In the present work, a comprehensive methodology to carry out the screening for novel natural functional compounds is presented. To do that, a new strategy has been developed including the use of unexplored natural sources (i.e., algae and microalgae) together with environmentally clean extraction techniques and advanced analytical tools. The developed procedure allows also estimating the functional activities of the different extracts obtained and even more important, to correlate these activities with their particular chemical composition. By applying this methodology it has been possible to carry out the screening for bioactive compounds in the algae Himanthalia elongata and the microalgae Synechocystis sp. Both algae produced active extracts in terms of both antioxidant and antimicrobial activity. The obtained pressurized liquid extracts were chemically characterized by GC-MS and HPLC-DAD. Different fatty acids and volatile compounds with antimicrobial activity were identified, such as phytol, fucosterol, neophytadiene or palmitic, palmitoleic and oleic acids. Based on the results obtained, ethanol was selected as the most appropriate solvent to extract this kind of compounds from the natural sources studied.

Subcritical water extraction at several temperatures ranging from 25 to 200 degrees C has been studied to selectively extract antioxidant compounds from rosemary leaves. An exhaustive characterization of the fractions obtained using subcritical water at different temperatures has been carried out by LC-MS, and the antioxidant activities of the extracts have been measured by a free radical method (DPPH). Results indicate high selectivity of the subcritical water toward the most active compounds of rosemary such as carnosol, rosmanol, carnosic acid, methyl carnosate, and some flavonoids such as cirsimaritin and genkwanin. The antioxidant activity of the fractions obtained by extraction at different water temperatures was very high, with values around 11.3 microg/mL, comparable to those achieved by SFE of rosemary leaves. A study of the effect of the temperature on the extraction efficiency of the most typical rosemary antioxidant compounds has been performed.

An experimental design has been used to optimize the extraction of antioxidants from the microalga Spirulina platensis using Accelerated Solvent Extraction (ASE) with four different solvents (hexane, petroleum ether, ethanol and water). The optimization of the main variables involved in the ASE process (extraction temperature and extraction time) has been performed by means of a Full Factorial (3 levels) design using as responses the extraction yield and the antioxidant activity of the extracts (determined as EC 50 , i.e. efficient concentration, using an in-vitro assay based on a free radical method). The parameters of the model, for each response variable, were estimated by multiple linear regression (MLR). The statistical analysis of the results allowed obtaining mathematical models able to predict the behavior of the different responses selected as a function of the main variables involved in the process. It could be observed that the optimum conditions that maximize yield and minimize EC 50 depend on the polarity of the solvent used to perform the extractions being the optimum temperature higher when using higher polarity solvents. The effect of extraction time was not as important as the effect of temperature but still had some influence mainly in its interaction with temperature. Ethanol was finally selected as the extracting solvent for its GRAS (generally recognized as safe) nature and because it provides the higher yields with medium antioxidant activities. The results presented in this work show the possibility of using a fast and easy process to recover natural antioxidants from natural sources such as microalgae.

The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO2 extraction from Rosmarinus officinalis L. were investigated. Gas chromatography-mass spectroscopy analysis of these fractions resulted in the identification of 33 compounds of the essential oil. The main components of these fractions were alpha-pinene, 1,8-cineole, camphor, verbenone, and borneol, constituting ca. 80% of the total oil. The antimicrobial activity was investigated by the disc diffusion and broth dilution methods against six microbial species, including gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the essential oil-rich fractions obtained showed antimicrobial activity against all of the microorganisms tested, with inhibition zones and minimal bactericidal and fungicidal concentration values in the range of 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. The most active fraction was the one obtained in experiment 4 (4% ethanol as modifier; extraction pressure, 25 MPa; extraction temperature, 60 degrees C). S. aureus was found to be the most sensitive bacteria to the rosemary extracts, whereas the least susceptible was A. niger. alpha-Pinene, 1,8-cineole, camphor, verbenone, and borneol standards also showed antimicrobial activity against all the microorganisms tested, borneol being the most effective followed by camphor and verbenone. In that way, it was confirmed that essential oil from experiment 4, with the best antimicrobial activity, presented the highest quantity of camphor, borneol, and verbenone.

a b s t r a c tIn this work, extraction of antioxidant carotenoids from Haematococcus pluvialis microalga, has been studied combining pressurized liquid extraction (PLE), using hexane and ethanol as extracting solvents, and analytical techniques such as thin layer chromatography (TLC) and HPLC with DAD. The effect of the extraction temperature (50, 100, 150 and 200 C) and the polarity of the solvent have been studied in terms of in vitro antioxidant activity and chemical composition considering two different morphological cells (green vegetative cells and red cysts). Results demonstrate that the extraction temperature had a positive influence in the extraction yield while its effect in the antioxidant activity was negative, lowering the activity of the extracts with an increase of the extraction temperature. The best yields were obtained with ethanol at the higher extraction temperature while the best antioxidant activity was also achieved using ethanol but at lower temperatures. Chemical composition was determined by TLC and HPLC with DAD. Several compounds were identified in the samples and concentration of astaxanthin was obtained. Results pointed out that the extracts contained different carotenoids in both, the green and the red phase, and that depending on its contribution a stronger antioxidant activity would be expected.

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In this work, extraction and characterization of compounds with antioxidant and antimicrobial activity from Haematococcus pluvialis microalga in red phase have been carried out. To do this, subcritical water extraction (SWE) has been combined with analytical techniques such as HPLC-DAD, HPLC-QqQ-MS and GC-MS and in vitro assays (i.e., for antioxidant and antimicrobial activity). The effect of the extraction temperature (50, 100, 150 and 200 degrees C) and solvent polarity has been studied in terms of yield and activity of the extracts. Results demonstrate that the extraction temperature has a positive influence in the extraction yield and antioxidant activity. Thus, the extraction yield achieved with this process was higher than 30% of dry weight at 200 degrees C as extraction temperature. Moreover, the extract obtained at 200 degrees C presented the highest antioxidant activity by far, while temperature does not seem to significatively affect the antimicrobial activity. Chemical composition was determined by HPLC-DAD, HPLC-QqQ-MS and GC-MS. Short chain fatty acids turned out to be responsible of the antimicrobial activity, whereas the antioxidant activity was correlated to vitamin E (present exclusively in the 200 degrees C extract), together with simple phenols, caramelization products and possible Maillard reaction products obtained during the extraction at high temperatures.

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