Antioxidative activity of mulberry leaf extract capable of inducing apoptosis in HepG2 cells

Abstract

The extracts from the leaves of mulberry (Morus alba L.) have been used in the oriental medicine to treat various disorders including cancer. The objectives of this study were (1) to partially characterize and determine the total phenolic content and antioxidant capacity of mulberry leaf extracts and (2) to investigate the effect of the extracts prepared from different four solvents (100% methanol, 50% aqueous methanol, 1-butanol and hot water) on cytotoxicity effect of human hepatoma HepG2 cells. High performance liquid chromatography coupled with photodiode array detection and mass spectrometry (HPLC-PDA-MS) were used. The organic extracts were found to contain compound of rutin, isoquercetin, kaempferol 3-rhanopyranosyl-glucopyranoside, quercetin 3-(6-malonylglucoside), astragalin and kaempferol 3-(6-malonylglucoside), and the hot water extract contained chlorogenic acid and caffeoyl quinic acid derivatives. The total phenolic contents of various extracts, measured by Folin-Ciocalteau assay were appeared to be in the range 1.80-3.52, 0.86-2.78, 0.46-1.50 g/100g dried leaves which were equivalent of chlorogenic acid, rutin, and gallic acid, respectively. All of the extracts inhibited 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide radical, hydroxyl radical and lipid peroxidation significantly in a dose-dependent manner. All the extracts had showed strong inhibition of cell proliferation of HepG2 cells. The 100% methanol extract appeared to possess higher cytotoxic effect to HepG2 cells than that of the normal human liver cells. Cell cycle distribution analysis and apoptosis, determined by flow cytometry, showed that the organic extracts caused cell cycle G2/M arrest and induced the caspases cascade for apoptosis. The hot water extract, on the other hand, showed very low effect. Among the proteins involved in the regulatory of cell cycle, the organic extracts reduced the enzyme level of topoisomerase IIα and increased that of p27Kip1 with no significant effect on p21Cip1/waf1. These results suggests that the phenolic-containing organic extracts of the mulberry leaves can inhibit HepG2 hepatoma cell population growth at G2/M phase through (i) inhibiting topoisomerase IIα, (ii) induced apoptosis processes, and (iii) induced the expression of p27Kip1. The mulberry leaf extracts, prepared from organic solvents, therefore, have anticancer potential to suppress liver cancer.