Background
Essential oils are often used in alternative medicine as analgesic and anti-inflammatory remedies. However, the specific compounds that confer the effects of essential oils and the molecular mechanisms are largely unknown. For example, linalool, a monoterpene compound commonly found as a major component of several essential oils has been reported to produce antinociception in two different pain models in mice although the mechanism of its analgesic effects is unknown [
1].
Transient receptor potential (TRP) channels respond to a wide variety of sensory stimuli, including temperature, nociceptive compounds, touch, osmolarity, and pheromones [
2‐
4]. TRPA1 is a TRP channel that functions as a receptor for noxious cold temperatures and allyl isothiocyanate (AITC), the pungent ingredient of mustard oil [
5‐
9]. Although the role of TRPA1 in sensing noxious cold and somatic mechanosensation
in vivo remains unsettled, especially in mammals [
6,
7,
10], TRPA1 is an established chemical nocisensor for a wide variety of reactive compounds. TRPA1 is a receptor for the irritation induced by parabens on the skin [
11] and for pain produced by alkaline pH [
12]. TRPA1 is also activated by flufenamic acid (FFA), 2-aminoethoxydiphenyl borate (2-APB), icilin, menthol, intracellular calcium or zinc ions [
8,
13‐
21]. However, menthol has different effects on TRPA1 in human and mouse. A previous study identified a bimodal action of mouse TRPA1 (mTRPA1) gating by menthol: submicromolar to low micromolar-concentrations of menthol cause robust channel activation, whereas higher concentrations lead to a reversible channel block. Such bimodal action is not observed on human TRPA1 (hTRPA1) [
22,
23]. TRPA1 has also been reported to be involved in inflammation produced by several airway irritants that cause asthma [
24,
25]. TRPA1 is an excitatory ion channel targeted by cold nociception and inflammatory pain. Therefore, TRPA1 is considered to be a promising target for use in identifying analgesic drugs [
26‐
31]. Moreover, TRPM8 is a thermosensitive receptor that detects cool temperatures and menthol [
32,
33], a natural non-reactive cooling compound, which is also involved in antinociception to some extent [
34,
35]. Menthol, the main ingredient of peppermint, is used for pain relief in daily life through TRPM8 activation [
35,
36]. However, high doses of menthol caused sensory irritation [
37] because it acts as a TRPA1 activator in humans [
23]. Camphor, another essential oil component, is now known to exert analgesic effects probably through inhibition of TRPA1 [
31] and activation of TRPM8 [
38]. However, camphor is not suited for use as an analgesic compound because it causes a warm and hot sensation [
39], probably through TRPV1 activation [
31]. Therefore, we thought an effective analgesic compound would activate TRPM8 and inhibit TRPA1, but not activate TRPV1.
Several TRP channels are known to be activated or inhibited by plant-derived substances, such as menthol and camphor, some of which are contained in essential oils. Essential oils have been used for a long time and their side effects are generally considered to be minimal. Accordingly, essential oils, especially ones acting on TRP channels, could be a promising source for the development of analgesic agents.
Therefore, we have been screening essential oils for the ability to activate human TRPM8 (hTRPM8) but not hTRPA1, distinct from menthol. Through the screening, we found that eucalyptus oil exhibited relatively high hTRPM8-activating ability with less activation of hTRPA1. Furthermore, 1,8-cineole, a main component of eucalyptus oil, was identified as a novel natural antagonist of hTRPA1.
Discussion
In this study, we screened several essential oils and fragrance chemicals to find substances that activate hTRPM8 but not hTRPA1, which presumably cause a comforting sensation. We identified 1,8-cineole as a substance with these properties although we used one concentration of 1,8-cineole (5 mM). Furthermore, 1,8-cineole could inhibit hTRPA1, suggesting this substance might act as an analgesic.
TRPA1 is an excitatory ion channel targeted by pungent irritants such as those from mustard oil and garlic and is thought to function in diverse sensory processes, including cold nociception and inflammatory pain. Therefore, TRPA1 is considered to be a promising target for use in identifying analgesic drugs. A natural analgesic compound that does not accelerate pain signaling is desirable for pharmaceutical or cosmetic pain relief. Several reports showed that TRPA1 antagonists, such as ruthenium red, HC-030031, AMG5445, A967079 and camphor, possess analgesic properties [
26‐
31]. Of these, camphor is the only naturally occurring compound and is often used in cosmetics because of its minimal adverse effects. However, camphor is not suited for use as an analgesic compound because it causes a warm and hot sensation [
39]. It has become clear that this warm and hot sensation is mediated through activation of TRPV1 [
31,
38]. Moreover, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia [
34,
35]. Although menthol, the main ingredient of peppermint, is used for pain relief in daily life through TRPM8 activation [
35], its ability to activate hTRPA1 restricts widespread use of menthol as an analgesic [
36]. Therefore, chemicals that activate TRPM8 and inhibit TRPA1, but do not activate TRPV1, would be ideal as analgesic agents.
