Honey, bees, and a hepatotoxic alkaloid echimidine

Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L, (Jane's Salvation, Patersons Curse), endemic to Australia. The plant is a great attractant for bees and as a result echimidine finds its way into honey. Because of its hepatotoxicity, echimidine became a target of new EU regulation requiring testing of imported honey. Under normal RP HPLC conditions echimidine produces always a sharp peak. Only recently, we discovered that this single peak represents actually a mixture of three alkaloids. Using relatively new “core-shell” RP HPLC column (Kinetex EVO C18, Phenomenex) in a buffer system we resolved “echimidine” into two well separated peaks. An NMR analysis proved that later eluting peak belonged indeed to echimidine, while the earlier peak contained two, largely unresolved alkaloids echihumiline (major) and hydroxymyoscorpine (minor). Each of them has been isolated before from other plants. All these alkaloids are isomeric C₂₀H₃₁NO₇ and produce in MS a single MH⁺, signal at m/e 398, in and their NMR spectra are similar, which explains why they have not been detected before. Examination of several of echimidine samples derived from different plant collections revealed that the contribution of the early peak varied from 13% to 43%. Considering that these alkaloids may contribute unequally to the hepatotoxicity, effects of both, purified echimidine and the mixture of echihumiline and hydroxymyoscorpine were compared in rat hepatocytes.