RESUMO
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 ß (GSK3ß) is overactivated and represents an interesting therapeutic target. Several flavonoids can inhibit GSK3ß and the purpose of this study was to search for the compounds in Diplotaxis harra which are able to modulate GSK3ß. 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 (PAR2)-stimulated IEC6 cells] and cancer (human Caco-2 cell line) intestinal cells. 50-100 µg/mL of fractions or compounds purified by HPLC were incubated with cells whose inhibited form of GSK3ß (Pser9 GSK3ß) 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 GSK3ß in PAR2-stimulated IEC6 cells. GSK3ß inhibition by the flavonoid-enriched D. harra fraction was dependent on PKC activation. The flavonoid-enriched D. harra fraction and its purified compound isorhamnetin-3,7-di-O-glucoside induced a 20% decrease of PAR2-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 GSK3ß inhibition.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Brassicaceae/química , Neoplasias do Colo/tratamento farmacológico , Flavonóis/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicosídeos/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Extratos Vegetais/farmacologia , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Flavonóis/isolamento & purificação , Flores , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicosídeos/isolamento & purificação , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/patologia , Espectroscopia de Ressonância Magnética , Metanol/química , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Solventes/química , Espectrometria de Massas em TandemRESUMO
Two new tetracyclic cucurbitane-type triterpene glycosides were isolated from an ethyl acetate extract of Citrullus colocynthis leaves together with four known cucurbitacins. Their structures were established on the basis of their spectroscopic data (mainly NMR and mass spectrometry). Evaluation of the in vitro cytotoxic activity of the isolated compounds against two human colon cancer cell lines (HT29 and Caco-2) and one normal rat intestine epithelial cell line (IEC6), revealed that one of the isolated compounds presented interesting specific cytotoxic activity towards colorectal cell lines.