Chemoprotective effects of Ulva lactuca (green seaweed) aqueous-ethanolic extract against subchronic exposure to benzo(a)pyrene by CYP1A1 inhibition in mice.

The protective effect of the supplementation with an aqueous-ethanolic extract obtained from Ulva lactuca (Delile) green seaweed on benzo[a] pyrene-induced damage in mice was evaluated. Animals were treated with oral doses of U. lactuca extract (100 and 400 mg/kg) for 9 weeks. They were exposed to 50 mg/kg of oral doses of benzo(a)pyrene starting from the second week and up to the fifth week. Groups treated with benzo(a)pyrene only (second to fifth weeks), sunflower oil (vehicle, 9 weeks), or U. lactuca extract (100 and 400 mg/kg, 9 weeks) were also included in the study. The treatment with 400 mg/kg of the extract ameliorated the oxidative damage, decreased IL-1ß and TNF-α levels, and favorably regulated the antioxidant defenses compared with benzo(a)pyrene-exposed group. The benzo(a)pyrene-induced DNA damage was also reduced, as it was evidenced by the lower micronucleus formation in U. lactuca extract-supplemented animals. The extract protected the hepatic tissue, and it reduced the liver activity/expression of CYP1A1. These results altogether suggested a chemoprotective effect of U. lactuca extract against benzo(a)pyrene-induced-toxicity in mice, probably associated with an inhibitory effect of carcinogen bioactivation.

Seasonal behavior of Thalassia testudinum (Hydrocharitaceae) metabolites.

The marine angiosperm Thalassia testudinum, commonly known as turtle grass, is a dominant seagrass that grows in the Caribbean Sea shelf associated to Syringodium filiforme. The hydroalcoholic extract of T. testudinum is rich in polyphenols; the most abundant metabolite in this extract is thalassiolin B, a glycosilated flavonoid with skin damage repairing properties, and antioxidant capacity among others. The present study aimed at generating information about the seasonal behavior of secondary metabolites, as well as to study the antioxidant capacity of the T. testudinum leaves extract, collected monthly during 2012 from the Northeast coastline of Havana, Cuba. For this study, spectrophotometric methods were used to determine the concentrations of polyphenols, flavonoids, anthocyanins, soluble carbohydrates and proteins, chlorophylls a and b, and antioxidant activity of the extracts. In general, results demonstrated seasonal variations of the analyzed parameters. Extracts prepared from the vegetal material collected in October and November showed the highest values of polyphenols (58.81 ± 1.53 and 52.39 ± 0.63 mg/g bs, respectivally) and flavonoids (44.12 ± 1.30 and 51.30 ± 0.67 mg/gdw, respectively). On the contrary, the lowest values of polyphenols were found in extracts of leaves collected in July and August (15.51 ± 0.84 and 13.86 ± 0.48 mg/g,respectively). In accordance with these results, the lower value of Inhibitory Concentration (IC50) was obtained to get a 50 % of maximal effect on free radical scavenging activity with the extracts prepared from leaves collected in October and November, and less significant IC50 was obtained from the extract prepared from leaves collected in August (5.63 mg/mL). A negative correlation (r= -0.694) was observed in this study between the content of polyphenols and the IC50 necessary to get the half of its antioxidant maximal effect. The high correspondence between the maximum values of polyphenols, flavonoids, carbohydrates and proteins in October and November, revealed a close relationship between these metabolites found in the extract of T. testudinum. Our hypothesis about the annual variation in the concentration of these metabolites was validated; and these results will support the correct harvesting of T. testudinum leaves for biotechnology and industrial purposes.

Repair of UVB-damaged skin by the antioxidant sulphated flavone glycoside thalassiolin B isolated from the marine plant Thalassia testudinum Banks ex König.

Daily topical application of the aqueous ethanolic extract of the marine sea grass, Thalassia testudinum, on mice skin exposed to UVB radiation resulted in a dose-dependent recovery of the skin macroscopic alterations over a 6-day period. Maximal effect (90%) occurred at a dose of 240 microg/cm(2), with no additional effects at higher doses. Bioassay-guided fractionation of the plant extract resulted in the isolation of thalassiolin B (1). Topical application of 1 (240 microg/cm(2)) markedly reduces skin UVB-induced damage. In addition, thalassiolin B scavenged 2,2-diphenyl-2-picrylhydrazyl radical with an EC(50) = 100 microg/ml. These results suggest that thalassiolin B is responsible for the skin-regenerating effects of the crude extract of T. testudinum.

