Macrophage and keratinocyte cell assays suggest that the supercritical CO2 extract of black soybean possesses anti-inflammatory and skin barrier-protective effects.

Atopic dermatitis (AD) is a prevalent inflammatory skin disorder with limited treatment options, highlighting the need for alternative therapeutic strategies. Traditionally, black soybean oil, prepared from Glycine max through high-temperature dry distillation, has been used in Asian medicine to relieve AD symptoms. However, this preparation method poses safety concerns due to the potential formation of carcinogenic by-products. To address these limitations, we prepared black soybean extract (BSE) using supercritical CO2 extraction and investigated its pharmacological effects and underlying mechanisms in AD-related cell models. Gas chromatography-mass spectrometry analysis identified five major fatty acids in BSE. In LPS-stimulated RAW264.7 macrophages, BSE reduced the production of pro-inflammatory mediators. In TNF-α/IFN-γ-stimulated HaCaT keratinocytes, BSE downregulated the mRNA levels of AD-associated alarmins, decreased apoptosis, and increased junction protein expression, suggesting its skin barrier-protective effects. Integrated network pharmacology and RNA sequencing analyses predicted that TNF signaling and its downstream MAPK and NF-κB pathways play key roles in BSE's effects. RT-qPCR validated that BSE downregulated the mRNA levels of genes involved in pro-inflammatory responses, immune activation, and skin barrier impairment. Western blotting results demonstrated that BSE inactivated MAPK and NF-κB signaling molecules, including ERK, p38, JNK, p65, and IκBα. Collectively, our findings demonstrate, for the first time, that BSE has anti-inflammatory and skin barrier-protective potential, with the inhibition of TNF-MAPK/NF-κB signaling pathways involved in BSE's effects. This study suggests that BSE has the potential to be used for managing AD-related inflammation and skin barrier dysfunction.