Rendimiento fisiológico de acuerdo a la tasa de crecimiento, contenido de pigmentos y proteínas de la macroalga parda Sargassum filipendula (Ochrophyta: Fucales) inducida a radiación UV en el laboratorio

UV radiation is a factor affecting the distribution and physiology of photosynthetic organisms in an aquatic ecosystem. Studies with macroalgae indicate diverse biological disturbances in response to UV radiation. This work aimed to study sensitivity of the brown macroalga Sargassum filipendula exposed to UV radiation: PAR (control), PAR+UVA+UVB(++) and PAR+UVA(++)+UVB. Changes in the physiological parameters growth rate, total soluble proteins, photosynthetic pigments and the UV-vis absorbing compounds were analysed after T0, T4, T7 and T10 (days) of UV exposure. Physiological parameters showed little variation between treatments and over time, suggesting that moderate UV radiation doses could regulate resistance responses to re-establish the cellular homoeostasis condition through activation of an antioxidant defence system, such as an overproduction of phenolic compounds. Responses recorded in S. filipendula would be related to acclimation mechanisms against acute UV radiation stress, triggering resistance responses to avoid serious damage to the metabolic machinery, activating control systems to maintain hormesis, and homoeostasis of deleterious actions of reactive species, similar to the phenomenon known as preparation for oxidative stress. Finally, UV-visible absorption spectra showed absorption bands evidencing the presence of mainly UV-absorbing compounds with photoprotective function, such as phlorotannins, flavonoids and carotenoids, which could provide adaptive advantages for organisms exposed to UV radiation.La radiación UV es un factor que afecta la distribución y la fisiología de los organismos fotosintéticos en el ecosistema acuático. Los estudios con macroalgas indican diversas alteraciones biológicas en respuesta a la radiación UV. Este trabajo tuvo como objetivo estudiar la sensibilidad de la macroalga parda Sargassum filipendula expuesta a radiación UV: PAR (control), PAR + UVA + UVB (++) y PAR + UVA (++) + UVB. Los cambios en la tasa de crecimiento, proteínas solubles totales, pigmentos fotosintéticos y compuestos absorbentes de UV-vis se analizaron después de T0, T4, T7 y T10 (días) de exposición a UV. Los parámetros fisiológicos mostraron poca variación entre los tratamientos y con el tiempo, lo que sugiere que dosis moderadas de radiación UV podrían regular las respuestas de resistencia para restablecer la condición de homeostasis celular a través de la activación del sistema de defensa antioxidante, como la sobreproducción de compuestos fenólicos. Las respuestas registradas en S. filipendula estarían relacionadas con mecanismos de aclimatación contra el estrés agudo por radiación UV, desencadenando respuestas de resistencia para evitar daños severos en la maquinaria metabólica, activando sistemas de control para mantener la hormesis y homeostasis de acciones deletéreas de especies reactivas, similar al fenómeno llamado preparación para el estrés oxidativo (POS). Finalmente, los espectros de absorción UV-visible mostraron bandas de absorción que evidencian la presencia de compuestos absorbentes de UV principalmente con función fotoprotectora, como los florotaninos, flavonoides y carotenoides que podrían proporcionar ventajas adaptativas para los organismos expuestos a la radiación UV

In vitro assay of nitrate reductase enzyme and effect of nitrate and phosphate availability in colour strains of Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