Gas Chromatography-Mass Spectrometry Analysis of Ulva fasciata (Green Seaweed) Extract and Evaluation of Its Cytoprotective and Antigenotoxic Effects.

The chemical composition and biological properties of Ulva fasciata aqueous-ethanolic extract were examined. Five components were identified in one fraction prepared from the extract by gas chromatography-mass spectrometry, and palmitic acid and its ethyl ester accounted for 76% of the total identified components. Furthermore, we assessed the extract's antioxidant properties by using the DPPH, ABTS, and lipid peroxidation assays and found that the extract had a moderate scavenging effect. In an experiment involving preexposition and coexposition of the extract (1-500 µg/mL) and benzo[a]pyrene (BP), the extract was found to be nontoxic to C9 cells in culture and to inhibit the cytotoxicity induced by BP. As BP is biotransformed by CYP1A and CYP2B subfamilies, we explored the possible interaction of the extract with these enzymes. The extract (25-50 µg/mL) inhibited CYP1A1 activity in rat liver microsomes. Analysis of the inhibition kinetics revealed a mixed-type inhibitory effect on CYP1A1 supersome. The effects of the extract on BP-induced DNA damage and hepatic CYP activity in mice were also investigated. Micronuclei induction by BP and liver CYP1A1/2 activities significantly decreased in animals treated with the extract. The results suggest that Ulva fasciata aqueous-ethanolic extract inhibits BP bioactivation and it may be a potential chemopreventive agent.

A novel sea anemone peptide that inhibits acid-sensing ion channels.

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.

Phytochemical analysis and antioxidant capacity of BM-21, a bioactive extract rich in polyphenolic metabolites from the sea Grass Thalassia testudinum.

The aqueous ethanol extract of Thalassia testudinum leaves (BM-21) is now being developed in Cuba as an herbal medicine due to its promising pharmacological properties. Although some interesting biological activities of BM-21 have already been reported, its chemical composition remains mostly unknown. Thus, we now describe the qualitative and quantitative analyzes of BM-21 using standard phytochemical screening techniques, including colorimetric quantification, TLC and HPLC analyses. Phytochemical investigation of BM-21 resulted in the isolation and identification of a new phenolic sulfate ester (1), along with ten previously described phenolic derivatives (2-11), seven of which have never been previously reported from the genus Thalassia. The structures of these compounds were established by analysis of their spectroscopic (1D and 2D NMR) and spectrometric (HRMS) data, as well as by comparison of these with those reported in the literature. Furthermore, BM-21 was found to exhibit strong antioxidant activity in four different free radical scavenging assays (HO*, RO2*, O2-* and DPPH*). Consequently, this is the first study which highlights the phytochemical composition of BM-21 and demonstrates that this product is a rich source of natural antioxidants with potential applications in pharmaceutical, cosmetic and food industries.

Combining multidimensional liquid chromatography and MALDI-TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer.

Sea anemones are sources of biologically active proteins and peptides. However, up to date few peptidomic studies of these organisms are known; therefore most species and their peptide diversity remain unexplored. Contrasting to previous venom peptidomic works on sea anemones and other venomous animals, in the present study we combined pH gradient ion-exchange chromatography with gel filtration and reversed-phase chromatography, allowing the separation of the 1-10 kDa polypeptides from the secretion of the unexplored sea anemone Phymanthus crucifer (Cnidaria/Phymanthidae). This multidimensional chromatographic approach followed by MALDI-TOF-MS detection generated a peptide fingerprint comprising 504 different molecular mass values from acidic and basic peptides, being the largest number estimated for a sea anemone exudate. The peptide population within the 2.0-3.5 kDa mass range showed the highest frequency whereas the main biomarkers comprised acidic and basic peptides with molecular masses within 2.5-6.9 kDa, in contrast to the homogeneous group of 4-5 kDa biomarkers found in sea anemones such as B. granulifera and B. cangicum (Cnidaria/Actiniidae). Our study shows that sea anemone peptide fingerprinting can be greatly improved by including pH gradient ion-exchange chromatography into the multidimensional separation approach, complemented by MALDI-TOF-MS detection. This strategy allowed us to find the most abundant and unprecedented diversity of secreted components from a sea anemone exudate, indicating that the search for novel biologically active peptides from these organisms has much greater potential than previously predicted.

