Assessment of the photoprotective potential and structural characterization of secondary metabolites of Antarctic fungus Arthrinium sp.

Interest in Antarctic fungi has grown due to their resilience in harsh environments, suggesting the presence of valuable compounds from its organisms, such as those presenting photoprotective potential, since this environment suffers the most dangerous UV exposure in the world. Therefore, this research aimed to assess the photoprotective potential of compounds from sustainable marine sources, specifically seaweed-derived fungi from Antarctic continent. These studies led to discovery of photoprotective and antioxidant properties of metabolites from Arthrinium sp., an endophytic fungus from Antarctic brown algae Phaeurus antarcticus. From crude extract, fractions A-I were obtained and compounds 1-6 isolated from E and F fractions, namely 3-Hydroxybenzyl alcohol (1), (-)-orthosporin (2), norlichexanthone (3), anomalin B (4), anomalin A (5), and agonodepside B (6). Compounds 1, 2, and 6 were not previously reported in Arthrinium. Fraction F demonstrated excellent absorbance in both UVA and UVB regions, while compound 6 exhibited lower UVB absorbance, possibly due to synergistic effects. Fraction F and compound 6 displayed photostability and were non-phototoxic to HaCaT cells. They also exhibited antioxidant activity by reducing intracellular ROS production induced by UVA in keratinocyte monolayers and reconstructed human skin models (resulting in 34.6% and 30.2% fluorescence reduction) and did not show irritation potential in HET-CAM assay. Thus, both are promising candidates for use in sunscreens. It is noted that Fraction F does not require further purification, making it advantageous, although clinical studies are necessary to confirm its potential applicability for sunscreen formulations.

Insights into agar and secondary metabolite pathways from the genome of the red alga Gracilaria domingensis (Rhodophyta, Gracilariales).

Gracilariales is a clade of florideophycean red macroalgae known for being the main source of agar. We present a de novo genome assembly and annotation of Gracilaria domingensis, an agarophyte alga with flattened thallus widely distributed along Central and South American Atlantic intertidal zones. In addition to structural analysis, an organizational comparison was done with other Rhodophyta genomes. The nuclear genome has 78 Mbp, with 11,437 predicted coding genes, 4,075 of which did not have hits in sequence databases. We also predicted 1,567 noncoding RNAs, distributed in 14 classes. The plastid and mitochondrion genome structures were also obtained. Genes related to agar synthesis were identified. Genes for type II galactose sulfurylases could not be found. Genes related to ascorbate synthesis were found. These results suggest an intricate connection of cell wall polysaccharide synthesis and the redox systems through the use of L-galactose in Rhodophyta. The genome of G. domingensis should be valuable to phycological and aquacultural research, as it is the first tropical and Western Atlantic red macroalgal genome to be sequenced.

Algal-Derived Halogenated Sesquiterpenes from Laurencia dendroidea as Lead Compounds in Schistosomiasis Environmental Control.

Schistosomiasis has been controlled for more than 40 years with a single drug, praziquantel, and only one molluscicide, niclosamide, raising concern of the possibility of the emergence of resistant strains. However, the molecular targets for both agents are thus far unknown. Consequently, the search for lead compounds from natural sources has been encouraged due to their diverse structure and function. Our search for natural compounds with potential use in schistosomiasis control led to the identification of an algal species, Laurencia dendroidea, whose extracts demonstrated significant activity toward both Schistosoma mansoni parasites and their intermediate host snails Biomphalaria glabrata. In the present study, three seaweed-derived halogenated sesquiterpenes, (-)-elatol, rogiolol, and obtusol are proposed as potential lead compounds for the development of anthelminthic drugs for the treatment of and pesticides for the environmental control of schistosomiasis. The three compounds were screened for their antischistosomal and molluscicidal activities. The screening revealed that rogiolol exhibits significant activity toward the survival of adult worms, and that all three compounds showed activity against S. mansoni cercariae and B. glabrata embryos. Biomonitored fractioning of L. dendroidea extracts indicated elatol as the most active compound toward cercariae larvae and snail embryos.

