Axinellamine E, One New Pyrrololactam Alkaloid from the South China Sea Sponge Axinella sp.

Chemical investigation of the EtOH extract of the sponge Axinella sp. collected from the South China Sea resulted in the identification of one new pyrrololactam alkaloid, axinellamine E (2), along with four known analogs (1, 3-5). Compound 1 was initially separated as enantiomers and was further separated to be optically pure compounds (1 a and 1 b) by a chiral column. The planar structure of compound 2 was determined mainly by 1D-, 2D-NMR, and HR-ESI-MS data analyses. Absolute configurations of 1 a and 1 b was defined by calculated ECD spectra method. All of the compounds were evaluated for their anti-inflammatory activities against nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells among which compound 1 showed weak activity at 40 µg/mL. Plausible biosynthetic pathways corresponding to aldisine analogs of 1, 2, 4, and 5 were also discussed.

Axinellasins A-D, Immunosuppressive Cycloheptapeptide Diastereomers, Discovered via a Precursor Ion Scanning-Supercritical Fluid Chromatography Strategy from the Marine Sponge Axinella species.

The precursor ion scanning-supercritical fluid chromatography (PI-SFC) method was applied to explore new methionine sulfoxide-containing cycloheptapeptides, axinellasins A-D (1-4), from the marine sponge Axinella sp. Their structures, including absolute configurations, were elucidated by detailed spectroscopic analyses and X-ray crystallography. The total synthesis of 4 was completed via an Fmoc solid/solution-phase synthesis. Compounds 1-4 exhibited immunosuppressive effects via inhibition of T and B cell proliferation, and 1 and 4 showed better inhibitory activities than their corresponding diastereomers.

Cultivation of fractionated cells from a bioactive-alkaloid-bearing marine sponge Axinella sp.

Sponges are among the most primitive multicellular organisms and well-known as a major source of marine natural products. Cultivation of sponge cells has long been an attractive topic due to the prominent evolutionary and cytological significance of sponges and as a potential approach to supply sponge-derived compounds. Sponge cell culture is carried out through culturing organized cell aggregates called 'primmorphs.' Most research culturing sponge cells has used unfractionated cells to develop primmorphs. In the current study, a tropical marine sponge Axinella sp., which contains the bioactive alkaloids, debromohymenialdisine (DBH), and hymenialdisine (HD), was used to obtain fractionated cells and the corresponding primmorphs. These alkaloids, DBH and HD, reportedly show pharmacological activities for treating osteoarthritis and Alzheimer's disease. Three different cell fractions were obtained, including enriched spherulous cells, large mesohyl cells, and small epithelial cells. These cell fractions were cultivated separately, forming aggregates that later developed into different kinds of primmorphs. The three kinds of primmorphs obtained were compared as regards to appearance, morphogenesis, and cellular composition. Additionally, the amount of alkaloid in the primmorphs-culture system was examined over a 30-d culturing period. During the culturing of enriched spherulous cells and developed primmorphs, the total amount of alkaloid declined notably. In addition, the speculation of alkaloid secretion and some phenomena that occurred during cell culturing are discussed.

Marine Fungus Aspergillus chevalieri TM2-S6 Extract Protects Skin Fibroblasts from Oxidative Stress.

The strain Aspergillus chevalieri TM2-S6 was isolated from the sponge Axinella and identified according to internal transcribed spacer (ITS) molecular sequence homology with Aspergillus species from the section Restricti. The strain was cultivated 9 days on potato dextrose broth (PDB), and the medium evaluated as antioxidant on primary normal human dermal fibroblasts (NHDF). The cultivation broth was submitted to sterile filtration, lyophilized and used without any further processing to give the Aspergillus chevalieri TM2-S6 cultivation broth ingredient named ACBB. ACCB contains two main compounds: tetrahydroauroglaucin and flavoglaucin. Under oxidative stress, ACCB showed a significant promotion of cell viability. To elucidate the mechanism of action, the impact on a panel of hundreds of genes involved in fibroblast physiology was evaluated. Thus, ACCB stimulates cell proliferation (VEGFA, TGFB3), antioxidant response (GPX1, SOD1, NRF2), and extracellular matrix organization (COL1A1, COL3A1, CD44, MMP14). ACCD also reduced aging (SIRT1, SIRT2, FOXO3). These findings indicate that Aspergillus chevalieri TM2-S6 cultivation broth exhibits significant in vitro skin protection of human fibroblasts under oxidative stress, making it a potential cosmetic ingredient.

Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea.

