Total Avenanthramides Research Articles

The synergistic effect of ascorbic and abscisic acids on enriching AVC-dominated avenanthramides in oat germination process

Avenanthramides are a group of antioxidants, of which avenanthramide C (AVC) shows the strongest antioxidant activity. Germination is conducive to the accumulation of total avenanthramides. However, it is unclear how to regulate the proportion of AVC in total avenanthramides by controlling the oat-germination process. To increase the proportion of AVC in total avenanthramides, single or combined treatments with abscisic (ABA) and ascorbic acid (Asc) during the steeping or germination period was performed. Compared to the control (541 μg/g dry weight), the 300 μM-ABA treatment during the steeping period significantly increased the proportion of AVC in total avenanthramides from 30.93 to 40.70%, which was accompanied by a remarkable increase in the content of total avenanthramides (1.8- fold). The content of total avenanthramides and the proportion of AVC within increased significantly (3.35-fold and by 9.28%, respectively) compared to the control when successively treated with 300 μM ABA during the steeping period and 20 mM Asc during the germination period. Moreover, ABA and Asc were synergistic in increasing the activities of hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and tyrosine ammonia-lyase (TAL), but reduced the activity of peroxidase (POD), which resulted in the accumulation of AVC-dominated avenanthramides. These findings provide novel information on the bio-concentration of AVC by regulation of the oat-germination process.

Simplified Analysis and Expanded Profiles of Avenanthramides in Oat Grains.

Uniquely, oats contain avenanthramides (AVAs), a group of phenolic alkaloids, exhibiting many health benefits. AVA analysis involves extraction with alcohol-based solvents and HPLC separation with UV and/or mass spectrometer detectors. There are many reported methods to extract AVAs. Almost all entail multiple extractions. The whole procedure is time- and labor-intensive. Furthermore, most quantifications are limited to three common AVAs (2f, 2p, 2c). The present study compared three extraction methods (all at 50 °C) for their effects on AVA concentrations and composition (% relative to total AVA) of oat grains. These included triplicate extractions with 80% ethanol containing 10 mM phosphate buffer (pH 2.0) (A), triplicate extractions with 80% ethanol (B), and a single extraction with 80% ethanol (C), while keeping solid/total solvent ratio at 1/60 (g/mL) and total extraction time of 60 min. Results showed that 80% buffered ethanol gave significantly lower AVA contents than 80% ethanol, while single and triplicate extractions with 80% ethanol produced the same extractability. However, the extraction method had no effect on AVA composition. Using 0.25 g sample size instead of 0.5 g saved extractants by half, without affecting AVA measurements. Consequently, a simplified method of extraction was developed, featuring Method C. The present study also expanded profiling individual AVAs beyond AVA 2c, 2p and 2f. Other AVAs identified and semi-quantified included 5p, 4p, 3f/4f, and 2pd. The simplified analysis was validated by measuring 16 selected oat grain samples. Some of these grains had relatively high contents of 4p, 3f/4f and 2pd, which have been considered minor AVAs previously.

Diversity of avenanthramide content in wild and cultivated oats

Background. Oat grains accumulate substantial amounts of various phenolic compounds that possess biological activity and have a potential to considerably increase health benefits of oats as a food. Avenanthramides (AVA) is an important group of these compounds due to their antioxidant, anti-itching, anti-inflammatory, antiproliferative activities.Materials and methods. Using combined HPLC and LC-MS analyses, we provide the first comprehensive review of the total avenanthramide content and composition in cultivated and wild oats. The AVA content was measured in 32 wild and 120 cultivated oat accessions obtained from the global collection of the N.I. Vavilov Institute of Plant Genetic Resources (VIR), St. Petersburg, Russia.Results and conclusion. The wild hexaploid A. sterilis L. had the highest total AVA content, reaching 1825 mg kg–1. Among cultivated accessions, naked oat cv. ‘Numbat’ (Australia) had the highest AVA content, 586 mg kg–1. The AVA composition exhibited a wide diversity among the analyzed samples. Accessions were identified where AVAs A, B and C, which are generally considered as major AVA, had a low percentage, and instead other AVAs prevailed. The AVA content in eight oat cultivars revealed significant annual changes in both the total AVA content and the proportions of individual AVAs. Using HPLC analyses, 22 distinguishable peaks in AVA extracts of oat seeds were detected and quantified. Several of these peaks, which have not been previously documented, presumably represent different AVAs. Further analyses are needed to detail these findings and to determine the specific AVA structures in oat grains.

