Local anesthetics reversibly block voltage-gated (voltage-dependent or voltage-sensitive) Na
+ channels (Nav channels) that are responsible for the initiation and propagation of action potentials of excitable cells in the peripheral nervous system and the cardiac system [
10]. Voltage-gated Na
+ channels are integral membrane proteins that are composed of a core α-subunit associated with one or more regulatory β-subunits (β1, β2, β3 and β4). The α-subunit not only forms the pore selectively permeable for Na
+ ions but also contains the binding or receptor site for local anesthetics, anti-arrhythmic drugs and several neurotoxins. Local anesthetics bind to such a site and cause occlusion of the pore, resulting in the blockade of Na
+ channels. At least nine distinct α-subunits (Nav1.1 to Nav1.9) have been cloned from mammalian Na
+ channels. Nav1.7, Nav1.8 and Nav1.9 are the channel isoforms of nociceptive neurons in the peripheral nervous system, and Nav1.1, Nav1.2, Nav1.3 and Nav1.6 are the isoforms in the central nervous system, whereas Nav1.4 and Nav1.5 are in skeletal muscle and heart, respectively [
18]. Since Nav1.7 and Nav1.8 isoforms play a crucial role in pain transmission, both channels are implicated as the targets for anesthetic and analgesic drugs. Based on their affinity for neurotoxin tetrodotoxin (TTX), Na
+ channel subtypes are divided into TTX-sensitive voltage-gated Na
+ channels (including Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6 and Nav1.7) and TTX-resistant voltage-gated Na
+ channels (including Nav1.5, Nav1.8 and Nav1.9), in which Nav1.8 and Nav1.9 are predominantly found in dorsal root ganglion neurons. While specific blockade of single or selected Na
+ channel subtypes in sensory neurons is considered to induce local anesthesia with less adverse effects, conventional local anesthetics and anti-arrhythmic drugs act on different Na
+ channel isoforms.
Local anesthetics introduced to clinical practice have a basic structure consisting of a lipophilic aromatic group, a positively chargeable amino terminus and an intermediate chain. In extracellular fluids, their molecules exhibit equilibrium between uncharged and charged forms, which is determined by the p
Ka of drugs and the pH of media. Once local anesthetics diffuse across membrane lipid bilayers, they re-exhibit equilibrium between uncharged and charged forms in intracellular fluids of cytoplasm. Their charged molecules exclusively interact with the receptor sites of Na
+ channels. In addition to membrane-embedded channel proteins, local anesthetics structure-specifically act on membrane-constituting lipids, directly depressing the functions of neuronal membranes and indirectly inhibiting the activity of Na
+ channels by modifying the physicochemical properties (fluidity, order, microviscosity or elasticity) of lipid membranes surrounding channel proteins [
11]. Since plant terpenoids, alkaloids and flavonoids have amphiphilic structures as well as local anesthetics, these phytochemicals are expected to affect the activity of voltage-gated Na
+ channels through the common mechanisms.
4.2. Essential Oils and Terpenoids
Essential oils, the concentrated liquids of a highly complex mixture of volatile phytochemicals, are generally extracted from aromatic plants, including citrus peel and caraway. In addition to the utility as perfume and flavor, the medicinal property has been suggested for essential oils from peppermint, lavender, eucalyptus, rosemary, thyme, nutmeg and chamomile. Terpenoids occupy more than 90% of phytochemicals contained in the essential oils. Their structures consisting of five-carbon (C5) isoprene units are classified by the number of C5 unit into monoterpenoids with 2 × C5 unit, sesquiterpenoids with 3 × C5 unit, diterpenoids with 4 × C5 unit and triterpenoids with 6 × C5 unit. Monoterpenoids are composed of acyclic (open-chain), monocyclic and bicyclic structures. Excellent reviews were recently published for the pharmacological effects of essential oils and their component terpenoids by de Sousa [
25] and Guimarães et al., [
26], and for the therapeutic benefits of essential oils by Djilani and Dicko [
27].
