Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties.

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

Rice Bran Phenolic Extracts Modulate Insulin Secretion and Gene Expression Associated with ß-Cell Function.

Oxidative stress is known to modulate insulin secretion and initiate gene alterations resulting in impairment of ß-cell function and type 2 diabetes mellitus (T2DM). Rice bran (RB) phenolic extracts contain bioactive properties that may target metabolic pathways associated with the pathogenesis of T2DM. This study aimed to examine the effect of stabilized RB phenolic extracts on the expression of genes associated with ß-cell function such as glucose transporter 2 (Glut2), pancreatic and duodenal homeobox 1 (Pdx1), sirtuin 1 (Sirt1), mitochondrial transcription factor A (Tfam), and insulin 1 (Ins1) in addition to evaluating its impact on glucose-stimulated insulin secretion. It was observed that treatment with different concentrations of RB phenolic extracts (25-250 µg/mL) significantly increased the expression of Glut2, Pdx1, Sirt1, Tfam, and Ins1 genes and glucose-stimulated insulin secretion under both normal and high glucose conditions. RB phenolic extracts favourably modulated the expression of genes involved in ß-cell dysfunction and insulin secretion via several mechanisms such as synergistic action of polyphenols targeting signalling molecules, decreasing free radical damage by its antioxidant activity, and stimulation of effectors or survival factors of insulin secretion.

Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release.

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

Coloured Rice Phenolic Extracts Increase Expression of Genes Associated with Insulin Secretion in Rat Pancreatic Insulinoma ß-cells.

Glucose-induced oxidative stress is associated with the overproduction of reactive oxygen species (ROS), which may dysregulate the expression of genes controlling insulin secretion leading to ß-cell dysfunction, a hallmark of type 2 diabetes mellitus (T2DM). This study investigated the impact of coloured rice phenolic extracts (CRPEs) on the expression of key genes associated with ß-cell function in pancreatic ß-cells (INS-1E). These genes included glucose transporter 2 (Glut2), silent mating type information regulation 2 homolog 1 (Sirt1), mitochondrial transcription factor A (Tfam), pancreatic/duodenal homeobox protein 1 (Pdx-1) and insulin 1 (Ins1). INS-1E cells were cultured in high glucose (25 mM) to induce glucotoxic stress conditions (HGSC) and in normal glucose conditions (NGC-11.1 mM) to represent normal ß-cell function. Cells were treated with CRPEs derived from two coloured rice cultivars, Purple and Yunlu29-red varieties at concentrations ranged from 50 to 250 µg/mL. CRPEs upregulated the expression of Glut2, Sirt1 and Pdx-1 significantly at 250 µg/mL under HGSC. CRPEs from both cultivars also upregulated Glut2, Sirt1, Tfam, Pdx-1 and Ins1 markedly at 250 µg/mL under NGC with Yunlu29 having the greatest effect. These data suggest that CRPEs may reduce ß-cell dysfunction in T2DM by upregulating the expression of genes involved in insulin secretion pathways.

Phenolic Compounds with Antioxidant Properties from Canola Meal Extracts Inhibit Adipogenesis.

The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p < 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.

The anti-inflammatory and antioxidant effects of acute consumption of pigmented rice in humans.

