In vitro total antioxidant capacity and anti-inflammatory activity of three common oat-derived avenanthramides
Introduction
More than 25 avenanthramide compounds in oats have been identified, although some of these have not been completely characterized. Structurally, all avenanthramides contain an anthranilic acid and a cinnamic acid, but the substitution patterns on the anthranilic acid and cinnamic acid distinguish them from each other (Bratt, Sunnerheim, Bryngelsson, Fagerlund, et al., 2003). The most abundant avenanthramides in oats are N-(3′,4′-dihydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2c), N-(4′-hydroxy-3′-methoxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2f), and N-(4′-hydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2p) (Fig. 1). These compounds differ by a hydroxyl group, methoxy group, or hydrogen on the 3′ carbon of the cinnamic acid (B) ring, which may account for differences in biological effects.
A large body of evidence suggests the involvement of ROS and RNS, including ROO, HO, O2−, 1O2, and ONOO− in the pathophysiology of aging and in chronic diseases (Finkel & Holbrook, 2000). In addition, free radicals can undergo interconversion. For example, O2− is converted to oxygen and H2O2 by superoxide dismutase, O2− also reacts with nitric oxide (NO) to form ONOO−, a more biologically damaging radical than either of the reactants (Beckman & Koppenol, 1996). In living cells, endogenous and exogenous antioxidants can neutralize and/or prevent the damage caused by ROS/RNS. These antioxidants include avenanthramides, readily bioavailable polyphenols in oats that have been shown to possess numerous beneficial properties, including anti-inflammatory, antiatherogenic, antiproliferative, anticancer, and anti-itch effects that may be useful in prevention of coronary heart disease, colon cancer, and skin irritation. These low-molecular-weight, soluble phenolic compounds are constitutive components of oat groats (Collins, 1986), hulls, bran (Emmons & Peterson, 2001), and leaves (Peterson & Dimberg, 2008), reaching a total level of from approximately 2 to 300 mg/kg in oat grains.
In vitro antioxidant activities of avenanthramides have been extensively studied (Bratt et al., 2003, Chu et al., 2013, Dimberg et al., 1993, Peterson et al., 2002) and found to be 10–30 times those of other phenolic antioxidants in oats (e.g., vanillin, caffeic acid). The avenanthramides 2c, 2p and 2f demonstrated antioxidant activity in the DPPH and β-carotene bleaching assays, with 2c showing the strongest antioxidant capacity of the three compounds (Peterson et al., 2002). In a linoleic acid model system, 2f displayed an antioxidant capacity approximately 20% that of α-tocopherol (Dimberg et al., 1993). In another study, all eight avenanthramides tested (1c, 1p, 1s, 1f, 2c, 2p, 2s, 2f) showed antioxidant activity in the linoleic acid model, and all but 1p showed activity in the DPPH model. The authors concluded that avenanthramides containing caffeic (1c, 2c) and sinapic acid (1s, 2s: 1s and 2s are not natural products) were the most effective antioxidants (Bratt et al., 2003).
The research data on the bioavailability of avenanthramides are limited. In one study, the bioavailability of avenanthramides 2c, 2p, and 2f was determined in six elderly subjects who had consumed an avenanthramide-enriched mixture. Maximum plasma concentrations after consuming 59 and 118 mg of avenanthramides were 167.5 and 559.6 nM, respectively (Chen, Milbury, Collins, & Blumberg, 2007). Recently, Koenig, Dickman, Wise, and Ji (2011) reported that 2c, 2f, and 2p can be detected in the circulating blood of rats following oral intake and are taken up by hepatic, cardiac, and skeletal muscle tissues. Although avenanthramide bioavailability in rats is low compared with that of humans and hamsters, the rank order of plasma concentration is the same: 2p ≫ 2f > 2c. A study investigating the in vivo antioxidant activity of avenanthramides in rats demonstrated that 2c attenuates ROS production in some tissues and increases the activity of certain antioxidative enzymes (Ji, Lay, Chung, Fuy, & Peterson, 2003). In addition, avenanthramides enhance antioxidant capacity in humans and act synergistically with vitamin C to protect against LDL-oxidation in hamsters (Chen, Milbury, Kwak, Collins, et al., 2004). In a human study, a total of 120 healthy individuals were randomly assigned to daily supplementation of oat avenanthramides (3.12 mg daily) or a placebo for 1 month (Liu, Yang, Hou, Yao, et al., 2011). In the group receiving avenanthramides, serum levels of superoxide dismutase and reduced glutathione increased by 8.4% and 17.9%, respectively (p < 0.05). These results suggest that the antioxidant effect of the avenanthramides is more associated with an indirect antioxidant response via increased activity of endogenous antioxidant enzymes in vivo (Dinkova-Kostova et al., 2007).
