Chilensosides E, F, and G-New Tetrasulfated Triterpene Glycosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida): Structures, Activity, and Biogenesis.

Three new tetrasulfated triterpene glycosides, chilensosides E (1), F (2), and G (3), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E (1) and F (2) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G (3) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3-a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2-excludes the possibility of this sugar chain's further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1-3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.

Absolute Stereochemistry and Cytotoxic Effects of Vismione E from Marine Sponge-Derived Fungus Aspergillus sp. 1901NT-1.2.2.

The metabolic profile of the Aspergillus sp. 1901NT-1.2.2 sponge-associated fungal strain was investigated using the HPLC MS technique, and more than 23 peaks in the HPLC MS chromatogram were detected. Only two minor peaks were identified as endocrocin and terpene derivative MS data from the GNPS database. The main compound was isolated and identified as known anthraquinone derivative vismione E. The absolute stereochemistry of vismione E was established for the first time using ECD and quantum chemical methods. Vismione E showed high cytotoxic activity against human breast cancer MCF-7 cells, with an IC50 of 9.0 µM, in comparison with low toxicity for normal human breast MCF-10A cells, with an IC50 of 65.3 µM. It was found that vismione E inhibits MCF-7 cell proliferation and arrests the cell cycle in the G1 phase. Moreover, the negative influence of vismione E on MCF-7 cell migration was detected. Molecular docking of vismione E suggested the IMPDH2 enzyme as one of the molecular targets for this anthraquinone derivative.

Structures and Biologic Activity of Chitonoidosides I, J, K, K1 and L-Triterpene Di-, Tri- and Tetrasulfated Hexaosides from the Sea Cucumber Psolus chitonoides.

Five new triterpene di-, tri- and tetrasulfated hexaosides (chitonoidosides I (1), J (2), K (3), K1 (4) and L (5)) were isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from a depth of 100-150 m. The structural variability of the glycosides concerned both the aglycones (with 7(8)- or 9(11)-double bonds) and carbohydrate chains differing from each other by the third sugar residue (Xyl or sulfated by C-6 Glc) and/or by the fourth-terminal in the bottom semi-chain-residue (Glc or sulfated by C-6 MeGlc) as well as by the positions of a sulfate group at C-4 or C-6 in the sixth-terminal in the upper semi-chain-residue (MeGlc). Hemolytic activities of these compounds 1-5 against human erythrocytes as well as cytotoxicity against human cancer cell lines, HeLa, DLD-1 and HL-60, were studied. The hexaosides, chitonoidosides K (3) and L (5) with four sulfate groups, were the most active against tumor cells in all the tests. Noticeably, the sulfate group at C-4 of MeGlc6 did not decrease the membranolytic effect of 5 as compared with 3, having the sulfate group at C-6 of MeGlc6. Erythrocytes were, as usual, more sensitive to the action of the studied glycosides than cancer cells, although the sensitivity of leukemia promyeloblast HL-60 cells was higher than that of other tumor cells. The glycosides 1 and 2 demonstrated some weaker action in relation to DLD-1 cells than against other tumor cell lines. Chitonoidoside K1 (4) with a hydroxyl at C 25 of the aglycone was not active in all the tests. The metabolic network formed by the carbohydrate chains of all the glycosides isolated from P. chitonoides as well as the aglycones biosynthetic transformations during their biosynthesis are discussed and illustrated with schemes.

New Tripeptide Derivatives Asperripeptides A-C from Vietnamese Mangrove-Derived Fungus Aspergillus terreus LM.5.2.

Three new tripeptide derivatives asterripeptides A-C (1-3) were isolated from Vietnamese mangrove-derived fungus Aspergillus terreus LM.5.2. Structures of isolated compounds were determined by a combination of NMR and ESIMS techniques. The absolute configurations of all stereocenters were determined using the Murfey's method. The isolated compounds 1-3 contain a rare fungi cinnamic acid residue. The cytotoxicity of isolated compounds against several cancer cell lines and inhibition ability of sortase A from Staphylococcus aureus of asterripeptides A-C were investigated.

