Untargeted Metabolomic to Access Chemical Differences Induced by Dual Endophyte Cultures Isolated from Euphorbia Umbellata.

Endophytic fungi live asymptomatically inside vegetal tissues, and such uncommon habitat contributes to their exceptional chemical diversity. Isolating natural products from endophytic fungi could fail due to silent biosynthetic gene clusters under ordinary in vitro culture conditions, and co-culturing has been assayed to trigger their metabolism. We carried out single and dual cultures with 13 endophyte strains isolated from Euphorbia umbellata leaves. Multivariate statistics applied to untargeted metabolomics compared the chemical profiles of all endophyte cultures. PCA analysis guided the selection of the Aspergillus pseudonomiae J1 - Porogramme brasiliensis J9 dual culture for its most significant chemical differentiation: Five compounds were putatively annotated in the J1-J9 culture according to UHPLC-HRMS data, kojic acid, haliclonol and its diastereoisomer, caffeic acid, and 2-(3,4-dihydroxyphenyl)acetaldehyde. Analysis by PLS-DA using VIP score showed that kojic acid displayed the most significative importance in discriminating single and dual J1-J9 cultures.

Fungal biocatalysts for labdane diterpene hydroxylation.

Labdane diterpenes and their derivatives have shown remarkable biological activities and are useful as chiral building blocks for the synthesis of a variety of bioactive compounds. There is great interest in developing biocatalyst technology to achieve regio- and stereoselective hydroxylation of unactivated C-H bonds in complex natural products, since the functionalization of unactivated C-H bonds generally requires hard reaction conditions and highly reactive oxidizing agents, which are limited regarding the control of regio- and stereoselectivity. Filamentous fungi are efficient biocatalysts capable of catalyzing a wide variety of hydroxylation reactions, and the use of whole cell biocatalysts provides advantages regarding cofactor regeneration and is much less expensive. Therefore, the goal of this study was to select biocatalysts to develop biotransformation processes that can be scalable under mild reaction conditions for hydroxylation of a labdane diterpene, 3ß-acetoxy-copalic acid, which contains the trans-decalin moiety and a side chain dienic system appropriate for the preparation of a variety of compounds. Biotransformation processes were carried out and five filamentous fungi were selected as capable of producing hydroxylated diterpenes at positions C-3, C-6, C-7 and C-18 of the trans-decalin moiety and C-13 of the side chain dienic system. Hydroxylation reactions occurred with regio- and stereoselectivity by using some fungi that produced only the 6α, 7α and 13α-hydroxyl derivatives. The chemical structures of the hydroxylated diterpenes were determined from spectrometric and spectroscopic data, and the relative stereochemistry of stereogenic centers was established from coupling constants, by NOE-diff experiments and/or by computational calculations.

Ferulic Acid and Naturally Occurring Compounds Bearing a Feruloyl Moiety: A Review on Their Structures, Occurrence, and Potential Health Benefits.

The ubiquitous compound 4-hydroxy-3-methoxycinnamic acid, also known as ferulic acid (FA), constitutes a bioactive ingredient of many foods that may offer beneficial effects against disorders related to oxidative stress, including cancer, diabetes, and neurodegenerative diseases. This review discusses the antioxidant properties of FA, establishing relationships to several biological activities already described for this natural product. Next, 387 naturally occurring compounds, all isolated from plants and published between 1990 and 2015, the structures of which bear 1 or more feruloyl moieties, are covered in this review along with their structural formulas, botanical sources, and bioactivities. The compounds' distribution, structural patterns, bioactivities, and perspectives on food research are also succinctly discussed.

Oxidative potential of two Brazilian endophytic fungi from Handroanthus impetiginosus towards progesterone.

Biotransformation has been successfully employed to conduct uncommon reactions, which would hardly be carried out by chemical synthesis. A wide diversity of compounds may be metabolized by fungi, leading to chemical derivatives through selective reactions that work under ecofriendly conditions. Endophytic fungi live inside vegetal tissues without causing damage to the host plant, making available unique enzymes for interesting chemical derivatization. Biotransformation of steroids by endophytic fungi may provide new derivatives as these microorganisms came from uncommon and underexplored habitats. In this study, endophytic strains isolated from Handroanthus impetiginosus leaves were assayed for biotransformation of progesterone, and its derivatives were identified through GC-EI-MS analysis. The endophyte Talaromyces sp. H4 was capable of transforming the steroidal nucleus selectively into four products through selective ene-reduction of the C4-C5 double bond and C-17 oxidation. The best conversion rate of progesterone (>90 %) was reached with Penicillium citrinum H7 endophytic strain that transformed the substrate into one derivative. The results highlight endophytic fungi's potential to obtain new and interesting steroidal derivatizations.

