Effect of rice bran polyphenols on biomarkers associated with cardiovascular disease and Type 2 diabetes

The project aimed to investigate the role of rice bran (RB) polyphenols in modulating biomarkers and risk factors associated with cardiovascular disease (CVD) and Type 2 diabetes (T2DM). Firstly, the effect of various processing conditions (heat stabilization treatments) on the phenolic composition of RB and its corresponding antioxidant activity were investigated. The results from this study established that stabilization via drum-drying resulted in a significant increase in measured total phenolic content and associated antioxidant activity. Mass spectrometry studies revealed ferulic acid, p-coumaric acid, caffeic acid, vanillic acid, syringic acid, sinapic acid, feruloyl glycoside, shikimic acid, tricin, and their isomers as the most predominant phenolic compounds present in drum-dried RB. Due to the increased antioxidant activity of the drum-dried RB sample, it was chosen for further in vitro and ex vivo analysis.
To determine the effect of RB polyphenols in modulating genes associated with antioxidant and anti-inflammatory pathways in vitro, a human umbilical vein endothelial cell (HUVEC) model under induced oxidative stress conditions was employed. HUVECs under oxidative stress were treated with varying concentrations of RB phenolic extracts (25, 50, 100, and 250 µg/mL). Using quantitative real-time polymerase chain reaction (qPCR), the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), intercellular adhesion molecule 1 (ICAM1), endothelial nitric oxide synthase (eNOS), ectonucleoside triphosphate diphosphohydrolase 1 (CD39), and ecto-5'-nucleotidase (CD73) genes were determined. Results obtained from this study revealed that treatment with RB extract down-regulated the expression of ICAM1, CD39, CD73, and NOX4 and up-regulated the expression of Nrf2, NQO1, HO1 and eNOS indicating an antioxidant/ anti-inflammatory effect of RB during endothelial dysfunction.
Furthermore, to investigate the impact of RB polyphenols on biomarkers associated with inflammation and free radical damage, a RAW264.7 mouse macrophage cell model stimulated with hydrogen peroxide and lipopolysaccharide was used. Following treatment with RB polyphenol (25, 50, 100, and 250 µg/mL) extracts, production of malondialdehyde, intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines (interleukin-12, p70, interferon-γ, interleukin-6) were significantly reduced.
Due to the observed antioxidant and anti-inflammatory effect of RB polyphenols, an ex vivo study was undertaken to examine if they had an impact on platelet function under oxidative stress conditions, a significant risk factor of vascular disorders. Blood samples obtained from healthy human volunteers were treated with varying concentrations of RB phenolic extracts (50, 100, 250 and 500 µg/mL). Hydrogen peroxide and ADP were used as an oxidative stress stimulant and platelet activator respectively. Biomarkers of platelet activation were evaluated through the measurement of platelet activation related conformational change (PAC-1) and de-granulation (CD62P/P-selectin). It was observed that RB phenolic extracts displayed significant antioxidant and anti-inflammatory activity, however, did not have an impact upon ADP induced platelet activation under oxidative stress conditions. Further investigations into the effect of RB polyphenol extracts on platelet stimulation via other activation pathways, such as collagen and thrombin, are warranted.
To evaluate the effect of RB phenolic extracts on risk factors of T2DM, a pancreatic β-cell model was used. β-cell function was investigated by evaluating the expression of candidate genes such as Glucose transporter 2 (Glut2), Pancreatic and Duodenal Homeobox 1 (Pdx1), Sirtuin 1 (Sirt1), Mitochondrial transcription factor A (Tfam) and Insulin 1 (Ins1). Gene expression was studied using qPCR. Additionally, the impact on glucose-stimulated insulin secretion was investigated using an enzyme-linked immunosorbent assay (ELISA). Treatment with different concentrations of RB extracts (25, 50, 100, and 250 µg/mL) resulted in a significant increase in the expression of Glut2, Pdx1, Sirt1, Tfam and Ins1 genes and glucose-stimulated insulin secretion under both normal and high glucose conditions. The outcomes from this study revealed that RB phenolic extracts can modulate the expression of genes involved in β-cell dysfunction and insulin secretion as a result of the synergistic action of polyphenols present in RB by targeting signalling molecules, reducing free radical damage by its antioxidant activity, and stimulation of effectors or survival factors of insulin secretion.
Finally, to evaluate the effect of RB phenolic extracts on glucose release in vitro, a novel starch digestibility model was utilized. The starch hydrolysis of a rice variety with a high glycemic index (GI) was pre-treated with RB phenolic extracts (7.5, 15 and 30 mg/mL) during cooking and with digestive enzymes. The results obtained from this study demonstrated a reduction in the rate of starch digestibility, possibly due to a direct interaction of the polyphenols present in the RB extracts with the starch and/ or with the enzymes. Since a steady release of glucose over time is essential for maintaining a moderate postprandial glycemic and insulinemic response, consumption of RB, which is abundant in GI lowering polyphenols, may be an appropriate method that can be used as a preventative measure to assist in controlled glucose release.
The outcomes highlighted in this project demonstrate that the bioactive constituents found in RB may play a key role in targeting specific mechanistic pathways associated with CVD and T2DM risk by virtue of its antioxidant and anti-inflammatory properties.