Ichthyotoxic Activity Research Articles

Larvicidal Activity of Geranylacetone Derivatives against Culex quinquefasciatus Larvae and Investigation of Environmental Toxicity and Non-Target Aquatic Species

A grindstone method based on Mannich condensation was used to synthesize geranylacetone derivatives (1a–1f). The method showed a high yield under milder reaction conditions. Analyses of the synthesized compounds were carried out by FTIR, 1H, 13C NMR, mass spectrometry, and elemental analysis. We synthesized and evaluated the larvicidal and ichthyotoxic activities of six compounds (1a–1f) in this study. Compound 1f (5,9-dimethyl-1-phenyl-3-(2-(3-phenylallylidene)hydrazinyl)deca-4,8-dien-1-one) was more active (LD50: 14.1 µg/mL) against the second instar larvae of Culex quinquefasciatus than geranylacetone (67.2 µg/mL), whereas the former caused 13.9% mortality at 100 µg/mL. Geranylacetone, in an antifeedant screening test, showed 53.1% against Oreochromis mossambicus within 24 h. The compound 1f showed high larvicidal activity against C. quinquefasciatus and was non-toxic to non-target aquatic species.

Naphthoquinone Derivatives from Diospyros maritima.

From the chloroform extract of the fresh fruits of Diospyros maritima BLUME (Ebenaceae), five new naphthoquinone derivatives, 2,7'-dimethyl-2',3-bijuglone (27), 2,7'-dimethyl-3,3'-bijuglone (28), 2,7'-dimethyl-6,8'-bijuglone (29), 7,7'-dimethyl-3,3'-ethylidenebijuglone (30), and 2',7-dimethyl-3,6'-ethylidenebijuglone (31), were isolated, in addition to twenty-one known naphthoquinone derivatives: plumbagin (4), droserone (5), 2,3-epoxyplumbagin (8), 3,3'-biplumbagin (9), chitranone (10), 3,8'-biplumbagin (11), elliptinone (12), maritinone (13), isozeylanone (14), methylene-3,3'-biplumbagin (15), ethylidene-3,3'-biplumbagin (16), ethylidene-3,6'-biplumbagin (17), ethylidene-6,6'-biplumbagin (18), 7-methyl-β-dihydrojuglone (19), 7-methyljuglone (20), 2,3-epoxy-7-methyljuglone (21), neodiospyrin (22), mamegakinone (23), ehretione (24), isoxylospyrin (25) and β-dihydroplumbagin (26). The structures of the new compounds were established by spectral analysis. The quinones obtained from the chloroform extract of the fruits were compared with previously reported quinones obtained from ethanol extracts. The quinones in the fruits were categorized in three groups: quinones from ethanol extract only, quinones from chloroform extract only, and quinones from both extracts. The six naphthoquinones, 19-21, 25, 26, and 29, were examined for their ichthyotoxic activity and germination inhibitory activity. Quinones 19-21, 26, and 29 showed ichthyotoxic activity against Japanese killifish (Oryzias latipes var.) at 10 ppm; quinones 19 to 21 and 26 showed germination inhibitory activity toward lettuce seeds (Lactuca sativa L. var. Great Lakes) at 100 ppm.

Zebrafish Embryo Toxicity Microscale Model for Ichthyotoxicity Evaluation of Marine Natural Products.

Marine organisms often protect themselves against their predators by chemical defensive strategy. The second metabolites isolated from marine organisms and their symbiotic microbes have been proven to play a vital role in marine chemical ecology, such as ichthyotoxicity, allelopathy, and antifouling. It is well known that the microscale models for marine chemoecology assessment are urgently needed for trace quantity of marine natural products. Zebrafish model has been widely used as a microscale model in the fields of environment ecological evaluation and drug safety evaluation, but seldom reported for marine chemoecology assessment. In this work, zebrafish embryo toxicity microscale model was established for ichthyotoxicity evaluation of marine natural products by using 24-well microplate based on zebrafish embryo. Ichthyotoxicity was evaluated by observation of multiple toxicological endpoints, including coagulation egg, death, abnormal heartbeat, no spontaneous movement, delayed hatch, and malformation of the different organs during zebrafish embryogenesis periods at 24, 48, and 72h post-fertilization (hpf). 3,4-Dichloroaniline was used as the positive control for method validation. Subsequently, the established model was applied to test the ichthyotoxic activity of the compounds isolated from corals and their symbiotic microbes and to isolate the bioactive secondary metabolites from the gorgonian Subergorgia mollis under bioassay guidance. It was suggested that zebrafish embryo toxicity microscale model is suitable for bioassay-guided isolation and preliminary bioactivity screening of marine natural products.

