Morusin

Based on Jung et al.’s paper [1 (link)], cell viability assay was conducted in HCT116 and SW480 CRCs by using MTT assay. Briefly, HCT116 and SW480 cells (1 × 10⁴ cells/well) were exposed to Morusin (0, 2.5, 5, and 10 μM) for 24 h and incubated with MTT (1 mg/mL) (Sigma-Aldrich, Saint Louis, MO, USA) for 2 h. The viability was calculated with optical density (OD) values as a percentage of viable cells in Morusin treated group versus untreated control. All assays were conducted in independent triplicates.

After one week period of acclimation the rats were divided into six groups (n = 8). All of the animals were fasted for 24 h prior to the induction of colitis. Colitis was induced by a single dose of TNBS (50 mg/kg; Sigma-Aldrich, Steinheim, Germany) dissolved in ethanol (50% v/v; total instilled volume: 1 mL/kg of rat body weight). TNBS was administered rectally using a rubber catheter inserted 8 cm proximal to the anus under light isoflurane anesthesia [14 (link)]. The rats were kept in a head-down position until they recovered from the anesthesia to prevent leakage of the instillation. The intact group received 0.9% saline instead of TNBS solution. Administration of the test compounds began one day after the induction of colitis and continued every 24 hours for five consecutive days. The compounds were suspended in a 10% gel of polyvinylpyrrolidone (PVP K30; Sigma-Aldrich) and were administered by gastric gavage at bolus doses of 12.5, 25, or 50 mg/kg of morusin and 50 mg/kg of sulfasalazine (Sigma-Aldrich). Rats in the untreated group received the vehicle (PVP K30) only. The animals were killed on the seventh day of the experiment by using an overdose of the veterinary euthanasia drug T61 (Intervet International B. V., Boxmeer, Netherlands).

Ultra-pure water (<5 µg/L TOC, (Total Organic Carbon) was obtained from a water purification system Arium 126 61316-RO, plus an Arium 611 UV unit (Sartorius, Goettingen, Germany). Methanol (HPLC grade) and formic acid (puriss. p.a. for mass spectrometry) from J.T. Baker (Phillipsburg, NJ, USA) were obtained. Folin-Ciocalteu (FC) reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride hexahydrate, 2,4,6-tris(2-pyridyl)-s-triazine, trolox, quercetin, gallic acid, DMSO, and HPLC standards (caffeic acid phenethyl ether (CAPE), galangin, morusin, naringenin, pinocembrin, rhamnetin and shikonin, with purity higher than 95% by HPLC) were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA) or Extrasynthèse (Genay, France). Cefotaxime was from Argentia^® (Bristol-Myers Squibb, Buenos Aires, Argentina). Mueller-Hinton broth was provided by Laboratorio Britania (Buenos Aires, Argentina).