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. 2021 Jan 18;10(1):133.
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

Urška Jug  1   2 , Katerina Naumoska  1 , Irena Vovk  1
Affiliations

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

Urška Jug et al. Antioxidants (Basel). .

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.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Logarithmic curves of the antioxidant activities of extracts of Japanese knotweed rhizome bark (JKRB) (n = 3) prepared with the following solvents and solvent mixtures: water (A), methanol (B), 80% methanol(aq) (C), acetone (D), 70% acetone(aq) (E), ethanol (F), 70% ethanol(aq) (G), and 90% ethyl acetate(aq) (H). The calculated values of IC50 are 3.561 (A), 3.715 (B), 3.469 (C), 2.632 (D), 3.350 (E), 2.893 (F), 3.503 (G), and 2.786 (H) µg mL−1 (obtained by GraphPad Prism 7 [60]).
Figure 2
Figure 2
Logarithmic curves plotting the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging effect (%) of ascorbic acid (A) and JKRB 70% ethanol(aq) extract (B) against the concentration, measured over time.
Figure 3
Figure 3
Ascorbic acid(aq) (50 µM) analyzed immediately after preparation (A) was subjected to 24 h of aging (B–E), stored in the refrigerator and protected from light (B,C) in plastic (B) and glass containers (C) or stored in daylight at room temperature (D,E) in plastic (D) and glass containers (E). The peak areas corresponding to ascorbic acid (tR 3.1 min, 254 nm) in aged solutions were compared to the peak area of ascorbic acid in the fresh solution. The intermediate precision of the method was 3% (n = 6).
Figure 4
Figure 4
SEC-HPLC-UV/Vis chromatogram at 280 nm (without post-column reaction) and at 517 nm (after post-column reaction with DPPH). The fractions and time intervals selected for fraction collection are marked. Fraction 8 was determined to be the strongest antioxidant due to the decrease in the absorbance at 517 nm.
Figure 5
Figure 5
The flavan-3-ol monomer identified by (−)ESI-MS based on the mass spectra and fragmentation patterns obtained for the signal at tR 6.4 min in the antioxidant fraction (FR 8) (A) and confirmed by (−)-epicatechin standard (B). Figure abbreviations: selected ion monitoring (SIM), and total ion current (TIC).
Figure 6
Figure 6
RP-HPLC-MS chromatograms of the antioxidant fraction (FR 8) in SIM mode (m/z 289), (−)-epicatechin and (+)-catechin standards (both in TIC mode; m/z 50–2000).
Figure 7
Figure 7
Matching the tRs of the antioxidant fraction FR 8 (A) and (−)-epicatechin (B), and the tRs of FR 9 (A) and (+)-catechin (B). Chromatograms were acquired at 280 nm using the SEC-HPLC method.
Figure 8
Figure 8
Chromatogram of FR 8 (track 1, 40 µL), (+)-catechin (track 2, 400 ng), (−)-catechin (track 3, 400 ng), and (−)-epicatechin (track 4, 400 ng) applied as 8 mm bands on the HPTLC cellulose plate developed with water, derivatized with DMACA reagent, and documented with illumination with white light.
Figure 9
Figure 9
Densitograms of FR 8 (40 µL) and monomeric flavan-3-ol standards (400 ng) scanned at 655 nm in the absorption/reflectance mode on the HPTLC cellulose plate developed with water and derivatized with DMACA.
Figure 10
Figure 10
Logarithmic curves plotting the DPPH scavenging effect (%) of (−)-epicatechin against the concentration, measured over time.
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