Preparative hplc

The ethanol extracts (118 g) was obtained after drying under vacuum condition at 50°C in a water bath, and then further extracted with ethyl acetate. These extracts were once again concentrated in a vacuum evaporator. The extracts was resolved using dichloromethane and isopropanol (1:2 v/v), then recrystallized at 4°C for 8 h. The recrystallization operation was performed three times until pure white crystals were obtained. The obtained filtrates were concentrated and recrystallized to obtain the mother liquor. The preparative-HPLC (Yilite,China) was used later for separation and the analytical-HPLC (1100, Agilent technologies, USA) for analysis to give compound (101 mg). The preparative-HPLC conditions contained water (A) and acetonitrile (B). The flow rate was 3 mL/min and injection volume was 500 μL. The gradient conditions were set as follows: 0–10% (B) in 0 to 5 min, 10–45% (B) in 5–10 min, 45% (B) in 10–30 min. The purification of limonin was calculated by area normalization method.

The purity of crude peptide was analyzed by
reverse-phase high performance liquid chromatography
(RP-HPLC; Agilent, USA). RP-HPLC was
done on an octadecylsilica (C18) column (4.6 mm
id×250 mm length, 5 μm) with TFA/water (0.1%
v/v) and TFA/ACN (0.1% v/v) as the mobile phase
with a linear gradient from 0 to 60% of TFA/ACN
over 30 minutes and flow rate of 1 mL/minute at
25˚C. RP-HPLC results were used to scale up and
purify the peptide. The peptide was purified by preparative
HPLC (Agilent, USA) with >95% purity
with the same mobile phase as RP-HPLC. Purified
peptide was lyophilized and stored at -20˚C until
future use in the experiments. For characterization
of the synthesized peptide, mass spectrum of the
peptide was obtained by quadropole LC-MS (Agilent,
USA) with an electrospray ionization system.