EP3116518A1

EP3116518A1 - Extract from microalgae comprising fucoxanthin, fucoxanthinol and fatty acids, process for its production and applications thereof

Info

Publication number: EP3116518A1
Application number: EP15711705.2A
Authority: EP
Inventors: Xavier Ortiz Almirall; Sonia TOURIÑO EIRIN; Xavier ALVAREZ MICO; Olga Durany Turk; Jordi SEGURA DE YEBRA; Jaume MERCADE ROCA
Original assignee: Greenaltech SL
Current assignee: Greenaltech SL
Priority date: 2014-03-14
Filing date: 2015-03-16
Publication date: 2017-01-18
Also published as: WO2015136123A1; EP2918278A1

Abstract

The invention relates to an algal extract comprising fucoxanthin, fucoxanthinol and fatty acids, its production process, and the use thereof in cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical applications.

Description

EXTRACT FROM MICRO ALGAE COMPRISING FUCOXANTHIN, FUC OXANTHINOL AND FATTY ACIDS, PROCESS FOR ITS PRODUCTION

AND APPLICATIONS THEREOF FIELD OF THE INVENTION

The invention relates to an algal extract from microalgae comprising fucoxanthin, fucoxanthinol and fatty acids, its production process, and the use thereof, especially in cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical applications.

BACKGROUND OF THE INVENTION

Inflammation forms part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Inflammation is considered as a mechanism of innate immunity. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. Chronic inflammation is involved in multiple diseases, such as periodontal disease, colitis, arthritis, atherosclerosis, Alzheimer's, asthma, multiple sclerosis, and inflammatory bowel diseases.

A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Inflammatory abnormalities are a large group of disorders that underlie a vast variety of diseases. The immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with etiological origins in inflammatory processes include cancer, atherosclerosis and ischaemic heart disease. Examples of disorders associated with inflammation include acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, or transplant rejection. Further, an inflammatory component is also present in atherosclerosis, allergies, myopathies, and cancer.

There are believed to be nearly 860 million Metabolic Syndrome patients in six major countries in the world. The Metabolic Syndrome is a common and complex disorder combining obesity, dyslipidemia, hypertension, and insulin resistance. It is a primary risk factor for diabetes and cardiovascular disease. Association between Metabolic Syndrome and inflammation disorders have been broad reported (Verh K., Acad Geneeskd Belg. 2008;70(3): 193-219; Faloia E. et al. Journal of Nutrition and Metabolism; Volume 2012 (2012), Article ID 476380, 7 pages). The number of obese people is increasing in Japan due to more Westernized and irregular dietary habits and lack of exercise resulting from a more convenient lifestyle. According to a trial calculation of the Ministry of Health, Labour and Welfare, approximately 190 million people (aged between 40 and 70) either have metabolic syndrome or will so in the future, which is one out of two men and one out of five women in the age group. Although obesity, high blood pressure, hyperlipidemia, and diabetes were formerly considered to be independent lifestyle diseases, accumulated visceral fat must also be considered as a common cause of them. Accumulated visceral fat increases free fatty acid in the blood and triggers hyperlipidemia and insulin resistance. Various physiologically active substances, namely adipocytokines, are secreted from visceral fat tissues. Excessively accumulated visceral fat has been confirmed to destroy the secretion balance or these substances and cause metabolic syndrome. White adipose tissues and brown adipose tissues are found in human fat tissues and perform different functions. White adipose tissues store excessive calories as fat. Increased white adipose tissues signify obesity. On the contrary, brown adipose tissues maintain body temperature at a certain level and consume excessive calories by degrading fat and generating heat. These activities are performed by uncoupling protein 1 (UCPl) existing selectively in mitochondrial inner membrane of brown adipose tissues. Various biogenic factors are involved in the expression of UCPl . Food components capsaicin, capsiate, and caffeine increase the UCPl expression by increasing the secretion of noradrenaline and EPA and DHA do the same by becoming PPARy ligand. However, in human bodies, the amount of brown adipose tissues decreases with age so the increase of brown adipose tissues does not necessarily contribute to the prevention of obesity. Therefore, the expression of UCPl in white adipose tissues is desired so that UCPl can accelerate the oxidation of white fat and conversion of energy to heat and in turn decrease white adipose tissues. A large variety of proteins are involved in inflammation, and any one of them is open to a genetic mutation which impairs or otherwise dysregulates the normal function and expression of that protein, being interleukin 8 (IL-8), also known as CXCL8, one of the relevant cell-derived mediators of the inflammatory process. IL-8 is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells.IL-8 has two primary functions. It induces chemotaxis in target cells [neutrophil granulocytes are the primary target cells of IL-8, although there are a relatively wide range of cells (endothelial cells, macrophages, mast cells and keratinocytes) that respond to this chemokine], causing them to migrate toward the site of infection. IL-8 also induces phagocytosis once they have arrived. IL-8 is also known to be a potent promoter of angiogenesis.

IL-8 is often associated with inflammation; in fact, IL-8 has been cited as a proinflammatory mediator in gingivitis and psoriasis. Further, it is believed that IL-8 plays a role in the pathogenesis of bronchiolitis, a common respiratory tract disease caused by viral infection. Interleukin-1 beta (IL-ip), a member of the interleukin 1 family of cytokines, is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The induction of cyclooxygenase-2 (PTGS2/COX2) by this cytokine in the central nervous system (CNS) is found to contribute to inflammatory pain hypersensitivity. Increased production of IL-Ιβ causes a number of different autoinflammatory syndromes (a set of disorders characterized by recurrent episodes of systemic and organ-specific inflammation), most notably the monogenic conditions referred to as cryopyrin-associated periodic syndrome (CAPS), due to mutations in the inflammasome receptor NLRP3 which triggers processing of IL- 1β.

Tumor necrosis factor alpha (TNFa) is a cell signaling protein involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activ ated macrophages, although it can be produced by many other ceil types such as CD4+ lymphocytes, NK ceils, neutrophils, mast cells, eosinophils, and neurons. Although its primary role is in the regulation of immune cells, TNFa, as an endogenous pyrogen, is able to induce fever and inflammation. Dysregulation of TNFa production has been implicated in a variety of human diseases including Alzheimer's Disease, cancer, major depression, and inflammatory bowel disease (IBD).

There are currently many anti-inflammatory/anti-pain agents that are used to treat inflammation and pain in patients. These agents generally include steroids and nonsteroidal anti-inflammatory drugs (NSAIDS). Steroids generally act to reduce inflammation by binding to the glucocorticoid receptor, whereas NSAIDS generally act to inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase (COX-2), thus inhibiting the catalysis of the formation of the inflammation messengers prostaglandins and thromboxane . These anti-inflammatory/anti-pain agents are widely used but can have many adverse side effects. Steroids have been shown to cause hyperglycemia, insulin resistance, diabetes, osteoporosis, cataracts, anxiety, depression, colitis, hypertension, ictus, erectile dysfunction, hypogonadism, hypothyroidism, amenorrhea, retinopathy, and teratogenic defects. NSAIDS have been shown to cause gastrointestinal adverse reactions (nausea, dyspepsia, gastric ulceration and bleeding, diarrhea), myocardial infarction, stroke, erectile dysfunction, renal adverse reactions (salt and fluid retention, hypertension, interstitial nephritis, nephrotic syndrome, acute renal failure, acute tubular necrosis), photosensitivity, teratogenic defects, premature birth, miscarriage, raised liver enzymes, headache, dizziness, hyperalaemia, confusion, bronchospasm, rashes, swelling, and irritable bowel syndrome.

Therefore, there remains a need for an anti-inflammatory agent that is effective in treating inflammation but reduces the risk of the above adverse reactions.

An option to satisfy that need comprises finding compounds from nature, which unlike the steroidal and non-steroidal anti-inflammatory agents have less, or preferably, no side effect, and yet which exhibit substantially equal anti-inflammatory action to the above anti-inflammatory agents. With regard to components derived from natural substances having an anti-inflammatory action, it has been reported that extracts from various plants such as an extract of bark of Yamamomo {Myrica rubra) exhibit a hexosaminidase release-inhibitory activity and extracts from leaves of Camellia japonica L., Camellia japonica L. cv. or Camellia sasanqua T. have a potent antiinflammatory action.

Nevertheless, the demand for finding compounds from nature, which unlike the steroidal and non-steroidal anti-inflammatory agents, have no side effect such as hormone action and cause no enteric disorders, and yet which exhibit equal antiinflammatory action to the above anti-inflammatory agents is still necessary. It is an objective of the present invention to provide such substances and anti-inflammatory agents utilizing them. Inventors have now found that microalgal extracts comprising fucoxanthin, fucoxanthinol and fatty acids exert an unexpectedly high anti-inflammatory activity. It has been reported that fucoxanthin promotes fat burning within fat cells in white adipose tissue and has strong anticancer activity, whereas fucoxanthinol exhibits suppressive effects on lipid accumulation during adipocyte differentiation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an overlaid HPLC chromatogram of the initial fucoxanthin, C-MEDPA [chemical derivatization method (Example 1)] and E-MEDPA [enzymatic derivatization method (Example 1)] reaction products.

Figure 2 shows an overlaid HPLC chromatogram of a Pavlova lutheri extract before and after the C-MEDPA derivatization process.

Figure 3 shows the HPLC chromatogram of an Isochrysis galbana extract before the C- MEDPA derivatization process (A) and after the C-MEDPA derivatization process (B).

Figure 4 shows the HPLC chromatogram of an /. galbana extract before the enzymatic E-MEDPA derivatization process (A) and after the E-MEDPA derivatization process with cholesterol esterase (B).

Figure 5 is a bar diagram showing the normalized results of the production of IL-8 by keratinocytes treated firstly (after 2 days of growth) with the extracts under examination and secondly (24 h later) challenged with PMA for 5 hours previous growth. Results are normalized to reduction in IL-8 production related to keratinocytes untrated after 2 days of growth and equally challenged with PMA (Untrated Control). Extract Treated Samples before PMA challenge: /. galbana extract containing fucoxanthin [EX 3 Crude extract 100% content in FUCO-A form)] and with extracts, at two concentrations, comprising fucoxanthin and related products (fucoxanthin and fucoxanthinol, and, optionally amarouciaxanthin A and/or isofucoxanthiol) obtained by subjecting /. galbana crude extracts to treatment with the C-MEDPA derivatization process or with the E-MEDPA derivatization process [Extracts: (i) EX3 C-MEDPA Extract-; FUCOs mixture; (ii) EX6 C-MEDPA Extract-; FUCOs mixture; and (iii) EX5 E-MEDPA Extract; FUCO-A-FUCO-OL mixture].

Figure 6 is a bar diagram showing the results of the production of IL-8 by keratinocytes treated firstly (after 2 days of growth) with some compositions and extracts and secondly (24 h later) challenged with PMA for 5 hours. Results were expressed as percentage using untreated control as 100%. Compositions: A: 2.5 μΜ Fucoxanthin (97% purity); B: 2.5 μΜ Fucoxanthinol (97% purity); C: 2.5 μΜ Fucoxanthin + 2.5 μΜ Fucoxanthinol (1 : 1) (both 97% purity); D: 2.5 μΜ Fucoxanthin + 0.5 μΜ Fucoxanthinol (5:1) (both 97% purity); and E: 0.08 μΜ Fucoxanthin (97% purity). Extracts: Ext. 1 : the extract of Example 3. a at 1% [methanol extract from /. galbana obtained according to Example 3, having Fucoxanthin 0.08 μΜ]; Ext. 2: the extract of Example 3.b at 1% [extract from /. galbana obtained according to Example 3, by C- MEDPA, having Fucoxanthin 0.04 μΜ + Fucoxanthinol 0.012 μΜ + Amauroxanthin A 0.016 μΜ + Isofucoxanthinol 0.008 μΜ (i.e., 0.08 μΜ in "carotenoids")]; and Ext. 3: the extract of Example 5 at 1 % [extract from /. galbana obtained according to Example 5, by E-MEDPA (lipase), having Fucoxanthin 0.052 μΜ + Fucoxanthinol 0.028 μΜ (i.e, 0.08 μΜ in "carotenoids")]. Figure 7 is a bar diagram showing the results of the production of IL-Ιβ and TNFa by

HaCat keratinocytes treated firstly (after 2 days of growth) with an /. galbana extract, C-MEDPA derivatized (as described in Example 3), and secondly (24 h later) challenged with LPS for 5 hours. Results were expressed as percentage using untreated control as 100%.

