New natural agonists of the transient receptor potential Ankyrin 1 (TRPA1) channel

The transient receptor potential (TRP) channels family are cationic channels involved in various physiological processes as pain, inflammation, metabolism, swallowing function, gut motility, thermoregulation or adipogenesis. In the oral cavity, TRP channels are involved in chemesthesis, the sensory chemical transduction of spicy ingredients. Among them, TRPA1 is activated by natural molecules producing pungent, tingling or irritating sensations during their consumption. TRPA1 can be activated by different chemicals found in plants or spices such as the electrophiles isothiocyanates, thiosulfinates or unsaturated aldehydes. TRPA1 has been as well associated to various physiological mechanisms like gut motility, inflammation or pain. Cinnamaldehyde, its well known potent agonist from cinnamon, is reported to impact metabolism and exert anti-obesity and anti-hyperglycemic effects. Recently, a structurally similar molecule to cinnamaldehyde, cuminaldehyde was shown to possess anti-obesity and anti-hyperglycemic effect as well. We hypothesized that both cinnamaldehyde and cuminaldehyde might exert this metabolic effects through TRPA1 activation and evaluated the impact of cuminaldehyde on TRPA1. The results presented here show that cuminaldehyde activates TRPA1 as well. Additionally, a new natural agonist of TRPA1, tiglic aldehyde, was identified and p-anisaldehyde confirmed.

Scientific RepoRtS | (2020) 10:11238 | https://doi.org/10.1038/s41598-020-68013-2 www.nature.com/scientificreports/ associated to the stimulation or inhibition of TRPA1 are various, as it has been shown to be expressed in numerous tissues as pancreatic β-cells 13 , intestinal enteroendocrine cells 14 , dorsal root ganglia (DRG) sensory neurons 15 or skin 16 . Moreover, TRPA1 is activated by a broad variety of natural molecules and has been associated to various physiological mechanisms as well as to the pungent, tingling, irritation and burning experience from their consumption 5 . Different classes of chemical compounds from plants or spices are able to induce TRPA1 activation. TRPA1 can be activated by the covalent binding of electrophile isothiocyanates found for example in wasabi, mustard or horseradish, or the covalent binding of thiosulfinates as diallyl sulfide or diallyl disulfide found in plants such as garlic or onion form the Allium genus. Some unsaturated aldehydes have shown to elicit as well big response of TRPA1 17 . Other aldehydes, irritant compounds of cigarette smoke, activates TRPA1 18 . Other foodborne activators of TRPA1 are found in the family of alkylamides as the non-specific agonist of TRPV1, hydroxa-α-sanshool, from Szechuan pepper 17 . Finally, members of the vanilloids have also been reported to activate TRPA1. Indeed, even though vanilloids have been initially reported to be canonical agonists of the TRPV sub-family, compounds like ethyl-vanillin 19 or 6-shogaol or 6-paradol 20 activate TRPA1. One particular TRPA1 agonist, cinnamaldehyde, is reported to impact metabolism. Cinnamaldehyde, the principal constituent of cinnamon oil, has shown positive impact on insulin sensitivity 21 and liver fat of obese mice 22 as well as lowering blood glucose level in diabetic mice 23 or high fat diet fed obese mice 21 , and reducing body weight gain 21,24 of obese mice. In humans, we have shown that after a single dose of cinnamaldehyde ingestion, post-prandial energy expenditure and fat oxidation were maintained higher than in placebo 25 . It could be speculated that these effects result from activation of TRPA1 26 . Indeed it has been shown that TRPA1 contribute to thermogenesis 26,27 . Recently, it has been reported that cuminaldehyde, a cumin compound structurally close to cinnamaldehyde, has an anti-obesity effect in diet induced obese rats 28 and an anti-hyperglycemic effect in a diabetic rat model 29,30 . Additionally, cumin seed oil has been shown to improve insulin sensitivity of patients with diabetes type II 31 . We speculated that this effect might be due to TRPA1 activation as hypothesized for cinnamaldehyde and investigated the response of TRPA1 to cuminaldehyde as well as to other natural aldehydes, anisaldehyde and tiglic aldehyde.

