eNOS-activity guided fractionation of vine leaves from Austria

Polyphenol fractions of Austrian red wine samples are reported to increase endothelial nitric oxide synthase (eNOS) activity in EA.hy926 endothelial cells [1]. The present investigation aims to identify further eNOS-activating fractions from red vine leaves. Two leaf samples of the variety Blaufränkisch from the region Neusiedlersee-Hügelland were selected for bio-assay guided fractionation using EA.hy926 endothelial cells and the [14C]L-arginine/[14C]L-citrulline conversion assay measuring eNOS activity. The first leaf sample was harvested late in September together with grapes as green vine leaves (GVL), and the other sample was collected as red vine leaves (RVL) five weeks later in the same vineyard. Both vine leaf samples were dried, pulverised, and extracted consecutively with dichloromethane and methanol using an accelerated solvent extractor. Further separation of the methanol-extract of the two samples was done by liquid-liquid partition with ethylacetate and water resulting in a polar fraction (PF) and an apolar fraction (AF). The methanol-extract of both vine leaf samples (GVL, RVL) showed the same level of enhanced eNOS activity at a concentration of 600µg/ml. The dichloromethane-extracts of GVL and RVL, tested in equal concentration, decreased the activity significantly. The AF of the samples revealed an activating effect on eNOS at 300µg/ml, whereas the complementary PF did not increase enzyme activity at the same concentration. Thus, the compounds responsible for eNOS activation seem to reside in the AF. Therefore, ongoing bio-assay guided fractionation of the AF will show which class of compounds is responsible for the observed increased eNOS activity.

References: 1. Donath O et al. (2008) Polyphenols Communications, 699–700.