An oxidation product of quercetin catalyzed by a crude enzyme preparation from red clover (Trifolium pratense L.); Its isolation and identification.

Abstract

We have reported that the nutritive value of protein isolate from red clover decreases with prolonged sundrying before isolation,1] and that this decrease is partly caused by nonenzymatic reactions of the proteins with quercetin (I) and its glucosylated derivative or rather theii oxidation products.2'3) Both methionine and lysine residues in red clover protein isolate are damaged during prolonged sun-drying.3) In this connection, wehave isolated 5,7,3 '^'-tetrahydroxy^-methoxy^^-flavandione 3-hydrate (II) as an oxidation product of I in methanol which is assumed to be a 2-0-methyl derivative oi 2,5,7,3 ',4'-pentahydroxy-3,4-flavandione 3-hydrate (III). Ill is probably a principle active substance responsible foi damage of the amino acid residues.4* An enzyme-catalyzed oxidation may be important in the formation of III ai early stages of sun-drying of red clover. This work is concerned with isolation of a main enzymatic oxidation product of I..To prepare a crude enzyme solution from red clover, theii leaves and stems were ground with acetone at -20°C in z Waring blender and immediately filtered on No. 2 filter paper to separate the residue. This operation was repeated until the filtrate became colorless. The residue thus obtained was washed with diethyl ether, dried in a vacuum desiccator, and obtained as acetone powder. About 2.5 g of the acetone powder was extracted with 75 ml of 0.05 m phosphate buffer (pH 7.0) containing 0.35 m NaCl and 1% sodium ascorbate with stirring at 5°C for 2hr. The extract after centrifugation at 10,000 x g for 30min was filtered through No. 2 filter paper, and the supernatant was put on a Sephadex G-25 column (3.50 x 30cm) equilibrated with 0.05 mphosphate buffer (pH 7). The effluent was collected between 80ml and 140ml and used as a crude enzyme solution. To 75ml of ethanol containing 30mg of quercetin were added 300ml of 0.067m phosphate buffer (pH 6.8) and 50ml of the crude enzyme solution, followed by incubation at 30°C for 4hr to oxidize the quercetin. This reaction did not occur in the absence of the enzyme solution. The mixture was extracted with ethyl acetate and the extract was chromatographedon a polyamide column

Oxidation of flavonols and flavonol glycosides by a hypodermal peroxidase isoenzyme from gamay rouge grape (Vitis vinifera) berries

“…The oxidation of quercetin by peroxidases leads to a vast array of oxidation products (Schreier and Miller 1985;Igarashi and Akai 1990). However, for short reaction times, the oxidation of quercetin and myricetin by grapevine peroxidase B, leads to only one absorbent product, as may be expected from the isobestic points in the consecutive UV spectra of the reaction media (Figs 1(A) and (B)), and from the application of the graphic analysis procedure of Coleman et al (1970) to a set of consecutive UV spectra monitoring the disappearance of substrate and the appearance of absorbent reaction products (Figs 2 and 3).…”