Seasonal and Organ-Specific Variations of Alkaloids in Buxus obtusifolia (Mildbr.) Hutch: A Multivariate LC/MS Study

Buxus obtusifolia (Mildbr.) Hutch is an evergreen shrub endemic to East Africa and is traditionally used to treat chest ailments. Our recent investigation on the dichloromethane leaf extract of this species yielded several aminosteroid alkaloids, some of which demonstrated promising in vitro antiprotozoal activity warranting more detailed studies on this interesting plant and its bioactive constituents. Given that abiotic factors are known to influence the biosynthesis and accumulation of plant secondary metabolites, this study aimed to investigate seasonal and organ-specific variability in the alkaloid profile of B. obtusifolia to gain insights into the dynamics of their formation and, potentially, obtain hints at the best times to harvest individual alkaloids. Consequently, leaf and twig samples were collected each month from the same population over a period of one year and analyzed using ultra-high-performance liquid chromatography coupled with positive-mode electrospray ionization double quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI⁺-QqTOF-MS/MS). The resulting data, after conversion to (<t_R:m/z>) variables, were analyzed by principal component analysis (PCA) to characterize variations in the metabolite profile. Evaluation of the first three principal components revealed clear differences between leaves and twigs, as well as subtle overall seasonal changes with some distinct dry-season clustering. A volcano plot was used to further analyze the differences between the minor constituents of the two organs. In total, 15 aminosteroid alkaloids were identified as key contributors to these differences. This represents the first seasonal and organ-specific phytochemical variability investigation in B. obtusifolia. Thus, this study offered the first valuable insights into the possible association of some abiotic factors and the phytochemical profile of this plant. Studies including further populations of this species from different locations will have to show whether the present findings allow general conclusions with respect to the investigated compounds' accumulation in response to external factors. Furthermore, the present results represent a basis to delineate the optimal harvest period for targeted isolation of larger quantities of bioactive aminosteroids for further development.