Isolation of highly polar galloyl glucoside tautomers from Saxifraga tangutica through preparative chromatography and assessment of their in vitro antioxidant activity

Abstract

In this work, the rapid and efficient preparation of isolated galloyl glucoside tautomer free radical inhibitors was investigated using Saxifraga tangutica as a raw material. Four highly polar galloyl glucoside tautomers, 3-O-galloyl-α-d-glucose ⇌ 3-O-galloyl-β-d-glucose (Fr2-1-1), 2-O-galloyl-α-d-glucose ⇌ 2-O-galloyl-β-d-glucose (Fr2-1-2/2-1-3), 1-O-galloyl-β-d-glucose (Fr2-2-1), and 6-O-galloyl-α-d-glucose ⇌ 6-O-galloyl-β-d-glucose (Fr2-3-1/Fr2-3-2), were obtained via two-step medium-pressure liquid chromatography (with solid loading instead of conventional liquid injection) and one-step high-performance chromatography coupled with on-line RPLC-DPPH techniques for targeted isolation. This separation integration technique not only increases sample intake and reduces time cost but also visualizes each step of targeted separation. All four compounds were isolated from the plant for the first time. In vitro antioxidant activity assays by DPPH (1,1‑diphenyl-2-picrylhydrazyl) revealed that Fr2-1-2/Fr2-1-3 (IC₅₀: 5.52 ± 0.32 μM), Fr2-2-1 (IC₅₀: 7.22 ± 0.57 μM), and Fr2-3–1/Fr2-3-2 (IC₅₀: 7.36 ± 0.25 μM) had superior free radical scavenging abilities and that both were superior to that of quercetin (IC₅₀: 18.61 ± 3.55 μM). Oxidative stress assays revealed that Fr2-1-2/Fr2-1-3 significantly inhibited oxidative stress damage in H₂O₂-induced HepG2 cells, decreased the level of ROS (P < 0.01) and protected hepatocytes. Combined with the current results, gallic acid showed greater antioxidant activity when H atoms were replaced at d-glucose –OH (C-2) than at the other three sites [–OH (C-1), –OH (C-6) and –OH (C-3)].