Seasonal behavior of Thalassia testudinum (Hydrocharitaceae) metabolites / Comportamiento estacional de los metabolitos de Thalassia testudinum (Hydrocharital: Hydrocharitaceae)

Abstract:The marine angiosperm Thalassia testudinum, commonly known as turtle grass, is a dominant sea- grass that grows in the Caribbean Sea shelf associated to Syringodium filiforme. The hydroalcoholic extract of T. testudinum is rich in polyphenols; the most abundant metabolite in this extract is thalassiolin B, a glycosilated flavonoid with skin damage repairing properties, and antioxidant capacity among others. The present study aimed at generating information about the seasonal behavior of secondary metabolites, as well as to study the antioxidant capacity of the T. testudinum leaves extract, collected monthly during 2012 from the Northeast coastline of Havana, Cuba. For this study, spectrophotometric methods were used to determine the concentrations of polyphenols, flavonoids, anthocyanins, soluble carbohydrates and proteins, chlorophylls a and b, and antioxidant activity of the extracts. In general, results demonstrated seasonal variations of the analyzed parameters. Extracts prepared from the vegetal material collected in October and November showed the highest values of polyphenols (58.81 ± 1.53 and 52.39 ± 0.63 mg/g bs, respectivally) and flavonoids (44.12 ± 1.30 and 51.30 ± 0.67 mg/g dw, respectively). On the contrary, the lowest values of polyphenols were found in extracts of leaves collected in July and August (15.51 ± 0.84 and 13.86 ± 0.48 mg/g, respectively). In accordance with these results, the lower value of Inhibitory Concentration (IC50) was obtained to get a 50 % of maximal effect on free radical scavenging activity with the extracts prepared from leaves collected in October and November, and less significant IC50 was obtained from the extract prepared from leaves collected in August (5.63 mg/mL). A negative correlation (r= -0.694) was observed in this study between the content of polyphenols and the IC50 necessary to get the half of its antioxidant maximal effect. The high correspondence between the maximum values of polyphenols, flavonoids, carbohydrates and proteins in October and November, revealed a close relationship between these metabolites found in the extract of T. testudinum. Our hypothesis about the annual variation in the concentration of these metabolites was validated; and these results will support the correct harvesting of T. testudinum leaves for biotechnology and industrial purposes. Rev. Biol. Trop. 64 (4): 1527-1535. Epub 2016 December 01.

Resumen:La angiosperma marina Thalassia testudinum, comúnmente conocida como "hierba tortuga", es un pasto marino dominante que crece en el Mar Caribe asociada a Syringodium filiforme. El extracto hidroalcohólico de T. testudinum es rico en polifenoles; el metabolito más abundante en este extracto es thalassiolina B, un flavonoide glicosilado con propiedades para la reparación de daños en la piel y la capacidad antioxidante, entre otros. El objetivo del presente estudio fue conocer el comportamiento estacional de los metabolitos secundarios, así como la capacidad antioxidante del extracto de hojas de T. testudinum recolectadas mensualmente durante el 2012, en la costa noreste de La Habana, Cuba. Para este estudio fueron empleados métodos espectrofotométricos para la determinación de la concentración de polifenoles, flavonoides, carbohidratos y proteínas solubles, clorofilas a y b y la actividad antioxidante del extracto. En sentido general, los resultados demostraron las variaciones estacionales de los parámetros analizados; los extractos preparados a partir de material vegetal recolectado en octubre y noviembre mostraron los mayores valores de polifenoles y flavonoides (44.12 ± 1.30 y 51.30 ± 0.67 mg/g bs respectivamente) y se encontraron los valores más bajos en los extractos de hojas recolectadas en julio y agosto. (15.51 ± 0.84 y 13.86 ± 0.48 mg/g respectivamente). De acuerdo con los resultados, se obtuvo el valor más bajo de la concentración inhibitoria (CI50 ) necesaria para obtener un 50 % de efecto máximo en la actividad de captación de radicales libres con los extractos preparados a partir de las hojas recolectadas en octubre y noviembre y la CI50 menos significativa se obtuvo a partir del extracto preparado a partir de las hojas recolectadas en agosto (5.63 mg/ml). Se observó una correlación negativa (r= -0,694) entre el contenido de polifenoles y la CI50 necesaria para obtener la mitad del efecto antioxidante máximo. La alta correspondencia que existe entre los valores máximos de polifenoles, flavonoides, carbohidratos y proteínas en octubre y noviembre reveló una estrecha relación entre estos metabolitos, encontrados en el extracto de T. testudinum. La hipótesis de la variación anual de la concentración de estos metabolitos fue validada. Estos resultados se tendrán en cuenta con el fin de seleccionar el momento de recolecta de las hojas de T. testudinum para su uso con fines biotecnológicos e industriales.