Thin-layer chromatography and β-elimination
Thin-layer chromatography and β-elimination analysis were performed to identify glycopeptides, following a previous report (Kuritani et al., 2020). A 5 mL of 0.5 M hydrochloric acid containing 20 mg of extract was heated at 120 °C for 4 h. The solution was loaded on thin-layer chromatography plates as 0.8 cm streaks. The plates were run in n-butanol/acetone/acetic acid/5% ammonia/water (4.5:1.5:1:1:1.2), dried, and stained by a chromogenic agent followed by re-drying and then heating in an oven at 105 °C for 5 min. The spots were visualized and photographed under sunlight.
Glycopeptide bond composition was identified by the β-elimination method (Zhu et al., 2020). The samples with or without alkali were dissolved in 0.4 M sodium hydroxide aqueous solution and held at 45 °C for 30 min incubation. The ultraviolet (UV) spectrum was measured. Alkali treatment was considered as the control.