The geographic position, highly fluctuating sea temperatures and hypersalinity make Persian Gulf an extreme environment. Although this unique environment has high biodiversity dominated by invertebrates, its potential in marine biodiscovery has largely remained untapped. Herein, we aimed at a detailed analysis of the metabolome and bioactivity profiles of the marine sponge Axinella sinoxea collected from the northeast coast of the Persian Gulf in Iran. The crude extract and its Kupchan subextracts were tested in multiple in-house bioassays, and the crude extract and its CHCl3-soluble portion showed in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium (Efm). A molecular networking (MN)-based dereplication strategy by UPLC-MS/MS revealed the presence of phospholipids and steroids, while 1H NMR spectroscopy indicated the presence of additional metabolites, such as diketopiperazines (DKPs). Integrated MN and 1H NMR analyses on both the crude and CHCl3 extracts combined with an antibacterial activity-guided isolation approach afforded eight metabolites: a new diketopiperazine, (-)-cyclo(L-trans-Hyp-L-Ile) (8); a known diketopiperazine, cyclo(L-trans-Hyp-L-Phe) (7); two known phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphocholine (1) and 1-O-octadecanoyl-sn-glycero-3-phosphocholine (2); two known steroids, 3ß-hydroxycholest-5-ene-7,24-dione (3) and (22E)-3ß-hydroxycholesta-5,22-diene-7,24-dione (4); two known monoterpenes, loliolide (5) and 5-epi-loliolide (6). The chemical structures of the isolates were elucidated by a combination of NMR spectroscopy, HRMS and [α]D analyses. All compounds were tested against MRSA and Efm, and compound 3 showed moderate antibacterial activity against MRSA (IC50 value 70 µg/mL). This is the first study that has dealt with chemical and bioactivity profiling of A. sinoxea leading to isolation and characterization of pure sponge metabolites.

Geographic range extensions of stalked, flabelliform sponges (Porifera) from eastern Canada with a new combination of a species of Plicatellopsis in the North Atlantic.

Three new species records, Axinella arctica (Vosmaer, 1885), Semisuberites cribrosa (Miklucho-Maclay, 1870), and Cladocroce spatula (Lundbeck, 1902), and one new combination, Plicatellopsis bowerbanki (Vosmaer, 1885) comb. nov. from eastern Canada are described. The four species have similar growth forms which are either fan or cup-shaped with obvious stalks. This is the first description of a member of the genus Plicatellopsis in the North Atlantic, and the second record of the genus in the northern hemisphere. The four species described here have a history of misidentification in eastern Canada and this work aims to guide future identifications in the region.

In vitro anti-proliferative activities of the sterols and fatty acids isolated from the Persian Gulf sponge; Axinella sinoxea.

PURPOSE: Marine sponges are rich sources of anticancer metabolites. Axinella sinoxea is a less studied sponge, found in the Larak Island's waters, of the Persian Gulf. In the present study, we have explored the cytotoxic properties and chemical constituents of A. sinoxea. METHODS: Repeated silica gel flash column chromatography of methanol extract of the Axinella sinoxea sponge, yielded fatty acid and sterol fractions. These fractions were analyzed by GC-MS and their anti-proliferative activities were evaluated by MTT assay against three human cancer cell lines including MOLT-4, MCF-7 and HT-29 as well as NIH/3 T3 fibroblast cells. The sterol-rich fractions were pooled and purified by HPLC and its sub fractions' cytotoxic activities were evaluated by MTT assay against MOLT-4 and NIH/3 T3 cells. RESULTS: The GC-MS spectral analysis of a fraction eluted with hexane: diethyl ether (90: 10), resulted in the identification of twelve fatty acids, including five linear chain saturated fatty acids; tetrdecanoic acid (1), pentadecanoic acid (3), hexadecanoic acid (5), heptadecanoic acid (7), and octadecanoic acid (10); one branched chain isoprenoid fatty acid, 4,8,12-trimethyltridecanoic acid (2); four monoenoic fatty acids; 9-hexadecenoic acid (4), 7-methyl-6-hexadecanoic acid (6), 9-octadecenoic acid (8) and 11-octadecenoic acid (9) and two polyunsaturated fatty acids; 5,8,11,14-eicosatetraenoic acid (11) and 4,7,10,13,16,19-docosahexaenoic acid (12). Spectral analysis of a non-polar fraction eluted with hexane: diethyl ether (85: 15), resulted in the identification of eight steroids including: cholesta-5,22-dien-3ß-ol (13), cholest-5-en-3ß-ol (14), ergosta-5,22-dien-3ß-ol (15), ergost-5-en-3ß-ol (16), stigmasta-5,22-dien-3ß-ol (17), γ-sitosterol (18), 33-norgorgosta-5,24(28)-dien-3ß-ol (19) and stigmasta-5,24(28)-dien-3ß-ol (20). Fatty acids-containing fraction was active against HT-29 cell line with IC50 26.52 ± 8.19 µg/mL, while the steroids-rich fraction was active against the three above mentioned cell lines with IC50 values of 1.20 ± 0.24, 4.12 ± 0.40 and 2.47 ± 0.31 µg/mL, respectively. All of the above-mentioned fractions and sub-fractions were inactive (IC50s > 50 µg/mL) when assayed against normal fibroblast cells. CONCLUSION: The present study suggests A. sinoxea as a potential natural source of cancer chemotherapeutics. Graphical abstract Cytotxic constituents of Axinella sinoxea.

