The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts / Efecto de los métodos de extracción sobre los fenoles totales y las actividades antioxidantes de los extractos de Caulerpa racemosa (Forsskal) J. Agardh

Background: The edible green algae Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae), also known as "sea grape", is an excellent source of phenolic compounds known for their activity to reduce free radicals. Objectives: The research aims to evaluate the total phenolic content and antioxidant activity of C. racemosa (70% ethanol extracts) obtained from different extraction methods, such as maceration, Soxhlet, and ultrasound. Methods: Total phenolics of the extracts were determined by the colorimetry method using the Folin-Ciocalteu reagent. Total phenol content was expressed as mg gallic acid equivalent (GAE) per g extract. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to assess the antioxidant activity of the extracts. Results: the ethanol extract of C. racemosa obtained from the ultrasonic methods had the highest phenolic content (39.38 mg GAE/g) compared to other extraction methods (Soxhlet was 36.88 mg GAE/g and maceration was 22.05 mg GAE/g). The IC50 value against DPPH of the C. racemosa ethanol extracts using ultrasonic, Soxhlet, and maceration was 352.95, 365.73, and 375.84 µg/mL, respectively. Conclusions: the variation of the extraction methods affected the total phenolics content of C. racemosa ethanol extracts and their antioxidant activity. We reported here the potential of C. racemosa extracts as an antioxidant raw material from marine plants for medicinal, nutraceutical, cosmetics, and food products; however, more research is needed.

Antecedentes: El alga verde comestible Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae), también conocida como "uva de mar", es una excelente fuente de compuestos fenólicos conocidos por su actividad para reducir los radicales libres. Objetivos: La investigación pretende evaluar el contenido fenólico total y la actividad antioxidante de C. racemosa (extractos de etanol al 70%) obtenidos a partir de diferentes métodos de extracción, como maceración, Soxhlet y ultrasonido. Métodos: Los fenoles totales de los extractos se determinaron por el método colorimétrico utilizando el reactivo de Folin-Ciocalteu. El contenido total de fenoles se expresó como mg de ácido gálico equivalente (GAE) por g de extracto. Para evaluar la actividad antioxidante de los extractos se utilizó el radical libre 2,2-difenil-1-picrilhidrazilo (DPPH). Resultados: el extracto etanólico de C. racemosa obtenido por ultrasonido presentó el mayor contenido fenólico (39,38 mg GAE/g) en comparación con otros métodos de extracción (Soxhlet fue de 36,88 mg GAE/g y maceración fue de 22,05 mg GAE/g). El valor IC50 frente a DPPH de los extractos etanólicos de C. racemosa mediante ultrasonido, Soxhlet y maceración fue de 352,95, 365,73 y 375,84 µg/mL, respectivamente. Conclusiones: la variación de los métodos de extracción afectó al contenido total de fenoles de los extractos etanólicos de C. racemosa y a su actividad antioxidante. Aquí reportamos el potencial de los extractos de C. racemosa como materia prima antioxidante a partir de plantas marinas para productos medicinales, nutracéuticos, cosméticos y alimenticios; sin embargo, se necesita más investigación.

Molecular characteristics of polysulfated fractions from the green seaweed Caulerpa cupressoides and actions on thrombin generation in vitro

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

Demografía de Caulerpa paspaloides var. Wudermannii (Bryopsidales: Caulerpaceae) en la zona costera de Campeche, México / Demography of Caulerpa paspaloides var. wudermanni (Bryopsidales: Caulerpaceae) in the coastal zone of Campeche, México

The subaquatic vegetation of Los Petenes, Campeche, Mexico, stands out due to its considerable floristic diversity, composed of a great variety of sea grasses and several species of the genus Caulerpa sp. This is a genus of ecological relevance, with the invasive species in the Mediterranean, with negative impact on several native sub-aquatic plants; nevertheless, little is known about the demography and population dynamics of Caulerpa species and their contribution to food webs. Thus the main objective of this study was to describe the demographics of Caulerpa paspaloides var. wudermanni, using the number of stolons, complete and incomplete fronds, the diameter of the stolons and the biomass. The information was used to determine the growth rate (λ) of this species. The study was conducted in the Biosphere Reserve of Los Petenes, which is located in the Northwest of the state of Campeche. The submerged aquatic vegetation (SAV) in the Petenes Biosphere consists of monospecific and mixed populations of seagrass species (Thalassia testudinum, Halodule wrightii and Syringodium filiforme). Although chlorophytes, brown algae and red algae, are fundamental elements in the specific composition of the SAV in Petenes, several species of Caulerpa are prominent because of their coverage and abundance. In May and June of 2010, significant differences in the quantity of stolons, their diameter, incomplete and complete fronds, and the size of the stolons and rhizomes, were observed. In 2010, the finite population growth rate (λ) was 2.38±0.1571 for individuals and 1.20±0.1356 for the population, and in 2011 the values of λ were 1.80±0.3608 and 1.35±0.1571, respectively. From these results it can be concluded that the population is growing; however, growth is controlled by biotic and abiotic factors. Despite there was no apparent threat, we suggest continuing the demographic studies of C. paspaloides var. wurdemannii, as well as of other species of the same genus, not only to detect invasion or explosive growth, but also their presence indicated low oxygen levels and high sulphates. Rev. Biol. Trop. 62 (2): 729-741. Epub 2014 June 01.

