Anti-inflammatory and anticancer effects of flavonol glycosides from Diplotaxis harra through GSK3β regulation in intestinal cells

Context and objective: Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) is traditionally used as an antidiabetic, anti-inflammatory or anticancer agent. In these pathologies, the glycogen synthase kinase 3 beta (GSK3 beta) is overactivated and represents an interesting therapeutic target. Several flavonoids can inhibit GSK3 beta and the purpose of this study was to search for the compounds in Diplotaxis harra which are able to modulate GSK3 beta. Materials and methods: Methanol extracts from D. harra flowers were prepared and the bio-guided fractionation of their active compounds was performed using inflammatory [protease-activated receptor 2 (PAR(2))-stimulated IEC6 cells] and cancer (human Caco-2 cell line) intestinal cells. 50-100 mu g/mL of fractions or compounds purified by HPLC were incubated with cells whose inhibited form of GSK3 beta (Pser9 GSK3b) and survival were analyzed by Western blot at 1 h and colorimetric assay at 24 h, respectively. LC-UV-MS profiles and MS-MS spectra were used for the characterization of extracts and flavonoids-enriched fractions, and the identification of pure flavonoids was achieved by MS and NMR analysis. Results: The methanol extract from D. harra flowers and its flavonoid-enriched fraction inhibit GSK3 beta in PAR(2)-stimulated IEC6 cells. GSK3 beta inhibition by the flavonoid-enriched D. harra fraction was dependent on PKC activation. The flavonoid- enriched D. harra fraction and its purified compound isorhamnetin-3,7di-O-glucosideinduced a 20% decrease of PAR(2)-stimulated IEC6 and Caco-2 cell survival. Importantly, normal cells (non-stimulated IEC6 cells) were spared by these treatments. Conclusion: This work indicates that flavonoids from D. harra display cytotoxic activity against inflammatory and cancer intestinal cells which could depend on GSK3 beta inhibition.