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.

Molecularly imprinted polymer membranes and thin films for the separation and sensing of biomacromolecules.

This review describes recent advances associated with the development of surface imprinting methods for the synthesis of polymeric membranes and thin films, which possess the capability to selectively and specifically recognize biomacromolecules, such as proteins and single- and double-stranded DNA, employing "epitope" or "whole molecule" approaches. Synthetic procedures to create different molecularly imprinted polymer membranes or thin films are discussed, including grafting/in situ polymerization, drop-, dip-, or spin-coating procedures, electropolymerization as well as micro-contact or stamp lithography imprinting methods. Highly sensitive techniques for surface characterization and analyte detection are described, encompassing luminescence and fluorescence spectroscopy, X-ray photoelectron spectroscopy, FTIR spectroscopy, surface-enhanced Raman spectroscopy, atomic force microscopy, quartz crystal microbalance analysis, cyclic voltammetry, and surface plasmon resonance. These developments are providing new avenues to produce bioelectronic sensors and new ways to explore through advanced separation science procedures complex phenomena associated with the origins of biorecognition in nature.

The Application of Template Selectophores for the Preparation of Molecularly Imprinted Polymers.

Molecularly imprinted polymers are versatile materials with wide application scope for the detection, capture and separation of specific compounds present in complex feed stocks. A major challenge associated with their preparation has been the need to sacrifice one mole equivalent of the template molecule to generate the complementary polymer cavities that selectively bind the target molecule. Moreover, template molecules can often be difficult to synthesise, expensive or lack stability. In this study, we describe a new approach, directed at the use of synthetic selectophores, chosen as readily prepared and low cost structural analogues with recognition groups in similar three-dimensional arrangements as found in the target molecule. To validate the approach, a comparative study of selectophores related to the polyphenolic compound (E)-resveratrol has been undertaken using traditional and green chemical synthetic approaches. These molecular mimic compounds were employed as polymer templates and also as binding analytes to interrogate the recognition sites associated with the molecularly imprinted polymers. Importantly, the study confirms that the use of selectophores has the potential to confer practical advantages, including access to more efficient methods for selection and preparation of suitable template molecules with a broader range of molecular diversity, as well as delivering imprinted polymers capable of recognizing the target compound and structurally related products.

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.

The Antioxidant and Anti-Inflammatory Properties of Rice Bran Phenolic Extracts.

Oxidative stress and inflammation are known to be linked to the development of chronic inflammatory conditions, such as type 2 diabetes and cardiovascular disease. Dietary polyphenols have been demonstrated to contain potent bioactivity against specific inflammatory pathways. Rice bran (RB), a by-product generated during the rice milling process, is normally used in animal feed or discarded due to its rancidity. However, RB is known to be abundant in bioactive polyphenols including phenolic acids. This study investigates the antioxidant and anti-inflammatory effects of RB phenolic extracts (25, 50, 100, and 250 µg/mL) on RAW264.7 mouse macrophage cells stimulated with hydrogen peroxide and lipopolysaccharide. Biomarkers of oxidative stress and inflammation such as malondialdehyde (MDA), intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-12, p70 (IL-12p70), and interferon-γ (IFN-γ) were measured in vitro. Treatment with RB extracts significantly decreased the production of MDA, intracellular reactive oxygen species, nitric oxide and pro-inflammatory cytokines (IL-6, IL-12p70, and IFN-γ) when compared to the control. It is proposed that RB phenolic extracts, via their metal chelating properties and free radical scavenging activity, target pathways of oxidative stress and inflammation resulting in the alleviation of vascular inflammatory mediators.

Parallel enrichment of polyphenols and phytosterols from Pinot noir grape seeds with molecularly imprinted polymers and analysis by capillary high-performance liquid chromatography electrospray ionisation tandem mass spectrometry.

This investigation describes an integrated workflow for the parallel extraction and recovery of polyphenols and phytosterols from Pinot noir grape seeds. Using (E)-resveratrol and stigmasterol as exemplars, the approach employs two different molecular imprinted polymers in tandem for the extraction of these compounds and their subsequent analysis by capillary high-performance liquid chromatography (capHPLC) interfaced with electrospray ionisation tandem mass spectrometry (ESI MS/MS). Information on the selectivity of the solid-phase extraction processes was obtained through analysis of the binding behaviour of (E)-resveratrol- and stigmasterol-imprinted polymers using structurally similar polyphenols or phytosterols with the extent of binding determined from the capHPLC-ion trap ESI MS/MS data. This study documents with Pinot noir grape seed extracts and optimised solid-phase extraction protocols that the (E)-resveratrol-templated MIP enabled a very high recovery (99%) of the health-beneficial polyphenol (E)-resveratrol with co-purification of procyanidin and catechin/epicatechin. Further, the stigmasteryl-3-O-methacrylate-templated polymer resulted in high recovery (96%) of the phytosterol stigmasterol with co-purification of campesteryl glycoside. The results also demonstrate that rapid and high-resolution capHPLC-ESI MS/MS methods can be used as part of the work flow for selectivity optimisation and monitoring of the performance of MIPs intended for use in the solid-phase extraction of bioactive molecules with nutraceutical properties from agricultural waste streams.

