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

K Waldbauer; M Zehl; E Urban; R McKinnon; VM Dirsch; B Kopp

doi:10.1055/s-0034-1394559

Planta Medica, Inhaltsverzeichnis

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

K Waldbauer ¹, M Zehl ¹, E Urban ², R McKinnon ¹, VM Dirsch ¹, B Kopp ¹

¹Department of Pharmacognosy, University of Vienna, Althanstrasse 14, 1090, Austria
²Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14,1090, Austria

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.

References:

[1] Shen Y, Croft K D, Hodgson J M, et al. Quercetin and its metabolites improve vessel function by inducing eNOS activity via phosphorylation of AMPK. Biochem Pharmacol 2012; 84(8): 1036 – 1044.

[2] Steinkamp-Fenske K, Bollinger L, Völler N. Ursolic acid from the Chinese herb Danshen (Salvia miltiorrhiza L.) upregulates eNOS and downregulates Nox4 expression in human endothelial cells. Atherosclerosis 2007; 195(1): 104 – 111.

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[4] O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000; 267: 5421 – 5426.