In-depth phytochemical and biological studies on potential AChE inhibitors in red and zigzag clover dry extracts using reversed-phase liquid chromatography (RP-LC) coupled with photodiode array (PDA) and electron spray ionization-quadrupole/time of flight-mass spectrometric (ESI-QToF/MS-MS) detection and thin-layer chromatography-bioautography.

In the study, Trifolium medium L. and T. pratense L. were used to obtain lyophilisates which were named as TML and TPL, respectively. The former clover taxon represents a little explored species, while the latter one is often found in dietary supplements and functional foods due to the content of isoflavones that alleviate various menopausal symptoms. Detailed phytochemical profiles of both lyophilisates were examined and compared using coupled chromatographic (RP-LC) and spectroscopic PDA/ESI-QToF/MS-MS methods. A total of 54 and 55 compounds were identified in TML and TPL, respectively. Close chemotaxonomic similarities were confirmed for both clover taxa examined, especially in terms of the most abundant isoflavones and hydroxycinnamates. For the first time, neurotropic polyphenols (AChE inhibitors) were identified in clover extracts using TLC-bioautography. In this group, caffeoylmalic acid exhibited significant AChE inhibitory effect confirming the potential of TPL and TML to support physiological functions of the endocrine and nervous systems.