Hulled Reins and hulless Lamont oats were dehulled and/or sequentially abraded to produce ten pearling fines and corresponding pearled kernels. Contents of nutrients (protein, oil, starch, beta-glucan, ash and other carbohydrates) and avenanthramides (AVA) 2p, 2c, 2f, and 5p in processing fractions and starting grains were measured. Results show that distribution patterns of nutrients varied with individual nutrients, but those of AVAs varied with variety and individual AVAs. In both varieties, from the surface to inner endosperms, protein and oil increased then decreased; ash and other carbohydrates decreased; starch increased; and beta-glucan unchanged except for the surface area. In Lamont oat, the four AVAs decreased, but in Reins oat, AVA 2p decreased while 2c, 2f and 5p increased, then decreased. Compared to whole grain, pearled oats not only contained lower AVAs, protein, oil, ash, and other carbohydrates and higher beta-glucan and starch but also had a different AVA composition.

Oats produce a group of natural products termed avenanthramides. These compounds are produced in both the vegetative tissue and the grain. They are produced in leaf tissue in response to crown rust infection and by chemical plant defense activators and likely other environmental stresses. Grain avenanthramide production tends to be constitutive but concentrations are highly variable and strongly influenced by environmental conditions. In this paper, we report the effect of a plant defense activator [benzothiadiazole (BTH)] on the temporal expression and tissue distribution of avenanthramides in the leaf, stem, root, panicle stem, glumes, lemma/palea, and filling grain in the oat plant. Hydroxycinnamoyl-CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT, a member of the BAHD acyltransferase family and the final enzyme in the biosynthetic pathway to the avenanthramides) activity is also determined in these tissues, as well as the relative expression ratios of HHT mRNA resulting from BTH treatment. Evidence for phloem transport of the avenanthramides is also presented. In summary, following BTH treatment, leaf tissue is the predominant location for avenanthramide biosynthesis. However, significant amounts are also found in the upper and lower stems, roots, panicle stems, and glumes. The lemma/palea and filling grain contained demonstrable but substantially lower amounts of avenanthramides. Avenanthramides were also detected in the phloem sap, indicating a source to sink transport of these metabolites following BTH treatment.

Oats, in addition to cholesterol-lowering properties, contain unique antioxidants called avenanthramides (Avns), which inhibit both inflammatory cytokines and adhesion molecules in endothelial cells in culture. This study evaluated the effects of Avns of oats on atherosclerosis in Ldlr-/- mice, one of the most commonly used atherosclerosis mouse models with their similar cholesterol distributions to humans. The Ldlr-/- mice were fed a low fat, high fat, high fat containing regular oat brans with low levels of Avns (HFLA), or high fat containing regular oat brans with high levels of Avns (HFHA) diet. After 16 weeks of intervention, blood cholesterol and extent of aortic lesions were evaluated. We found that both oat-based diets reduced high fat diet-induced atheroma lesions in the aortic valve (p < 0.01). Furthermore, the effects of oat-based diets are more profound in HFHA mice than mice fed HFLA. Total plasma cholesterol levels were similarly reduced in both oat-supplemented mice. We concluded that oat bran diets reduce atheroma lesions and higher levels of Avns further reduce aortic lesions compared to regular oat bran. These preliminary in vivo data indicate that consumption of oats bran, with high Avns, has demonstrable beneficial effects on prevention of cardiovascular disease.