La vegetación subacuática de Los Petenes, Campeche, México, se destaca por su gran diversidad florística, compuesto por una gran variedad de pastos marinos y varias especies del género Caulerpa sp. Caulerpa es un género de relevancia ecológica, con especies como la Caulerpa taxifolia, que es invasiva en el Mediterráneo, con un impacto negativo en varias plantas sub-acuáticas nativas, pero poco se sabe acerca de la demografía y la dinámica poblacional de las especies de Caulerpa y su contribución a las redes alimenticias. El objetivo general es conocer la demografía de Caulerpa sp., por medio del número de estolones, frondas completas e incompletas, el diámetro de los estolones y la biomasa. El estudio se realizó en la Reserva de la Biosfera de Los Petenes, en Campeche, México. La Vegetación Acuática Sumergida (VAS) en la Biosfera de los Petenes está constituida por poblaciones tanto monoespecíficas como mixtas de las especies de pastos marinos Thalassia testudinum, Halodule wrightii y Syringodium filiforme. Aunque las especies de algas clorófitas, feofitas y rodófitas son elementos fundamentales de la VAS, destacan por su cobertura y abundancia las especies de Caulerpa. En mayo y junio del 2010, hubo diferencias significativas de la cantidad de estolones, su diámetro, frondas completas e incompletas y el tamaño de los estolones como el de los ejes erectos. En el 2010 el valor de la tasa finita de crecimiento poblacional (l) fue de 2.38±0.15 con el método de las marcas y con el de la biomasa 1.20±0.13 y en el 2011 los valores fueron de 1.80±0.36 con las marcas y de 1.35±0.15 con la biomasa. Los resultados indican que la población está creciendo, pero es controlada por factores bióticos y abióticos. A pesar de que no se detecta la especie como una amenaza aparente, sugerimos que se continúen estos estudios demográficos sobre C. paspaloides var. wurdemannii e incluso de otras especies del mismo género. No solo para detectar una invasión o un crecimiento explosivo, sino porque la presencia de estas especies no indican condiciones bajas en oxígeno y altas en sulfatos.

Physical factors contributing to the benthic dominance of the alga Caulerpa sertularioides (Caulerpaceae, Chlorophyta) in the upwelling Bahía Culebra, north Pacific of Costa Rica / Factores físicos que contribuyen a la dominancia bentónica del alga Caulerpa sertularioides (Caulerpaceae, Chlorophyta) en la surgencia Bahía Culebra, Pacífico norte de Costa Rica

Caulerpa sertularioides has been spreading in Bahía Culebra, a seasonal upwelling bay in the north Pacific of Costa Rica, since 2001. The survey was carried out from December 2003 to March 2005, in several locations around Bahía Culebra, located inside the Gulf of Papagayo. This study investigated spatial and temporal patterns, percent coverage, monthly growth rate, reproductive adaptations, and morphological variations of frond length and stolon diameter of Caulerpa sertularioides, at different environmental physical and chemical factors at the bay. The alga extended to depths of 23 m on a variety of substrates. The stolons extended quickly, with a maximum growth rate of 31.2 cm month-1. This alga grows mainly by fragmentation of its fronds and stolons; nevertheless it can also reproduce sexually by releasing gametes in the water column. These two modes of spreading promote the adaptation of this opportunistic species to environmental, chemical, and physical changes at this bay. At the same time the alga showed variations in the length of its fronds and stolons, adapting to conditions such as depth and season. Average percent cover and frond density increased during the dry season when the upwelling of nutrients and cold water occurs. In the rainy season the average percent cover and frond density decreased; however there was a peak in September, when high precipitation resulted in runoff into the bay of nutrient-rich waters. The morphological and physiological plasticity of C. sertularioides, in synergy with its predominant clonal propagation and sexual reproduction provided this species with a great adaptability to changes in temperature and nutrient concentration at Bahía Culebra.