The Prospective Use of Brazilian Marine Macroalgae in Schistosomiasis Control.

Schistosomiasis is a parasitic disease that affects more than 250 million people. The treatment is limited to praziquantel and the control of the intermediate host with the highly toxic molluscicidal niclosamide. Marine algae are a poorly explored and promising alternative that can provide lead compounds, and the use of multivariate analysis could contribute to quicker discovery. As part of our search for new natural compounds with which to control schistosomiasis, we screened 45 crude extracts obtained from 37 Brazilian seaweed species for their molluscicidal activity against Biomphalaria glabrata embryos and schistosomicidal activities against Schistosoma mansoni. Two sets of extracts were taxonomically grouped for metabolomic analysis. The extracts were analyzed by GC-MS, and the data were subjected to Pattern Hunter and Pearson correlation tests. Overall, 22 species (60%) showed activity in at least one of the two models. Multivariate analysis pointed towards 3 hits against B. glabrata veliger embryos in the Laurencia/Laurenciella set, 5 hits against B. glabrata blastula embryos, and 31 against S. mansoni in the Ochrophyta set. Preliminary annotations suggested some compounds such as triquinane alcohols, prenylated guaianes, dichotomanes, and xenianes. Despite the putative identification, this work presents potential candidates and can guide future isolation and identification.

HPLC/HR-MS-Based Metabolite Profiling and Chemometrics: A Powerful Approach to Identify Bioactive Compounds from Abarema cochliacarpos.

Despite its importance as a medicinal plant, there is a lack of studies that assessed the chemical composition of A. cochliacarpos extracts. Herein, we used a metabolite profiling approach and chemometrics as a powerful strategy to correlate the chemical composition with the antioxidant activity of A. cochliacarpos extracts. Extracts obtained with ethyl acetate showed greater antioxidant activity and higher total phenolic content than extracts obtained with hexane. The chemical composition was assessed by HPLC/HR-MS and it encompassed fatty alcohols, terpenoids, phenolic derivatives, lipids, carotenoid-like compounds, alkaloids, flavonoids, polyketides, and glycerophospholipids. Chemometrics successfully differentiated not only the chemical composition of extracts in response to the nature of the extraction solvent and the botanical part used during extraction but also it allowed us to associate the chemical composition with the antioxidant activity of the extracts, which might be particularly helpful for drug discovery and development programs.

Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model.

Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a "mitochondrial-targeted" action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl)ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes.

Re-investigation of the fragmentation of protonated carotenoids by electrospray ionization and nanospray tandem mass spectrometry.

RATIONALE: Carotenoids are polyene isoprenoids with an important role in photosynthesis and photoprotection. Their characterization in biological matrices is a crucial subject for biochemical research. In this work we report the full fragmentation of 16 polyenes (carotenes and xanthophylls) by electrospray ionization tandem mass spectrometry (ESI-CID-MS/MS) and nanospray tandem mass spectrometry (nanoESI-CID-MS/MS). METHODS: Analyses were carried out on a quadrupole time-of-flight (QTOF) mass spectrometer coupled with a nanoESI source and on a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with an ESI source. The formulae of the product ions were determined by accurate-mass measurements. RESULTS: It is demonstrated that the fragmentation routes observed for the protonated carotenoids derive essentially from charge-remote fragmentations and pericyclic rearrangements, such as electrocyclic and retro-ene eliminations (assisted or not by a sigmatropic hydrogen shift). All mechanisms are dependent on cis-trans isomerization through the formation of several conjugated polyene carbocation intermediates. Some specific ions for the carotenoid epoxides were justified through formation of cyclic oxonium ions. CONCLUSIONS: Complete fragmentation pathways of protonated carotenoids by ESI- and nanoESI-CID-MS/MS provided structural information about functional groups, polyene chain and double bonds, and contribute to identification of carotenoids based on MS/MS fragmentation patterns. Copyright © 2016 John Wiley & Sons, Ltd.