A variety of monoterpenoids have been suggested to modulate the activity of voltage- and ligand-gated ion channels [
28]. Zalachoras et al., [
29] determined the conduction changes of frog sciatic nerve fibers to compare the local anesthetic activity of five monoterpenes: acyclic linalool, monocyclic
p-cymene, and bicyclic eucalyptol (1,8-cineol), α-pinene and fenchone (
Figure 3). They placed the isolated nerves in a three-chambered recording bath and monitored compound action potentials (CAPs). Their results indicated that 7.5–30 mM linalool and 30 mM fenchone exert local anesthetic effects as well as 3.5–30 mM lidocaine, while the effects of eucalyptol, α-pinene and
p-cymene were minor.
Lavender (
Lavandula angustifolia, Lamiaceae) has been traditionally used as a medicinal herb with relaxant and antispasmodic activity. Ghelardini et al., [
30] showed that lavender essential oil and its major components linalool and linalyl acetate (
Figure 3) reduce the electrically evoked contractions of rat phrenic-hemidiaphragm at 0.1 μg/mL to 1 mg/mL as well as procaine and lidocaine. In their following experiments, lavender essential oil, linalool and linalyl acetate depressed rabbit conjunctival reflexes by administrating 0.03–2.5 mg/mL in the conjunctival sac. Such effects were evident 5 min after 2.5 mg/mL administration and diminished within 15 min. Leal-Cardoso et al., [
31] revealed that linalool reversely blocks the excitability of rat sciatic nerves and inhibits the voltage-gated Na
+ currents of rat dorsal root ganglion neurons at sub-micromolar concentrations. Linalool exists as (−)-enantiomer or (+)-enantiomer in essential oils because it has a chiral center at the 3-position (
Figure 3). (−)-Linalool predominantly occurs in essential oils from lavender, laurel (
Laurus nobilis, Lauraceae) and basil (
Ocimum basilicum, Lamiaceae), but (+)-linalool in ones from coriander (
Coriandrum sativum, Apiaceae). Peana et al., [
32] reported the comparative anti-inflammatory effects of (−)-enantiomer, racemate and acetyl ester of linalool. In a carrageenan-induced hind paw edema model of rats, (−)-linalool (25–75 mg/kg, s.c.) reduced the edemas more effectively than racemic linalool and linalyl acetate.
Monocyclic monoterpene menthol is contained in essential oils from peppermint (
Mentha piperita, Lamiaceae) and spearmint (
Mentha spicata, Lamiaceae) that are used for aromatherapy, mouthwash, toothpaste and topical preparations to relieve irritation and inflammation. Among eight stereoisomers, menthol naturally occurs as a (−)-enantiomer of the 1
R,2
S,5
R configuration (
Figure 3). Galeotti et al., [
33] comparatively evaluated the local anesthetic activity of (−)-menthol and (+)-menthol and their structural analog thymol and (−)-menthone (oxidized menthol). (−)-Menthol and (+)-menthol reduced the electrically evoked contractions of rat phrenic nerve-hemidiaphragm at 0.1–100 ng/mL as well as procaine, although thymol and (−)-menthone were not effective even at 1 μg/mL. When applied to the rabbit conjunctival sac at 30–300 μg/mL, (−)-menthol and (+)-menthol increased the number of stimuli necessary to provoke the reflex, but neither thymol nor (−)-menthone. Both menthol enantiomers produced these effects 5 min after treatments. Gaudioso et al., [
34] studied the effects of micromolar menthol on different Na
+ channel subtypes of rat dorsal root ganglion neurons by a patch clamp method. They indicated that menthol inhibits TTX-resistant Nav1.8 and Nav1.9 and TTX-sensitive Na
+ channels in a concentration-, voltage- and frequency-dependent manner. Pan et al., [
35] examined the action of menthol on pain hypersensitivity induced by inflammation. Their results suggested that menthol is a nonselective analgesic to act on both peripheral and central pain targets.