Desde el 2001 se ha observado una propagación del alga verde Caulerpa sertularioides en Bahía Culebra, zona de afloramiento costero, en el Pacífico norte de Costa Rica. El muestreo se llevó acabo entre Diciembre 2003 a marzo 2005, en varias localidades de Bahía Culebra. En este estudio se presentan los patrones de distribución, cobertura, tasa mensual de crecimiento, adaptaciones reproductivas y variaciones morfológicas del largo de la fronda y diámetro del estolón de Caulerpa sertularioides, a diferentes factores ambientales físico-químicos en Bahía Culebra. Esta alga se extiende hasta profundidades de 23 m, en una gran variedad de sustratos. Los estolones se extienden rápidamente, con un crecimiento máximo de 31.2 cm mes-1. Esta alga se propaga principalmente por la fragmentación de sus frondas y estolones, así mismo se reproduce sexualmente liberando gametos a la columna de agua. Estos modos de reproducción promueven la adaptación de esta especie oportunista, a los cambios ambientales, tanto químicos, como físicos, de la bahía. Al mismo tiempo esta alga presenta variaciones en el largo de sus frondas y el diámetro del rizoma, adaptándose a diferentes profundidades y condiciones de la época del año. El porcentaje de cobertura y densidad de frondas aumentan durante la época seca, cuando emergen aguas frías y nutrientes por el afloramiento costero. Por otro lado, en la época lluviosa estas medidas decrecen, sin embargo se presenta un pico en setiembre, cuando la precipitación aumenta y llega una carga extra de nutrientes a la bahía por escorrentía. La plasticidad morfológica y fisiológica de C. sertularioides, en sinergia con su propagación clonal, proveen a esta alga con una gran adaptabilidad a los cambios en temperatura y nutrientes de Bahía Culebra.

Polissacarídeos sulfatados isolados das clorofíceas Caulerpa racemosae Caulerpa cupressoides – extração, fracionamento e atividadeanticoagulante / Sulfated polysaccharides isolated from Caulerpa racemosa and Caulerpa cupressoides (Chlorophyceaes) – extraction, fractionation and anticoagulant activity

A incidência de doenças cardiovasculares e os efeitos adversos da heparinoterapiatêm motivado a busca por novos agentes terapêuticos e os polissacarídeos sulfatados (PS) de algasmarinhas têm sido reportados como fontes alternativas para tal. Objetivou-se avaliar o potencialanticoagulante dos PS totais (PST) isolados e fracionados das clorofíceas Caulerpa racemosa eCaulerpa cupressoides. Inicialmente, os PST foram extraídos com papaína em tampão acetato desódio 0,1 M (pH 5,0) contendo cisteína 5 mM e EDTA 5 mM, seguidos de fracionamento emcoluna de troca iônica de DEAE-celulose com gradiente de NaCl. As frações obtidas foramanalisadas por eletroforese em gel de agarose a 0,5% e a atividade anticoagulante, mensurada pelotempo de tromboplastina parcial ativada (TTPA), usando-se plasma humano normal ecomparada a uma curva-padrão de heparina (193 UI mg-1). Verificaram-se semelhantes perfiscromatográficos entre os PS de ambas as espécies, porém com padrões de mobilidades distintasquando as frações foram comparadas por eletroforese. Os PS modificaram o TTPA, cujasatividades anticoagulantes foram de apenas 21,23 e 24,36 UI mg-1, quando eluídos com 0,75 M desal para C. racemosa e C. cupressoides, respectivamente. Portanto, PS anticoagulantes isolados dasclorofíceas C. racemosa e C. cupressoides resultaram em efeitos anticoagulantes inferiores aos daheparina. Estudos comparativos dessas moléculas também são sugeridos como ferramentasauxiliares na identificação de algas do mesmo gênero.

The incidence of cardiovascular diseases and adverse effects from heparintherapy have led to asearch for new therapeutic agents, and the sulfated polysaccharides (SP) of seaweeds have beenreported as alternative sources. The aim of this work was to evaluate the anticoagulant potentialof total SP (TSP) isolated and fractionated from Caulerpa racemosa and Caulerpa cupressoides(Chlorophyceaes). Initially, the TSP were extracted with papain in 0.1 M sodium acetate buffer(pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by fractionation on ionexchange DEAE-cellulose column with NaCl gradient. The obtained fractions were analyzed by0.5% agarose gel electrophoresis and the anticoagulant activity measured by the activated partialthromboplastin time (APTT) using normal human plasma, and compared to a standard heparincurve (193 IU mg-1). Similar chromatographic profiles of SP were shown on both species, butwith distinct mobility patterns, when the SP fractions were compared by electrophoresis. SPeluted with 0.75 M of NaCl modified the APTT, whose anticoagulant activities were only 21.23and 24.36 IU mg-1 for C. racemosa and C. cupressoides, respectively. Therefore, anticoagulant SPisolated from chlorophyceaes showed effects inferior to heparin, and comparative studies of thesemolecules are also suggested as auxiliary tools in the identification of algae of the same genus.