Mitchell L. Wise

Avenanthramides are bioavailable and accumulate in hepatic, cardiac, and skeletal muscle tissue following oral gavage in rats.

Avenanthramides (AVA), polyphenols found exclusively in oats ( Avena sativa L.), may play a role in the anti-inflammatory and antiatherogenic activity of oats. The bioavailability of AVA has been demonstrated previously, but its distribution at the organ and tissue level and the extent of conjugation following ingestion have been unexplored. Synthetic AVA was administered to 24 rats by oral gavage, whereas 6 control rats received saline. AVA concentrations were measured via HPLC in plasma, liver, heart, and gastrocnemius (GAS) obtained over a 12 h period (0, 2, 4, 12 h; n = 6 at each time point). Samples were extracted with and without glucuronidase-sulfatase to assess the level of conjugation. We conclude that AVA are bioavailable to the blood circulation following oral ingestion in the rat and reach peripheral tissues where they can be taken up by various organs differentially. With AVA remaining in the organs for up to 12 h, it seems possible to maintain an increased level of AVA in the rat via repeated feedings.

In vitro total antioxidant capacity and anti-inflammatory activity of three common oat-derived avenanthramides

To better understand mechanisms underlying the health benefits of oats, the free radical scavenging capacities of oat avenanthramides 2c, 2f, and 2p and their ability to inhibit NF-κB activation were evaluated. The antioxidant capacities of 2c, 2f, and 2p against peroxyl radicals, hydroxyl radicals, superoxide anion, singlet oxygen, and peroxynitrite were determined by using ORAC, HORAC, SORAC, SOAC, and NORAC assays, respectively. The total antioxidant capacity of 2c was approximately 1.5-fold those of 2f and 2p. Total antioxidant capacity was primarily attributable to SORAC and ORAC for 2c (>77%, p<0.05), and to ORAC and SOAC for 2f. ORAC accounted for approximately 32% of total antioxidant capacity in 2p. EC50 values for inhibiting TNF-α-induced NF-κB activation in C2C12 cells were 64.3, 29.3, and 9.10 μM for 2c, 2f, and 2p, respectively. Differences in antioxidant capacities and ability to inhibit NF-κB among the avenanthramides could be ascribed to structural variations.

Effect of Chemical Systemic Acquired Resistance Elicitors on Avenanthramide Biosynthesis in Oat (Avena sativa)

Oats produce a group of phenolic antioxidants termed avenanthramides. These metabolites are, among food crops, unique to oats and have shown, in experimental systems, certain desirable nutritional characteristics such as inhibiting atherosclerotic plaque formation and reducing inflammation. Avenanthramides occur in both the leaves and grain of oat. In the leaves they are expressed as phytoalexins in response to crown rust (Puccina coronata) infection. The experiments reported here demonstrate that avenanthramide levels in vegetative tissue can be enhanced by treatment with benzothiadiazole (BTH), an agrochemical formulated to elicit systemic acquired resistance (SAR). The response to BTH was dramatically stronger than those produced with salicylic acid treatment. The roots of BTH treated plants also showed a smaller but distinct increase in avenanthramides. The dynamics of the root avenanthramide increase was substantially slower than that observed in the leaves, suggesting that avenanthramides might be transported from the leaves.

Phytochemicals Attenuating Aberrant Activation of β-Catenin in Cancer Cells

Phytochemicals are a rich source of chemoprevention agents but their effects on modulating the Wnt/β-catenin signaling pathway have remained largely uninvestigated. Aberrantly activated Wnt signaling can result in the abnormal stabilization of β-catenin, a key causative step in a broad spectrum of cancers. Here we report the modulation of lithium chloride-activated canonical Wnt/β-catenin signaling by phytochemicals that have antioxidant, anti-inflammatory or chemopreventive properties. The compounds were first screened with a cervical cancer-derived stable Wnt signaling reporter HeLa cell line. Positive hits were subsequently evaluated for β-catenin degradation, suppression of β-catenin nuclear localization and down-regulation of downstream oncogenic targets of Wnt/β-catenin pathway. Our study shows a novel degradation path of β-catenin protein in HeLa cells by Avenanthramide 2p (a polyphenol) and Triptolide (a diterpene triepoxide), respectively from oats and a Chinese medicinal plant. The findings present Avenanthramide 2p as a potential chemopreventive dietary compound that merits further study using in vivo models of cancers; they also provide a new perspective on the mechanism of action of Triptolide.