The anti-inflammatory and antiatherogenic properties of avenanthramides have also been investigated. Avenanthramides are able to suppress vascular endothelial cell expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin), thereby preventing monocyte adhesion to human aortic endothelial cell monolayers and down-regulating the production of inflammatory cytokines and chemokines (e.g., IL-6, IL-8, and MCP-1) (Liu, Zubik, Collins, Marko, & Meydani, 2004). By up-regulating the p53-p21cip1 pathway and inhibiting the phosphorylation of retinoblastoma protein, 2c inhibits smooth muscle cell proliferation in humans and increases nitric oxide production, two key factors in the prevention of atherosclerosis (Nie, Wise, Peterson, & Meydani, 2006). An avenanthramide enriched oat extract as well as synthetic 2c and its methyl ester decreased proinflammatory cytokine production and adhesion molecule expression in human aortic endothelial cells by inhibiting NF-κB activation (Guo, Wise, Collins, & Meydani, 2008). In keratinocytes, the anti-inflammatory activity of avenanthramides at concentrations as low as 1 ppb prevented the degradation of IκB and decreased phosphorylation of the p65 subunit (Sur, Nigam, Grote, Liebel, & Southall, 2008). Treating cells with avenanthramides inhibited tumor necrosis factor-alpha (TNF-α)-induced NF-κB activity and subsequently reduced interleukin-8 release.
Phenolic compounds exert their antioxidant activity through two main mechanisms: by acting as hydrogen atom donators or as metal ion chelators. The antioxidant capacities of phenolics depends on the number and arrangement of hydroxyl groups, the nature of the substituents in the ring structures, ionization state, steric hindrance, and the stability of the resulting phenoxy radicals. Although the antioxidant activity of oat avenanthramides has been extensively documented, no studies have investigated the antioxidant capacities of 2c, 2f, and 2p in scavenging different types of free radicals, nor has their relative effect in inhibiting the activation of NF-κB been evaluated. The objective of this work was therefore to evaluate the free radical scavenging capacity of synthesized 2c, 2f, and 2p and assess their ability to inhibit TNF-α-induced NF-κB activation in vitro.
Section snippets
Chemicals and apparatus
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), dihydrorhodamine 123 (DHR-123), and disodium fluorescein (3′,6′-dihydroxyspiro[isobenzofuran-1[3H],9′[9H]-xanthen]-3-one) were purchased from Sigma–Aldrich (St. Louis, MO, USA), and 3-morpholinosydnonimine, hydrochloride (SIN-1) was obtained from Toronto Research Chemicals (North York, ON, Canada). The compound 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals USA (Richmond, VA, USA).
Peroxyl radical scavenging capacity
The ORAC assay was used to determine the antioxidant capacities of 2c, 2f, or 2p (5 μM) against peroxyl radicals, which are the most common free radicals in vivo and are highly reactive and unstable. The fluorescein decay curves induced by AAPH are shown in Fig. 2(a), revealing the strongest scavenging capacity for 2c, as indicated by AUC. ORAC values were 36,818 ± 3366 μmol TE/g for 2c, 17,860 ± 1939 μmol TE/g for 2f, and 23,301 ± 1551 μmol TE/g for 2p. Taking the total antioxidant capacity to be the sum
Conclusions
In this study we evaluated the antioxidant capacities of oat avenanthramides 2c, 2f, and 2p against common free radicals, and the ability of these compounds to inhibit TNF-α-induced NF-κB activation in mouse myoblast C2C12 cells. Using multiple assays (NORAC, SORAC, ORAC, SOAC, HORAC), we found that 2c has the highest total antioxidant capacity of the three avenanthramides (p < 0.05), which was attributable mainly to SORAC and ORAC; total antioxidant capacity did not differ significantly between
Conflict of interest
This project was funded by PepsiCo, Inc., the maker of Quaker Oats™. YiFang Chu and Jun Yang are employees of PepsiCo, Inc. The views expressed in this manuscript are those of the authors and do not necessarily reflect the position or policy of PepsiCo, Inc.
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