Eugenol as a Promising Molecule for the Treatment of Dermatitis: Antioxidant and Anti-inflammatory Activities and Its Nanoformulation

Abstract

Contact dermatitis produces an inflammatory reaction primarily via stimulation of keratinocytes and cells of the immune system, which promote the release of cytokines, reactive oxygen species (ROS), and other chemical mediators. Eugenol (EUG, phenylpropanoid of essential oils) has attracted attention due to its anti-inflammatory properties, as well as antioxidant effect. On the other hand, it is volatile and insoluble and is a skin irritant. In this case, nanostructured systems have been successfully employed as a drug carrier for skin diseases since they improve both biological and pharmaceutical properties of active compounds. The cytotoxic, antioxidant, and anti-inflammatory effects of EUG were assessed in human neutrophils and keratinocytes. Additionally, polymeric nanocarries (NCEUG) were prepared to improve the chemical and irritant characteristics of EUG. EUG presented apparent safety and antioxidant and anti-inflammatory effects on human neutrophils, but presented cytotoxic effects on keratinocytes. However, the nanocapsules were able to reduce its cytotoxicity. An in vivo experiment of irritant contact dermatitis (ICD) in mice induced by TPA showed that NCEUG reduced significantly the ear edema in mice when compared to the EUG solution, as well as the leukocyte infiltration and IL-6 level, possibly due to better skin permeation and irritancy blockage. These findings suggest that EUG is a promising bioactive molecule, and its nanoencapsulation seems to be an interesting approach for the treatment of ICD.

Figures

Figure 1

Evaluation of EUG toxicity on MTT test in human neutrophils. Data from three to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). Triton X-100 (Tx, 0.02%) was used as cytotoxic standard. ∗vs. HBSS: nontreated cells. p < 0.05 (ANOVA and Tukey as the post hoc test).

Figure 2

Effect of EUG on the percentage of measurement of membrane integrity of human neutrophils as monitored by flow cytometry using the sensitive fluorochrome propidium iodide. Neutrophils were cultured for 30 min in the presence of EUG at various concentrations (10–50 μg/mL). Values are presented as means ± SEM of 3 separated experiments in triplicate and by analysis of 10,000 events. The control group consists of cells treated with vehicle (DMSO 1%). ∗vs. HBSS: nontreated cells. p < 0.001 (ANOVA and Tukey as the post hoc test).

Figure 3

Effects of eugenol (EUG) on the release of human neutrophil myeloperoxidase (MPO) stimulated by phorbol myristate acetate (PMA). Freshly isolated cells (2.5 × 106) were preincubated with indicated concentrations of EUG prior to the addition of PMA (0.1 μg/mL). Indomethacin (36 μg/mL) was used as positive control. Data are expressed as percentages of inhibition by EUG on the release of MPO. Numbers represent mean ± SEM. Data from three to eight samples.

Figure 4

Evaluation of antioxidant activity of EUG in human neutrophils by chemiluminescence (CL). The inhibitory effect of EUG in human neutrophil oxidative metabolism was assessed by luminol (LumCL) (a) or lucigenin (LucCL) (b). Data from three to eight samples. ∗vs. control (DMSO), p < 0.05, and ∗∗∗vs. control (DMSO), p < 0.001 (ANOVA and Tukey as the post hoc test).

Figure 5

Size distribution by photon correlation spectroscopy of the eugenol-loaded nanocapsules (NCEUG) and the blank formulation (NCB).

Figure 6

Evaluation of EUG and NCEUG toxicity on MTT test for 24 hours, 48 hours, and 72 hours in human neutrophils. Data from two to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). ∗vs. DMEM: untreated cells. p < 0.05 (ANOVA and Tukey as the post hoc test).

Figure 7

Effects from EUG and NCEUG on TPA-induced acute edema in mice. Swiss mice (25 to 30 g) were treated topically with EUG or NCEUG (0.04, 0.08, and 0.16 mg/ear), NCB (blank formulations), dexamethasone (DEXA; 0.05 mg/ear), or acetone (vehicle control) with topical application of TPA (2.5 μg/ear) on the surface of the left ear of mice. Values represent mean ± SEM from 8 animals per group. a vs. the normal group; b vs. control; c vs. NCB group; p < 0.05 (ANOVA and Tukey's test).

Figure 8

Effect of EUG on myeloperoxidase activity induced by TPA in mice. Swiss mice (25 to 30 g) were treated topically with EUG or NCEUG (0.04, 0.08, and 0.16 mg/ear), dexamethasone (DEXA; 0.05 mg/ear), or acetone (vehicle control) with topical application of TPA (2.5 μg/ear) on the surface of the left ear of mice. Values represent mean ± SEM the amount of MPO in U/mL. 8 animals were used per group. a vs the normal group; b vs control group; c vs NCB; p < 0.05 (ANOVA and Tukey's test).

Figure 9

Antiedematogenic activity from EUG and NCEUG induced by TPA in mice. Swiss mice (25 to 30 g) were treated topically with EUG or NCEUG (0.04, 0.08, and 0.16 mg/ear), NCB (blank formulations), dexamethasone (DEXA; 0.05 mg/ear), or acetone (vehicle control) with topical application of TPA (2.5 μg/ear) on the surface of the left ear of mice. Values represent mean ± SEM from 8 animals per group. a vs the normal group; b vs control; c vs NCB group; p < 0.05 (ANOVA and Tukey's test).

Figure 10

The effect of NCEUG from EUG and NCEUG on IL-6 (a) and KC (b) levels in ear tissue homogenates induced by TPA in mice. Swiss mice (25 to 30 g) were treated topically with EUG or NCEUG (0.04, 0.08, and 0.16 mg/ear), NCB (blank formulations), dexamethasone (DEXA; 0.05 mg/ear), or acetone (vehicle control) with topical application of TPA (2.5 μg/ear) on the surface of the left ear of mice. Values represent mean ± SEM from 8 animals per group. ∗vs control group; p < 0.05 (ANOVA and Dunnett's test).