Cytotoxic Drimane-Type Sesquiterpenes from Co-Culture of the Marine-Derived Fungi Aspergillus carneus KMM 4638 and Beauveria felina (=Isaria felina) KMM 4639.

Chemical investigation of a coculture of the marine-derived fungi Beauveria felina KMM 4639 and Aspergillus carneus KMM 4638 led to the identification of three new drimane-type sesquiterpenes, asperflavinoids B, D and E (2, 4, 5), and nine previously reported related compounds. The structures of these compounds were established using spectroscopic methods and by comparison with known analogues. We also investigated the cytotoxic activity of the isolated compounds against several cancer and normal cell lines. Asperflavinoid C (3) and ustusolate E (9) exerted a significant effect on human breast cancer MCF-7 cell viability, with IC50 values of 10 µM, and induced in caspase-dependent apoptosis and arrest of the MCF-7 cell cycle in the G2/M phase in these cells.

The Isolation, Structure Elucidation and Bioactivity Study of Chilensosides A, A1, B, C, and D, Holostane Triterpene Di-, Tri- and Tetrasulfated Pentaosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida).

Five new triterpene (4,4,14-trimethylsterol) di-, tri- and tetrasulfated pentaosides, chilensosides A (1), A1 (2), B (3), C (4), and D (5) were isolated from the Far-Eastern sea cucumber Paracaudina chilensis. The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The structural variability of the glycosides concerned the pentasaccharide chains. Their architecture was characterized by the upper semi-chain consisting of three sugar units and the bottom semi-chain of two sugars. Carbohydrate chains of compounds 2-5 differed in the quantity and positions of sulfate groups. The interesting structural features of the glycosides were: the presence of two sulfate groups at C-4 and C-6 of the same glucose residue in the upper semi-chain of 1, 2, 4, and 5 and the sulfation at C-3 of terminal glucose residue in the bottom semi-chain of 4 that makes its further elongation impossible. Chilensoside D (5) was the sixth tetrasulfated glycoside found in sea cucumbers. The architecture of the sugar chains of chilensosides A-D (1-5), the positions of sulfation, the quantity of sulfate groups, as well as the aglycone structures, demonstrate their similarity to the glycosides of the representatives of the order Dendrochirotida, confirming the phylogenetic closeness of the orders Molpadida and Dendrochirotida. The cytotoxic activities of the compounds 1-5 against human erythrocytes and some cancer cell lines are presented. Disulfated chilensosides A1 (2) and B (3) and trisulfated chilensoside C (4) showed significant cytotoxic activity against human cancer cells.

In Vitro Anti-Orthohantavirus Activity of the High-and Low-Molecular-Weight Fractions of Fucoidan from the Brown Alga Fucus evanescens.

The Hantaan orthohantavirus (genovariant Amur-AMRV) is a rodent-borne zoonotic virus; it is the causative agent of haemorrhagic fever with renal syndrome in humans. The currently limited therapeutic options require the development of effective anti-orthohantavirus drugs. The ability of native fucoidan from Fucus evanescens (FeF) and its enzymatically prepared high-molecular-weight (FeHMP) and low-molecular-weight (FeLMP) fractions to inhibit different stages of AMRV infection in Vero cells was studied. The structures of derivatives obtained were determined using nuclear magnetic resonance (NMR) spectroscopy. We found that fucoidan and its derivatives exhibited significant antiviral activity by affecting the early stages of the AMRV lifecycle, notably virus attachment and penetration. The FeHMP and FeLMP fractions showed the highest anti-adsorption activity by inhibiting AMRV focus formation, with a selective index (SI) > 110; FeF had an SI of ~70. The FeLMP fraction showed a greater virucidal effect compared with FeF and the FeHMP fraction. It was shown by molecular docking that 2O-sulphated fucotetrasaccharide, a main component of the FeLMP fraction, is able to bind with the AMRV envelope glycoproteins Gn/Gc and with integrin ß3 to prevent virus-cell interactions. The relatively small size of these sites of interactions explains the higher anti-AMRV activity of the FeLMP fraction.

Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima.

Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10-100 kDa and 50-100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.

