2.8. Topical Anti-Inflammatory Activity

Acute inflammation in the ear pinna was induced by instilling 20 μL of TPA dissolved in acetone (2.5 μg/ear). Mice were divided into 9 groups with 6 animals per group. TPA (2.5 μg/ear) dissolved in 20 μL of acetone was applied to the inner and outer surfaces of mouse ears with the aid of a micropipette [34]. Treatments, ACEOs (5.0, 1.0, and 0.2%), CIN (2.5, 0.5, and 0.1%), and TPN (10, 2, and 0.4%) or indomethacin (0.5 mg/ear) was applied after the TPA induction. Four h later, the thickness of the ear was measured using a digital screw gauge. Six h after the treatment, the animals were euthanized for the collection of tissue. Ear biopsies (5 mm diameter punches) were weighed, homogenized in PBS (pH 7.5) with 1 mM EDTA, and centrifuged (10,000 ×g for 15 min) for the collection of supernatant which were used for the quantification of various cytokines.

For the assessment of skin inflammation, biopsies from control and treated ears of mice in each treatment group were collected and fixed in 4% formaldehyde (0.1 M phosphate buffer, pH 7.4). Subsequently, the tissues were dehydrated, blocked in paraffin, and serially sliced at a thickness of 5.0 μM using a microtome (Leica Microsystems, USA). The sections were stained with hematoxylin-eosin (H & E), and a representative section from each group of animals was selected to show the histopathological changes. The selected sections were analyzed by light microscopy (Leica Microsystems, USA), and the images were captured at 10x and 40x magnifications. The measurements of ear thickness and epidermal thickness (μm) were acquired by the Image Processing software of Leica Microsystems.

MPO activity was quantified according to the method proposed by Pulli et al. [35]. Briefly, the homogenate was added with a mixture containing 80 mM·PBS (pH 5.4), 0.22 M·PBS (pH 5.4), and 0.017% hydrogen peroxide. The reaction was initiated by the addition of 20 μL of 18.4 mM TMB in dimethylsulphoxide. The plate was incubated at 37°C for 4 min followed by the addition of 1.46 M sodium acetate (pH 3.0) to stop the reaction. The absorbance at 620 nm was measured using a plate reader to determine the enzyme activity. MPO activity was expressed as absorbance of TPA-treated ear tissue homogenate ÷ absorbance of ACEO-treated ear tissue homogenate.

The procedure described by Lee and Jeong [36] was followed. Briefly, nociception was induced by injecting 20 μL of 2.5% formalin in 0.9% saline in the subplantar region of the right hind paw. Mice (n = 6, per group) were pretreated topically with ACEOs (5%, 1%, and 0.25%), CIN (2.5, 0.5, and 0.1%), TPN (10, 2, and 0.4%), 1% diclofenac cream, and vehicle for 1 h prior to injecting formalin. These mice were individually placed in a transparent glass chamber for observation. The amount of time spent licking and flinching of the injected paw was indicative of pain. The number of flinches and lickings after injection of formalin was counted during 0 to 5 min (early phase) and 20 to 30 min (late phase). Antinociception was considered as a statistically substantial reduction in the time spent in licking and flinching of the injected paw in comparison with the control group. Edema was estimated by measuring the paw volume before and after 4 h of formalin injection using plethysmometer (IITC, Life Scientific Instruments, Woodland Hills, CA, USA). The percentage reduction in paw volume by ACEOs was calculated in comparison with the formalin-treated group.