Quinic

All standard compounds used for chromatographic quantifications (47885-U, 2-deoxyglucose, α, β, γ, and δ tocopherols, oxalic, quinic, malic, ascorbic, citric, and fumaric acids, from Sigma-Aldrich (St. Louis, MO, USA); tocol, from Matreya (Pleasant Gap, PA, USA); chlorogenic acid, p-coumaric acid, apigenin-6-glucoside, apigenin-7-glucoside, and luteolin-6-C-glucoside, from Extrasynthèse (Genay, France); and aloin, from Alfa Aesar (Ward Hill, MA, USA)) and bioactivity assays (trolox, kojic acid, dexamethasone, ellipticine, streptomycin, and ketoconazole, from Sigma-Aldrich) had a purity level of at least 95%. All other reagents were of analytical grade and purchased from common sources.

Organic acids were determined based on protocols described by Sánchez-Mata et al. [46 (link)]. Extraction was performed with 0.5 g of sample in 25 mL of 3% m-phosphoric acid and analyzed using an HPLC-UV methodology. The HPLC equipment used was a liquid chromatograph (Micron Analítica, Madrid, Spain) equipped with a Sphereclone ODS (2) 250 * 4.60 mm, 5 µm Phenomenex column, isocratic pump (model PU-II), an AS-1555 automatic injector (Jasco, Tokyo, Japan), and a UV-visible detector (Thermo Separation Spectra Series UV100, Waltham, MO, USA), 215 nm for organic acids. The mobile phase was 1.8 mM H₂SO₄ (pH = 2.6), with a flow rate of 0.4 mL/min for organic acids, and injection volume was 100 µL for samples and serial volumes for the standard curve (20, 30, 40, 50, 60, 70, 80, 90, and 100 µL). The compounds were identified by chromatographic comparisons with authentic standards (quinic (0.152 mg/mL), ascorbic (0.155 mg/mL), malic (0.403 mg/mL), fumaric (0.254 mg/mL) and citric acids (0.307 mg/mL), all from Sigma, St. Louis, MO, USA), using linear calibration curves of all compounds for quantification purposes. All data were analyzed using Biocrom 2000 3.0 software (Biocrom, Madrid, Spain).

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).