Isolation of Five New Flavonoids from Melicope triphylla

Five new flavonoids, 5,8-dihydroxy-3,7-dimethoxy-3',4'-methylenedioxyflavone (1), 7-hydroxy-3,5-dimethoxy-3',4'-methylenedioxyflavone (2), 7-(2,3-dihydroxy-3-methylbutoxy)-3,5-dimethoxy-3',4'-methylenedioxyflavone (3), 7-(2,3-dihydroxy-3-methylbutoxy)-3,3',4',5-tetramethoxyflavone (4), and 7-(2,3-dihydroxy-3-methylbutoxy)-3,3',4',5,8-pentamethoxyflavone (5), were isolated from the leaves of Melicope triphylla. In addition, six known flavonoids were detected: 3,4',5-trihydroxy-3',7,8-trimethoxyflavone (6), 5,7-dihydroxy-3-methoxy-3',4'-methylenedioxyflavone (7), 4',5,7-trihydroxy-3,3'-dimethoxyflavone (8), 4',7-dihydroxy-3,3',5,8-tetramethoxyflavone (9), 4',7-dihydroxy-3,3',5-trimethoxyflavone (10), and 4',5,7-trihydroxy-3,3',8-trimethoxyflavone (11). The new compound structures were determined by spectroscopic methods. Compounds 1-5 did not exhibit any ichthyotoxic activity against Japanese killifish (medaka in Japanese) (Oryzias latipes var.) at 10 ppm.

Isolation of Six New Flavonoids from <i>Melicope triphylla</i>

Six new flavonoids--5-hydroxy-3,8-dimethoxy-3',4':6,7-bismethylenedioxyflavone (1), 3,3',4',5-tetramethoxy-7-(3-methylbut-2-enyloxy)flavone (2), 7-(2-hydroxy-3-methylbut-3-enyloxy)-3,3',4',5-tetramethoxyflavone (3), 7-(2-hydroxy-3-methylbut-3-enyloxy)-3,5-dimethoxy-3',4'-methylenedioxyflavone (4), 7-(2-hydroxy-3-methylbut-3-enyloxy)-3,3',4',5,8-pentamethoxyflavone (5), and 7-(2-hydroxy-3-methylbut-3-enyloxy)-3,5,8-trimethoxy-3',4'-methylenedioxyflavone (6)--were isolated from the leaves of Melicope triphylla. In addition, six already known flavonoids were also detected: 5-hydroxy-3,6,7-trimethoxy-3',4'-methylenedioxyflavone (7), 5,7-dihydroxy-3,3',4',8-tetramethoxyflavone (8), 4',5-dihydroxy-3,3',7,8-tetramethoxyflavone (9), 3,5,6,7,8-pentamethoxy-3',4'-methylenedioxyflavone (10), 3,5,6,7-tetramethoxy-3',4'-methylenedioxyflavone (11), and 3,3',4',5,6,7,8-heptamethoxyflavone (12). The structures of the new compounds were established by spectroscopic methods. Compound 2 displayed ichthyotoxic activity against Japanese killifish (medaka in Japanese) (Oryzias latipes var.) at 10 ppm.

ChemInform Abstract: Cyercenes, Novel Polypropionate Pyrones from the Autotomizing Mediterranean Mollusc Cyerce cristallina.

Abstract Seven novel pyrones of polypropionate biosynthetic origin have been isolated from the dorsal appendages (cerata) of the autotomizing ascoglossan mollusc Cyerce cristallina , and their structure established by means of spectroscopic techniques. The new compounds exhibited ichthyotoxic activity. Their possible involvement in the mollusc defensive mechanisms, as well as in regenerative processes following the autotomy, is hypothesized.

