Novel Antifouling and Antimicrobial Compound from a Marine-Derived Fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC₅₀ of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 μg ml⁻¹ to 3.81 μg ml⁻¹ while the LC₅₀ was 266.68 μg ml⁻¹ for B. amphitrite cyprids; EC₅₀ ranged from 0.67 μg ml⁻¹ to 0.78 μg ml⁻¹, and LC₅₀ was 2.64 μg ml⁻¹ for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 μg per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.     

Antifouling Activity of Secondary Metabolites Isolated from Chinese Marine Organisms

Biofouling results in tremendous economic losses to maritime industries around the world. A recent global ban on the use of organotin compounds as antifouling agents has further raised demand for safe and effective antifouling compounds. In this study, 49 secondary metabolites, including diterpenoids, steroids, and polyketides, were isolated from soft corals, gorgonians, brown algae, and fungi collected along the coast of China, and their antifouling activity was tested against cyprids of the barnacle Balanus (Amphibalanus) amphitrite. Twenty of the compounds were found to inhibit larval settlement significantly at a concentration of 25 μg ml^-1. Two briarane diterpenoids, juncin O (2) and juncenolide H (3), were the most promising non-toxic antilarval settlement candidates, with EC₅₀ values less than 0.13 μg ml^-1 and a safety ratio (LC₅₀/EC₅₀) higher than 400. A preliminary structure—activity relationships study indicated that both furanon and furan moieties are important for antifouling activity. Intriguingly, the presence of hydroxyls enhanced their antisettlement activity.     

Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC₅₀ range of 0.08–1.49 μg ml^?1. Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC₅₀ values were <10 μg ml^?1. The LC₅₀/EC₅₀ ratios of almost all of the synthesized compounds were >10². The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species,the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC₅₀ = 0.82 μg ml^?1) and B. neritina (EC₅₀ = 3 μg ml^?1). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml^?1 and 10 μg ml^?1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Synthesis and Fungicidal Activities of New 1,2,4‐Triazolo[1,5‐a]pyrimidines

A series of new acetohydrazone‐containing 1,2,4‐triazolo[1,5‐a]pyrimidine derivatives were designed and synthesized for the purpose of searching for novel agrochemicals with higher fungicidal activity. Their in vitro fungicidal activities against Rhizoctonia solani were evaluated, and the most promising compound, 2‐[(5,7‐dimethyl[1,2,4]triazolo[1,5‐a]pyrimidin‐2‐yl)sulfanyl]‐2′‐[(2‐hydroxyphenyl)methylidene]acetohydrazide ( 2‐17 ), showed a lower EC₅₀ value (5.34 μg ml^?1) than that of commercial carbendazim (EC₅₀=7.62 μg ml^?1). Additionally, compound 2‐17 was also found to display broad‐spectrum fungicidal activities, and its EC₅₀ value (4.56 μg ml^?1) against Botrytis cinereapers was very similar to that of carbendazim. Qualitative structure–activity relationships (QSARs) of the synthesized compounds were also discussed.     

Reversible anti-settlement activity against Amphibalanus (=Balanus) amphitrite,Bugula neritina,and Hydroides elegans by a nontoxic pharmaceutical compound,mizolastine

Mizolastine, an antihistamine pharmaceutical, was found to significantly inhibit larval settlement of the barnacle Amphibalanus (=Balanus) amphitrite, the bryozoan Bugula neritina, and the polychaete Hydroides elegans with EC₅₀ values of 4.2, 11.2, and 4.1 µg ml^?1, respectively. No toxicity against the larvae of these three species was observed at the concentration range tested during incubations with mizolastine. To determine whether the anti-settlement activity of mizolastine is reversible, recovery bioassays using these three species were conducted. More than 70% of the larvae that had been exposed for 4 h to mizolastine at concentrations four-fold greater than their respective EC₅₀ values completed normal metamorphosis. The results of the recovery bioassay provide evidence that the anti-settlement effect of mizolastine is reversible in addition to being nontoxic. The anti-settlement activities of several intermediates of the synthesis process of mizolastine were also examined. One of the intermediates, 2-chloro-1-(4-fluorobenzyl)-1H-benzo[d]imidazole, inhibited larval settlement and metamorphosis with low toxicity. These results may improve the understanding of the key functional group responsible for the anti-settlement activity of mizolastine.     

Bioactive Steroid Derivatives and Butyrolactone Derivatives from a Gorgonian‐Derived Aspergillus sp. Fungus

Six steroid derivatives, 1 – 6 , and five butyrolactone derivatives, 7 – 11 , were isolated from the fermentation broth of a gorgonian‐derived Aspergillus sp. fungus. Their structures were elucidated on the basis of NMR and MS spectral data. Compound 1 is a new, highly conjugated steroid. The NMR and MS data of 7 and 8 are reported for the first time, as their structures were listed in SciFinder Scholar with no associated reference. Compounds 1, 4, 5 , and 8 – 11 inhibited the larval settlement of barnacle Balanus amphitrite with EC₅₀ values ranging from 0.63 to 18.4 μg ml^?1. Butyrolactone derivatives 7 and 8 showed pronounced antibacterial activities against Staphylococcus aureus with the same MIC values as the positive control ciprofloxacin (MIC 1.56 μM for all three compounds).     

