Identification of Two Anthocyanidin Reductase Genes and Three Red-Brown Soybean Accessions with Reduced <i>Anthocyanidin Reductase 1</i> mRNA, Activity, and Seed Coat Proanthocyanidin Amounts

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (<i>ANR1</i> and <i>ANR2</i>) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (−)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced <i>ANR1</i> gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced <i>ANR1</i> activity in vitro. These results suggest that defects in the <i>ANR1</i> gene can be associated with red-brown soybean grain color. These results suggest that suppressing <i>ANR1</i> gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains

Metabolites analysis of selected hot water extracts based on stimulating glucose uptake activity.

<p>PCA scores (5A), OPLS-DA scores (5B) S-plot (5C) of UPLC-QTOF metabolome of HWE of Cree plants. Those stimulating glucose transport (<i>R</i>. <i>gromenlandicum</i>, <i>R</i>. <i>tomentosum</i>, and <i>S</i>. <i>purpurea</i>) grouped seperately from inactive ones (<i>K</i>. <i>angusfolia</i> was found to be an outlier and was excluded from the process). The 95% confidence interval for each group is given. In the S-plot, the metabolome of the active plants was compared with the inactive plants to identify discriminant biomarkers with <i>K</i>. <i>angustifolia</i> excluded. In Fig 5D, quercetin 3-O-α-L-arabinopyranoside (Q3A, 50 μM) stimulated GU in C2C12 cells, 140% compared with vehicle control.</p

Effect of selected extracts on the modulation of insulin and AMPK pathway in H4IIE cells.

<p>The cells were treated for 18h with vehicle control, insulin (100 nM), EE or HWE plants extracts. Metformin (400 μM, 18 hours), AICAR (2 mM, 2 hours) or insulin (100 nM, 18 hours) was used as positive controls for AMPK or insulin pathways, respectively. Phosphorylation of AMPK (4A) and of Akt (4D) was measured by western blot and results (4B, 4E) expressed as means ± SE for 3 separate experiments, normalized to the vehicle-treated condition. # Denotes EE samples significantly different from vehicle control (p < 0.05), one-way ANOVA and post hoc Dunnett's test. $ Denotes HWE samples significantly different from vehicle control (p < 0.05), one-way ANOVA and post hoc Dunnett's test. Correlation results (4C) were analyzed by linear regression and the equation was y = -45.418x + 285.3 (R = 0.48, <i>p</i> < 0.05).</p

Effect of selected extracts on expression of GLUT4, Insulin and AMPK pathway in C2C12 cells.

<p>Cells were differentiated and treated for 18 hours with vehicle or with EE and HWE of the 5 selected plants. Metformin (400 μM, 18 hours), AICAR (2 mM, 2 hours) or insulin (100 nM, 30 min) was applied as positive controls for the AMPK or insulin pathways, respectively. GLUT4 (3A), phosphorylation of AMPK (3D), phosphorylation of Akt (3G) were measured by western blot and results (3B, 3E, 3H) were expressed as means ± SE for 3 separate experiments, normalized to the vehicle-treated condition. # Denotes EE samples significantly different from vehicle control (p < 0.05), one-way ANOVA and post hoc Dunnett's test. $ Denotes HWE samples significantly different from vehicle control (<i>p</i> < 0.05), one-way ANOVA and post hoc Dunnett's test. * (<i>p</i> < 0.05), **(<i>p</i> < 0.01) and *** (<i>p</i> < 0.001) denote significant differences between EE and HWE counterpart, two-way ANOVA. Correlation results (3C, 3F) were analyzed by linear regression and the equations were y = 32.06x + 52.95 (R = 0.80, <i>p</i> < 0.05), y = 11.602x + 84.825 (R = 0.46, <i>p</i> < 0.05), respectively.</p

List of investigated plant species and the concentrations of the extracts tested in cells.

<p>List of investigated plant species and the concentrations of the extracts tested in cells.</p

Effects of extracts on muscle glucose transport.

<p>C2C12 skeletal muscle cells were treated with either 0.1% DMSO (vehicle), Metformin (400 μM), EE and HWE (at concentrations described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135721#pone.0135721.t001" target="_blank">Table 1</a>) for 18 hours, or with insulin (100 nM) for 30 min. Results represent means ± SE for 3 separate experiments, normalized to the vehicle-treated condition. # Denotes EE samples significantly different from vehicle control (<i>p</i> < 0.05), one-way ANOVA and post hoc Dunnett's test. $ Denotes HWE samples significantly different from vehicle control (p < 0.05), one-way ANOVA and post hoc Dunnett's test. * (<i>p</i> < 0.05), **(<i>p</i> < 0.01) and *** (<i>p</i> < 0.001) denote significant differences between respective EE and HWE counterparts, two-way ANOVA.</p

Antidiabetic Compounds from <i>Sarracenia purpurea</i> Used Traditionally by the Eeyou Istchee Cree First Nation

Through ethnobotanical surveys, the CIHR Team in Aboriginal Antidiabetic Medicines identified 17 boreal forest plants stemming from the pharmacopeia of the Cree First Nations of Eeyou Istchee (James Bay region of Northern Quebec) that were used traditionally against diabetes symptoms. The leaves of <i>Sarracenia purpurea</i> (pitcher plant), one of the identified Cree plants, exhibited marked antidiabetic activity in vitro by stimulating glucose uptake in C2C12 mouse muscle cells and by reducing glucose production in H4IIE rat liver cells. Fractionation guided by glucose uptake in C2C12 cells resulted in the isolation of 11 compounds from this plant extract, including a new phenolic glycoside, flavonoid glycosides, and iridoids. Compounds <b>6</b> (isorhamnetin-3-<i>O</i>-glucoside), <b>8</b> [kaempferol-3-<i>O</i>-(6″-caffeoylglucoside], and <b>11</b> (quercetin-3-<i>O</i>-galactoside) potentiated glucose uptake in vitro, which suggests they represent active principles of <i>S. purpurea</i> (EC₅₀ values of 18.5, 13.8, and 60.5 μM, respectively). This is the first report of potentiation of glucose uptake by compounds <b>6</b> and <b>8</b>, while compound <b>11</b> (isolated from <i>Vaccinium vitis</i>) was previously shown to enhance glucose uptake. Treatment of H4IIE liver cells with the new compound <b>1</b>, 6′-<i>O</i>-caffeoylgoodyeroside, decreased hepatic glucose production by reducing glucose-6-phosphatase enzymatic activity (IC₅₀ = 13.6 μM), which would contribute to lowering glycemia and to the antidiabetic potential of <i>S. purpurea.</i