Kumamoto et al., [
36,
37,
38] carried out a series of studies to compare the effects of various monoterpenoids on nerve conduction and transient receptor potential channels by recording CAPs in frog sciatic nerves. Acyclic citral (a mixture of
trans-geranial and
cis-neral), citronellol, citronellal, (−)-linalool, racemic linalool and geraniol; monocyclic (−)-menthol, (+)-menthol, thymol, carvacrol (thymol isomer), α-terpineol, (−)-menthone, (+)-menthone, (−)-carvone, (+)-carvone and (+)-pulegone; terpenoid phenol eugenol; bicyclic (−)-borneol, (+)-borneol, eucalyptol (1,8-cineol) and 1,4-cineol; and terpenoid ester linalyl acetate, geranyl acetate and bornyl acetate (
Figure 3) reduced CAP peak amplitudes with IC
50 values of 0.34–7.2 mM. These monoterpenoids also inhibited nerve conduction by blocking TTX-sensitive voltage-gated Na
+ channels involved in CAP production, but not activating transient receptor potential channels. The inhibition of CAPs was greatest in carvacrol and thymol, followed by citronellol, bornyl acetate and citral. In the structure and activity relationship, the CAP-inhibitory potency generally varied in the order of being phenols (carvacrol, thymol and eugenol) ≥ aldehydes (citral and citronellal) ≥ esters (bornyl acetate, geranyl acetate and linalyl acetate) ≥ alcohols (citronellol, geraniol, (+)-menthol, (−)-menthol, (+)-borneol, racemic linalool, (−)-borneol, (−)-linalool and α-terpineol) ≥ ketones ((+)-pulegone, (−)-carvone, (−)-menthone, (+)-carvone and (+)-menthone) > bicyclic monoterpenes (eucalyptol and 1,4-cineol). When comparing IC
50 values (0.34–0.93 mM) to reduce CAP peak amplitudes in frog sciatic nerves, the local anesthetic effects of carvacrol, thymol, citronellol, bornyl acetate, citral, citronellal, geranyl acetate, geraniol, linalyl acetate and (+)-menthol were almost equivalent to those of levobupivacaine, ropivacaine, lidocaine and cocaine [
39,
40,
41].
In in vitro experiments of Joca et al., [
42] using rat nerve samples, carvacrol reversely blocked the excitability of sciatic nerves with an IC
50 value of 0.5 mM and reduced the voltage-gated Na
+ currents of dorsal root ganglion neurons with an IC
50 value of 0.37 mM. Cavalcante Melo et al., [
43] confirmed its in vivo effects on mice. Carvacrol (50–100 mg/kg, p.o.) significantly inhibited nociception in acetic acid-induced abdominal constriction, formalin injection and hot plate tests. Such effects were not reversed by naloxone and
l-arginine, indicating that neither the opioid system nor the nitric oxide pathway is responsible for antinociception by carvacrol. Gonçalves et al., [
44] reported that (−)-carvone reduced the excitability of rat sciatic nerves at 10 mM to block about 50% CAP and that (−)-carvone (100–200 mg/kg, i.p.) was effective in inhibiting acetic acid-induced writhing and formalin-induced hind paw nociception of mice.
Leal-Cardoso and his colleagues [
45,
46,
47,
48,
49,
50] performed a series of in vitro experiments using nerve samples from rats to determine the local anesthetic activity of monoterpenoid components in
Croton nepetaefolius (Euphorbiaceae),
Ocimum basilicum (Lamiaceae) and
Ravensara anisata (Lauraceae). These plants used in folk medicine are rich in essential oils containing estragole and anethole (
Figure 3). Both monocyclic monoterpenes directly inhibited Na
+ channels and blocked the excitability of peripheral nerves. Estragole inhibited total Na
+ currents and TTX-resistant Na
+ currents in dorsal root ganglion neurons with IC
50 values of 3.2 and 3.6 mM, respectively, suggesting that it affects the excitability of peripheral nerves as well as local anesthetics [
46]. Among monoterpenoids contained in
Lippia alba (Verbenaceae) and
Croton nepetaefolius (Euphorbiaceae), citral inhibited CAPs in sciatic nerves with an IC
50 value of 0.23 mM [
48]. Eucalyptol reversely blocked the excitability of sciatic nerves and superior cervical ganglion neurons by acting on Na
+ channels directly [
49,
50].
