Supelguard

The extracts were prepared using 0.2 g of dry leaf matter at 35°C with 4 mL of methanol for HPLC (Neon^®) in an ultrasound bath for 30 min. The filtration was performed through a membrane Millipore (Advantec HP020AN−20 μm). Then, chromatographic analysis was performed using a Shimadzu HPLC with an SPD-M20A photodiode matrix detector (λ = 254 nm) and an LC18 column (25 cm × 4.6 mm, 5 μm, Supelcosil™) coupled to a 2 cm LC18 precolumn (Supelguard, Supelco). The mobile phase consisted of Milli-Q water (A) and acetonitrile (B) at a flow rate of 0.6 ml/min. The oven was adjusted to 30°C. The sample injection volume was 20 μL, and the detection was 254 nm, following a method adapted from Morais et al. (2018 (link)). The furanocumarin concentration present in the extracts was calculated from a four data point calibration curve psoralen and bergapten standard, respectively. The concentration was expressed in micrograms of metabolite per gram of leaf matter (μg/g⁻¹ LM). The yield was calculated as the dry weight (g) of the leaves multiplied by the production of the metabolite (μg/g⁻¹).

The quantification of sugars and organic acids was performed by Agilent 1100 high-performance liquid chromatography (HPLC) with ChemStation software by installing a Supelcogel C610H column, 30 cm × 7.8 mm; and a pre-column Supelguard, 5 cm × 4.6 mm (Supelco, Bellefonte, PA, USA). For the organic acids measurement, a diode array detector (DAD) (Diode Aray DAD G1315A) set at 210 nm was used, while a refractive index detector (RID) (G-1362-A) was employed for sugar content determination. Reference standards were used for organic acids (L-ascorbic acid, malic acid, citric acid, oxalic acid, acetic acid, lactic acid, and succinic acid) and sugars (glucose, fructose, and sucrose) supplied by Sigma Aldrich (Poole, Dorsert, UK) with calibration curves with R² ≥ 0.999. The results obtained correspond to the mean values (=5) and are expressed in g 100 mL⁻¹.

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).