ObjectivesAvenanthramides (AVA) are a group of diphenolic acids that are found only in oats and have anti‐inflammatory and antioxidant effects. Although previous publications have reported AVA are bioavailable in humans, our study is the first to examine whether AVA are bioavailable in humans after acute consumption of oat cookies made with natural and non‐treated oat flour. We examined the metabolic fate of orally ingested oat AVA by measuring plasma AVA concentrations and their pharmacological characteristics.MethodsSixteen male and female non‐obese subjects were enrolled in a randomized, crossover trial. Subjects received three cookies made with oat flour containing high (229.6 mg/kg, H‐AVA) or low (32.7 mg/kg, L‐AVA) AVA on separate occasions. The H‐AVA cookie (7.2 mg/cookie; 21.6 mg total) contained 5.09 mg AVA‐A, 8.67 mg AVA‐B and 7.85 mg AVA‐C, whereas the L‐AVA (1.03 mg/cookie; 3.09 mg total) contained 0.57 mg AVA‐A, 0.95 mg AVA‐B and 1.55 mg AVA‐C. Subjects consumed the cookies in the morning after 10 h fast and blood samples were collected at 0 (baseline), 0.5, 1, 2, 3, 5 and 10h. Plasma total (conjugated and free) AVA were quantified with Ultra Performance Liquid Chromatography Quadrupole Time‐of‐Flight Mass Spectrometer (UPLC Q‐TOF‐MS), and pharmacokinetic (PK) parameters were calculated and presented as mean±SD. Half life time (T1/2) was calculated through logarithmic transformation and elimination constant (Kel) was calculated using equation Kel=0.693/T1/2. At least three time points in elimination phase were required to calculate T1/2. All plasma concentrations used for estimation of PK parameters were above assay limit of quantitation.ResultsAVA‐A, ‐B and ‐C were present at the peak concentration (Tmax) in plasma at 2.50±1.2 h, 2.04±0.9 h and 2.29±1.0 h in H‐AVA and at 1.80±1.3 h, 1.50±1.5 h and 1.32±1.0 h in L‐AVA, respectively. Maximal plasma concentrations (Cmax) for AVA‐A, ‐B and ‐C were 8.39±4.2 ng/ml, 8.44±2.3 ng/ml and 4.26±2.0 ng/ml in H‐AVA, and 1.98±1.3 ng/ml, 2.43±1.1 ng/ml and 1.33±0.7 ng/ml in L‐AVA, respectively (P<0.001). H‐AVA cookies led to significantly higher AVA‐A and ‐B than ‐C concentrations (P<0.05). Half‐life (T1/2), the time taken to fall to half initial value was 2.16±0.6 h, 4.23±0.8 h and 2.71±1.2 h in H‐AVA, and 2.44±0.8 h, 4.60±2.7 h and 2.87±1.4 h in L‐AVA, respectively (P<0.05, AVA‐B vs. AVA‐A or AVA‐C in H‐AVA).ConclusionsAVA found naturally in oats are bioavailable in humans. AVA‐B has the slowest elimination rate and highest half‐life compared to AVA‐A and AVA‐C, while AVA‐C has the lowest absorption rate.Support or Funding InformationThis study is sponsored by PepsiCo Nutrition.

Avenanthramides are phenolic antioxidants produced in oat (Avena sativa L.) in response to fungal infection and, as shown in greenhouse experiments, by treatment with plant defense activators, such as benzothiadiazole (BTH). Presented here are the results of application of BTH under field conditions. Field trials were conducted in 2013 using four application rates, approximating manufacturer's recommendations, on two oat cultivars and in 2014 using three application rates on three oat cultivars. The results showed a subtle but demonstrable effect of increased avenanthramide production in vegetative tissue and mature grain and enhanced resistance to crown rust infection. Gene expression of pathogenesis related protein‐1 (PR‐1), phenylalanine ammonia lyase (PAL) and hydroxycinnamoyl‐CoA:hydroxyanthranilate hydroxycinnamoyl‐CoA transferase (HHT) was also monitored by RT‐qPCR. Both PR‐1 and PAL expression were demonstrably up‐regulated in the field trials; HHT was not. However, greenhouse experiments showed all three genes to be elicited with 63‐, 9‐, and 3‐fold increases, respectively. Thus, field application of plant defense activators can effectively enhance crown rust resistance and avenanthramide production in oat by up‐regulating certain metabolic pathways; their practical application, however, will require substantial investigative efforts.

