Antiedematogenic activity of the indole derivative n-salicyloyltryptamine in animal models

The N-salicyloyltryptamine (NST) is an indole derivative compound analogue to the alkaloid N-benzoyltryptamine. In the present study, the antiedematogenic activity of NST was investigated in animal models. Firstly, the acute toxicity for NST was assessed according to the OECD Guideline no. 423. The potential NST-induced antiedematogenic activity was evaluated by carrageenan-induced paw edema in rats, as well as by dextran-, compound 48/80-, histamine-, serotonin-, capsaicine-, and prostaglandin E2-induced paw edema in mice. The effect of NST on compound 48/80-induced ex vivo mast cell degranulation on mice mesenteric bed was investigated. No death or alteration of behavioral parameters was observed after administration of NST (2000 mg/kg, i.p.) during the observation time of 14 days. The NST (100 and 200 mg/kg, i.p.) inhibited the carrageenan-induced edema from the 1st to the 5th hour (**p<0.01; ***p<0.001). The edematogenic activity induced by dextran, compound 48/80, histamine, serotonin, capsaicin, and prostaglandin E2 was inhibited by NST (100 mg/kg, i.p.) throughout the observation period (**p<0.01; ***p<0.001). The pretreatment with NST (50, 100 or 200 mg/kg, i.p) attenuates the compound 48/80-induced mast cell degranulation (**p<0.01; ***p<0.001). Thus, the inhibition of both mast cell degranulation and release of endogenous mediators are probably involved in the NST-induced antiedematogenic effect.


INTRODUCTION
Inflammation involves the participation of a wide sort of biological mediators, such as histamine, serotonin, bradykinin, and prostaglandins. These inflammatory agents promote vasodilation and neutrophil migration from blood towards inflamed tissues (Hajare et al. 2001).
The interaction between the chemical mediators triggers a sequence of molecular events, Therefore, the present study aims to investigate the antiedematogenic activity induced by N-salicyloyltryptamine (NST) in animal models of acute inflammation, as well as the possible mechanisms of action.

ACUTE TOXICOLOGICAL EVALUATION IN MICE
The toxicological assessment was performed according to the OECD Guideline no. 423 (OECD 2002). Female Swiss mice were separated into groups of 3 animals, and then treated with NST at the dose of 2000 mg/kg (i.p.). Afterwards, the animals were submitted to clinical and behavioral observation during 8 hours during the first day of treatment. Afterwards, the animals were daily observed during 2 h in a period of 14 days.
The edema was expressed as the difference between the final thickness (mm) and the initial thickness (mm) of the paw (Winter et al. 1962). In order to carring out the next protocols of NST-induced antiedematogenic activity, the effective dose of 100 mg/kg was chosen.

INVESTIGATION OF NST EFFECT ON PAW EDEMA INDUCED BY DIF-FERENT PHLOGISTIC AGENTS IN MICE.
Initially, male and female Swiss mice were divided into 4 groups (n=6), and pretreated (i.p.) with vehicle, NST (100 mg/kg) or indomethacin (10 mg/kg). After 30 min, 50μL (1.0 mg/ mL) of dextran, compound 48/80, histamine, serotonin, capsaicin or prostaglandin E2 was administered in the right hind paw (i.pl.). The Sham group (untreated control group) was intraperitoneally treated with vehicle, and after 30 min, 50 μL of sterile saline was administered by subplantar region. The thickness of paws was recorded by digital caliper (Pantec®) immediately before the administration of phlogistic agents (t0), and each 30 min during 2 h.
The edema was expressed as the difference between the final thickness (mm) and the initial thickness (mm) of the paw (Winter et al. 1962).

INVESTIGATION OF NST EFFECT ON THE EX VIVO COMPOUND 48/80-INDUCED MAST CELL DEGRANULATION IN RATS.
The animals were divided into 6 groups (n=6), and then intraperitonally treated with vehicle, NST (50, 100 and 200 mg/kg) or ketotifen (2 mg/kg). After 2 h, the animals were euthanized, the mesenteric bed was removed and placed in tubes containing Ringer solution (10 mL). Afterwards, the mast cell degranulation was induced by incubating the mesenteric beds with 100 μL of compound 48/80 (0.4 μg/mL). The Sham group was pretreated with vehicle, and the mesenteric beds were incubated with 100 μL of distilled water instead of compound 48/80. After 30 minutes of incubation, the mesenteric beds were mounted on glass slides and stained with 0.1% toluidine blue. The intact and degranulated mast cells were counted by randomly choosing 10 fields of each slide. The results were expressed as percentage of degranulated mast cells (Norton 1954).

STATISTICAL ANALYSES
The results were expressed as mean ± standard error of the mean (S.E.M.). The Oneway ANOVA followed by Tukey's post-test was applied for comparison between groups with only one independent variable, and the Two-way ANOVA followed by Bonferroni post-test between groups with more than one independent variable. The level of significance was considered when *p<0.05. All statistical analyses were performed using Prism® version 6.01 software (GraphPad Software, San Diego, CA, USA).

ACUTE TOXICOLOGICAL ASSESSMENT IN MICE
The NST at the dose of 2000 mg/kg (i.p.) showed neither apparent signal of toxicity nor deaths of the animals after 14 days of observation. Therefore, the DL50 value was not possible to be determined.

EFFECT OF NST ON CARRAGEENAN-INDUCED PAW EDEMA IN RATS
The pretreatment of animals with NST (100 mg/kg, i.p.) significantly inhibited the by carrageenan-induced paw edema during the 1st, 3rd, 4th, and 5th hour (*p<0.05), as well as during the 2nd hour with higher level of significance (**p<0.01), when compared with vehicle.
Similarly, the pretreatment with NST (200 mg/kg, i.p.) also significantly decreased the formation of edema during the 1st hour (*p<0.05), and the maximal inhibition was obtained from 2nd to 5th hour of observation (***p<0.001) when compared with vehicle. The indomethacin (10 mg/kg, i.p.) significantly inhibited the formation of edema from the 1st hour (*p<0.05), and remained until the 6th hour (***p<0.001) when compared with vehicle. The NST at the dose of 50 mg/kg (i.p.) did not show significant inhibition of paw edema. Furthermore, any dose of NST inhibited the formation of edema at the 6th hour (Fig. 2, Table I).  , i.pl.). The values are expressed as mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001 when compared with vehicle group. Two-way ANOVA followed by Bonferroni's post-hoc test.

EFFECT OF NST ON SEROTONIN-INDUCED PAW EDEMA IN MICE
The treatment of mice with NST (100 mg/kg) reduced the formation of serotonin-induced paw edema (1 mg/mL) by 38.3%, 40.4% (*p<0.05), and 60.4% (**p<0.01) at times of 30, 60, and 90 min, respectively, when compared with vehicle. A significant inhibition of edema by 65.5%, 59.5%, and 62.5% (***p<0.001) was observed in mice treated with indomethacin (10 mg/kg) during the first 90 minutes compared the vehicle group. However, any treatment was able to significantly reverse the formation of edema during the last hour of observation (*p>0.05) (Fig. 3d).

DISCUSSION
The indole core structure is present in many natural substances mainly derived from plants (Williams 2001  In conclusion, the present study demonstrated that NST is able to decrease the formation of paw edema induced by different phlogistic agents, and the mast cells degranulation, which represent a sort of relevant experimental models of inflammation widely applied in the investigation of novel natural and synthetic products with therapeutic potential in the treatment of inflammatory diseases. Thus, the inhibition of release or action of mediators might be underlying the NST-induced anti-inflammatory effects.