Bioactivity-guided identification and isolation of a major antimicrobial compound in Cistus creticus subsp. creticus leaves and resin ladano

Cistus creticus subsp. creticus is a shrubby Mediterranean plant used since ancient times in folk medicine for the treatment of different diseases. C. creticus extracts and resin contain different types of secondary metabolites, such as terpenoids (predominantly labdane type diterpenoids), and phenylpropanoids. Growth conditions seem to influence the content of labdane-type diterpenes and flavan-3-ols in leaves of C. creticus subsp. creticus. Histochemical staining of leaves' trichomes and comprehensive phytochemical characterization of resin, leaves and their exudates, indicated that long-stalked capitate trichomes of C. creticus subsp. creticus, grown both in vitro (IV) and in greenhouse (GH), are capable of producing bioactive oleoresin-related terpenoids and phenylpropanoids compounds. Bioactivity-guided approach was implemented in search for the major antibacterial compound in C. creticus resin against two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus cereus and Micrococcus flavus). Bioautographic assay on TLC plates with separated components of Cistus resin extract, revealed a pronounced zone of microbial growth inhibition, corresponded to a highly active compound with Rf values of 0.45, structurally characterized and identified as ent-3 beta-acetoxy-13-epi-manoyl oxide. This finding opens the route for focusing on isolation and functional characterization of genes involved in the biosynthesis of ent-3 beta-acetoxy-13-epi-manoyl oxide and its precursor ent-3 beta-hydroxy-13-epi-manoyl oxide, with the aim to establish sustainable in vitro biotechnological protocols for its large-scale production in homologous and heterologous plant and microbial hosts.

Automated summary

Cistus creticus subsp. creticus, a Mediterranean shrub, is traditionally used in folk medicine and contains various secondary metabolites. The trichomes of the plant produce bioactive compounds, including the antibacterial compound ent-3 beta-acetoxy-13-epi-manoyl oxide. This discovery provides a foundation for further research on the antibacterial properties and biosynthesis of this compound.