Extraction, distribution, characterization and bioactivities of phenolics in millet grains

Chandrasekara, Gamage Anoma Priyanganie (2011) Extraction, distribution, characterization and bioactivities of phenolics in millet grains. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Seven millet varieties grown in Sri Lanka (proso, foxtail, finger (Ravi), finger (local), kodo, and little) and India (pearl) were used in this study. Soluble and bound phenolics obtained by solvent extraction were used to evaluate antioxidant, DNA scission and enzyme inhibitory and antiproliferative activities by several chemical and biological methods. Distribution of phenolics and their antioxidant activity was assessed in dehulled grains and hulls separated by air classification. Phenolic compounds in free, soluble conjugates and insoluble bound fractions were identified and quantified. In addition, accessibility of phenolics in the digestive tract was determined using a simulated in vitro digestion model and colonic fermentation. The contents of total phenolics (TPC),total flavonoids (TFC) and proanthocyanidins (PC) as well as scavenging activities of test compounds were evaluated using superoxide, hydroxyl, peroxyl, 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical, H₂O₂, and singlet oxygen. Different concentrations of extract were used to determine their IC₅₀ values. In addition, the ferrous chelating and reducing activities of extracts were measured. The highest phenolic content of soluble phenolics of millet grains was obtained with 70% (v/v) acetone for 25 minutes under reflux conditions. The TPC of soluble and insoluble bound phenolic fractions of millet varieties were 7.2 - 32.4 and 2.2-81.6 μmol ferulic acid eq/g defatted meal, respectively. This demonstrates that bound phenolics are important contributors to antioxidant potential of millets. Kodo millet showed the highest soluble phenolic content followed by finger (local), finger (Ravi), little, foxtail, pearl and proso millets. Millets with dark brown pigmented testa and pericarp possessed a higher soluble phenolic content than those with white or yellow testa and pericarp. The TFC of soluble and insoluble extracts were 1.2-33.7 and 0.3-4.5 μmol catechin eq (CE)/g defatted meal, respectively. The soluble extracts of finger (local) millet had the highest PC (311.28 μmol CE/g defatted meal). All varieties exhibited effective radical, H₂O₂ and singlet oxygen inhibition, reducing activity as well as ferrous ion chelating power which varied according to the assay employed and the variety used. Several hydroxybenzoic and hydroxycinnamic acids and flavonoids were identified and quantified by HPLC analysis. In general, ferulic and ρ-coumaric were found to be the major phenolic acids followed by protocatechuic, caffeic, sinapic, ρ-hydroxybenzoic, gentisic, and vanillic acids and their contents depended on the variety and the form present in the grain matrix. Flavonoids identified were flavones, flavan-3-ol monomers and dimers, and flavanonols. All seven varieties effectively inhibited oxidation in a pork model system, and stripped corn oil. At a concentration of 0.5 mg/ml all extracts inhibited oxidation by 16-88% in the linoleic acid emulsion system. All phenolic extracts inhibited Cu (II)-induced human LDL and liposome oxidation and DNA scission. Millet extracts at 0.05 mg/mL inhibited LDL cholesterol oxidation by 1-41% and exhibited a dose-dependent inhibition of DNA scission. The antiprolifeative studies showed that kodo millet extract inhibited HT-29 cells proliferation in the range of 75-100% after 4 days of incubation. The hulls from dehulled millets had higher TPC and antioxidant activity compared to their whole, dehulled and cooked grain counterparts. The phenolic extracts of dehulled millet grains, following in vitro enzymic digestion under simulated gastrointestinal conditions and colonic fermentation, showed effective antioxidant activity and were accessible under physiological conditions.

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