Phenolic Compound Biotransformation by Trametes versicolor ATCC 200801 and Molecular Docking Studies.

The filamentous fungus Trametes versicolor is a rich source of laccase (Tvlac). Laccases catalyze reactions that convert substituted phenol substrates into diverse derivatives through aromatic oxidation. We investigated methyl p-coumarate, methyl ferulate, and methyl caffeate biotransformation by Trametes versicolor ATCC 200801. Despite substrate similarity, the biotransformation reactions varied widely. Only methyl p-coumarate was converted into three derivatives. We isolated and identified the chemical structures of such derivatives by NMR and IR analysis. Hydroxylation, methylation, and hydrolysis were the main reactions resulting from the studied biotransformation. We also analyzed the interactions between Tvlac (PDB ID: 1GYC) and the three phenolic substrates by molecular docking simulations. The substituents in the phenol ring influenced substrate conformation and orientation in the Tvlac site. The biotransformation reaction selectivity correlated with the different binding energies to the Tvlac site. Our results demonstrated that docking studies successfully predict the biotransformation of cinnamic acid analogs by T. versicolor.

Inactivation of ß-Lapachone Cytotoxicity by Filamentous Fungi that Mimic the Human Blood Metabolism.

BACKGROUND AND OBJECTIVES: ß-Lapachone is a drug candidate in phase II clinical trials for treatment of solid tumors. The therapeutic efficacy of ß-lapachone is closely related to its metabolism, since this o-naphthoquinone produces cytotoxic effect after intracellular bioreduction by reactive oxygen species formation. The aim of this study was to produce ß-lapachone human blood phase I metabolites to evaluate their cytotoxic activities. METHODS: The biotransformation of ß-lapachone was performed using Mucor rouxii NRRL 1894 and Papulaspora immersa SS13. The metabolites were isolated and their chemical structures determined from spectrometric and spectroscopic data. Cell cytotoxicity assays were carried out with ß-lapachone and its metabolites using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. RESULTS: Microbial transformation of ß-lapachone by filamentous fungi resulted in the production of five metabolites identical to those found during human blood metabolism, a novel metabolite and a product stated before only in a synthetic procedure. The analysis of the results showed that ß-lapachone metabolites were not cytotoxic for the neoplastic cell line SKBR-3 derived from human breast cancer and the normal human fibroblast cell line GM07492-A. The cytotoxic activity assay against the neoplastic cell line SKBR-3 revealed that the lowest half-maximal inhibitory concentration (IC50) values of these ß-lapachone metabolites were 33- to 52-fold greater than IC50 values of ß-lapachone. CONCLUSIONS: The cytotoxic activity of ß-lapachone in vivo may be reduced due to its swift conversion in blood.

Microbial Metabolism of Atovaquone and Cytotoxicity of the Produced Phase I Metabolite.

BACKGROUND AND OBJECTIVES: Atovaquone is a hydroxynaphthoquinone with selective action in the mitochondrial respiratory chain of malaria parasite. It is employed for both the treatment and prevention of malaria, in a combination with proguanil. The aim of this study was to elucidate the in vitro metabolites from atovaquone and to evaluate their cytotoxic activities. METHODS: The biotransformation of atovaquone was performed using Mucor rouxii NRRL 1894, Cunninghamella echinulata var. elegans ATCC 8688a and C. elegans ATCC 10028b, which have been reported as microbial models of mammalian drug metabolism. Experiments were also carried out with two probiotic strains from the human intestinal tract: Bifidobacterium sp. and Lactobacillus acidophilus. The phase I metabolite was isolated, its chemical structure was elucidated and its toxicity was evaluated using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. Cell cytotoxicity assays were also carried out with atovaquone. RESULT: Only the fungi were able to convert atovaquone to metabolite trans-3-[4'-(4″-chlorophenyl)cyclohexyl)-1,2-dioxo-dihydro-1H-indene-3-carboxylic acid. The metabolite displayed 50 % inhibitory concentration (IC50) values of 110.20 ± 2.2 and 108.80 ± 1.5 µmol/L against breast cancer cell line SKBR-3 and fibroblasts cell line GM07492-A, respectively. The IC50 values of atovaquone were 282.30 ± 1.8 and 340.50 ± 1.4 µmol/L against breast cancer and normal fibroblasts cell lines, respectively. CONCLUSIONS: The produced metabolite was more toxic than atovaquone and was not selective to normal or cancer cell lines. The present study is the first to report the production of atovaquone metabolite.