Results
Activation of TRPA1 dependent current by selected compounds. Using whole cell patch-clamp technique in CHO-cells expressing hTRPA1 under the induction of transcription by tetracycline, we tested if the three studied flavors, anisaldehyde, cuminaldehyde and tiglic aldehyde ( Fig. 1) activate TRPA1. An inward current was recorded in hTRPA1 expressing cells clamped at resting membrane potential (− 80 mV) under the addition of 5/50 μL of 3 mM cuminaldehyde (Fig. 2a, right panel red trace), 5 mM anisaldehyde (Fig. 2 b, right panel red trace) or 25 mM tiglic aldehyde (Fig. 2c, right panel red trace). The observed inward current was quickly inactivated. None of the vehicle buffer (ethanol, DMSO or electrophysiology buffer) at corresponding concentration induced an inward current in hTRPA1 expressing cells (Fig. 2a-c, right panel black traces). To verify the specificity of these activated currents, non-induced hTRPA1 stably transfected cells where stimulated with same compounds. None of them elicited any inward currents in the absence of hTRPA1 expression (Fig. 2a-c, left panel red traces). To establish the voltage dependence of the activation of hTPRA1 by the three flavors, voltage ramp protocols (Fig. 3a) were applied to set up current-voltage (I-V) relationships (Fig. 3). I-V curve under cuminaldehyde (Fig. 3b), anisaldehyde (Fig. 3c) or tiglic aldehyde (Fig. 3d) showed the TRPA1 characteristic outward rectifying current and a reverse potential close to zero that could be observed under cinnamaldehyde or cold stimulation 32 .
Dose-dependent activation of TRPA1. By ratiometric calcium imaging in hTRPA1 expressing CHO cells, we confirmed the activation of hTRPA1 by cuminaldehyde, anisaldehyde and tiglic aldehyde (Fig. 4d). Doing dose-response curves, we determined half-maximum activation (EC 50 ) concentration of 0.91 mM for anisaldehyde, 0.72 mM for cuminaldehyde and 1.49 mM for tiglic aldehyde. Compared to a maximum activation of hTRPA1 obtained by a stimulation with 100 μM of cinnamaldehyde, both anisaldehyde and cuminaldehyde reach equivalent maximum activation with concentrations of 5 mM and 10 mM, respectively. Tiglic www.nature.com/scientificreports/ aldehyde reach its maximum level of hTRPA1 activation at about 5 mM with represent only 80% of its activation obtained by 100 μM of cinnamaldehyde (Fig. 4d). HC030031 blocked the responses of anisaldehyde (Fig. 4a), tiglic aldehyde (Fig. 4b) and cuminaldehyde (Fig. 4c). Residual activation can be observed at high concentrations of anisaldehyde upon inhibition with HC030031 ( Fig. 4a, gray square). This activation could be explained by a non-specific activation of CHO cells since same kind of activation can be observed in non-hTRPA1-expressing CHO (non-induced CHO-hTRPA1) cells (Fig. 4a, open circle). To be noted that at high concentrations, close to 10 mM, non-specific activation of non-hTRPA1-expressing CHO can be observed with anisaldehyde and cuminaldehyde ( Fig. 4a,c, open circle). To summarize, by ratiometric calcium imaging, we could show that anisaldehyde, cuminaldehyde and tiglic aldehyde activate hTRPA1 in a dose-dependent manner with lower affinity than reported for cinnamaldehyde, indeed compared to the reported EC 50 of 61 μM for cinnamaldehyde 32 we reported here EC 50 ~ 11, ~ 24 and ~ 24 times higher for cuminaldehyde, anisaldehyde and tiglic aldehyde, respectively. Excepted for tiglic aldehyde, maximum activity comparable to cinnamaldehyde could be reach.

