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.

中文翻译：

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（-）-Epicatechin-由抗氧化活性引导级分确定的日本虎杖根茎树皮提取物的抗氧化活性的重要贡献者

用八种不同溶剂或混合溶剂（水，甲醇，80％甲醇_（aq），丙酮，70％丙酮_（aq），乙醇，70％乙醇_（aq）和90 _）制备的日本虎杖根茎树皮提取物的抗氧化活性使用2，2-二苯基-1-吡啶并肼基（DPPH）自由基清除测定法测定％乙酸乙酯_{（水溶液）}。所有提取物的低半数最大抑菌浓度（IC ₅₀）值（2.632–3.720 µg mL ^-1）在已知的抗氧化剂抗坏血酸在t ₀（3.115 µg mL ^-1）的IC ₅₀值范围内。由于提取率最高（〜44％），所以乙醇_{（水溶液）的}含量为70％选择用于提取物制备的进一步研究。计算出的其抗氧化活性的IC ₅₀值保持稳定至少14天，而抗坏血酸的IC ₅₀随时间增加。稳定性研究表明，容器材料对于将抗坏血酸_（aq）溶液在冰箱中进行遮光保护至关重要。开发了尺寸排阻-高效液相色谱（SEC-HPLC）-紫外和反相（RP）-HPLC-UV结合多级质谱（MS ⁿ）来分离70％乙醇_{（aq）的方法}提取物和进一步的化合物鉴定。在最有效的抗氧化剂SEC馏分中，通过在线后SEC-HPLC-DPPH测定确定了表儿茶素，白藜芦醇丙二酰己糖苷及其来源片段（白藜芦醇和白藜芦醇乙酰己糖苷）通过RP-HPLC进行了初步鉴定-MS ⁿ。此外，还通过两种正交方法进一步确认了表儿茶素，即SEC-HPLC-UV和高效薄层色谱（HPTLC）结合光密度测定法。最终，后一种技术实现了（-）-表儿茶素的鉴定。（-）-Epicatechin表现出有效且稳定的时间依赖性抗氧化活性（IC ₅₀值为〜1.5 µg mL ^-1）至少持续14天。