Ethyl acetate fraction from Nymphaea hybrida Peck modulates inflammatory responses in LPS-stimulated RAW 264.7 cells and acute inflammation murine models

J Ethnopharmacol. 2021 Apr 6:269:113698. doi: 10.1016/j.jep.2020.113698. Epub 2020 Dec 15.


Ethnopharmacological relevance: Nymphaea hybrida Peck is used as a traditional medicinal herb for treating pain and inflammatory diseases, and known for its ornamental value and as a hot drink. However, the effects of N. hybrida polar fractions on lipopolysaccharide (LPS)-induced in vitro inflammation model and acute inflammation murine models have yet to be evaluated.

Aim of the study: The aim of this study was to elucidate the anti-inflammatory effects of N. hybrida ethanol extract (NHE) and its polar fractions: petroleum ether (PE), methylene chloride (MC), ethyl acetate (EA), methanol (ME), and water (WA). The underlying molecular mechanisms of active fraction in LPS-stimulated RAW 264.7 murine macrophages were further investigated.

Material and methods: Fractions with potential anti-inflammatory effects were screened using direct nitric oxide (NO) radical scavenging and cyclooxygenase (COX)-2 inhibition assays in vitro. The anti-inflammatory properties of potential fraction were evaluated in LPS-stimulated RAW264.7 cells, xylene-induced ear edema, carrageenan-induced paw edema and xylene-induced Evans blue exudation of acute inflammation murine models. The regulation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were investigated using western blotting and immunofluorescence.

Results: Compared to other polar fractions, NHE-EA displayed higher phenol and flavonoid content, and exerted greater activity in direct NO radical scavenging and COX-2 inhibition assay in vitro. NHE-EA markedly decreased the levels of inflammatory mediators, NO and prostaglandin E2 (PGE2), by suppressing the over-expression of inducible nitric oxide synthase (iNOS) and COX-2 in LPS-stimulated RAW264.7 cells. The NHE-EA fraction dose-dependently alleviated over-elevation of LPS-associated intracellular calcium and decreased the abnormal secretion of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and interferon-γ (IFN-γ). The combination with NHE-EA effectively attenuated the activation and nuclear translocation of NF-κB p65, and the phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases of MAPK pathways. NHE-EA could significantly ameliorate the degree of swelling of the mice ear and paw, the skin exudation of Evans blue and the excessive secretion of inflammatory cytokines.

Conclusion: Our results demonstrated that NHE-EA was the most active polar fraction of N. hybrida extracts. It inhibited the LPS-associated inflammatory response by blocking the activation of NF-κB and MAPKs pathways in RAW264.7 cells. It also effectively alleviated the inflammatory response of acute inflammation. These results indicated the role of NHE-EA as adjuvants and their potential role in alternative strategy for the treatment of inflammatory diseases.

Keywords: Anti-inflammation; Extracts; MAPKs; NF-κB; RAW264.7 cells.

MeSH terms

  • Acetates / chemistry*
  • Acute Disease
  • Animals
  • Anti-Inflammatory Agents / isolation & purification*
  • Anti-Inflammatory Agents / pharmacology*
  • Anti-Inflammatory Agents / therapeutic use
  • Calcium / metabolism
  • Capillary Permeability / drug effects
  • Carrageenan / toxicity
  • Cell Survival / drug effects
  • Cyclooxygenase 2 / drug effects
  • Cyclooxygenase 2 / metabolism
  • Cytokines / metabolism
  • Dinoprostone / metabolism
  • Disease Models, Animal
  • Edema / chemically induced
  • Edema / drug therapy
  • Edema / pathology
  • Inflammation / drug therapy*
  • Inflammation / metabolism
  • Lipopolysaccharides / toxicity
  • MAP Kinase Signaling System / drug effects
  • Male
  • Mice
  • Mice, Inbred ICR
  • NF-kappa B p50 Subunit / drug effects
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type II / metabolism
  • Nymphaea / chemistry*
  • Otitis / chemically induced
  • Otitis / drug therapy
  • Otitis / pathology
  • Plant Extracts / chemistry
  • Plant Extracts / isolation & purification*
  • Plant Extracts / pharmacology*
  • Plant Extracts / therapeutic use
  • RAW 264.7 Cells
  • Xylenes / toxicity


  • Acetates
  • Anti-Inflammatory Agents
  • Cytokines
  • Lipopolysaccharides
  • NF-kappa B p50 Subunit
  • Plant Extracts
  • Xylenes
  • Nfkb1 protein, mouse
  • Nitric Oxide
  • ethyl acetate
  • Carrageenan
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Ptgs2 protein, mouse
  • Cyclooxygenase 2
  • Dinoprostone
  • Calcium