Antioxidant and anti-proliferative properties of the phenolic-rich fraction obtained from cauline leaves of Isatis tinctoria L. (Brassicaceae)

Isatis tinctoria L. (Brassicaceae) is a biennial species with erect stem, basal leaves oblong-lanceolate and cauline sagittate; the yellow flowers are clustered in racemes. In Italy the plant grows wild mainly in Southern and North-western parts of the country, as well as on the major islands. Our previous investigations highlighted some biological properties of polar extracts from the leaves (cauline and basal) collected in different times of the year, and the flowers of I. tinctoria grown wild around Acireale (Catania, Sicily, Italy) (1, 2). The obtained results indicated the good antioxidant and cytotoxic potential of the hydroalcoholic extract from the cauline leaves collected in January (It-J). In continuation of our studies, the present work has been focused on the evaluation of the antioxidant and anti-proliferative activities of the phenolic-rich fraction, phytochemically characterized, obtained from cauline leaves of I. tinctoria picked in January. The leaves were lyophilized and sequentially extracted with dichloromethane and 70% methanol at a temperature of 50 °C; then, the hydroalcoholic extract was suspended in distilled water and subjected to solvent-solvent partitioning with ethyl acetate in a separating funnel to obtain the phenolic-rich ethyl acetate fraction (ItJ-EAF). The total phenolic, total flavonoid and condensed tannin contents of ItJ-EAF were determined spectrophotometrically (3). The HPLC-PDA-ESI-MS qualitative analysis of the fraction led to the identification of 20 compounds belonging to the groups of flavonoids and phenolic acids, and revealed a phenolic profile superimposable to that of the crude extract. Flavone glycosides were quantitatively the most abundant constituents, and vicenin-2 resulted the main flavonoid. The antioxidant properties of ItJ-EAF were examined by different in vitro systems: DPPH, reducing power, and ferrous ions chelating activity assays (3). Butylated hydroxytoluene (BHT) and ethylenediaminetetraacetic acid (EDTA) were used as reference standards. The fraction exhibited radical scavenging activity (IC50 = 0.6657  0.0024 mg/ml) and reducing power (3.87  0.71 ASE/ml), whereas it did not show any chelating activity. The potential cytotoxicity of ItJ-EAF was tested using Artemia salina lethality bioassay (3); the fraction resulted non-toxic to brine shrimp larvae (LC50>1000 µg/ml). The anti-proliferative effect of ItJ-EAF was evaluated in vitro on three human anaplastic thyroid carcinoma (ATC) cell lines (C643, CAL-62 and 8505C) by MTT assay. The fraction showed low activity against C643 cells, whereas a reduction of proliferation rate was observed in 8505C and CAL-62 cells in a concentration- and time-dependent way. At 48 h after treatment, about 65% growth inhibition was found in 8505C cells (p<0.001 vs untreated cells). The strongest anti-proliferative effect was observed in CAL-62 cells; particularly, after 48 h exposure, ItJ-EAF markedly inhibited cell growth, causing nearly 80% reduction of viability at the highest tested concentration (100 μg/ml) (p<0.0001 vs untreated cells). Taken these results together, it can be concluded that I. tinctoria cauline leaves collected in January represent a source of phenolic compounds with antioxidant and anti-proliferative activity, which could be potentially used as natural agents for health promotion. Acknowledgments The authors wish to thank the “University of Messina” within the “Research and Mobility” Project. 1) M.F. Taviano, S. Ragusa, G. Paterniti Mastrazzo, A. Melchini, L.P. Buongiorno, P. Dugo, F. Cacciola, M.L. Guzman, H.-T. Hsu, G. Galletti, N. Miceli (2014) 109° Congresso della Società Botanica Italiana (International Plant Science Conference). Florence, 2-5 September. 2) N. Miceli, A. Filocamo, S. Ragusa, G. Paterniti Mastrazzo, R.F. De Rose, V. Maggisano, A. Melchini, M.F. Taviano (2015) 110° Congresso della Società Botanica Italiana (II International Plant Sciences Conference). Pavia, 14-18 September. 3) N. Miceli, L.P. Buongiorno, M.G. Celi, F. Cacciola, P. Dugo, P. Donato, L. Mondello, I. Bonaccorsi, M.F. Taviano (2016) Natural Product Research, 30(11), 1229-1239.