Figure 8 is a bar diagram showing the results of the relative lipolytic activity (%) of an algal extract ("Extract -1A"), at two different concentrations, obtained from Thalassiosira pseudonana after treatment with the C-MEDPA derivatization process. Figure 9 shows GC chromatograms of the /. galbana extracts obtained after treatment with the C-MEDPA or E-MEDPA derivatization process in Examples 2, 3 and 5. DETAILED DESCRIPTION OF THE INVENTION

The inventors of the instant invention have found that an algal extract from microalgae comprising fucoxanthin and fucoxanthinol together with other algal components, such as, for example, further carotenoids such as amarouciaxanthin A and/or isofucoxanthinol, and lipids, such as fatty acids, among others, exerts an unexpectedly high anti-inflammatory activity, as it is shown in Examples 9-11, wherein the production of the pro-inflammatory chemokine mediator, interleuquin 8 (IL-8), is strongly repressed in keratinocytes treated with said algal extract and challenged with PMA (phorbol-12-myristate- 13 -acetate) [Examples 9-10] as well as the production of the pro-inflammatory cytokines interleuquin- 1 beta (IL-Ιβ) and tumor necrosis factor alpha (TNFa) is strongly repressed in keratinocytes treated with said algal extract and challenged with LPS (lipopolysaccharide) [Example 11]. Said algal extract is produced by subjecting the biomass obtained from a culture of fucoxanthin producing microalgae to a chemical or enzymatic derivatization process on fucoxanthin without the need of isolating and purifying fucoxanthin from the production medium.

Thus, the present invention relates to an algal extract from microalgae comprising fucoxanthin and fucoxanthinol together with other microalgal components, particularly, fatty acids, its production process and its applications. Some applications relate to its use as anti-inflammatory agent whereas some other applications relate to the uses of the components thereof; by illustrative, it is well-known that fucoxanthin and fucoxanthinol can be used as anti-obesity agents since they have a fat burner effect, or as antiangiogenic agents, etc. Thus, said algal extract can be used in a lot of cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical applications.

Algal extract of the invention In an aspect, the invention relates to an algal extract, hereinafter referred to as the "algal extract of the invention", comprising fucoxanthin and fucoxanthinol, together with other algal components, particularly, fatty acids, wherein said algal extract is obtained by a process comprising: a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; and

b) reacting the fucoxanthin previously obtained with:

b.l) a base under conditions for hydrolysis of at least a portion of fucoxanthin to fucoxanthinol; or, alternatively, with b.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting at least a portion of fucoxanthin into fucoxanthinol.

As used herein, the term "algal extract" refers to a product obtained from microalgae, for example, by subjecting a microalgae culture to specific treatments. The components present in an algal extract will vary depending on the microalgae and the treatments applied thereon.

As used herein, the term "fucoxanthin" refers to the acetic acid [(15',3i?)-3-hydroxy-4- [QE,5E E,9E, 1 IE, 13E, 15E)- 18-[( l_lS,4_lS,6i?)-4-hydroxy-2,2,6-trimethyl-7-oxa- bicyclo[4.1.0]heptan-l-yl]-3,7,12,16-tetramethyl-17-oxooctadeca-l,3,5,7,9,l l,13,15- octaenylidene]-3,5,5-trimethycyclohexyl]ester compound of formula

Fucoxanthin

Fucoxanthin is a xanthophyll that can be found as an accessory pigment in the chloroplasts of a great number of algae (both macroalgae and microalgae), for example, brown algae and most heterokonts, giving them a brown or olive-green color. Some metabolic and nutritional studies carried out on rats and mice indicate that fucoxanthin promotes fat burning within fat cells in white adipose tissue by increasing the expression of thermogenin. Further, it has been reported to have strong anticancer activity [Masashi Hosokawa et al., Biochimica et Biophysica Acta, 1675, pp. 113-119 (2004)]. Additional uses of fucoxanthin include its use in, for example, treatment of skin pigmentation, metabolic syndrome management, medical treatment and prevention of virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma, treatment of hyperuricemia, osteoporosis and depression, as well as anticancer agent, anti-neoplastic agent, neovascularization inhibitor, adiponectin production accelerator, cholesterol lowering agent, antihypertensive agent or antiallergic agent.

Fucoxanthin can be present in the algal extract of the invention in a very broad concentration range. Nevertheless, in a particular embodiment, fucoxanthin is present in the algal extract of the invention at a concentration comprised between about 0.001% and about 90% by weight with respect to the total weight of the algal extract of the invention, preferably between 0.01% and 75% by weight, more preferably between 0.1%) and 45% by weight, still more preferably between 1% and 15% by weight. In some specific embodiments, the algal extract of the invention comprises between 1% and 15%) by weight of fucoxanthin, such as, for example, between 1% and 10% by weight, between 2% and 4% by weight or between 5% and 10% by weight. In some other specific embodiments, the algal extract of the invention comprises between 0.001%) and 5% by weight of fucoxanthin, such as, for example, between 0.002%> and

0.5%) by weight, between 0.2% and 5% by weight, or between 0.5% and 5% by weight. Other specific embodiments include algal extracts of the invention comprising between 0.2% and 4% by weight of fucoxanthin or between 0.4% and 1.3% by weight of fucoxanthin.

As used herein, the term "fucoxanthinol" refers to the deacetylated form of fucoxanthin,

1. e., a substance in that the -OCOCH3 group of fucoxanthin is substituted by a -Oi l group, of formula

Fucoxanthin is hydrolyzed into fucoxanthinol in the gastrointestinal tract before absorption in the intestine. Fucoxanthinol can be produced from isolated fucoxanthin by enzymatic hydrolysis with a porcine pancreas lipase [WO 2007060811 Al]. Fucoxanthinol exhibits suppressive effects on lipid accumulation during adipocyte differentiation. The suppressive effect of fucoxanthinol is stronger than that of fucoxanthin. Additional uses of fucoxanthinol include treatment of osteosarcoma, skin pigmentation, as well as anticancer agent or anti-neoplastic agent. Fucoxanthinol can be present in the algal extract of the invention in a very broad concentration range. Nevertheless, in a particular embodiment, fucoxanthinol is present in the algal extract of the invention at a concentration comprised between about 0.001% and about 90% by weight with respect to the total weight of the algal extract of the invention, preferably between 0.01% and 75% by weight, more preferably between 0.1%) and 45% by weight, still more preferably between 1% and 15% by weight. In some specific embodiments, the algal extract of the invention comprises 1% to 15% by weight of fucoxanthinol, such as, for example, between 1% and 10% by weight, between 2% and 4% by weight or between 5% and 10% by weight. In some other specific embodiments, the algal extract of the invention comprises between 0.001% and 5%> by weight of fucoxanthinol, such as, for example, between 0.002%> and 0.5% by weight, or between 0.5% and 5% by weight.

The algal extract of the invention also contains other microalgal components. As it is used herein, the term "other microalgal components" include compounds other than fucoxanthin and fucoxanthinol that may be present in the algal extract of the invention, wherein said compounds are present in the fucoxanthin producing unicellular algae and are soluble in the solvent used for producing the algal extract of the invention. Said components include naturally occurring compounds in said unicellular algae, typically in fucoxanthin producing unicellular algae, such as metabolites, carotenes, carotenoids, chlorophylls, lipids, phytosterols and polypehnols, and the like, as well as non-naturally occurring compounds in said microalgae, for example, in case of engineered (e.g., transformed, etc.) microalgae that produce non-naturally occurring compounds in said microalgae; alternatively, said compounds may be produced during the process for producing the algal extract of the invention. In a particular embodiment, the solvent used for producing the algal extract of the invention is an alcohol, such as ethanol, and the algal extract of the invention can contain carotenes, such as, for example, β- 5 carotene, etc., carotenoids, such as, for example, amarouciaxanthin A, isofucoxanthinol, diadinoxanthin, diazoxanthin, etc., lipids, such as, for example, fatty acids (saturated fatty acids and/or unsaturated fatty acids), including long chain fatty acids.

As used herein, the term "amarouciaxanthin A" refers to the (3'S,5'R,6S,6'R,8'R)-3',5',6- l o trihydroxy-4,7'-didehydro-5',6,7,8-tetrahydro-P,P-carotene-3,8-dione of formula

Amarouciaxanthin A can be separated, for example, from acetone extracts from 15 Amaroucium pliciferum with silica gel column chromatography using acetone n-hexanc (1 :9, v/v) [Mi-Jin et aL J. Agric. Food Chem. 2011, 59, 1 46 1652].

Amarouciaxanthin A can be used as an agent to supress adipocyte differentiation on 3T3-L1 cells (Yim et al, J. Agric. Food Chem. 2011 Mar 9;59(5): 1646-52).

20

Amarouciaxanthin A can be present, or not, in the algal extract of the invention. Thus, in a particular embodiment, the algal extract of the invention does not contain amarouciaxanthin A. Alternatively, if present, the concentration of amarouciaxanthin A in the algal extract of the invention can vary broadly. Thus, in a particular embodiment, 25 the algal extract of the invention contains amarouciaxanthin A in a concentration between more than 0% by weight and about 90% by weight with respect to the total weight of the algal extract of the invention, preferably between about 0.001% and about 80%) by weight, more preferably between 0.01% and 70%> by weight, still more preferably between 0.1% and 50%> by weight, still more preferably between 0.05%> and 30%) by weight,even still more preferably between 1% and 15% by weight. In some specific embodiments, the algal extract of the invention comprises 1% to 15% by weight of amarouciaxanthin A, such as, for example, between 0.02%> and 5% by weight, or between 0.6%> and 4% by weight.

As used herein, the term "isofucoxanthinol" refers to the (3S,5R,3'S,5'R,6'R)-3',5'3',6'- tetrahydroxy-6 ' ,7 ' -didehydro-5 ,8,5 ' ,6 ' -tetrahydro-P,P-carotene-8-one of formula

Isofucoxanthinol can be prepared from fucoxanthinol by alkaline treatment with 1% potassium hydroxide (KOH) for 35 min at room temperature [Mi-Jin et al., J. Agric. Food Chem. 2011, 59, 1646 1652].

Isofucoxanthinol can be present, or not, in the algal extract of the invention. Thus, in a particular embodiment, the algal extract of the invention does not contain isofucoxanthinol. Alternatively, if present, the concentration of isofucoxanthinol in the algal extract of the invention can vary broadly. Thus, in a particular embodiment, the algal extract of the invention contains isofucoxanthinol in a concentration between more than 0% by weight and about 10% by weight with respect to the total weight of the algal extract of the invention, preferably between about 0.001% and about 8% by weight, more preferably between 0.01% and 5% by weight, still more preferably between 0.1% and 1.5% by weight. In some specific embodiments, the algal extract of the invention comprises between 0.02% and 5% by weight, or between 0.1% and 1.5% by weight. Illustrative, non-limitative, examples of lipids which can be present in the algal extract of the invention include fatty acids (i.e., carboxylic acids with a long aliphatic tail (chain), usually consisting of 4 to 28 carbon atoms, which is either saturated or unsaturated), including polyunsaturated fatty acids (PUFAs), i.e., fatty acids that contain more than one double bond in their backbone (although some monounsaturated omega-9 fatty acids are also considered as PUFAs). According to the length of its chain, fatty acids are usually categorized as (i) short-chain fatty acids (SCFA), i.e., fatty acids with aliphatic tails of fewer than 6 carbons; (ii) medium-chain fatty acids (MCFA), i.e., fatty acids with aliphatic tails of 6 to 12 carbons, which can form medium-chain triglycerides; (iii) long-chain fatty acids (LCFA), i.e., fatty acids with aliphatic tails of 13 to 22 carbons; and (iv) very long-chain fatty acids (VLCFA), i.e., fatty acids with aliphatic tails longer than 22 carbons.

