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

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 (IC ₅₀) values (2.632–3.720 µg mL ⁻¹) for all the extracts were in the range of the IC ₅₀ value of the known antioxidant ascorbic acid at t ₀ (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 IC ₅₀ value calculated for its antioxidant activity remained stable for at least 14 days, while the IC ₅₀ 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 (MS ⁿ) 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-MS ⁿ. 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 (IC ₅₀ value ~1.5 µg mL ⁻¹) for at least 14 days.