Volatile substances from adult extracts induce larval settlement of the barnacle balanus amphitrite *

The volatile substance extracted from conspecific adults induces larval settlement of the barnacle Balanus amphitrite. The settlement inducing activity of the volatile fractions was checked monthly from June, 1997 to December, 1998. Both water soluble extracts and volatile fractions from the barnacle were prepared by the steam distillation. The active cue in the volatile fraction was always extracted with n‐pentane under acidic conditions, although settlement inducing activity varied with the sample. GC‐MS analysis of the active and inactive pentane fractions revealed 1, 2, 3‐trimethyl‐benzene as the settlement inducing substance. Commercially available 1, 2, 3‐trimethylbenzene also showed high settlement inducing activity at a concentration of 0.8 × 10^‐12 M (100 pg 1^‐1). This substance was detected at concentrations of more than 7 ng g^‐1 of wet barnacle (equivalent to 0.6 × 10^‐12M, equal to 70pg1^‐1) by GC analysis. These results indicate that 1,2,3‐trimethylbenzene in the volatile fractions acts as a chemical cue for larval settlement. Monthly variation in the settlement inducing activity was observed, which synchronized with the breeding season of the barnacle. This observation suggests that the barnacle produced the chemical cue in the gonad during maturation or accumulated it from the environment.     

Variation in toxicity of copper pyrithione among populations and families of the barnacle,Balanus amphitrite

Inter- and intra-population variation in the toxicity of the antifouling biocide copper pyrithione (CuPT) was examined for nauplius larvae of the barnacle Balanus amphitrite. Nauplii were collected from brooding adults from four sites within the Newport River estuary (NC), chosen based on an initial estimation of recent and historical human activities that affect local contamination levels. Each site was characterized for the presence of polycyclic aromatic hydrocarbons and for the frequency of gastropod imposex, an indicator of contamination by organotins. Sensitivity of nauplii to CuPT varied significantly across the sites/populations, with LC₅₀ values ranging from 4.0 μg l^?1 to 6.1 μg l^?1. Larvae from the most contaminated site were the most sensitive to CuPT. Intrapopulation variation in toxicity was investigated by exposing nauplius larvae from 15 maternal families to a fixed concentration of CuPT (6.1 μg l^?1). Variation in larval mortality among the families was significant, ranging from 15.1% to 98.9%.     

Novel Antifoulants: Inhibition of Larval Attachment by Proteases

We investigated the effect of commercially available enzymes (α-amylase, α-galactosidase, papain, trypsin, and lipase) as well as proteases from deep-sea bacteria on the larval attachment of the bryozoan Bugula neritina L. The 50% effective concentrations (EC₅₀) of the commercial proteases were 10 times lower than those of other enzymes. Crude proteases from six deep-sea Pseudoalteromonas species significantly decreased larval attachment at concentrations of 0.03 to 1 mIU ml⁻¹. The EC₅₀ of the pure protease from the bacterium Pseudoalteromonas issachenkonii UST041101-043 was close to 1 ng ml⁻¹ (0.1 mIU ml⁻¹). The protease and trypsin individually incorporated in a water-soluble paint matrix inhibited biofouling in a field experiment. There are certain correlations between production of proteases by bacterial films and inhibition of larval attachment. None of the bacteria with biofilms that induced attachment of B. neritina produced proteolytic enzymes, whereas most of the bacteria that formed inhibitive biofilms produced proteases. Our investigation demonstrated the potential use of proteolytic enzymes for antifouling defense.     

Isethionic Acid and Floridoside Isolated from the Red Alga,Grateloupia turuturu,Inhibit Settlement of Balanus amphitrite Cyprid Larvae

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-α-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml^?1).     

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food‐grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02–2.5 μg·ml^?1) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC₅₀ values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 μg·ml^?1 respectively, while values of nisin found were 0.50 and 1.04 μg·ml^?1. In the LDH assay, the EC₅₀ values were 0.65 and 0.62 μg·ml^?1 for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.     

7,8-secolignans from Schisandra wilsoniana and their anti-HIV-1 activities

Two new 7,8‐secolignans, marphenols A and B ( 1 and 2 , resp.), together with a known related derivative, 7,8‐secoholostylone B ( 3 ), were isolated from the stems of Schisandra wilsoniana. The structures of 1 and 2 were elucidated by spectroscopic methods, including extensive 1D‐ and 2D‐NMR techniques. The anti‐HIV‐1 activities of 1 – 3 were evaluated. Compound 1 inhibited HIV‐1_IIIB‐induced syncytia formation with an EC₅₀ value of 0.55 μg ml^?1. It reduced p24 antigen expression in acutely HIV‐1_IIIB‐infected C8166 cells and primary isolate HIV‐1_TC‐2‐infected peripheral blood mononuclear cells (PBMCs), with EC₅₀ values of 3.34 and 0.52 μg ml^?1, respectively. It showed no effects on the HIV‐1_IIIB replication in chronically infected H9 cells as well as fusion inhibition.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

Bioactive Diphenyl Ether Derivatives from a Gorgonian‐Derived Fungus Talaromyces sp.

