ALS-L1023 was prepared from M. officinalis leaves (Alfred Galke
GmbH, Harz, Germany) using an activity-guided fractionation. The
dried Melissa leaves were extracted with aqueous ethanol and the
extract was filtered and concentrated. The concentrated ethanol
extract was further fractionated with ethyl acetate, concentrated
and dried to obtain ALS-L1023 in a dried powder form. ALS-L1023
was standardized with two reference compounds of rosmarinic
acid and caffeic acid by high-performance liquid chromatography
(HPLC), then dissolved in 100% DMSO and used for in vitro tests.
HPLC analysis was carried out on Hitachi LC-organizer. The column
was a Vydac protein & peptide C18 reversed-phase column (5 μm,
250 4.6 mm2
) and thermostated at 30 °C. The composition of
mobile phase A and B was 5% formic acid in water and methanol,
respectively. Elution was initially with mobile phase A for 5 min,
changed to linear gradient to mobile phase B for 20 min and eluted
with mobile phase B for 10 min. The flow rate was 1.0 ml/min, the
sample injection volume was 10 μl and UV detection was carried
out at 285 nm. The retention times of caffeic acid and rosmarinic
acid were 4.18 and 7.33 min, respectively, as shown in HPLC
chromatogram (Fig. 1).