Maria Teresa Villela Romanos

Glycolipids from macroalgae: potential biomolecules for marine biotechnology?

Brown, red and green algae from the Southeastern coast of Brazil were successively extracted with chloroform/methanol 2:1 and 1:2 (v/v). The crude lipid extract was partitioned according to Folch and the lower phase enriched in glycolipids was fractionated on a silica gel column chromatography eluted with chloroform, acetone and methanol. Three major orcinol-reactive bands present in the acetone and methanol fractions were detected by thin-layer chromatography with chromatographic mobilities corresponding to sulfoglycolipids and glycosyldiacylglycerols. These fractions exhibited potent antiviral activity against HSV-1-ACVs and HSV-1-ACVr and present low toxicity for cell cultures. Purification and identification of these bioactive glycolipids will be necessary in order to elucidate their primary structures and mechanism of action.

Structural Characterization and Anti-HSV-1 and HSV-2 Activity of Glycolipids from the Marine Algae Osmundaria obtusiloba Isolated from Southeastern Brazilian Coast

Glycolipids were extracted from the red alga Osmundaria obtusiloba from Southeastern Brazilian coast. The acetone insoluble material was extracted with chloroform/methanol and the lipids, enriched in glycolipids, were fractionated on a silica gel column eluted with chloroform, acetone and then methanol. Three major orcinol-positive bands were found in the acetone and methanol fractions, being detected by thin layer chromatography. The structures of the corresponding glycolipids were elucidated by ESI-MS and 1H/13C NMR analysis, on the basis of their tandem-MS behavior and HSQC, TOCSY fingerprints. For the first time, the structure of sulfoquinovosyldiacylglycerol from the red alga Osmundaria obtusiloba was characterized. This molecule exhibited potent antiviral activity against HSV-1 and HSV-2 with EC50 values of 42 µg/mL to HSV-1 and 12 µg/mL to HSV-2, respectively. Two other glycolipids, mono- and digalactosyldiacylglycerol, were also found in the alga, being characterized by ESI-MS/MS. The structural elucidation of algae glycolipids is a first step for a better understanding of the relation between these structures and their biological activities.

Antiviral Sulfoquinovosyldiacylglycerols (SQDGs) from the Brazilian Brown Seaweed Sargassum vulgare

Total lipids from the Brazilian brown seaweed Sargassum vulgare were extracted with chloroform/methanol 2:1 and 1:2 (v/v) at room temperature. After performing Folch partition of the crude lipid extract, the lipids recovered from the Folch lower layer were fractionated on a silica gel column eluted with chloroform, acetone and methanol. The fraction eluted with methanol, presented a strong orcinol-positive band characteristic of the presence of sulfatides when examined by TLC. This fraction was then purified by two successive silica gel column chromatography giving rise to fractions F4I86 and F4II90 that exhibited strong activity against herpes simplex virus type 1 and 2. The chemical structures present in both fractions were elucidated by ESI-MS and 1H/13C NMR analysis HSQC fingerprints based on their tandem–MS behavior as sulfoquinovosildiacylglycerols (SQDGs). The main SQDG present in both fractions and responsible for the anti-herpes activity observed was identified as 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol.

In vitro and in vivo studies into the biological activities of 1,10-phenanthroline, 1,10-phenanthroline-5,6-dione and its copper(II) and silver(I) complexes

1,10-Phenanthroline (phen, 5), 1,10-phenanthroline-5,6-dione (phendione, 6), [Cu(phendione)3](ClO4)2·4H2O (12) and [Ag(phendione)2]ClO4 (13) are highly active, in vitro, against a range of normal and cancerous mammalian cells, fungal and insect cell lines, with the metal complexes offering a clear enhancement in activity. Cytoselectivity was not observed between the tumorigenic and non-tumorigenic mammalian lines. In in vivo tests, using Galleria mellonella and Swiss mice, all four compounds were well tolerated in comparison to the clinical agent, cisplatin. In addition, blood samples taken from the Swiss mice showed that the levels of the hepatic enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), remained unaffected. Immunocompromised nude mice showed a much lower tolerance to 13 and, subsequently, when these mice were implanted with Hep-G2 (hepatic) and HCT-8 (colon) human-derived tumors, there was no influence on tumor growth.

Evaluation of cytotoxicity and degree of conversion of glass ionomer cements reinforced with resin

The objective of the present study was to evaluate the cytotoxicity and degree of monomer conversion of resin-reinforced glass ionomer cements (RGIC) over different time periods. Four RGICs: Fuji Ortho LC (FOLC), Fuji Ortho Band (FOB), Orthoglass (OGL), and Multicure Glass Ionomer (MCI) were evaluated for cytotoxicity in fibroblastic L929 cells and for their degree of monomer conversion over different time periods. Three control groups were also analysed: positive control (C+), consisting of Tween 80 cell detergent; negative control (C-), consisting of phosphate-buffered saline; and cell control (CC), consisting of cells exposed to any material. To evaluate the cytotoxicity, the dye-uptake technique was used and the degree of conversion was evaluated using infrared spectroscopy. The data obtained were analysed by analysis of variance and the Tukeys test. The results showed cytotoxicity of the RGICs at 1 and 24 hours; the viability values of these materials were statistically different from the C- and CC groups (P < 0.05). After 48 hours, the FOLC group was statistically similar to the CC and C- groups but different from the others. At 1 hour, there was no difference in the degree of conversion between the FOLC and OGL groups (P > 0.05) or between the FOB and MCI (P < 0.05) groups. However, at 48 hours, the FOLC group had greater conversion values than the other groups (P < 0.05). There is a direct relationship between the degree of conversion and RGIC cytotoxicity. Following initial polymerization, cytotoxicity decreases and, consequently, the degree of conversion of the material increases.

