(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation

Urška Jug; Katerina Naumoska; Irena Vovk

doi:10.3390/antiox10010133

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation

Antioxidants (Basel). 2021 Jan 18;10(1):133. doi: 10.3390/antiox10010133.

Authors

Urška Jug^{1

2}, Katerina Naumoska¹, Irena Vovk¹

Affiliations

¹ Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
² Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.

Abstract

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol_(aq), acetone, 70% acetone_(aq), ethanol, 70% ethanol_(aq), and 90% ethyl acetate_(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC₅₀) values (2.632-3.720 µg mL^-1) for all the extracts were in the range of the IC₅₀ value of the known antioxidant ascorbic acid at t₀ (3.115 µg mL^-1). Due to the highest extraction yield (~44%), 70% ethanol_(aq) was selected for the preparation of the extract for further investigations. The IC₅₀ value calculated for its antioxidant activity remained stable for at least 14 days, while the IC₅₀ of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid_(aq) solution in a refrigerator. Size exclusion-high-performance liquid chromatography (SEC-HPLC)-UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSⁿ) were developed for fractionation of the 70% ethanol_(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSⁿ. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (-)-epicatechin. (-)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC₅₀ value ~1.5 µg mL^-1) for at least 14 days.

Keywords: DPPH derivatization; DPPH test; Polygonum cuspidatum; Reynoutria; flavan-3-ols; invasive species; phenolic compounds; size-exclusion chromatography; stilbenes; vitamin C.

Abstract

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