The antioxidant activity of aqueous spinach extract: chemical identification of active fractions

In previous studies we have elucidated the presence of powerful. natural an tioxidants (NAO) in water and demonstrated their biological activity in bot h in vitro and in vivo systems. In the present study. the chemical identity of several of these antioxidant components is presented. Spinach leaves we re extracted with water and the 20,000 g supernatant which contained the an tioxidant activity was extracted with a water:acetone (1:9) solution. The 2 0,000 g supernatant obtained was, further purified on reverse phase HPLC us ing C-8 semi-preparative column. Elution with 0.1%, TFA resulted in five hy drophilic peaks. Elution with acetonitrile in TFA resulted in seven additio nal hydrophobic peaks. All the peaks were detected at 250 nm. All the fract ions obtained showed antioxidant activity when tested using three different assays. Based on H-1 and C-13 NMR spectroscopy four of the hydrophobic fra ctions were identified as glucuronic acid derivatives of flavonoids and thr ee additional fractions as trans and cis isomers of p-coumaric acid and oth ers as meso-tartarate derivatives of p-coumaric acid. The present study dem onstrates for the first time the presence of both flavonoids and p-coumaric acid derivatives as antioxidant components of the aqueous extract of spina ch leaves. (C) 2001 Published by Elsevier Science Ltd.