Supplementary Material for: O. fragrans flower ameliorates compound 48/80-induced pseudo-allergic reactions in vitro and in vivo

Introduction: O. fragrans flower is a highly popular edible flower due to its long-lasting aroma. Although it has been reported to possess multiple activities, no study focused on its effects on Mrgpr-mediated mast cell activation.
Methods: The ethanol extract of O. fragrans flower (OFE) was prepared and the analyzed by the NaNO2-Al(NO3)3-NaOH colorimetric method and HPLC. Compound 48/80 (C48/80) was used for activating Mrgpr signaling. Supernatant β-hexosaminidase level was assayed by using the fluorescence probe 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide. The levels of cytoplasmic Ca2+ (Ca2+[c]) and intracellular mitochondrial Ca2+ (Ca2+[m]) were determined by fluorescence probes Fluo-3 AM and rhod-2/AM, respectively. The Ca2+ level outside the mitochondria was indicated by the fluorescence probe Calcium Green-5N. C48/80-induced vascular leakage, scratching behavior, and hypothermia models were used for evaluating the in vivo effects of OFE.
Results: The highly water-soluble flavonoid-rich OFE significantly inhibited C48/80-induced degranulation in LAD2 cells and primary rat peritoneal mast cells (RPMCs). It could rapidly and reversibly decrease Ca2+[c] level independent of the activation of Mrgpr signaling. Among four classical pathways responsible for Ca2+[c] removal, OFE failed to affect PMCA, SERCA, and NCX, but promoted the uptake of mitochondrial Ca2+. In vivo, OFE alleviated C48/80-induced vascular leakage, scratching, as well as hypothermia.
Conclusion: This study indicates, for the first time, that OFE is a mast cell stabilizer. It decreases Ca2+[c] to suppress mast cell degranulation by directly enhancing mitochondrial Ca2+ uptake. These findings may unveil a potential medicinal value of O. fragrans flower as a promising candidate for relieving mast cell activation-related diseases.