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The purpose of this study was to determine the antiallergic effects of AF-343, a mixture of natural plant extracts from Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on rat basophilic leukemia (RBL-2H3) cells. The inhibitory effects on cell degranulation, proinflammatory cytokine secretion, and reactive oxygen species (ROS) production were studied in compound 48/80-treated RBL-2H3 cells. The bioactive compounds in AF-343 were also identified by HPLC–UV. AF-343 was found to effectively suppress compound 48/80-induced β-hexosaminidase release, and interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production in RBL-2H3 cells. In addition, AF-343 exhibited DPPH free radical scavenging effects in vitro (half-maximal inhibitory concentration (IC₅₀) = 105 μg/mL) and potently inhibited compound 48/80-induced cellular ROS generation in a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay. Specifically, treatment with AF-343 exerted stronger antioxidant effects in vitro and antiallergic effects in cells than treatment with three single natural plant extracts. Furthermore, AF-343 was observed to contain bioactive compounds, including catechin, aurantio-obtusin, and chicoric acid, which have been reported to elicit antiallergic responses. This study reveals that AF-343 attenuates allergic responses via suppression of β-hexosaminidase release, IL-4 and TNF-α secretion, and ROS generation, perhaps through mechanisms related to catechin, aurantio-obtusin, and chicoric acid. The results indicate that AF-343 can be considered a treatment for various allergic diseases. Full article