Noninvasive evaluation of heavy metal uptake and storage in micoralgae using a fluorescence resonance energy transfer-based heavy metal biosensor.

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg2+ followed by Cd2+≈Pb2+>Zn2+>Cu2+. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na+ and Mg2+). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb2+>Cd2+) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.

Microalgae for economic applications: advantages and perspectives for bioethanol.

Renewable energy has attracted significant interest in recent years as a result of sustainability, environmental impact, and socio-economic considerations. Given existing technological knowledge and based on projections relating to biofuels derived from microalgae, microalgal feedstock is considered to be one of the most important renewable energy sources potentially available for industrial production. Therefore, this review examines microalgal bioethanol technology, which converts biomass from microalgae to fuel, the chemical processes involved, and possible ways of increasing the bioethanol yield, such as abiotic factors and genetic manipulation of fermenting organisms.

Antioxidant properties and UV absorbance pattern of mycosporine-like amino acids analogs synthesized in an environmentally friendly manner.

In current study, we report efficient and clean procedure for preparing mycosporine-like amino acids (MMAs) analogs and evaluate their ultraviolet absorbance properties and antioxidant activities. The ultraviolet radiation absorbance patterns of the compounds were recorded and then used to define their molar absorptivities. The antioxidant activities were assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and superoxide radical scavenging assays. Eight of nine compounds showed good activity against superoxide radicals, as only one of the analogs exhibited a measurable IC50 in the DPPH assay.

Bioactive compounds from Vellozia pyrantha A.A.Conc: A metabolomics and multivariate statistical analysis approach.

The chemical composition of V. pyrantha resin (VpR) and fractions (VpFr1-7 and VpWS) were assessed by LC-MS and NMR. Twenty-eight metabolites were identified, including 16 diterpenoids, seven nor-diterpenoids, one fatty acid, one bis-diterpenoid, one steroid, one flavonoid, and one triterpenoid. The pharmacological potential of VpR, VpFr1-7, and isolated compounds was assessed by determining their antioxidant, antimicrobial, and cytotoxic activities. VpFr4 (IC50 = 205.48 ± 3.37 µg.mL-1) had the highest antioxidant activity, whereas VpFr6 (IC50 = 842.79 ± 10.23 µg.mL-1) had the lowest. The resin was only active against Staphylococcus aureus (MIC 62.5 µg.mL-1) and Salmonella choleraesius (MIC and MFC 500 µg.mL-1), but fractions were enriched with antibacterial compounds. V. pyrantha resin and fractions showed great cytotoxic activity against HCT116 (IC50 = 20.08 µg.mL-1), HepG2 (IC50 = 20.50 µg.mL-1), and B16-F10 (12.17 µg.mL-1) cell lines. Multivariate statistical analysis was used as a powerful tool to pinpoint possible metabolites responsible for the observed activities.

The application of "double isolation" in Fourier transform ion cyclotron resonance sustained off-resonance irradiation collisionally-induced dissociation tandem mass spectrometry to remove labile isobaric impurities.

This study reports the application of "double isolation" in sustained off-resonance irradiation collisionally-induced dissociation tandem mass spectrometry (SORI-CID-MS/MS) to remove radio- frequency (RF) fragment ions of very close mass isobaric ions (0.02 m/z apart). Analyses were performed with a fraction of a biological extract isolated from a macroalgae containing the mycosporine-like amino acid asterina-330. Direct isolation of the precursor ion by narrowing the isolation window proved ineffective as it impinged upon the required ion thus substantially reducing its intensity. By increasing the correlated sweep time, ejection efficiency of the isolation was improved, but caused the unwanted side-effect of RF fragmentation of labile ions. Finally, by skipping the ion activation step and performing a second isolation (in the MS(3) module) the RF fragments from the first isolation were removed leaving a very pure isolation of the required precursor ion and allowed a very clean CID fragmentation. We demonstrated that the m/z 272.1351 ion is derived from the loss of NH(3) from m/z 289.1620 isobaric impurity and is not related to asterina-330. This application represents a powerful tool to remove unwanted ions in the MS/MS spectrum that result from fragmentation of isobaric ions.

