Flavonoid glucuronides isolated from spinach inhibit IgE-mediated degranulation in basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

Yuta Morishita ,Ryohei Yamamoto ,Emi Saito ,Ryoma Fujikawa ,Masanori Kuroyanagi ,Eri Takemura ,Osamu Shirota

doi:10.15761/imm.1000119

Abstract

We isolated three active constituents from an aqueous extract of spinach leaves by evaluating the inhibitory activity of IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells, and determined their chemical structures. These compounds, referred to as SO-1, SO-2 and SO-3, were assessed to be flavonol glucuronides with the same fundamental structure. Among them, SO-1 exhibited the most potent granulation-inhibitory activity at the concentrations of 0.5~10 µg/ml. This inhibitory effect was completely exhibited by its aglycon, which was prepared by the enzymatic treatment of β-glucuronidase and the chromatographic isolation. These compounds hardly showed cytotoxicity at the same concentration range in RBL-2H3 cell culture. To reveal its inhibitory mechanism, we examined the effects of SO-1 on the signal transduction of antigen-stimulated degranulation in the cells. Both SO-1 and its aglycon reduced the elevation of intracellular Ca 2+ concentration in a dose-dependent manner and also effectively inhibited the activation of tyrosine kinases such as Syk and Lyn, which underlay in the upstream of intracellular signal transduction followed by antigen stimulation. Furthermore, both SO-1 and its aglycon remarkably suppressed the passive cutaneous anaphylaxis (PCA) reaction induced in ICR mouse ear, as compared with the authentic anti-allergic drugs. In this PCA reaction, the aglycon exerted more potent inhibitory activity than SO-1, indicating that SO-1 also functions in vivo through the hydrolysis to the aglycon in the gastrointestinal tract. Together with these results, we conclude that spinach contains the promising anti-allergic flavonoid glucuronide, suggesting that this compound and its original vegetable may be beneficial to prevent or ameliorate allergic constitutions of humans.

Highlights

The number of type 1 allergy patients has been increasing in the world, especially in developed and industrialized countries
We demonstrate the inhibitory effect of an aqueous extract from spinach leaves on IgE-mediated degranulation in RBL2H3 cells and the isolation of three active compounds based on the biological evaluation test
The aqueous extract of spinach leaves showed an apparent inhibition of IgE-mediated degranulation in RBL-2H3 cells by using the sequentially diluted samples (Figure 1)

Summary

Introduction

The number of type 1 allergy patients has been increasing in the world, especially in developed and industrialized countries. This is one of most important healthcare problems to be resolved. Type I allergy exerts symptoms of acute inflammation reactions like asthma and rhinitis. Type 1 allergy reaction is mediated by the specific binding of polyvalent allergens to the corresponding IgE receptors (Fcε R1) on the surface of mast cells and basophils [1,2]. The antigenbinding to the cells immediately triggers the intracellular signal cascade leading to the final release of chemical mediators, such as histamine, serotonin, arachidonic acid metabolites, proteases, heparin, and so on, from intracellular granules, followed by the occurrence of acute allergic symptoms. Mast cell plays a critical role in the allergic reaction [3,4], indicating that the regulation of such hypersensitivity of the cells is considered to be very important for the prevention and/or amelioration of allergic constitution of humans

Methods

Results

Conclusion