Gentisic acid

Pure chemical standards of selected plant secondary metabolites were acquired from commercial sources. trans-anethole (99%), benzaldehyde (≥99.5%), caffeic acid (98%), caffeine (pure), δ-3-carene (90%), carvacrol (98%), trans-β-caryophyllene (≥98.5%), catechin (98%), citral (95%), citronellal (96%), p-coumaric acid (98%), p-cymene (99%), 1-decanol (≥98%), 1-dodecanol (≥98%), 1-tridecanol (97%), 1-undecanol (99%), eugenol (99%), geraniol (98%), isopulegol (99%), limonene (97%), linalool (98%), methyl salicylate (≥99%), menthol (99%), 2-octyl-1-decanol (97%), piperitone (analytical standard), α-pinene (≥99%), β-pinene (analytical standard), 3-octanol (analytical standard), pulegone (96%), quercetin (≥95%), sabinene (75%), α-terpineol (≥96%), terpinen-4-ol (≥95%), γ-terpinene (97%), thymol (99%), and 2-undecanone (99%) were acquired from Sigma-Aldrich (Lisboa, Portugal); ferulic acid (for research only), gallic acid (for research only), and gentisic acid (for research only) were acquired from Extrasynthèse (Genay, France). All compounds were diluted in acetone (99.8%, Carl Roth GmbH + Co. KG.Portugal) to an initial concentration of 200 mg/mL. Phytochemical stock solutions were stored at −20 °C until used. The commercially available nematicide oxamyl (AFROMYL^®, Epagro) was also tested at 2 mg/mL in water.

Air-dried powdered roots, aerial parts (stems and leaves), and flower heads (15 g) were extracted with 80% MeOH (1:20 w/v) by sonication (80 kHz, ultra-sound bath Biobase UC-20C) for 15 min (×2) at room temperature. Then, the extracts were concentrated in vacuo and lyophilized (lyophilizer Biobase BK-FD10P) to yield crude extracts as follows: flower heads 3.54 g, aerial parts 2.82 g and roots 2.14 g. The lyophilized extracts (1 mg) were dissolved in 80 % methanol (10 mL). An aliquot (2 mL) of each extract solution was filtered through a 0.45 μm syringe filter disc (Polypure II, Alltech, Lokeren, Belgium) and subjected to UHPLC–HRMS analyses.3.3. Chemicals
Acetonitrile and formic acid for LC–MS, and HPLC grade methanol were purchased from Fisher Scientific (Hampton, NY, USA).
The authentic standards used for compound identification were obtained as follows: gentisic acid, vanillic acid, protocatechuic acid, quercetin, luteolin, apigenin, genkwanin, apigenin 7-O-glucoside, kaempferol 3-O-glucoside, luteolin 7-O-glucoside and kaempferol 3-O-rutinoside, from Extrasynthese (Genay, France); caffeic acid, neochlorogenic acid, 3,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid and hispidulin were supplied from Phytolab (Vestenbergsgreuth, Germany); chlorogenic acid acaciin and pectolinarin were purchased from Sigma-Aldrich (St. Louis, MO, USA).