Processing optimisation, molecular characterisation, and anti-cancer activities of arabinoxylans extracted from corn bran in vitro

Abstract

Background: Arabinoxylans (AXs) are the main non-starch polysaccharides that widely exist in by-products of cereal processing. The anti-cancer activities, such as inhibiting cancer cell proliferation and promoting apoptosis, of AXs which are extracted from various cereal materials have been reported recently. However, the possible relationship between the structures and anti-cancer activities of AXs have not yet been established and proposed. The anti-cancer mechanisms of AXs are unknown. The aims of this study were to develop and optimise the AXs combined extraction treatment from selected cereals, characterise the molecular structure and measure the anti-cancer activities of AXs extracts in vitro to explore the possible relationship between their structures and anti-cancer activity, and propose the potential mechanisms. Methods: The alkaline (A) extraction method, hydrothermal (H) extraction method, and a low concentration alkaline-assisted hydrothermal (LAH) extraction method were used to extract AXs from cereal materials. The high-performance liquid chromatography (HPLC) methods were used to characterise the molecular structure and monocular composi6on of extracted AXs. The anti-cancer effects of extracted AXs on the viability, proliferation cycle and apoptosis of human gastric adenocarcinoma cell line (AGS) cells were assessed in vitro using flow cytometry. The mRNA expressions of selected genes related to cell growth and apoptosis control were measured by RT-qPCR analysis and the protein expressions were tested by Western blot assay. Results: Under the optimum extraction condi6ons of LAH, the extraction rate of AXs reached 33.31%, which is about 7 6mes of H extraction (4.65%) and no significant difference when compared with that of A-AXs (32.34%) (p ≥ 0.05). Compared to molecular characteristics of AAXs., LAH-AXs extracts have a lower percentage (23.68%) of low molecular weight range (0.01KDa to 10.00KDa), which is 29.71%, and a higher ferulic acid (FA) content (0.64 mg/g), which is 7 6mes compared to A-AXs (0.09mg/g). In vitro studies showed that both LAH-AXs and A-AXs at optimised concentra6on of 50 μg /ml were able to significantly inhibit the proliferation of AGS and significantly promote apoptosis in AGS cells in 24 hrs treatment (p ≤0.05). Furthermore, in the examination of the mechanism of AXs anti-cancer activities, the cell cycle analysis results demonstrated that both LAH-AXs and A-AXs treatment significantly increased the accumula6on of AGS cells in the S phase, which is 1.4-fold compared with negative control (p ≤ 0.05). Furthermore, both LAH-AXs and A-AXs significantly up-regulated the mRNA expressions of cyclin-dependent kinase (CDK) inhibitor p21 and pro-apoptosis genes Bad and Bid. Differently, LAH-AXs significantly up-regulated caspase-8 mRNA expression (2.33-fold) and pERK protein expression levels (1.4-fold) compared with negative control (p ≤ 0.05), whereas A-AXs did not show a significant impact in this regard. Conclusions: At optimal conditions for LAH-AXs-C extraction, the extraction rate can reach levels comparable to those achieved with high alkaline concentrations (8%). Molecular characterisations indicate variations in molecular distribution, molecular composition and FA content between LAH-AXs-C and A-AXs-C. The distinct effects of LAH-AXs-C and A-AXs-C on the cell cycle arrest occurring at different stages. A decrease in cell viability and induction of apoptosis in AGS cells, may be attributed to the differences in their structures. The underlying mechanisms of their anti-cancer activities possibly involve regulation through the Bcl-2 family signalling pathway and the ERK signalling pathway, which are suggested by the up-regulation of mRNA expressions level of p21 and the pro-apoptosis genes Bad, Bid and protein pERK. Moreover, the upregulation in caspase-8 (2.33-fold) expression indicates the activation of extrinsic pathway. This study optimised the extraction method for AXs, which significantly increased the extraction rate (р≤0.05). It also characterised the structure of AXs and explored their potential anti-cancer effects, providing an initial understanding of the relationship between the structure and bioactivities of AXs at the molecular level.