Some serotonin amides, as N‐behenoyl‐5‐hydroxytryptamine (C22:0‐5‐HT), are present in the surface wax of coffee beans and few studies show their possible biological activity. Thus, the aim of this work was to evaluate the anti‐inflammatory effect of C20:0‐5‐HT in the model of carrageenan‐induced inflammation into the subcutaneous air pouch (SAP), production of reactive oxygen species (ROS) by PMA‐stimulated leukocytes and MTT cell viability assay healing effect on the contraction model of the lesion in diabetic mice. C22:0‐5‐HT was synthesized by the group of Professor Claudia Rezende, Chemistry Institute, UFRJ. Female Webster mice (20–25g; n= 6–8) received oral administration of C22:0‐5‐HT (0.1, 1, 3 or 10 mg/kg) and were evaluated in model of SAP. Mice received carrageenan injection into the SAP and after 24 h were euthanized and the exsudate from SAP were collected for measurement of cell count. For the evaluation of ROS, leukocytes from the SAP were collected and incubated with C22:0‐5‐HT (0.1, 1 or 3 μM) for 1 h. Cells were incubated with 10 nM phorbol myristate acetate and 2 mM 2′‐7′‐dichlorodihidrofluoescein diacetate. The results were expressed as DCF‐DA fluorescence. Cell viability was measured by the MTT assay using RAW 264.7 mouse macrophages. For the analysis of healing activity female Webster mice (30–35g, n=10–12) received alloxan (65 mg/kg, iv) and after 7 days were anesthetized with ketamine and xylazine. The dorse was trichotomized and an area of 10 mm in diameter was exposed. The animals received topical administration (3 mg/animal) for 14 days and photos were taken on days 0, 3, 7, 10 and 14 to follow the retraction of the lesion. The image was processed in ImageJ program and the results are presented as the mean ± standard deviation of the wound area and expressed as arbitrary units (AU). Statistical significance between groups was determined by ANOVA followed by Bonferroni's test (*p<0.05) and the number of the animal research ethical committee is DFBCICB015‐04/16. C22:0‐5‐HT showed anti‐inflammatory activity in the three higher doses tested with reduction on cell migration (vehicle= 60.8 ± 8 × 106 cells/mL versus 61.7 ± 7 × 106 cells/mL, 31.2 ± 6* × 106 cells/mL, 37.1 ± 7* × 106 cells/mL, and 30.4 ± 6* × 106 cells/mL), with the doses of 0.1, 1, 3, and 10 mg/kg, respectively. It was also observed reduction in levels of ROS by PMA‐stimulated leukocytes in all concentrations tested (vehicle= 197,237.4 ± 8 fluorescence versus 98,852.1 ± 9* fluorescence, 87,951.3 ± 8.* fluorescence, 77,932.1 ± 7* fluorescence) and did not reduce the cell viability. Was observed improvement in healing of the treated mice on days 10 and 14 after injury. Naïve: day 0 = 40.1 ± 3.1 AU; 3 = 29.2 ± 5.1 AU; 7 = 13.1 ± 3.4 AU; 10 = 6.1 ± 2.2 AU; 14 = 3.2 ± 1.1 AU; Diabetic mice: day 0 = 41.3 ± 5.1 AU; 3 = 30.2 ± 5.4 AU; 7 = 20.1 ± 4.4 AU; 10 = 13 ± 3.5 AU; 14 = 7.2 ± 2 AU; Diabetic mice C22:0‐5‐HT‐treated (3 mg/animal) day 0 = 37.4 ± 6 AU; 3 = 30.9 ± 4 AU; 7 = 14.1 ± 3 AU; 10 = 2.7 ± 0.9* AU; 14 = 0.3 ± 0.1* AU. The results demonstrate that C22:0‐5‐HT produces anti‐inflammatory and healing effect. The mechanism(s) still under investigation.Support or Funding InformationThis study was financed in part by the CNPq and FAPERJ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract

The C18 5‐HT, a new Nβ‐alkanoyl‐5‐hydroxytryptamide is naturally found in the surface wax of coffee beans (Speer et al., 2006) and has antinociceptive effect (Giorno et al., 2018). Some amides of the serotonin class demonstrated an anti‐inflammatory effect by inhibiting the expression of caspases participants in inflammatory process (Meijerink et al., 2013)The C18 5‐HT, a new Nβ‐alkanoyl‐5‐hydroxytryptamide is naturally found in the surface wax of coffee beans (Speer et al., 2006) and has antinociceptive effect (Giorno et al., 2018). Some amides of the serotonin class demonstrated an anti‐inflammatory effect by inhibiting the expression of caspases participants in inflammatory process (Meijerink et al., 2013). In this study, we have investigated the in vitro anti‐inflammatory activity of C18 5‐HT, a new fatty acid amide of serotonin. RAW 264.7 murine macrophage cells stimulated with 1 μg/mL of E. coli lipopolysaccharide (LPS) were treated with different concentrations of C18 5‐HT (0.01, 0.03, 0.1, 0.3, 1 and 3 μM) for 24 h or 48h to evaluated cell viability by MTT (3‐(4,5‐dimethylthiazol‐2‐yl) ‐2,5‐difenltetrazolium). These LPS‐stimulated cells were also treated with 5‐HT C18 (0.1, 0.3, or 1 μM) to assess nitric oxide (NO) levels and cytokines (TNF‐α, IL‐1β, IL‐6 and IL‐10). The results showed that the cell viability of cells was significantly reduced only after 24h incubation with 3 μM of C18 5‐HT. LPS caused fold increase in NO production (49.7 ± 1.3 μM). Pretreatment of LPS‐activated cells with C18 5‐HT significantly suppressed NO production at 0.3 and 1 μM concentrations (39.9 ± 2.6* μM and 32.5 ± 3.1* μM, respectively versus vehicle‐treated group=49.7 ± 1.3 μM). C18 5‐HT also decreased the levels of TNF‐α (0.3 μM= 1,448.1± 376.6*; 1 μM= 1,083.1 ± 298.4* versus vehicle‐treated group = 2,231.9 ± 256.5 pg/mL and IL‐1 β (0.1 μM= 2,699 ± 597.3; 0.3 μM= 1,265.4 ± 438.8; 1 μM= 647.4 ± 187.5 versus vehicle‐treated group=5632.4 ± 1324.2 pg/mL) in a dose‐dependent manner. The compound also decreased significantly IL‐6 levels in the 1 μM concentration, promoting a reduction of 58.3 % when compared to vehicle‐treated group (1 μM=197.6 ± 46.7* versus vehicle‐treated group=474.4 ± 128.3 pg/mL) and increased the level of IL‐10 at 1 μM concentration (1 μM=4,393.5 ± 723.7* versus vehicle‐treated group= 2,514.2 ± 373.1 pg/mL). We can conclude that the treatment with C18 5‐HT did not reduce the viability of cells and that C18 5‐HT effectively inhibits pro‐inflammatory markers such as NO and cytokines (TNF‐α, IL‐1β and IL‐6). Data suggest that this substance could be used as new prototype for the development of new anti‐inflammatory drugs.Support or Funding InformationFinancial support: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES)/Finance Code 001, CNPq and FAPERJ.Technical Support: Alan MinhoAnimal donation: Institute Vital BrazilThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.