Ghelardini et al., [
51] compared the local anesthetic activity of components in essential oils from medicinal herb marjoram (
Origanum majorana, Lamiaceae) and anise (
Pimpinella anisum, Apiaceae) by in vitro and in vivo experiments. α-Terpineol and anethole reduced the electrically evoked contractions of rat phrenic nerve-hemidiaphragm at 0.001–1 μg/mL in a concentration-dependent manner, but not citronellal, (−)-carvone, (+)-carvone, α-terpinene, eugenol and
trans-cinnamaldehyde (
Figure 3). Both active monoterpenes also increased the number of stimuli required to evoke rabbit conjunctival reflex at 10–100 μg/mL. Quintans-Júnior et al., [
52] examined in vivo effects of several monoterpenes by acetic acid-induced writhing and formalin-injected hind paw licking tests using mice.
para-Cymene (50–200 mg/kg, i.p.) showed the greatest antinociceptive effect in both tests, followed by acyclic geranyl acetate and bicyclic (+)-camphene (
Figure 3).
Myrrh, a resin secreted by plants of the genus
Commiphora (Burseraceae), has traditionally been used for treating wounds and toothache. Dolara et al., [
53] chromatographed the extracts from
Commiphora molmol to specify one fraction (280 μg/mL) with about a half anesthetic potency of 1% procaine by a rabbit conjunctival reflex test. They isolated two phytochemicals from the active fraction and identified them as furanodiene-6-one and methoxyfuranoguaia-9-ene-8-one (
Figure 3). Both sesquiterpenes selectively and reversibly blocked Na
+ channels in an electrophysiological experiment using rat cardiac myocytes.
The bicyclic sesquiterpene (−)-β-caryophyllene (
Figure 3) naturally occurs in essential oils from clove (
Syzygium aromaticum, Myrtaceae), hop (
Humulus lupulus, Cannabaceae), wild sweet basil (
Ocimum campechianum, Lamiaceae) and oregano (
Origanum vulgare, Lamiaceae). Ghelardini et al., [
54] reported that β-caryophyllene exerted in vitro and in vivo local anesthetic effects comparable to those of procaine. Beta-caryophyllene reduced the electrically evoked contractions of rat phrenic nerve-hemidiaphragm at 0.1 ng/mL to 1.0 μg/mL and increased the number of stimuli necessary to provoke rabbit conjunctival reflex at 10 μg/mL to 1.0 mg/mL.
As one of pharmacological mechanisms for terpenoids, Mendanha et al., [
55] investigated the interaction with biological membranes by electron paramagnetic resonance spectroscopy. All the tested nerolidol, menthol, pulegone, carvone, (+)-limonene, α-terpineol and eucalyptol (
Figure 3) mechanistically interacted with mouse fibroblast and human erythrocyte membranes to increase their fluidity with the potency being sesquiterpene nerolidol greater than other monoterpenoids. Yin et al., [
56] and Nowotarska et al., [
57] revealed that bicyclic borneol and monocyclic carvacrol act on phospholipid bilayers to cause membrane fluidization as well as geraniol. Reiner et al., [
17] verified the interactivity of thymol, eugenol and carvacrol with egg phosphatidylcholine unilamellar vesicles by
1H-nuclear magnetic resonance spectroscopy. Their results indicated that these monoterpenoids are inserted into lipid bilayers to locate in the region between the choline polar group, the glycerol and the first atoms of the acyl chains. Tsuchiya and Mizogami [
58] characterized the membrane effects of terpenoid phenols that increase the fluidity of neuro-mimetic membranes at 1–10 μM with the relative potency being thymol > carvacrol > eugenol as well as 50–200 μM bupivacaine and lidocaine. These phytochemicals penetrated into lipid bilayers with preference to the deeper hydrophobic region of membranes. Thymol and eugenol achieve the concentrations of 10–100 μM in dental pulps and dentins adjacent to the pulp space. Since both terpenoids show local anesthetic and analgesic effects at such micromolar concentrations together with acting on neuronal membranes, they are frequently applied to clinical dentistry as a sedative for toothache, pulpitis and dental hyperalgesia.
Sarmento-Neto et al., [
59] recently published a review that focused on the antinociceptive potentials of essential oils from 31 plant species and their major component terpenoids.
