Quantitative Removal of Pyrrolizidine Alkaloids from Essential Oils by the Hydrodistillation Step in Their Manufacturing Process.

Pyrrolizidine alkaloids are naturally occurring toxins produced by certain weeds that can, if accidentally co-harvested, contaminate plant-based food, feed, and herbal medicinal products. Focusing on herbal medicinal products, the presence of pyrrolizidine alkaloids is restricted by regulatory prescribed thresholds to assure patient safety. Among the multitude of different herbal active substances utilized in herbal medicinal products, the class of pharmaceutically effective essential oils is considered to exhibit a negligible contribution to pyrrolizidine alkaloid contamination. Within the present investigation, this hypothesis should be scientifically scrutinized. For this purpose, an experimental set-up was chosen that reproduces the typical manufacturing step of hydrodistillation. Essential oils of eucalyptus and lemon were selected exemplarily and spiked with 3 representative pyrrolizidine alkaloids (retrorsine, retrorsine-N-oxide, and lycopsamine), whereupon hydrodistillation was performed. Analysis of the resulting distillates by LC-MS/MS proved that artificially added pyrrolizidine alkaloids were removed completely. Moreover, quantitative pyrrolizidine alkaloid recovery in the aqueous phases was observed. Hence, it was experimentally confirmed that herbal medicinal products employing hydrodistilled essential oils of pharmaceutical quality are intrinsically free of pyrrolizidine alkaloids due to the particularities of their manufacturing process. Furthermore, it can be concluded from theoretical considerations that essential oils produced by cold pressing have a negligible risk of carrying pyrrolizidine alkaloid contamination. Our findings provide a strong indication that the requirement for analytical pyrrolizidine alkaloid testing of essential oils for pharmaceutical use should be fundamentally reconsidered.

Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2.

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M-U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed.