Honey, bees, and a hepatotoxic alkaloid echimidine

Abstract

Experimental E. plantagineum grown in Connecticut was extracted with MeOH and after removal of MeOH, the resulting oleoresin was defatted by partitioning between hexane-MeOH-water (10:9:1). The lower phase was partially concentrated and diluted with water. The pH was adjusted to 2 by adding H2SO4 and zinc dust was added to reduce the N-oxides during stirring. After 20 hr the reaction mixture was made alkaline with Na2CO3 and the alkaloids were extracted with ethyl acetate. This extract was again partitioned agains acidic water and the aqueous layer was re-extracted after addition of Na2CO3. This extract was evaporated to produce a crude mixture of pyrrolizidine alkaloids. Echimidine was separated from other alkaloids by Centrifugal Partition Chromatography (FCPC Kromaton A100, Rousellet Robatel, Fr.) using chloroform as the mobile phase and citrate buffer at pH 5.6 as the stationary phase. Analytical HPLCs were performed on a Kinetex EVO C18, 5µm, 100°A, 150 x 4.6 mm, and preparative on a Kinetex EVO C18, 5µm, 100°A, 150 x 21.2 mm column (Phenomenex). The mobile phase buffer was the same as for the preparative HPLC. The samples were eluted from the analytical column on a gradient from 5% acetonitrile to 16% acetonitrile, followed by a column wash with 29% acetonitrile. The flow rate was 1.0 ml/minute. The UV absorbance signal on the diode array detector was monitored at 210 nm. The mobile phase contained a buffer of 32 mM lithium phosphate, adjusted to pH 7.2 with phosphoric acid. The column was equilibrated in 32 mM lithium phosphate, pH 7.2/acetonitrile (9:1) and run in a gradient from 10% to 20% MeCN over 14.4 min at 20 ml/min. For preparative runs, samples of 60 mg were injected in DMF/methanol/water/acetic acid (1:1:1:0.003) to a concentration of 100 mg/ml. Fig. 2b shows how the cuts were determined. The total of 1.3 grams of CPC-fractionated material was loaded in 23 preparative HPLC runs.