Banana peel (BP) has potent antioxidative properties; however, the anti-inflammatory potential of BP and its related bioactive components remain unclear. This study used solvent extraction and gas chromatography–mass spectrometry (GC–MS) to isolate and identify the active fractions and compounds in BP. BP was extracted with 95% ethanol (BP-95E) and partitioned with an ethyl acetate (EA) and water mixture to obtain the BP-95E-EA and BP-95E-H2O fractions. The BP-95E-EA fractions were further partitioned with n-hexane (Hex) and methanol (MeOH) mixtures to obtain BP-95E-EA-Hex and BP-95E-EA-MeOH subfractions, and the BP-95E-H2O fractions were partitioned with n-butanol (BuOH) to obtain BP-95E-H2O-BuOH subfractions and the H2O residual. The results show that the BP-95E-H2O-BuOH subfractions exhibited the most potent inhibition of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion while down-regulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. In this active subfraction, five non-polyphenol compounds were identified, namely, 5-hydroxyethyl furfural (5-HMF), guaiol, oleic acid, linoleic acid, and oleamide. 5-HMF, guaiol, and oleamide were the most effective at suppressing IL-6 and TNF-α secretion. The in vivo immunomodulatory action of BP was evaluated in an LPS-induced endotoxemia model of BALB/c mice. Oral administration of BP-95E-H2O-BuOH extracts (42 and 166 mg/kg b.w.) for two weeks lowered the serum levels of IL-6 and TNF-α and normalized the activated T-cell population, as evidenced by an increase in CD3CD69 and decrease in IFN-γ/IL-4 (Th1/Th2) in mice with systemic inflammation. Our findings reveal that BP exhibits anti-inflammatory and T-cell immunomodulatory effects that may contribute to delaying endotoxemia-associated disorders.
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