Inhibitory Effect of Extracts of Brazilian Marine Algae on Human T-Cell Lymphotropic Virus Type 1 (HTLV-1)-Induced Syncytium Formation In Vitro

Extracts from four species of Brazilian marine algae collected from the Rio de Janeiro State coast were screened to determine the inhibitory effect on HTLV-1-induced syncytium formation. Before performing the syncytium inhibition assay the 50% cytotoxic dose (CyD50) of the algal extracts was evaluated. The antiviral test was carried out in HeLa cells co-cultured with HTLV-I infected T-cell line (C91/PL cells) in the presence of marine algal extracts in the concentration inferior to that corresponding to the CyD50. It was observed that co-cultured cells exposed to Ulva fasciata extract showed 60.2% syncytium inhibition at a concentration of 2.5%. At 5% concentration, Sargassum vulgare and Vidalia obtusiloba extracts presented 78.8 and 76% syncytium inhibition, respectively. The best inhibitory activity was observed with Laminaria abyssalis that presented 100% syncytium inhibition at a concentration of 2.5%. This work shows that extracts of marine algae, mainly L. abyssalis extract, are able to inhibit the cell-to-cell contact essential for the spreading of the virus and could be useful to prevent the infection.

Structural and Functional Characterization of a Multifunctional Alanine-Rich Peptide Analogue from Pleuronectes americanus

Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets.

Antiviral activity of extracts from Brazilian seaweeds against herpes simplex virus

Organic extracts of 36 species of marine algae (sixteen species of Rhodophyta, eight species of Ochrophyta and twelve species of Chlorophyta) from seven locations on the Brazilian coast were evaluated for their anti-HSV-1 and anti-HSV-2 activity resistant to Acyclovir (ACV). Activity tests in crude extracts, followed by the identification of the major compounds present, were performed for all species. The chemical profiles of all crude extracts were obtained by 1H-NMR and 13C-NMR spectroscopy. The percentage of extracts with antiviral activity was higher for HSV-1 (86.1%) than for HSV-2 (55.5%). The green algae Ulva fasciata and Codium decorticatum both showed the highest activity (99.9%) against HSV-1, with triacylglycerols and fatty acids as the major components. The red alga Laurencia dendroidea showed good activity against HSV-1 (97.5%) and the halogenated sesquiterpenes obtusol and (-)-elatol were identified as the major components in the extract. Against HSV-2, the green alga Penicillus capitatus (Chlorophyta) and Stypopodium zonale (Ochrophyta) were the most active (96.0 and 95.8%). Atomaric acid, a meroditerpene, was identified as the major secondary metabolite in the S. zonale extract. These results reinforce the role of seaweeds as important sources of compounds with the potential to enter into the pipeline for development of new drugs against herpes simplex.

In Vitro Anti-HMPV Activity of Meroditerpenoids from Marine Alga Stypopodium zonale (Dictyotales)

In this paper, we evaluated the antiviral activity against HMPV replication of crude extract of the marine algae Stypopodium zonale and of two meroditerpenoids obtained from it, atomaric acid and epitaondiol, and a methyl ester derivative of atomaric acid. Their selectivity indexes were 20.78, >56.81, 49.26 and 12.82, respectively. Compared to ribavirin, the substances showed a relatively low cytotoxicity on LLC-MK2 cells, with a significant antiviral activity, inhibiting at least 90% of viral replication in vitro, which demonstrates the potential of these marine natural products to combat infections caused by HMPV in vitro.

Investigation of biotechnological potential of sponge‐associated bacteria collected in Brazilian coast

Marine bacteria are a rich source of structurally unique natural compounds, several of which have shown a wide variety of biological activities. In this study, the metabolites present in the culture supernatants of the eight sponge‐associated bacteria were extracted using ethyl acetate, and all extracts showed activity against Staphylococcus aureus. Subsequently, the extracts of the Pseudomonas fluorescens H40 and H41, and Pseudomonas aeruginosa H51 were subjected to solvent partitioning, and the active fractions were submitted to chromatographic separation. Three different active fractions were obtained, one of which was identified as diketopiperazine cyclo‐(L‐Leu‐L‐Pro). This substance was bactericidal for Staph. aureus and Ps. aeruginosa and showed cytotoxic activity against HEp‐2 tumour cells. Putative gene fragments coding for the type I polyketide synthase (PKS‐I) and nonribosomal peptide synthetase (NRPS) domains were PCR‐amplified from five and three strains, respectively. The results suggest that sponge‐associated bacteria analysed in this study may represent a potential source for production of antimicrobial substances against bacterial pathogens of medical importance.