Diazepam

Nitrocellulose paper (Hybond ECL, 0.45 mm) was supplied by Amersham Pharmacia Biotech United Kingdom Ltd. (Buckinghamshire, United Kingdom). Ketamine was from Parke-Davis (São Paulo, SP, Brazil), diazepam and thiopental were from Cristália (Itapira, SP, Brazil). Anti-phospho-IRβ (rabbit polyclonal, SC-25103), anti-IRβ (rabbit polyclonal, SC-711) and NOS2 (mouse polyclonal, SC-7271) antibodies were from Santa Cruz Biotechnology, Inc. Anti-phospho-Akt (rabbit polyclonal, #9271), anti-alpha tubulin (rabbit polyclonal, #2144), and anti-Akt (rabbit polyclonal, #9272) were from Cell Signaling Technology (Beverly, MA, USA). Insulin came from Eli Lilly and Company (Indianapolis, IN, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The PILAB compounds, azide, and triazoles were kindly donated by the Laboratório de Produtos Naturais e Transformações Químicas (IQ-UFRJ, Rio de Janeiro, Brazil). Acetylsalicylic acid (Sigma, Saint Louis, MO, USA), morphine, flumazenil, midazolam, and diazepam (Cristália, Itapira, Brazil) were freshly prepared in dimethyl sulfoxide (DMSO, Cristália) minutes prior to the experiments. Naloxone (Cristália), formaldehyde (Isofar, Duque de Caxias, Brazil), nor-binalthophimine, naltrindole, and CTOP and pentobarbital sodium salt (Tocris Bioscience, Minneapolis, MN, USA) were dissolved in distilled water.

Deuterated dimethyl sulfoxide (DMSO-d₆ purity > 99%), Folin-Ciocalteu reagent, Butylated hydroxytoluene (BHT 99% purity), 2,2-Diphenyl-2-picryl hydrazyl (DPPH purity ≥ 99%), gallic acid and rutin, quercetin (purity of standards ≥ 99%) were all purchased from Sigma-Aldrich. Merck acetonitrile (HPLC purity ≥ 99.9%) was obtained from Merck. The anhydrous sodium carbonate (Na₂CO₃) was obtained from Synth. Aluminum chloride hexahydrate (AlCl₃.6H₂O) was purchased from VETEC. Isoflurane 100% and diazepam (5mg/mL) were obtained from Cristália, heparin 5000 IU/mL from Blaú—Cristal Pharma laboratory, sodium chloride (NaCl 0.9%) was purchased from Sanobiol. The other chemical reagents were purchased from VETEC.

To evaluate the motor performance, mice were submitted to the rotarod test, as previously described [32 (link)]. The rotarod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The bar rotated at a constant speed of 8 revolutions per min. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 120 s [26 (link)]. At the test day, mice from different experimental groups were placed on a rotating rod and the resistance to falling was measured up to 120 s. Mice treated with diazepam (10 mg/kg; Cristália, Itapira, Brazil), the reference drug of the test, were placed on a rotating rod 1 h after treatment. The results are expressed as the average time (s) the animals remained on the rotarod in each group.

Since the behaviors evaluated in nociceptive tests depend on the integrity of the motor capacity of the mice, the motor performance was monitored throughout the experimental period using the rotarod test. We followed the methods of Santos et al. [49 (link)], and the method description partly reproduces their wording. The rotarod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The bar rotated at a constant speed of 8 revolutions per min. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 120 s. On the test day, mice from different experimental groups were placed on a rotating rod, and the resistance to falling was measured up to 120 s. Mice treated with diazepam (10 mg/kg; Cristália, Itapira, Brazil), the reference drug of the test, were placed on a rotating rod one hour after treatment. The results are expressed as the average time (s) the animals remained on the rotarod in each group.

To evaluate the motor performance, mice were submitted to the rota-rod test, as previously described [29 (link)]. The rota-rod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. On the test day, mice from different experimental groups were placed on the rotating rod (eight revolutions per min) and the falling avoidance was measured for up to 120 s. Mice treated with diazepam (10 mg/kg; Cristália, Itapira, SP, Brazil), the test reference drug, were placed on a rotating rod 1 h after treatment. The results were analyzed as the average time (s) the animals remained on the rota-rod in each group.

In the present study riparins was synthesized from substituted synthetic benzoic acids using methodology previously described by Barbosa-Filho and co-workers [19 ]. Riparin IV (chemical name N-[8′-(4′-methoxyphenylethyl)]-3,4,5-trimethoxybenzoylamide), is a synthetic amide modeled on the natural riparins isolated from Aniba riparina. Dexamethasone, indomethacin, complete Freund’s adjuvant (CFA), lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were obtained from Sigma Chemical Company (St. Louis, MO, USA). Diazepam and morphine were obtained from Cristália (Itapira, SP, Brazil). Riparins were dissolved in 5% Tween 80 in distilled water. indomethacin was dissolved in Tris HCl 0.1 M pH 8.0 plus distilled water. Dexamethasone was dissolved in ethanol 10% in distilled water, and remaining substances were dissolved directly in distilled water. Test compounds were administrated by intraperitoneal (ip) route 40 min before testing, and the control group only received vehicle.