Comparison of Anti-Inflammatory Effects of Flavonoid-Rich Common and Tartary Buckwheat Sprout Extracts in Lipopolysaccharide-Stimulated RAW 264.7 and Peritoneal Macrophages

Abstract

Buckwheat sprouts have been widely consumed all around world due to their great abundance of bioactive compounds. In this study, the anti-inflammatory effects of flavonoid-rich common buckwheat sprout (CBS) and tartary buckwheat sprout (TBS) extracts were evaluated in lipopolysaccharide- (LPS-) stimulated RAW 264.7 murine macrophages and primary peritoneal macrophages from male BALB/c mice. Based on the reversed-phase HPLC analysis, the major flavonoids in CBS were determined to be C-glycosylflavones (orientin, isoorientin, vitexin, and isovitexin), quercetin-3-O-robinobioside, and rutin, whereas TBS contained only high amounts of rutin. The TBS extract exhibited higher inhibitory activity as assessed by the production of proinflammatory mediators such as nitric oxide and cytokines including tumor necrosis factor-α, interleukin- (IL-) 6, and IL-12 in LPS-stimulated RAW 264.7 macrophages than CBS extract. In addition, TBS extract suppressed nuclear factor-kappa B activation by preventing inhibitor kappa B-alpha degradation and mitogen-activated protein kinase phosphorylation in LPS-stimulated RAW 264.7 macrophages. Moreover, the TBS extract markedly reduced LPS-induced cytokine production in peritoneal macrophages. Taken together, these findings suggest that TBS extract can be a potential source of anti-inflammatory agents that may influence macrophage-mediated inflammatory disorders.

Figure 1

HPLC chromatograms of flavonoids in (a) common and (b) tartary buckwheat sprouts at 350 nm. Peak number 1, orientin; 2, isoorientin; 3, vitexin; 4, isovitexin; 5, quercetin-3-O-robinobioside; 6, rutin.

Figure 2

Inhibitory effects of common buckwheat sprout (CBS) and tartary buckwheat sprout (TBS) extracts on (a) NO production and (b) levels of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages. RAW 264.7 macrophages were incubated for 24 h, and exposed to sprout extracts together with LPS (1 μg/mL) for 24 h. Levels of iNOS and COX-2 expression were determined using Western blot assay. Relative protein levels are expressed as the percentage of intensity to the cells treated with LPS alone, which was set to 100%. Data represent the means ± standard deviation of three independent experiments. ### indicates p < 0.001 in comparison to untreated controls; significant difference was determined using unpaired Student's t-test. ∗∗∗ indicates p < 0.001 in comparison to cells treated with LPS alone by one-way ANOVA followed by Dunnett's test for multiple comparison.

Figure 3

Inhibitory effect of common and tartary buckwheat sprout extracts on LPS-induced (a) IL-6, (b) IL-12, and (c) TNF-α cytokine production and LPS-induced (d) IL-6, (e) IL-12, and (f) TNF-α mRNA expression in RAW 264.7 macrophages. RAW 264.7 macrophages were exposed to sprout extracts together with LPS (1 μg/mL). Level of cytokine expression in the culture media was measured using ELISA. mRNA levels were analyzed by real-time RT-PCR. Data represent the means ± standard deviation of three independent experiments. ^##p < 0.01 and ^###p < 0.001 in comparison with untreated controls; significant difference was determined using unpaired Student's t-test. ††† indicates p < 0.001 in comparison to cells treated with LPS alone (unpaired Student's t-test). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 in comparison with cells treated with LPS alone by one-way ANOVA followed by Dunnett's test for multiple comparison.

Figure 4

Effect of common buckwheat sprout (CBS) and tartary buckwheat sprout (TBS) extracts on LPS-stimulated phosphorylation of IκB-α in RAW 264.7 macrophages. RAW 264.7 macrophages were incubated for 24 h and exposed to LPS (1 μg/mL) with sprout extracts for 24 h. Levels of phosphorylated IκB-α were determined using Western blot assay. Relative protein levels are expressed as the percentage of intensity to the cells treated with LPS alone, which was set to 100%. Data represent the means ± standard deviation of three independent experiments. ### indicates p < 0.001 in comparison with untreated controls; significant difference was determined using unpaired Student's t-test. ^∗p < 0.05 and ^∗∗∗p < 0.001 in comparison with cells treated with LPS alone by one-way ANOVA followed by Dunnett's test for multiple comparison.

Figure 5

Effect of common buckwheat sprout (CBS) and tartary buckwheat sprout (TBS) extracts on LPS-stimulated (a) MAPK and (b) MAPK kinase 4 activation. RAW 264.7 macrophages were incubated for 24 h and exposed to CBS or TBS extracts with LPS (1 μg/mL) for 24 h. Whole protein was determined using Western blot assay. Relative protein levels are expressed as the percentage of intensity to the cells treated with LPS alone, which was set to 100%. Data represent the means ± standard deviation of three independent experiments. † indicates p < 0.05 in comparison with cells treated with LPS alone (unpaired Student's t-test). ### indicates p < 0.001 in comparison with untreated controls (unpaired Student's t-test). ^∗∗p < 0.01 and ^∗∗∗p < 0.001 in comparison with cells treated with LPS alone by one-way ANOVA followed by Dunnett's test for multiple comparison.

Figure 6

Inhibitory effects of common and tartary buckwheat sprout extracts on the secretion of (a) IL-6, (b) IL-12, and (c) TNF-α cytokines in LPS-induced peritoneal macrophages of male BALB/c mice. Mouse peritoneal macrophages were exposed to LPS (100 ng/mL) with sprout extracts. Levels of cytokine expression in culture media were measured by ELISA. Data represent the means ± standard deviation of three independent experiments. ### indicates p < 0.001 in comparison with untreated controls; significant difference was determined using unpaired Student's t-test. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 in comparison with cells treated with LPS alone by one-way ANOVA followed by Dunnett's test for multiple comparison.