4.3. Alkaloids
Unlike conventional local anesthetics to act on Na
+, K
+ and Ca
2+ channels, neurotoxins specifically block voltage-gated Na
+ channels [
60], so they should be an ideal local anesthetic. Natural neurotoxins, many of which belong to plant alkaloids, potentially produce long-duration local anesthesia. Some alkaloids isolated from plants of the genera
Aconitum and
Delphinium have been applied as analgesic and anti-inflammatory agents in Chinese medicine.
Dzhakhangirov et al., [
61] investigated the surface and infiltration anesthetic effects of
Aconitum and
Delphinium alkaloids by a rabbit corneal reflex test to drop sample solutions (0.01–1%) into the conjunctival sac and by a cat neck trunk anesthesia method to inject 0.1 mL sample solutions (0.1–0.5%) intracutaneously and subcutaneously. Lappaconitine, sepaconitine, ranaconitine, septephine, artecorine, 6-
O-benzoylheteratisine and tadzhaconine (
Figure 4) showed greater potency and longer duration of anesthesia than procaine and lidocaine.
At least six receptor sites on voltage-gated Na
+ channels have been identified for neurotoxin alkaloids. Plant alkaloid aconitine, lappaconitine and bulleyaconitine A (
Figure 4) bind to the receptor site 2 to block Na
+ conduction, whereas pufferfish toxin TTX and shellfish toxin saxitoxin, to the receptor site 1. Site 2 neurotoxins and local anesthetics have overlapping but non-identical binding regions [
62]. The most abundant neurotoxins in
Aconitum and
Delphinium plants are aconitine-like alkaloids that activate Na
+ channels and shift a conformational equilibrium toward the activated state, whereas the other alkaloids block voltage-gated Na
+ channels. Despite the structural similarity, aconitine activates voltage-gated Na
+ channels, but lappaconitine blocks. The structure and activity relationship indicates that alkaloids with a benzoyl ester side chain at the 14-position and at the 4-position act as an agonist and a blocker of voltage-gated Na
+ channels, respectively.
Gutser et al., [
63] compared the local anesthetic effects of
Aconitum alkaloids. They intravenously administered alkaloid samples to mice and after 5 min, injected 5% formaldehyde (20 μL, s.c.) to induce hyperalgesia, followed by monitoring nociception-related behaviors during the subsequent 30 min. Antinociceptive ED
50 values in the early phase (0–15 min) and the late phase (15–30 min) were 0.028 mg/kg and 0.027 mg/kg for aconitine and 0.097 mg/kg and 0.077 mg/kg for 3-acetylaconitine. Both alkaloids showed high affinity for the site 2 of rat synaptosomal Na
+ channels. In contrast, lappaconitine was less effective to show antinociceptive ED
50 values of 2.7 mg/kg and 2.9 mg/kg in the early phase and the late phase, respectively. Aconitine and 3-acetylaconitine are speculated to inhibit neuronal conduction by persistent depolarization, whereas lappaconitine, to block Na
+ channels like local anesthetics. Wang et al., [
64] reported analgesic and anti-inflammatory effects of lappaconitine, which were verified by a mouse acetic acid-induced writhing test, a mouse hot plate test, and rat paw and mouse ear edema models.
Wang et al., [
65] confirmed the in vitro effects of bulleyaconitine A on neuronal voltage-gated Na
+ channels under the whole cell patch-clamp configuration of rat pituitary GH
3 cells expressing Nav1.1, 1.2, 1.3 and 1.6 isoforms. Next, they injected bulleyaconitine A in a volume of 0.2 mL into the sciatic notch of the left hind limb of rats after inhalational anesthesia with sevoflurane and determined changes in sensory and motor functions. Bulleyaconitine A blocked both sensory and motor function of the sciatic nerves at 0.375 mM, although it induced hyperexcitability and cardiac arrhythmia following the sciatic nerve block. Co-injection of 0.375 mM bulleyaconitine A with 2% lidocaine or epinephrine (1:100,000) decreased such systemic toxicity and prolonged the nerve-blocking duration to ~4 h. In their following study [
66], the effects of bulleyaconitine A on voltage-gated Na
+ channels were evaluated by recording Na
+ currents of human embryonic kidney cells expressing different Nav isoforms and by measuring the cutaneous trunci muscle reflex after subcutaneous (via dorsal skin) injection of 0.6 mL test solution to rats. Bulleyaconitine A blocked Nav1.7 and Nav1.8 Na
+ currents at 10 μM and induced the complete nociceptive blockade lasting for ~3 h at 0.125 mM. When co-injecting 0.125 mM bulleyaconitine A with 0.5% lidocaine/epinephrine (1:200,000), the duration of cutaneous analgesia was increased from 3 h to 24 h without adverse effects.
