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.

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.

In vitro antiplasmodial activity, cytotoxicity and chemical profiles of sponge species of Cuban coasts.

Aqueous and organic fractions from the crude extracts of 17 sponge species collected at Boca de Calderas, Havana, Cuba were analysed. The organic fractions of Mycale laxissima, Clathria echinata and Agelas cerebrum exhibited values of concentrations causing 50% inhibition of in vitro growth of Plasmodium berghei (IC50) of 42.3 ± 5.1, 52 ± 9.7 and 60.3 ± 10.6 µg/mL, respectively, while their selectivity indexes for fibroblast cell lines were 9.45, 4.24 and 8.7, correspondingly. These fractions reduced parasitemia of infected Balb/c mice as well. Selective cytotoxicity indexes against tumour HeLa cells focused an interest on the aqueous fraction of M. laxissima (>7.12) and organic fractions of Polymastia nigra (5.95), A. cerebrum (5.48) and Niphates erecta (>4.2). Triterpenoids/steroids and alkaloids detected in the organic fractions of M. laxissima, C. echinata and A. cerebrum should be isolated for future investigation.

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.

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.

Photoprotecting action and phytochemical analysis of a multiple radical scavenger lipophilic fraction obtained from the leaf of the seagrass Thalassia testudinum.

The apolar fraction F1 of Thalassia testudinum was chemically characterized by gas chromatography-mass spectrometry, which led to the identification of 43 metabolites, all of them reported for the first time in the genus Thalassia. More than 80% of the F1 composition was constituted by aromatic metabolites including the major components 1,1-bis(p-tolyl)ethane (6.0%), 4,4'-diisopropylbiphenyl (4.8%) and a 1,1-bis(p-tolyl)ethane isomer (4.7%). This lipophilic fraction was assayed for its antioxidant effects and skin protective action. In vitro assays showed that F1 strongly scavenged DPPH* (IC(50) 312.0 ± 8.0 µg mL(-1)), hydroxyl (IC(50) 23.8 ± 0.5 µg mL(-1)) and peroxyl radical (IC(50) 6.6 ± 0.3 µg mL(-1) ), as well as superoxide anion (IC(50) 50.0 ± 0.7 µg mL(-1)). Also, F1 markedly inhibited the spontaneous lipid peroxidation (LPO) in brain homogenates (IC(50) 93.0 ± 6.0 µg mL(-1)) and the LPS-stimulated nitrite generation on RAW624.7 macrophages (58.6 ± 3.2%, 400 µg mL(-1)). In agreement with these findings, its topical application at 250 and 500 µg cm(-2) strikingly reduced skin damage on mice exposed to acute UVB radiation by 45% and 70%, respectively and significantly attenuated the LPO developed following the first 48 h after acute exposure to UVB irradiation, as manifested by the decreased malondialdehide level and by the increased of reduced gluthatione content. Our results suggest that F1 may contribute to skin repair by attenuating oxidative stress due to its antioxidant activity.

[Antioxidant effect and polyphenol content of Syringodium filiforme (Cymodoceaceae)]. / Efecto antioxidante y contenido polifenólico de Syringodium filiforme (Cymodoceaceae).

The marine phanerogam Syringodium filiforme, known as "manatee grass", is a common species that grows in coastal areas associated to Thalassia testudinum. With the aim to describe some of its possible chemical characteristics, this study was performed with a sample of 1.2 kg, collected in March 2009, in Guanabo beach, Havana, Cuba. The sample was dried (less than 12% humidity) and a total extract prepared; other three extracts were prepared with the use of solvents of increasing polarity. The phytochemical screening and analytical determinations of each fraction were undertaken Total polyphenol content was determined using pyrogallol as reference's standard; chlorophyll a and b and anthocyanin content were also quantified. Total extract and fractions antioxidant activity were evaluated by using the free radical scavenging activity assay with 1,1-Diphenyl-2-Picrylhydrazyl reactive (knowing as DPPH's method). The phytochemical screening of the different extracts detected the presence of high concentrations of flavonoids, phenols, terpenes, antocyanins, reducing sugars and alkaloids. The total extract and methanol fraction showed significant free radical scavenging properties, while the petroleum ether fraction showed moderate activity, and the chloroform fraction and the aqueous soluble precipitate (residual salt) obtained didn't show antioxidant properties against free radicals. The results of this work confirmed the potentialities of this species for biological purposes.

