## Discussion  
The use of SO extracts for voice care is widespread. Singers, actors and other professional users of the voice use commercial products containing SO (often in combination with other botanicals) for this purpose, and recently clinical studies [7] confirmed its efficacy in the treatment of vocal discomfort. Despite the long history of use and the phytochemical knowledge of Sisyphrium, its mechanism of action on the voice is still unclear. The effect on pharyngeal irritation is suggested to be related to \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & & & **TRPA1** & \\ \hline \multirow{2}{*}{**cpd**} & \multirow{2}{*}{**Name**} & \multicolumn{2}{c}{**Efficacy**} & \multirow{2}{*}{**Potency**} & **IC\({}_{50}\) Inhibition** \\  & & & **(\%AITC 100\(\mu\)M)** & **EC\({}_{50}\) uM** & **TRPA1 uM** \\ \hline
**5** & (\(-\))-Sinigrin & \(<\)10 & NA & \(>\)100 \\
**1** & Gluco-putranjivine & \(<\)10 & NA & \(>\)100 \\
**2** & Isopropyl & \(108.8\pm 1.6\) & 5.9 \(\pm\) 0.4 & 10.8 \(\pm\) 0.4 \\  & isothi & \(98.0\pm 1.0\) & 4.9 \(\pm\) 0.2 & 12.1 \(\pm\) 0.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: In vitro assays of isolated compounds on transient receptor potential ankyrin 1 (TRPA1) receptor. Figure 2: Panel (**a**): dose-related effect of isopropyl-thiocyanate (IPITC) and 2-butyl-liothiocyanate (2-BITC) on elevation of intracellular Ca\({}^{2+}\) in human embryonic kidney (HEK)-293 cells stably transfected with rat TRPA1 (rTREA1-HEK-293). Data are expressed as percentages of the effect observed with ally-lioshiocyanate (AITC, 100 uM). Panel (**b**): dose-related effect of 5-min pre-incubation of rTREA1-HEK-293 cells with IPITC and 2-BITC on the AITC (100 uM)-induced elevation of intracellular Ca\({}^{2+}\). Data are expressed as percentages of the effect observed with AITC (100 uM) alone. the content of mucilage (10.9-13.5%) [7], and [PERSON] [14] highlighted that the extract of _Sisymbrium officinal_ possesses a myorelaxant activity, particularly against the agents involved in the inflammatory process. Nevertheless, a specific biological target for its active principles has never been identified. Several literature reports confirm that the TRPA1 ion channel is a polymodal sensor involved in mediating inflammatory pain signals. Plenty of plants containing TRPA1 agonists are used for their anti-inflammatory and/or anti-nociceptive features, among which are turmeric, cinnamon, clove, ginger and many more. Plants in the Brassicaceae family are rich in TRPA1 active compounds, and some of them are already recognized as potent agonists, as with the AITC contained in mustard. Also, other isothiocyanate compounds, including benzyl-, phenylethyl-, isopropyl- and methylisothiocyanate, have been previously shown to activate the human TRPA1 channel by calcium imaging, but the activity was expressed only as a range [23; 25; 32]. Our results indicate that both the ITCs contained in SO are potent agonists of the TRPA1 ion channel. In vitro, the efficacy of IPITC (compound **2**) is even higher than that of AITC, the reference standard compound for this chemical class. Compound **4**, 2-BITC, has been tested for the first time and its efficacy turned out to be high as well, although a little lower than that of AITC. The potency values of the two ITCs from SO have been compared to that of many natural agonists of the TRPA1 ion channel owing to their different chemical families, which have been recently reported by some researchers [33] (Figure 3). The two ITCs that are present in SO extracts are among the highest-potency agonists, which include oleocanthal, curcumin and umbelliferone, whereas other well-known agonists such as cynnamaldehyde and 6-gingerol are less potent. The involvement of sulphur derivatives in the therapeutic action of SO, as well as of many other medicinal plants in the Brassicaceae family, is usually expressed as a matter of fact.