*2.2. Induction of Tissue Eosinophilia in Peridinin-Treated Mice Sensitized with PCl*

We examined whether the migration of eosinophils to the site of inflammation was suppressed by administering peridinin or fucoxanthin. We measured the number of eosinophils in the ear section of BALB/cAJc1 mice and compared it to the number in the negative control group (sensitized, but not challenged with PCl) and the hydrocortisone group (68.5% suppression). Peridinin by either route of administration, paint (79.9% suppression) or i.p. (60.3% suppression), significantly decreased the number of eosinophils at the site of inflammation (ear section) at 48 h after antigen challenge. In contrast, fucoxanthin did not inhibit the number of eosinophils that migrated to the site of inflammation at 48 h after the antigen challenge (Figure 3A,B). Eosinophils were not observed in the negative control group without the challenge with PCl. Eosinophils were more abundant after the challenge of sensitized mice with PCl. In the group treated with hydrocortisone by painting at 3 h before the challenge, the number of eosinophils was lower than that of the positive control. Groups treated with peridinin either by painting or i.p. showed lower number of eosinophils than the positive control. There were no significant difference between the hydrocortisone-treated group and peridinin-treated group. In contrast, the fucoxanthin-treated group, either by painting or i.p., did not have a lower number of eosinophils compared with the 

**Figure 2.** Suppression of delayed-type hypersensitivity (DTH) responses by peridinin and fucoxanthin in BALB/cAJc1 mice. Mice were sensitized with picryl chloride (PCl) after pretreatment with cyclophosphamide. Two weeks after 10 ΐg of peridinin or fucoxanthin or hydrocortisone in 10 ΐL of olive oil was painted onto the ears (per earlobe), 50 ΐg of peridinin or fucoxanthin in 100 ΐL of olive oil was administered i.p. (intraperitoneally) to these mice 3 h before the antigen challenge. Ear thickness was measured with a dial thickness gauge before (0 h) and after the antigen challenge (4, 24 and 48 h). Each value for each treatment group is expressed as the mean ± SD ( *<sup>n</sup>* = 10). Pos cont, positive control (sensitized and challenged); Hydroc, hydrocortisone; Peri, peridinin; Fuco, fucoxanthin; Peri i.p., peridinin administered intraperitoneally; Fuco i.p., fucoxanthin administered intraperitoneally; Neg cont, negative control (sensitized, but not challenged). \* An asterisk indicates a significant difference between the treatment group and the positive control ( *p* < 0.01, Tukey–Kramer's *post hoc* test). Asterisks are only shown for groups showing suppression compared to the positive control at 24 or 48 h after the antigen challenge. 

*2.3. Suppression of the Percentage of Eosinophils in Peripheral Blood in Peridinin-Treated Mice Sensitized with PCl* 

We measured and compared the percentage of eosinophils among white blood cells in the peripheral blood of individual mice between the non-challenged and hydrocortisone-treated groups and found the effect of treatment to result in 83.1% suppression. Peridinin administered by either route, paint (82.2% suppression) or i.p. (78.4% suppression), significantly suppressed the number of eosinophils in peripheral blood at 48 h after the antigen challenge. In contrast, fucoxanthin did not suppress the number of eosinophils in peripheral blood at 48 h after the antigen challenge (Figure 4). These results suggest that peridinin, but not fucoxanthin, suppressed the proliferation of eosinophils. In this DTH model, serum levels of IFN- · and IL-5 were increased 48 h after the antigen challenges, and peridinin did not suppress the serum level of either cytokine significantly in either route of administration (IFN- ·, negative control, 37.8 ± 14; positive control, 104.4 ± 28; peridinin painted, 94 ± 32; peridinin i.p., 72.6 ± 31. IL-5: negative control, 12.3 ± 8.5; positive control, 18.6 ± 3.5; peridinin painted, 25.0 ± 0.1; peridinin i.p., 23.2 ± 12. Results are expressed as the mean (picogram per millimeter) ± SD). The serum level of IL-17 was not increased in this experiment [16]. Thus, we hypothesized that the production of eotaxin, the most potent chemo-attractant of the eosinophils, may be suppressed at the site of inflammation. 

**Figure 3.** The effects of peridinin or fucoxanthin on the number of eosinophils in ear sections. Mice were sensitized with PCl, as described in the Experimental Section. Two weeks after the sensitization, 10 ΐg of peridinin, fucoxanthin or hydrocortisone was painted onto the ears and 50 ΐg of peridinin or fucoxanthin was administered i.p. to these mice 3 h before the antigen challenge. At 48 h after the antigen challenge, tissue  specimens were fixed and stained with hematoxylin/eosin solution to count the number of eosinophils. (**A**) The number of eosinophils in each section from each group is shown. (**B**) Representative section photomicrographs from each group of mice are shown. Each value is expressed as the mean ± SD (*<sup>n</sup>* = 10). Pos cont, positive control; Hydroc, hydrocortisone; Peri, peridinin; Fuco, fucoxanthin; Peri i.p., peridinin intraperitoneally; Fuco i.p., fucoxanthin intraperitoneally; Neg cont, negative control. \* An asterisk indicates a significant difference between the treatment group and the positive control (*p* < 0.01, Tukey–Kramer's *post hoc* test). 