Efecto antioxidante y contenido polifenólico de Syringodium filiforme (Cymodoceaceae)

Antioxidant effect and polyphenol content of Syringodium filiforme (Cymodoceaceae). The marine phanerogam Syringodium filiforme, known as "manatee grass", is a common species that grows in coastal areas associated to Thalassia testudinum. With the aim to describe some of its possible chemical characteristics, this study was performed with a sample of 1.2kg, collected in March 2009, in Guanabo beach, Havana, Cuba. The sample was dried (less than 12% humidity) and a total extract prepared; other three extracts were prepared with the use of solvents of increasing polarity. The phytochemical screening and analytical determinations of each fraction were undertaken Total polyphenol content was determined using pyrogallol as reference´s standard; chlorophyll a and b and anthocyanin content were also quantified. Total extract and fractions antioxidant activity were evaluated by using the free radical scavenging activity assay with 1,1- Diphenyl-2-Picrylhydrazyl reactive (knowing as DPPH´s method). The phytochemical screening of the different extracts detected the presence of high concentrations of flavonoids, phenols, terpenes, antocyaninns, reducing sugars and alkaloids. The total extract and methanol fraction showed significant free radical scavenging properties, while the petroleum ether fraction showed moderate activity, and the chloroform fraction and the aqueous soluble precipitate (residual salt) obtained didn’t show antioxidant properties against free radicals. The results of this work confirmed the potentialities of this species for biological purposes. Rev. Biol. Trop. 59 (1): 465-472. Epub 2011 March 01.

La fanerógama marina Syringodium filiforme, conocida comúnmente como "Hierba Manatí", crece en los fondos marinos tropicales asociada con la especie Thalassia testudinum. Este estudio fue realizado con una muestra de S. filiforme (1.2kg peso húmedo) recolectada en la playa de Guanabo en La Habana, Cuba en Marzo 2009. La muestra fue secada hasta humedad inferior al 12% y se realizaron tres extracciones con solventes de polaridad creciente previamente al tamizaje fitoquímico y a las determinaciones analíticas. A partir del extracto total y en las fracciones se detectó la presencia de grupos químicos, fundamentalmente estructuras típicas de los flavonoides, por las absorbancias obtenidas en los espectros ultravioleta-visible. En el tamizaje fitoquímico se corroboró la existencia de altas concentraciones de flavonoides, triterpenos, polifenoles, antocianinas, azúcares reductores y alcaloides en los diferentes extractos obtenidos. El contenido de polifenoles totales de S. filiforme, se cuantificó mediante el uso de pirogalol como patrón de referencia; así como el contenido de clorofilas a y b, y el nivel de antocianinas. La actividad antioxidante se determinó por el método reducción del 1,1-difenil-2-picrilhidrazilo (DPPH). El extracto total y la fracción metanólica mostraron elevada actividad antioxidante en correspondencia con la concentración de polifenoles, mientras que la fracción de éter de petróleo mostró actividad moderada y la fracción clorofórmica y el precipitado remanente no muestran actividad antioxidante significativa. Estos resultados predicen las potencialidades de la especie para su uso con fines biológicos.

Antinociception produced by Thalassia testudinum extract BM-21 is mediated by the inhibition of acid sensing ionic channels by the phenolic compound thalassiolin B.

BACKGROUND: Acid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats. RESULTS: Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21. CONCLUSIONS: To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

Polar alkaloids from the Caribbean marine sponge Niphates digitalis.

A method involving flash chromatography, semi-preparative phenylhexyl RP HPLC-DAD-ELSD combined with analytic polar-RP HPLC-DAD, was applied to separate and purify six highly nitrogenated bases and a bicyclic amidine alkaloid, the major components of the marine sponge Niphates digitalis. Their structures were identified as 1,8-diazabicyclo[5.4.0]undec-7-ene (1), deoxycytidine (2), phenylalanine (3), adenosine (4), deoxyguanosine (5), adenine (6) and thymidine (7) on the basis of spectroscopic data analyses. This is the first report of these compounds in a marine sponge belonging to the Niphates genus and the first evidence of the presence of 1 from a natural source.

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.