Specific activation.
Since the selected compounds of this study might produce spicy sensation 33 , we evaluated their capacity to activate the related receptor hTRPV1 activated by pungent ingredients from spices like capsaicin. By ratiometric calcium imaging on hTRPV1 expressing cells, we evaluated the activation of hTRPV1 by doses of anisaldehyde, cuminaldehyde and tiglic aldehyde from 1 μM up to 10 mM, and compared to the doseresponse of hTRPV1 to capsaicin (Fig. 5). Compared to the activation of hTRPV1 by capsaicin, no comparable activation could be observed with any of the flavors tested. We could only notice a slight activation upon highest doses of anisaldehyde and cuminaldehyde, which was also observed in hTRPA1 expressing CHO cells and noninduced CHO-hTRPA1 cells (Fig. 4a,c) and interpreted as non-specific to CHO cells. To summarize, no specific activation of hTRPV1 could be recorded under stimulation by anisaldehyde, cuminaldehyde or tiglic aldehyde.

Sensory profile of selected ingredients.
The selected compounds of this study ( Fig. 1) are both reported as flavors and selected for their potential activation of TRPA1. As a confirmation of their capability to stimulate the sensory receptor TRPA1, they have been evaluated in sensory tasting to describe their sensory profile (Table 1). Evaluated concentrations were selected based on safety consideration in the range of flavoring usage in gelatin/pudding food categories. Anisaldehyde evaluated at 47.94 ppm (352 μM) was not reported as tingling but characterized by an herbaceous and anise flavor with a strong persistency of the aromas. Cuminaldehyde at 29 ppm (195 μM) was described as strong in cumin or spicy aroma. Chemesthetic description as irritant, pungent, warm or metallic was associated to the tasted concentration of cuminaldehyde as well as a tingling, burning, metallic sensation in persistency. Tiglic aldehyde at 5 ppm (60 μM) was associated to a tingling or cooling sensation of low overall intensity, an onion after taste and a rubber, fat, almond or syrup-like aroma.
Taken together the sensory description show that both cuminaldehyde and tiglic aldehyde are associated to the characteristic chemesthetic sensation produce by the activation of TRPA1 in the trigeminal nerve, which is not the case for anisaldehyde at the given concentration.

Discussion
In the present study, we reported that three natural compounds, cuminaldehyde, p-anisaldehyde and tiglic aldehyde from spice's origin (cumin, anise and onion/garlic, respectively) are able to activate hTRPA1 specifically but with lower affinity than the well described compound of cinnamon oil, cinnamaldehyde. Indeed, we found for cuminaldehyde, p-anisaldehyde and tiglic aldehyde EC 50 of 0.72 mM, 0.91 mM and 1.49 mM respectively, compared to an EC 50 of close to 60 μM 32 for cinnamaldehyde. P-anisaldehyde, as well found in Korean Mint 34 , was also described as a TRPA1 agonist 34 . We report here, for p-anisaldehyde, an EC 50 of 0.91 mM similar to the published one (0.55 mM) 34 but, contrary to Moon et al. 34 , we found that p-anisaldehyde reach the similar  www.nature.com/scientificreports/ maximum activation (efficacy) as cinnamaldehyde and can be consider as a full agonist. One risk for agonists of lower affinity is the lack of specificity due to higher concentration needed to get similar lever of receptor activation. By calcium imaging, we observed non-specific activation of TRPA1 at high concentrations of cuminaldehyde and anisaldehyde that might be due to cellular toxicity at high doses and a cellular damage induced calcium release. As many TRPA1 agonists have a non-specific effect reported on TRPV1 17 , we verified the cross-activation on TRPV1 (Fig. 5) and confirmed the specificity of these agonists on TRPA1 vs. TRPV1. However, we cannot exclude other non-evaluated non-specific effects at high concentration. Cuminaldehyde, p-anisaldehyde and tiglic aldehyde activated subpopulations of sensory neurons from rat DRG. We observed that the concentrations used to obtain recordable responses in DRG neurons were higher than in hTRPA1 expressing CHO cells. This might be explained by species differences in the pharmacology of primate and rodent TRPA1 already described for several compounds 35 . It is worth noting also that diverse populations of neurons responded to anisaldehyde, cuminaldehyde or tiglic aldehyde: neurons responding to both or only capsaicin or cinnamaldehyde, or neurons responding to neither capsaicin or cinnamaldehyde. The diverse profile of DRG neurons responding to these three tested compounds might reflect the already described high neural diversity in DRG 36 or trigeminal ganglion neurons 37 and variance in gene expression profile among it. Still, a functional role for each neural profile is not clear.
