## 4 Discussion  
AD is a common chronic inflammatory skin disease with an incidence from 3-20% in children and 2-5% in adults worldwide [22; 23]. Accumulating evidence suggests that AD is associated with various neuropsychiatric comorbidities, such as ADHD, anxiety, and depression, which reduce the quality of life of AD patients significantly [24]. Recent studies indicated that AD exhibits a transient association with the later onset of ADHD and approximately 7% of total AD patients suffer from ADHD symptoms [25; 26]. Therefore, managing both skin symptoms and psychiatric disorders might be a more effective approach for AD treatment. This study revealed the inhibitory effects of GMP on AD-skin inflammation and AD-induced hyperactivity behavior in DNCB-treated mice, suggesting its potential in the drug development for AD. DNCB-induced cutaneous inflammation in mice was reported as an appropriate model for AD studies [27]. In this study, the topical application of DNCB to the back skin of BALB/c mice induced AD-like symptoms, such as redness, dryness, and excoriation. On the other hand, the GMP treatment could ameliorate these symptoms in AD mice significantly. Epidermal hyperplasia and infiltration of inflammatory cells in skin lesions are typical characteristics of AD [28]. These results indicated that the topical treatment of GMP alleviated DNCB-induced epidermal thickening and infiltration of mast cells and eosinophils in skin lesions. The roles of mast cells in allergic disease, including AD,  
Figure 7: Analysis of chemical constituents of GMP by HPLC. (**A**) Structures of picrasin B and quassin. HPLC chromatograms of the standard compounds (**B**) and GMP (**C**). were well characterized in previous studies [29; 30]. Mast cells are generally activated by binding IgE to FccRI receptors on the cell surface, and activated mast cells can secrete a range of inflammatory molecules, such as histamine, TNF-\(\alpha\), and VEGF [31]. Interestingly, GMP could decrease both IgE and VEGF levels in DNCB-treated mice. Pretreatment with GMP also reduced SP-induced production of VEGF in HMC-1 cells, suggesting the inhibitory effects of GMP on the mast cell response in AD. In addition to mast cells, macrophages, another innate immune cell type, play a pivotal role in developing AD. The accumulation of macrophages is observed in acute and chronic AD inflamed skin. In the early stages of inflammation, macrophages have pro-inflammatory functions, such as antigen presentation and the production of inflammatory cytokines [32]. These results suggest that GMP inhibited the LPS-induced TNF-\(\alpha\) production in Raw264.7 macrophages. These results suggest the suppressive effects of GMP on the innate immune system for the treatment of AD symptoms. T lymphocytes, particularly T helper cells, play crucial roles in the pathogenesis of AD [33]. The infiltration of CD4+ T cells into the dermis was observed in AD skin lesions [34]. In the early phase of AD, Th2 cells are prominent infiltrates in lesional skin. The recruitment of Th2 lymphocytes into AD lesions might be triggered by several chemokines, such as MDC and TARC, mainly produced by keratinocytes [35]. A previous study reported that the levels of these Th2 chemokines were elevated in AD patients compared to healthy subjects [36]. Infiltrated Th2 cells secrete many inflammatory cytokines, including IL-4, IL-5, and IL-10, to promote skin inflammation and skin barrier disruption in AD [37]. In this study, GMP reduced the expression of IL-10 in skin lesions of AD mice and suppressed the production of TARC in TI-stimulated HaCaT keratinocytes, suggesting the effects of GMP on the Th2 response in AD. In addition to Th2 cells, Th1 and Th17 cells, as well as their effector cytokines are involved in the progression of AD. Th1 cells can produce various cytokines, such as IFN-\(\gamma\), TNF-\(\alpha\), and IL-2, while Th17 cells are well known for the production of IL-17 [38]. IL-17 triggers the Th2 response and B cell differentiation to promote an inflammatory response in AD [39; 40].