Bioactivity guided isolation of apigenin from Stachys lavandulifolia Vahl. in mice with anxiolytic effects

Introduction: Stachys lavandulifolia is traditionally used for the treatment of anxiety. Our previous study showed that the ethyl acetate fraction of the plant had substantial anxiolytic action in mice. The present study was aimed to investigate the main constituent responsible for the observed effects. Methods: Ethyl acetate extract was fractionated using column chromatography. Yielded fractions (FR۱-۴) at ۵۰ mg/kg, and diazepam at ۰.۵ mg/kg were tested on the elevated plus-maze (EPM). Bioactive fraction was subjected to more purification on repeated chromatography columns. The isolated compound was identified based on ۱H-NMR, ۱۳C-NMR, and ESI-Mass spectra. Results: In the time spent in open arm, Fr۱, and Fr۳ did not show any significant effect on mice behavior, Fr۲ marginally decreased the percentage of spent time by -۸.۴%, and Fr۴ significant increased in the time spent in the open arms by ۱۵.۷%. In the open arm entry number Fr۱, and Fr۲ did not show any significant effect on mice behavior, Fr۳ marginally increased the percentage of open arm entries by ۷.۹ %, and Fr۴ significantly increased the open arm entry by ۱۸.۲%. The bioactive fraction (Fr.۴) was subjected to more purification. Phytochemical analysis of Fr۴ lead to the identification of ۴’,۵,۷-trihydroxyflavon (apigenin). Conclusion: Semi-polar sub-fraction of S. lavandulifolia showed anxiolytic effects by increased time spent and the entry numbers in the open arms comparable to diazepam in the EPM model in mice. Bioactivity-guided isolation leads to the characterization of apigenin with flavone structure as its active constituent. Hence, it might be introduced as a new anxiolytic agent.