Trans cinnamic acid

Phenylalanine-ammonia lyase (PAL) activity was analyzed according to Boo et al. (2011) (link). Samples (0.5 g) were collected from the same leaves used to analyze total phenolic concentration and antioxidant capacity and were stored at −80°C prior to PAL activity analysis. The sample was ground in liquid nitrogen and extracted with 10 ml of 25 mm borate buffer (pH 8.8) and 2 ml of 3 mm β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, United States). The extract was centrifuged at 2,000g for 20 min. After centrifugation, the supernatant was mixed with 2.5 mm borate buffer (pH 8.8) and 2.5 ml of 10 mm L-phenylalanine (Sigma-Aldrich, St. Louis, MO, United States) and incubated at 40°C for 2 h. The addition of 0.1 ml HCl stopped the reaction, and the absorbance was measured at 290 nm using a spectrophotometer. A standard curve using trans-cinnamic acid (calibrated at 0, 0.0625, 0.125, 0.25, 0.5, and 1 mm; Sigma-Aldrich, St. Louis, MO, United States) equivalent in mmol per hour per gram of fresh weight (mmol trans-cinnamic acid· h⁻¹·g⁻¹ FW) was used to determine PAL activity.

Phenylalanine ammonia lyase (PAL) activity was measured using the method described by Martinez-Tellez and Lafuente [51 ]. The enzyme activity was determined spectrophotometrically by measuring the production of trans-cinnamic acid from L-phenylalanine. Enzyme extract (10 μL) was incubated at 40°C with 12.1 mM L-phenylalanine (90 μL, Sigma) prepared in 50 mM Tris-HCl, (pH 8.5). After 15 min of reaction, trans-cinnamic acid yield was estimated by measuring increase in the absorbance at 290 nm. The standard curve was prepared using trans-cinnamic acid as standard (Sigma) and the PAL activity was expressed as nM trans-cinnamic acid/μg protein/h.

Chemicals were of analytical grade and obtained from Sigma‐Aldrich (Taufkirchen, Germany) unless otherwise stated. HPLC LC–MS grade and supergradient HPLC grade acetonitrile and HPLC LC–MS grade methanol were obtained from VWR Chemicals (Darmstadt, Germany). Formic acid was purchased from Carl Roth (Karlsruhe, Germany), and Formic acid Optima LC–MS grade from Fisher Chemicals (Waltham, USA). Trans‐ferulic acid (purity 99%), caffeic acid (purity ≥98%), p‐coumaric acid (purity ≥98%), trans‐cinnamic acid (purity 99%), l‐tryptophan (purity ≥98%), and 3,4‐dimethoxycinnamic acid (purity 99%) were obtained from Sigma‐Aldrich (Taufkirchen, Germany). Nonanedioic acid (azelaic acid) (purity ≥98%), octanedioic acid (suberic acid) (purity ≥98%), (9, 10, 13)‐trihydroxy octadecenoic acid (purity >99%), and (9,12,13)‐trihydroxy octadecenoic acid (purity >99%) were purchased from Larodan (Solna, Sweden).