Oat avenanthramides induce heme oxygenase-1 expression via Nrf2-mediated signaling in HK-2 cells

SCOPE Numerous studies have shown that avenanthramides (AVAs), unique compounds found in oats, are strong antioxidants, though the mechanism of action remains unclear. Here, we investigated whether AVAs affect heme oxygenase-1 (HO-1) expression through the activation of Nrf2 translocation. METHODS AND RESULTS We investigated the effects AVA 2c, 2f, and 2p on HK-2 cells, and found that AVAs could significantly increase HO-1 expression in both a dose- and time-dependent manner. Furthermore, we found that AVA-induced HO-1 expression is mediated by Nrf2 translocation. The addition of N-acetylcysteine (NAC), but not specific inhibitors of p38 (SB202190), PI3K (LY294002), and MEK1 (PD098059) attenuated AVA-induced HO-1 expression, demonstrating an important role for reactive oxygen species, but not PI3K or MAPK activation, in activating the HO-1 pathway. Moreover, hydrogenation of the double bond of the functional α,β-unsaturated carbonyl group of AVAs eliminated their effects on HO-1 expression, suggesting that this group is crucial for the antioxidant activity of AVAs. CONCLUSION Our results suggest a novel mechanism whereby AVAs exert an antioxidant function on human health. Further investigation of these markers in human is warranted to explore the beneficial health effects of whole grain oat intake.

Barley and Oat β-Glucan Content Measured by Calcofluor Fluorescence in a Microplate Assay

Of the various components of barley and oats, one constituent of the cereal cell wall, (1→3,1→4)-β-D-glucan (β-glucan), is important to a wide range of interests. Malting barley breeders strive to develop lines that modify well, yielding malts with low soluble β-glucan levels in Congress worts (less than the American Malting Barley Association guidelines of 100–120 ppm) (http:// www.ambainc.org/ni/Guidelines.pdf) to meet brewers’ needs for low-viscosity/high-filterability wort in the brewhouse. Similarly, barley grain with reduced β-glucan content is more suitable as poultry feed than barley because of a higher β-glucan content (Bergh et al 1999) due to feed digestibility and related issues. In contrast, the U.S. Food and Drug Administration recognizes benefits to human coronary health from including soluble β-glucan in the diet. β-glucan from oat and barley reduced both blood cholesterol levels as well as the glycemic index of foods (Wood 2002, Baik and Ullrich 2008). With the increased interest in barley β-glucan as a food component, there is a need for convenient, cost-effective procedures to analyze the grain for β-glucan content. Probably the most widelyused method for analyzing barley (and oat) β-glucan content in research laboratories is the enzymatic method (McCleary and Codd, 1991) used in Approved Method 32-23 (AACC International 2000). Essentially, the mixed-linkage, linear β-glucan polymers are extracted from grain flour, hydrolyzed to monosaccharides by sequential enzymatic reactions (endo-(1→3,1→4)-βD-glucan 4-glucanohydrolase (lichenase), followed by β-glucosidase), and finally coupled to color generation using glucose oxidase and a chromogenic substrate. Kits providing the principle reagents for this method are commercially available from Megazyme (www.megazyme.com) and require only relatively simple laboratory instruments (spectrophotometer, centrifuge, pipettes, water bath, etc.). However, the enzymatic method does include a number of manipulations, increasing the labor requirement for the method. Also, the costs for the reagent kits can be significant for large sample sets, making use of the kits less feasible for routine high-throughput analysis. A second method for β-glucan determination measures fluorescence increase upon the binding of Calcofluor to high molecular weight β-glucans in grain extracts (Li et al 2008). Commonly, Calcofluor methods for β-glucan analysis involve use of flow injection analysis (FIA). In FIA, an autosampler is used to inject a fixed volume of sample into a stream of buffer or water, which is subsequently mixed with a second stream of Calcofluor reagent. A fluorescence detector measures the increase in fluorescence in the sample bolus in the combined streams due to interaction of the sample β-glucan with the Calcofluor reagent. The sample βglucan is quantified by comparing sample peak area or peak height with a standard curve generated from injections of a dilution series of β-glucan standards. This protocol, while procedurally simpler than the enzymatic method, requires more extensive instrumentation in its most common implementation. Dedicated commercial instrumentation for such flow injection analysis or comparable segmented flow analysis can cost in excess of

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

100,000. Home-built versions of such FIA instrumentation can be more affordable, although still requiring significant initial expense and maintenance costs. As a result, the FIA instrumentation is commonly found only in high-volume dedicated quality analysis laboratories. Apart from the cost of instrumentation, the Calcofluor methodology requires relatively inexpensive reagents and significantly less labor. A third alternative method, using a dye-binding (colorimetric) procedure analogous to the Calcofluor method that can be read in a microplate spectrophotometer, has been adopted as EBC Method 4.16.3 High Molecular Weight β-Glucan Content of Wort: Colorimetric Method (see Freijee 2005). However, the kit for the colorimetric method (GlucaTest from NovaBiotec Dr. Fechter GmbH, www.novabiotec.de) is not commercially available in North America at the present time. In this note, we show that the Calcofluor method can be adapted to a fluorescent microplate reader, providing a simple means of measuring β-glucan in cereal grains and malt, using inexpensive reagents, and affordable, commonly available instrumentation.

Avenanthramides: Chemistry and Biosynthesis

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.