Description and molecular phylogeny of Axinella nayaritensis n. sp. (Porifera: Axinellida) from East Pacific and remarks about the polyphyly of the genus Axinella.

Axinella nayaritensis n. sp. is a typical species of the genus Axinella in spiculation (oxeas and styles), skeletal arrangement (axial condensation from which radiate an extra-axial plumoreticulated skeleton), and external form (arborescent). The new species is orange, branching, up to 25 cm in height, and which usually live in soft bottoms. One of the most conspicuous characteristic of the new species is its aquiferous system, which is formed by a system of superficial canals and meandering ridges which run around the entire length of the branches. The species is compared with Axinella polycapella from Gulf of Mexico, which is very close in morphology, and which it shares a high similarity in the 18S ribosomal RNA region. It was also compared with Axinella aruensis, a very different species from a morphological point of view, which is distributed throughout the northern Australia, Indonesia and Papua New Guinea as both species share a high similarity in the COI mitochondrial gene region. The new species was included in the clade named Axinella by Gazave et al. (2010) for 18S and COI, where also are included eight Axinella-species. Species in this clade are typically arborescent with a choanosomal skeleton differentiated between axial and extra-axial regions. Axinella nayaritensis n. sp. constitute the first record of an Axinella for the east Pacific coast.

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization.

In search of alternative and safer sources of collagen for biomedical applications, the marine demosponges Axinella cannabina and Suberites carnosus, collected from the Aegean and the Ionian Seas, respectively, were comparatively studied for their insoluble collagen, intercellular collagen, and spongin-like collagen content. The isolated collagenous materials were morphologically, physicochemically, and biophysically characterized. Using scanning electron microscopy and transmission electron microscopy the fibrous morphology of the isolated collagens was confirmed, whereas the amino acid analysis, in conjunction with infrared spectroscopy studies, verified the characteristic for the collagen amino acid profile and its secondary structure. Furthermore, the isoelectric point and thermal behavior were determined by titration and differential scanning calorimetry, in combination with circular dichroism spectroscopic studies, respectively.

Cytotoxic Guanidine Alkaloids from a French Polynesian Monanchora n. sp. Sponge.

Four bicyclic and three pentacyclic guanidine alkaloids (1-7) were isolated from a French Polynesian Monanchora n. sp. sponge, along with the known alkaloids monalidine A (8), enantiomers 9-11 of known natural product crambescins, and the known crambescidins 12-15. Structures were assigned by spectroscopic data interpretation. The relative and absolute configurations of the alkaloids were established by analysis of (1)H NMR and NOESY spectra and by circular dichroism analysis. The new norcrambescidic acid (7) corresponds to interesting biosynthetic variation within the pentacyclic core. All compounds exhibited antiproliferative and cytotoxic efficacy against KB, HCT116, HL60, MRC5, and B16F10 cancer cells, with IC50 values ranging from 4 nM to 10 µM.

N-Formyl-7-amino-11-cycloamphilectene, a marine sponge metabolite, binds to tubulin and modulates microtubule depolymerization.

The importance of protein-small molecule interaction in drug discovery, medicinal chemistry and biology has driven the development of new analytical methods to disclose the whole interactome of bioactive compounds. To accelerate targets discovery of N-formyl-7-amino-11-cycloamphilectene (CALe), a marine bioactive diterpene isolated from the Vanuatu sponge Axinella sp., a chemoproteomic-based approach has been successfully developed. CALe is a potent anti-inflammatory agent, modulating NO and prostaglandin E2 overproduction by dual inhibition of the enhanced inducible NO synthase expression and cyclo-oxygenase-2 activity, without any evidence of cytotoxic effects. In this paper, several isoforms of tubulin have been identified as CALe off-targets by chemical proteomics combined with bio-physical orthogonal approaches. In the following biological analysis of its cellular effect, CALe was found to protect microtubules against the colcemid depolymerizing effect.

