Lívia Soman de Medeiros

UHPLC-MS/MS determination of ochratoxin A and fumonisins in coffee using QuEChERS extraction combined with mixed-mode SPE purification.

A method was developed for simultaneous determination of the mycotoxins: ochratoxin A (OTA) and fumonisins B2 (FB2), B4 (FB4), and B6 (FB6) in green, roasted, and instant coffee. Extraction was performed by QuEChERS (quick, easy, cheap, effective, rugged, and safe) under acidic conditions followed by mixed-mode reversed phase-anion exchange solid phase extraction. OTA and FB2 were detected at levels down to 0.5 and 2 μg/kg by UHPLC-MS/MS and quantitated via isotope dilution using U-(13)C-labeled FB2 and OTA as internal standards. Mixing 20% isopropanol in the acetonitrile of the acidic UHPLC gradient system increased the signal intensity by 50% and decreased the ion-suppression with 50-75% in roasted coffee samples. About half of the roasted coffee samples (n = 57, from 9 countries) contained detectable levels of OTA, however, with only 5 samples above the EU regulatory limit of 5 μg/kg and the highest with 21 μg/kg. None of the 25 instant coffee samples contained OTA above the EU regulatory level of 10 μg/kg. Nonetheless, the toxin could be detected in 56% of the analyzed instant coffee samples. Fumonisins were not detected in any of the roasted or instant coffee samples (n = 82). However, in the green coffee samples (n = 18) almost half of the samples were positive with a maximum value of 164 μg/kg (sum of FB2, FB4, and FB6). This discrepancy between green coffee and processed coffees indicated that the fumonisins decompose during the roasting process, which was confirmed in roasting experiments. Here fumonisins could not be detected after roasting of the green, 164 μg/kg coffee, sample. Under the same conditions, OTA was reduced from 2.4 to 0.5 μg/kg.

An antimicrobial alkaloid and other metabolites produced by Penicillium sp. An endophytic fungus isolated from Mauritia flexuosa L. f.

The alkaloid glandicoline B (1) and six other compounds: ergosterol (2), brassicasterol (3), ergosterol peroxide (4), cerevisterol (5), mannitol (6) and 1-O-α-D-glucopyranoside (7) were isolated from Penicillium sp. strain PBR.2.2.2, a fungus from Mauritia flexuosa roots. The structures of the isolated metabolites were established by spectral analysis. MeOH extract of the fungal mycelium at 500 µg mL-1 exhibited antimicrobial activity against Staphylococcus aureus and the compound 1 at 100 µg mL-1 was active against S. aureus, Micrococcus luteus and Escherichia coli. The relationship between the bioactive properties of the fungus PBR.2.2.2 and those achieved for glandicoline B, as well the potential of this substance as bactericide is discussed.

An antimicrobial diketopiperazine alkaloid and co-metabolites from an endophytic strain of Gliocladium isolated from Strychnos cf. toxifera

From an endophytic strain of Gliocladium sp. isolated from the Amazonian plant Strychnos cf. toxifera, we obtained the diketopiperazine alkaloid cyclo-(glycyl-L-tyrosyl)-4,4-dimethylallyl ether (1), the steroids ergosterol (2), ergosterol peroxide (3), cerevisterol (4) and the citric acid (5). The AcOEt extract of the fermented broth by Gliocladium sp. showed potent activity against the cancer cell lines MDA-MB435 (human breast cancer cells), HCT-8 (human colorectal cancer cells) and SF-295 (human glioblastoma cancer cells). Compound 1 exhibited a strong antimicrobial activity against Micrococcus luteus at a concentration of 43.4 µM.

Mauritic acid: a new dammarane triterpene from the roots of Mauritia flexuosa L.f. (Arecaceae)

A new dammarane triterpene named mauritic acid (1) was isolated from the roots of Mauritia flexuosa L.f. The complete structural assignment of this new compound was elucidated from spectroscopic methods. Moreover, this compound was evaluated for its cytotoxicity against human cancer cell lines (OVCAR-8, PCM3, NCIH358M and different leukaemia cell strains). The mauritic acid presented significant cytotoxicity against OVCAR-8, PCM3 and NCIH358M cell lines with IC50 3.02, 2.39 and 6.19 μM, respectively. The triterpenes 1 and 2 were also tested for their antimicrobial activity against 15 strains of microorganisms, including fungi and bacteria, with the best minimal inhibitory concentration values ranging from 50.8 to 203.5 μM.

