## 4 Discussion
The major representative compound of _Lonicera caerulea_ fruit is C3G of the anthocyanin series [77]. Therefore, we measured the content of C3G, a representative anthocyanin, in HR2302-30E. C3G content of HR2302-30E was found to be 7.02 \(\pm\) 0.17 mg/g. C3G accounts for 70-90% of the anthocyanin profile and is present at 0.68-41.16 mg/mL in _Lonicera caerulea_ fruit [51; 59; 70; 78; 79; 80]. Allergy is the overreaction of the immune system to a generally harmless substance, accompanied by an inflammatory response. Mast cell activation is the response to the cross-linking of allergens to the IgE/Fc\(\epsilon\)RI complex, which induces allergic immune responses. FceRI receptor consists of an \(\alpha\)-subunit to which IgE binds and \(\beta\)- and \(\gamma\)-subunits involved in intracellular signaling. Upon receptor aggregation, initial phosphorylation of ITAM in the \(\beta\)- and \(\gamma\)-subunits is caused by Lyn and Fyn, respectively, which are Src family kinases. Activated ITAM serves as a docking site for the activation of Syk [81; 82; 83]. Incubation of RBL-2H3 cells with IgE in vitro increases the expression of FccRI on the cell surface [84; 85]. Moreover, FccRI aggregate formation is activated by Src family kinases, such as Lyn, Fyn, and Syk [82]. HR2302-30E and C3G significantly decreased the expression of FccRI\(\beta\) and FccRIy and inhibited the phosphorylation of Src, Fyn, and Syk in anti-DNP-IgE-activated RBL-2H3 mast cells. These results indicate that HR2302-30E and C3G inhibit mast cell activation by blocking the binding sites of FccRI and interfering with the signaling pathways of Src, Fyn, and Syk. Tyrosine-phosphorylated Syk induces the activation of downstream signaling proteins, such as LAT, PLCy, and PKC8. Phosphorylated LAT provides a binding site for PLCy, which regulates the elevation of intracellular Ca\({}^{2+}\) levels and activation of PKC8 [86; 87]. Phosphorylation of PLCy and PKC8 stimulates the secretion of histamine, \(\beta\)-hexosaminidase, and inflammatory cytokines, which induce allergic responses [88]. Activation of Fyn and Syk induces MAPK. MAPK activation plays a key role in the production of inflammatory mediators, including cytokines, and recruitment of immune cells [89]. MAPK activation of ERK regulates the proliferation, migration, and differentiation of mast cells under allergic conditions, as well as the activation of JNK and p38, which produces inflammatory cytokines and mediators in abnormally activated mast cells [65]. It is the major mechanism regulating allergic inflammation. In the present work, HR2302-30E and C3G inhibited the phosphorylation of PLCy and PKC8. Additionally, HR2302-30E and C3G significantly suppressed the phosphorylation of p38, ERK, and JNK in mast cells with abnormally activated MAPK signaling pathways. These findings suggest that HR2302-30E and C3G may inhibit the release of allergy mediators, such as Ca\({}^{2+}\), histamine, and \(\beta\)-hexosaminidase by blocking the phosphorylation of PLCy and PKC8. Furthermore, by suppressing the MAPK signaling pathway, they may block the inflammatory response, thereby contributing to their anti-allergic and anti-inflammatory effects. Our study demonstrated the antiallergic effects of HR2302-30E and C3G, particularly through the regulation of various signaling pathways (FccRI, Src, Fyn, Syk, PLCy, PKC8, MAPK), which modulate the release of \(\beta\)-hexosaminidase and histamine in RBL-2H3 mast cells. These results suggest the potential of HR2302-30E and C3G as natural products for regulating mast cell activation in allergy treatments. Increased intracellular calcium levels play a key role in stimulating mast cell activation and extracellular secretion of granules [90]. Activated mast cells release allergic mediators, such as histamine, \(\beta\)-hexosaminidase, serotonin, and protease, via immediate degranulation and induce allergic inflammation via cytokine gene expression [91].