Characterization and quantification of karlotoxins by liquid chromatography–mass spectrometry

Karlodinium veneficum is a cosmopolitan dinoflagellate with a worldwide distribution in mesohaline temperate waters. The toxins from K. veneficum, or karlotoxins (KmTxs), which have been implicated in fish kill events, have been purified from monoalgal cultures, and shown to possess hemolytic, cytotoxic and ichthyotoxic activities. Three karlotoxins (KmTx 1–1, KmTx 1–3 and KmTx 2) have been isolated from two different North American strains of K. veneficum and characterized using liquid chromatography–mass spectrometry (LC–MS). KmTx 1 karlotoxins have a UV absorption maximum ( λ max 225 nm) at lower wavelengths than KmTx 2 karlotoxins ( λ max 235 nm). The exact masses and predicted empirical formulae for the karlotoxins (KmTx 1–1, 1308.8210, C 67H 120O 24; KmTx 1–3, 1322.8637, and C 69H 126O 23; KmTx 2, 1344.7938, C 67H 121ClO 24) were determined using high resolution mass spectrometry. Although the individual toxins produce a single peak in reverse phase high performance liquid chromatography (HPLC), MS revealed congeners co-eluting within each peak. These congeners could be separated under normal phase chromatography and revealed a single hydroxylation being responsible for the mass differences. Multistage MS (MS n ) showed that the three KmTxs and their congeners share a large portion of their structures including an identical 907 amu core fragment. These data were used to develop a quantitative LC–MS assay for karlotoxins from cultures and environmental samples. The sensitivity afforded by MS detection compared to UV absorbance allowed toxin quantification at 0.2 ng when injected on column. Aqueous solutions of karlotoxins were found to quantitatively adsorb to PTFE and nylon membrane filters. Aliquots from whole cultures or environmental samples could be concentrated and desalted by adsorption to PTFE syringe filters and karlotoxins eluted with methanol for analysis by LC–MS. This simplified solid phase cleanup afforded new data indicating that each karlotoxin may also exist as sulfated derivatives and also provided a rapid detection method for karlotoxin from environmental samples and whole cultures.

163 Identification of Euglenoids That Produce Ichthyotoxin(S) (Euglenophyta)

Diatoms, dinoflagellates, pelagiophytes, prymnesiophytes, and cyanobacteria are the only divisions of microalgae known to produce toxins. We now report toxin production by freshwater members of the genus Euglena. Fish mortalities (sheepshead minnows, catfish, striped bass, and tilapia) have been observed following exposure in the field to Euglena blooms and in the laboratory when exposed to unialgal isolates of two species of Euglena (E. sanguinea Ehrenberg and E. granulata (Klebs) Lemm.). Three toxic fractions have been isolated from unialgal isolates of both species, and include both water soluble and lipophilic compounds having ichthyotoxic activity. The toxins are stable at −80°C for at least 60 days and are heat stable to 30°C. Erratic swimming behavior of fish suggests a neurological toxin. This is the first report of fish kills by any freshwater algal taxa from both field and laboratory studies.

Three New Naphthoquinone Derivatives from Diospyros maritima Blume.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond

A dinoflagellate bloom was found associated with a fish kill event in a South Carolina brackish water retention pond. A multi-analytical approach was used to confirm the identity of the bloom dinoflagellate and evaluate its potential toxicity. Karlodinium micrum was confirmed through light microscopy, pigment profile comparisons, species-specific PCR, and gene sequence data. Necropsy findings on several fish were suggestive of an acute kill event. Toxicity of filtrate from bloom samples was tested by a hemolytic assay using rainbow trout ( Oncorhynchus mykis) erythrocytes and an ichthyotoxicity assay using larval zebrafish ( Danio rerio). Hemolytic activity was measurably high (>80% hemolysis) in both whole filtrate and fractionated filtrate (from the 80% MeOH C 18 column elution). This fraction also demonstrated high ichthyotoxic activity as exposed fish experienced rapid death. These results implicate toxic K. micrum as a causative factor in fish death in a non-aquaculture brackish pond associated with a housing development, and extend recent findings linking this species to fish kills in aquaculture ponds.

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp ( Paleomonetes pugio; adult 2–3 cm), blue crab ( Callinectes sapidus; juvenile 4–7 cm) and brine shrimp ( Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish . We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.

Three new naphthoquinone derivatives from Diospyros maritima Blume.

Three new naphthoquinone derivatives, 6-(1-ethoxyethyl)plumbagin (16), ethylidene-3,3'-biplumbagin (17), and ethylidene-3,6'-biplumbagin (18), were isolated, in addition to six known naphthoquinones, isozeylanone (10), 3,3'-biplumbagin (11), chitranone (12), methylene-3,3'-biplumbagin (13), 2,3-epoxyplumbagin (14), and 3,8'-biplumbagin (15), from the fruits of Diospyros maritima Blume (Ebenaceae). The structures of the new compounds were established by spectroscopic methods. The eight naphthoquinones 11-18 were examined for ichthyotoxic activity and germination inhibitory activity. The quinones 11, 12, and 14-16 showed strong ichthyotoxic activity and the quinone 14 mild germination inhibitory activity.

