Biological properties of the polar extracts from leaves and flowers of Isatis tinctoria L. (Brassicaceae) growing in Sicily

Isatis tinctoria L. (woad) is a herbaceous biennial species belonging to Brassicaceae family. It does grow wild mainly in Southern and North-western Italy, as well as on the major Italian islands. Rural people living around Vulcan Etna (Sicily, Italy) consume boiled flower buds of this plant as ingredients for salads and omelets (1). Historically, I. tinctoria has been cultivated for the production of indigo dye (blue colour) in Europe and it has also been used as medicinal plant. However its use as natural dye has declined, due to the import of cheaper indigo and the disappearance of woad cultures. Nowadays I. tinctoria and its closely related species Isatis indigotica Fort., are employed in traditional Chinese medicine (2). In the last decades, in vivo and in vitro studies highlighted the anti-inflammatory and anti-cancer properties of lipophilic extracts and isolated compounds. In particular some alkaloids including tryptanthrin, indirubin and indolinone have been studied for their beneficial effects. However few investigations have been carried out for studying the potential biological properties of polar constituents. In continuation of our studies on I. tinctoria, this work was designed to define and compare some biological properties of the polar constituents of flowers and leaves of I. tinctoria grown wild around Acireale (Catania, Sicily, Italy). Moreover the correlation between biological effects and phenolic compounds was investigated. Cauline leaves were collected in January (It-J) and April (It-A), basal leaves (It-B) in November and flowers (It-F) were harvested in April. All plant material was lyophilized and sequentially extracted with dichloromethane and methanol 70%. The total phenolic, total flavonoid and condensed tannin content of hydroalcoholic extracts was determined spectrophotometrically (3). The total phenolics ranged from 99.36 ± 0.17 (It-A) to 68.60 ± 0.50 mg GAE/g (It-B); the flavonoids ranged from 41.70 ± 0.46 (It-F) to 12.44 ± 0.14 mg QE/g (It-J); the condensed tannins ranged from 27.59 ± 0.21 (It-A) to 10.92 ± 0.47 mg CE/g (It-F). The antioxidant properties of the extracts were examined by different in vitro tests (3). In the DPPH test It-F extract resulted the most active with an IC50 value of 0.437 ± 0.003 mg/mL, whilst It-A extract showed the best activity in reducing power (ASE/mL= 1.546 ± 0.006) and ferrous ions chelating activity assays (IC50= 0.564 ± 0.011 mg/mL). Linear regression analysis revealed a positive relationship between total phenolic content and both reducing power and chelating activity, and between tannins and reducing power. Finally a weak relationship with total flavonoids was highlighted for DPPH only. The potential cytotoxic activity of It-J, It-A, It-B and It-F extracts was tested using Artemia salina lethality bioassay (3); all extracts did not display any cytotoxicity against brine shrimp larvae (LC50>1000 µg/mL). The anti-proliferative effect of I. tinctoria extracts was evaluated in vitro on three human anaplastic thyroid carcinoma (ATC) cell lines (CAL-62, C-643 and 8505C). By MTT assay, a reduced proliferation rate of all cancer cell lines in a concentration- and time-dependent way was observed. In particular, after 48 h of treatment, approximately 20% of growth inhibition (p<0.05 vs control) was observed in 8505C cells with 0.1-0.5-1 mg/mL of It-J, It-A and It-F, and a stronger inhibiting effect (about 50%) was obtained using It-B at the same concentrations. Similar results were observed in the C-643 cells. The strongest anti-proliferative effect was observed in CAL-62 cells; particularly after 48 h exposure to It-B at the concentration of 1 mg/mL, a decrease in cell growth of 80% was observed (p<0.001 vs untreated cells). The obtained results suggest that there isn’t correlation between the anti-proliferative activity of I. tinctoria extracts against ATC cells and the antioxidant phenolic compounds. We previously characterized the glucosinolate profile of It-J and It-A extracts (2); at present LC/MS analysis of glucosinolates contained in It-B and It-F extracts is ongoing in order to establish whether these bioactives are associated with their anti-proliferative effects. 1) S. Galletti, M. Bagatta, L. Ragusa, S. Argento, F. Branca, R. Iori (2012) 6th International Symposium on Brassica and 18th Crucifer Genetics Workshop. Catania, 12-16 November. 2) M.F. Taviano, S. Ragusa, G. Paterniti Mastrazzo, A. Melchini, LP. Buongiorno, P. Dugo, F. Cacciola, M.L. Guzman, H.-T. Hsu, G. Galletti, N. Miceli (2014) 109° Congresso della Società Botanica Italiana. Florence, 2-5 September. 3) M.F. Taviano, A. Marino, A. Trovato, V. Bellinghieri, T.M. La Barbera, A. Güvenç, M.M. Hürkul, R. De Pasquale, N. Miceli (2011) Pharm. Biol. 49(10), 1014-1022.