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 . 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 , its ability to activate hTRPA1 restricts widespread use of menthol as an analgesic . 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 . Furthermore, 1,8-cineole inhibits the chemical nociception produced by several irritants, and has an anti-inflammatory efficacy in patients with severe asthma . 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 . 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 .
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 . 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 ; ii) camphor inhibits hTRPA1 ; 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.