## 4 Materials and Methods
### In Vitro Assays with _rTRP Ion Channels_  
Compound effects on intracellular Ca\({}^{2+}\) concentration ([Ca\({}^{2+}\)],) were determined using the selective intracellular fluorescent probe Fluo-4. Assays of the rat TRPA1-mediated elevation of intracellular Ca\({}^{2+}\) in transfected HEK-293 cells were performed as described [36]. Briefly, human embryonic kidney (HEK-293) cells (stably transfected with rat TRPA1 or not transfected) were cultured in EMEM + 2 mM Glutamine + 1 % non-essential amino acids (NEAA) + 10 % FBS and maintained at 37 \({}^{\circ}\)C with 5 % CO\({}_{2}\). On the day of the experiment, the cells were loaded with Fluo-4 AM (4 \(\upmu\)M) for 1 h at room temperature, rinsed and resuspended in [PERSON]'s solution (145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl\({}_{2}\), 1.2 mM MgCl\({}_{2}\), 10 mM d-glucose, and 10 mM HEPES, pH 7.4), and finally transferred to a quartz cuvette of a spectrofluorimeter (Perkin-Elmer LS50B, Perkin Elmer Inc, Buckinghamshire, UK); \(\lambda_{\text{EX}}=488\) nm, \(\lambda_{\text{EM}}=516\) nm) equipped with a PTP-1 Fluorescence Peltier System (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) under continuous stirring. Cell fluorescence before and after the addition of various concentrations of test compounds was measured, normalizing the effects against the response to ionomycin (4 uM). The values of the effect on [Ca\({}^{2+}\)]i in not-transfected HEK-293 cells were used as a baseline and subtracted from the values obtained from transfected cells. Agonist efficacy was expressed as a percentage of the effect on [Ca\({}^{2+}\)]i observed with 100 uM allyisothiocyanate (AITC). The potency of the compounds (EC\({}_{50}\) values) was determined as the concentration required to produce half-maximal increases in [Ca\({}^{2+}\)]i. Antagonist/desensitizing behavior as evaluated against TRPA1 agonist AITC 100 uM by adding the compound directly into the quartz cuvette 5 min before the stimulation of cells with the agonist. IC\({}_{50}\) is expressed as the concentration exerting a half-maximal inhibition of an agonist effect, taking as 100% the effect on [Ca\({}^{2+}\)]i exerted by the agonist alone. Dose-response curves are fitted by a sigmoidal regression with a variable slope. Curve fitting and parameter estimation were performed with a GraphPadPad Prism (GraphPad Software Inc., San Diego, CA). Three independent experiments were done for each determination; all determinations were performed at least in triplicate. The time-course spectra showing the increase of intracellular Ca\({}^{2+}\) upon application of the compounds are available in Supplementary Material (S10). The following are available online. Figure S1: GCMS Total Ion Chromatogram (TIC) of volatiles in SO. Figure S2: Mass spectra of volatiles in SO. Figure S3: synthetic scheme for the synthesis of glucoputranijivine. Figure S4: analytical data for intermediates in the synthesis of glucoputranijivin. Figure S5: HPLC chromatogram of compound **1**. Figure S6: \({}^{1}\)HNMR spectra of compound **1**. Figure S7: \({}^{13}\)CNMR spectra of compound **1**. Figure S8: HRMS spectra of compound **1**. Figure S9: IR spectrum of compound **1**. Figure S10: Representative traces of [Ca\({}^{2+}\)]i increase evoked by IPITC (a), 2-BITC (b) and AITC (c, for comparison) at 100 uM in HEK293 cells over-expressing rat TRPA1. Conceptualization, A.B. and G.B. ; Methodology, A.B., G.B., L.D.P. ; Formal Analysis, G.B., M.G., N.Z., P.D.N., A.S.M., Writing--Original Draft Preparation, A.B., G.B., L.D.P., P.D.N., Writing--Review & Editing, A.B., G.B., L.D.P. ; Project Administration, A.B. ; Funding Acquisition, A.B. This research was funded by Fondazione Cariplo, Universita degli Studi di Milano and Conservatorio G. Verdi di Milano, grant project \"Erisimo a Milano\", number 1653-2017. We thank [PERSON] and our students [PERSON], [PERSON] and [PERSON] for technical assistance. The authors declare no conflict of interest.