Eclipse xdb c18

Plant extracts were filtered through a 0.2 mm membrane and 20 μL were injected in triplicate into a reverse phase column (Zorbax Eclipse XDB-C18, 60 Å, 5 μm, 250 × 4.6 mm) using a Waters HPLC system (Waters Corporation, Milford, MA, USA). The mobile phase consisted of solvent A (acetonitrile) and solvent B (0.0125 N acetic acid). The elution was as follows: isocratic from 0 to 2 min with 5% A and 95% B, gradient condition from 2 to 5 min starting with 5% to 15% and ending with a gradient conditions 5 to 20 min starting with 15% A and ending with 50% gradient conditions of 20 to 25 min starting with 50% A and ending with 5% isocratic conditions of 25 to 35 min with 5% A and 95% B. The flow rate was 1 mL/min, the absorbance was measured at maximum length of 280 nm and 20 µL of sample were injected. Quantification was performed by external standardization using protocatechuic acid, gallic acid, caffeic acid, rosmarinic acid, p-coumaric acid, quercetin, naringenin, catechin, kaempferol, and rutin.

Cellular nucleotides were extracted according to published procedures^{75 (link)}. Briefly, 1 × 10⁶ cells were washed with phosphate-buffered saline and quenched with liquid nitrogen, and then vigorously mixed with methanol and acetonitrile (1:1, v:v). After incubation on ice for 15 min, the samples were centrifuged at 12,000× g at 4 °C for 10 min. Cellular nucleotides were separated and quantified using a C18 column (Agilent Eclipse XDB-C18, 4.6 × 250 mm, average particle size 5 μm) assembled on the Waters Alliance e2695 Separations Module (Milford, MA, USA). Acetonitrile (5%) and 50 mM KH₂PO₄ (pH 6.5) containing 10 mM tetrabutylammonium bromide were used as mobile phase A, and acetonitrile was used as mobile phase B. All samples were separated in the mobile phase at a flow rate of 1 mL/min for 30 min at 22 °C. No degradation of individual nucleotides or changes in the ratios of nucleotide mixtures was detected during the experiments. The GTP concentration in the samples was calculated based on the slope of the calibration curves generated using pooled authentic samples (to mimic the matrix), and guanosine-5′-triphosphoric acid disodium salt was used as a standard.