Diversity and antibacterial activity of bacteria cultured from Mediterranean Axinella spp. sponges.

AIMS: Evaluation of the diversity and antibacterial activity of bacteria cultivated from Mediterranean Axinella sponges and investigating the influence of culture conditions on antibacterial activity profiles of sponge bacteria. METHODS AND RESULTS: Based on 16S rRNA gene sequence analysis, the 259 bacteria isolated from the three Mediterranean Axinella sponges A. cannabina, A. verrucosa and A. polypoides belonged to 41 genera from the four phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria and included five potential newly cultured genera. In antagonistic streak assays, 87 isolates (34%) from 13 genera showed antibacterial activity towards at least one of the 10 environmental and laboratory test bacteria. The extracts and filtrates of 22 isolates grown under three different culture conditions were less often active as the isolates in the corresponding antagonistic streak assays. Changes in antibacterial activity profiles were isolate- and culture condition-specific. CONCLUSIONS: Axinella sponges are a good source to cultivate phylogenetic diverse and hitherto novel bacteria, many of which with antibacterial activity. Analysis of induced antibacterial activities might enhance the role of sponge bacteria in efforts to isolate new antibiotics in the future. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to investigate the diversity and antibacterial activity of bacteria isolated from A. cannabina and A. verrucosa. It highlights the potential importance of induced activity and the need for employing multiple culture conditions in antibacterial screening assays of sponge-associated bacteria.

Luteivirga sdotyamensis gen. nov., sp. nov., a novel bacterium of the phylum Bacteroidetes isolated from the Mediterranean sponge Axinella polypoides.

A novel aerobic bacterium, designated strain PIII.02(T), was isolated from a Mediterranean sponge (Axinella polypoides) collected off the Israeli coast near Sdot Yam. The non-motile cells were Gram-staining-negative, oxidase-positive and catalase-positive. The orange pigment of colonies growing on marine agar was neither diffusible nor flexirubin-like. Strain PIII.02(T) grew at 15-35 °C, at pH 6.0-9.0, with 2.0-7.0 % (w/v) NaCl, and with 1.0-8.0 % (w/v) sea salts. The predominant fatty acids were iso-C15 : 0, iso-C16 : 1 H, iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and C16 : 1ω7c. The major respiratory quinone was MK-7. The genomic DNA G+C content of the novel strain was 38.1 mol%. Results from 16S rRNA gene sequence analysis indicated that strain PIII.02(T) was distantly related to established members of the phylum Bacteroidetes. The established species found to be most closely related to the novel strain was Persicobacter diffluens NCIMB 1402(T) (87.6 % 16S rRNA gene sequence similarity). Based on the phenotypic and chemotaxonomic data and the results of the phylogenetic analyses, strain PIII.02(T) represents a novel species of a new genus, for which the name Luteivirga sdotyamensis gen. nov., sp. nov. is proposed. The type strain is PIII.02(T) ( = ATCC BAA-2393(T)  = LMG 26723(T)).

Fulvitalea axinellae gen. nov., sp. nov., a member of the family Flammeovirgaceae isolated from the Mediterranean sponge Axinella verrucosa.

The yellow-pigmented, non-motile, Gram-negative, strictly aerobic, rod-shaped bacterial strain VI.18(T) was isolated from the Mediterranean sponge Axinella verrucosa collected off the coast near Sdot Yam, Israel. Results from 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flammeovirgaceae. The highest nucleotide similarity (91.4 %) occurred with Aureibacter tunicatorum A5Q-118(T). The predominant cellular fatty acids of strain VI.18(T) were iso-C15 : 0 (56.0 %), iso-C17 : 1ω9c (22.8 %) and C16 : 0 (7.4 %) and its major respiratory quinone was MK-7. The DNA G+C content was 47.5 mol%. The strain could readily be distinguished from its phylogenetically closest relatives by phenotypic, physiological and chemotaxonomic properties. On the basis of the data from the present polyphasic study, we propose a novel genus and species within the family Flammeovirgaceae, with the name Fulvitalea axinellae gen. nov., sp. nov. Strain VI.18(T) ( = ATCC BAA-2395(T)  = LMG 26722(T)) is the type strain of Fulvitalea axinellae.

A new taurine derivative from South China Sea marine sponge Axinella sp.

A new (1) and two known (2 and 3) acylated taurine derivatives were isolated from the South China Sea marine sponge Axinella sp. The structures of the compounds were determined on the basis of spectral analysis. Compounds 1-3 did not inhibit the growth of hepatoma carcinoma cell (HepG2), lung carcinoma cell (A549), human breast carcinoma (MCF-7), no-small cell lung cancer (NCI-H460) and human nasopharyngeal carcinoma cell lines.