Biochemical composition of two red seaweed species grown on the Brazilian coast.

BACKGROUND: Algae species have been used as an important source of food because they are highly nutritive considering their vitamin, protein, mineral, fiber, essential fatty acid and carbohydrate contents. However, a large number of seaweeds have been poorly studied, especially Brazilian species. Two red macroalgae species from the Brazilian coast (Plocamium brasiliense and Ochtodes secundiramea) were assessed with respect to their total lipid, fatty acid, total nitrogen, protein, amino acid and total carbohydrate contents. RESULTS: The total lipid contents (dry weight) were 36.3 and 35.4 g kg(-1); fatty acid contents were 9.3 and 12.1 g kg(-1); total nitrogen contents were 37.4 and 24.9 g kg(-1); protein contents were 157.2 and 101.0 g kg(-1); amino acid contents were 127.5 and 91.4 g kg(-1); and total carbohydrate contents were 520.3 and 450.7 g kg(-1) for P. brasiliense and O. secundiramea, respectively. CONCLUSION: Considering these compositions, both algae species were determined to have sources of protein, essential amino acids and carbohydrates similar to the edible seaweeds Laminaria japonica and Palmaria palmata.

Planting Season Impacts Sugarcane Stem Development, Secondary Metabolite Levels, and Natural Antisense Transcription.

To reduce the potentially irreversible environmental impacts caused by fossil fuels, the use of renewable energy sources must be increased on a global scale. One promising source of biomass and bioenergy is sugarcane. The study of this crop's development in different planting seasons can aid in successfully cultivating it in global climate change scenarios. The sugarcane variety SP80-3280 was field grown under two planting seasons with different climatic conditions. A systems biology approach was taken to study the changes on physiological, morphological, agrotechnological, transcriptomics, and metabolomics levels in the leaf +1, and immature, intermediate and mature internodes. Most of the variation found within the transcriptomics and metabolomics profiles is attributed to the differences among the distinct tissues. However, the integration of both transcriptomics and metabolomics data highlighted three main metabolic categories as the principal sources of variation across tissues: amino acid metabolism, biosynthesis of secondary metabolites, and xenobiotics biodegradation and metabolism. Differences in ripening and metabolite levels mainly in leaves and mature internodes may reflect the impact of contrasting environmental conditions on sugarcane development. In general, the same metabolites are found in mature internodes from both "one-year" and "one-and-a-half-year sugarcane", however, some metabolites (i.e., phenylpropanoids with economic value) and natural antisense transcript expression are only detected in the leaves of "one-year" sugarcane.

Combination of a multiplatform metabolite profiling approach and chemometrics as a powerful strategy to identify bioactive metabolites in Lepidium meyenii (Peruvian maca).

Lepidium meyenii is an edible plant that has been used as a nutritional supplement worldwide due to its medicinal properties. However, most of the studies have focused on the pharmacological activities of the extracts rather than their chemical composition. Herein, we used a combination of a multiplatform metabolite profiling approach and chemometrics to identify bioactive metabolites in L. meyenii. Extracts obtained with ethyl acetate and ethanol showed the promising antioxidant, anti-glioma and antibacterial activities. Sixty metabolites were identified by HPLC-MS, whereas fifteen were identified by GC-MS. Partial least squares discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA), and Variable Importance in Projection (VIP) successfully discriminated extracts obtained in different organic solvents from in natura dry roots and commercial product samples of L. meyenii. Additionally, correlation analysis allowed us to pinpoint potential candidates responsible for each biological activity tested for the extracts, which could be extrapolate for other food-related species.

