Isolation, purification and identification of antioxidants in an aqueous aged garlic extract

An aqueous aged garlic extract (AGE) was prepared by soaking sliced garlic in water for 20 days at room temperature (23-25 °C). In order to locate the antioxidant ingredients of the aqueous AGE, an activity-guided fractionation approach using ABTS assay, DPPH assay and FRAP assay were conducted to guide the fractionation by means of extraction, column chromatography and semi-preparative HPLC. Some phenols and organosulfur compounds were identified as antioxidants in AGE by GC-MS. Furthermore, UV, IR, ESI-MS, NMR and specific rotation experiments led to the identification of l-phenylalanine, l-tryptophan, (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as the major antioxidants in the AGE. The EC50 values of these purified tetrahydro-β-carboline derivatives were 0.625-1.334 μmol/mL and 1.063-2.072 μmol/mL in ABTS assay and DPPH assay, respectively. It is the first time for us to identify (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as an in vitro antioxidant in AGE.