Coumaric acid

For total phenolics extraction from algae and subsequent quantification, aliquots of algal cultures containing about 120 × 10⁶ cells were harvested by centrifugation (600× g, 10 min) and stored at −20 °C until extraction. For the preparation of extracts, algal biomass was mixed with methanol and then exposed to mechanical rupture as follows: 15 min of continuous vortexing, followed by 30 min in an ultrasound bath, and finally, further 10 min-long vortexing ([75 (link)], with modifications). Then, samples were centrifugated (1500× g, 10 min), and the supernatants were harvested for the quantification with the Folin–Ciocalteu reaction described in [75 (link)]. Briefly, 100 µL of samples were left to react for 5 min with 750 µL of Folin–Ciocalteu reagent (Merck, Darmstadt, Germany; 1:10 in distilled water); then, the mixture was added with 750 µL of Na₂CO₃ (60 gL⁻¹) and let incubate for 1.5 h at RT. The mixtures were measured at 725 nm using the same Pharmacia spectrophotometer described above. The total phenolics concentration of the methanolic extracts was calculated on the basis of a calibration curve prepared using coumaric acid (Sigma Chemicals Co., St. Louis, MO, USA) as the standard and was expressed as mg equivalents of coumaric acid over total DW of algae cultures (mg_Coum.Ac.Eq. g_DW⁻¹) [76 (link)].

The phenolic compounds were identified and quantified using ultra-high-efficiency liquid chromatography (UHPLC) coupled to a diode array detector (DAD) with wavelength monitoring between 200 and 800 nm. A Shimadzu Shim-pack XR-ODS column (Shimadzu, Kyoto, Japan) with a particle size of 2.2 µm and a particle size of 2 mm × 75 mm was used. The injection rate employed was 2 µL with a 0.4 mL/min flow rate. The gradient used consisted of (A) water with 0.1% acetic acid (v/v) and (B) acetonitrile with 0.1% acetic acid (v/v). Identification was performed using analytical standards (3,4-hydroxycinnamic acid, catechin, gallic acid, ferulic acid, coumaric acid, maleic acid, rutin, quercertin, apigenin, and vanillin) obtained from Merck (Darmstadt, Germany) A curve was developed with the analytical standards at concentrations between 1 and 1000 µg/mL for quantification.

Cyanidin, peonidin 3-o-glucoside, malvidin-3-o-glucoside, quercetin, quercetin 3-o-glucoside, coumaric acid, gallic acid, syringic acid, epicatechin, catechin, and kaempferol (all analytical standard) were from Merck (Milan, Italy). Methanol, ethanol, and acetonitrile (all LC/MS grade solvents), ortophospforic acid 85%, Folin–Ciocalteu reagent and sodium carbonate were bought from Carlo Erba (Milan, Italy). Water was purified through a Milli-Q system from Millipore (conductivity: 18.2 MΩ cm at 25 °C; Milford, MA, USA).