stably transfected, trigeminal ganglion neurons. IC 50  = 0.5 mM. [ 131 ] ↓NaV, Dental primary afferent neurons. IC 50  = 0.6 mM Alleviates neuropathic pain in SNL at 50 µg [ 29 ][ 30 ] Dodoneine Phenolic Agelanthus dodoneifolius ↓L-type CaV Rat cardiac myocytes. IC 50  = 10 mM [ 115 ] Gastrodin Gastrodia Gastrodia   IC 50 NaV1.7 = 25.87 µM, HEK cells. IC 50 NaV1.8 = 67.5 µM, DRGs. Alleviates peripheral neuropathic pain and reverts hyperexcitability. [ 56 ] [ 6 ]-gingerol and [ 6 ]-shogaol Phenolic Ginger, rhizome ↓NaV1.8 ↓Nav1.1 ↓NaV1.3 ↓NaV1.6 ↓NaV1.7 ↓CaV2.2 IC 50 NaV1.8 = 45 µM. Analgesic effect in a rat oral ulcerative mucositis model at 300 µM and 150 µM. [ 125 ] - Attenuates pain-associated behavior in rats with SNL at 100–400 mg/kg. [ 31 ] (-)-hardwickiic acid ((-)-HDA) Diterpenoid Croton californicus (Euphorbiaceae) ↓NaV TTX-S Rat DRGs tested with 20 µM. [ 57 ] ↓NaV1.1, NaV 1.3, NaV1.5 HEK cells tested with 20 µM.   Alleviates HIV- and chemotherapy-induced neuropathy at 2 µg/5 µl. Hautriwaic acid (HTA) Diterpenoid Eremocarpus setigerus (Euphorbiaceae) ↓NaV TTX-S Rat DRGs tested with 20 µM. [ 57 ] Alleviates HIV- and Chemotherapy-induced neuropathy at 2 µg/5 µl. Imperatorin Furocoumarin Angelica biserrata ↓NaV1.7 IC 50  = 28 nM. Analgesic activity in thermal pain and formalin-induced pain in mice. [ 32 ][ 93 ] Lappaconitine Diterpene alkaloid Aconitum sinimontanum roots NaV1.5 HEK cells (Acute 30, 60, 100 µM). Irreversible block. [ 61 ] ↓NaV1.7 HEK cells tested with 30 µM. IC 50  = 27.67 µM. [ 62 ] ↓NaV1.3, ↓NaV1.4, ↓NaV1.5, ↓NaV1.8 HEK cells tested with 100 µM. Lavender essential oil Silexan, a standardized lavender oil preparation Monoterpenoid Lavender stoechas and angustifolia   Antihyperalgesia in mice with SNI at 100 mg/kg. ↓pERK1/2 and JNK1. [ 33 ] ↓CaV3.2 ↓CaV2.2 ↓CaV2.1 CHO cells, tested with 1 or 10 µg/ml. Murine synaptosomes and hippocampal neurons tested with 1 µM. Decrease of anxiety-related behavior in humans at 80 mg/d. [ 34 ][ 35 ][ 36 ] Linalool essential oil Monoterpenoid Lavender stoechas and angustifolia ↓CaV3.2 HEK cells. IC 50  = 84 µM. [ 138 ] ↓NaV Olfactory receptor cells (ORCs). IC 50  = 0.56 mM [ 133 ] Methyl eugenol Phenylpropene Asari Radix et Rhizoma ↓NaV1.7 Transfected CHO cells. IC 50  = 295 μM [ 37 ] Magnolol Polyphenolic Bark of Magnolia officinalis ↓NaV NG108-15 cells. IC 50  = 15 and 30 µM at −70 and −100 mV, respectively [ 38 ] TTX-STTX-R DRG neurons, TTX-S IC 50  = 9.4 μM. TTX-R IC 50  = 7 μM [ 78 ] Naringenin Flavonoid Citrus, tomatoes, and figs ↓NaV1.8 ↓CaV2.2 Rat DRG neurons tested with 100 µM. Reverses mechanical allodynia (pSNL), in males at 30 mg/kg. [ 142 ] Paeonol Methoxybenzene Paeonia suffruticosa ↓NaV total Guinea pig ventricular myocytes. IC 50  = 17 µM. [ 80 ] Peimine Alkaloid Fritillaria ↓NaV1.7 IC 50 47.2 µM [ 39 ] Physalin F Steroidal Physalis acutifolia ↓CaV2.2 ↓CaV2.3 Rat DRG neurons tested with 1 µM. Antinociceptive effect on paclitaxel-induced peripheral neuropathy and SNL at 2 µg/5 µl. [ 119 ] L-Tetrahydropalmatine, protopine, and dehydrocorydaline Alkaloids Cordyalis yanhusuo ↓NaV1.5 ↓NaV1.7 L-Tetrahydropalmatine, IC 50  = 7.05 µM. CHO cells stably expressing NaV1.7 and NaV1.5. Analgesic effects