By illustrative, the algal extract of the invention can contain saturated or unsaturated fatty acids such as, for example, caprylic acid (C8:0), capric acid (C10:0), undecanoic acid (CI 1 :0), lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:0), myristoleic acid (C14: l n5), pentadecanoic acid (C15:0), cz^'s- 10-pentadecenoic acid (C 15 : 1 n5), palmitic acid (C16:0), palmitoleic acid (C16: l n7), heptadecanoic acid (C17:0), cz^'s- 10-heptadecanoic acid (C17: l n7), stearic acid (C18:0), elaidic acid (CI 8: It n9), oleic acid (CI 8: 1c n9), linolelaidic acid (C18:2t n6), linoleic acid [LA] (C18:2c n6), gamma-linoleic acid [GLA] (C18:3 n6), arachidic acid (C20:0), alpha- linolenic acid [ALA] (C18:3 n3), cis-l 1-eicosenoic acid (C20: l n9), heneicosanoic acid (C21 :0), cz^'s- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), behenic acid (C22:0), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17- eicosatrienoic acid (C20:3 n3), euricic acid (C22: l n9), tricosanoic acid (C23:0), cis- 5,8,11, 14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16-docosadienoic acid (C22:2 n6), lignoceric acid (C24:0), nervonic acid (C:24: l n9), cis-4,7,10,13,16- docosapentaenoic acid (C22:5 n6), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3), etc.

In a particular embodiment, the algal extract of the invention comprises a fatty acid selected from the group consisting of capric acid (C10:0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16: l n7), oleic acid (C18: lc n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), cw-13,16-docosadienoic acid (C22:2 n6), cw-4,7,10,13,16,19- docosahexanoic acid [DHA] (C22:6 n3), and mixtures thereof.

In another particular embodiment, the algal extract of the invention comprises a PUFA selected from the group consisting of linolelaidic acid (C18:2t n6), linoleic acid [LA] (C18:2c n6), gamma-linoleic acid [GLA] (C18:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), cz^'s- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,l l,14-eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), cis-5, 8, 11, 14, 17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16- docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof.

In a particular embodiment, the algal extract of the invention comprises a fatty acid selected from the group consisting of palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha- linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cw-5,8,11,14,17- eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof. According to this embodiment, the algal extract of the invention may comprise monounsaturated fatty acids (MUFAs) and/or PUFAs, advantageoulsy a combination of both MUFAs and PUFAs. In a preferred embodiment, the algal extract of the invention comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8 : 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (CI 8:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8, 11, 14, 17-eicosapentaenoic acid [EPA] (C20:5 n3) and cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3). The "other algal components" can be present in the algal extract of the invention in a very broad concentration range. Nevertheless, in some particular embodiments, the algal extract of the invention comprises 1% to 99.998% by weight of other algal components, for example, between 10% and 99.5% by weight, between 20%> and 99.5% by weight, between 30%> and 99.5% by weight, between 40% and 99.5% by weight, between 45% and 99.5% by weight, between 50% and 99.5% by weight, between 55% and 99.5% by weight, between 60% and 99.5% by weight, between 70% and 99.5% by weight, between 80% and 99.0% by weight, between 90% and 99.0% by weight. In some specific embodiments, the algal extract of the invention comprises between 80% and 98.5%) by weight, between 89.7% and 96.9% by weight, or between 79.9% and 94.94% by weight, of the other algal components. The amounts in which the different components that may be present in the other algal components fraction can vary broadly depending among other things on the microalgae, the solvent used for producing the extract, the extraction conditions, etc.

In a particular and preferred embodiment, the other algal component fraction that is present in the algal extract of the invention comprises one or more fatty acids (including, for example, in a mix of free fatty acids, in a mix of mono-, di- or triglycerides (mono-di- or triacylglycerols), in a mix of esterified fatty acids, or even in a mix of free fatty acids together with mono-, di- or triglycerides and/of partially esterified fatty acids), including one or more saturated fatty acid such as caprylic acid (C8:0),capric acid (C10:0), undecanoic acid (CI 1 :0), lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:0), pentadecanoic acid (C13:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), heneicosanoic acid (C21 :0), behenic acid (C22:0), tricosanoic acid (C23:0), lignoceric acid (C24:0), etc. and/or one or more unsaturated fatty acid, including monounsaturated fatty acids (MUFAs), such as myristoleic acid (C14: l n5), cz^'s- 10-pentadecenoic acid (C 15 : 1 n5), palmitoleic acid (C16: l n7), cz^'s- 10-heptadecanoic acid (C17: l n7), elaidic acid (C18: lt n9), oleic acid (C18: lc n9), cz^'s- 11-eicosenoic acid (C20: l n9), euricic acid (C22: l n9), nervonic acid (C:24: l n9), etc., and preferably one or more PUFAs, such as the above mentioned PUFAs, advantageously n-3 polyunsaturated fatty acids such as ALA, cis- 11,14,17-eicosatrienoic acid (C20:3 n3), EPA, DHA, etc., n-6 polyunsaturated fatty acids such as linolelaidic acid, LA, GLA, cis- 11,14-eicosadienoic acid (C20:2 n6), cis- 8,11,14-eicosatrienoic acid (C20:3 n6), ARA, cz^'s-13,16-docosadienoic acid (C22:2 n6), cis-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), etc., and any combination thereof. The presence of PUFAs, especially DHA, appears to increase the absorption and efficacy of fucoxanthin. Thus in a particular embodiment, the algal extract of the invention comprises at least one fatty acid, either a saturated fatty acid or an unsaturated fatty acid. In another particular embodiment, the algal extract of the invention comprises at least a PUFA, for example, a polyunsaturated omega-3 (n-3) fatty acid, a polyunsaturated omega-6 (n-6) fatty acid, etc. In another particular embodiment, the algal extract of the invention comprises two or more fatty acids, the fatty acids being saturated fatty acids or unsaturated fatty acids including PUFAs, in any combination thereof. In a preferred embodiment, the algal extract of the invention comprises a fatty acid selected from the group consisting of capric acid (C10:0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (C18:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16-docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16,19- docosahexanoic acid [DF1A] (C22:6 n3), and any combination thereof. In another particular embodiment, the algal extract of the invention comprises a PUFA selected from the group consisting of linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8:2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (CI 8:3 n3), cz^'s- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis- 11,14,17-eicosatrienoic acid (C20:3 n3), cis- 5,8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16-docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cz^'s-4,7,10,13,16,19- docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof. In another particular embodiment, the algal extract of the invention comprises a fatty acid selected from the group consisting of palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), czs-5,8,11,14,17- eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof. The fatty acids can be present in the algal extract of the invention (as a component of the "other algal components" fraction) in a very broad concentration range. As it has been previously mentioned, the specific fatty acids and their amounts that may be 5 present on the algal extract of the invention can vary depending, among other features, from the microalgae from which the algal extract of the invention is obtained; by illustrative, it is well-known that Navicula jeffreyae, Nitzschia closterium and Thalassiosira pseudonana are rich in EPA (about 21, 24 and 19% respectively of the total fatty acids), the latter having also good percentages of DHA (4%); Isochrysis sp. is o rich in DHA (about 8%) but only has trace amounts of EPA; and Pavlova lutheri has a similar percentage of DHA, but is also rich in EPA (20%). Nevertheless, in a particular embodiment, fatty acids are present in the algal extract of the invention at a concentration comprised between about 1% and about 50%> by weight with respect to the total weight of the algal extract of the invention, preferably between 2% and 40% by5 weight, more preferably between 10% and 40% by weight, still more preferably between 15% and 40%> by weight, even more preferably between 15% and 35% by weight.

The fatty acids can be present in the algal extract of the invention in a mix of free fatty o acids, and/or in a mix of mono-, di- or triglycerides, and/or in a mix of esterified fatty acids; nevertheless, in preferred embodiments, the algal extract of the invention comprises a portion of the fatty acids as free fatty acids. Thus, in some specific embodiments, the algal extract of the invention comprises at least 1% by weight, usually 5% by weight, normally 10% by weight, preferably at least 15% by weight, 5 more preferably at least 20% by weight, still more preferably, at least 25% by weight of free fatty acids with respect to the algal extract, and up to 50% by weight, usually up to 45%) by weight, commonly up to 40%, normally up to 35% by weight of free fatty acids with respect to the algal extract of the invention. Advantageously, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and0 45%) by weight, usually between 10% and 40% by weight of free fatty acids with respect to the algal extract of the invention. In preferred embodiments, a portion of the fatty acids are PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises at least 1% by weight, usually 5% by weight, normally 10% by weight, preferably at least 15% by weight, more preferably at least 20% by weight, still more preferably at least 25% by weight of PUFAs and up to 50%> by weight, usually up to 45% by weight, commonly up to 40%, normally up to 35% by weight of PUFAs with respect to the algal extract. Advantageously, the algal extract of the invention comprises between 1% by weight and 40%) by weight, typically between 5% and 35% by weight of PUFAs with respect to the algal extract of the invention. Said PUFAs are present in the algal extract of the invention in a mix of free PUFAs, and/or in a mix of mono-, di- or triglycerides (wherein the fatty acid is a PUFA), and/or in a mix of esterified PUFAs; preferably a portion of said PUFAs is present as free PUFAs in the algal extract of the invention. Thus, advantageously, in some embodiments, the algal extract of the invention comprises between 1% and 40% by weight, typically between 5% and 35% by weight, usually between 10% and 30% by weight, of free PUFAs with respect to the algal extract of the invention.

In further embodiments, a portion of the fatty acids are a combination of MUFAs and PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and 40% by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention. In a particular embodiment, said combination of MUFAs and PUFAs comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis- 5,8,11,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cz^'s-4,7,10,13, 16,19- docosahexanoic acid [DHA] (C22:6 n3). Said MUFAs and PUFAs can be present in the algal extract of the invention as free MUFAs and/or PUFAs, and/or in any esterified form thereof. Thus, in some embodiments, the algal extract of the invention comprises between 1% and 40% by weight, typically between 3% and 25% by weight, usually between 5% and 15% by weight, of free MUFAs with respect to the algal extract of the invention whereas the amount of free PUFAs with respect to the algal extract of the invention has been previously mentioned.

Measurement of the total fatty acids can be performed by conventional methods for the measurement of fatty acids, for example, by gas cromatography. Acccording to this method, the fatty acids are transformed into free fatty acid by the action of a base. Subsequently, the free fatty acids are converted into the fatty acid methyl esthers by the use of a large excess of anhydrous methanol in the presence of a catalyst, boron trifluoride (Morrison et a I J Lipid Res i 964, 5.600).

The measurement of free fatty acids can be performed by conventional methods for the quantitative measurement of free fatty acids. Numerous methods have been described for the quantitative measurement of free fatty acids. They include, for example, (i) chemical titration methods [e.g., methods based on determining the number of mg of KOH required to neutralize the fatty acids contained in 1 g of the sample to be analyzed; methods based on the use of m-cresol as indicator and titration with NaOH (Ke PJ et al., Anal Chim Acta 1978, 99, 387), etc.]; (ii) thermometric titration methods [e.g., by Catalyzed Endpoint Thermometric Titrimetry or CETT (Smith TK, J Am Oil Chem Soc 2003, 80, 21-24), etc.]; (iii) measurement of metal-fatty acid complexes [e.g., methods based on the ability of fatty acids to form complexes with some metals (Cu, Co) and to be detectable by spectrophotometry such as the radiochemical assay of the complex of fatty acids with ⁶⁰Co (Ho RJ et al, Anal Biochem 1969, 31, 426-436)]; methods based on the formation of fatty acid-copper complexes (Tinnikov AA et al., Clin Chim Acta 1999, 281, 159; Wawrik B et al, J Microbiol Methods 2010, 80, 262; Chen Y et al., Anal Chim Acta 2012, 724, 67), etc.]; (iv) enzymatic methods [e.g., methods based on the introduction of acyl-CoA synthetase that enable to quantify fatty acids in a sample in coupling the formation of activated fatty acids with three enzymatic reactions which lead to a fluorimetric detection of NADH consumption (Jebens E et al. Scand J Clin Lab Invest 1992, 52, 717), etc.]; (v) methods using a fatty acid binding protein [e.g., methods based on the use of a fluorescent probe ADIFAB, composed of a fatty acid binding protein derivatized with the fluorescent molecule acrylodan (Richieri GV et al. Biochemistry 1993, 32, 7574; Richieri GV et al. Moll Cell Biochem 1995, 192, 87-94; US 5,470,714; etc.]; and (vi) spectroscopic methods [e.g., a Fourier- transform infrared spectroscopic technique has been developed for the non-invasive measurement of saturated and unsaturated fatty acid compositions (Yoshida S, Lipid Technol 2008, 20, 184); etc.].

