Vascular permeability assay in ICR mice Male ICR mice weighing 18–22 g were pretreated with dexamethasone (5 mg/kg) or C12 (5 or 15 mg/kg) intraperitone- ally. Control animals received a similar volume (200 mL/kg) of saline. After 15 min, 200 mL of 10% azovan blue solution in saline was i.v.-injected and 300 mL of acetic acid (0.7%) was i.p.-injected, respectively. Then, 30 min after acetic acid injection, the peritoneal fluid was collected in sterile and heparinized PBS (1 mL) solution. The total of the collection was evaluated by optical density assay (620 nm). Carrageenan-induced paw oedema in mice Male ICR mice weighing 18–22 g were divided into four groups (n = 6 in each), that were submitted with respect to the treatment by the i.p. route with saline, Dexamethasone (5 mg/kg, Xianju Pharma, Zhejiang, China) or C12 (5 or 15 mg/kg), 15 min before oedema induction by subcutaneous injection of carrageenan (300 mg/paw) in the right paw. The left paw was injected with an equal volume of saline as a negative control, and both paws were monitored by a procedure similar to that described elsewhere [19]. The paw oedema was measured by a electronic micrometer and calculated by the difference between paw volumes 4 h after paw oedema induction. Chemically-induced inflammatory abdominal constrictions in mice Abdominal constrictions to the i.p. injection of acetic acid (0.6%) were monitored. The procedure was similar to that previously described by de Campos et al. (18). Male ICR mice weighing 18–22 g were pretreated with Dexamethasone (5 mg/kg) or C12 (5 or 15 mg/kg) intraperitoneally 15 min before the injection of acetic acid. Control animals received a similar volume (10 mL/kg) of saline. After challenge, pairs of mice were placed in separate boxes, and the number of abdominal constrictions was cumulatively counted. References 1. Brevetti G, Giugliano G, Brevetti L, Hiatt WR (2010) Inflammation in peripheral artery disease. 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C12 Exerts Anti-Inflammatory Effects Formalin-induced inflammatory nociception in mice The procedure used was similar to that previously described by Correa and Calixto [30]. Male ICR mice weighing 18–22 g were pretreated intraperitoneally with C12 (5 or 15 mg/kg) or Dexamethasone (5 mg/kg) 15 min before pain induction. Control animals received only saline. 20 mL of 2.5% formalin in PBS was injected under the paw surface of the right hind paw. The mice (control and treated groups) were observed simultaneously from 0– 30 min following formalin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered indicative of pain. Statistical analysis The results are presented as means6SD. The statistical significance of differences between groups was obtained by means of analysis of variance followed by the post-test of comparison. P values less than 0.05 (p,0.05) were considered indicative of significance. Nitrite determination and cell viability were per- formed in triplicate. All experiments were repeated at least three times. Supporting Information Figure S1 Toxicity assay of C12. Male ICR mice weighing 18–22 g were randomly seperated into 4 groups (n = 10 in each group) and were treated with C12 at 5, 10, 20, or 40 mg/kg by i.p. administration. Survival of the mice was monitored for 14 days. (DOC) The toxicity of C12 in vivo. Text S1 (DOC) Author Contributions Conceived and designed the experiments: GL YW. Performed the experiments: CY YP JL YW YZ HZ. Analyzed the data: GL XL SY. Contributed reagents/materials/analysis tools: GL FY XL. Wrote the paper: GL YW. Synthesized and prepared the compound C12 used in this study: GL FY. 12. Jeon KI, Xu X, Aizawa T, Lim JH, Jono H, et al. (2010) Vinpocetine inhibits NF-kappaB-dependent inflammation via an IKK-dependent but PDE-indepen- dent mechanism. Proc Natl Acad Sci U S A 107: 9795–9800. 13. 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Cell Signal 13: 85–94. 18. Rittirsch D, Flierl MA, Day DE, Nadeau BA, McGuire SR, et al. (2008) Acute lung injury induced by lipopolysaccharide is independent of complement activation. J Immunol 180: 7664–7672. 19. Paulino N, Rodrigues NC, Pardi PC, Suarez JA, dos Santos RP, et al. (2009) Evaluation of anti-inflammatory effect of synthetic 1,5-bis(4-acetoxy-3-methox- yphenyl)-1,4-pentadien-3-one, HB2. Bioorg Med Chem 17: 4290–4295. 20. Oliveira C, Navarro-Xavier RA, Anjos-Vallota EA, Martins JO, Silveira VL, et al. (2010) Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions. Br J Pharmacol 161: 899–910. 21. Olajide OA, Makinde JM, Okpako DT (2003) Evaluation of the anti- inflammatory property of the extract of Combretum micranthum G. Don (Combretaceae). Inflammopharmacology 11: 293–298. PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24377