The consumption of oatmeal and oat bran has been shown to reduce total and low density lipoprotein (LDL) cholesterol levels in plasma, which are major risk factors for coronary heart disease (CHD). Oats, in addition to containing soluble fiber (β‐glucan), are a good source of protein and lipids as well as several antioxidants including vitamin E, phytic acid, phenolics, and Avenanthramides (Avns), unique soluble bioactive compounds present only in oats. Avns can inhibit inflammation in arterial endothelium by suppressing NF‐κB, which regulates expression of several inflammatory cytokines. We have reported that Avns suppressed endothelial expression of chemokines (MCP‐1, IL‐8), pro‐inflammatory cytokines (IL‐1 and IL‐6) and several adhesion molecules, all of which suggest oats’ potential anti‐inflammatory and anti‐atherogenic properties. In agreement with our in vitro observations, supplementing a high fat diet of LDLr−/− mice with feed containing 27% or 40% oats significantly reduced aortic lesion development. However, this effect of an oat‐based diet cannot be attributed only to its cholesterol lowering effect; rather, it is due to the additive/synergistic anti‐inflammatory interaction with oat Avns. To elucidate Avns’ contribution to the inhibition of atherogenesis, we supplemented a high fat diet of LDLr−/− mice with regular oats or false‐malted oats for 16 wks. Avns’ concentration as measured by LC/MS‐MS in the regular oat diet was 10 ppm; in the false‐malted oat diet, it was 451 ppm. Food intake was not different between the groups, but high fat‐fed mice gained body weight significantly (p<0.05). Both oat‐based diets (malted or false‐malted) significantly (p<0.05) reduced high fat diet‐induced atheroma lesions in the aortic tricuspid valve, but there was no difference in the extent of lesions between these two groups. However, LDLr−/− mice fed a high fat diet containing false‐malted oats with high levels of Avns had significantly (p<0.05) lower numbers of lesions in the descending aorta than mice fed a low fat diet, a high fat diet, or a high fat diet supplemented with regular oats. A non‐significant trend toward reduction of expression of VCAM‐1 in the lesions of aortic valves of mice fed high fat diet containing high levels of Avns was observed. Importantly, reduction of blood total cholesterol levels was the same in both oat‐supplemented groups, suggesting that false‐malted oats, with their higher concentrations of Avns, contributed more to the reduction of aortic lesions. These preliminary in vivo data suggest that consuming oats, especially false‐malted oats, may be beneficial in the prevention of cardiovascular disease.Support or Funding InformationSupported by USDA‐ARS agreement #58‐1950‐0‐014

Laboratory and human studies have shown that avenanthramides (AVAs), unique compounds found in oats, are strong antioxidants. However, their specific mechanism of action remains unclear. The objective was to investigate the abilities of the three major AVAs in oats (2c, 2p, 2f) to activate Nrf2 nuclear translocation leading to the activation of phase II detoxification genes in vitro. The three AVAs significantly increased heme oxygenase‐1 (HO‐1) expression in HK‐2 cells in both a dose‐ and time‐dependent manner. Mechanistic studies show that HO‐1 expression induced by AVAs is mediated via Nrf2 translocation. All three AVAs stimulated Nrf2 nuclear translocation in both a dose‐ and time‐dependent manner. The addition of N‐acetylcysteine (NAC) suppressed the HO‐1 expression induced by 2c, 2f, and 2p, establishing that ROS plays a role on the up‐regulation of HO‐1 by AVAs. Pretreatment of cells with inhibitors of p38, PI3K, and MEK1 did not attenuate AVA‐induced HO‐1 expression, suggesting that HO‐1 expression is not associated with PI3K or MAPK activation. Hydrogenation of the double bond of the functional α,β‐unsaturated carbonyl group of AVAs eliminated their effects on HO‐1 expression, suggesting a role for the functional α,β‐unsaturated carbonyl group in the mechanism of action of AVAs. These results show the ability of AVAs to induce HO‐1 expression in HK‐2 cells. This action appears to be mediated by Nrf2 activation and is not associated with PI3K or MAPK activation.

The Genome-Wide Association Studies approach was used to detect Quantitative Trait Loci associated with tocochromanol concentrations using a panel of 1,466 barley accessions. All major tocochromanol types- α-, β-, δ-, γ-tocopherol and tocotrienol- were assayed. We found 13 single nucleotide polymorphisms associated with the concentration of one or more of these tocochromanol forms in barley, seven of which were within 2 cM of sequences homologous to cloned genes associated with tocochromanol production in barley and/or other plants. These associations confirmed a prior report based on bi-parental QTL mapping. This knowledge will aid future efforts to better understand the role of tocochromanols in barley, with specific reference to abiotic stress resistance. It will also be useful in developing barley varieties with higher tocochromanol concentrations, although at current recommended daily consumption amounts, barley would not be an effective sole source of vitamin E. However, it could be an important contributor in the context of whole grains in a balanced diet.

