Fungal Sterol Analyses by Gas Chromatography-Mass Spectrometry Using Different Derivatives.

Sterols are the main components of the fungal membrane. Their study can be used to describe the chemical biodiversity among the strains and species or to work on antifungal treatment. Those molecules can be analyzed by gas chromatography coupled with mass spectrometry (GC-MS) as free molecules or after derivation as acetate or trimethylsilyl ether (TMSi). Here we describe how to extract sterols from fungal biomass according to its physiological form and the culture conditions (liquid and solid media). The different methodologies that can be used to obtain free sterols, acetate, and/or TMSi derivatives are presented. Identification keys using the fragmentation at 70 eV are also described.

Steroids from Marine-Derived Fungi: Evaluation of Antiproliferative and Antimicrobial Activities of Eburicol.

The most common sterol in fungi is ergosterol, which has frequently been investigated in human pathogenic fungal strains. This sterol, and others isolated from fungal strains, has also demonstrated cytotoxicity against cancer cell lines and antimicrobial activities. Marine fungi can produce high amounts of bioactive compounds. So, a screening was performed to study sterol composition using GC/MS in 19 marine fungal strains and ergosterol was always the major one. One strain, Clonostachys rosea MMS1090, was selected due to its high amount of eburicol and a one strain many compounds approach was performed on seven culture media to optimize its production. After purification and structural identification by NMR, eburicol was assessed against four cancer cell lines, MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549, and seven human pathogenic bacteria Staphylococcus aureus, Bacillus sp., Bacillus cereus, Listeria ivanovii, Escherichia coli, Citrobacter freundii and Salmonella spp. The most significant activity was cytotoxicity against MCF-7 cells (2 µM). This is the first report of such an accumulation of eburicol in the marine fungal strain C. rosea confirming its potential in the production of bioactive lipids.

Lipid Composition, Fatty Acids and Sterols in the Seaweeds Ulva armoricana, and Solieria chordalis from Brittany (France): An Analysis from Nutritional, Chemotaxonomic, and Antiproliferative Activity Perspectives.

Lipids from the proliferative macroalgae Ulva armoricana (Chlorophyta) and Solieria chordalis (Rhodophyta) from Brittany, France, were investigated. The total content of lipids was 2.6% and 3.0% dry weight for U. armoricana and S. chordalis, respectively. The main fractions of S. chordalis were neutral lipids (37%) and glycolipids (38%), whereas U. armoricana contained mostly neutral lipids (55%). Polyunsaturated fatty acids (PUFA) represented 29% and 15% of the total lipids in U. armoricana and S. chordalis, respectively. In both studied algae, the phospholipids were composed of PUFA for 18%. In addition, PUFA were shown to represent 9% and 4.5% of glycolipids in U. armoricana and S. chordalis, respectively. The essential PUFA were 16:4n-3, 18:4n-3, 18:2n-3, 18:2n-6, and 22:6n-3 in U. armoricana, and 20:4n-6 and 20:5n-3 in S. chordalis. It is important to notice that six 2-hydroxy-, three 3-hydroxy-, and two monounsaturated hydroxy fatty acids were also identified and may provide a chemotaxonomic basis for algae. These seaweeds contained interesting compounds such as squalene, α-tocopherol, cholest-4-en-3-one and phytosterols. The antiproliferative effect was evaluated in vitro on human non-small-cell bronchopulmonary carcinoma line (NSCLC-N6) with an IC50 of 23 µg/mL for monogalactosyldiacylglycerols isolated from S. chordalis and 24 µg/mL for digalactosyldiacylglycerols from U. armoricana. These results confirm the potentialities of valorization of these two species in the fields of health, nutrition and chemotaxonomy.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 µM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 µM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

Antimalarial activity of axidjiferosides, new ß-galactosylceramides from the African sponge Axinyssa djiferi.

The marine sponge, Axinyssa djiferi, collected on mangrove tree roots in Senegal, was investigated for glycolipids. A mixture containing new glycosphingolipids, named axidjiferoside-A, -B and -C, accounted for 0.07% of sponge biomass (dry weight) and for 2.16% of total lipids. It showed a significant antimalarial activity, with a 50% inhibitory concentration (IC50) of 0.53 ± 0.2 µM against a chloroquine-resistant strain of Plasmodium falciparum. They were identified as homologous ß-galactopyranosylceramides composed of 2-amino-(6E)-octadec-6-en-1,3,4-triol, and the major one, axidjiferoside-A (around 60%), contained 2-hydroxytetracosanoic acid. Cytotoxicity was studied in vitro on human cancer cell lines (multiple myeloma, colorectal adenocarcinoma, glioblastoma and two lung cancer NSCLC-N6 and A549). Results of this investigation showed that axidjiferosides are of interest, because they proved a good antiplasmodial activity, with only a low cytotoxicity against various human cell lines and no significant antitrypanosomal and antileishmanial activity. Thus, it seems that galactosylceramides with a ß anomeric configuration may be suitable in searching for new antimalarial drugs.

Antiproliferative activity against human non-small cell lung cancer of two O-alkyl-diglycosylglycerols from the marine sponges Myrmekioderma dendyi and Trikentrion laeve.

Glycolipids of Myrmekioderma sponges contain Myrmekiosides, a new family of glycolipids with a unique structure of mono-O-alkyl-diglycosylglycerols. This report deals with the identification and biological activity of the new Myrmekioside E from Myrmekioderma dendyi. Its structure has been elucidated from spectroscopic data and chemical degradation studies. It contained a glycerol backbone linked to xylose and N-acetylglucosamine, and an alkyl long-chain with a terminal alcohol group. A related glycolipid, Trikentroside, known in the sponge Trikentrion laeve, was subjected to a comparative biological evaluation. Both glycolipids inhibit proliferation of two human non-small cell lung cancer cell lines (NSCLC-N6 and A549).

Cytotoxicity on human cancer cells of ophidiacerebrosides isolated from the African starfish Narcissia canariensis.

The starfish Narcissia canariensis harvested from the coasts off Dakar, Senegal, was investigated for glycolipids (GL). This report deals with the isolation, characterization and biological activity of a fraction F13-3 separated from the GL mixture and selected according to its ability to inhibit KB cell proliferation after 72 hours of treatment. Firstly, a GL mixture F13 was obtained that accounted for 1.36% of starfish biomass (dry weight) and 0.36% of total lipids. The fraction F13-3 obtained from F13 contained three homologous GL identified as peracetylated derivatives on the basis of chemical and spectroscopic evidence. These contained a ß-glucopyranoside as sugar head, a 9-methyl-branched 4,8,10-triunsaturated long-chain aminoalcohol as sphingoid base and amide-linked 2-hydroxy fatty acid chains. The majority (63%) had an amide-linked 2-hydroxydocosanoic acid chain and was identified as the ophidiacerebroside-C, firstly isolated from the starfish Ophidiaster ophidiamus. The minor components of F13-3 differed by one more or one less methylene group, and corresponded to ophidiacerebroside-B and -D. We found that F13-3 displayed an interesting cytotoxic activity over 24 hours on various adherent human cancerous cell lines (multiple myeloma, colorectal adenocarcinoma and glioblastoma multiforme) with an IC(50) of around 20 µM.

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum.

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.