A second species of ichthyotoxic Pfiesteria (Dinamoebales, Dinophyceae)

A second toxic species within the family Pfiesteriaceae, Pfiesteria shumwayae Glasgow & Burkholder sp. nov., is described from the New River Estuary and the Neuse Estuary of the Albemarle-Pamlico Estuarine Ecosystem, USA. The species is polymorphic and multiphasic, with flagellated, amoeboid and cyst stages. The flagellated zoospores (diameter 8–24 μm) have permanently condensed chromosomes (mesokaryotic nucleus); a chrysophyte-like cyst (diameter 6–25 μm)with organic scales and bracts; and thin thecal plates arranged in a Kofoidian series of Po, cp, X, 4′, la, 6″, 6c, 4s, 5″′, 2″″. The benthic filopodial (filose), lobopodia1 (lobose) and rhizopodial amoeboid stages (5–250 μm) have an outer covering that ranges from rough to smooth in texture, depending on the stage of origin and the prey source. Pfiesteria shumwayae amoebae have a normal eukaryote nucleus and cysts of multiple sizes (diameter 4–25 μm) with a reticulate outer covering. Toxic strains of the two Pfiesteria species have overlapping distributions in the mid-Atlantic and southeastern United States and Scandinavia, with toxic P. shumwayae also having been verified from New Zealand. Pfiesteria shumwayae is similar to P. piscicida in its complex life cycle, general nutrition, attraction to live fish prey, and ichthyotoxic activity that is stimulated by the presence of live fish or their fresh tissues and excreta. However, it can be distinguished from P. piscicida morphologically by having six precingular plates and a four-sided la plate, as well as genetically, on the basis of its ISS ribosomal DNA sequence.

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus, the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.

Ichthyotoxic phloroglucinol derivatives from Dryopteris fragrans and their anti-tumor promoting activity.

Two new ichthyotoxic compounds, aspidin PB (8) and dryofragin (9), along with three known phloroglucinol derivatives (1-3) and five terpenoids, were isolated from the whole herbs of Dryopteris fragrans by toxicity-directed fractionation using Oryzias latipes (Japanese name; medaka). The structures of the new compounds were determined by spectroscopic methods including 2D NMR techniques. Amongst the isolates, aspidin PB (8), dryofragin (9), and 1-5 exhibited potent ichthyotoxic activity against medaka with a median tolerance limit (TLm after 24 h) of 1.2-4.3 microg/ml. These compounds which are toxic to fish also had a potent inhibitory effect on the activation of Epstein-Barr virus early-antigen (EBV-EA) induced by tetradecanoyl phorbol 13-acetate, which is an in vitro short-term assay for anti-tumor promoting agents. Aspidin BB (2) and albicanol (4), which exhibited strong inhibitory effects on the EBV-EA activation, significantly suppressed an in vivo two-stage carcinogenesis on mouse skin.

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6–11× higher hemolytic activity and 10× higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic α-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3–4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic α-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Gs 1.

Iridal-type triterpenoids with ichthyotoxic activity from belamcanda chinensis

Ichthyotoxic activity-guided fractionation of the hexane and ether extracts of Belamcanda chinensis (Iridaceae) has resulted in the isolation of eleven iridal-type triterpenoids including six new compounds, 3-O-tetradecanoyl-16-O-acetylisoiridogermanal (4), 3-O-decanoyl-16-O-acetylisoiridogermanal (5), belachinal (7), anhydrobelachinal (9), epianhydrobelachinal (10), and isoanhydrobelachinal (11). Structures of the new iridals were determined by spectral and chemical methods. The absolute configuration of isoiridogermanal (1) at C-16 was determined to be R by the modified Mosher's method. Of these compounds, 16-O-acetylisoiridogermanal (3), 7, and spiroiridal (12) exhibited potent ichthyotoxic activity against killie-fish (Oryzias latipes).

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin.

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin

Biological Activity of Crude Extract and Saponin Pseudoginsenoside-RT1 Derived from the Fruit of Randia siamensis

Ursolic acid (1), pseudoginsenoside-RP 1 (2), and pseudoginsenoside-RT 1 (3) were isolated from the fruit of Randia siamensis. 13 C NMR spectroscopy was particularly useful in confirming the structures of these components. Studies with rats showed that saponin 3 caused a decrease in blood pressure, an increase in heart rate and an increase in spontaneous contractility of the uterus. Also, a crude ethanol extract of the fruit of R. siamensis exhibited acute ichthyotoxic activity.