The pathogenesis of lifestyle diseases has been significantly correlated to high levels of oxidative stress and pro-inflammation. The antioxidant and anti-inflammatory properties of polyphenols in coloured rice varieties could have potential to neutralize oxidative stress and modulate inflammatory responses. A cross-over design, randomised, dietary intervention human clinical trial was conducted on a pre-screened healthy population (n = 24) investigating the antioxidant and anti-inflammatory potential of pigmented rice (purple, red and brown) varieties. Post baseline blood samples collection volunteers consumed a serve of cooked pigmented rice. Blood samples were collected at 30-minutes, 1, 2 and 4-hours post rice consumption. A one-week wash-out period between each supplementation bout (rice variety) was conducted. Blood and biochemical parameters were analysed on baseline blood samples. Antioxidant activity, malondialdehyde (MDA) and a pro-inflammatory cytokine panel were analysed on the blood samples collected. Post purple rice consumption, antioxidant activity increased (p < 0.0001) by 70.5% and maintained elevated for all time points. The red rice variety Yunlu29, significantly (p < 0.005) reduced MDA levels by 9.2% at the 30-minute time point. Purple rice demonstrated a significant (p < 0.05) decrease by 4.0% at the 30-minute time point only. Purple rice significantly decreased TNF-α levels at the 1-hour (p < 0.05) and 4-hour (p < 0.005) time points by 21.9% and 25.4% respectively. IL-6 concentrations were significantly reduced at 1 and 2-hour post Purple (p < 0.05; 11.7%) and Yunlu29 (red) (p < 0.01; 14.1%) consumption respectively. The brown rice variety did not affect any parameters tested. The outcomes of this study, highlight that polyphenols found in pigmented rice may play a key role in targeting specific oxidative stress and inflammatory therapeutic pathways. Pigmented rice varieties may serve as a potential functional food in reducing risk factors associated with lifestyle diseases.

Rice Bran Phenolic Compounds Regulate Genes Associated with Antioxidant and Anti-Inflammatory Activity in Human Umbilical Vein Endothelial Cells with Induced Oxidative Stress.

Oxidative stress, inflammation and endothelial dysfunction are associated with the development of cardiovascular and metabolic diseases. Phenolic extracts derived from rice bran (RB) are recognised to have antioxidant and anti-inflammatory potential. However, the underlying mechanisms remain unknown. Therefore, this study aimed to evaluate the ability of RB-derived phenolic extracts to modulate genes associated with antioxidant and anti-inflammatory pathways in human umbilical vein endothelial cells (HUVECs) under induced oxidative stress conditions. HUVECs under oxidative stress were treated with varying concentrations of RB phenolic extracts (25-250 µg/mL). Using quantitative real-time polymerase chain reaction, the expression of candidate genes that regulate antioxidant and anti-inflammatory pathways were determined. This included 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). Phenolic extracts derived from RB down-regulated the expression of four genes, ICAM1, CD39, CD73 and NOX4 and up-regulated the expression of another four genes, Nrf2, NQO1, HO1 and eNOS, indicating an antioxidant/ anti-inflammatory effect for RB against endothelial dysfunction.

Coloured rice-derived polyphenols reduce lipid peroxidation and pro-inflammatory cytokines ex vivo.

Rice-derived polyphenols have been demonstrated to alleviate obesity-related oxidative stress and inflammation. The aim of the study was to investigate if coloured rice polyphenol extracts (PE) reduce malondialdehyde, interleukin-6 and tumour necrosis factor-α (TNF-α) levels in obese individuals ex vivo. Malondialdehyde and pro-inflammatory cytokines were quantified using high-performance liquid chromatography and flow cytometry respectively. Fasting blood samples were treated with PE from three coloured rice varieties (purple, red and brown rice) at varying concentrations (10, 20, 50, 100, 200 and 500 µg mL-1). PE treatment demonstrated a dose-dependent reduction in malondialdehyde and TNF-α levels. Purple PE reduced plasma malondialdehyde concentration by 59% compared to red (21%) and brown (25.5%) rice PE. Brown rice PE at 50 µg mL-1 reduced TNF-α levels by 98% compared to red (80%) and purple rice PE (74%). Rice PE did not modulate plasma interleukin-6 concentrations. Coloured rice may be of therapeutic benefit as a potential functional food alternative in targeting specific pathways associated with obesity.

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils.

Medium-chain fatty acids (MCFA, C6-14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl-CoA-dependent pathway for the synthesis of MFCA-rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

Effects of roasting on phenolic composition and in vitro antioxidant capacity of Australian grown faba beans (Vicia faba L.).

