Bioassay-guided isolation of insect repellent compounds from Ligusticum porteri root extract

In our program of screening natural products against the pests of medical and veterinary importance, ethanolic extract of the roots of Ligusticum porteri J.M.Coult. & Rose showed significant repellency against mosquitoes. The extract was then fractionated to test different fractions to identify the active repellent compounds. This testing resulted in the isolation of different compounds including (Z)-3-butylidenephthalide, (E)-3-butylidenephthalide, and a mixture of (E/Z)-3-butylidenephthalide. Biting deterrence of all these compounds was similar to N,N-diethyl-3-methylbenzamide (DEET) against Aedes aegypti (L.) in Klun and Debboun (K & D) bioassay. (E/Z)-3-butylidenephthalide which is a mixture of the two compounds was further tested in Ali & Khan (A & K) bioassay. Based on these data repellency of this compound was similar whereas the MED values of the mixtures of (E/Z)-3-butylidenephthalide with carotol were lower (6.25 + 6.25 = 12.5 mu g/cm(2)) than individual treatments (25 mu g/cm(2)). In in vivo (direct skin application bioassay), (E/Z)-3-butylidenephthalide showed excellent repellency. The residual repellency of (E/Z)-3-butylidenephthalide at 8 and 16 % application rates was 4.5 and 10-h respectively which was equal to or better than DEET with the residual time of 5 and 9-h, respectively. The mixture of (E/Z)-3-butylidenephthalide with carotol (8 + 8 %) increased the residual repellency by 2-h (44 %) as compared to (E/Z)-3-butylidenephthalide alone at a dose of 8 %. These data indicated that (E/Z)-3-butylidenephthalide is an effective mosquito repellent that is stable and has a long shelf life. The activity of this compound is extraordinary and residual time is comparable to DEET. In vivo data demonstrated an enormous potential of (E/Z)-3-butylidenephthalide as a repellent that can be developed for commercial use. However, (E/Z)-3-butylidenephthalate was found in lower amounts of the L. porteri essential oil.