Phytochemical Investigation and Reproductive Capacity of the Bulgarian Endemic Plant Species Marrubium friwaldskyanum Boiss. (Lamiaceae)

Marrubium friwaldskyanum Boiss (Lamiaceae) is a Bulgarian endemic species. Overall, the essential oil (EO) composition of M. friwaldskyanum was different from that of the other Marrubium species reported in the literature. The main EO constituents of M. friwaldskyanum were (E)-caryophyllene, germacrene D, and caryophyllene oxide. The effect of the harvest stage was significant only on alpha-copaene, (E)-caryophyllene, caryophyllene oxide, and tau-muurolol. The concentration of alpha-copaene (1.26-1.83% range of the total oil), (E)-caryophyllene (31-41%), caryophyllene oxide (6.4-11.8%), and tau-muurolol (1.3-2.8%) were the highest at 2-3 pair of leaves or before flowering and lower at flowering. The harvest stage did not significantly affect the concentrations of the other six identified EO compounds beta-bourbonene (1.1%), alpha-humulene (2.8%), germacrene D (23.3%), bicyclogermacrene (2.85%), delta-cadinene (1.1%), and spathulenol (2.8%). In a separate experiment, grinding of the biomass prior to EO extraction had a significant effect only on the concentrations of D-limonene (0.24-3.3%) and bicyclogermacrene (3.6-9.1%). Grinding in water or without water, maceration, and addition of Tween(R)20 had rather small effects on the EO profile. The identified EO constituents and their mean concentrations in this experiment were (E)-caryophyllene (25.4%), germacrene D (17.6%), caryophyllene oxide (9.1%), spathulenol (6.5%), tau-muurolol (5.0%), carvacrol (3.9%), alpha-copaene (2.5%), beta-bourbonene (2.5%), delta-cadinene (2.4%), alpha-humulene (1.8%), and Z-beta-farnesene (1.3%). Embryological studies observed anther and the development of the male gametophyte and ovule and development of the female gametophyte of M. friwaldskyanum. Furthermore, pollen and seed viability assays were conducted, and mass spectrometry-based metabolomics analysis of an extract from shoots revealed the presence of 45 natural products, identified as flavonoids, phenolic acids, and (tri)terpenoids. Overall, the phytochemistry and some of the microscopic analyses distinguished this endemic species from other species in Marrubium.