Chemical Profiling (HPLC-NMR & HPLC-MS), Isolation,and Identification of Bioactive Meroditerpenoids from the Southern Australian Marine Brown Alga Sargassum paradoxum

A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6–8, and 11–14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5–7, and 10–12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.     

Red Algae (Rhodophyta) from the Coast of Madagascar: Preliminary Bioactivity Studies and Isolation of Natural Products

Several species of red algae (Rhodophyta) from the coastal regions of Madagascar have been investigated for their natural products. The most abundant compound was cholesterol (5) in combination with a series of oxidized congeners. The brominated indoles 1–3 along with the sesquiterpene debilone (4) have been isolated from Laurencia complanata. For the first time, debilone (4) has been obtained from a marine plant. From the methanol extract of Calloseris sp., we have achieved the second isolation of the unusual A-ring contracted steroids (−)-2-ethoxycarbonyl-2β-hydroxy-A-nor-cholest-5-en-4-one (9) and phorbasterone B (10). The crude extracts of Laurencia complanata exhibited antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans.     

Cytotoxicity,Fractionation and Dereplication of Extracts of the Dinoflagellate Vulcanodinium rugosum,a Producer of Pinnatoxin G

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of “fast-acting toxins”, its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC₅₀ values of 0.38 µg mL⁻¹ and 0.19 µg mL⁻¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL⁻¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.     

The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity

In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation.     

Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

高压消解-氢化物发生-原子荧光光谱法同时测定海藻中汞和砷

采用高压消解-氢化物发生-原子荧光光谱法(HG-AFS)同时测定了5种不同海藻的汞和砷含量以考察海藻的食用安全性和海水污染情况。结果表明:汞、砷的线性范围分别为0~5.0、0~20.0μg/L,相关系数分别为0.9997、0.9999,检出限分别为5.0×10-4、0.01 mg/kg,相对标准偏差分别为2.8%、0.5%,加标回收率分别为92.7%~98.1%、96.6%~104.8%。高压消解方法操作简单、快速省时、消解完全,HG-AFS法操作简便、分析速度快、灵敏度高,可对汞和砷同时测定。对5种海藻中的汞和砷测定比较发现,不同海藻汞、砷含量存在一些差异,但都低于限量标准,以石花菜中汞和砷含量最低。褐藻门富集汞的能力强于红藻门,但褐藻门和红藻门富集砷的能力没有明显的界限。该研究的5种海藻均满足消费要求,海水质量较好。     

Overview on Biological Activities and Molecular Characteristics of Sulfated Polysaccharides from Marine Green Algae in Recent Years

Among the three main divisions of marine macroalgae (Chlorophyta, Phaeophyta and Rhodophyta), marine green algae are valuable sources of structurally diverse bioactive compounds and remain largely unexploited in nutraceutical and pharmaceutical areas. Recently, a great deal of interest has been developed to isolate novel sulfated polysaccharides (SPs) from marine green algae because of their numerous health beneficial effects. Green seaweeds are known to synthesize large quantities of SPs and are well established sources of these particularly interesting molecules such as ulvans from Ulva and Enteromorpha, sulfated rhamnans from Monostroma, sulfated arabinogalactans from Codium, sulfated galacotans from Caulerpa, and some special sulfated mannans from different species. These SPs exhibit many beneficial biological activities such as anticoagulant, antiviral, antioxidative, antitumor, immunomodulating, antihyperlipidemic and antihepatotoxic activities. Therefore, marine algae derived SPs have great potential for further development as healthy food and medical products. The present review focuses on SPs derived from marine green algae and presents an overview of the recent progress of determinations of their structural types and biological activities, especially their potential health benefits.     

Production of Lectins from Marine Algae: Current Status,Challenges, and Opportunities for Non-Destructive Extraction

Marine algae are an excellent source of novel lectins. The isolation of lectins from marine algae expands the diversity in structure and carbohydrate specificities of lectins isolated from other sources. Marine algal lectins have been reported to have antiviral, antitumor, and antibacterial activity. Lectins are typically isolated from marine algae by grinding the algal tissue with liquid nitrogen and extracting with buffer and alcohol. While this method produces higher yields, it may not be sustainable for large-scale production, because a large amount of biomass is required to produce a minute amount of compound, and a significant amount of waste is generated during the extraction process. Therefore, non-destructive extraction using algal culture water could be used to ensure a continuous supply of lectins without exclusively disrupting the marine algae. This review discusses the traditional and recent advancements in algal lectin extraction methods over the last decade, as well as the steps required for large-scale production. The challenges and prospects of various extraction methods (destructive and non-destructive) are also discussed.     

