Waters 2545 quaternary gradient module

The acid-cleaved peptides were air-dried overnight. The peptides were washed with ice-cold diethyl ether by ultrasonicating at 4 °C for 30 min. The peptides were then centrifuged at 7000× g at 4 °C for 15 min to pelletize. The supernatant was discarded, and the ether wash was repeated two more times. The remaining ether was air-dried overnight. The peptides were resuspended in a solution of 0.1% (v/v) TFA in water and sonicated until completely solubilized. Palmitic acid tail peptides were solubilized in 0.1% (v/v) TFA in methanol. The peptide solution was filtered through a 0.22 µm membrane and separated using reverse phase high performance liquid chromatography (HPLC; Waters 2545 quaternary gradient module, Waters 2996 photodiode array detector, Waters fraction collector 3, and Luna Phenomenex C8 column) using 0.1% (v/v) TFA in water and 0.1% (v/v) TFA in acetonitrile as the mobile phases. Acetonitrile was substituted with methanol for peptides with palmitic acid tails. The collected peptide solutions were lyophilized and stored desiccated at −20 °C until use.

The flavonoids of the herbal SGR formula were analysed by HPLC. Chromatographic separation was performed on a Waters 2545 quaternary gradient module (Waters Corp., MA, USA) coupled with 2998 photodiode array (PDA) detector and equipped with an online degasser. An autosampler was used for solvent delivery. The measurements were performed on a SunFire analytical C18 column (250 mm × 4.6 mm, Waters, USA) at a column temperature of 25°C. The solvent system was a gradient of solvent A (aqueous phosphoric acid, 0.2%) and solvent B (methanol): initial composition, 78% B; linear gradient to 30% B from 0 min to 40 min; initial composition, 22% A; linear gradient to 70% B from 0 min to 40 min; and washing and reconditioning the column. The injection volume was 20 μL for each sample solution. The elution was run at a flow rate of 1 mL/min, and the UV spectra were monitored in the range of 200 nm to 400 nm. The HPLC chromatogram wavelength of 290 nm was selected for the quantitative analysis of SGR.