Some illustrative, non-limitative examples of algal extracts of the invention are described below wherein all the percentages are referred to the total weight of the algal extract of the invention. In a particular embodiment, the algal extract of the invention comprises:

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

- and

1%) to 99.998%) by weight of other algal components.

In a particular embodiment, the algal extract of the invention comprises:

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol; and

1%) to 99.998%) by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises:

1%) to 10%) by weight of fucoxanthin;

0.1% to 10% by weight of fucoxanthinol; and

- 80%) to 98.5%o by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises:

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

- more than 0% to 90% by weight of amarouciaxanthin A; and

1 % to 99.998%) by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises: - 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

- more than 0% to 10% by weight of isofucoxanthinol; and

1 % to 99.998% by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

- more than 0% to 90% by weight of amarouciaxanthin A;

- more than 0% to 10%> by weight of isofucoxanthinol; and

1%) to 99.998%) by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises

- 0.001 % to 15 % by weight of fucoxanthin;

- 0.001 % to 15 % by weight of fucoxanthinol;

- 0%) to 15%) by weight of amarouciaxanthin A; and

- 55% to 99.998%) by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises

- 0.001 % to 15 % by weight of fucoxanthin;

- 0.001 % to 15 o by weight of fucoxanthinol;

- 0%o to 15%o by weight of amarouciaxanthin A; and

- 0%) to 10%o by weight of isofucoxanthinol;

- 45%o to 99.998% by weight of other algal components.

In another particular embodiment, the algal extract of the invention comprises

- 2%o to 4%o by weight of fucoxanthin;

- 0.4%o to 0.8%) by weight of fucoxanthinol;

0.6% to 4% by weight of amarouciaxanthin A;

- 0.1 % to 1.5%o by weight of isofucoxanthinol; and

- 89.7%o to 96.9%o by weight of other algal components. In another particular embodiment, the algal extract of the invention comprises:

- 5% to 10% by weight of fucoxanthin;

- 0.02%) to 0.1 % by weight of fucoxanthinol;

0.02% to 5% by weight of amarouciaxanthin A;

- 0.02%) to 5%> by weight of isofucoxanthinol; and

79.9% to 94.94% by weight of other algal components.

Any of the above particular embodiments of the algal extract of the invention, preferably comprises one or more fatty acids within the other algal components fraction, in an amount such that said fatty acids present in the algal extract of the invention are at a concentration comprised between about 1% and about 50% by weight with respect to the total weight of the algal extract of the invention, preferably between 2%) and 40%> by weight, more preferably between 10%> and 40%> by weight, still more preferably between 15% and 40%> by weight, even more preferably between 15% and 35%) by weight.

In preferred embodiments of the invention, a portion of said free fatty acids are PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises at least 1%) by weight, usually at least 5% by weight, normally at least 10% by weight, preferably at least 15% by weight, more preferably at least 20% by weight, still more preferably at least 25% by weight of PUFAs and up to 50% by weight, usually up to 45%) by weight, commonly up to 40%, normally up to 35% by weight of PUFAs with respect to the algal extract. Advantageously, the algal extract of the invention comprises between 1% by weight and 40% by weight, typically between 5% and 35% by weight of PUFAs with respect to the algal extract of the invention.

In further embodiments, a portion of the fatty acids are a combination of MUFAs and PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and 40% by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention. In a particular embodiment, said combination of MUFAs and PUFAs comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis- 5,8,11,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cz^'s-4,7,10,13, 16,19- docosahexanoic acid [DHA] (C22:6 n3).

In a particular embodiment, the algal extract of the invention comprises:

1% to 10% by weight of fucoxanthin;

- 0.05% to 1%) by weight of fucoxanthinol; and

- 89%o to 98.95%o by weight of other algal components,

wherein said other algal components comprise at least one fatty acid at a concentration between about 1%> and about 50%> by weight with respect to the total weight of the algal extract of the invention, preferably between 2% and 45%) by weight, more preferably between 3%> and 40%> by weight, still more preferably between 4% and 35% by weight, even more preferably between 5%> and 30%> by weight.

In a particular embodiment, said fatty acid is selected from the group consisting of caprylic acid (C8:0), capric acid (CI 0:0), undecanoic acid (CI 1 :0), lauric acid (CI 2:0), tridecanoic acid (C13:0), myristic acid (C14:0), myristoleic acid (C14: l n5), pentadecanoic acid (CI 5:0), cz^'s- 10-pentadecenoic acid (C 15 : 1 n5), palmitic acid (C16:0), palmitoleic acid (C16: l n7), heptadecanoic acid (C17:0), cz^'s- 10-heptadecanoic acid (C17: l n7), stearic acid (C18:0), elaidic acid (C18: lt n9), oleic acid (C18: lc n9), linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (C18:3 n6), arachidic acid (C20:0), alpha-linolenic acid [ALA] (C18:3 n3), cis- 11-eicosenoic acid (C20: l n9), heneicosanoic acid (C21 :0), cis-l 1,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), behenic acid (C22:0), arachidonic acid [APvA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), euricic acid (C22: l n9), tricosanoic acid (C23:0), cis-5, 8,11,14,17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16-docosadienoic acid (C22:2 n6), lignoceric acid (C24:0), nervonic acid (C:24: l n9), cis-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3), and any combination thereof, preferably a fatty acid selected from the group consisting of capric acid (C10:0), undecanoic acid (CI 1 :0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16: l n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), alpha- linolenic acid [ALA] (C18:3 n3), cz^'s-5,8,l l,14,17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16- docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3), and any combination thereof.

In another particular embodiment, said fatty acid is a fatty acid selected from the group consisting of capric acid (C10:0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (C18:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16-docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16,19- docosahexanoic acid [DHA] (C22:6 n3), and mixtures thereof. In another particular embodiment, said fatty acid comprises a PUFA selected from the group consisting of linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (C18:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), cis- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), cis- 5,8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16-docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cz^'s-4,7,10,13,16,19- docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof.

In another particular embodiment, said fatty acid comprises a fatty acid selected from the group consisting of palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), czs-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof.

In preferred embodiments, the algal extracts of the invention comprise between 1% and 40% by weight, typically between 5% and 35% by weight of PUFAs with respect to the algal extract of the invention. In further embodiments, the algal extracts of the invention comprise between 1% and 50% by weight, typically between 5% and 40%> by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention.

In another embodiment, the algal extract of the invention comprises fucoxanthin and fucoxanthinol, together with other algal components, wherein said other algal components comprise between about 1% and about 50%> by weight of fatty acids with respect to the total weight of the algal extract, and wherein said algal extract is obtained by a process comprising: a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; and

b) reacting the fucoxanthin previously obtained with:

b. l) a base under conditions for the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol, and at least of a portion of mono-, di- or tiglycerides or esterified fatty acids present in the fucoxanthin producing microalgae biomass to fatty acids; or, alternatively, with b.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting at least a portion of fucoxanthin into fucoxanthinol, and at least of a portion of mono-, di- or tiglycerides or esterified fatty acids present in the fucoxanthin producing microalgae biomass into fatty acids.

The fatty acids can be present in the algal extract of the invention in a mix of free fatty acids, and/or in a mix of mono-, di- or triglycerides, and/or in a mix of esterified fatty acids; nevertheless, in preferred embodiments, the algal extract of the invention comprises a portion of the fatty acids as free fatty acids. Thus, as mentioned above, in a particular embodiment, the algal extract of the invention comprises at least 1% by weight, usually, 5% by weight, normally 10%> by weight, preferably at least 15% by weight, more preferably at least 20% by weight, still more preferably, at least 25% by weight of free fatty acids. In preferred embodiments, a portion of said free fatty acids are PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises between 1% and 40% by weight, typically between 5% and 35% by weight, usually between 10%> and 30% by weight, of free PUFAs with respect to the algal extract of the invention. Said PUFAs, as mentioned above, can be present in the algal extract of the invention in a mix of free PUFAs, and/or in a mix of mono-, di- or triglycerides (wherein the fatty acid is a PUFA), and/or in a mix of esterified PUFAs; preferably a portion of said PUFAs is present as free PUFAs in the algal extract of the invention. In further embodiments, the algal extracts of the invention comprise between 1%) and 50%) by weight, typically between 5% and 40%> by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention. In a preferred embodiment, the algal extract of the invention comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3) and czs-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3).

As mentioned above, the algal extract of the invention is obtained by a process which comprises:

a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; and

b) reacting the fucoxanthin previously obtained with:

As used herein, the term "microalga" includes a large and diverse group of simple, typically autotrophic unicellular organisms, i.e., microscopic algae, typically found in freshwater and marine systems.

According to the invention, the microalga must produce fucoxanthin, either naturally or artificially (i.e., as a result of a process for transforming non-fucoxanthin producing algae into fucoxanthin-producing algae). Consequently, any fucoxanthin producing microalga can be used to produce the algal extract of the invention.

In a particular embodiment, the fucoxanthin producing microalga is a microalgae belonging to the phylum Heterokontophyta (i.e., unicellular heterokonts), Haptophyta or Dinophyta.

In a more particular embodiment, the fucoxanthin producing alga is a microalga belonging to a class selected from the group consisting of Rapidophyceae, Bacillariophyceae (Diatomeas), Crysophyceae, Pavlophyceae, and Prymnesiophyceae.

Still in a more particular embodiment, the fucoxanthin producing alga is a microalga that belongs to a genus selected from the group consisting of Isochrysis, Thalassiosira, Navicula, Pavlova, Ochromonas, Phaeodactylum, Odontella, Skeletonema, Chaetoceros, Prymnesium, Nitzschia, Dinobryon, Synura, Chrysochromulina, Ochrosphaera, Cylindrotheca, Chromulina, Mallomonas, and Emiliania.

Illustrative, non-limitative, examples of fucoxanthin producing algae include Isochrysis aff. galbana, Thalassiosira pseudonana, Navicula incerta, Pavlova lutheri, Ochromonas sp., Phaeodactylum tricornutum, Odontella aurita, Isochrysis galbana, Isochrysis sp., Pavlova gyrans, Skeletonema costatus, Chaetoceros gracilis, Chaetoceros calcitrans, Prymnesium parvum, Nitzschia heufleriana, Nitzschia sp., Dinobryon sp., Synura uvella, Synura petersenii, Chrysochromulina brevifikum, Ochrosphaera neapolitana, Cylindrotheca fusiformis, Chromulina neblosa, Mallomonas asmundae, Ochromonas danica, Ochromonas spherocystis, and Emiliania huxleyi, preferably, /. aff. galbana, T. pseudonana, N. incerta, P. lutheri, and Ochromonas sp.

The term "biomass", as used herein, includes biological material comprising, or deriving from, living or recently living organisms. By extension, the term includes not only the biological material or organic matter which constitutes an organism, but also the biological material or organic matter generated in a biological process, spontaneous or not spontaneous (i.e., provoked). Thus, the expression "fucoxanthin producing microalgae biomass" refers to biomass comprising fucoxanthin producing microalgae.

According to step a), the fucoxanthin producing microalgae biomass is cultured under conditions that allow the production of fucoxanthin, thereby obtaining an algal extract comprising fucoxanthin and fucoxanthinol, and other algal components from the fucoxanthin producing microalgae biomass. The conditions for culturing fucoxanthin producing microalgae can vary widely depending, among other factors, on the specific fucoxanthin producing microalga elected for carrying out the process for producing the algal extract of the invention.

In a particular embodiment, the fucoxanthin producing microalga is a photoautotroph organism (i.e., an organism capable of synthesizing its own food from inorganic substances using light as energy source and is capable of using carbon dioxide as its principal source of carbon). Thus, according to this particular embodiment, the fucoxanthin producing microalgae biomass can be obtained by photoautotrophic growth.

In another particular embodiment, the fucoxanthin producing microalga is a mixotroph organism (i.e., an organism that can use a mix of different sources of energy and carbon). Thus, according to this particular embodiment, the fucoxanthin producing microalgae biomass can be obtained by mixotrophic growth.

For use within the context of the present invention, the fucoxanthin producing microalga can be collected from the natural medium or can be cultured in a photobioreactor.