Identification of bioactive metabolites from corn silk extracts by a combination of metabolite profiling, univariate statistical analysis and chemometrics.

Corn silk has been widely used as a nutritional and medicinal supplement due to its pharmacological properties, but there is a lack of studies that correlate the extracts' chemical composition with their biological activities. Herein, we performed the large-scale chemical characterization of corn silk extracts and used chemometrics to correlate the chemical composition with the biological activities of the extracts. Twenty-two metabolites were identified by High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), whereas twelve were identified by Gas Chromatography coupled to Mass Spectrometry (GC-MS). Chemometrics allowed us to discriminate extracts obtained in different organic solvents from in natura and commercial product samples and to pinpoint potential candidate metabolites for the antioxidant and anti-glioma activities. Two flavone glycosides (7 and 8), along with a O-methylated anthocyanidin (26) seems to be the main contributors for the biological activities of the corn silk extracts.

Bioactivity and composition of lipophilic metabolites extracted from Antarctic macroalgae.

Macroalgae comprise a vast group of aquatic organisms known for their richness in phytochemicals. In this sense, the lipophilic profile of five Antarctic seaweed species was characterized by chromatographic and spectroscopic analysis and their antioxidant and antimicrobial potential was evaluated. Results showed there were 31 lipophilic substances, mainly fatty acids (48.73 ± 0.77 to 331.91 ± 10.79 mg.Kg-1), sterols (14.74 ± 0.74 to 321.25 ± 30.13 mg.Kg-1), and alcohols (13.07 ± 0.04 to 91.87 ± 30.07 mg.Kg-1). Moreover, Desmarestia confervoides had strong antioxidant activity, inhibiting 86.03 ± 1.47% of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical at 1 mg.mL-1. Antimicrobial evaluation showed that extracts from Ulva intestinalis, Curdiea racovitzae, and Adenocystis utricularis inhibited the growth of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Salmonella typhimurium (ATCC 14028) from concentrations of 1.5 to 6 mg.mL-1. Therefore, the evaluated brown, red, and green macroalgae contained several phytochemicals with promising biological activities that could be applied in the pharmaceutical, biotechnological, and food industries.

In Vitro Evaluation of the Photoprotective Potential of Quinolinic Alkaloids Isolated from the Antarctic Marine Fungus Penicillium echinulatum for Topical Use.

Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability < 30%), which indicates their very low acute photoirritation and high photosafety potential in humans. Viridicatin was considered weakly photoreactive, while viridicatol showed no photoreactivity; both compounds inhibited UVA-induced ROS generation in HaCaT cells, although viridicatol was not able to protect the RHS model against UVA-induced ROS production. Thus, the results highlighted the photoprotective and antioxidant potential of metabolites produced by P. echinulatum which can be considered a new class of molecules for photoprotection, since their photosafety and non-cytotoxicity were predicted using recommended in vitro methods for topical use.

The antileishmanial activity of the antarctic brown alga Ascoseira mirabilis Skottsberg.

Leishmaniasis is a group of diseases that have limited and high toxic therapeutic options. Herein, we evaluated the antileishmanial potential and cytotoxicity of hexanic extract obtained from the Antarctic brown alga Ascoseira mirabilis using bioguided fractionation against Leishmania amazonensis and murine macrophages, which was fractionated by SPE, yielding seven fractions (F1-F7). The fraction F6 showed good anti-amastigote activity (IC50 = 73.4 ± 0.4 µg mL-1) and low cytotoxicity (CC50 > 100 µg mL-1). Thus, in order to identify the bioactive constituent(s) of F6, the fraction was separated in a semipreparative HPLC, yielding four fractions (F6.1-F6.4). F6.2 was the most bioactive fraction (IC50 = 66.5 ± 4.5 µg mL-1) and GC-MS analyses revealed that the compounds octadecane, propanoic acid, 1-monomyristin and azelaic acid correspond to 61% of its composition. These data show for the first time the antileishmanial potential of the Antarctic alga A. mirabilis.