Staining tumor cells with biotinylated ACL-I, a lectin isolated from the marine sponge, Axinella corrugata.

Axinella corrugata lectin 1 (ACL-1) was purified from aqueous extracts of the marine sponge, Axinella corrugata. ACL-1 strongly agglutinates native rabbit erythrocytes. The hemagglutination is inhibited by N-acetyl derivatives, particularly N, N', N"-triacetylchitotriose, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine and N-acetyl-D-galactosamine. We investigated the capacity of biotinylated ACL-1 to stain several transformed cell lines including breast (T-47D, MCF7), colon (HT-29), lung (H460), ovary (OVCAR-3) and bladder (T24). ACL-I may bind to both monosaccharides and oligosaccharides of tumor cells, N-acetyl-D-galactosamine, and N-acetyl-D- glucosamine glycan types. The lectins are useful, not only as markers and diagnostic parameters, but also for tissue mapping in suspicious neoplasms. In addition, they provide a better understanding of neoplasms at the cytological and molecular levels. Furthermore, the use of potential metastatic markers such as lectins is crucial for developing successful tools for therapy against cancer. We observed that biotinylated ACL-I stains tumor cells and may hold potential as a probe for identifying transformed cells and for studying glycan structures synthesized by such cells.

Aureivirga marina gen. nov., sp. nov., a marine bacterium isolated from the Mediterranean sponge Axinella verrucosa.

Two bacterial strains, VI.14 and VIII.04(T), were isolated from the Mediterranean sponge Axinella verrucosa collected off the Israeli coast near Sdot Yam. The non-motile, aerobic, Gram-negative isolates were oxidase-negative and catalase-positive, and formed golden-brown colonies on marine agar 2216. The pigment was neither diffusible nor flexirubin-like. Strain VIII.04(T) grew at 15-37 °C, at pH 6.0-9.0, in the presence of 20-50 g NaCl l(-1) and 20-80 g sea salts l(-1), The spectrum was narrower for strain VI.14, with growth at pH 7.0-8.0. and in the presence of 30-50 g NaCl l(-1) and 30-70 g sea salts l(-1). The predominant fatty acid (>50 %) in both strains was iso-C15 : 0, and the major respiratory quinone was MK-6. The DNA G+C content was 30.7 and 31.1 mol% for VIII.04(T) and VI.14, respectively. Results from 16S rRNA sequence similarity and phylogenetic analyses indicated that both strains are closely related to members of the family Flavobacteriaceae within the phylum Bacteroidetes, with as much as 91.7 % 16S rRNA sequence similarity. On the basis of data from the polyphasic analysis, we suggest that the strains represent a novel species in a new genus within the family Flavobacteriaceae, for which the name Aureivirga marina gen. nov., sp. nov. is proposed. Strain VIII.04(T) ( = ATCC BAA-2394(T) = LMG 26721(T)) is the type strain of Aureivirga marina.

Further investigation of the Mediterranean sponge Axinella polypoides: isolation of a new cyclonucleoside and a new betaine.

An exhaustive exploration into the metabolic content of the Mediterranean sponge Axinella-polypoides resulted in the isolation of the new betaine 5 and the new cyclonucleoside 8. The structures of the new metabolites were elucidated by spectroscopic methods assisted by computational methods. The analysis also provided evidence that the sponge does not elaborate pyrrole-imidazole alkaloids (PIAs) but, interestingly, it was shown to contain two already known cyclodipeptides, compounds 9 (verpacamide A) and 10.

Biological activities of ACL-I and physicochemical properties of ACL-II, lectins isolated from the marine sponge Axinella corrugata.

Lectin II from the marine sponge Axinella corrugata (ACL-II) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel, followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-II is a lectin with an Mr of 80 kDa and 78 kDa, estimated by SDS-PAGE and by FPLC on Superose 12 HR column, respectively. ACL-II mainly agglutinates native rabbit erythrocytes and this hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but is inhibited by d-galactose, chitin and N-acetyl derivatives, with the exception of GalNAc. ACL-II is stable for up to 65 °C for 30 min, with a better stability at a pH range of 2 to 6. In contrast, ACL-I displays a strong mitogenic and cytotoxic effect.

New tetromycin derivatives with anti-trypanosomal and protease inhibitory activities.

Four new tetromycin derivatives, tetromycins 1-4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001(T) cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV M(pro), and PL(pro). The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with K(i) values in the low micromolar range.