In a particular embodiment, the fucoxanthin producing microalga is cultured in a photobioreactor in a suitable medium, under a suitable luminous intensity, at a suitable temperature. Practically any medium suitable for growing microalgae can be used; nevertheless, illustrative, non-limitative examples of said media include: f/2 (Guillard and ryther 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8:229-39), Erds (Tompkins et al., 1995. Culture Collection of Algae and Protozoa. Catalog of Strains. Ambleside, UK, 204 pp.) or K/2 (Keller et al., 1987 Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23:633-8). The luminous intensity can vary widely, nevertheless, in a particular embodiment, the luminous intensity is comprised between 25 and 150 μιηοΐ fotons m^"2 s^"1. The temperature can vary usually between about 17°C and about 30°C. The culture can be performed in the absence of aeration or with aeration. In a particular embodiment, the culture is carried out without aeration. In another embodiment, the culture is performed with aeration, for example, with air or with up to 5% C0₂ enriched air, at a rate of delivery comprised between more than 0 and 1 L/min.

The fucoxanthin contained in the product resulting from step a) is subjected to an alkaline chemical treatment or to an enzymatic hydrolysis [step b)]. If necessary, the product resulting from step a) can be subjected to a treatment, for example, cell lysis and an extractive method before applying the alkaline chemical or enzymatic hydrolysis. It will depend on the nature of the microalgae biomass used. Lysis of microalgae cells can be performed by conventional methods for lysing said type of cells knwon by the skilled person in the art. Thus, in step b. l), fucoxanthin produced as in step a) is subjected to an alkaline treatment (saponification) under conditions that allow the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol, thereby fucoxanthinol is obtained by chemical hydrolysis of fucoxanthin. The skilled person in the art will understand that at least a portion of other compounds which are present in the extract, specifically fatty acids, for example in the form of mono-, di- or triglycerides or esterified fatty acids, will also be hydrolysed.

The base can be an inorganic base or an organic base. Illustrative, non-limitative, examples of inorganic bases include ammmonia (NH₃), alkaline metals or alkaline earth metals hydroxides or carbonates, such as, for example, KOH, Na₂C0₃, etc. Illustrative, non- limitative, examples of organic bases include N-heterocyclic compounds [e.g., benzimidazol, imidazole, piperidine, pyridine, etc., and derivatives thereof, such as 4- (dimethylaminopyridine), di-tert-butylpyridine, 2,6-lutidine, etc., 1,5-diazabicyclo [4.3.0]non-5-ene, l,8-diazabicyclo[5.4.0]undec-7-ene, etc.], amines [e.g., methylamine, ethylamine, dimethylamine, diethylamine, diisopropylamine, N,N- diisopropylmethylamine, N-ethyldiisopropylamine, 2-(2chloro-6-fluorophenyl) ethylamine, choline, etc.], organolithiums [e.g., methyllithium, n-butyllithium, sec- butyllithium, tert-butyllithium, hexyllithium, etc.], Grignard reagents [e.g., butylmagnesium chloride, sec-butylmagnesium chloride, methylmagnesium bromide, ethylmagnesium bromide, 1,1-dimethylpropylmagnesium chloride, 2,2- dimethylpropylmagnesium chloride, methylmagnesium bromide, hexylmagnesium chloride, etc.], tetraalkylammonium hydroxides [e.g., tetramethylammonium hydroxide, tetramethylammonium hydroxide, diethyldimethylammonium hydroxide, benzyltrimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, etc.], phosphonium hydroxides [e.g., 2,8,9-trimethyl-2,5,8,9-tetraaza-l-phosphabicyclo [3.3.3]undecane, etc.], metal alkoxides [e.g., lithium methoxide, lithium ethoxide, lithium tert-butoxide, lithium isopropoxide, barium tert-butoxide, potassium tert- butoxide, magnesium di-tert-butoxide, etc.], metal amides [e.g., lithium diethylamide, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, lithium dicyclohexylamide, lithium dimethylamide, etc.], metal silanoates [e.g., lithium trimethylsilanoate, etc.], phosphazene bases [e.g., phosphazene base Pi-t-Bu, etc.]. In a preferred embodiment, the inorganic base is KOH or ΝΗ₄ΟΗ, and the organic base is lithium tert-butoxide.

The base can be added to the fucoxanthin containing medium at any appropriate molar ratio with respect to fucoxanthin. In any case, the base will be added in a ratio with respect to fucoxanthin that allows the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol. Usually the base is added at a ratio higher than the corresponding stoichiometric ratio because a portion of the base can react with other co-extracted algal components such as, for example, lipids or chlorophylls (this also allows the hydrolysis of at least a portion of mono-, di- or tiglycerides or esterified fatty acids present in the fucoxanthin producing microalgae biomass to fatty acids). Thus, the presence of said components should be taken into account when preparing the base to be added to the reaction medium. The reaction between fucoxanthin and the base is preferably carried out in the dark, in a suitable reaction medium comprising an organic solvent, at a suitable temperature, and for a suitable period of time (reaction time) so that at least a portion of fucoxanthin is converted to fucoxanthinol. Illustrative, non-limitative, examples of organic solvents include alcohols (e.g., methanol, ethanol, etc.), ethers (e.g., tetrahydrofuran (THF), etc.), ketones (e.g., acetone, etc.), etc. The temperature can vary usually between about 4°C and about 30°C. The period of time must be sufficient to guarantee that at least a portion of fucoxanthin is converted to fucoxanthinol; although it can vary within a broad range, in a particular embodiment, the reaction time between fucoxanthin and the base is comprised between about 2 minutes and 24 hours, preferably between 2 minutes and 12 hours, more preferably between 5 minutes and 6 hours, still more preferably between 10 minutes and 3 hours, usually between about 10 minutes and about 120 minutes. The reaction can be carried out under inert atmosphere or not. Thus, in a particular embodiment, the reaction is carried out under inert atmosphere. In another particular embodiment, the reaction is not carried out under inert atmosphere.

As it will be evident for the skilled person in the art, varying the hydrolysis conditions, it is possible to regulate or control the conversion of fucoxanthin to fucoxanthinol and co-hydrolisis of other compounds that are present in the extract such as, for example, fatty acids in the form of mono-, di- or triglycerides or esterified fatty acids. In fact, varying the amount and type of base, reaction medium, reaction time and reaction temperature it is possible to control (e.g., increase) the conversion of fucoxanthin to fucoxanthinol. In all cases, the reaction is performed in the dark. Further, depending on the base and the reaction time, the reaction can be performed (or not) under inert atmosphere. By illustrative, when fucoxanthin is hydrolyzed using 4 g Γ¹ of KOH in ethanol, at 25°C during 10 minutes under a non-inert atmosphere, the final algal extract may contain 3% by weight of fucoxanthin, 0.3% by weight of fucoxanthinol, 1.3% by weight of amarouciaxanthin A and 1% by weight of iso fucoxanthinol. On the other hand, when fucoxanthin is hydrolyzed using 6 g Γ¹ of KOH in methanol, at 0°C during 60 minutes under inert atmosphere, the final algal extract may contain 5% by weight of fucoxanthin, 1.5% by weight of fucoxanthinol, 2% by weight of amarouciaxanthin A and 1% by weight of iso fucoxanthinol. In addition, this chemical treatment will also hydrolyze at least a portion of the mono-, di-, or triglycerides and esterified fatty acids which are present in the microalga and, as a consequence thereof, free fatty acids will be released.

5 In step b.2), fucoxanthin produced as in step a) is subjected to enzymatic hydrolysis with an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions that allow the conversion of at least a portion of fucoxanthin to fucoxanthinol, thereby fucoxanthinol is obtained by enzymatic hydrolysis of fucoxanthin. The skilled person in the art will understand that at least a portion of other o compounds which are present in the extract, specifically fatty acids, for example in the form of mono-, di- or triglycerides or esterified fatty acids, will also be enzymatically hydrolysed.

Enzymes that catalyze the conversion of fucoxanthin to fucoxanthinol include5 hydrolases (i.e., enzymes that act on ester bonds) included within class EC 3.1 class.

Illustrative, non-limitative, examples of enzymes suitable or use in the process of the invention include lipases and cholesterol esterase. A lipase is an enzyme that catalyzes the hydrolysis of fats (lipids) and is considered as a subclass of esterases. A cholesterol esterase is an enzyme that catalyzes the hydrolysis of sterol esters into their component o sterols and fatty acids. Preferred enzymes for use in the process of the invention include a pancreas lipase, such as a porcine pancreas lipase, or a cholesterol esterase.

As it will be evident for the skilled person in the art, the enzymes that catalyze the conversion of fucoxanthin to fucoxanthinol, namely, lipase and cholesterol esterase, 5 will also hydrolyze fatty acids in the form of mono-, di- or triglycerides or esterified fatty acids thus releasing fatty acids.

Once finished the alkaline chemical treatment or enzymatic hydrolysis performed in step b), an algal extract comprising fucoxanthin and fucoxanthinol, and other algal0 components is obtained. Among these other algal components, free fatty acids are usually present. These free fatty acids derive from the microalgae used for producing the algal extract of the invention. Usually said fatty acids will be present in said microalgae in the form of mono-, di- or triglycerides, or even in other esterified forms, and due to the chemical or enzymatic treatment applied thereon said mono-, di- or triglycerides, or said other esterified forms, will be total or partially hydrolyzed and the corresponding free fatty acids will be released. Thus, the algal extract of the invention contains all or a portion of the fatty acids present in the microalgae used for producing said algal extract of the invention in a form (as free fatty acids) other than the form in which they are present in said naturally occurring microalgae.

If desired, the algal extract comprising fucoxanthin, fucoxanthinol and other microalgal components so obtained is separated (removed) by conventional techniques including, for example, decantation after formation of a biphasic system, and, if desired, dried by rotoevaporation to dryness in order to obtain a substantially dry algal extract comprising fucoxanthin, fucoxanthinol and other microalgal components, such as an algal extract of the invention, or concentrated by rotoevaporation and addition of a matrix oil, for example a vegetal (vegetable) oil (e.g., corn oil, etc.).

Thus, in a particular embodiment, the product resulting from the alkaline chemical treatment or enzymatic hydrolysis performed in step b) is treated with an aqueous saline solution in order to form a biphasic system comprising an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol and fatty acids in different forms (e.g., free fatty acids, mono-, di- or triglycerides and other fatty acids esters), together with other compounds in the extract. Virtually any non-toxic, water-soluble salt, can be used, preferably a non-toxic, water-soluble salt, that increases polarity of the aqueous phase, e.g., an aqueous solution comprising NaCl, KC1, NaHPC"3, etc. The organic phase is separated from the aqueous phase by any suitable technique, for example, decantation, centrifugation, etc., and, if desired, dried by rotoevaporation to dryness, in order to obtain a substantially dry algal extract comprising fucoxanthin, fucoxanthinol and other microalgal components, such as an algal extract of the invention.

In a particular embodiment, the algal extract of the invention obtained according to the process of the invention comprises fucoxanthin, fucoxanthinol, and, optionally, amarouciaxanthin A and/or isofucoxanthinol, together with other algal components, especially one or more fatty acids, such as those previously mentioned.

In another particular embodiment, the algal extract of the invention obtained according to the process of the invention comprises:

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

0% to 90% by weight of amarouciaxanthin A;

- 0%) to 10%) by weight of isofucoxanthinol; and

- 1%) to 99.998%) by weight of other algal components.

In a particular embodiment, the algal extract of the invention so obtained comprises one or more fatty acids within the other microalgal components fraction, in an amount such that said fatty acids present in the algal extract of the invention are at a concentration comprised between about 1% and about 50%> by weight with respect to the total weight of the algal extract of the invention, preferably between 2% and 40% by weight, more preferably between 10%> and 40%> by weight, still more preferably between 15% and 40%) by weight, even more preferably between 15% and 35% by weight. The fatty acids can be present in the algal extract of the invention in a mix of free fatty acids, and/or in a mix of mono-, di- or triglycerides, and/or in a mix of esterified fatty acids; nevertheless, in preferred embodiments, the algal extract of the invention comprises a portion of the fatty acids as free fatty acids. Thus, in particular embodiments, the algal extract of the invention comprises at least 1% by weight, usually, 5% by weight, normally 10% by weight, preferably at least 15% by weight, more preferably at least 20%) by weight, still more preferably, at least 25% by weight of free fatty acids with respect to the algal extract of the invention. Advantageously, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and 45% by weight, usually between 10% and 40% by weight of free fatty acids with respect to the algal extract of the invention.