Comprehensive analysis of oat avenanthramides using hybrid quadrupole-Orbitrap mass spectrometry: Possible detection of new compounds.

Avenanthramides (AVNs) are constituents unique to oats and have many outstanding health benefits. AVNs are antioxidants and possess anti-inflammatory, antifungal and antibacterial activity. The number of known AVNs increased recently because of the latest developments in high-resolution tandem mass spectrometry (HRMS/MS) techniques. Oat seed extract from 10 oat cultivars was analysed using ultra-high-performance liquid chromatography (UHPLC) and Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (HRMS/MS) with positive heated electrospray ionization. Thirty-five AVNs were identified and characterized in seed extracts, and the structures of 10 novel AVNs were tentatively elucidated, among which were AVNs bearing a cinamoyl or sinapoyl moiety. These AVNs are reported in oats for the first time. The method was validated using AVN standards (AVNs 2c, 2f and 2p), with limits of detection and quantitation at low picomole levels. Recovery of AVN standards varied from 83% to 106%, and relative standard deviations ranged from 2% to 9%. The total AVNs in the selected oat varieties ranged from 36.0 to 302.5 μg/g (dry weight), with AVN 2c, AVN 2f and AVN 2p representing approximately 65%-70% of that total. Our comprehensive method for detecting the full avenanthramide spectrum can contribute to better understanding the chemical and biological properties of individual AVNs for utilization in developing new oat cultivars and novel functional foods.

Enhancement of γ-aminobutyric acid, avenanthramides, and other health-promoting metabolites in germinating oats (Avena sativa L.) treated with and without power ultrasound

Power ultrasound as an emerging processing technology has been investigated for stimulating seeds to enhance germination and accumulation of health-promoting metabolites, such as γ-aminobutyric acid (GABA) and phenolic compounds. This work was undertaken to evaluate the effects of power ultrasound (25 kHz) on the nutritional properties of germinated oats, and the microstructure of oat groats after treatment. The changes in the external and internal microstructures of the ultrasound-treated oats kernel were investigated using Environmental Scanning Electron Microscopy (ESEM) and 3D X-ray Micro Computed Tomography (Micro-CT). Physicochemical properties of oats including GABA, free sugars, avenanthramides, total phenolic content, and antioxidant capacities were enhanced after germination. Furthermore, the power ultrasound treatment for 5 min after soaking significantly enhanced the GABA (48–96 h), alanine (24–96 h), succinic acid (48–72 h), total phenolic content (24 h), and total avenanthramides (24 h) in the germinated oats.

Determination of selected phenolic acid and majoritarian avenanthramides in different varieties of naked oats (Avena sativa L.) grown in Slovakia

Abstract Oats are important cereals. Oats are a good source of protein and lipids, polyphenolics, phenolic acids, flavonoids and avenanthramides. Avenanthramides is phenolic group, which is unique in oats and have antioxidant activity, anti-inflammatory, anti-atherogenic and anti-proliferative effect. The aim of study is determination of the majoritarian avenanthramides (2c, 2p and 2f) and phenolic acids (p-coumaric and ferulic) in selected varieties of oat (Avena sativa L.) grown in two consecutive years using the HPLC method. The oats were exposed to ultrasound supported extraction (two 15 min cycles).The simultaneous separation was performed using C18 type of stationary phase. The method showed a good linearity in the concentration range 0.04 - 5.24 μg/mL for p-coumaric acid, 0.04 - 5.13 μg/mL for ferulic acid, 0.19 - 24.5 μg/mL for avenanthramide 2c, 0.53 - 17.1 μg/mL for avenanthramide 2p, 0.8 - 25.6 μg/mL for avenanthramide 2f. Correlation coefficients were higher than 0.9997. Detector operated at a wavelength 320 nm. The repeatability of the method was evaluated in three concentration levels with satisfactory results for each analyte. The content of both phenolic acids is significantly lower (50- - 100-times) compared to the total content of avenanthramides in both years’ harvests for all analyzed varieties. Content of total avenanthramides was the highest in varieties Racoon (723.28 mg/kg) followed by Oliver (578.59 mg/kg) and Kamil (384.17 mg/kg).

Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa).

Avenanthramides are amides, with a phenylalkenoic acid (PA) and an anthranilic acid (AA) subunit, which are secondary metabolites of oat. Oat seeds were germinated, extracted, and the avenanthramides analysed by a combination of UHPLC with ion trap and high resolution ESI-MS. Typical fragmentation pathways with corresponding diagnostic fragments belonging to the PA and AA subunits were identified and summarised in a decision guideline. Based on these findings 28 unique avenanthramides were annotated in the oat seed(ling) extracts, including the new avenanthramide 6f (with a 4/5-methoxy AA subunit). Avenanthramide content increased by 25 times from seed to seedling. Avenanthramides 2p, 2c, and 2f, which are commonly described as the major avenanthramides, represented less than 20% of the total content in the seedlings. Future quantitative analyses should, therefore, include a wider range of avenanthramides to avoid underestimation of the total avenanthramide content.

