Phytochemical characterization and cytotoxic properties of the polar extracts from the leaves of Isatis tinctoria L. collected in Sicily

The spontaneous flora of Sicily includes several species belonging to the Brassicaceae family, potential source of bioactive compounds. Isatis tinctoria L. (woad) is a herbaceous biennial species which grows wild mainly in Southern and North-western Italy, as well as on the major islands. The stems are erect, up to 120 cm in height, simple below and branched above. Basal leaves are oblong-lanceolate and long-petioled. Cauline leaves, narrower than basal and gradually reduced upwards, are simple, entire, sagittate, usually amplexicaul, with acute auricles (1). I. tinctoria L. has been cultivated and used in Europe since antiquity for production of the blue dye indigo and as medicinal plant, until it fell into oblivion due to the import of cheaper indigo and the disappearance of woad cultures. Nowadays I. tinctoria L. is employed, together with the closely related Isatis indigotica Fort., in traditional Chinese medicine (2). This study was designed to characterize the phytochemical profile and to investigate the cytotoxic properties of the polar constituents of I. tinctoria L. leaves collected at different times of the year, January (It-J) and April (It-A). Cauline leaves, picked from I. tinctoria L. grown wild around Acireale (Catania, Sicily), were lyophilized and sequentially extracted with dichloromethane and methanol 70%. The HPLC-PDA-ESI-MS analysis of It-J and It-A hydroalcoholic extracts revealed a similar phenolic fingerprint, being flavonoids the most abundant constituents, although some differences in the content of individual compounds were found; vicenin-2 was the main flavonoid in both extracts. Glucosinolates (GLSs) were identified by LC/MS analysis, and the indole GLS glucobrassicin was detected as the major compound in It-J only. The anti-proliferative effect of It-J and It-A extracts was evaluated in vitro on human leukemia cell line (MOLM-13). After the treatment for 24 and 48 hours, to assess viability, cells were stained with Annexin V and 7-amino-actinomycin D and fluorescence was evaluated by flow cytometry. It-J extract demonstrated good cytotoxic effect at both time points, with an IC50 of 0.3 mg/mL at 24 hours and of 0.2 mg/mL at 48 hours. It-A extract didn’t prove to be active and could barely reach an IC50 value even after a longer treatment. The potential cytotoxic activity of It-J and It-A was tested using Artemia salina lethality bioassay, too (3); both extracts did not display any cytotoxicity against brine shrimp larvae (LC50 > 1000 µg/mL). In the last decades, antioxidant compounds have received increased attention from nutritionists and researchers for their potential activities in the prevention of several degenerative diseases such as cancer. The antioxidant and cancer protective properties of polyphenols have been well documented in several studies (3). In a previous work, we showed the in vitro antioxidant activity of I. tinctoria L. leaves hydroalcoholic extracts, higher for It-A than It-J, which seems to be related to their total phenolic content (99.36 ± 0.17 mg GAE/g and 79.00 ± 0.64 mg GAE/g, respectively) (4). In spite of this, our experimental data show that there isn’t correlation between the anti-proliferative activity of I. tinctoria L. leaves extracts against MOLM-13 cells and the antioxidant phenolic compounds. It’s well known that isothiocyanates, bioactive compounds derived from the hydrolysis of GLSs, possess chemopreventive properties in a variety of cell and animal models (5). Hence, it can be hypothesized that GLSs are involved, almost in part, in the cytotoxic activity of It-J extract. A more extensive analysis is ongoing to better dissect the promising cytotoxic properties of It-J extract and further investigate its anti-cancer applications.