Isolation, Purification, and Characterization of the Antinociceptive Fraction From Caspian Cobra ( <i>Naja naja oxiana</i> ) Snake Venom by Chromatographic and Electrophoretic Methods and Using Animal Model of Formalin‐Induced Pain

ABSTRACT Neurotoxins isolated from the venom of cobras have been shown to alleviate both acute and chronic pain. The venom of the Caspian cobra ( Naja naja oxiana ) snake, which is a highly venomous member of the Elapidae family, has demonstrated analgesic effects. This study presents the behavior, purification, and isolation of the antinociceptive fraction from the Naja venom for the first time in an experimental pain model. To obtain the fraction of interest, the venom was fractionated using size exclusion (gel filtration) on Sephadex‐G50, ion‐exchange chromatography on carboxymethyl‐sepharose, and RP‐HPLC. After each step, fractions were assessed for protein concentration using the Lowry method, the median lethal dose via the Spearman–Karber method, and antinociceptive properties by intraplantar injection of a 5% formalin solution (50 µL) into the right hind paw following intraperitoneal administration of the venom and its fractions in rats. Normal saline and morphine (5 mg/kg) were used as negative and positive controls (standard), respectively. The purity level of isolated fractions was confirmed by RP‐HPLC and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The protein content and the lethal dose of the venom were determined to be 96% and 414.9 µg/kg, respectively. The F3 fraction obtained from gel filtration, F38 from the ion‐exchange chromatography, and F381 fraction collected from the RP‐HPLC C18 column showed significant analgesic activity against formalin 5% as a nociceptive agent compared to the saline‐administered control. The median lethal doses of F3, F38, and F381 were 2985, 2448, and 1380 µg/kg, respectively. This study successfully isolated and purified an antinociceptive fraction (F381) from N. naja oxiana venom, demonstrating its ability to inhibit formalin‐induced pain in rats. Suggesting its potential antinociceptive activity in animal models of pain.