Sensory features of these compounds are not predictive of their pharmacological activities on TRPA1. Indeed, even though cuminaldehyde and tiglic aldehyde were tasted at concentrations below EC 50 , their sensory properties were associated to chemesthetic descriptive such as tingling, burning or metallic that could be attributed to the stimulation of hTRPA1 in trigeminal fibers, which was not the case for anisaldehyde. The differences in chemesthetic sensation might be due to the degradation in the saliva or their capability to diffuse into the epithelium. Discrepancy in sensory properties and in vitro activity on a TRP channel has been previously described for TRPA1 38 . Indeed, the in vitro recording of only TRPA1 activity in a heterologous cellular expression was not able to fully explain the perception of sensory irritation produced by an environmental pollutant. The sensory perception should rather result from the activation of a class of receptors and/or cross-interactions between them 38 , which might be the case as well for the coding of chemesthetic sensation produced by chemicals from spices.
As cinnamaldehyde 21 , cuminaldehyde 28 after 5 or 6 weeks, respectively, of daily ingestion, has shown to promote reduction of body weight gain and lower level of glucose in blood of diet induced obese rodents. Additionally to cuminaldehyde, Haque et al. 28 evaluated the impact of thymol on the metabolism of high fat diet induced obese rats which show to have as well potent anti-obesity effect while not affecting glycaemia. They observed effects that were more significant when cuminaldehyde and thymol were combined than thymol or cuminaldehyde alone. As both cinnamaldehyde 32 and thymol 39 are known to activate TRPA1 and this study reports as well the activation of TPRA1 by cuminaldehyde, it can be speculated that their metabolic effect is partially due to TRPA1 activation, even though nor the effect of cinnamaldehyde neither of cuminaldehyde and thymol on metabolism are understood. Additionally, concurring data can be found in the characterization of other plant extracts activity, as gingerol shown both anti-obesity action 40 , and TRPA1 activation 41 . However, the proposed anti-obesity or anti-hyperglycemic mechanisms of action of gingerol and cuminaldehyde are through the inhibition of α-amylase 40 or α-glucosidase, reducing the intestinal absorption of carbohydrates, and aldose reductase 42 inhibition, respectively. Reinforcing the hypothesis that alternative explanation than activation of TRPA1 may occur in the metabolic effects of cinnamaldehyde and cuminaldehyde is the comparison of in vivo efficient doses to their receptor's potency. Indeed, doses with in vivo impact on metabolism in rodent were, for cuminaldehyde reported by Haque et al. 28 and for cinnamaldehyde reported in our previous study 21 , respectively 12 mg/kg bw/ day and 250 mg/kg bw/day; as the potency of cinnamaldehyde on TRPA1 is higher than cuminaldehyde, we would expect, if the metabolic effects would be due to TPRA1, to have same in vivo effect with lower doses of cinnamaldehyde compared to cuminaldehyde.
Cuminaldehyde demonstrated as well a glucose dependent insulin secretagogue activity in diabetics rats 30 . Interestingly the antidiabetic drug glibenclamide induces insulin release via inhibition of K(ATP) channels in pancreatic β-cell, but also activates TRPA1 43 . As the activation of TRPA1, expressed in pancreatic β-cells, induces insulin release 13 , it might be speculated that the in vivo secretagogue effect of cuminaldehyde is due to TRPA1 stimulation.
