Algae are important sources of various bioactive compounds with different physi­ological effects (toxic or curative) on human health. Many of them possess antioxi­dant, antimicrobial, and antiviral activities that are important for the protection of algal cells against stress conditions. The discovery of new analytical methods and techniques is important for the study of metabolites in algae and similar organisms with respect to their applications in pharmacology and the food industry [132].

4.1 Carotenoids

Carotenoids are prominent for their distribution, structural diversity, and various functions. More than 600 different naturally occurring carotenoids are now known, not including cis and trans isomers, all derived from the same basic C40 isoprenoid skeleton by modifications, such as cyclization, substitution, elimination, addition, and rearrangement. The different carotenoids have been isolated and characterized from natural sources as plants [43, 187], algae [142,143], bacteria [183,191], yeast [119], and fungi [70].

Carotenoids play a key role in oxygenic photosynthesis, as accessory pigments for harvesting light or as structural molecules that stabilize protein folding in the photosynthetic apparatus. Carotenoids are powerful antioxidants. The beneficial effects of carotenoids have been well documented from the numerous clinical and epidemiological studies in various populations. Due to its high antioxidant activity, carotenoids have been proposed as cancer prevention agents [173], potential life extenders [88], and inhibitors of ulcer [74], heart attack, and coronary artery disease [151, 194].

All photosynthetic eukaryotes are able to synthesize lycopene, a C40 polyene, which is the precursor of two different carotenoid synthesis pathways, the b, e-carotene and the b, b-carotene pathways [1694. Xanthophylls are oxidation products of carotenes; diversification of xanthophylls increases by the inclusion of allene or acetylene groups. Allenic and acetylenic carotenoids are highly represented in algae, and at least 30 different carotenoids have been identified in this group [169]. The distribution of carotenoids having different molecular structures or the presence of specific biosynthesis pathways can be an index for algae classification. For example, the major carotenoids that occur in seaweeds (Fig. 1) include b-carotene, lutein, violax-
anthin, neoxanthin, and zeaxanthin in green algae (Chlorophytes); a — and b-caro — tene, lutein, and zeaxanthin in red seaweeds (Rhodophytes) and b-carotene, violaxanthin, and fucoxanthin in brown algae (Phaeophytes).

Carotenoid composition of algae can present great variations mainly related to environmental factors, such as water temperature, salinity, light, and nutrients avail­able. Most of the environmental parameters vary according to season, and the changes in ecological conditions can stimulate or inhibit the biosynthesis of several nutrients, such as carotenoids. For example, D. salina is a green microalga, well known for being one of the main natural sources of b-carotene. Under particular conditions, this microalga is able to produce b-carotene up to 14% of its dry weight. Moreover, the particular growing conditions able to maximize the production of b-carotene at industrial scale have been investigated [48-50, 84, 198, 118, 206]. Because b-carotene may play important roles in preventing degenerative diseases due to its associated antioxidant activity, different procedures have been studied, not only for the production of this compound but also for its extraction and isolation [66, 97, 106, 118]. The most widely employed technique has probably been SFE. The low polarity characteristics of the supercritical CO2 make this solvent appropri­ate for the b-carotene extraction from this microalga [66, 97, 106, 118].

Other example is the green microalgae H. pluvialis that produces chlorophylls a and b and primary carotenoids, namely, b-carotene, lutein, violaxanthin, neoxan — thin, and zeaxanthin, while it has the ability to accumulate, under stress conditions, large quantities of astaxanthin, up to 2-3% on a dry weight basis [150]. Using this carotenogenesis process, it undergoes different changes in cell physiology and morphology, giving as a result large red palmelloid cells [76, 204] . Astaxanthin is present in lipid globules outside the chloroplast, its functions in the cell include protection against light-related damage by reducing the amount of light available to the light-harvesting pigmented protein complexes. These pigments possess powerful biological activities, including antioxidant capacity [19], ulcer preven­tion [74] as well as immunomodulation and cancer prevention [130]. In fact, the extraction of astaxanthin has been thoroughly investigated. Different methods have been tested, including neat supercritical CO2 [189] or supercritical CO2 with differ­ent cosolvents [127], PLE [25, 67], MAE [203], direct extraction with vegetable oils [76] or solvents [75], or even treating cells with various solvents and organic acids at 70°C before acetone extraction, with the aim to facilitate the astaxanthin extraction from the thick cell wall without affecting the original astaxanthin esters profile [166].

Fucoxanthin is the most characteristic pigment of brown algae, and is also one of the most abundant carotenoids in nature [61] , accounting for more than 10% of estimated total natural production of carotenoids [103]. Fucoxanthin is an oxygen­ated carotenoid that is very effective in inhibiting cell growth and inducing apopto­sis in human cancer cells [60, 87]; it also has anti-inflammatory [172], antioxidant [158], antiobesity [99], and antidiabetic [100] properties.