Description: Abstract ETHNOPHARMACOLOGICAL RELEVANCE: The oleoresin of Brazilian Copaifera reticulata is a traditional remedy used for the treatment of skin and urinary tract infections, respiratory diseases, rheumatism, ulcer and tumours; thus, playing an important role in the primary health care of the indigenous population. AIM: As most previous pharmacological tests used the crude oleoresin and only a few studies so far dealt with enriched fractions or pure chemically defined compounds, the aim of this study was to systematically evaluate the antimicrobial and cytotoxic properties of the Copaifera reticulata oleoresin and to assign traditional uses to specific secondary metabolites. MATERIALS AND METHODS: The oleoresin, as well as its neutral and acidic fractions were tested for their activity against six cancer cell lines, two clinically relevant bacterial strains, and two dermatophytes. Both fractions were analysed by GC-MS and UHPLC-ELSD, respectively. The antibacterial acidic phase was further fractionated by preparative chromatography to purify and characterize the compounds responsible for the observed pharmacological effect. RESULTS: Whereas no cytotoxic activity was detected, the crude oleoresin and its acidic fraction showed antibacterial activity against gram-positive bacteria Enterococcus faecium (IC50 values 4.2 and 4.8 µg/mL, respectively) and methicillin-resistant Staphylococcus aureus (MRSA, IC50 values 5.3 and 7.2 µg/mL, respectively). Purification of the acidic fraction of the C. reticulata oleoresin yielded two dicarboxylic diterpene acids and the four main diterpene acids, comprising three different diterpene scaffolds. Interestingly, the activity was not restricted to a particular diterpene-type but rather depended on the compounds' lipophilicity, with the most active constituent showing IC50 values of 1.6 (E. faecium) and 2.5 µg/mL (MRSA), respectively. Furthermore, ent-polyalthic acid, the major diterpenoid, was significantly active against dermatophytes with IC50 values of 6.8 µg/mL (Trichophyton rubrum) and 4.3 µg/mL against (T. mentagrophytes). CONCLUSION: The present study proved the antimicrobial effects of the C. reticulata oleoresin and its diterpenoid constituents, confirming its wide use in folk medicine for the treatment of skin and urinary tract infections. The inhibitory activity of copaiba diterpenoids against dermatophytic fungi as well as the gram-positive bacteria E. faecium and MRSA is being reported for the first time, providing potential lead structures for the treatment of these clinically relevant bacterial strains.

Description: Onychomycosis caused by, e.g., Trichophyton rubrum or Candida albicans is the most common human nail disease with a worldwide prevalence of more than 10%. The therapeutic efficacy of topical antimycotics for the treatment of onychomycosis proved to be inadequate in numerous studies on patients. The main reasons are, above all, the poor bioavailability of the active ingredients in the nail compartment, causing the requirement for extremely long application periods and correspondingly high demands on adherence by the patient. In the present study, we aimed to develop a more effective and prompt photodynamic approach for the treatment of onychomycosis. The principle of photodynamic therapy (PDT) for onychomycosis has already been investigated. However, these studies used photosensitizers such as methylene blue, which were neither optimized for their keratinophilic features nor for their bioavailability in the nail. Hence, we initiated a screening campaign using T. rubrum and C. albicans cell-based assays, infected bovine keratin models, and keratin-penetrating irradiation to identify suitable hit compounds for a PDT approach toward onychomycosis. Here, we report on the discovery of Henna/Lawson-derived keratinophilic naphthazarines that act as highly potent PDT antimycotic photosensitizers with photoresponsiveness when irradiated by light at a keratin-permeable wavelength (〉500 nm, e.g., compounds 10 and 11 with PDT-IC50 = 1 and 3 nM, respectively, against T. rubrum), hence with superior efficacy than the positive controls nystatin and clotrimazole. Notably, our photodynamic approach not only affected the actual pathogens but also prevented reinfection of keratin models within 10 days, suggesting an additional efficacy against fungal spores. Compared to established concepts, our proposed PDT approach using the novel naphthazarine photosensitizers could enable an effective, precise, and sustainable therapy option for the future treatment of onychomycosis.

Description: The Antarctic deep-sea sponge Latrunculia (Latrunculia) biformis Kirkpatrick, 1908 (Class Demospongiae Sollas, Order Poecilosclerida Topsent, Latrunculiidae Topsent) was selected for chemical analyses due to its potent anticancer activity. Metabolomic analysis of its crude extract by HRMS/MS-based molecular networking showed the presence of several clusters of pyrroloiminoquinone alkaloids, i.e., discorhabdin and epinardin-type brominated pyridopyrroloquinolines and tsitsikammamines, the non-brominated bis-pyrroloiminoquinones. Molecular networking approach combined with a bioactivity-guided isolation led to the targeted isolation of the known pyrroloiminoquinone tsitsikammamine A (1) and its new analog 16,17-dehydrotsitsikammamine A (2). The chemical structures of the compounds 1 and 2 were elucidated by spectroscopic analysis (one-dimensional (1D) and two-dimensional (2D) NMR, HR-ESIMS). Due to minute amounts, molecular modeling and docking was used to assess potential affinities to potential targets of the isolated compounds, including DNA intercalation, topoisomerase I-II, and indoleamine 2,3-dioxygenase enzymes. Tsitsikammamines represent a small class of pyrroloiminoquinone alkaloids that have only previously been reported from the South African sponge genus Tsitsikamma Samaai & Kelly and an Australian species of the sponge genus Zyzzya de Laubenfels. This is the first report of tsitsikammamines from the genus Latrunculia du Bocage and the successful application of molecular networking in the identification of comprehensive chemical inventory of L.biformis followed by targeted isolation of new molecules. This study highlights the high productivity of secondary metabolites of Latrunculia sponges and may shed new light on their biosynthetic origin and chemotaxonomy