Jug Urška, Naumoska Katerina, Vovk Irena
Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.
Antioxidants (Basel). 2021 Jan 18;10(1):133. doi: 10.3390/antiox10010133.
The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol, acetone, 70% acetone, ethanol, 70% ethanol, and 90% ethyl acetate), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC) values (2.632-3.720 µg mL) for all the extracts were in the range of the IC value of the known antioxidant ascorbic acid at t (3.115 µg mL). Due to the highest extraction yield (~44%), 70% ethanol was selected for the preparation of the extract for further investigations. The IC value calculated for its antioxidant activity remained stable for at least 14 days, while the IC 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 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) were developed for fractionation of the 70% ethanol 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. 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 value ~1.5 µg mL) for at least 14 days.
使用2,2-二苯基-1-苦基肼（DPPH）自由基清除试验，测定了用八种不同溶剂或溶剂混合物（水、甲醇、80%甲醇、丙酮、70%丙酮、乙醇、70%乙醇和90%乙酸乙酯）制备的虎杖根茎皮提取物的抗氧化活性。所有提取物的低半数最大抑制浓度（IC）值（2.632 - 3.720μg/mL）处于已知抗氧化剂抗坏血酸在t时的IC值（3.115μg/mL）范围内。由于提取率最高（约44%），选择70%乙醇制备提取物用于进一步研究。其抗氧化活性计算得到的IC值至少14天保持稳定，而抗坏血酸的IC值随时间增加。稳定性研究表明，容器材料对于冰箱中避光储存抗坏血酸溶液非常重要。分别开发了尺寸排阻-高效液相色谱（SEC-HPLC）-紫外和反相（RP）-HPLC-紫外联用多级质谱（MS），用于70%乙醇提取物的分离和进一步的化合物鉴定。在使用在线柱后SEC-HPLC-DPPH试验测定的最有效的抗氧化SEC馏分中，通过RP-HPLC-MS初步鉴定出表儿茶素、白藜芦醇丙二酰己糖苷及其源内碎片（白藜芦醇和白藜芦醇乙酰己糖苷）。此外，通过两种正交方法SEC-HPLC-紫外和高效薄层色谱（HPTLC）结合密度测定法进一步确认了表儿茶素。最后，后一种技术鉴定出了（-）-表儿茶素。（-）-表儿茶素表现出至少14天的高效且稳定的时间依赖性抗氧化活性（IC值约1.5μg/mL）。
