Marine organisms, particularly cyanobacteria, are important resources for the production of bioactive secondary metabolites for the treatment of human diseases. In this study, a bioassay-guided approach was used to discover metabolites with lipid-reducing activity. Two chlorophyll derivatives were successfully isolated, the previously described 13²-hydroxy-pheophytin a (1) and the new compound 13²-hydroxy-pheofarnesin a (2). The structure elucidation of the new compound 2 was established based on one- and two-dimensional (1D and 2D) NMR spectroscopy and mass spectrometry. Compounds 1 and 2 showed significant neutral lipid-reducing activity in the zebrafish Nile red fat metabolism assay after 48 h of exposure with a half maximal effective concentration (EC₅₀) of 8.9 ± 0.4 µM for 1 and 15.5 ± 1.3 µM for 2. Both compounds additionally reduced neutral lipid accumulation in 3T3-L1 multicellular spheroids of murine preadipocytes. Molecular profiling of mRNA expression of some target genes was evaluated for the higher potent compound 1, which indicated altered peroxisome proliferator activated receptor gamma (PPARγ) mRNA expression. Lipolysis was not affected. Different food materials (Spirulina, Chlorella, spinach, and cabbage) were evaluated for the presence of 1, and the cyanobacterium Spirulina, with GRAS (generally regarded as safe) status for human consumption, contained high amounts of 1. In summary, known and novel chlorophyll derivatives were discovered from marine cyanobacteria with relevant lipid-reducing activities, which in the future may be developed into nutraceuticals.

Obesity is a complex disease resulting in several metabolic co-morbidities and is increasing at epidemic rates. The marine environment is an interesting resource of novel compounds and in particular cyanobacteria are well known for their capacity to produce novel secondary metabolites. In this work, we explored the potential of cyanobacteria for the production of compounds with relevant activities towards metabolic diseases using a blend of target-based, phenotypic and zebrafish assays as whole small animal models. A total of 46 cyanobacterial strains were grown and biomass fractionated, yielding in total 263 fractions. Bioactivities related to metabolic function were tested in different in vitro and in vivo models. Studying adipogenic and thermogenic gene expression in brown adipocytes, lipid metabolism and glucose uptake in hepatocytes, as well as lipid metabolism in zebrafish larvae, we identified 66 (25%) active fractions. This together with metabolite profiling and the evaluation of toxicity allowed the identification of 18 (7%) fractions with promising bioactivity towards different aspects of metabolic disease. Among those, we identified several known compounds, such as eryloside T, leptosin F, pheophorbide A, phaeophytin A, chlorophyll A, present as minor peaks. Those compounds were previously not described to have bioactivities in metabolic regulation, and both known or unknown compounds could be responsible for such effects. In summary, we find that cyanobacteria hold a huge repertoire of molecules with specific bioactivities towards metabolic diseases, which needs to be explored in the future.

Vitamin K isoforms are known as co-factors for the synthesis of blood-clotting proteins, but several other bioactivities were reported.

Cyanobacteria have demonstrated their therapeutic potential for many human diseases. In this work, cyanobacterial extracts were screened for lipid reducing activity in zebrafish larvae and in fatty-acid-overloaded human hepatocytes, as well as for glucose uptake in human hepatocytes and ucp1 mRNA induction in murine brown adipocytes. A total of 39 cyanobacteria strains were grown and their biomass fractionated, resulting in 117 chemical fractions. Reduction of neutral lipids in zebrafish larvae was observed for 12 fractions and in the human hepatocyte steatosis cell model for five fractions. The induction of ucp1 expression in murine brown adipocytes was observed in six fractions, resulting in a total of 23 bioactive non-toxic fractions. All extracts were analyzed by untargeted UPLC-Q-TOF-MS mass spectrometry followed by multivariate statistical analysis to prioritize bioactive strains. The metabolite profiling led to the identification of two markers with lipid reducing activity in zebrafish larvae. Putative compound identification using mass spectrometry databases identified them as phosphatidic acid and aromatic polyketides derivatives—two compound classes, which were previously associated with effects on metabolic disorders. In summary, we have identified cyanobacterial strains with promising lipid reducing activity, whose bioactive compounds needs to be identified in the future.

Obesity is one of the major public health concerns in our societies. This condition increases the risk of developing several chronic diseases, such as diabetes (T2DM) and cardiovascular diseases, among others. Complementary strategies are necessary for the treatment of obesity, which can include pharmacological treatments along with lifestyle adjustments. Cyanobacteria are an ancient group of gram-negative photosynthetic prokaryotes regarded as a prolific source of biologically active natural compounds. In this work, a bioassay-guided isolation strategy is used to explore the potential of secondary metabolites from cyanobacteria with beneficial activities towards obesity. Therefore, three strains of cyanobacteria belonging to Chroococcales and Synechococcales orders were cultured and harvested. Their freeze-dried biomass was extracted using a mixture of dichloromethane and methanol (2:1). The resultant organic extract was submitted to vacuum liquid chromatography, to generate fractions, which were tested against zebrafish Nile red fat metabolism assay. Afterwards, more (sub)fractions were originated, either with flash chromatography, preparative TLC, reverse-phase HPLC, or normal phase solid-phase extraction. The chemical nature of the fractions was followed by spectroscopic techniques (NMR). At the moment, some fractions of strains display interesting anti-obesity activities, by reducing 40% to 60% of the neutral lipids in the zebrafish larvae, with statically significant difference from control. Work is ongoing to isolate and purify the active compounds, in order to elucidate their chemical structures.

Environmental and lifestyle adaptations over the last decades have contributed to enhanced man´s longevity, however it also paved the ground for different diseases to unfold. Today one of the main public health challenges is obesity and its related metabolic dysfunctions. In recent years, the pharmacological options are reported as being insufficient, therefore, the discovery and pharmacological development of new therapeutic approaches are required to overcome this epidemic. Vitamin K are a fat-soluble family of compounds implicated in a number of essential biological functions. Vitamin K1 and K2 are two naturally occurring compounds, while vitamin K3 is the most common synthetic form. A bioactivity-guided approach was used with the Nile red fat metabolism assay in zebrafish larvae to successfully isolate an analog of vitamin K1 for the first time from Tychonema sp. LEGE 07196. The structure confirmation was based on NMR spectroscopy and mass spectrometry. This analog was first isolated in 1965 from the cyanobacteria Synechococcus elongatus, but little is known on its bioactivity. The anti-obesity effects of all vitamin K forms, including the K1-analog, were studied. The compounds did not cause any general toxicity or malformations and showed significant neutral lipid-reducing activity after 48 h of exposure. The different vitamin forms displayed different levels of activity which shows the importance of the naphthoquinone ring, as well as the impact of the saturation and polarity of the aliphatic side-chain for the structure–activity relationship. Metabolomics approaches were employed to establish the distinct bioavailability and biotransformation of the different forms of vitamins in the organisms. The metabolite profiling was achieved using different databases and MetaboAnalyst was used for associated analysis. The organism has a clear preference to transform the various vitamins into K1 and K1-analog forms, regardless of the supplementation, and each exposed vitamin significatively altered the expression of different metabolites indicating that different metabolic targets are involved. This work is ongoing, and the final aim is to understand the effects of vitamin K family on obesity and related co-morbidities, which could lay the ground to develop a future nutraceutical with lipid reduction activity.