In preferred embodiments, a portion of said free fatty acids are PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises at least 1% by weight, usually 5% by weight, preferably 10% by weight, more preferably at least 15% by weight, still more preferably at least 20% by weight of PUFAs and up to 50% by weight, usually up to 45% by weight, commonly up to 40%, normally up to 35% by weight of PUFAs with respect to the algal extract. Advantageously, the algal extract of the invention comprises between 1% by weight and 40% by weight, typically between 5% and 35% by weight of PUFAs with respect to the algal extract of the invention. Said PUFAs can be present in the algal extract of the invention in a mix of free PUFAs, and/or in a mix of mono-, di- or triglycerides (wherein the fatty acid is a PUFA), and/or in a mix of esterified PUFAs; preferably a portion of said PUFAs is present as free PUFAs in the algal extract of the invention.

In further embodiments, a portion of the fatty acids are a combination of MUFAs and PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and 40% by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention. In a particular embodiment, said combination of MUFAs and PUFAs comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis- 5,8,11,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cz^'s-4,7,10,13, 16,19- docosahexanoic acid [DHA] (C22:6 n3). Said MUFAs and PUFAs can be present in the algal extract of the invention as free MUFAs and/or PUFAs, and/or in any esterified form thereof.

The composition of the algal extract of the invention depends on several factors, for example, on the microalgae used as starting material, the solvent used in the extraction step, as well as on the treatment (chemical or enzymatic) applied on the product resulting from step a). Thus, if said product is subjected to the alkaline chemical treatment the algal extract of the invention may contain in addition to fucoxanthin and fucoxanthinol, amaurociaxanthin A and/or isofucoxanthinol, together with other microalgal components such as diadinoxanthin, diazoxanthin, β-carotene, chlorophylls, lipids, such as fatty acids and PUFAs, depending on the solvent used in the production of the extract. However, if the product resulting from step a) is subjected to the enzymatic hydrolysis the algal extract of the invention does not contain amaurociaxanthin A or iso fucoxanthinol, but it contains fucoxanthin and fucoxanthinol, together with, optionally, other microalgal components such as diadinoxanthin, diazoxanthin, β-carotene, chlorophylls, polyphenols, phytosterols, lipids, such as fatty acids and PUFAs, depending on the solvent used in the production of the extract.

The invention also contemplates the possibility of subjecting the product resulting from step a) to a suitable preparation treatment in order to prepare fucoxanthin for the alkaline chemical treatment or enzymatic hydrolysis to be performed in step b) prior to perform step b). This option will be described below in connection with the process of the invention.

Process for producing the algal extract of the invention

In another aspect, the invention relates to a process, hereinafter referred to as the "process of the invention", for producing an algal extract comprising fucoxanthin and fucoxanthinol, together with other microalgal components, particularly fatty acids (i.e., the algal extract of the invention), which comprises: a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; and b) reacting fucoxanthin previously obtained with: b.l) a base under conditions for hydrolysis of at least a portion of fucoxanthin to fucoxanthinol; or, alternatively, with b.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting at least a portion of fucoxanthin to fucoxanthinol. The above mentioned algal extract, as it has been previously mentioned, is a product obtained from microalgae.

The term "microalga" has been previously defined in connection with the algal extract of the invention and it is incorporated herein by reference. Briefly, as used herein, the term "microalga" includes a large and diverse group of simple, typically autotrophic unicellular organisms, i.e., microscopic algae, typically found in freshwater and marine systems. According to the invention, the microalga must produce fucoxanthin, either naturally or artificially (i.e., as a result of a process for transforming non-fucoxanthin producing algae into fucoxanthin-producing algae). Consequently, any fucoxanthin producing microalga can be used in the process of the invention. In a particular embodiment, the fucoxanthin producing microalga is a microalga belonging to the phylum Heterokontophyta (i.e., unicellular hetrerokonts), Haptophyta or Dinophyta.

In a more particular embodiment, the fucoxanthin producing microalga is a microalga belonging to a class selected from the group consisting of Rapidophyceae, Bacillariophyceae (Diatomeas), Crysophyceae, Pavlophyceae, and Prymnesiophyceae.

Illustrative, non-limitative, examples of fucoxanthin producing algae include Isochrysis aff. galbana, Thalassiosira pseudonana, Navicula incerta, Pavlova lutheri, Ochromonas sp., Phaeodactylum tricornutum, Odontella aurita, Isochrysis galbana, Isochrysis sp., Pavlova gyrans, Skeletonema costatus, Chaetoceros gracilis, Chaetoceros calcitrans, Prymnesium parvum, Nitzschia heufleriana, Nitzschia sp., Dinobryon sp., Synura uvella, Synura petersenii, Chrysochromulina brevifikum, Ochrosphaera neapolitana, Cylindrotheca fusiformis, Chromulina neblosa, Mallomonas asmundae, Ochromonas danica, Ochromonas spherocystis, and Emiliania huxleyi, preferably, /. off. galbana, T. pseudonana, N. incerta, P. lutheri, and Ochromonas sp.

The term "biomass" has also been previously defined in connection with the algal extract of the invention and it is incorporated herein by reference; briefly, it includes biological material comprising, or deriving from, living or recently living organisms. By extension, the term includes not only the biological material or organic matter which constitutes an organism, but also the biological material or organic matter generated in a biological process, spontaneous or not spontaneous (i.e., provoked). Thus, the expression "fucoxanthin producing microalgae biomass" refers to biomass comprising fucoxanthin producing microalgae.

According to step a) of the process of the invention, the fucoxanthin producing microalgae biomass is cultured under conditions for producing (i.e., that allow the production of) fucoxanthin, thereby obtaining an algal extract comprising fucoxanthin and fucoxanthinol, and other algal components from the fucoxanthin producing microalgae biomass.. The conditions for culturing fucoxanthin producing algae can vary widely depending, among other factors, on the specific fucoxanthin producing microalga elected for carrying out the process of the invention. In a particular embodiment, the fucoxanthin producing microalga is a photoautotroph organism (i.e., an organism capable of synthesizing its own food from inorganic substances using light as energy source and is capable of using carbon dioxide as its principal source of carbon). Thus, according to this particular embodiment, the fucoxanthin producing microalgae biomass can be obtained by photoautotrophic growth. In another particular embodiment, the fucoxanthin producing microalga is a mixotroph organism (i.e., an organism that can use a mix of different sources of energy and carbon). Thus, according to this particular embodiment, the fucoxanthin producing microalgae biomass can be obtained by mixotrophic growth.

For use within the context of the present invention, the fucoxanthin producing microalga can be collected from the natural medium or can be cultured in a photobioreactor. In a particular embodiment, the fucoxanthin producing microalga is cultured in a photobioreactor in a suitable medium, under a suitable luminous intensity, at a suitable temperature. Practically any medium suitable for growing microalgae can be used; nevertheless, illustrative, non-limitative examples of said media include: f/2 (Guillard and ryther 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8:229-39), Erds (Tompkins et al., 1995. Culture Collection of Algae and Protozoa. Catalog of Strains. Ambleside, UK, 204 pp.) or K/2 (Keller et al., 1987 Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23:633-8). The luminous intensity can vary widely, nevertheless, in a particular embodiment, the luminous intensity is comprised between 25 and 150 μιηοΐ fotons m^"2 s^"1. The temperature can vary usually between about 17°C and about 30°C. The culture can be performed in the absence of aeration or with aeration. In a particular embodiment, the culture is carried out without aeration. In another embodiment, the culture is performed with aeration, for example, with air or with up to 5% C0₂ enriched air, at a rate of delivery comprised between more than 0 and 1 L/min.

The fucoxanthin contained in the product resulting from step a) of the process of the invention is subjected to an alkaline chemical treatment or to an enzymatic hydrolysis [step b)]. If necessary, the product resulting from step a) can be subjected to a treatment, for example, cell lysis and extractive method, before applying the alkaline chemical or enzymatic hydrolysis. It will depend on the nature of the microalgae biomass used. Lysis of microalgae cells can be performed by conventional methods for lysing said type of cells knwon by the skilled person in the art. Thus, in step b.l) of the process of the invention, fucoxanthin produced as in step a) is subjected to an alkaline treatment (saponification) under conditions for (i.e., that allow) the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol. The skilled person in the art will understand that at least a portion of other compounds which are present in the extract, specifically fatty acids, for example in the form of mono-, di- or triglycerides or esterified fatty acids, will also be hydrolysed.

The base can be an inorganic base or an organic base, as mentioned above in connection with the algal extract of the invention. Illustrative, non-limitative, examples of inorganic bases include ammmonia (N¾), alkaline metals or alkaline earth metals hydroxides or carbonates, such as, for example, KOH, Na₂C03, etc. Illustrative, non- limitative, examples of organic bases include N-heterocyclic compounds [e.g., benzimidazol, imidazole, piperidine, pyridine, etc., and derivatives thereof, such as 4- (dimethylaminopyridine), di-tert-butylpyridine, 2,6-lutidine, etc., 1,5-diazabicyclo [4.3.0]non-5-ene, l,8-diazabicyclo[5.4.0]undec-7-ene, etc.], amines [e.g., methylamine, ethylamine, dimethylamine, diethylamine, diisopropylamine, N,N- diisopropylmethylamine, N-ethyldiisopropylamine, 2-(2chloro-6-fluorophenyl) ethylamine, choline, etc.], organolithiums [e.g., methyllithium, n-butyllithium, sec- butyllithium, tert-butyllithium, hexyllithium, etc.], Grignard reagents [e.g., butylmagnesium chloride, sec-butylmagnesium chloride, methylmagnesium bromide, ethylmagnesium bromide, 1,1 -dimethylpropylmagnesium chloride, 2,2- dimethylpropylmagnesium chloride, methylmagnesium bromide, hexylmagnesium chloride, etc.], tetraalkylammonium hydroxides [e.g., tetramethylammonium hydroxide, tetramethylammonium hydroxide, diethyldimethylammonium hydroxide, benzyltrimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, etc.], phosphonium hydroxides [e.g., 2,8,9-trimethyl-2,5,8,9-tetraaza-l-phosphabicyclo [3.3.3]undecane, etc.], metal alkoxides [e.g., lithium methoxide, lithium ethoxide, lithium tert-butoxide, lithium isopropoxide, barium tert-butoxide, potassium tert- butoxide, magnesium di-tert-butoxide, etc.], metal amides [e.g., lithium diethylamide, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, lithium dicyclohexylamide, lithium dimethylamide, etc.], metal silanoates [e.g., lithium trimethylsilanoate, etc.], phosphazene bases [e.g., phosphazene base Pi-t-Bu, etc.]. In a preferred embodiment, the inorganic base is KOH or NH₄OH, and the organic base is lithium tert-butoxide.

The base can be added to the fucoxanthin containing medium at any appropriate molar ratio with respect to fucoxanthin. In any case, the base will be added in a ratio with respect to fucoxanthin that allows the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol. Usually the base is added at a ratio higher than the corresponding stoichiometric ratio because a portion of the base can react with other co-extracted algal components such as, for example, lipids (e.g., mono-, di- or triglycerides or fatty acids esters) or chlorophylls (this also allows the hydrolysis of at least a portion of mono-, di- or tiglycerides or esterified fatty acids present in the fucoxanthin producing microalgae biomass to fatty acids). Thus, the presence of said components should be taken into account when preparing the base to be added to the reaction medium. The reaction between fucoxanthin and the base is carried out in the dark, in a suitable reaction medium comprising an organic solvent, at a suitable temperature, and for a suitable period of time (reaction time) so that at least a portion of fucoxanthin is converted to fucoxanthinol. Illustrative, non-limitative, examples of organic solvents include alcohols (e.g., methanol, ethanol, etc.), ethers (e.g., tetrahydrofuran (THF), etc.), ketones (e.g., acetone, etc.), etc. The temperature can vary usually between about 4°C and about 30°C. The period of time must be sufficient to guarantee that a portion of fucoxanthin is converted to fucoxanthinol; although it can vary within a broad range, in a particular embodiment, the reaction time between fucoxanthin and the base is comprised between about 2 minutes and 24 hours, preferably between 2 minutes and 12 hours, more preferably between 5 minutes and 6 hours, still more preferably between 10 minutes and 3 hours, usually between about 10 minutes and about 120 minutes. The reaction can be carried out under inert atmosphere or not. Thus, in a particular embodiment, the reaction is carried out under inert atmosphere. In another particular embodiment, the reaction is not carried out under inert atmosphere.

