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Characterization of phenolic compounds using UPLC–HRMS and HPLC–DAD and anti-cholinesterase and anti-oxidant activities of Trifolium repens L. leaves


This study was aimed to investigate the phenolic profile of methanolic extract of Trifolium repens L. leaves using LC–HRMS and LC–DAD and its anti--cholinesterase and anti-oxidant activities. The n-Hexane (Tr.Hex), chloroform (Tr.Chf), ethanol (Tr.Et), methanol (Tr.Cme), and aqueous (Tr.Aq) extracts were tested for anti-cholinesterases and anti-oxidant potentials. Twenty-nine phenolic compounds were quantified and identified. The highest amount present were of tyrosol (11.1 mg/g), quercetin-3-glucuronide (5.36 mg/g), formononetin-7-glucoside (5.03 mg/g), quercetin-3-O-glucoside (4.71 mg/g), 3,4-di-O-caffeoylquinic acid (4.69 mg/g), formononetin-7-glucoside-acetate (4.29 mg/g), quercetin-3-O-glucoside (4.26 mg/g), and formononetin (3.64 mg/g). Tr.Chf and Tr.Et were most active against AChE, BChE, and free radicals. Tr.Chf exhibited IC50 of 15 and 21 µg/mL against AChE and BChE, respectively. In the anti-oxidant study, Tr.Chf showed IC50 of 42 and 25 µg/mL against ABTS and DPPH radicals, respectively. The current results of anti-cholinesterase and anti-radical studies on T. repens further signify its potential application in neurodegenerative disorders.

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Correspondence to Alam Zeb.

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Ahmad, S., Zeb, A., Ayaz, M. et al. Characterization of phenolic compounds using UPLC–HRMS and HPLC–DAD and anti-cholinesterase and anti-oxidant activities of Trifolium repens L. leaves. Eur Food Res Technol 246, 485–496 (2020).

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  • Trifolium repens
  • Phenolic compounds
  • Anti-cholinesterase activity
  • Anti-oxidant activity