Antioxidant and anti-inflammatory activity of the extracts from the ancient apple variety “Mela Rosa dei Monti Sibillini”

This study was carried out to assess the antioxidant and anti-inflammatory activities of the methanolic extracts from the “Mela Rosa dei Monti Sibillini”, an ancient apple variety cultivated in the area of the Sibillini mountains, Marche region, Central Italy. Nine apple peel and pulp samples were lyophilized after homogenization with liquid nitrogen or dried at 45°C, then were extracted using methanol. Extracts were analysed by HPLC-DAD-MS for 20 secondary metabolites (flavan-3-ols, flavonols, dihydrochalcones, hydroxycinnamic acids and triterpene acids) [1-2]. For comparative purposes, another traditional apple cultivar from the southern Italy (Annurca) and 2 commercial apple varieties (Golden Delicious and Granny Smith) were analysed as well. The results revealed that the peel dried samples were richer in triterpene acids (ursolic and oleanolic acids) than the peel lyophilised samples which contained higher amounts of phenolic compounds (epicatechin, procyanidin B2 and phloridzin). Compared with Annurca and commercial samples, the Mela Rosa dei Monti Sibillini contained higher levels of the afore-mentioned polyphenols constituents. The apple extracts were assayed for antioxidant capacity using different methods, i.e. DPPH, ABTS, FRAP, ORAC and Folin-Ciocalteau [3]. The peels extracts were far more potent than the pulp ones. As well as, the lyophilized material showed higher antioxidant activity than dried material. In a general way, the DPPH and ABTS indicated low IC50 values of some samples (lyophilized material) from the Mela Rosa dei Monti Sibillini respect to the other apple varieties. The antioxidant power of the Mela Rosa dei Monti Sibillini has also been revealed by the FRAP and the Folin assays. To better characterize the biological activity of the apple peel extracts, a mouse microglial cell line (BV2) was used. First of all, a cytotoxicity test was carried out by MTT assay treating cells with different concentrations (1-100 µg/mL) of the extracts. The latter did not influence cell viability up to 10 µg/mL. To investigate the antioxidant/anti-inflammatory activity of the extracts, LPS-treated BV2 microglia cells were exposed to different concentrations of the extracts, and reactive oxygen species (ROS) levels were measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Lyophilized extracts obtained by Mela Rosa dei Monti Sibillini significantly reduced ROS compared to LPS treated cells. The anti-inflammatory activity of the extracts was studied evaluating the expression, by RT-PCR, of pro-inflammatory molecules and enzymes: interleukin 1- (IL-1), tumour necrosis factor  (TNF-), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) [4-5]. In agreement with the DCFH-DA assay results, lyophilized extracts obtained by Mela Rosa dei Monti Sibillini, were the most effective in reducing the expression of these inflammatory molecules. These results support the possible use of this ancient apple variety as a source of nutraceuticals and functional foods useful to manage oxidative stress and inflammations. At the same time, they represent an important stage for the valorisation and promotion of this old traditional apple in Italy. References [1] Laghari AH, Memon S, Nelofar A, Khan KM, Yasmin A. Determination of free phenolic acids and antioxidant activity of methanolic extracts obtained from fruits and leaves of Chenopodium album. Food Chemistry. 2011;126(4):1850-1855. [2] Caprioli G, Alunno A, Beghelli D, Bianco A, Bramucci M, Frezza C, Tirillini B. Polar constituents and biological activity of the berry-like fruits from Hypericum androsaemum L. Frontiers in plant science. 2016;7:232. [3] Caprioli G, Iannarelli R, Innocenti M, Bellumori M, Fiorini D, Sagratini G, Quassinti L. Blue honeysuckle fruit (Lonicera caerulea L.) from eastern Russia: phenolic composition, nutritional value and biological activities of its polar extracts. Food & function. 2016;7(4):1892-1903. [4] Lau FC, Bielinski DF, Joseph JA. Inhibitory effects of blueberry extract on the production of inflammatory mediators in lipopolysaccharide‐activated BV2 microglia. Journal of neuroscience research. 2007;85(5):1010-1017. [5] Bak MJ, Truong VL, Kang HS, Jun M, Jeong WS. Anti-inflammatory effect of procyanidins from wild grape (Vitis amurensis) seeds in LPS-induced RAW 264.7 cells. Oxidative medicine and cellular longevity. Oxid Med Cell Longev. 2013;2013:409321.