Cholesterol-lowering effects of policosanol in rabbits

Policosanol is a natural mixture of higher primary aliphatic alcohols isolated and purified from sugar cane (Saccharum officinarum, L.) wax, whose main component is octacosanol. Policosanol (5-200 mg/kg) orally administered for 4 weeks to normocholesterolemic New Zealand rabbits significantly reduced total cholesterol and low density lipoprotein cholesterol (LDL-C) serum levels in a dose dependent manner. Serum triglyceride levels of treated and control animals were significantly different, but the reduction observed was not dose-dependent. High density lipoprotein cholesterol (HDL-C) levels remained unchanged. Results indicate that the reduction in total cholesterol values induced by policosanol is mainly mediated through a decrease in LDL-C levels

Cholesterol-lowering effects of policosanol in rabbits

Policosanol is a natural mixture of higher primary aliphatic alcohols isolated and purified from sugar cane (Saccharum officinarum, L.) wax, whose main component is octacosanol. Policosanol (5-200 mg/kg) orally administered for 4 weeks to normocholesterolemic New Zealand rabbits significantly reduced total cholesterol and low density lipoprotein cholesterol (LDL-C) serum levels in a dose dependent manner. Serum triglyceride levels of treated and control animals were significantly different, but the reduction observed was not dose-dependent. High density lipoprotein cholesterol (HDL-C) levels remained unchanged. Results indicate that the reduction in total cholesterol values induced by policosanol is mainly mediated through a decrease in LDL-C levels(AU)

Efecto del Policosanol sobre el perfil lipídico de conejos normocolesterolemicos / Effect of policosanol upper lipid profile of normocholesterolemic rabbits

En este trabajo se estudio el efecto de la administración oral de Policosanol (mezcla de alcoholes de alto molecular cuyo componente fundamental es el octacosanol) sobre los lípidos y lipoproteínas séricos de conejos Nueva Zelanda normocolesterolémicos. El tratamiento oral con Policosanol (5 mg/kg/día) durante 4 semanas provocó una disminución significativa de los niveles séricos de colesterol, triglicéridos y LDL-C, sin producir cambios significativos en los valores de HDL-. El contenido de colesterol hepático de los animales tratados resultó significativamente menor que el de los controles

Estudio químico del Heliotropium Curassavicum L que crece en Cuba / Cheical study of Heliotropium Curassavicum L growing in Cuba

Como parte del programa de tamizaje antitumoral de plantas cubanas del Departamento de Bioquímica del Instituto Nacional de Oncología y Radiobiología, se inició el estudio de la especie Heliotropium Curassavicum L, la que resultó activa en el tamizaje primario de leucemia, 1 210, y hepatoma, 22. Al realizar el estudio fitoquímico de la misma se aislaron los componentes mayoritarios heliocurassavicina y heliocurassavinina, los que fueron caracterizados espectroscópicamente

Estudio químico del Heliotropium Curassavicum L que crece en Cuba / Chemical study of Heliotropium Curassavicum L growing in Cuba

Como parte del programa de tamizaje antitumoral de plantas cubanas del Departamento de Bioquímica del Instituto Nacional de Oncología y Radiobiología, se inició el estudio de la especie Heliotropium Curassavicum L, la que resultó activa en el tamizaje primario de leucemia, 1 210, y hepatoma, 22. Al realizar el estudio fitoquímico de la misma se aislaron los componentes mayoritarios heliocurassavicina y heliocurassavinina, los que fueron caracterizados espectroscópicamente

Aplicación de la técnica de cromatografía en capa delgada a 32 cepas del M- habana / Application of thin-layer chromatography to 32 strains of M. habana

Se informa que se estudiaron 32 cepas del M. habana aisladas de pacientes sintomáticos respiratorios y se comparan por la técnica de cromatografía en capa delgada. Se plantea que la extracción de lípidos se hizo aplicando el método de diacetilación. Se trabaja con los lípidos intactos y los lípidos diacetilados. Se concluye que esta especie micobacteriana pertencente al Grupo I (según Brennan), pues sus lípidos son álcalis resistentes

Aplicación de la técnica de cromatografía en capa delgada a 32 cepas del M habana

Se informa que se estudiaron 32 cepas del M habana aisladas de pacientes sintomáticos respiratorios y se comparan por la técnica de cromatografía en capa delgada. Se plantea que la extracción de lípidos se hizo aplicando el método de diacetilación. Se trabaja con los lípidos intactos y los lípidos diacetilados. Se concluye que esta especie micobacteriana pertenece al grupo I (según Brennan), pues sus lípidos son álcalis resistentes(AU)