Phenolic acid bound arabinoxylans extracted from Little and Kodo millets modulate immune system mediators and pathways in RAW 264.7 cells

Abstract

The immunomodulating effect of Phenolic acid bound arabinoxylans (PCA-AXs) extracted from Little (PCA-AX-L) and Kodo (PCA-AX-K) millet seeds in RAW 264.7 cells were investigated. The PCA-AXs were extracted from millets and their chemical characterization were carried out by GC-MS, HPLC, and FT-IR. The immunomodulatory effect of PCA-AXs in RAW 264.7 cells were investigated by estimating ROS, NO, and cytokines TNF-α, IL-1β, IL-6, and evaluation of molecular mechanism by q-PCR & western blotting techniques. The xylose: arabinose ratio of PCA-AX-L and PCA-AX-K were 1.48:1.0 and 2.26:1.0, respectively. The phenolic acids content was higher in PCA-AX-K than PCA-AX-L determined by HPLC. FT-IR analysis confirms the presence of α-glucosidic linkage with the degree of substitution of xylan backbone by arabinose residues. The evaluation of immunomodulating effect of PCA-AXs revealed that the PCA-AX-L-treated cells showed higher release of NO, ROS and cytokines than PCA-AX-K-treated cells. The mRNA expressions of TNF-α, iNOS and COX-2 were upregulated by PCA-AX-L and downregulated by PCA-AX-K in dose-dependent manner. Furthermore, in western blotting, the ERK and NF-κB were found to be activated by PCA-AX-L and inhibited by PCA-AX-K. Our findings suggest that the high branched arabinoxylans of PCA-AX-L could modulate the immune response in RAW 264.7 cells through activation of ERK and NF-κB signaling pathways and acts as an immunostimulant. The higher phenolic content in PCA-AX-K could modulate the immune response by downregulation of ERK and NF-κB signaling pathways and thus, it could act as an immunomodulator. PRACTICAL APPLICATIONS: Millets are the richest source of arabinoxylans in which they are known to be bound with phenolic acids (PCA-AX). Arabinoxylans derived from rice and wheat is known immunomodulators. This study was focused to evaluate the immunomodulatory property of PCA-AX derived from two different millets little and kodo. The study results clearly indicated the immune stimulatory action of PCA-AX-L and immunomodulatory action of PCA-AX-K. The explored mechanism indicated that the PCA-AXs modulate NF-κB & ERK pathways for their immunomodulatory action.

Keywords: NF-ĸB and ERK pathways; RAW 264.7 cells; immunomodulating effect; millets; phenolic acid bound arabinoxylans (PCA-AXs).