A enzima nitrato redutase (NR) catalisa a redução do nitrato a nitrito e controla a taxa de assimilação do nitrato. O ensaio in vitro da nitrato redutase foi otimizado para a linhagem selvagem (marrom, MA) e para a linhagem deficiente em ficoeritrina (verde-clara, VC) de Hypnea musciformis. As duas linhagens foram cultivadas em temperatura de 23 ± 2°C, fotoperíodo de 14 horas, irradiância de 60-90µmol fótons m-2s-1, e meio composto por água do mar esterilizada (30ups) enriquecida com a solução de von Stosch na concentração de 50% (VSES/2). As condições ótimas de ensaio para ambas as linhagens foram: 40µM de NADH; 10min de incubação do extrato bruto (EB) e 100µL de EB. A atividade ótima da NR ocorreu em 4 e 2mM de nitrato para a linhagem VC e MA, respectivamente. As linhagens VC e MA apresentaram, respectivamente, constante aparente de Michaelis-Menten (K M) para NADH de 0,2068 e 0,0837 µM, e K M para nitrato de 0,0492 e 0,0294mM. Os resultados indicam que a NR da linhagem MA tem maior afinidade pelo substrato do que a NR da linhagem VC de H. musciformis. Os experimentos para avaliar os efeitos da disponibilidade de nitrato (5 a 105µM) e nitrato e fosfato (0,5 a 25,5µM, com a relação N:P de 4:1) mostraram que a atividade da NR das linhagens VC e MA não aumentou com a adição de nitrato no meio, o que pode estar relacionado com o estado nutricional dessas algas. A atividade da NR foi maior nos tratamentos com adição de fosfato do que naqueles com adição de apenas nitrato, indicando que esse nutriente é importante para os processos metabólicos relacionados a atividade da NR.The enzyme nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite and controls the rate of nitrate assimilation. The in vitro assay of NR was optimized for the wild strain (brown, MA), and the phycoerythrin-deficient strain (light-green, VC) of Hypnea musciformis. Both strains were cultured at temperature of 23 ± 2°C, photoperiod of 14h, irradiance of 60-90 µmol photons m-2s-1, with medium composed by sterilized seawater (salinity 30 psu) with 50% von Stosch's enrichment solution (VSES/2). The optimal conditions for in vitro assay of NR were: 40µM of NADH; 10min of incubation of crude extracts (EB), and 100µL of EB to both strains. Optimal activity of NR occurred at 4 and 2mM of nitrate to the VC and MA strains, respectively. The VC and MA strains showed, respectively, Michaelis-Menten constants (K M) for NADH of 0.2068 and 0.0837µM, and K M for nitrate of 0.0492 and 0.0294mM. The results indicate that the NR of MA strain has higher affinity by the substrate than the NR of VC strain of H. musciformis. Experiments on the effects of availabilities of nitrate (5 to 105µM) and nitrate and phosphate (0.5 to 25.5µM, with a N:P relation of 4:1) showed that NR activity of VC and MA strain did not increase with the addition of nitrate to the medium, what can be related with their nutritional state. The NR activity was higher in treatments with phosphate addition than those with only nitrate addition, indicating that this nutrient is important to metabolic processes related to the NR activity.CNPqFAPES

Standardization of a protocol to extract and analyze chlorophyll a and carotenoids in Gracilaria tenuistipitata Var. Liui. Zhang and Xia (Rhodophyta)

Chlorophyll a and carotenoids are important pigments in photosynthesis. Several studies have been published describing extraction and analysis protocols of these pigments, mainly in vascular plant species. This study standardizes an extraction and analysis protocol of these substances in Gracilaria tenuistipitata var. liui, a red seaweed. Apical portions grown in vitro were triturated in liquid nitrogen. Extracts were prepared in 1.5 mL solvent and centrifuged. Quantitative and qualitative analyses of pigments were performed by UV/visible light spectrophotometry and high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC-MS). The parameters assessed were: minimum biomass, best extraction solvent, and number of extraction steps. Methanol was the most efficient solvent, and 50 mg fresh biomass was the amount of sample indicated, submitted to one single extraction step. No significant differences were observed in levels of these pigments by UV-visible light spectrophotometry and HPLC. However, HPLC or HPLC-MS are required to identify the different carotenoids present in this seaweed species.Clorofila a e carotenoides são importantes pigmentos da fotossíntese. Na literatura são encontrados vários protocolos de extração e análise desses pigmentos utilizando, principalmente, plantas vasculares. O objetivo deste estudo foi padronizar uma metodologia de extração e análise dessas substâncias em uma macroalga vermelha, Gracilaria tenuistipitata var. liui. Amostras de talos gametofíticos cultivados in vitro foram trituradas em nitrogênio líquido, extraídas em 1,5 mL de solvente, centrifugadas e os pigmentos analisados quantitativamente e qualitativamente através de espectrofotometria de UV/visível, cromatografia liquida de alta eficiência (CLAE) e CLAE-acoplada a espectrometria de massas (CLAE-EM). Foram testados os parâmetros massa mínima, solvente para extração e número de extrações. Dentre os solventes testados, o metanol foi o mais eficiente, sendo 50mg de material fresco a massa mínima indicada para ser submetida a somente uma extração. Não foram encontradas diferenças significativas na quantificação desses pigmentos comparando-se os dados obtidos em espectrofotometria de UV/visível com os de CLAE. No entanto, para a identificação dos diferentes carotenoides e suas quantificações são necessárias CLAE ou CLAE-EM.The authors thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for their financial support (2010/02948-3), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for PBT fellowship

Brazilian Beach-Cast Seaweeds: Antioxidant, Photoprotection and Cytotoxicity Properties.