Avenanthramide Aglycones and Glucosides in Oat Bran: Chemical Profile, Levels in Commercial Oat Products, and Cytotoxicity to Human Colon Cancer Cells.

Avenanthramides (AVAs), unique phytochemicals in oat, have attracted an increasing amount of attention due to their outstanding health benefits. However, the chemical profile and the levels of AVAs in commercial oat products as well as their health benefits have not been examined in detail. In the present study, a total of 29 AVA aglycones and AVA glucosides were identified and characterized from oat bran using NMR (1D and 2D NMR) and LC-MS techniques. Among them, 17 novel AVA glucosides were reported in oat bran for the first time. The most abundant AVA glucoside, 2c-3'- O-glc, had a similar growth inhibitory activity with the major AVA, 2c, against HCT-116 and HT-29 human colon cancer cells, indicating glucosylation does not affect the growth inhibitory effects of AVAs. Furthermore, the levels of all individual AVAs in 13 commercial oat products were analyzed using HPLC-MS/MS. The total AVAs contents in various oat products ranged from 9.22 to 61.77 mg/kg (fresh weight).

Analysis of avenanthramides in oat products and estimation of avenanthramide intake in humans

Avenanthramides are phenolic compounds found only in oats, and are of interest due to suggested bioactivities, including anti-inflammatory effects and induction of apoptosis. The objective of this work was to optimise a high performance liquid chromatography (HPLC) method for analysis of avenanthramides in food, and analyse the avenanthramide content in 45 oat fractions and products. The optimised HPLC method was based on triplicate extraction of 100 mg sample with 1 ml 80% ethanol in phosphate buffer (pH 2.8) and used gallacetophenone as an internal standard. Avenalumic acid-derived avenanthramide homologues 2fd and 2pd were also present, making up to 20% of the total avenanthramides detected in oats. The amounts of avenanthramides detected in oat products was 2–82 µg/g. It was estimated that mean avenanthramide intake among oat consumers ranges from 0.3 to 2.1 mg/day, considerably lower than the amount used in studies that have investigated biological effects of avenanthramides in humans.

Ultrasound-assisted extraction from defatted oat (Avena sativa L.) bran to simultaneously enhance phenolic compounds and β-glucan contents: Compositional and kinetic studies

In this research, the influence of ultrasonic application and temperature on extraction yields of free, esterified, bound phenolics, and β-glucan from defatted oat bran was investigated. Ultrasonic-assisted extraction (UAE) and conventional extraction (CE) were performed at different temperature. Extracts kinetics were monitored by determining the total phenolic content (TPC), antioxidant capacity (ORAC), and total avenanthramides of free phenolic compounds by mathematical model. HPLC-DAD was used to identify and quantify the main phenolic compounds. The results suggested that phenolic extraction yields of UAE was faster and higher than that of CE for free phenolics, with fitting to mathematically model (MRPD < 6%) well, whereas the bound fractions decreased. Besides, the TPC, ORAC and total avenanthramides of free phenolics were significantly improved by increasing the temperature in both UAE and CE, whereas the bound were significantly decreased. β-Glucan yields pretreated in UAE were approximately 37% higher than that in CE.

Phenolic contents, cellular antioxidant activity and antiproliferative capacity of different varieties of oats

The objectives of this research were to determine the phenolic contents, oxygen radical absorbance capacities (ORAC), cellular antioxidant activities (CAA), and antiproliferative capacities of nine oat varieties and four brans in China. Of all varieties, Longyan 3 and Beiyan 1 exhibited the highest total avenanthramides (146.94±7.31 and 120.95±6.66µg/g, respectively) and ORAC values (21.03±0.56 and 21.18±1.45µM Trolox/g, respectively), while Shaotong exhibited the highest total phenolic acids (143.52±9.42µg/g) and CAA values (33.38±1.74µM quercetin/100g). The EC50 of antiproliferative capacities ranged from 167.31±6.42 to 233.42±21.31mg/mL, with the lowest in Beixiao 8 while the highest in Jinyan 8. ORAC values correlated with avenanthramides while CAA values correlated with phenolic acids. Moreover, phenolic contents, antioxidant properties, and antiproliferative capacities of oat brans was higher than that of corresponding whole oats in most cases.

