## 4 Discussion
Further studies are needed to clarify the exact mechanisms by which compounds in the fRGM exert beneficial effects on the progression of AD. The pathophysiology of AD is multi-factorial with immune alteration, skin barrier dysfunction, and environmental factors [38]. Skin barrier dysfunction increases the penetration of allergens and pathogens, leading to immune alteration and further disruption of barrier function [38, 39]. The secondary lymphoid tissues, including the spleen and lymph nodes, are the main regions exposed to various allergens in the chronic stage of AD, which further differentiates naive T cells into effector T cells, and stimulates B cells to produce IgE [18, 40]. Our DNCB-induced AD-like model showed hypertrophic changes of the spleen and lymph node with increased inflammatory cytokines in previous and current studies [26]. These changes were involved in hyperplasia of the white pulp cells of the spleen and follicular cells of the lymph node, probably by increasing leukocytes and differentiating into effector lymphocytes after the induction of DNCB [40]. However, fRGM inhibited systemic immune activation in both lymphoid tissues by inhibiting the production of Th1-derived mediators and the development of the germinal center related to IgE production, suggesting therapeutic potential in chronic AD. Chronic inflammation of AD elevates free radicals and reactive oxygen and nitrogen species (RONS), similar to other chronic inflammatory diseases, and RONS-generated regional inflammation can further activate allergic responses [41, 42]. Natural products generally possess polyphenic compounds and flavonoids that act as direct free radical scavengers against superoxide anions and inhibitors of lipid peroxidation [18]. Indeed, natural antioxidant ingredients attribute to inhibition of IgE production, inflammatory progression, and enhancement of epidermal permeability barrier function in AD [43], and topical antioxidants, furfuryl palmitate and its derivatives, are considered clinical replacements for topical corticosteroids and calcineurin inhibitors [44]. Here, fRGM improved antioxidant capacities in AD-like skin lesions by increasing antioxidants (GSH) and reducing lipid peroxidation (MDA and 4-HNE), superoxide anion, and RONS-induced protein nitration (NT). The fRGM contained polyphenic compounds and flavonoids despite small contents, and the antioxidant activities could be enhanced via fermentation with effective microorganisms [22, 23, 45]. Although the DEXA group had little effect on skin levels of GSH, MDA, and superoxide anions, the potent anti-inflammatory effects may be involved in reducing the RONS-induced products (NT and 4-HNE). Furthermore, immunoreactivity for apoptotic markers (Cas-3 and PARP) in the AD-like skin lesion was reduced by treatments of DEXA and fRGM, suggesting anti-apoptosis related to the anti-inflammatory effects rather than antioxidant effects. However, given that RONS damages DNA and cellular biomolecules, and the most significant target is the MAPK pathway [46], the antioxidant properties of fRGM may contribute to additional anti-allergic effects by inhibiting inflammatory responses and apoptotic changes. RGM has been considered only a byproduct of ginseng extraction processing. Previously, fRGM had undetectable contents of toxic substances (e.g., Pb, As, Hg, Sb, Cd), and topical treatment has shown hair-growth promoting effects with no side effects [25]. Here, topical treatment of fRGM, particularly at higher doses, alleviated the AD-like lesions by inhibiting IgE production, inflammatory progress, and apoptotic changes with enhanced antioxidant activities, similar to the anti-AD effects of red ginseng. The fRGM contained various bioactive components, including ginensosides, particularly Rb1, Rb2, Rg2, Rg3, and Rh1, as well as polyphenols and flavonoids [25], and further studies are needed to find indicator substances involving the anti-AD properties.