Polyhydroxylated Steroids from the South China Sea Soft Coral Sarcophyton sp. and Their Cytotoxic and Antiviral Activities

Chemical investigation on the soft coral Sarcophyton sp. collected from the South China Sea yielded three new polyhydroxylated steroids, compounds (1–3), together with seven known ones (4–10). Their structures were established by extensive spectroscopic methods and comparison of their data with those of the related known compounds. All the isolates possessed the 3β,5α,6β-trihydroxylated steroidal nucleus. The cytotoxicities against selected HL-60, HeLa and K562 tumor cell lines and anti-H1N1 (Influenza A virus (IAV)) activities for the isolates were evaluated. Compounds 2, 3 and 5–8 exhibited potent activities against K562 cell lines with IC₅₀ values ranging from 6.4 to 10.3 μM. Compounds 1, 6–8 potently inhibited the growth of HL-60 tumor cell lines, and 6 also showed cytotoxicity towards HeLa cell lines. In addition, preliminary structure-activity relationships for the isolates are discussed. The OAc group at C-11 is proposed to be an important pharmacophore for their cytotoxicities in the 3β,5α,6β-triol steroids. Compounds 4 and 9 exhibited significant anti-H1N1 IAV activity with IC₅₀ values of 19.6 and 36.7 μg/mL, respectively.     

Secosteroids and Norcembranoids from the Soft Coral Sinularia nanolobata

Two new 9,11-secosteroids, 22α-acetoxy-24-methylene-3β,6α,11-trihydroxy-9,11-seco-cholest-7-en-9-one (1) and 11-acetoxy-24-methylene-1β,3β,6α-trihydroxy-9,11-seco-cholest-7-en-9-one (2), as well as two known norcembranoids, 5-epi-sinuleptolide (3) and sinuleptolide (4), were isolated from the soft coral Sinularia nanolobata. The structures of these metabolites were elucidated on the basis of extensive spectroscopic analysis. The anti-HCMV (human cytomegalovirus) activity of 1–4 and its cytotoxicity against selected cell lines were evaluated.     

Bioactive Polyoxygenated Steroids from the South China Sea Soft Coral,Sarcophyton sp

Seven new polyoxygenated steroids (1–7) were isolated together with seven known analogues (8–14) from the South China Sea soft coral, Sarcophyton sp. The structures of the new compounds were identified on the basis of extensive spectroscopic analysis and comparison with reported data. All the steroids are characterized with 3β,5α,6β-hydroxy moiety, displaying carbon skeletons of cholestane, ergostane, gorgostane and 23,24-dimethyl cholestane. In the in vitro bioassay, metabolites exhibited different levels of antimicrobial activity against bacterial species Escherichia coli and Bacillus megaterium, and fungal species Microbotryum violaceum and Septoria tritici. No inhibition was detected towards microalga Chlorella fusca. Preliminary structure-activity analysis suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane moiety at the side chain may also contribute to the bioactivity.     

Topsensterols A–C,Cytotoxic Polyhydroxylated Sterol Derivatives from a Marine Sponge Topsentia sp.

Three new polyhydroxylated sterol derivatives topsensterols A–C (1–3) have been isolated from a marine sponge Topsentia sp. collected from the South China Sea. Their structures were elucidated by detailed analysis of the spectroscopic data, especially the NOESY spectra. Topsensterols A–C (l–3) possess novel 2β,3α,4β,6α-tetrahydroxy-14α-methyl Δ⁹⁽¹¹⁾ steroidal nuclei with unusual side chains. Compound 2 exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC₅₀ value of 8.0 μM. Compound 3 displayed cytotoxicity against human erythroleukemia cell line K562 with an IC₅₀ value of 6.0 μM.     

Neuritogenic and Neuroprotective Effects of Polar Steroids from the Far East Starfishes Patiria pectinifera and Distolasterias nipon

The neuritogenic and neuroprotective activities of six starfish polar steroids, asterosaponin Р₁, (25S)-5α-cholestane-3β,4β,6α,7α,8,15α,16β,26-octaol, and (25S)-5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol (1–3) from the starfish Patiria pectinifera and distolasterosides D₁–D₃ (4–6) from the starfish Distolasterias nipon were analyzed using the mouse neuroblastoma (NB) C-1300 cell line and an organotypic rat hippocampal slice culture (OHSC). All of these compounds enhanced neurite outgrowth in NB cells. Dose-dependent responses to compounds 1–3 were observed within the concentration range of 10–100 nM, and dose-dependent responses to glycosides 4–6 were observed at concentrations of 1–50 nM. All the tested substances exhibited notable synergistic effects with trace amounts of nerve growth factor (NGF, 1 ng/mL) or brain-derived neurotrophic factor (BDNF, 0.1 ng/mL). Using NB cells and OHSCs, it was shown for the first time that starfish steroids 1–6 act as neuroprotectors against oxygen-glucose deprivation (OGD) by increasing the number of surviving cells. Altogether, these results suggest that neurotrophin-like neuritogenic and neuroprotective activities are most likely common properties of starfish polyhydroxysteroids and the related glycosides, although the magnitude of the effect depended on the particular compound structure.     

