Luna 5 m c18 2

HPLC was used to identify the presence of glyconjugate products. The HPLC protocol utilized a C₁₈ column (150 × 4.6 mm i.d.: Luna® 5 µm C18(2), Phenomenex, Torrance, CA) with an LC-10Avp system (Shimadzu, Kyoto, Japan), and a detection wavelength of 190~700 nm. The mobile phase consisted of 1% acetic acid in water (eluent A) and 100% acetonitrile (eluent B), with a flow rate of 1.0 mL/min. The elution program was executed as follows: 10–25% B for 0–5 min, 25–50%B for 5–10 min, 50–80%B for 10–17 min, 80–30% for 17–20 min, 30–10% for 20–21 min and termination at the 22 min. The Ultra Performance Liquid Chromatography (UPLC)-MS/MS system was utilized as previously described^{49 (link)}.
In order to identify the glycosylation position of substrates, the kaempferol glucoside products were purified by Silica gel column chromatography and collected. The samples were evaporated and freeze-dried and dissolved in deuterated methanol. The NMR spectra of the kaempferol and its glucoside were acquired on a Bruker AVANCE AV 400-MHz NMR spectrometer at 22 °C. The data were processed and analyzed using MestReNova v. 5.2.5 software.

HPLC-diode array detection analysis was performed on the DCM extract using an Agilent 1,100 series (Agilent, Waldbronn, Germany) instrument equipped with photo diode array, autosampler, column thermostat and degasser. A Phenomenex: Luna 5 µm C₁₈ (2) (150 × 4.6 mm; 5 μm particle size) column was used as the stationary phase. Water containing 0.1% of formic acid (A) and acetonitrile (B) served as mobile phases at a flow rate of 1 ml/min. Gradient elution was applied as follows: Initial ratio 95% A: 5% B, keeping for 10 min, changed to 90% A: 10% B in 10 min, changed to 70% A: 30% B in 10 min, to 50% A: 50% B in 10 min, maintaining for 0.5 min and back to initial ratio in 0.5 min. Temperature was set to 30°C. Extract or standards were dissolved in HPLC grade methanol (2 mg/ml) and the injection volume was 20.0 μl. Chromatograms were recorded at 254 nm.

High-performance liquid chromatography (HPLC)–diode array detection analysis of the extracts (hexane, DCM, and ethanol) was carried out with an Agilent 1100 series (Agilent, Waldbronn, Germany) instrument equipped with a photodiode array, an autosampler, a column thermostat, and a degasser. The stationary phase consisted of A Phenomenex: Luna 5 µm C₁₈ 2) (150 × 4.6 mm; 5 μm particle size) column. Water containing 0.1% of formic acid (A) and acetonitrile (B) served as mobile phases at a flow rate of 1 ml/min. The gradient elution was as follows: initial ratio 95% A:5% B, maintained for 10 min, changed to 90% A:10% B in 10 min, changed to 70% A:30% B in 10 min, to 50% A:50% B in 10 min, maintained for 0.5 min, and back to initial ratio in 0.5 min. The temperature was set to 30°C, injection volume was 20.0 μl, and chromatograms were recorded at 254 nm (Erukainure et al., 2020 (link)).