Faba bean phenolic compounds encompassed phenolic acids, flavonols, proanthocyanidins and anthocyanins. Roasting faba beans for 120 min decreased the total phenolic, flavonoid and proanthocyanidin contents by 42, 42 and 30%, respectively. Roasting beans for 120 min decreased the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, total equivalent antioxidant capacity and ferric reducing antioxidant power by 48, 15 and 8%, respectively. High performance liquid chromatography-post column derivatisation revealed the generation of new phenolic compounds as a result of roasting. Antioxidant mechanism of bean less-polar phenolic compounds was largely based on free radical scavenging activity. The bean phenolic compounds with reducing capability were heat stable. Roasted faba bean extracts (70% acetone, v/v) were fractionated into relatively polar and non-polar fractions; the latter contributed the majority of the antioxidant capacity. The extracts from beans with different seed coat colours differed in their phenolic compositions, which suggest different levels of potential benefits to health. Although roasting initially lowers the bean antioxidant capacity, prolonged roasting at 150 °C for 60 min and longer causes generation of new phenolic compounds and an increased antioxidant capacity. The findings encourage a wider ultilisation of faba beans for human foods particularly in baked/roasted products.

Effects of soaking, boiling and autoclaving on the phenolic contents and antioxidant activities of faba beans (Vicia faba L.) differing in seed coat colours.

The Australian grown faba beans of different seed coat colours were either soaked, boiled or autoclaved, and analysed for phenolic contents and antioxidant activity using an array of reagent-based assays. Soaking, boiling and autoclaving were shown to lower the level of active compounds in faba beans. A significant amount of active compounds was leached to the soaking and cooking medium. Boiling was a better method in retaining active compounds in beans than autoclaving. The boiled beans had more active compounds than those of resulting cooking broths, which was the opposite observation when autoclaving. The buff-genotypes had a similar level of active compounds to red- and green-genotypes. The high performance liquid chromatography-post column derivatisation (HPLC-PCD) system detected a dense collection of high antioxidant HPLC peaks ('humps') in extracts of raw, soaked and boiled beans. The present findings encouraged consumption of faba beans together with cooking broth for the maximum potential health benefits.

In vitro investigations of the potential health benefits of Australian-grown faba beans (Vicia faba L.): chemopreventative capacity and inhibitory effects on the angiotensin-converting enzyme, α-glucosidase and lipase.

The functional properties, including antioxidant and chemopreventative capacities as well as the inhibitory effects on angiotensin-converting enzyme (ACE), α-glucosidase and pancreatic lipase, of three Australian-grown faba bean genotypes (Nura, Rossa and TF(Ic*As)*483/13) were investigated using an array of in vitro assays. Chromatograms of on-line post column derivatisation assay coupled with HPLC revealed the existence of active phenolics (hump) in the coloured genotypes, which was lacking in the white-coloured breeding line, TF(Ic*As)*483/13. Roasting reduced the phenolic content, and diminished antioxidant activity by 10-40 % as measured by the reagent-based assays (diphenylpicrylhydrazyl, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and oxygen radical absorbance capacity) in all genotypes. Cell culture-based antioxidant activity assay (cellular antioxidant activity) showed an increase of activity in the coloured genotypes after roasting. Faba bean extracts demonstrated cellular protection ability against H2O2-induced DNA damage (assessed using RAW264.7 cells), and inhibited the proliferation of all human cancer cell lines (BL13, AGS, Hep G2 and HT-29) evaluated. However, the effect of faba bean extracts on the non-transformed human cells (CCD-18Co) was negligible. Flow cytometric analyses showed that faba bean extracts successfully induced apoptosis of HL-60 (acute promyelocytic leukaemia) cells. The faba bean extracts also exhibited ACE, α-glucosidase and pancreatic lipase inhibitory activities. Overall, extracts from Nura (buff-coloured) and Rossa (red-coloured) were comparable, while TF(Ic*As)*483/13 (white-coloured) contained the lowest phenolic content and exhibited the least antioxidant and enzyme inhibition activities. These results are important to promote the utilisation of faba beans in human diets for various health benefits.