The potential of seaweed extracts in cosmetic applications have increased with novel development and market. Beneficial biological properties include the ability to struggle aging and anticancer agents. This research aimed to evaluate antioxidant, photoprotective and cytotoxic properties of extracts from beach-cast seaweed species from the Brazilian coast in order to propose an appropriate use of this sustainable resource. Analysis of antioxidant capacity, UV/VIS absorption, quantification of UV photoprotectors, total content of C, N and S, development of cosmetic creams and evaluation of the cytotoxicity activity against tumoral cells lines were performed. The highest antioxidant capacity was found in extracts of brown seaweeds, followed by red seaweeds, with the lowest activity detected in the green seaweed. Same pattern was observed for phenolic compounds. The extracts did not show cytotoxicity activity against healthy human cells. Other forms of extraction and incorporation of the extract into the base creams should be evaluated, the isolation and purification of substances could increase the efficiency of the photoprotective capacity. The species were highly promising and proved to be natural sources of antioxidants and substances with cytotoxicity activity against tumoral cells; therefore, they could be exploited as functional ingredients with specific applications for different types of industries. The application of seaweed extracts in combination with other natural ingredients can help in the design of new cosmetics against the negative effects of UV radiation, in addition to having the great advantage of not presenting toxicity to health or the environment because they are biodegradable.Funding for open access publishing: Universidad Málaga/CBUA. // TBH thanks to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; 140144/2017-0) for financial support and PhD scholarship and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Code 001) for funding the postgraduate program in Botany at the Institute of Bioscience and PhD sandwich scholarship (88887.368014/2019-00). FC thanks FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo; 2018/18015-8) for financial support and CNPq for research productivity grants (303937/2015-7; 303493/2018-6). This work is part of the international research project BMBF 031B0284 (023/IVV-113816). FLF thanks the financial support by the Ministry of Sciences and Innovation (BlueMaro Project, PID2020-116136RB-I0) and by the Government of Andalusia (Facco Project - UMA18-FEDER JA-162 and NAZCA Project -P20-004589)

STANDARDIZATION OF A PROTOCOL TO EXTRACT AND ANALYZE CHLOROPHYLL A AND CAROTENOIDS IN Gracilaria tenuistipitata VAR. LIUI. ZHANG AND XIA (RHODOPHYTA)

A B S T R A C T Chlorophyll a and carotenoids are important pigments in photosynthesis. Several studies have been published describing extraction and analysis protocols of these pigments, mainly in vascular plant species. This study standardizes an extraction and analysis protocol of these substances in Gracilaria tenuistipitata var. liui, a red seaweed. Apical portions grown in vitro were triturated in liquid nitrogen. Extracts were prepared in 1.5 mL solvent and centrifuged. Quantitative and qualitative analyses of pigments were performed by UV/visible light spectrophotometry and high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC-MS). The parameters assessed were: minimum biomass, best extraction solvent, and number of extraction steps. Methanol was the most efficient solvent, and 50 mg fresh biomass was the amount of sample indicated, submitted to one single extraction step. No significant differences were observed in levels of these pigments by UV-visible light spectrophotometry and HPLC. However, HPLC or HPLC-MS are required to identify the different carotenoids present in this seaweed species. R E S U M O Clorofila a e carotenoides são importantes pigmentos da fotossíntese. Na literatura são encontrados vários protocolos de extração e análise desses pigmentos utilizando, principalmente, plantas vasculares. O objetivo deste estudo foi padronizar uma metodologia de extração e análise dessas substâncias em uma macroalga vermelha, Gracilaria tenuistipitata var. liui. Amostras de talos gametofíticos cultivados in vitro foram trituradas em nitrogênio líquido, extraídas em 1,5 mL de solvente, centrifugadas e os pigmentos analisados quantitativamente e qualitativamente através de espectrofotometria de UV/visível, cromatografia liquida de alta eficiência (CLAE) e CLAE-acoplada a espectrometria de massas (CLAE-EM). Foram testados os parâmetros massa mínima, solvente para extração e número de extrações. Dentre os solventes testados, o metanol foi o mais eficiente, sendo 50mg de material fresco a massa mínima indicada para ser submetida a somente uma extração. Não foram encontradas diferenças significativas na quantificação desses pigmentos comparando-se os dados obtidos em espectrofotometria de UV/visível com os de CLAE. No entanto, para a identificação dos diferentes carotenoides e suas quantificações são necessárias CLAE ou CLAE-EM