It has been as well shown in human, that after a single dose ingestion of cinnamaldehyde energy expenditure and fat oxidation are maintained higher than in placebo condition 25 . Moreover, chronic ingestion of cinnamaldehyde reduces visceral adipose tissue in rodents and increase UCP1 expression in brown adipose tissue (BAT) 24 . It has been proposed that TRPA1 could increase metabolism through cold sensing and thermogenesis stimulation in BAT 44 . It might be as well speculated that long term anti-diabetic effects of TRPA1 agonists as cinnamaldehyde and cuminaldehyde are due to increased energy expenditure and fat oxidation.
As cinnamaldehyde or cuminaldehyde, several natural solutions like capsaicin from red chili or curcumin 45 have been proposed for the improvement of obese or diabetic conditions. Natural solutions with history of food usage could easily be included in the daily diet and complement the efficacy of drug therapy or help, associated to lifestyle modifications, to prevent obesity and diabetes. More broadly, natural solutions could be foreseen for all TRPA1 associated improvement of health disorder that could be associated, as vasodilatation 46 or improvement of swallowing disorders in elderly 10 . However, regarding some discrepancy between potency of TRPA1 agonist and their in vivo effective doses on metabolism, more pre-clinical evidence are needed to confirm the potential of this pathway to target metabolic activities.
Additionally, clinical evidences and assessment of side effects, as noxious irritation 47 related to TRPA1 stimulation, are needed to conclude on the real opportunity to use TRPA1 agonists as complement to preventive or therapeutic approaches. Electrophysiology. The day before the experiment TRPA1 cells were induced using a Ham's F12 medium supplemented with 10% Fetal Bovine Serum, 100 units/ml Penicillin-Streptomycin and 1 μγ/ml Tetracycline Hydrochloride (Sigma Aldrich) to allow the expression of the human TRPA1. The day of the experiment cells were harvested using 5 min treatment with Accutase (Sigma Aldrich) and kept in suspension in extracellular medium (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate and 10 mM Hepes/NaOH, ph 7.4, 298 mOsmol).
All electrophysiological data were collected in the whole-cell configuration using the Port-a-Patch automated system (Nanion). Intracellular solution is 50 mM CsCl, 10 mM NaCl, 60 mM Cs-Fluoride, 20 mM EGTA and 10 mM Hepes/CsOH, ph 7. www.nature.com/scientificreports/ 2 mM CaCl2, 5 mM d-glucose monohydrate and 10 mM Hepes/NaOH, ph 7.4, 298 mOsmol. Microchips (Nanion) of 2-5 mOhm were used. Voltage-clamp recordings were obtained using EPC 10 patch-clamp amplifier (HEKA) and PatchMaster software (HEKA) and PatchControl software (Nanion). Recordings were performed at room temperature at an holding potential of − 80 mV. Series resistances, and fast and slow capacitance transients were compensated by the patch-clamp amplifier. Only cells with leak currents below 100 pA and resistance around 1 GOhm were analyzed.
Sensory evaluation. For safety concern, the doses of compounds used for technical sensory evaluation were the maximum reported use level in food categories related to gelatins and pudding by Burdock 33 . Anisaldehyde was evaluated at 47.94 ppm (352 μM), Cuminaldehyde at 29 ppm (195 μM) and Tiglic aldehyde at 5 ppm (60 μM) in TUC at nectar viscosity (Resource ThickenUp Clear 2.4 g in 200 ml of water). Compounds were first prepared as stock solution in ethanol (Tiglic aldehyde: 20 mg/ml, Cuminaldehyde: 116 mg/ml, p-anisaldehyde: 192 mg/ml). Seven voluntary panelists were asked to assess the sensory characteristics of the samples and conclude on the most obvious shared sensory descriptors.