Mitigation of H2O2 induced Reactive Oxygen species and Proinflammatory cytokines by Buckwheat (Fagopyrum tataricum) rutin in cultured RAW264.7 cells

Abstract

Background

Buckwheat rutin, sourced from the Himalayan region of Nepal, exhibits significant free radical-scavenging potential. The rutin fraction derived from a tartary buckwheat seeds cultivar NGRC03731 exerts protective effects by preventing cellular damage and regulating proinflammatory cytokines in the mouse macrophage cell line RAW264.7.

Methods

The rutin fraction antioxidant properties were investigated using DPPH and ABTS methods in a cell-free system. RAW 264.7 cell viability following exposure to rutin fraction and standard rutin in the presence or absence of H₂O₂ was determined using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Free radical generation was evaluated through DCFDA staining. Pro-inflammatory cytokine gene expression levels were assessed using real-time PCR, while the corresponding protein levels were measured using Enzyme linked immunosorbent assay (ELISA).

Results

The rutin fraction markedly reduced NO and PGE2 levels. The viability of RAW 264.7 cells exposed to H₂O₂ and co-treated with the rutin fraction retained cell viability. Moreover, the rutin fraction alone (200 μg/mL) did not alter the morphology of RAW 264.7 cells, and reduced cellular damage in the presence of H₂O₂. TNF-α, COX-2a, IL-1, IL-6, IL-8, and IL-10 gene expression was significantly downregulated as well the cytokine protein levels of TNF-α, IL-1β, IL-8, IL-6, and IL-10 were markedly down regulated in RAW 264.7 cells treated with the rutin fraction.

Conclusion

The results of our study demonstrate the remarkable anti-inflammatory properties of the buckwheat rutin fraction in suppressing inflammatory responses.