Identification of methylated avenanthramides in human plasma

Avenanthramides (AVs) are phytochemicals unique to oats consisting of an anthranilic acid and hydroxycinnamic acid linked by a pseudo‐peptide bond. We have demonstrated AV bioavailability to be low in hamsters and humans, suggesting they may be metabolized by phase II enzymes to facilitate their rapid excretion. Thus, we sought to identify the presence of phase II metabolites of 6 dominant AV isomers (A, B, C, O, P, and Q). AV‐A, ‐B, and ‐C differ in the moiety at position 3 (A: ‐H; B: ‐CH3O; and C: ‐OH) of the cinnamic acid ring, and AV‐O, ‐P, and ‐Q correspond to A, B, and C in terms of position 3 but differ in the length of the pseudo‐peptide linkage. We conducted a placebo‐controlled, crossover study in 10 healthy adults (60.4 y, BMI = 25.3). Blood was collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 24 h after consumption of a muffin made with either 20 g AV‐enriched oat bran (containing 46.2 mg total AVs) or regular oat bran. After removal of lipids with hexane, AV phase II metabolites in the defatted plasma were extracted twice with ethyl acetate, dried under N2, and reconstituted in 55% methanol for analysis on an Agilent UPLC‐QToF‐MS equipped with an Agilent Zorbax Extend C‐18 RR HT column (2.1 × 50 mm, 1.8 μm). Utilizing the MassHunter Qualitative Analysis software, we identified with a confidence score ≥80 for aglycones AV‐O and ‐A, and ≥90 for methylated AV‐O. Their detection is consistent with plasma AV results obtained after the removal of the glucuronide and sulfate conjugates that AV‐O and ‐A were the most abundant isomers. AV‐A was detected at fewer time points than AV‐O probably due to its much lower circulating concentrations. AV‐A had a maximum concentration (Cmax) of 2.2 ± 1.6 ng/mL with the time to reach Cmax (Tmax) at 1.9 ± 0.8 h. The Cmax for AV‐O was 6.4 ± 3.9 ng/mL and the Tmax was 2.6 ± 0.8 h, as well as 14.0 ± 8.2 ng/mL AV‐O equivalents and 1.9 ± 0.7 h for methylated AV‐O. Though AVs are extensively transformed to phase II metabolites, our analyses reveal the presence of AV‐O and ‐A aglycones in the circulation. Ours is the first report of a methylated AV metabolite following acute AV consumption. Future studies are warranted to explore the potential bioactivity of methylated AVs.Support or Funding InformationSupported by USDA and PepsiCo

Antioxidant Property of Naked Oat Core Collections

Naked oat(Avena nuda L.) originated in China,has a high values in nutrition and medicine.Besides dietary fiber and nutrients,various natural antioxidants,such as phenolic compounds,phenolic acids,avenanthramides(unique to oats) and phytosterols are also contained in naked oat.All these compounds possess potential health-promoting properties,partly attributing to their antioxidant activity.Up to now,China has built the core of naked oat germplasm with the greatest diversity of naked oat types.The objective of this study was to determine the total phenol(TP),total avenanthramides(TAV),β-glucan contents and the antioxidant activity of oat cultivars from main productive areas of China as well as Former Soviet Union and North America.The correlation of antioxidant activities with contents of these components was also statistically investigated,which is benefit for oat breeding.The antioxidant activities,contents of TP,TAV,and β-glucan of 120 naked oat accessions were evaluated by Folin-Ciocalteu method,HPLC analysis and enzymatic method,respectively.Meanwhile,the antioxidant capacity was determined by 1,1-Diphenyl-2-picrylhydrazyl radical(DPPH) scavenging ability method.Significantly genotypic variations were observed,and contents of TP,TAV,and β-glucan were 4.34–179.98 mg L-1,250.99–807.88 mg kg-1,25.21–347.55 mg kg-1,and 0.59–9.69% respectively.The 120 accessions were clustered into six groups using K-mean cluster method,which consisted of 20,39,2,22,12,and 25 accessions respectively.There were significantly positive correlations between antioxidant activity and the contents of TP and TAV.However,the correlation between β-glucan and DPPH was not significant.These results indicated that avenanthramides contributed greatly to the antioxidant property in naked oat grains.A relative wide variations in TAV and TP contents and compositions were mainly due to differences among cultivars.Duji from Sichuan province and Yumai 38 from Qinghai province with high contents of TP and TAV are expected to be used in further quality breeding of naked oat.