Triterpenoic acids from apple – Activators of endothelial nitric oxide synthase?

Abstract

Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Several substances present in apples such as quercetin and ursolic acid have been shown to increase the activity of endothelial nitric oxide synthase (eNOS) [1, 2]. As these compounds are also present in pomace, a bioassay-guided fractionation was performed to obtain apple pomace fractions with an enriched potential for eNOS activation. Methanolic/water (70/30) extracts from dried apple pomaces produced by Accelerated Solvent Extraction (ASE) were fractionated by column chromatography on polystyrene (DIAION-HP20 ®) using water/ethanol (90/10) and ethanol for the elution of polar (PF) and apolar fractions (APF). The influence of the obtained APF on eNOS activity was measured by the 14C-L-arginine to 14C-L-citrulline conversion assay (ACCA) [3] in endothelium-derived EA.hy926 cells and their metabolic activity was quantified using resazurin [4]. Phytochemical analysis was performed by HPLC-ELSD-DAD and LC-MS detection using reference compounds. Fractions showing a significant increase of eNOS activity at tested concentrations of 50 µg/mL, were enriched in tri-, di- and monohydroxy-triterpenoic acids with ursane and oleane skeleton. For the most active fraction a triterpenoic acid content of 65.9% was measured. Nine known triterpenoic acids were identified by MS- and NMR- studies as euscaphic acid [37.6% of total triterpenoic acids], annurcoic acid [8.0%], pomolic acid [8.5%], cuneataol [3.0%], maslinic acid [2.3%], corosolic acid [1.4%], ursolic acid [0.8%], oleanolic acid [0.6%] and pirolonic acid [0.2%]). Furthermore, one new triterpenoic acid – (2β,3β)-3,25-epoxy-2,3,19-trihydroxy-urs-12-en-28-oic acid [33.0%] – was identified.