Biologically Active Metabolites from the Marine Sediment-Derived Fungus Aspergillus flocculosus.

Four new compounds were isolated from the Vietnamese marine sediment-derived fungus Aspergillus flocculosus, one aspyrone-related polyketide aspilactonol G (2), one meroterpenoid 12-epi-aspertetranone D (4), two drimane derivatives (7,9), together with five known metabolites (1,3,5,6,8,10). The structures of compounds 1-10 were established by NMR and MS techniques. The absolute stereoconfigurations of compounds 1 and 2 were determined by a modified Mosher's method. The absolute configurations of compounds 4 and 7 were established by a combination of analysis of ROESY data and coupling constants as well as biogenetic considerations. Compounds 7 and 8 exhibited cytotoxic activity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells.

Asperindoles A⁻D and a p-Terphenyl Derivative from the Ascidian-Derived Fungus Aspergillus sp. KMM 4676.

Four new indole-diterpene alkaloids asperindoles A⁻D (1⁻4) and the known p-terphenyl derivative 3″-hydroxyterphenyllin (5) were isolated from the marine-derived strain of the fungus Aspergillus sp., associated with an unidentified colonial ascidian. The structures of 1⁻5 were established by 2D NMR and HRESIMS data. The absolute configurations of all stereocenters of 1⁻4 were determined by the combination of ROESY data, coupling constants analysis, and biogenetic considerations. Asperindoles C and D contain a 2-hydroxyisobutyric acid (2-HIBA) residue, rarely found in natural compounds. Asperindole A exhibits cytotoxic activity against hormone therapy-resistant PC-3 and 22Rv1, as well as hormone therapy-sensitive human prostate cancer cells, and induces apoptosis in these cells at low-micromolar concentrations.

The carboxymethylated derivative of laminaran from brown alga Saccharina cichorioides: Structure, anticancer and anti-invasive activities in 3D cell culture.

Polysaccharides' derivatives are promising biologically active compounds for biotechnology, nutrition, industries, and are becoming increasingly important in medicine and pharmacy. Laminaran from brown alga Saccharina cichorioides (ScL) was chemically modified to obtain the carboxymethylated derivative (ScLCM) with improved structure and bioactivity. ScLCM was identified as (1 â†’ 3)-ß-D-glucan with -CH2-COOH groups at some positions 2, 4, and 6 of glucose residues. The anticancer activity of ScLCM was studied on the models of viability and invasion of 3D human melanoma SK-MEL-28, breast cancer T-47D, and colorectal carcinoma DLD-1 cells in comparison with native laminaran or its sulfated or aminated derivatives. ScLCM had the highest anticancer and anti-invasive effects among investigated polysaccharides. ScLCM significantly suppressed the viability and invasion of 3D SK-MEL-28 cells via the regulation of the activity of matrix metalloproteinase 9 (MMP 9) and protein kinases of ERK/MAPK signaling pathway. These findings may contribute to the reported anticancer effects of algal polysaccharides' derivatives.

Production of high- and low-molecular weight fucoidan fragments with defined sulfation patterns and heightened in vitro anticancer activity against TNBC cells using novel endo-fucanases of the GH107 family.

Fucoidans are complex fucose-containing sulfated polysaccharides with pronounced anticancer effects. Their structure-anticancer activity relationships are difficult to determine due to fucoidans' complex, often irregularities-including structures. Fucoidan-active enzymes can be used for this propose. We have investigated two new recombinant endo-fucanases FWf3 and FWf4 from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T that belong to the 107 family of glycoside hydrolases (GH). Both enzymes cleaved α-(1→4)-glycosidic bonds but in fucoidan fragments with different sulfation patterns. FWf3 is the first characterized endo-fucanase that cleaves glycosidic bonds between 2O- and 2,4diO-sulfated L-fucose residues. The obtained endo-fucanases were used to produce low- and high-molecular weight fucoidan derivatives with different sulfate group locations. Low- and high-molecular weight fucoidan derivatives rich with 2,4diO-sulfation were shown to inhibit MDA-MB-231 cell colony formation more efficiently than the native fucoidan and the derivatives sulfated otherwise. Such derivatives effectively suppressed the mitochondrial membrane potential of MDA-MB-231 cells and reduced the expression of the glucose transporter 1 (GLUT1). Co-treatment of MDA-MB-231 cells with the fucoidan derivatives and oligomycin (an OXPHOS inhibitor) resulted in a synergistic anticancer effect. The data obtained demonstrate, that fucoidan and its 2,4diO-sulfated derivatives can be an effective adjunct in TNBC therapy targeting cell metabolism.