Cytotoxic,Cytostatic and HIV-1 PR Inhibitory Activities of the Soft Coral Litophyton arboreum

Bioassay-guided fractionation using different chromatographic and spectroscopic techniques in the analysis of the Red Sea soft coral Litophyton arboreum led to the isolation of nine compounds; sarcophytol M (1), alismol (2), 24-methylcholesta-5,24(28)-diene-3β-ol (3), 10-O-methyl alismoxide (4), alismoxide (5), (S)-chimyl alcohol (6), 7β-acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24-methylcholesta-5,24(28)-diene-3β,7β,19-triol (9). Some of the isolated compounds demonstrated potent cytotoxic- and/or cytostatic activity against HeLa and U937 cancer cell lines and inhibitory activity against HIV-1 protease (PR). Compound 7 was strongly cytotoxic against HeLa cells (CC₅₀ 4.3 ± 0.75 µM), with selectivity index of SI 8.1, which was confirmed by real time cell electronic sensing (RT-CES). Compounds 2, 7, and 8 showed strong inhibitory activity against HIV-1 PR at IC₅₀s of 7.20 ± 0.7, 4.85 ± 0.18, and 4.80 ± 0.92 µM respectively. In silico docking of most compounds presented comparable scores to that of acetyl pepstatin, a known HIV-1 PR inhibitor. Interestingly, compound 8 showed potent HIV-1 PR inhibitory activity in the absence of cytotoxicity against the cell lines used. In addition, compounds 2 and 5 demonstrated cytostatic action in HeLa cells, revealing potential use in virostatic cocktails. Taken together, data presented here suggest Litophyton arboreum to contain promising compounds for further investigation against the diseases mentioned.     

Philinopgenin A,B, and C,Three New Triterpenoid Aglycones from the Sea Cucumber Pentacta quadrangulasis

Three new triterpenoid aglycones named Philinopgenin A (1), B (2), and C (3) were isolated from the acid hydrolysate of the crude glycoside mixture prepared from the whole sea cucumber Pentacta quadrangulasis Lesson. The corresponding structures were determined as 16β-acetoxyholosta-8(9), 24(25)-diene-3β-ol (1), 20, 25-epoxy-lanosta-9(11)-ene-3β-ol 18(16)–lactone (2) and 16β-acetoxyholosta-9(11), 24(25)-diene-3β-ol (3), respectively, on the basis of spectral evidence.     

Two Novel Tyrosinase Inhibitory Sesquiterpenes Induced by CuCl2 from a Marine-Derived Fungus Pestalotiopsis sp. Z233

Two new sesquiterpenes, 1β,5α,6α,14-tetraacetoxy-9α-benzoyloxy-7βH-eudesman-2β,11-diol (1) and 4α,5α-diacetoxy-9α-benzoyloxy-7βH-eudesman-1β,2β,11,14-tetraol (2), were produced as stress metabolites in the cultured mycelia of Pestalotiopsis sp. Z233 isolated from the algae Sargassum horneri in response to abiotic stress elicitation by CuCl₂. Their structures were established by spectroscopic means. New compounds 1 and 2 showed tyrosinase inhibitory activities with IC₅₀ value of 14.8 µM and 22.3 µM.     

Structural Characteristics and Anticancer Activity of Fucoidan from the Brown Alga Sargassum mcclurei

Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-l-Fucp(2,4SO₃⁻)-(1→3)-α-l-Fucp(2,4SO₃⁻)-(1→ motif with 1,4-linked 3-sulfated α-l-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal β-d-Galp residues or chains of alternating sulfated 1,3-linked α-l-Fucp and 1,4-linked β-d-Galp residues, which have been identified in galactofucans for the first time. Both α-l-Fucp and β-d-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of β-d-Galp) and potentially the C-3 of terminal β-d-Galp, 1,4-linked β-d-Galp and 1,4-linked α-l-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.     

Antimalarial Activity of Axidjiferosides,New β-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 β-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 β anomeric configuration may be suitable in searching for new antimalarial drugs.     

