Eugenol

Eugenol, β-escin, curcumin, berberine hydrochloride, sclareol, capsaicin, parthenolide, ellagic acid, glutathione, L-ascorbic acid were from Sigma–Aldrich (United Kingdom); osthole and pterostilbene were from Stratech (United Kingdom) and mitoquinol from Cayman Chemical Company (United Kingdom); all other NPs were components of the Puretitre natural compound library from Caithness Biotechnologies (United Kingdom). All of the above except Eugenol (70% ethanol), glutathione, and L-ascorbic acid (dH₂O) were dissolved in dimethyl sulfoxide (DMSO, Sigma United Kingdom) and added to growth media from the following stock solutions prepared in those solvents: Eugenol, 500 mM; glutathione, 375 mM, L-ascorbic acid, 500 mM; osthole, 200 mM; pterostilbene, 200 mM; β-escin, 50 mM; curcumin, 50 mM; berberine hydrochloride, 200 mM; sclareol, 130 mM; capsaicin, 200 mM; parthenolide, 130 mM; ellagic acid, 33.3 mM, mitoquinol, 2.94 mM.

•NO generation in leaf tissues was monitored by constant potential amperometry with a NO-selective disc-type electrode^{34 (link),35 (link),37 (link)–40 (link)}. The electrode was prepared by electropolymerizing a poly-eugenol thin film on a cleaned Pt disc^{37 (link)} and repeatedly scanning the potential between −0.2 and 0.6 V in a 10 mM solution of eugenol (Merck) in 0.1 M NaOH. The modified electrode was then conditioned at a constant potential of 0.9 V in a phosphate buffer (pH 7.4) until a stable background current was reached. Electrochemical monitoring of NO generation in leaf tissue extracts and cell suspensions was performed as described previously^{37 (link)}. The current was recalculated into concentration units based on a calibration curve (ISO-NO Mark II instruction manual, World Precision Instruments) constructed by measuring current responses to the addition of freshly prepared •NO aqueous solutions generated in situ from the reaction of iodide with nitrite in acid solution within the range of 0.3–100 μM (Extended Data Fig. 1g,h; ISO-NO Mark II instruction manual, World Precision Instruments)^{41 (link)}.

The minimum inhibitory concentration (MIC) for eugenol (Sigma–Aldrich, St. Louis, MO, United States) was determined by the antifungal microdilution susceptibility standard test proposed by the CLSI M27-A3 method (Institute Clinical and Laboratory Standards, 2008 ). The inoculum was prepared in sterile saline and the transmittance of the suspensions was adjusted to 75–77% (530 nm), followed by further dilution in RPMI-1640 buffered with MOPS (Sigma–Aldrich, St Louis, MO, United States) medium to achieve 1.0 × 10³ to 5.0 × 10³ CFU/mL. The final concentrations ranged from 2 to 1024 mg/L for eugenol. The plates were incubated at 35°C for 72 h. The MIC for eugenol was determined visually as 100% growth inhibition when compared to the control. The results were confirmed by adding the salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma–Aldrich, St Louis, MO, United States) (5.0 mg/mL) to determine the reduction in the metabolic cell activity. Briefly, the plates were incubated at 35°C for 3 h and DMSO was added before spectrophotometric reading at 490 nm. The MIC endpoint for interpreting the results was 100% of reduction in metabolic activity for eugenol compared with the control. The isolate Candida parapsilosis ATCC 22019 was used as a quality control. All the tests were performed in duplicate for each strain.