Structure and chemopreventive activity of fucoidans from the brown alga Alaria angusta.

Six fucoidan fractions were isolated from the brown alga Alaria angusta. Structures of enzymatic hydrolysis products of the fraction 1AaF2 (Fuc:Gal ~ 1:1; 33 % of sulfates) by fucanase from Wenyingzhuangia fucanilytica were studied by chemical and instrumental (NMR spectroscopy and mass-spectrometry) methods. It was shown that 1AaF2 consisted of two structurally different fucoidans: a sulfated 1,3;1,4-α-L-fucan and an enzyme-resistant sulfated and acetylated complex fucogalactan (Fuc:Gal ~ 1:2; 19 % of sulfates) 1AaF2_HMP containing extended 1,3-linked fucose and 1,3/1,4-linked galactose fragments (up to 5 residues). The fractions 1AaF2 and 1AaF2_HMP were a non-cytotoxic, possessed dose-dependent chemopreventive effect on EGF-induced neoplastic cell transformation of mouse normal epidermal JB6 Cl41 cells and inhibited the colony formation of human melanoma SK-MEL-28 cells.

Pectins from the sea grass Enhalus acoroides (L.f.) Royle: Structure, biological activity and ability to form nanoparticles.

Two pectins from the seagrass Enhalus acoroides (L.f.) Royle were isolated for the first time. Their structures and biological activities were investigated. NMR spectroscopy showed one of them to consist exclusively from the repeating →4-α-d-GalpUA→ residue (Ea1), while the other had a much more complex structure that also included 1→3-linked α-d-GalpUA residues, 1→4-linked ß-apiose residues and small amounts of galactose and rhamnose (Ea2). The pectin Ea1 showed noticeable dose-dependent immunostimulatory activity, the Ea2 fraction was less effective. Both pectins were used to create pectin-chitosan nanoparticles for the first time, and the influence of pectin/chitosan mass ratio on their size and zeta potential was investigated. Ea1 particles were slightly smaller than Ea2 particles (77 ± 16 nm vs 101 ± 12 nm) and less negatively charged (-23 mV vs -39 mV). Assessment of their thermodynamic parameters showed that only the second pectin could form nanoparticles at room temperature.

Two GH16 Endo-1,3-ß-D-Glucanases from Formosa agariphila and F. algae Bacteria Have Complete Different Modes of Laminarin Digestion.

There is a comparative analysis of primary structures and catalytic properties of two recombinant endo-1,3-ß-D-glucanases from marine bacteria Formosa agariphila KMM 3901 and previously reported F. algae KMM 3553. Both enzymes had the same molecular mass 61 kDa, temperature optimum 45 °C, and comparable ranges of thermal stability and Km. While the set of products of laminarin hydrolysis with endo-1,3-ß-D-glucanase from F. algae was stable of the reaction with pH 4-9, the pH stability of the products of laminarin hydrolysis with endo-1,3-ß-D-glucanase from F. agariphila varied at pH 5-6 for DP 2, at pH 4 and 7-8 for DP 5, and at pH 9 for DP 3. There were differences in modes of action of these enzymes on laminarin and 4-methylumbelliferyl-ß-D-glucoside (Umb), indicating the presence of transglycosylating activity of endo-1,3-ß-D-glucanase from F. algae and its absence in endo-1,3-ß-D-glucanase from F. agariphila. While endo-1,3-ß-D-glucanase from F. algae produced transglycosylated laminarioligosaccharides with a degree of polymerization 2-10 (predominately 3-4), endo-1,3-ß-D-glucanase from F. agariphila did not catalyze transglycosylation in our lab parameters.