Isolation of Araguspongine M,a New Stereoisomer of an Araguspongine/Xestospongin alkaloid,and Dopamine from the Marine Sponge Neopetrosia exigua Collected in Palau

A new stereoisomer of an araguspongine/xestospongin alkaloid, named araguspongine M (1), has been isolated together with 12 known compounds, araguspongines B (2) and D (3), dopamine, three galactosyl diacylglycerols, 24-methyl cholesterol, 5,6-dihydrocholesterol, β-sitosterol, and three 5α,8α-epidioxy sterols (11–13), from the marine sponge Neopetrosia exigua (formerly Xestospongia exigua) collected in Palau. The structure of 1 was assigned on the basis of its spectral data analysis. This is the first report on the isolation of dopamine from a marine sponge. This compound may be produced by an endosymbiotic Synechococcus-like cyanobacterium. Compounds 1–3 and 11–13 showed cytotoxicity against HL-60 at IC₅₀’s of 5.5, 5.5, 5.9, 22.4, 9.5, and 9.6 μM, respectively. The possible biosynthesis origin of the isolated metabolites is discussed.     

Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion,Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB₂, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O₂⁻ production, followed by a progressive decrease in O₂⁻ release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB₂, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O₂⁻ as well as neuroinflammatory TXB₂, MMP-9, cytokines and chemokines.     

New α-Glucosidase Inhibitory Triterpenic Acid from Marine Macro Green Alga Codium dwarkense Boergs

The marine ecosystem has been a key resource for secondary metabolites with promising biological roles. In the current study, bioassay-guided phytochemical investigations were carried out to assess the presence of enzyme inhibitory chemical constituents from the methanolic extract of marine green alga—Codium dwarkense. The bioactive fractions were further subjected to chromatographic separations, which resulted in the isolation of a new triterpenic acid; dwarkenoic acid (1) and the known sterols; androst-5-en-3β-ol (2), stigmasta-5,25-dien-3β,7α-diol (3), ergosta-5,25-dien-3β-ol (4), 7-hydroxystigmasta-4,25-dien-3-one-7-O-β-d-fucopyranoside (5), 7-hydroxystigmasta-4,25-dien-3-one (6), and stigmasta-5,25-dien-3β-ol (7). The structure elucidation of the new compound was carried out by combined mass spectrometry and 1D (¹H and ¹³C) and 2D (HSQC, HMBC, COSY, and NOESY) NMR spectroscopic data. The sub-fractions and pure constituents were assayed for enzymatic inhibition of alpha-glucosidase. Compound 1 showed significant inhibition at all concentrations. Compounds 2, 3, 5, and 7 exhibited a dose-dependent response, whereas compounds 4–6 showed moderate inhibition. Utilizing such marine-derived biological resources could lead to drug discoveries related to anti-diabetics.     

Gageostatins A–C,Antimicrobial Linear Lipopeptides from a Marine Bacillus subtilis

Concerning the requirements of effective drug candidates to combat against high rising multidrug resistant pathogens, we isolated three new linear lipopeptides, gageostatins A–C (1–3), consisting of hepta-peptides and new 3-β-hydroxy fatty acids from the fermentation broth of a marine-derived bacterium Bacillus subtilis. Their structures were elucidated by analyzing a combination of extensive 1D, 2D NMR spectroscopic data and high resolution ESIMS data. Fatty acids, namely 3-β-hydroxy-11-methyltridecanoic and 3-β-hydroxy-9,11-dimethyltridecanoic acids were characterized in lipopeptides 1 and 2, respectively, whereas an unsaturated fatty acid (E)-7,9-dimethylundec-2-enoic acid was assigned in 3. The 3R configuration of the stereocenter of 3-β-hydroxy fatty acids in 1 and 2 was established by Mosher’s MTPA method. The absolute stereochemistry of amino acid residues in 1–3 was ascertained by acid hydrolysis followed by Marfey’s derivatization studies. Gageostatins 1–3 exhibited good antifungal activities with MICs values of 4–32 µg/mL when tested against pathogenic fungi (R. solani, B. cinerea and C. acutatum) and moderate antibacterial activity against bacteria (B. subtilis, S. aeureus, S. typhi and P. aeruginosa) with MICs values of 8–64 µg/mL. Futhermore, gageostatins 1–3 displayed cytotoxicity against six human cancer cell lines with GI₅₀ values of 4.6–19.6 µg/mL. It is also noteworthy that mixed compounds 1+2 displayed better antifungal and cytotoxic activities than individuals.     