Evaluation of herbicidal potential of depsides from Cladosporium uredinicola, an endophytic fungus found in Guava fruit

Two natural products produced by Cladosporium uredinicola, an endophytic fungus isolated from guava fruit, were evaluated for their effects on photosynthesis. Both of them inhibited electron flow (basal, phosphorylating, and uncoupled) from water to methylviologen (MV), acting as Hill reaction inhibitors in freshly lysed spinach thylakoids. These polyketides, belonging to depsides class, inhibited partial reactions of photosystem II (PS II) electron flow from water to 2,5-dichloro-1,4-benzoquinone (DCBQ), from water to sodium silicomolybdate (SiMo Na+), and partially inhibited electron flow from 1,5-diphenylcarbazide (DPC) to 2,6-dichloroindophenol (DCPIP). These results established that the depsides sites of inhibition are located on the donor and acceptor sides of PS II, between P680 and QA. Chlorophyll a fluorescence measurements corroborated this mechanism of action. None of the tested compounds inhibited photosystem I (PS I) electron transport.

Dereplication-guided isolation of depsides thielavins S-T and lecanorins D-F from the endophytic fungus Setophoma sp.

Dereplication methodology using UHPLC-DAD-QTOFMS was applied during the metabolic profiling investigation of the endophyte Setophoma sp., a fungus isolated from symptomless guava fruits. The approach performed allowed a fast analysis of the microbial secondary metabolites. From this fungus, seven highly C-alkylated depsides were isolated and identified as polyketides thielavins S, T, U and V and lecanorins D, E and F. Their structures were elucidated through spectroscopic methods including NMR, HRMS and especially with assistance of HRMS/MS experiments. The compounds were tested for quorum sensing regulation activity in the virulence gene expression of Staphylococcus aureus, but no inhibitory effect was detected. Nevertheless, moderate antibacterial activity was encountered in three of tested depsides, particularly with thielavin T, whose MIC was 6.25 μg/mL against S. aureus.

Dichlorinated and Brominated Rugulovasines, Ergot Alkaloids Produced by Talaromyces wortmannii

UHPLC-DAD-HRMS based dereplication guided the detection of new halogenated alkaloids co-produced by Talaromyces wortmannii. From the fungal growth in large scale, the epimers 2,8-dichlororugulovasines A and B were purified and further identified by means of a HPLC-SPE/NMR hyphenated system. Brominated rugulovasines were also detected when the microbial incubation medium was supplemented with bromine sources. Studies from 1D/2D NMR and HRMS spectroscopy data allowed the structural elucidation of the dichlorinated compounds, while tandem MS/HRMS data analysis supported the rationalization of brominated congeners. Preliminary genetic studies revealed evidence that FADH2 dependent halogenase can be involved in the biosynthesis of the produced halocompounds.

HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL−1, and the limit of detection was found to be 0.03 μg·mL−1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source.

Rapid Detection of ACTG- and AK-Toxins in Alternaria alternata by LC-ESI-MS/MS Analysis and Antifungal Properties of Citrus Compounds

A methanol-soluble fraction of the dichloromethane extract from the culture broth of A. alternata AT4303 strain afforded the following five known mycotoxins: alternariol, alternariol monomethyl ether, altenusin, altenuene and altertoxin I. A hexane-soluble fraction of the dichloromethane extract yielded the steroids ergosterol and ergosterol peroxide, and alternariol monomethyl ether. The concentrated ethyl acetate extract afforded the nucleoside uridine, uracil and inosine. The micro-extracts obtained from this strain in solid media were analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and the following five known toxins were identified: ACTG-C, D, E and F and AK-toxin II. The major toxins produced by the tangerine pathotype ACT-toxins appear to be absent in strain AT4303. However, the low concentration of ACT-toxins appears to be responsible for their lack of detection in this study. Some compounds isolated from citrus plants were tested for in vitro activity against this strain. The results indicated that 17.3 µM apigenin-7-O-rutinoside inhibited by 80% conidial germination and appressorium development.

Understanding the cytotoxic effects of new isovanillin derivatives through phospholipid Langmuir monolayers

Twenty-one isovanillin derivatives were prepared in order to evaluate their cytotoxic properties against the cancer cell lines B16F10-Nex2, HL-60, MCF-7, A2058 and HeLa. Among them, seven derivatives exhibited cytotoxic activity. We observed that for obtaining smaller IC50 values and for increasing the index of selectivity, two structural features are very important when compared with isovanillin (1); a hydroxymethyl group at C-1 and the replacement of the hydroxyl group at C-3 by different alkyl groups. As the lipophilicity of the compounds was changed, we decided to investigate the interaction of the cytotoxic isovallinin derivatives on cell membrane models through Langmuir monolayers by employing the lipids DPPC (1,2-diplamitoyl-sn-glycero-3-phosphocoline) and DPPS (1,2-diplamitoyl-sn-glycero-3-phosphoserine). The structural changes on the scaffold of the compounds modulated the interaction with the phospholipids at the air-water interface. These results were very important to understand the biophysical aspects related to the interaction of the cytotoxic compounds with the cancer cell membranes.