in formalin-induced pain model. [ 67 ] Puerarin Isoflavonoid glycoside Kudzu root ↓NaV1.8 through β1 ↓NaV1.7 Rat DRG neurons. IC 50  = 481.5 µM. Analgesic effect in paclitaxel-induced neuropathic pain at 24 mg/kg. [ 85 ] Qishenkel Luteolin, cryptotanshinone, licochalcone A, and glycyrrhetinic acid – Mixture ↓CaV1.2 Pig cardiac myocytes tested with 0.33 g/kg. Luteolin, cryptotanshinone, licochalcone A, and glycyrrhetinic acid. IC 50  = 1.5, 5.7, 4.3, and 12.8 µM, respectively. [ 40 ] Quercetin, Quercitrin, Hyperin, Rutin Flavonoids Acanthopanax senticosus ↓L-type CaV Rat ventricular myocytes tested with 283.12 µg/mL. [ 121 ] Radix paeoniae rubra (paeoniflorin, benzoylpaeniflorin, albiflorin, lactiflorin, oxypaeoniflorin, paeonin, paeoniflorigenone, paenoside, paeonolide, paeonol, galloylpaeoniflorin, gallotannin) – Mixture ↓Total NaV Rat hippocampal CA1 neurons tested with 0.8 mg/ml. [ 50 ] Rosmarinic acid Phenolic acids Rosmarinic officinalis ↓CaV3.2 IC 50  = 53.5 µg/ml. [ 138 ] Saikosaponins A Pentacyclic triterpenoid Bupleurum Chinese root ↓NaV1.7 IC 50  = 28.6 nM. Analgesic activity in thermal pain and formalin-induced pain in mice. [ 32 ][ 93 ] Sophoraflavanone G Flavonoid Root of Sophora flavescens ↓CaV3.1 ↓CaV3.2 HEK cells, IC 50 CaV3.1 = 1.4 µM. HEK cells IC 50 CaV3.2 = 0.75 µM. Alleviates mechanical allodynia. [ 43 ] Sophoraflavanone G Flavonoid Root of Sophora ↓HVA NG108-15 cells IC 50 HVA = 1.8 µM [ 43 ] Sophoraflavanone G Vincapusine, Vincarodine, Serpentine Flavonoid Monoterpene indole alkaloid Root of Sophora Bulbus Fritillaria Catharanthus roseus - Murine models of inflammatory or neuropathic pain. Inhibits acetic-acid-induced writhing response in the formalin test, paclitaxel-induced neuropathic pain (3 or 1.5 mg/kg p.o.). [ 43 ][ 124 ] ↓CaV3.1 HEK-293 cells transiently transfected. Vicapusine, IC 50  = 11.83 µM. Vincarodine, IC 50  = 14.3 µM. Serpentine, IC 50  = 14.54 µM. Composition and source of natural products, their ion channel targets and effects on heterologous cells and neurons, and analgesic actions in vivo.

Insufficient understanding of the efficacies and adverse reactions of natural products and their molecular mechanisms still represents an important obstacle in modern drug development as well as the clinical translation of naturally derived compounds. Nevertheless, these difficulties can be circumvented by large natural product screening libraries, improved fractionation strategies, and techniques for structural elucidation of active fractions from complex natural product precursors. Together, these approaches afford the accurate exploration of the biological activity of previously inaccessible sources of natural products. Nature offers a great source of bioactive molecules with activity toward VGSCs and VGCCs. As reviewed in this manuscript, there are many promising drug candidates directed to these channels that have a natural provenance. The present review is intended to show that the chemical diversity of natural products is well suited to provide the core scaffolds for future drugs that may allow the treatment of pathological conditions related to VGSCs and VGCCs malfunction, particularly, but not limited to, chronic pain.