Isolation, Characterization, Complete Structural Assignment, and Anticancer Activities of the Methoxylated Flavonoids from Rhamnus disperma Roots

Abstract

Different chromatographic methods including reversed-phase HPLC led to the isolation and purification of three <i>O</i>-methylated flavonoids; 5,4'-dihydroxy-3,6,7-tri-<i>O</i>-methyl flavone (penduletin) (<b>1</b>), 5,3'-dihydroxy-3,6,7,4',5'-penta-<i>O</i>-methyl flavone (<b>2</b>), and 5-hydroxy-3,6,7,3',4',5'-hexa-<i>O</i>-methyl flavone (<b>3</b>) from <i>Rhamnus disperma</i> roots. Additionlly, four flavonoid glycosides; kampferol 7-<i>O</i>-<i>α</i>-L-rhamnopyranoside (<b>4</b>), isorhamnetin-3-<i>O</i>-<i>β</i>-D-glucopyranoside (<b>5</b>), quercetin 7-<i>O</i>-<i>α</i>-L-rhamnopyranoside (<b>6</b>), and kampferol 3, 7-di-<i>O</i>-<i>α</i>-L-rhamnopyranoside (<b>7</b>) along with benzyl-<i>O</i>-<i>β</i>-D-glucopyranoside (<b>8</b>) were successfully isolated. Complete structure characterization of these compounds was assigned based on NMR spectroscopic data, MS analyses, and comparison with the literature. The <i>O</i>-methyl protons and carbons of the three <i>O</i>-methylated flavonoids (<b>1</b>-<b>3</b>) were unambiguously assigned based on 2D NMR data. The occurrence of compounds <b>1</b>, <b>4</b>, <b>5</b>, and <b>8</b> in <i>Rhamnus disperma</i> is was reported here for the first time. Compound <b>3</b> was acetylated at 5-OH position to give 5-<i>O</i>-acetyl-3,6,7,3',4',5'-hexa-<i>O</i>-methyl flavone (<b>9</b>). Compound <b>1</b> exhibited the highest cytotoxic activity against MCF 7, A2780, and HT29 cancer cell lines with IC₅₀ values at 2.17 µM, 0.53 µM, and 2.16 µM, respectively, and was 2-9 folds more selective against tested cancer cell lines compared to the normal human fetal lung fibroblasts (MRC5). It also doubled MCF 7 apoptotic populations and caused G₁ cell cycle arrest. The acetylated compound <b>9</b> exhibited cytotoxic activity against MCF 7 and HT29 cancer cell lines with IC₅₀ values at 2.19 µM and 3.18 µM, respectively, and was 6-8 folds more cytotoxic to tested cancer cell lines compared to the MRC5 cells.