Phenolic-Containing Organic Extracts of Mulberry (Morus alba L.) Leaves Inhibit HepG2 Hepatoma Cells Through G2/M Phase Arrest, Induction of Apoptosis, and Inhibition of Topoisomerase IIα Activity

Abstract

The entire plant of Morus alba L. (Family Moraceae), or mulberry, possesses medical benefits, including anticancer properties. In this study, we investigated the effect of mulberry leaf extracts on the human hepatoma HepG2 cell line, which is related to hepatocellular carcinoma. Mulberry leaf extracts were prepared using four solvents, each with different polarities: 100% methanol (MeOH), 50% aqueous MeOH, 1-butanol (BuOH), and hot water (W). The phenolic profile, total polyphenol content, antioxidant capacity, and effect on human hepatoma HepG2 cells of the leaf extracts were analyzed by examining cytotoxicity, cell cycle progression, apoptosis, expression of topoisomerase IIα, and proteins involved in cell cycle progression. High-performance liquid chromatography-mass spectrometry analysis revealed that 100% MeOH, 50% MeOH, and BuOH extracts contained rutin, isoquercetin, and various derivatives of kaempferol and quercetin glycosides as their major constituents; the W extract contained primarily chlorogenic acid and caffeoylquinic acid derivatives. Total phenolic content based on rutin equivalents was 17.1%, 9.6%, 8.3%, and 6.5% of dry 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activities were 70.0%, 45.8%, 41.0%, and 33.6%, and 50% inhibitory concentration values were 33.1, 79.4, 35.6, and 204.2 μg/mL for HepG2 cell proliferation inhibition for 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. MeOH extracts caused cell cycle G2/M arrest and induced the caspase cascade and apoptosis, but the W extract had very little effect on cell cycle progression. MeOH extracts reduced the level of topoisomerase IIα but increased the level of p27(Kip1), with no significant effect on p21(Cip1/waf1). Therefore, we concluded that phenolic-containing organic extracts of mulberry leaves inhibit the growth of HepG2 hepatoma cells through coordinated actions of inducing cell cycle arrest in the G2/M phase (with p27(Kip1) protein expression), inhibiting topoisomerase IIα activity, and inducing cell apoptosis by activation of caspases.

Protective Effect of Chlorogenic Acid against Aβ-Induced Neurotoxicity

“…Therefore, the decrease in Bax or active caspase-3 expression and increase in Bcl-2 expression can provide a neuroprotective effect by inhibiting Aβ-induced apoptosis. For example, organic extracts of mulberry leaves containing mainly phenolic compounds, including chlorogenic acid, inhibited apoptosis in HepG2 hepatoma cells by arresting the cell cycle in the G2/M phase (Granado-Serrano et al, 2007;Naowaratwattana et al, 2010). In addition, chlorogenic acid exhibited an anti-apoptotic effect on methylglyoxyal-induced neuroblastoma cell apoptosis (Neuro-2A cells) (Huang et al, 2008).…”

Morus rubra Extract Induces G1 Cell Cycle Arrest and Apoptosis in Human Lung and Prostate Cancer Cells

“…21 M. rubra is one of the most important species of the genus Morus, with fruits having substantial levels of phenolics, anthocyanins, carotenoids and ascorbic acid. 3 Many studies have investigated the cytotoxic effects of different berry species, [8][9][10] however to the authors' best knowledge there is no study reporting cytotoxic effect of M. rubra in the literature. It is adviced that in vitro experiments are helpful and advisor for determining first biological effect of a natural or syntetic product.…”

“…Hovewer, higher extract concentrations which cause an arrest of cells at the G 2 /M phase of the cell cycle and modify microtubule organization, 35 while Naowaratwattana et al reported that M. alba leaf extract inhibits the proliferation of HepG2 cells in a dose-dependent manner by inducing cell cycle arrest at G 2 /M phase, caspase activity and inhibiting topoisomerase IIα activity. 9 Recently, Kollar et al reported that prenylated flavonoids from Morus alba inhibit the growth of human monocytic leukemia (THP-1) cells through inducing G 1 cell cycle arrest and apoptotic pathway. 36 We determined that AMRE induced cell cycle arrest at G 0 /G 1 phase in both A549 and PC-3 cells in a concentration dependent manner.…”

“…Seeram et al reported that fresh fruit extracts of various berries have cytotoxic effects on human oral, breast, prostate and colon cancer cells, 8 while Naowaratwattana et al demonstrated that M. alba leaf extract inhibits the proliferation of HepG2 cells in a dose-dependent manner by inducing cell cycle arrest at G 2 /M phase, caspase activity and inhibiting topoisomerase IIα activity. 9 Recently, Deepa et al demonstrated that M. alba extract induces apoptosis in HCT-15 and MCF-7 cells and downregulation of inducible nitric oxide synthase is suggested as a possible apoptotic mechanism. 10 However, to the authors' best knowledge there is no study reporting cytotoxic effect of M. rubra in the literature.…”

Anti–microbial principles of selected remedial plants from Southern India

“…Anti-microbial properties have been described by previous science fair investigators from some natural extracts, and the majority of these studies focus on crude extracts [9] . Morus species have been a rich food source, and the leaf extracts of Morus Alba (M. alba) are used for anticancer and antioxidants properties [10,11] . Bombax seeds are useful for treating gonorrhea and chronic cystitis, bark for improving skin color, flowers for making tea and silk for cotton production.…”