Rice Bran Derived Bioactive Compounds Modulate Risk Factors of Cardiovascular Disease and Type 2 Diabetes Mellitus: An Updated Review.

Cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) are two chronic diseases that have claimed more lives globally than any other disease. Dietary supplementation of functional foods containing bioactive compounds is recognised to result in improvements in free-radical-mediated oxidative stress. Emerging evidence indicates that bioactive compounds derived from rice bran (RB) have therapeutic potential against cellular oxidative stress. This review aims to describe the mechanistic pathways behind CVD and T2DM development and the therapeutic potential of polyphenols derived from RB against these chronic diseases.

A GC-MS untargeted metabolomics approach for the classification of chemical differences in grape juices based on fungal pathogen.

Fungal bunch rot of grapes leads to production of detrimental flavour compounds, some of which are well characterised but others remain unidentified. The current study uses an untargeted metabolomics approach to classify volatile profiles of grape juices based on the presence of different fungal pathogens. Individual grape berries were inoculated with Botrytis cinerea, Penicillium expansum, Aspergillus niger or A. carbonarius. Grape bunches were inoculated and blended with healthy fruit, to provide 10% (w/w) infected juice. Juices from the above sample batches were analysed by GC/MS. PLS-DA of the normalised summed mass ions indicated sample classification according to pathogen. Compounds identified from those mass ion matrices that had high discriminative value for classification included 1,5-dimethylnaphthalene and several unidentified sesquiterpenes that were relatively higher in B. cinerea infected samples. A. niger and A. carbonarius samples were relatively higher in 2-(4-hexyl-2,5-dioxo-2,5-dihydrofuran-3-yl)acetic acid, while P. expansum samples were higher in γ-nonalactone and m-cresol.

Sequential molecularly imprinted solid-phase extraction methods for the analysis of resveratrol and other polyphenols.

Molecularly imprinted polymers (MIPs) templated with either the phytoalexin, (E)-resveratrol, or its structural analog, 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide, have been used in tandem for the sequential extraction of (E)-resveratrol from aqueous peanut meal extracts in high purity and in near quantitative yields. Re-processing of the (E)-resveratrol-depleted peanut meal extract with the 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide imprinted MIP yielded additional polyphenolic components, identified as A-type procyanidins. Tandem liquid chromatography-electrospray ionization mass spectrometry confirmed the identity and purity of the isolated products. This study documents the advantages of tandem approaches with MIPs for the solid phase extraction and analysis of multiple bioactive compounds present in complex biomass waste streams.

Selectivity mapping of the binding sites of (E)-resveratrol imprinted polymers using structurally diverse polyphenolic compounds present in Pinot noir grape skins.

This investigation describes a general procedure for the selectivity mapping of molecularly imprinted polymers, using (E)-resveratrol-imprinted polymers as the exemplar, and polyphenolic compounds present in Pinot noir grape skin extracts as the test compounds. The procedure is based on the analysis of samples generated before and after solid-phase extraction of (E)-resveratrol and other polyphenols contained within the Pinot noir grape skins using (E)-resveratrol-imprinted polymers. Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation tandem mass spectrometry (ESI MS/MS) was then employed for compound analysis and identification. Under optimised solid-phase extraction conditions, the (E)-resveratrol-imprinted polymer showed high binding affinity and selectivity towards (E)-resveratrol, whilst no resveratrol was bound by the corresponding non-imprinted polymer. In addition, quercetin-3-O-glucuronide and a dimer of catechin-methyl-5-furfuraldehyde, which share some structural features with (E)-resveratrol, were also bound by the (E)-resveratrol-imprinted polymer. Polyphenols that were non-specifically retained by both the imprinted and non-imprinted polymer were (+)-catechin, a B-type procyanidin and (-)-epicatechin. The compounds that did not bind to the (E)-resveratrol molecularly imprinted polymer had at least one of the following molecular characteristics in comparison to the (E)-resveratrol template: (i) different spatial arrangements of their phenolic hydroxyl groups, (ii) less than three or more than four phenolic hydroxyl groups, or (iii) contained a bulky substituent moiety. The results show that capillary RP-HPLC in conjunction with ESI MS/MS represent very useful techniques for mapping the selectivity of the binding sites of imprinted polymer. Moreover, this procedure permits performance monitoring of the characteristics of molecularly imprinted polymers intended for solid-phase extraction of bioactive and nutraceutical molecules from diverse agricultural waste sources.

Recovery of ergosterol from the medicinal mushroom, Ganoderma tsugae var. Janniae, with a molecularly imprinted polymer derived from a cleavable monomer-template composite.

