Gloriosa superba L. (Liliaceae) seeds, known as "kalihari" (Hindi), were phytochemically investigated for colchicine (well known for gout treatment) and other related alkaloid content. Colchicine, 2- demethylcolchicine, 3-demethylcolchicine, and N-formyl-N-deacetylcolchicine were alkaloids isolated from the seeds. The isolated samples have been standardized for their purity with respect to the reference standard using HPLC. The structures were confirmed by NMR spectroscopy and were analyzed by spiking them along with colchicine reference by HPLC. The purity of colchicine, 2- demethylcolchicine, 3- demethylcolchicine and N-formyl-N-deacetylcolchicine were 99.82, 96.78, 98.71, and 98.13% respectively. The compounds were subjected to an anti-inflammatory study by using the formaldehyde inflammagen-induced inflammation model. Oral administration of colchicine at 2, 4, and 6 mg/kg body weight resulted in 48.9, 68.7, and 79.1% inhibition respectively, while 30.9% inhibition was seen in the phenylbutazone 100 mg/kg treated group once daily for a period of 4 days. The results clearly indicated that the colchicine is more effective as an anti-inflammatory agent compared with phenylbutazone, the standard drug used in the study, whereas the oral administration of 6 mg/kg body weight of 2- demethylcolchicine, 3-demethylcolchicine and N-formyl-N-deacetylcolchicine showed very poor activity (41.6, 40.4, and 41.1% activity respectively).