2.11. Acute Inflammation Model

To assess anti-inflammatory activity during acute inflammation, ear swelling assays were conducted in mouse models as described by Inoue et al. [20]. This approach is considered a relatively reliable method for in vivo anti-inflammatory activity assessment [19, 21]. The samples were administered with a pipette (20 μL/ear) to the inside and outside of the right ear of each mouse. DMSO was used as a vehicle and was therefore used as a negative control. After 10 min, arachidonic acid (AA) (1 mg/ear) was applied to the right ear at the same site. After edema formation by AA administration, ear thickness was measured using a micrometer (Mitutoyo Mfg., Japan). For histologic analyses, lung tissue samples were fixed with 10% neutral phosphate-buffered formalin and embedded in paraffin using a Tissue-Tek® TEC™ 5 Tissue Embedding Console System (Sakura Finetek®, Torrance, CA, USA). The embedded lung tissue samples were sectioned at a 4 μm thickness and then stained with hematoxylin and eosin (H & E) to assess the severity of the ear edemas.