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

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.

[3] Schmitt CA, Handler N, Heiss EH, Erker T, Dirsch VM. No evidence for modulation of endothelial nitric oxide synthase by the olive oil polyphenol hydroxytyrosol in human endothelial cells. Atherosclerosis 2007; 195(1): 58 – 64.

[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.