Three new diphenyl ether derivatives, talaromycins A–C ( 1 – 3 , resp.), together with six known analogs, 4 – 9 , were isolated from a gorgonian‐derived fungus, Talaromyces sp. The structures of the new compounds were determined by analysis of extensive NMR spectroscopic data. All of the isolated metabolites, 1 – 9 , were evaluated for their cytotoxic and antifouling activities. Compound 4 exhibited pronounced cytotoxicity against the tested human cell lines with the IC₅₀ values ranging from 4.3 to 9.8 μM . Compounds 3, 5, 8 , and 9 showed potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite with the EC₅₀ values ranging from 2.2 to 4.8 μg/ml.     

Mosquito larvicidal efficacy of phenolic acids of seaweed Chaetomorpha antennina (Bory) Kuetz. against Aedes aegypti

Mosquito larvicidal and repellent activities of phenolic acids of Chaetomorpha antennina (Bory) Kuetz. against the third instar larvae of Aedes aegypti were investigated. The larval mortality was observed after 24 h exposure. Results of mosquito larvicidal tests revealed that insoluble bound phenolic acids and soluble conjugated phenolic acid fractions of C. antennina had an excellent inhibitory effect against A. aegypti and its LC₅₀ values were 23.4 and 44.6 μg ml⁻¹, respectively. The repellency assay of insoluble bound phenolic acids and soluble conjugated phenolic acid fractions of C. antennina, at 10 μg cm⁻² concentration gave 100% protection up to 120 min. The results indicate that phenolic acids of C. antennina have a wide spectrum of larvicidal and repellent activities against Aedes aegypti.     

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1

Aims: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. Methods and Results: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV‐1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44·1%) have disclosed antiviral activity against one or more of the viruses assayed with EC₅₀ values in the range of 23·2 ± 2·5–422·7 ± 10·9 μg ml^?1. Conclusions: Twelve of the 34 extracts (35·3%) might be considered promising sources of antiviral natural products, as they have shown EC₅₀ ≤ 100 μg ml^?1. The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. Significance and Impact of the Study: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.     

Anti-Barnacle Activities of Isothiocyanates Derived from β-Citronellol and Their Structure–Activity Relationships

Antifouling agents with low toxicity are in high demand for sustaining marine industries and the environment. This study aimed to synthesize 15 isothiocyanates derived from β-citronellol and evaluate their antifouling activities and toxicities against cypris larvae of the barnacle Amphibalanus amphitrite. The synthesized isothiocyanates exhibited effective antifouling activities (EC₅₀=0.10–3.33 μg mL⁻¹) with high therapeutic ratios (LC₅₀/EC₅₀ >30). Four isothiocyanates with an amide or isocyano group showed great potential as effective antifouling agents (EC₅₀=0.10–0.32 μg mL⁻¹, LC₅₀/EC₅₀=104–833). The enantiomers of the isothiocyanates only slightly differed in their antifouling activities. These results may serve as a basis for further research and development of β-citronellol-derived isothiocyanates as effective low-toxic antifouling agents. To the best of our knowledge, this study is the first to report the antifouling activities of isothiocyanates derived from accessible natural products.     

Synthesis and Antioxidant Activity of New Norcantharidin Analogs

New norcantharidin analogs were designed and obtained as compounds with biological activity. As a starting material, exo‐7‐oxabicyclo[2.2.1]heptane‐2,3‐dicarboxylic acid anhydride was used. Three groups of compounds: dicarboximides, triazoles and thiazolidines were obtained in multistep reactions. The ¹H‐ and ¹³C‐NMR spectra were used to confirm the structures of all obtained products and they were in agreement with the proposed structure of substances. All derivatives were screened for their antioxidant activity. The most promising group was dicarboximides ( 1 – 4 , 6 ). Derivatives 2–4 displayed antioxidant activity with EC₅₀=7.75–10.89 μg/ml, which may be comparable to strong antioxidant Trolox (EC₅₀=6.13 μg/ml). Excellent activity with EC₅₀=10.75 μg/ml also presented norcantharidin analog with 1,2,4‐triazole system ( 12 ).     

Antifouling potential of the marine microalga Dunaliella salina

Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC₅₀ value against Skeletonema costatum was 58.9 μg ml^?1. The isolated purified extract was tested for antifouling activity, the EC ₅₀ value against S. costatum was 21.2 μg ml^?1, and the LC₅₀ against Balanus amphitrite larvae was 18.8 μg ml^?1. Subsequently, both UHR–TOF–MS and GC–MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.