With respect to local anesthetic
Aconitum and
Delphinium alkaloids, Turabekova et al., [
67] recently published an excellent report on the relationship between structure and activity to antagonize voltage-gated Na
+ channels.
Tsuchiya [
68] examined the membrane effects of β-carboline alkaloids on lipid bilayer membranes by a fluorescence polarization method. He demonstrated that tetrahydroharman and tetrahydronorharman (
Figure 4) interact with biomimetic membranes to show biphasic effects that increase the membrane fluidity at high micromolar concentrations but decrease at low nanomolar concentrations. There is the possibility that these membrane-interacting alkaloids may counteract the mechanistic membrane effects of local anesthetics to affect their anesthetic efficacy at physiologically-relevant ~15 nM [
69].
4.4. Flavonoids
Flavonoids are polyphenolic phytochemicals that ubiquitously occur in edible and medicinal plants. A number of flavonoids are structurally derived from the parent compound with a tricyclic (C
6–C
3–C
6) skeleton, sharing a benzene ring A condensed with a heterocyclic six-membered ring C that carries a phenyl ring B at the 2-position for flavonoids (
Figure 5). Based on a structural variation of C ring and its substituents, flavonoids are divided into several subclasses of flavone, flavonol (3-hydroxyflavone), flavanone (dihydroflavone), flavanonol (dihydroflavonol), flavanol (catechin), anthocyanidin with the backbone of 1-benzopyrylium instead of 1-benzopyran-4-one, chalcone lacking the C ring, and isoflavone with the B ring at the 3-position.
Wu et al., [
70] recorded patch-clamp whole-cell currents of mouse dorsal root ganglionic neuronal cells and demonstrated that polyphenols extracted from red wine inhibit voltage-gated Na
+, K
+ and Ca
2+ channels with IC
50 values of 2.5, 4.0 and 0.8–1.5 μg/mL, respectively. They referred to active substances as flavonoids like quercetin, (+)-catechin, (−)-epicatechin and anthocyanins (mostly 3-glucosides of anthocyanidins) and the stilbenoid resveratrol (
Figure 5).
(−)-Epigallocatechin-3-gallate and (−)-epigallocatechin (
Figure 5) are best known as the active components in green tea (the product of
Camellia sinensis, Theaceae), which possess antiproliferative, antitumor, antimicrobial, antithrombotic, anti-inflammatory, anti-allergic, apoptosis-inducing and antioxidant property. Kim et al., [
71] examined their effects on Na
+ currents in rat dorsal root ganglion neurons. (−)-Epigallocatechin-3-gallate inhibited both TTX-sensitive and TTX-resistant Na
+ currents more potently than (−)-epigallocatechin.
In in vitro studies of Paillart et al., [
72], genistein and daidzein (
Figure 5) blocked voltage-sensitive Na
+ channels in cultured rat brain neurons with IC
50 values of 60 and 195 μM, respectively. Their effects were not mediated by tyrosine kinase inhibition that is well-known as the pharmacological mechanism of isoflavonoids.
Garcinia kola (Guttiferae) is a medicinal plant used for laryngitis, cough and liver disease. Its seeds predominantly contain kolaviron [
73], a bioflavonoid complex consisting of 3,8-linked flavanone dimers such as GB-1, GB-2 and kolaflavanone (
Figure 5). Tchimene et al., [
74] evaluated the local anesthetic activity of ethanol extracts from
Garcinia kola seeds and their flavonoid components by an intradermal wheal assay using guinea pigs. GB-1 induced 92% local anesthesia at 10 mg/mL (i.d.), being comparable to the effect of lidocaine (0.66 mg/kg, i.d.).