Anti-inflammatory activity of hydroxycinnamic acid derivatives isolated from corn bran in lipopolysaccharide-stimulated Raw 264.7 macrophages

Abstract

In this study, the effect of the 80% ethanolic extract of corn bran (EECB) on inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells was investigated. The EECB inhibited LPS-induced NO production and iNOS expression in a dose-dependent manner. Four hydroxycinnamic acid derivatives (HADs), including two free cinnamic acids, p-coumaric acid (CA) and ferulic acid (FA), and their conjugate phenolic amides, p-dicoumaroyl-putrescine (DCP) and diferuloylputrescine (DFP), were found to be present in the EECB by LC–MS analysis, and DFP (378.66 μg/g) was the predominant phenolic compound, followed by DCP (7.83 μg/g) > CA (5.58 μg/g) > FA (1.84 μg/g). The four HADs significantly inhibited NO production and iNOS expression in a dose-dependent manner. Among the four HADs tested, DFP showed the most potent inhibition on NO production and iNOS mRNA and protein expression, followed by DCP > FA ⩾ CA. DFP also exhibited the strongest inhibition on LPS-induced iNOS and NF-κB luciferase activity, which was followed by DCP ⩾ FA (CA) > CA (FA). Thus, these results suggest that phenolic amides in the corn bran may be a potential source of natural anti-inflammatory agents.

Introduction

Inflammation is a complex process that occurs in the body in response to cell damage and vascularized tissues. The inflammatory responses are controlled through cytokines and lipid mediators including prostaglandins and leukotrienes that are produced by inflammatory cells, such as macrophages and neutrophils (Ross and Auger, 2002). However, chronic inflammation caused by pro-inflammatory mediators is known to cause a variety of chronic inflammation-derived diseases, such as rheumatoid arthritis, diabetes, atherosclerosis, and cancer (Ljung et al., 2006, Tada, 2011, Watanabe et al., 2000). Therefore, inhibition of the production of pro-inflammatory mediators is an important target in the treatment of various inflammatory diseases. The LPS-treated Raw 264.7 murine macrophage model has been widely used to study inflammatory responses. Exposure of Raw 264.7 macrophages to external bacterial toxins like lipopolysaccharide (LPS) has been extensively shown to stimulate the secretion of nitric oxide (NO), which is produced by the inducible isoforms of nitric oxide synthase (iNOS) (MacMicking et al., 1997, Moncada et al., 1991, Jew et al., 2003, Lee et al., 2005). NF-κB has been shown to be an essential nuclear factor in the regulation of iNOS transcription via its binding to iNOS promoter in macrophage cells (Chiang et al., 2005). Thus, many studies have evaluated the effects of anti-inflammatory agents on the direct inhibition of NO production and iNOS expression, and suppression of NF-κB regulating transcription factor. More recently, many studies have been directed at identifying anti-inflammatory agents from natural sources (Kazlowska et al., 2010, Heo et al., 2010), because of the side effects of associated with synthetic anti-inflammatory drugs, including aspirin, indomethacin, and ibuprofen (Makins and Ballinger, 2003, Dogné et al., 2006). In particular, phenolic compounds, such as cinnamic acids, flavonoids, stilbenoids, and lignans, are being widely evaluated as potential effective alternatives to synthetic anti-inflammatory agents (Rahman et al., 2006, Middleton, 1998, Cíz et al., 2008).

Corn bran, which is obtained as a byproduct during the corn dry milling process, is rich in several functional lipid constituents including unsaturated fatty acids, tocopherols, phytosterols, dietary fibers and carotenoid pigments, etc. (Plate and Gallaher, 2005, Rose et al., 2010). In addition, corn bran has recently received renewed interest as a functional phytochemical source due to its high levels of hydroxycinnamic acid derivatives (HADs), including p-coumaric acid (CA), ferulic acid (FA), and their phenolic amides, such as diferuloylputrescine (DFP), p-coumaroylferuloylputrescine (CFP), and p-dicoumaroylputrescine (DCP) (Moreau et al., 2001). In particular, phenolic amides exhibit many biological activities, including antidiabetic (Niwa et al., 2003), inhibition of aflatoxin biosynthesis (Mellon and Moreau, 2004), and antioxidant and anti-melanogenic activities (Choi et al., 2007). The aqueous extract of corn husk, and arabinoxylan and lutein of corn has been reported to possess anti-inflammatory activity (Owoyele et al., 2010, Odabasoglu et al., 2008, Ogawa et al., 2005, Rafi and Shafaia, 2007). However, the anti-inflammatory activity of HADs including phenolic amides in corn bran has not yet been systematically evaluated.

The purpose of the present study was to investigate the anti-inflammatory activity of phenolic amides (DCP and DFP) and their parent cinnamic acids (CA and FA), against LPS-induced inflammatory response in Raw 264.7 macrophages.