We found that activation of hTRPA1 induced by several agonists with different activation mechanisms can be inhibited by 1,8-cineole. Moreover, 1,8-cineole activated hTRPM8 and hTRPV3, but not hTRPA1, hTRPV1 or hTRPV2. It was recently shown that both peripheral and central activation of TRPM8 could produce an analgesic effect that specifically reverses the sensitization of behavioral reflexes elicited by peripheral nerve injury [
34‐
36]. From this point of view, 1,8-cineole appears to be an ideal natural analgesic that activates hTRPM8 and inhibits hTRPA1.
1,8-cineole is known to act as an agonist of the TRPM8 channel with lower efficacy and potency (3.4 ± 0.4 mM) on TRPM8 than menthol [
32,
38]. 1,8-cineole also activates the TRPV3 channel in mice, but not the western clawed frog TRPV3 [
45]. Furthermore, 1,8-cineole inhibits the chemical nociception produced by several irritants, and has an anti-inflammatory efficacy in patients with severe asthma [
51]. The present study suggests that the known analgesic and anti-inflammatory actions of 1,8-cineole can be attributed to its TRPM8-activating and TRPA1-inhibiting abilities.
1,8-cineole has a fresh smell and elicits a cooling sensation when ingested or applied to the skin and is a common additive in flavorings, food, mouthwashes and cough suppressants. 1,8-cineole is also often used in aromatherapy, as a stimulant in skin baths, by the pharmaceutical industry in drug formulations to enhance percutaneous penetration and as a decongestant and antitussive [
52‐
54]. Experimental data have shown that 1,8-cineole is an analgesic and anti-inflammatory agent with beneficial effects for patients with severe asthma [
51]. Although inhibitory effects of 1,8-cineole on the formation of prostaglandins and cytokines by stimulated monocytes have been observed
in vitro, the molecular targets and mechanisms of the analgesic effect of 1,8-cineole remain unclear [
55].
In a human study, we examined whether 1,8-cineole could inhibit sensory irritation caused by octanol and menthol with senseitive volunteers. Because of the clinical setting, especially in the cosmetic research field, both menthol and octanol are well-known chemicals causing skin irritation, and neither cinnnamaldehyde nor allicin is used for human skin studies. The result that 1,8-cineole, whose ability to activate TRPM8 is lower than menthol, inhibited menthol-evoked skin irritation clearly suggests that the inhibitory effects of 1,8-cineole are probably due to inhibition of TRPA1 but not activation of TRPM8.
The inhibitory effects of 1,8-cineole on menthol-induced hTRPA1 activation was a little greater than those for AITC- or FFA-induced hTRPA1 activation (Figure
6B, D, F). Menthol has bimodal action through transmembrane domain 5 of TRPA1 in some species [
22]. Therefore, the similarity between the molecular structures of menthol and 1,8-cineole (Figure
4A) suggests that 1,8-cineole could act on the same domain of TRPA1 as menthol, although the structural basis for menthol-evoked hTRPA1 activation is not known. Four compounds with similar structures (Figure
4A) exhibited different effects on hTRPM8 and hTRPA1: i) menthol and 1,4-cineole activate both hTRPM8 and hTRPA1 [
56]; ii) camphor inhibits hTRPA1 [
31]; iii) 1,8-cineole activates hTRPM8 and inhibits hTRPA1 (Figures
2,
3,
4). The fact that the four compounds exhibit promiscuous effects on hTRPM8 and hTRPA1 suggests that more detailed analyses would lead to a better understanding of the structural basis for the action of these compounds on TRPM8 and TRPA1.
Conclusions
1,8-cineole was found to be a rare natural antagonist of hTRPA1. 1,8-cineole activates hTRPM8 but inhibits hTRPA1 activated by several agonists. Moreover, the sensory irritation caused by octanol or menthol, TRPA1 agonists, was inhibited by concomitant 1,8-cineole application in humans. Thus, the analgesic and anti-inflammatory effects of 1,8-cineole might be related to its capacity to inhibit TRPA1 activity, suggesting there may be many effective uses for 1,8-cineole based on its unique action on TRPM8 and TRPA1.
Methods
Molecular cloning
Full-length hTRPA1, hTRPM8, hTRPV1, and hTRPV2 were obtained from Life Technologies (Carlsbad, CA, USA) and hTRPV3 was generously provided by Dr. Hwang (Korea University). cDNAs were cloned into the pcDNA3.1 vector.
