Shikimic

The following standards were used for determination of sugars and organic acids: sucrose, fructose and glucose, as well as citric, malic, oxalic, tartaric and fumaric, ascorbic acid from Fluka Chemie (Buchs, Switzerland); shikimic and quinic acid from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Standards for phenolic compounds were acquired from Sigma-Aldrich Chemicals (3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, luteolin-4'-O-glucoside, quercetin-3-O-rutinoside).
Zeaxanthin, chlorophyll a and b, β-carotene and lutein were from Sigma-Aldricht Chemie (Steinheim, Germany) and neoxanthin, violaxanthin, antheraxanthin and β-cryptoxanthin from DHI LAB Product (Hørsholm, Denmark). Methanol for the extraction of phenolics was obtained at Sigma-Aldrich Chemicals. The chemicals for the mobile phase were HPLC-MS grade acetonitrile, sulphuric acid and formic acid from Sigma-Aldrich Chemicals. Water for the mobile phase was double distilled and purified with a Milli-Q system (Millipore, Bedford, MA, USA).

Sugars and organic acids were identified and quantified according to Hernández [15 (link)], with some modifications. Approximately 1 g of sample was diluted in 5 mL of phosphate buffer (pH 7.8), homogenized by Ultra-TurraxTM (IKA L004640, Staufen, Germany) for 1 min, and centrifuged at 15,000× g for 10 min. Finally, samples were filtered through a 0.45 μm Millipore filter. For the determination of the content of sugars and organic acids on samples, an HPLC (high-performance liquid chromatograph) Hewlett-Packard series 1100 (Hewlett-Packard, Wilmington, DE, USA) was used. The elution buffer consisted of 0.1% phosphoric acid with a flow rate of 0.5 mL/min.
Sugars and organic acids were isolated using a Supelco column (Supelcogel TM C-610H column 30 cm × 7.8 mm, Supelco, Inc., Bellefonte, PA, USA) and a precolumn Supelguard (5 cm × 4.6 mm; Supelco), and the absorbance was measured at 210 nm using a diode-array detector (DAD). Standards of sugars (glucose, fructose, sucrose, raffinose, maltitol, and sorbitol) and organic acids (oxalic, citric, tartaric, malic, quinic, shikimic, succinic and fumaric) were obtained from Sigma (Poole, UK). Calibration curves were used for the quantification of sugars and organic acids, showing good linearity (R² = 0.999). Results for both organic acids and sugars were expressed as concentrations g/L of dry weight (dw).

Acetonitrile, formic acid, methanol, DPPH (1,1-diphenyl-2 picrylhydrazyl radical), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), acetic acid, phloroglucinol, arbutin, caffeic acid, betulinic, oleanolic and ursolic acid, fructose, glucose, sorbitol and sucrose, and malic, oxalic, citric, shikimic, succinic, and fumaric acids were purchased from Sigma-Aldrich (Steinheim, Germany). Quercetin 3-O-glucoside, kaempferol 3-O-glucoside, cyanidin 3-O-glucoside, isorhamnetin 3-O-glucoside, apigenin 7-O-glucoside, chlorogenic, quinic, p-coumaric and ferulic acids, (+)-catechin, and procyanidin B2 were purchased from Extrasynthese (Lyon, France). Sodium hydroxide (CAS 1310-73-2), iodine (CAS 7553-56-2), and potassium iodide (CAS 7681-11-0) were purchased from CHEMPUR (Piekary Slaskie, Poland).