Cellular Antioxidant Effect of Four Bromophenols from the Red Algae,Vertebrata lanosa

Three known bromophenols, 2,3-dibromo-4,5-dihydroxybenzylaldehyde (1), 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-hydroxymethyldiphenylmethane (2) and bis(2,3-dibromo-4,5-dihydroxylbenzyl) ether (3), and one new one, 5,5″-oxybis(methylene)bis(3-bromo-4-(2′,3′-dibromo-4′,5′-dihydroxylbenzyl)benzene-1,2-diol) (4), were isolated from an extract of the red alga, Vertebrata lanosa. The antioxidant activity of these four bromophenols was examined using one biochemical and two cellular assays: Oxygen Radical Absorbance Capacity (ORAC), Cellular Antioxidant Activity (CAA) and Cellular Lipid Peroxidation Antioxidant Activity (CLPAA) assays. Compound 2 distinguished itself by showing potent activity, having a better antioxidant effect than luteolin in both the CAA and CLPAA assays and of quercetin in the CLPAA assay. Although several bromophenols are known to be potent antioxidants in biochemical assays, this is the first time their cellular antioxidant activity has been demonstrated.     

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A₅₆₅/A₂₈₀) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K₂HPO₄ (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.     

Application of HPLC-NMR in the Identification of Plocamenone and Isoplocamenone from the Marine Red Alga Plocamium angustum

A combination of on-line HPLC-NMR and off-line chemical investigations has resulted in the identification of the previously reported polyhalogenated monoterpene plocamenone, together with the new structural analogue isoplocamenone from the crude extract of the marine alga Plocamium angustum. On-flow and stop-flow HPLC-NMR analyses (including the acquisition of WET 2D NMR spectra) rapidly assisted in the identification of the major component plocamenone and in the partial identification of its unstable double bond isomer isoplocamenone. Conventional off-line isolation and structural characterization techniques were employed to unequivocally confirm both structures, leading to a structural revision for plocamenone, as well as to obtain sufficient quantities for biological testing.     

Emerging Strategies and Integrated Systems Microbiology Technologies for Biodiscovery of Marine Bioactive Compounds

Marine microorganisms continue to be a source of structurally and biologically novel compounds with potential use in the biotechnology industry. The unique physiochemical properties of the marine environment (such as pH, pressure, temperature, osmolarity) and uncommon functional groups (such as isonitrile, dichloroimine, isocyanate, and halogenated functional groups) are frequently found in marine metabolites. These facts have resulted in the production of bioactive substances with different properties than those found in terrestrial habitats. In fact, the marine environment contains a relatively untapped reservoir of bioactivity. Recent advances in genomics, metagenomics, proteomics, combinatorial biosynthesis, synthetic biology, screening methods, expression systems, bioinformatics, and the ever increasing availability of sequenced genomes provides us with more opportunities than ever in the discovery of novel bioactive compounds and biocatalysts. The combination of these advanced techniques with traditional techniques, together with the use of dereplication strategies to eliminate known compounds, provides a powerful tool in the discovery of novel marine bioactive compounds. This review outlines and discusses the emerging strategies for the biodiscovery of these bioactive compounds.     

三亚红沙港环境特征及赤潮生物调查

2006年和2007年对三亚红沙港的自然环境概况、污染物来源和养殖状况及赤潮生物进行调查分析,为热带港湾赤潮可持续防控研究提供依据.结果表明:三亚红沙港是一个典型的热带半封闭型港湾;沿岸地表径流很少;污染物来源有养殖污染、生活污染和船舶污染,网箱养殖污染和沿岸池塘养殖污染是红沙港海域污染的主要来源;该海域赤潮生物种类非常丰富,共38种,主要种类为硅藻和甲藻,硅藻占68%,甲藻占26%;浮游植物主要优势种为骨条藻(Skeletonema sp.)和角毛藻(Chaetoceros sp.).该海域丰富的赤潮生物种类构成了该海域赤潮发生的潜在因素.     

Effect of Temperature and Light Intensity on the Polar Lipidome of Endophytic Brown Algae Streblonema corymbiferum and Streblonema sp. In Vitro

The effect of temperature and light intensity on the polar lipidome of endophytic brown algae Streblonema corymbiferum and Streblonema sp. in vitro was investigated. More than 460 molecular species have been identified in four glycoglycerolipids classes, five phosphoglycerolipids classes and one betaine lipid class. The lipids glucuronosyldiacylglycerol and diacylglyceryl-N,N,N-trimethyl-homoserine were found in the algae of the order Ectocarpales for the first time. A decrease in cultivation temperature led to an increase in the unsaturation level in all classes of polar lipids. Thus, at low temperatures, the content of 18:4/18:4 monogalactosyldiacylglycerol (MGDG), 20:5/18:4 digalactosyldiacylglycerol (DGDG), 18:3/16:0 sulfoquinovosyldiacylglycerol (SQDG), 18:3/18:3 and 18:3/18:4 phosphatidylglycerol (PG), 20:4/20:5 and 20:5/20:5 phosphatidylethanolamine (PE), 14:0/20:5, 16:0/20:5 and 20:5/20:5 phosphatidylcholine (PC), 20:5/20:4 phosphatidylhydroxyethylglycine and 18:1/18:2 DGTS increased. At high temperatures, an increase in the content of chloroplast-derived MGDG, DGDG and PG was observed. Both low and high light intensities caused an increase in 20:5/18:3 MGDG and 18:3/16:1 PG. At low light intensity, the content of DGDG with fatty acid (FA) 18:3 increased, and at high light intensity, it was with FA 20:5. The molecular species composition of extraplastid lipids also showed a dependence on light intensity. Thus, the content of PC and PE species with C20-polyunsaturated FA at both sn-positions, 18:1/18:1 DGTS and 16:0/18:1 phosphatidylinositol increased. Low light intensity induced a significant increase in the content of chloroplast-derived 18:1/16:1 phosphatidylethanolamine.     