A semi-covalent imprinting strategy has been developed for the synthesis of molecularly-imprinted polymers specific for the fungal sterol, ergosterol, a biological precursor of vitamin D2. This imprinting approach involved a novel post-synthesis cleavable monomer-template composite, namely ergosteryl methacrylate, and resulted in the formation of an imprinted polymer that selectively and efficiently recognized ergosterol through non-covalent interactions. The derived molecularly-imprinted polymer and the corresponding non-imprinted polymer were systematically evaluated for their selectivity towards ergosterol via static and dynamic binding studies using various ergosteryl esters (e.g. ergosteryl-cinnamate, -ferulate, -coumarate, -ferulate acetate and -acetate, respectively) as competitors. Moreover, the binding capacity of the molecularly imprinted polymer for ergosterol was enhanced when the sample loading conditions involved the use of partially aqueous solvent mixtures, such as acetonitrile/water (9:1 (v/v) or 8:2 (v/v)). These attributes were exploited in a solid-phase extraction format, whereby ergosterol was obtained with excellent recoveries from an extract of the fruiting body powder of the medicinal fungus Ganoderma tsugae var. Janniae.

A comparison of covalent and non-covalent imprinting strategies for the synthesis of stigmasterol imprinted polymers.

Non-covalent and covalent imprinting strategies have been investigated for the synthesis of stigmasterol imprinted polymers. The synthesized molecularly imprinted polymers (MIPs) were then evaluated for their recognition and selectivity towards stigmasterol via static and dynamic batch-binding assays and their performance measured against control non-imprinted polymers (NIPs). MIPs prepared using the conventional non-covalent imprinting method displayed little to no binding affinity for stigmasterol under various conditions. In contrast, the application of a covalent imprinting approach using the novel post-synthetically cleavable monomer-template composite stigmasteryl-3-O-methacrylate resulted in the fabrication of a MIP that successfully recognized stigmasterol in both organic and partially aqueous environments. The affinity and selectivity of the covalently prepared MIP was enhanced when undertaken in a partially aqueous environment consisting of an acetonitrile/water (9:1, v/v) solvent mixture. These features have been exploited in a molecularly imprinted solid-phase extraction (MISPE) format, wherein the preferential retention of stigmasterol (with an imprint factor of 12) was demonstrated with 99% recovery in comparison to cholesterol (imprint factor of 6) and ergosterol (imprint factor of 4) while in the presence of several closely related steryl analogues.

Rapid solid-phase extraction and analysis of resveratrol and other polyphenols in red wine.

Red wine has long been credited as a good source of health-beneficial antioxidants, including the bioactive polyphenols catechin, quercetin, and (E)-resveratrol. In this paper, we report the application of reusable molecularly imprinted polymers (MIPs) for the selective and robust solid-phase extraction (SPE) and rapid analysis of (E)-resveratrol (LOD=8.87×10(-3) mg/L, LOQ=2.94×10(-2) mg/L), along with a range of other polyphenols from an Australian Pinot noir red wine. Optimization of the molecularly imprinted solid-phase extraction (MISPE) protocol resulted in the significant enrichment of (E)-resveratrol and several structurally related polyphenols. These secondary metabolites were subsequently identified by RP-HPLC and µLC-ESI ion trap MS/MS methods. The developed MISPE protocol employed low volumes of environmentally benign solvents selected according to the Green Chemistry principles, and resulted in the recovery of 99% of the total (E)-resveratrol present. These results further demonstrate the potential of generic protocols for the analysis of target compound with health beneficial properties within the food and nutraceutical industries using tailor-made MIPs.

Enrichment of (E)-resveratrol from peanut byproduct with molecularly imprinted polymers.

Molecularly imprinted solid phase extraction (MISPE) has been employed to isolate and concentrate bioactive polyphenols from peanut press waste. To this end, a molecularly imprinted polymer (MIP) templated with the phytoalexin (E)-resveratrol has been prepared via self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Subsequent molecular interrogation of the MIP binding sites demonstrated preferential structural selectivity for (E)-resveratrol with respect to other structurally related naturally occurring compounds. This selectivity was subsequently exploited to achieve substantial sample cleanup of peanut press waste under aqueous conditions with significant enrichment of (E)-resveratrol (>60 fold) requiring minimal sample preparation.

Preparation of molecularly imprinted polymers for the selective recognition of the bioactive polyphenol, (E)-resveratrol.

(E)-Resveratrol imprinted polymers have been rationally designed with the aid of molecular modelling and NMR spectroscopic titration techniques to determine the optimal ratio of the template to functional monomer for polymer formation. Based on this approach, (E)-resveratrol imprinted polymers were prepared via non-covalent self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Polymerisation in the presence of a cross-linker resulted in rigid block copolymers that had selective capacities towards (E)-resveratrol (e.g. 14 µmol/g) when compared to the non-imprinted reference polymer. The selectivity of these MIPs was also examined using several structurally related polyphenolic compounds to determine the influence of polyphenolic hydroxyl number and position on binding and molecular recognition.