As it will be evident for the skilled person in the art, varying the hydrolysis conditions, it is possible to regulate or control the conversion of fucoxanthin to fucoxanthinol and co-hydrolisis of other compounds that are present in the extract such as, for example, fatty acids in the form of mono-, di- or tri-glycerides or esterified fatty acids. In fact, varying the amount and type of base, reaction medium, reaction time and reaction temperature it is possible to control (e.g., increase) the conversion of fucoxanthin to fucoxanthinol. In all cases, the reaction is performed in the dark. Further, depending on the base and the reaction time, the reaction can be performed (or not) under inert atmosphere. By illustrative, when fucoxanthin is hydrolyzed using 4 g Γ¹ of KOH in ethanol, at 25°C during 10 minutes under a non-inert atmosphere, the final algal extract may contain 3% by weight of fucoxanthin, 0.3% by weight of fucoxanthinol, 1.3% by weight of amarouciaxanthin A and 1% by weight of iso fucoxanthinol. On the other hand, when fucoxanthin is hydrolyzed using 6 g Γ¹ of KOH in methanol, at 0°C during 60 minutes under inert atmosphere, the final algal extract may contain 5% by weight of fucoxanthin, 1.5% by weight of fucoxanthinol, 2% by weight of amarouciaxanthin A and 1% by weight of iso fucoxanthinol. In addition, this chemical treatment will also hydrolyze at least a portion of the mono-, di-, or triglycerides and esterified fatty acids which are present in the microalga and, as a consequence thereof, free fatty acids will be released.

In step b.2) of the process of the invention, fucoxanthin produced as in step a) is subjected to enzymatic hydrolysis with an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting (i.e., that allow the conversion of) at least a portion of fucoxanthin to fucoxanthinol, thereby fucoxanthinol is obtained by enzymatic hydrolysis of fucoxanthin. The skilled person in the art will understand that at least a portion of other compounds which are present in the extract, specifically fatty acids, for example in the form of mono-, di- or triglycerides or esterified fatty acids, will also be enzymatically hydrolysed.

Enzymes that catalyze the conversion of fucoxanthin to fucoxanthinol include hydrolases (i.e., enzymes that act on ester bonds) included within class EC 3.1 class. Illustrative, non-limitative, examples of enzymes suitable or use in the process of the invention include lipases and cholesterol esterase. A lipase is an enzyme that catalyzes the hydrolysis of fats (lipids) and is considered as a subclass of esterases. A cholesterol esterase is an enzyme that catalyzes the hydrolysis of sterol esters into their component sterols and fatty acids. Preferred enzymes for use in the process of the invention include a pancreas lipase, such as a porcine pancreas lipase, or a cholesterol esterase. As it will be evident for the skilled person in the art, the enzymes that catalyze the conversion of fucoxanthin to fucoxanthinol, namely, lipase and cholesterol esterase, will also hydrolyze fatty acids in the form of mono-, di- or triglycerides or esterified fatty acids thus releasing fatty acids. Once finished the alkaline chemical treatment or enzymatic hydrolysis performed in step b), an algal extract comprising fucoxanthin and fucoxanthinol and other algal components, is obtained. Among these other algal components, free fatty acids are usually present. These free fatty acids derive from the microalgae used for producing the algal extract of the invention. Usually said fatty acids will be present in said microalgae in the form of mono-, di- or triglycerides or other esterified forms and due to the chemical or enzymatic treatment applied thereon said mono-, di- or triglycerides or said other esterified forms will be total or partially hydrolyzed and the corresponding free fatty acids will be released. Thus, the algal extract of the invention contains all or a portion of the fatty acids present in the microalgae used for producing said algal extract of the invention in a form (as free fatty acids) other than the form in which they are present in said naturally occurring microalgae.

If desired, the algal extract comprising fucoxanthin and fucoxanthinol obtained is separated (removed) by conventional techniques including, for example, decantation after formation of a biphasic system, and, if desired, dried by rotoevaporation to dryness in order to obtain a substantially dry algal extract comprising fucoxanthin and fucoxanthinol, such as an algal extract of the invention, or concentrated by rotoevaporation and addition of a matrix oil, for example a vegetal (vegetable) oil (e.g., corn oil, etc.).

Thus, in a particular embodiment, the product resulting from the alkaline chemical treatment or enzymatic hydrolysis performed in step b) is treated with an aqueous saline solution in order to form a biphasic system comprising an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol and fatty acids in different forms (e.g., free fatty acids, mono-, di- or triglycerides and other fatty acids esters), together with other compounds in the extract. Virtually any non-toxic, water-soluble salt, can be used, preferably a non-toxic, water-soluble salt, that increases polarity of the aqueous phase, e.g., an aqueous solution comprising NaCl, KC1, NaHPC"3, etc. The organic phase is separated from the aqueous phase by any suitable technique, for example, decantation, centrifugation, etc., and, if desired, dried by rotoevaporation to dryness, in order to obtain a substantially dry algal extract comprising fucoxanthin and fucoxanthinol, such as an algal extract of the invention.

In a particular embodiment, the algal extract of the invention obtained according to the process of the invention comprises fucoxanthin and fucoxanthinol, and, optionally, amarouciaxanthin A and/or isofucoxanthinol, together with other algal components.

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

- 0% to 90% by weight of amarouciaxanthin A;

- 0%) to 10%) by weight of isofucoxanthinol; and

1%) to 99.998%) by weight of other algal components.

In another particular embodiment, the algal extract of the invention so obtained comprises one or more fatty acids within the other algal components fraction, in an amount such that said fatty acids present in the algal extract of the invention are at a concentration comprised between about 1% and about 45% by weight with respect to the total weight of the algal extract of the invention, preferably between 2% and 40% by weight, more preferably between 10% and 40% by weight, still more preferably between 15% and 40%> by weight, even more preferably between 15% and 35% by weight. The fatty acids can be present in the algal extract of the invention in a mix of free fatty acids, and/or in a mix of mono-, di- or triglycerides, and/or in a mix of esterified fatty acids; nevertheless, in preferred embodiments, the algal extract of the invention comprises a portion of the fatty acids as free fatty acids. Thus, in particular embodiments, the algal extract of the invention comprises at least 1% by weight, usually, 5% by weight, normally 10% by weight, preferably at least 15% by weight, more preferably at least 20% by weight, still more preferably, at least 25% by weight of free fatty acids with respect to the algal extract, and up to 50%> by weight, usually up to 45%) by weight, commonly up to 40%>, normally up to 35% by weight of free fatty acids with respect to the algal extract of the invention. Advantageously, the algal extract of the invention comprises between 1% and 50%> by weight, typically between 5% and 45%) by weight, usually between 10% and 40% by weight of free fatty acids with respect to the algal extract of the invention.

In preferred embodiments, a portion of the fatty acids are PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises at least 1% by weight, usually 5% by weight, normally 10% by weight, preferably at least 15% by weight, more preferably at least 20% by weight, still more preferably at least 25% by weight of PUFAs and up to 50% by weight, usually up to 45% by weight, commonly up to 40%, normally up to 35% by weight of PUFAs with respect to the algal extract. Advantageously, the algal extract of the invention comprises between 1% by weight and 40%) by weight, typically between 5% and 35% by weight of PUFAs with respect to the algal extract of the invention. Said PUFAs can be present in the algal extract of the invention in a mix of free PUFAs, and/or in a mix of mono-, di- or triglycerides (wherein the fatty acid is a PUFA), and/or in a mix of esterified PUFAs; preferably a portion of said PUFAs is present as free PUFAs in the algal extract of the invention. Thus, advantageously, in some embodiments, the algal extract of the invention comprises between 1% and 40% by weight, typically between 5% and 35% by weight, usually between 10% and 30% by weight, of free PUFAs with respect to the algal extract of the invention. In further embodiments, a portion of the fatty acids are a combination of MUFAs and PUFAs; thus, in further specific embodiments, the algal extract of the invention comprises between 1% and 50% by weight, typically between 5% and 40% by weight, usually between 10% and 35% by weight, of a combination of MUFAs and PUFAs with respect to the algal extract of the invention. In a particular embodiment, said combination of MUFAs and PUFAs comprises palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cis- 5,8,11,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cz^'s-4,7,10,13, 16,19- docosahexanoic acid [DHA] (C22:6 n3). Said MUFAs and PUFAs can be present in the algal extract of the invention as free MUFAs and/or PUFAs, and/or in any esterified form thereof. Thus, in some embodiments, the algal extract of the invention comprises between 1%> and 40%> by weight, typically between 3%> and 25% by weight, usually between 5% and 15% by weight, of free MUFAs with respect to the algal extract of the invention whereas the amount of free PUFAs with respect to the algal extract of the invention has been previously mentioned. Other algal extracts included within the scope of the algal extract of invention obtainable according to the process of the invention include the algal extracts previously disclosed in connection with the algal extract of the invention and are included herein by reference. The composition of the algal extract of the invention depends on several factors, for example, on the algae used as starting material, the solvent used in the extraction step, as well as on the treatment (chemical or enzymatic) applied on the product resulting from step a) of the process of the invention. Thus, if said product is subjected to the alkaline chemical treatment the algal extract of the invention may contain in addition to fucoxanthin and fucoxanthinol, amaurociaxanthin A and/or isofucoxanthinol, together with other algal compounds such as diadinoxanthin, diazoxanthin, β-carotene, chlorophylls, lipids, such as fatty acids and PUFAs, depending on the solvent used in the production of the extract. However, if the product resulting from step a) is subjected to the enzymatic hydrolysis the algal extract of the invention does not contain amaurociaxanthin A or isofucoxanthinol, but it contains fucoxanthin and fucoxanthinol, together with, optionally, other algal components such as diadinoxanthin, diazoxanthin, β-carotene, chlorophylls, polyphenols, phytosterols, lipids, such as fatty acids and PUFAs, depending on the solvent used in the production of the extract

The invention also contemplates the possibility of subjecting the product resulting from step a) of the process of the invention to a suitable preparation treatment in order to prepare fucoxanthin for the alkaline chemical treatment or enzymatic hydrolysis to be performed in step b) prior to perform step b) of the process of the invention.

Thus, in a particular embodiment, the algal biomass that contains fucoxanthin (among other algal components) obtained by culturing fucoxanthin producing algae biomass under conditions that allow the production of fucoxanthin [i.e., the product resulting from step a) of the process of the invention] is removed from the culture medium, optionally dried, and subjected to a treatment, hereinafter referred to as "preparation treatment" comprising: i) contacting said removed algal biomass containing fucoxanthin with an organic solvent in order to obtain an organic extract comprising fucoxanthin; and, if desired, ii) drying said organic extract comprising fucoxanthin from step i) to obtain a residue comprising fucoxanthin; and iii) dispersing (suspending) said residue comprising fucoxanthin from step ii) in an organic solvent.

The algal biomass that contains fucoxanthin (among other algal components) obtained by culturing fucoxanthin producing algae biomass under conditions that allow the production of fucoxanthin can be removed by conventional means and techniques including, for example, filtration, centrifugation, fiocculation or decantation. Further, if desired, the removed algal biomass containing fucoxanthin can be dried by freeze- drying, also known as lyophilisation or cryodesiccation or dried during 24 hours under vacuum at 50°C. The optionally dried algal biomass containing fucoxanthin is then contacted with an organic solvent in order to obtain an organic extract comprising fucoxanthin [step i)]. Practically any organic solvent in which fucoxanthin is at least partially soluble can be used; illustrative, non-limitative, examples of said organic solvents include alcohols (e.g., methanol, ethanol, etc.), ethers (e.g., diethyl ether, THF, etc.), etc. The organic extract comprising fucoxanthin is then dried to obtain a residue comprising fucoxanthin [step ii)] by rotoevaporation, and the resulting residue is dispersed in an organic solvent wherein fucoxanthin is at least partially soluble such as, for example, an alcohol (e.g., methanol, ethanol, etc.), an ether (e.g., diethyl ether, THF, etc.), etc., thus rendering an organic suspension comprising fucoxanthin. The resulting product can be subjected to the alkaline treatment or enzymatic hydrolysis as defined in step b) of the process of the invention.