Cell culture
HEK293T cells were maintained in DMEM (WAKO Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS (Biowest SAS, Caille, France), 100 units/mL penicillin (Life Technologies Corp., Carlsbad, CA, USA), 100 μg/mL streptomycin (Life Technologies Corp.), and 2 mM L-glutamine (GlutaMAX, Life Technologies Corp.) at 37°C in 5% CO2. For Ca2+-imaging, 1 μg plasmid DNA containing hTRPA1, hTRPV1, hTRPV2, hTRPV3 or hTRPM8 in pcDNA3 in OPTI-MEM medium (Life Technologies Corp.) were transfected into HEK293T cells using Lipofectamine Plus Reagent (Life Technologies Corp.). After incubating for 3 to 4 h, the cells were reseeded on coverslips and further incubated at 37°C in 5% CO2.
Animals
Male C57BL/6 mice (4–5 weeks, SLC, Shizuoka, Japan) were used. Animals were housed in a controlled environment (12 h light/dark cycle, room temperature 22–24°C, 50–60% relative humidity) with free access to food and water. All procedures involving the care and use of animals were approved by The Institutional Animal Care and Use Committee of National Institutes of Natural Sciences and performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 85–23, revised 1985).
Preparation of primary mouse DRG neurons
Mouse dorsal root ganglions (DRGs) were dissected from mice, incubated with 1.25 mg/mL collagenase (Sigma-Aldrich) at 37°C for 15 min, and dissociated using mechanical trituration. After filtration with a cell strainer (70 μm, BD, Franklin Lakes, USA), cells were plated on poly-D-lysine-coated coverslips and incubated in medium (MEM supplemented with 10% FBS, penicillin, streptomycin, and l-glutamine) containing nerve growth factor (100 ng/mL).
Human subjects
Japanese male subjects in their 20s and 30s were selected as participants to eliminate confounding factors that may influence the perception of sensitive skin, including race, age, gender, and hormonal and psychosocial interactions. To evaluate sensory irritation, we selected skin sensitive male volunteers. Female volunteers were excluded because of possible hormonal influences. Ethics approval and informed consent was obtained from all participants.
Ca2+-imaging
Ca2+-imaging was performed 1 day after transfection. HEK293T cells on coverslips were mounted in an open chamber and superfused with standard bath solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.4). Cytosolic-free Ca2+ concentrations in HEK293T cells were measured by dual-wavelength fura-2 (Molecular Probes, Invitrogen Corp.) microfluorometry with excitation at 340/380 nm and emission at 510 nm. The fura-2 ratio image was calculated and acquired using the IP-Lab imaging processing system (Scanalytics Inc., Fairfax, VA USA).
Electrophysiology
Whole-cell patch-clamp recordings were performed 1 day after transfection. The standard bath solution was the same as that used in the Ca2+-imaging experiments, and extracellular Ca2+ was removed and 5 mM EGTA was added for the recording of AITC-, menthol- and FFA-induced current responses. The pipette solution contained 140 mM KCl, 5 mM EGTA, 10 mM HEPES, pH 7.4 (adjusted with KOH). Data from whole-cell voltage-clamp recordings were sampled at 10 kHz and filtered at 5 kHz for analysis (Axon 200B amplifier with pCLAMP software, Axon Instruments, Sunnyvale, CA, USA). Membrane potential was clamped at −60 mV and voltage ramp-pulses from −100 to +100 mV (500 ms) were applied every 5 sec. All experiments were performed at room temperature.
Sensory irritation tests
The study was conducted at a temperature of 21–23°C and a relative humidity of 45-55%. Areas of skin were cleaned with a wet towel and acclimatized for 10 min prior to testing. Blind randomized half-region (left vs. right) trials were performed with two different samples applied to the neck region. A total of 200 μl of base was applied. The subjects evaluated pricking, stinging, burning and itching sensations after 1, 3, 5, 7 and 10 min of compound/chemical application in accordance with the criteria summarized in Table
1. The total sensory irritation scores were calculated for the entire period.
Table 1
Sensory irritation scores
| 5 | Unbearable intense sensation |
Itching | 4 | |
Slightly unusual | 3 | Distinct sensation |
Stinging pain | 2 | |
Burning sensation | 1 | Obscure sensation |
Data analysis
Data in all figures are shown as means ± standard error of the mean (SEM). Statistical significance of effects of 1,8-cineole on several TRP channels were evaluated using ANOVA followed by two-tailed multiple t-test with Bonferroni correction, inhibitory effects of 1,8-cineole on mouse DRG neuron activation were made by using ANOVA with the Bonferroni’s post-hoc test. Sensory irritation tests were evaluated using a Wilcoxon signed-rank test.
Competing interests
The authors have no financial or other relationship that could lead to a conflict of interest.
Authors' contributions
MT, FF, KU and MT designed the experiments, and MT, FF, KU performed the in vitro experiments and analysis. SY, MS, CH and MS performed the human study. MT, FF, KU and MT prepared the manuscript. All authors read and approved the final manuscript.