Bioactive Metabolites from Mangrove Endophytic Fungus Aspergillus sp. 16-5B

Chemical investigation of the endophytic fungus Aspergillus sp. 16-5B cultured on Czapek’s medium led to the isolation of four new metabolites, aspergifuranone (1), isocoumarin derivatives (±) 2 and (±) 3, and (R)-3-demethylpurpurester A (4), together with the known purpurester B (5) and pestaphthalides A (6). Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The absolute configuration of Compound 1 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra, and that of Compound 4 was revealed by comparing its optical rotation data and CD with those of the literature. The structure of Compound 6 was further confirmed by single-crystal X-ray diffraction experiment using CuKα radiation. All isolated compounds were evaluated for their α-glucosidase inhibitory activities, and Compound 1 showed significant inhibitory activity with IC₅₀ value of 9.05 ± 0.60 μM. Kinetic analysis showed that Compound 1 was a noncompetitive inhibitor of α-glucosidase. Compounds 2 and 6 exhibited moderate inhibitory activities.     

Calcarides A–E,Antibacterial Macrocyclic and Linear Polyesters from a Calcarisporium Strain

Bioactive compounds were detected in crude extracts of the fungus, Calcarisporium sp. KF525, which was isolated from German Wadden Sea water samples. Purification of the metabolites from the extracts yielded the five known polyesters, 15G256α, α-2, β, β-2 and π (1–5), and five new derivatives thereof, named calcarides A–E (6–10). The chemical structures of the isolated compounds were elucidated on the basis of one- and two-dimensional NMR spectroscopy supported by UV and HRESIMS data. The compounds exhibited inhibitory activities against Staphylococcus epidermidis, Xanthomonas campestris and Propionibacterium acnes. As the antibacterial activities were highly specific with regard to compound and test strain, a tight structure-activity relationship is assumed.     

Effects of n-3 Polyunsaturated Fatty Acids (ω-3) Supplementation on Some Cardiovascular Risk Factors with a Ketogenic Mediterranean Diet

Background: the ketogenic diet (KD) has become a widely used nutritional approach for weight loss. Some of the KD’s positive effects on metabolism and cardiovascular risk factors are similar to those seen after n-3 polyunsaturated fatty acids (ω-3) supplementation. We hypothesized that a ketogenic Mediterranean diet with phytoextracts combined with ω-3 supplementation may have increased positive effects on cardiovascular risk factors and inflammation. Methods: We analyzed 34 male overweight subjects; aged between 25 and 65 years who were overall healthy apart from overweight. The subjects followed a ketogenic diet protocol for four weeks; with (KDO3) or without (KD) ω-3 supplementation. Results: All subjects experienced a significant loss of body weight and body fat and there was no significant differences between treatment (body weight: KD—4.7 kg, KDO3—4.03 kg, body fat KD—5.41 kg, KDO3—5.86 kg). There were also significant decreases in total cholesterol, LDL-c, and glucose levels. Triglycerides and insulin levels decreased more in KDO3 vs. KD subjects, with a significant difference. All the investigated inflammatory cytokines (IL-1β, IL-6, TNF-α) decreased significantly in KDO3 subjects whilst only TNF-α showed a significant decrease in KD subjects over the 12 month study period. No significant changes were observed in anti-inflammatory cytokines (IL-10 and IL-1Ra), creatinine, urea and uric acid. Adiponectin increased significantly only in the KDO3 group. Conclusions: ω-3 supplementation improved the positive effects of a ketogenic Mediterranean diet with phytoextracts on some cardiovascular/metabolic risk factors and inflammatory state.     

Territrem and Butyrolactone Derivatives from a Marine-Derived Fungus Aspergillus Terreus

Seventeen lactones including eight territrem derivatives (1–8) and nine butyrolactone derivatives (9–17) were isolated from a marine-derived fungus Aspergillus terreus SCSGAF0162 under solid-state fermentation of rice. Compounds 1–3 and 9–10 were new, and their structures were elucidated by spectroscopic analysis. The acetylcholinesterase inhibitory activity and antiviral activity of compounds 1–17 were evaluated. Among them, compounds 1 and 2 showed strong inhibitory activity against acetylcholinesterase with IC₅₀ values of 4.2 ± 0.6, 4.5 ± 0.6 nM, respectively. This is the first time it has been reported that 3, 6, 10, 12 had evident antiviral activity towards HSV-1 with IC₅₀ values of 16.4 ± 0.6, 6.34 ± 0.4, 21.8 ± 0.8 and 28.9 ± 0.8 μg·mL⁻¹, respectively. Antifouling bioassay tests showed that compounds 1, 11, 12, 15 had potent antifouling activity with EC₅₀ values of 12.9 ± 0.5, 22.1 ± 0.8, 7.4 ± 0.6, 16.1 ± 0.6 μg·mL⁻¹ toward barnacle Balanus amphitrite larvae, respectively.