New Antibacterial Chloro-Containing Polyketides from the Alga-Derived Fungus Asteromyces cruciatus KMM 4696.

Six new polyketides acrucipentyns A-F (1-6) were isolated from the alga-derived fungus Asteromyces cruciatus KMM 4696. Their structures were established based on spectroscopic methods. The absolute configurations of acrucipentyn A was assigned by the modified Mosher's method and ROESY data analysis. Acrucipentyns A-E were identified to be the very first examples of chlorine-containing asperpentyn-like compounds. The cytotoxic and antimicrobial activities of the isolated compounds were examined. Acrucipentyns A-F were found as antimicrobial agents, which inhibited sortase A enzyme activity, bacterial growth and biofilm formation of Staphylococcus aureus and decreased LDH release from human keratinocytes HaCaT in S. aureus skin infection in an in vitro model.

Relationship between the structure of a highly regular fucoidan from Fucus evanescens and its ability to form nanoparticles.

Chitosan/fucoidan nanoparticles were created using two fucoidans from the Fucus evanescens algae. One of them was a regular fucoidan obtained for the first time from the alga harvested at the reproductive growth stage, using only standard extraction methods, without additional modifications. Its structure was established via NMR spectroscopy to consist of the repeating →3)-α-L-Fucp-(2,4SO3-)-(1 â†’ 4)-α-L-Fucp-(2SO3-)-(1→ fragment. Such fragment also coustituted 55% of the other fucoidan's structure, however it also included long sequences of α-L-fucopyranose residues sulfated only at C2. The nanoparticles were re-dispersed in water and the influence of fucoidan/chitosan mass ratio on the nanoparticles' size and zeta potential was investigated. 3D models of the regular fucoidan and chitosan's sections were created and their molecular docking was performed, showing that either polymer could occupy the exterior of the complex, depending on their ratio. Thermodynamic parameters of fucoidan-chitosan binding process were accessed, with the results indicating that significant conformational changes of fucoidan and chitosan molecules take place during the interaction, presumably to allow for more effective binding.

Enzymatic transformation and anti-tumor activity of Sargassum horneri fucoidan.

Structure of the fucoidan from Sargassum horneri and products of its enzymatic transformation with molecular weight over 20 kDa were investigated. Fucoidan was hydrolyzed by recombinant fucoidanase FFA1 and its fraction of higher molecular weight was fractionated using anion-exchange chromatography, resulting in three sulphated polysaccharides of various molecular weight (63-138 kDa). Their structures were analyzed using NMR spectroscopy, showing the fucoidan (ShF) to be a branched polysaccharide with the backbone consisting of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment and side chains including the α-l-Fucp-1→2-α-l-Fucp-1→ or α-l-Fucp-1→3-α-l-Fucp(4SO3-)-1→ fragments attached to the main chain at C4. The fragment F3 differing by molecular weight and side chain from other fucoidans fragments possessed the most significant anticancer and radiosensitizing activities.

Citriperazines A-D produced by a marine algae-derived fungus Penicillium sp. KMM 4672.

Four new diketopiperazine alkaloids, citriperazines A-D were isolated from algae-derived Penicillium sp. KMM 4672. The structures of compounds 1-4 were determined using spectroscopic methods. The absolute configurations of compounds 1 and 4 were established by comparison of calculated and experimental ECD spectra. The cytotoxicity of compounds 1-4 against several human prostate cell lines was evaluated.

Auroglaucin-related neuroprotective compounds from Vietnamese marine sediment-derived fungus Aspergillus niveoglaucus.

Two new auroglaucin-derived compounds, niveoglaucins A (1) and B (2), together with four known related compounds were isolated from extract of the marine sediment-derived strain of Aspergillus niveoglaucus. The structures of these compounds were determined by 1D and 2D NMR spectroscopy and high resolution MS. The plausible biosynthetic pathway was proposed for new compounds 1 and 2. The neuroprotective activity in 6-OHDA-induced Parkinson's disease cell model was shown for niveoglaucin A (1).