Salicylic acid

The experimental model involved macrophage cells seeded in 6-well plates (2 × 10⁵ cells/well) and allowed to adhere overnight. The study design included the following experimental groups: (1) cells treated only with LPS; (2) cells treated with SE FAE; (3) cells pre-treated with SE FAE and consequently challenged with LPS. For control groups, we used untreated cells (blank); salicylic acid-treated cells (positive, anti-inflammatory control) and cells pre-treated with salicylic acid and consequently challenged with LPS.
Cells were pre-treated with SE FAE with increasing concentrations of 2.5%, 5% and 10% v/v (0.25 mg DW/mL, 0.5 mg DW/mL, 1 mg DW/mL, respectively) or salicylic acid (100 μM) (Merck, Germany) dissolved in DMEM (with 4.5 g/L glucose, w/o phenol red and L-glutamine) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin/100 µg/mL streptomycin mixture and 2 mM L-glutamine. After 24 h cells were treated with 200 ng/mL LPS (Escherichia coli 026:B6, Sigma-Aldrich, Taufkirchen, Germany) or not, by the simple refreshing of culture media and incubated for additional 24 h. Following the last incubation period, the cells were lysed and total RNA or total protein were extracted and subjected to subsequent analyses. All treatments were performed in triplicate.

Three elicitation models were used for the elicitation of E. californica suspension cultures. Simple salicylic acid elicitation (SA) at final concentrations of 4, 6, and 8 mg/L in growth media, the model included combinations of SA (4, 6, 8, mg/L) with L-tyrosine at final concentration 1 mM simultaneously added into growth media and pre-treatment of suspension cultures with L-tyrosine (1 mmol/L) 24 h before salicylic acid elicitation (sequential treatment).
Stock solutions were prepared by dissolution of 0.4 g of salicylic acid (Merck, Germany) and 0.575 g of L-tyrosine hydrochloride (Sigma-Aldrich, St. Louis, MS, USA) in 100 mL of sterile distilled water. Adequate volumes of both stock solutions were aseptically dosed to the growth media of suspension cultures through 0.22 µm syringe membrane filters (Millipore, Merck, Germany) to achieve the desired concentrations. The presence of an elicitor and precursor were omitted in control samples. All samples were prepared in triplicate. Suspension cultures were maintained on the orbital rotator (110 rpm) at 24 °C and relative humidity of 70%. Plant material was harvested 24, 48 and 72 h after elicitor treatment. Phytomass was finally separated from growth medium by vacuum filtration, lyophilized and stored at −20 °C.

The following standards were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany): catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, gallocatechin gallate, procyanidin B1 and B2, procyanidin A2, procyanidin C1, kaempferol, quercetin, rutin, salicylic acid, catechin-2,3,4–¹³C₃ 99 atom % ¹³C (98 % CP), gallocatechin-2,3,4–¹³C₃ ≥ 99 atom % ¹³C (≥97 % CP), catechin-2,3,4–¹³C₃ gallate ≥99 atom % ¹³C (≥97 % CP), salicylic acid-D₄ certified reference material. Naringenin was purchased from Thermo Scientific (Waltham, MA, U.S.A).
The following chemicals were purchased from Sigma-Aldrich (Merck-Millipore, Merck KGaA, Darmstadt, Germany): dimethyl sulfoxide (DMSO), hydrochloric acid (HCl, 37 %), formic acid (FA, LiChropur 98–100 % LCMS grade), 2 M Folin-Ciocalteu′s phenol reagent (47641-500 ML-F), gallic acid (G7384-100G) and β-glucuronidase type H-1 from Helix pomatia. Sodium bicarbonate (S6014-500G, Fluka), methanol (85681.320, VWR), and acetonitrile (ACN, HiPerSolv Chromanorm) were purchased from VWR Chemicals (Radnor, PA, U.S.A.). Sodium acetate was obtained from Merck (Darmstadt, Germany) and glacial acetic acid from Fluka/Sigma-Aldrich (Thermo Fisher Scientific, Waltham, MA, U.S.A; Merck KGaA, Darmstadt, Germany).