An Overview of the Medical Applications of Marine Skeletal Matrix Proteins

In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.     

Preliminary Study on the Activity of Phycobiliproteins against Botrytis cinerea

Phycobiliproteins (PBPs) are proteins of cyanobacteria and some algae such as rhodophytes. They have antimicrobial, antiviral, antitumor, antioxidative, and anti-inflammatory activity at the human level, but there is a lack of knowledge on their antifungal activity against plant pathogens. We studied the activity of PBPs extracted from Arthrospira platensis and Hydropuntia cornea against Botrytis cinerea, one of the most important worldwide plant-pathogenic fungi. PBPs were characterized by using FT-IR and FT-Raman in order to investigate their structures. Their spectra differed in the relative composition in the amide bands, which were particularly strong in A. platensis. PBP activity was tested on tomato fruits against gray mold disease, fungal growth, and spore germination at different concentrations (0.3, 0.6, 1.2, 2.4, and 4.8 mg/mL). Both PBPs reduced fruit gray mold disease. A linear dose–response relationship was observed for both PBPs against disease incidence and H. cornea against disease severity. Pathogen mycelial growth and spore germination were reduced significantly by both PBPs. In conclusion, PBPs have the potential for being also considered as natural compounds for the control of fungal plant pathogens in sustainable agriculture.     

Antiplatelet and Anticoagulant Effects of Diterpenes Isolated from the Marine Alga,Dictyota menstrualis

Cardiovascular diseases represent a major cause of disability and death worldwide. Therapeutics are available, but they often have unsatisfactory results and may produce side effects. Alternative treatments based on the use of natural products have been extensively investigated, because of their low toxicity and side effects. Marine organisms are prime candidates for such products, as they are sources of numerous and complex substances with ecological and pharmacological effects. In this work, we investigated, through in vitro experiments, the effects of three diterpenes (pachydictyol A, isopachydictyol A and dichotomanol) from the Brazilian marine alga, Dictyota menstrualis, on platelet aggregation and plasma coagulation. Results showed that dichotomanol inhibited ADP- or collagen-induced aggregation of platelet-rich plasma (PRP), but failed to inhibit washed platelets (WP). In contrast, pachydictyol A and isopachydictyol A failed to inhibit the aggregation of PRP, but inhibited WP aggregation induced by collagen or thrombin. These diterpenes also inhibited coagulation analyzed by the prothrombin time and activated partial thromboplastin time and on commercial fibrinogen. Moreover, diterpenes inhibited the catalytic activity of thrombin. Theoretical studies using the Osiris Property Explorer software showed that diterpenes have low theoretical toxicity profiles and a drug-score similar to commercial anticoagulant drugs. In conclusion, these diterpenes are promising candidates for use in anticoagulant therapy, and this study also highlights the biotechnological potential of oceans and the importance of bioprospecting to develop medicines.     

Purification,Chemical Characterization and Immunomodulatory Activity of a Sulfated Polysaccharide from Marine Brown Algae Durvillaea antarctica

An immunomodulatory polysaccharide (DAP4) was extracted, purified, and characterized from Durvillaea antarctica. The results of chemical and spectroscopic analyses demonstrated that the polysaccharide was a fucoidan, and was mainly composed of (1→3)-α-l-Fucp and (1→4)-α-l-Fucp residues with a small degree of branching at C-3 of (1→4)-α-l-Fucp residues. Sulfate groups were at C-4 of (1→3)-α-l-Fucp, C-2 of (1→4)-α-l-Fucp and minor C-6 of (1→4)-β-d-Galp. Small amounts of xylose and galactose exist in the forms of β-d-Xylp-(1→ and β-d-Gal-(1→. The immunomodulatory activity of DAP4 was measured on RAW 264.7 cells, the results proved that DAP4 exhibited excellent immunomodulatory activities, such as promoted the proliferation of spleen lymphocytes, increased NO production, as well as enhanced phagocytic of macrophages. Besides, DAP4 could also produce better enhancement on the vitality of NK cells. For the high immunomodulatory activity, DAP4 might be a potential source of immunomodulatory fucoidan with a novel structure.