Higher concentrations of anthocyanins in vegetables are important for attractive appearance and may offer health benefits for consumers. The red color of onion (Allium cepa) bulbs is due primarily to the accumulation of anthocyanins. The goal of this study was to identify chromosome regions that significantly affect concentrations of anthocyanins and soluble solids in onion bulbs. Segregating haploid plants from the cross of yellow (OH1) and red (5225) inbreds were asexually propagated and bulbs were produced in replicated trials across three environments. Concentrations of soluble solids were measured at 30 days after harvest and quantitative analyses revealed a significant region on chromosome 5. Analyses using a binary model for segregation of red versus yellow bulbs revealed a significant region on chromosome 7 and two regions linked in repulsion phase on chromosome 4. These results are consistent with the complementary two-locus model previously proposed to control red versus yellow bulb colors in onion. The region on chromosome 7 mapped to the same location as the R locus, and the regions on chromosome 4 may correspond to the L and L2 loci. The intensity of red bulb color was assessed visually by a panel of evaluators and by amounts of anthocyanins [peonidin 3-glucoside and cyanidin 3-(6″-malonoyl-laminaribioside)] measured by high-performance liquid chromatography. Quantitative analyses using a normal model revealed significant quantitative trait loci on chromosomes 1, 4 and 8 affecting anthocyanin concentrations, and yellow onion contributed beneficial genetic variation to enhance red bulb color. Significant correlations were observed between these anthocyanin concentrations and panel scores, indicating that visual selection should be effective for increasing anthocyanin levels in onion bulbs. These selected populations may be more attractive to consumers, potentially provide health benefits from increased anthocyanin consumption, and be a source of natural colorants.

A renewed interest in breeding barley specifically for food end-uses is being driven by increased consumer interest in healthier foods. We conducted association mapping on physicochemical properties of barley that play a role in food quality and processing including grain hardness, polyphenol oxidase activity, total phenolics, amylose content, and β-glucan. We used 3,069 elite two-row and six-row spring barley breeding lines from eight US breeding programs and 2,041 SNP markers for association mapping. Marker–trait associations were identified using a mixed model that incorporated population structure and kinship. We detected two previously identified QTL for grain hardness on chromosome 2H in the telomeric region of 5H along with two novel regions on 4H and 6H. For amylose content, we detected marker–trait associations on 7H from 0.63 to 30 cM. We detected four regions on chromosomes 1H, 2H, 3H, and 4H associated with polyphenol oxidase activity. The chromosome 2H region co-localized with the two previously mapped polyphenol oxidase genes PPO1 and PPO2, and the regions on chromosomes 1H, 3H, and 4H QTL were novel. For total phenolics, we identified three significant regions on 3H, 4H, and 5H. Two regions on 2H and 7H were associated with β-glucan. Both previously identified and novel QTL are segregating in elite US breeding germplasm. Only three of the 24 SNPs that were associated with traits using either the two-row or six-row mapping panel were identified in both panels. Nine SNPs were detected in the individual two-row or six-row panels that were not detected in the analysis using the complete panel and accounting for population structure. The distribution of favorable alleles at these loci that underpin food quality across the breeding programs suggests several strategies to use markers to improve barley for food uses.

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.

Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.

Phytochemicals are rich source of chemoprevention agents but their effects on modulating the Wnt/β‐catenin signaling pathway and attenuating cancer stem cell proliferation 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, antiinflammatory 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 present in wholegrain oats. To further investigate the underlying mechanism of the action, we isolated cervical cancer stem cells and confirmed multiple stem cell surface biomarkers using fluorescence‐activated cell sorting analysis. Our continuing and future goals are to evaluate effects of Avenanthramides and other phytochemicals on growth of isolated cancer stem cell like populations as well as in animal models of carcinogenesis and metastasis. Research support came from National Institutes of Health and SD Agriculture Experiment Station.

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.

Oats are gaining increasing scientific and public interest for their purported antioxidant-associated health benefits. Most reported studies focused on specific oat extracts or particular oat components, such as β-glucans, tocols (vitamin E), or avenanthramides. Studies on whole oats with respect to antioxidant and anti-inflammatory activities are still lacking. Here the antioxidant and anti-inflammatory activities from whole oat groats of seven common varieties were evaluated. All oat varieties had very similar oxygen radical absorption capacity compared with other whole grains. In an anti-inflammatory assay, oat variety CDC Dancer inhibited tumor necrosis factor-α induced nuclear factor-kappa B activation by 27.5% at 2mg/ml, whereas variety Deiter showed 13.7% inhibition at a comparable dose. Avenanthramide levels did not correlate with the observed antioxidant and anti-inflammatory activities. Further investigations are needed to pinpoint the specific antioxidant and anti-inflammatory compounds, and potential synergistic and/or matrix effects that may help explain the mechanisms of oat’s anti-inflammatory actions.