extraction, isolation and characterization of compounds from euphorbia prostrata and their analgesic properties

Abstract

Pain is a symptom that results from particular physiological processes as injurious stimuli characteristic of cell injury or disease. Several herbs including Phyllanthus lawii, Diallium guineense, Jathropha gossypiofolia linn, Pangamia pinnata linn and Papaver somniferrum have been reported for use to relieve various aches, such as headaches, toothache, lower back pain and neuralgia. The objectives of this study were to extract, isolate, characterize and determine the analgesic efficacy of compounds from Euphorbia prostrata. The plant was collected from its natural habitat in Uasin-Gishu County and samples processed as whole plant by air drying before grinding. Serial extraction was carried out by soaking ground powder in three organic solvents (hexane, ethyl acetate and acetone) of increasing polarity each for 48 hours before filtering and rotary evaporation to dryness. Testing for analgesic activity was then carried out by Tail Immersion Model using albino rats. The active extracts were packed on silica gel column for chromatographic separation. Thin Layer Chromatography (TLC) was used to monitor the fractions with the help of visualization reagents. Further bioassay was carried out for the isolated fractions before purification by crystallization. Pure isolated fraction with the highest analgesic activity was then characterized by UV-Vis, Fourier Transform-Infra Red spectroscopy (FT-IR) and Gas Chromatography-Mass Spectroscopy (GC-MS). Results showed that ethyl acetate crude extracts afforded best yield of 5.80%. The highest analgesic activity was from ethyl acetate crude extract which significantly (p> 0.05) increased the pain reaction time (PRT) to10.40 seconds compared to positive control (Declofenac) that was 8.04seconds. Isolation by column chromatography afforded four fractions: H 1 , H 2 , H 3 and H 4 from hexane crude extract and five fractions: E1, E 2 , E 3 , E 4 and E 5 from ethyl acetate crude extracts. Further bioassay and purification of the fractions was done to yield fraction A which had optimum PRT of 6.21 seconds and UV λ max of 259 nm. By using spectroscopic methods, fraction A showed steroidal characteristics. This was confirmed from FT-IR strong peaks of the fraction A at 3480 cm -1 (OH group, alcohols), 3150-3400 cm -1 (C-H stretching, aliphatic), 1650 cm -1 (C=C, alkene), 1400 cm -1 (CH 2 ), 1050 cm -1 (cycloalkane) and 870 cm -1 (C-H vibration for unsaturated part) as characteristic functional groups for steroids. GC-MS results showed pronounced molecular peaks at m/z 412, 414 and 426 for retention time of 12.26, 13.04 and 13.21 minutes respectively. With the help of data obtained, fraction A is most probably having a mixture of stigmasterol, β-sitosterol and cholest-5-en-3-ol, 24 propylidene. Analgesic activity of Euphorbia prostrata is attributed to the presence of steroids. E. prostrata is worth consideration for exploration in the quest for lead compounds in drug discovery particularly analgesics.