Anti-oxidant and anti-inflammatory effects of rice bran and green tea fermentation mixture on lipopolysaccharideinduced RAW 264.7 macrophages

Yun-Hee Rhee,Chun Hee Rhee,Phil-Sang Chung,Jin-Chul Ahn

doi:10.4314/tjpr.v16i12.19

Abstract

Purpose: To investigate the anti-inflammatory and anti-oxidant properties of an enzyme bath of Oryza sativa (rice bran) and Camellia sinensis O. Kuntz (green tea) fermented with Bacillus subtilis (OCB). Methods: The anti-oxidant effects of OCB were assessed by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay and flow cytometry. The anti-inflammatory effects of OCB were assessed by a nitric oxide (NO) assay. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were used to quantify expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The major compounds of OCB were identified using high performance liquid chromatography (HPLC) analysis. Results: OCB had no cytotoxic effect on LPS-stimulated macrophages or peripheral blood mononuclear cells up to 1 mg/mL. OCB displayed anti-oxidant effects comparable to those of ascorbic acid and reduced reactive oxygen species (ROS) levels in target cells. OCB treatment of LPSstimulated mavrophages decreased nitric oxide (NO), NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and key pro-inflammatory cytokine expressions, suggesting that OCB acts as an anti-oxidant and anti-inflammatory agent by reducing ROS levels and inhibiting pro-inflammatory mediators. The main effector compounds in OCB were epicatechin gallate, cathechin, synigrin acid, epicathechin, epigallocatechin gallate, rutin, and isoquercetin, which are known anti-oxidants. Conclusion: OCB fermentation product may be used as synergistic adjuvant therapy for inflammatory diseases. Keywords: Rice bran, Green tea, Bacillus subtilis, Enzyme bath, Anti-oxidant, Anti-inflammation

Highlights

Enzyme baths have traditionally been used in Asian countries to treat various neurological disorders
We examined the cytotoxicity of OCB extract on both primary peropheral blood mononuclear cells (PBMCs) and RAW 264.7 macrophages
We observed no cytotoxic effects of OCB on mouse PBMCs in the presence or absence of LPS

Summary

INTRODUCTION

Enzyme baths have traditionally been used in Asian countries to treat various neurological disorders. We examined the effect of metabolites of rice bran, green tea, and microbial enzymes from fermentation on oxidative stress and pro-inflammatory cytokine production in cultured cells. Kuntz (green tea) fermented from Bacillus subtilis (Enzyme LAPA Co,) and extracted with ethanol (OCB; Figure 1). We identified key components in the fermentation products of OCB using high-performance liquid chromatography (HPLC) Together these data elucidate a mechanism for the therapeutic. The cytotoxic effect of OCB on RAW 264.7 cells and PBMCs was evaluated by the MTT assay. Cells were treated same as well as ROS anaylsis, and followed by measurement of total GSH and glutathione disulfide (GSSG) using a GSH/GSSG ratio detection assay kit. The cells were treated with various concentrations of OCB with or without 1 μg/mL LPS for 24 h, and the supernatant was removed.

RESULTS

DISCUSSION

CONCLUSION

Conflict of interest