"Nulichal" Barley Extract Suppresses Nitric Oxide and Pro-Inflammatory Cytokine Production by Lipopolysaccharides in RAW264.7 Macrophage Cell Line

Abstract

The cultivar "Nulichal," a type of naked waxy barley (Hordeum vulgare L.), was developed by the National Institute of Crop Science, Rural Development Administration, Korea, in 2010. In this study, we investigated the anti-inflammatory and antioxidant properties of the "Nulichal" ethanol extract (NRE) using various assays. The NRE exhibited a total phenolic content of 7.55±0.30 mg gallic acid equivalent/g and a flavonoid content of 1.74±0.08 mg rutin equivalent/g. Cell viability assays showed no toxicity of NRE on RAW264.7 macrophage cells up to concentrations of 500 μg/mL. The NRE (300 and 500 μg/mL) significantly reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). It also down-regulated the mRNA expression and protein levels of inducible NO synthase and cyclooxygenase-2 in a dose-dependent manner. Moreover, the NRE treatment significantly decreased the levels of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, and their mRNA expression compared to LPS treatment alone. The NRE demonstrated strong free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals in a dose-dependent manner. The ferric reducing antioxidant power assay also showed increased antioxidant activity with increasing NRE concentrations. These findings suggest that the NRE can be used as a functional food with anti-inflammatory and antioxidant properties.

Keywords: antioxidant; barley; inflammation; nitric oxide.

Fig. 1

Effects of the “Nulichal” ethanol extract (NRE) on cell viability and nitric oxide levels in lipopolysaccharides-induced RAW264.7 macrophages. Data are presented as mean±SE of three independent experiments. Values not sharing a common letter (a-c) indicate significant between-group differences (P<0.05). CON, control; NRE100, 100 μg/mL of NRE; NRE300, 300 μg/mL of NRE; NRE500, 500 μg/mL of NRE.

Fig. 2

Effects of the “Nulichal” ethanol extract (NRE) on inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression and their protein expression in lipopolysaccharides (LPS)-induced RAW264.7 macrophages. Data are presented as mean±SE of three independent experiments. The protein expression data were normalized with β-actin and calculated as a ratio to the vehicle value. Values not sharing a common letter (a-c) indicate significant between-group differences (P<0.05). CON, control; NRE300, 300 μg/mL of NRE; NRE500, 500 μg/mL of NRE.

Fig. 3

Effects of the “Nulichal” ethanol extract (NRE) on tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels and their mRNA expression in lipopolysaccharides (LPS)-induced RAW264.7 macrophages. Data are presented as mean±SE of three independent experiments. Values not sharing a common letter (a-d) indicate significant between-group differences (P<0.05). CON, control; NRE100, 100 μg/mL of NRE; NRE300, 300 μg/mL of NRE; NRE500, 500 μg/mL of NRE.

Fig. 4

Effects of the “Nulichal” ethanol extract (NRE) on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals scavenging activities and half-maximal inhibitory concentration (IC₅₀) values. Data are presented as mean±SE of three independent experiments. Values not sharing a common letter (a-d) indicate significant between-group differences (P<0.05).

Fig. 5

Effects of the “Nulichal” ethanol extract (NRE) on ferric reducing antioxidant power (FRAP) activity. Data are presented as mean±SE of three independent experiments. Values not sharing a common letter (a-f) indicate significant between-group differences (P<0.05). AsA, ascorbic acid.