In another particular embodiment, the algal biomass that contains fucoxanthin (among other algal components) obtained by culturing fucoxanthin producing algae biomass under conditions that allow the production of fucoxanthin [i.e., the product resulting from step a) of the process of the invention] is not removed from the culture medium. This option can be followed when the treatment selected comprises the alkaline chemical treatment of fucoxanthin. Thus, according to this embodiment, the appropriate reagents and solvents for performing said treatment are added to the culture medium. In another particular embodiment, the algal biomass that contains fucoxanthin obtained by culturing fucoxanthin producing algae biomass under conditions that allow the production of fucoxanthin [i.e., the product resulting from step a) of the process of the invention] is removed from the culture medium. This option is particularly useful when the treatment selected comprises the enzymatic hydrolysis of fucoxanthin (wherein the enzymatic hydrolysis is carried out in the resulting extract) or when the treatment selected comprises the alkaline chemical treatment of fucoxanthin. Thus, according to this embodiment, the appropriate enzymes, or alternatively alkaline reagents, for performing the corresponding treatments are added to the culture medium. In a particular embodiment of the process of the invention, the invention contemplates a process for producing an algal extract comprising fucoxanthin and fucoxanthinol, and, optionally, amarouciaxanthin A and/or isofucoxanthinol, together with other algal components (i.e., the algal extract of the invention), hereinafter referred to as the "process ΓΠ of the invention", which comprises: a) culturing fucoxanthin producing algae biomass under conditions for producing (that allow the production of) fucoxanthin; b) removing the algal biomass comprising fucoxanthin resulting from step a); c) contacting the algal biomass containing fucoxanthin resulting from step b) with an organic solvent under conditions for (that allow) obtaining an organic extract comprising fucoxanthin; and, if desired, drying said organic extract comprising fucoxanthin to obtain a residue comprising fucoxanthin; and dispersing said residue comprising fucoxanthin in an organic solvent; d) contacting the product resulting from step c) with fucoxanthin with: d.l) a base under conditions for (that allow) the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol; or, alternatively, with d.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting (that allow the conversion of) at least a portion of fucoxanthin to fucoxanthinol; e) adding an aqueous saline solution to the product resulting from step d) under conditions for forming (that allow the formation of) an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol; f) removing and, if desired, drying said organic phase comprising fucoxanthin and fucoxanthinol, to render an extract comprising fucoxanthin and fucoxanthinol. The particulars of the fucoxanthin producing algae biomass to be used in the process [1] of the invention have been previously described in connection with the process of the invention and are incorporated herein by reference. According to step b) of the process [1] of the invention, the algal biomass comprising fucoxanthin resulting from step a) is removed, thus obtaining a removed algal biomass comprising fucoxanthin, by conventional techniques including, for example, filtration, floculation or centrifugation; if desired, the resulting product can be dried by, for example, freeze-drying or during 24 hours under vacuum at 50°C.

Step c) of the process [1] of the invention corresponds to the "preparation treatment" whose particulars have been previously described in connection with the process of the invention and are incorporated herein by reference. Step d) of the process [1] of the invention corresponds to step b) of the process of the invention whose particulars have been previously described in connection with the process of the invention and are incorporated herein by reference.

According to step e) of the process [1] of the invention, an aqueous saline solution is added to the product resulting from step d) under conditions that allow the formation of an aqueous phase and an organic phase, thereby forming an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol. Virtually any non-toxic, water-soluble salt, can be used, preferably a non-toxic, water- soluble salt, that increases polarity of the aqueous phase, e.g., an aqueous solution comprising NaCl, KC1, NaHP0₃, etc.

The product resulting from step e) of the process [1] of the invention, the organic phase comprising fucoxanthin and fucoxanthinol is separated from the aqueous phase, thus obtaining a separated orgabnic phase, by conventional techniques including, for example, centrifugation, decantation, and the organic phase is dried by rotoevaporation to dryness in order to render an algal extract comprising fucoxanthin and fucoxanthinol, such as an algal extract of the invention. In another particular embodiment of the process of the invention, the invention contemplates a process for producing an algal extract comprising at least 37% by weight of fucoxanthin and 25% by weight of fucoxanthinol, and, optionally, amarouciaxanthin A and/or isofucoxanthinol, together with other algal components (i.e., an algal extract of the invention), hereinafter referred to as the "process [2] of the invention", which comprises: a) culturing a fucoxanthin producing microalgal under conditions for producing (that allow the production of) fucoxanthin; b) removing, and optionally drying, said microalgal biomass comprising fucoxanthin resulting from step a); c) contacting said microalgal biomass comprising fucoxanthin from step b) with ethanol under conditions for (that allow) obtaining an ethanol extract comprising fucoxanthin; d) drying said ethanol extract comprising fucoxanthin from step c) to obtain a solid residue comprising fucoxanthin; e) dispersing said solid residue comprising fucoxanthin from step d) in diethyl ether; f) contacting the suspension resulting from step e) with a base in a medium comprising an alcohol; g) adding an aqueous sodium chloride solution to the product resulting from step f) under conditions that allow the formation of an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol; h) removing said organic phase comprising fucoxanthin and fucoxanthinol from step g); i) washing said removed organic phase comprising fucoxanthin and fucoxanthinole; and j) drying said organic phase comprising fucoxanthin and fucoxanthinol to render an extract comprising at least 37% by weight of fucoxanthin and 25% by weight of fucoxanthinol.

The particulars of the fucoxanthin producing microalgae biomass to be used in the process [2] of the invention have been previously described in connection with the process of the invention and are incorporated herein by reference. In a particular embodiment, said fucoxanthin producing microalga is a microalga belonging to the phylum Heterokontophyta, Haptophyta or Dinophyta, such as a microalga selected from the group consisting of Isochrysis aff. galbana, Thalassiosira pseudonana, Navicula incerta, Pavlova lutheri, Ochromonas sp., Phaeodactylum tricornutum, Odontella aurita, Isochrysis galbana, Isochrysis sp., Pavlova gyrans, Skeletonema costatus, Chaetoceros gracilis, Chaetoceros calcitrans, Prymnesium parvum, Nitzschia heufleriana, Nitzschia sp., Dinobryon sp., Synura uvella, Synura petersenii, Chrysochromulina brevifikum, Ochrosphaera neapolitana, Cylindrotheca fusiformis, Chromulina neblosa, Mallomonas asmundae, Ochromonas danica, Ochromonas spherocystis, and Emiliania huxleyi, preferably, /. aff. galbana, T. pseudonana, N. incerta, P. lutheri, and Ochromonas sp.

In a particular embodiment, the fucoxanthin producing microalga is cultured in a photobioreactor in a suitable medium, such as f/2, under a luminous intensity comprised between 25 and 150 μιηοΐ fotons m^"2 s^"1, at a temperature between about 17°C and about 30°C, optionally with aeration. In a particular embodiment, the culture is carried out with aeration, for example, with air or with up to 5% C0₂ enriched air, at a rate of delivery comprised between more than 0 and 1 L/min.

According to step b) of the process [2] of the invention, the microalgal biomass comprising fucoxanthin resulting from step a) is removed, thereby obtaining a removed algal biomass comprising fucoxanthin, by conventional techniques including, for example, filtration, floculation or centrifugation, and, optionally, dried by freeze-drying or during 24 hours under vacuum at 50°C.

The dry microalgal biomass resulting from step c) is contacted with ethanol in step c) of the process [2] of the invention under conditions that allow (i.e., for) obtaining an ethanol extract comprising fucoxanthin, thereby obtaining an ethanol extract comprising fucoxanthin. Said ethanol extract comprising fucoxanthin is dried [step d) of the process [2] of the invention], thereby obtaining a residue comprising fucoxanthin, by conventional methods, for example by rotoevaporation to dryness in order to obtain a residue comprising fucoxanthin. The residue comprising fucoxanthin is dispersed [step e) of the process [2] of the invention] in an ether, such as diethyl ether, thus obtaining a diethyl ether extract comprising fucoxanthin.

The diethyl ether extract comprising fucoxanthin resulting from step d) is subjected to an alkaline hydrolysis [step f)] by treatment con a base, such as an alkaline hydroxide, preferably NaOH or KOH, in a medium comprising an organic solvent, such as an alcohol, preferably methanol, in inert atmosphere, at dark, at a temperature comprised between about 0° and about 60°C, thereby provoking the conversion of at least a portion of fucoxanthin to fucoxanthinol.

According to step g) of the process [2] of the invention, an aqueous salt solution comprising sodium chloride is added to the product resulting from step f) comprising fucoxanthin and fucoxanthinol under conditions that allow the formation of (i.e., for forming) an aqueous phase and an organic phase, thereby forming an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol. The organic phase comprising fucoxanthin and fucoxanthinol is separated [step h)] from the aqueous phase, thereby obtaining a removed organic phase, by conventional means, for example by decantation, and the organic phase is washed with an aqueous sodium chloride solution [step i)] thereby obtaining a washed organic phase, and dried [step j)], thereby obtaining a dry algal extract, by conventional means, for example by rotoevaporation to dryness to render an algal extract comprising at least 37% by weight of fucoxanthin and 25% by weight of fucoxanthinol. Applications of the algal extract of the invention

The algal extract comprising fucoxanthin and fucoxanthinol, together with other algal components, especially fatty acids, can be used in a lot of applications. Some applications relate to its use as anti-inflammatory agent whereas some other applications relate to the uses of the components thereof; by illustrative, it is well- known that fucoxanthin and fucoxanthinol can be used as anti-obesity agents since they have a fat burner effect, as antiangiogenic agents, as anti-neoplastic agents, as neovascularization inhibitors, as adiponectin production accelerators, as cholesterol lowering agents, as antihypertensive agents, as antiallergic agents, etc., as well as in the treatment of skin pigmentation, metabolic syndrome management, and in the treatment and/or prevention of virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma, hyperuricemia, osteoporosis, depression, etc. Thus, said algal extract can be used in a lot of cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical applications.

Therefore, in a particular embodiment, the algal extract of the invention can be used as an adiponectin production accelerator (e.g., for prevention and/or treatment of obesity, siabetes, coronary arterial disease, hypertension, etc.), an antiallergic agent, an antiangiogenic agent (e.g., for prevention and/or treatment of an angiogenic-mediated disease), an antibacterial agent (e.g., for prevention and/or treatment of a bacterial disease), an anticancer agent (e.g., for prevention and/or treatment of cancer, etc.), an antidiabetic agent (e.g., for prevention and/or treatment of diabetes), an antifungal agent (e.g., for prevention and/or treatment of a fungal disease), an antiinflamatory agent (e.g., for prevention and/or treatment of an inflammation-mediated disease), an antimalarial agent (e.g., for prevention and/or treatment of an malaria), an antineoplastic agent (e.g., for prevention and/or treatment of an neoplastic-mediated disease), an antiobesity agent (e.g., for prevention and/or treatment of obesity, namely morbid obesity, or for controlling obesity in non-therapeutical applications), an antioxidant agent, an antihypertensive agent (e.g., for prevention and/or treatment of hypertension), a bone protective agent (e.g., for prevention and/or treatment of arthritis, osteoporosis, etc.), a cerebrovascular protective agent (e.g., for prevention and/or treatment of Alzheimer Disease, Parkinson, etc.), a cholesterol lowering agent (e.g., for prevention and/or treatment of dyslipidemias), an hepatoprotective agent (e.g., for prevention and/or treatment of cirrhosis, liver cancer, etc.), an ocular protective agent, a neovascularization inhibitor (e.g., for prevention and/or treatment of an neovascularization-mediated disease), a skin-protective agent (e.g., for protecting skin against UV-B radiation photodamage and UV-B radiation induced skin pigmentation) as well as in the prevention and/or treatment of depression, hyperuricemia, inflammatory diseases (i.e., diseases having an inflammatory component, such as, for example, gastrointestinal inflammatory diseases (Crohn's Disease, etc.), gingivitis, joint/muscle recovery from overexertion, osteoarthritis, psoriasis, rheumatoid arthritis, etc.), metabolic syndrome, osteoporosis, skin pigmentation, virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma, etc. Thus, the algal extract of the invention can be used in a lot of cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical applications.

Therefore, in an aspect, the invention relates to the use of the algal extract of the invention as a cosmetic. In another aspect, the invention relates to the use of the algal extract of the invention as a cosmeceutical. In another aspect, the invention relates to the use of the algal extract of the invention as a food (foodstuff). In another aspect, the invention relates to the use of the algal extract of the invention as a nutraceutical. In another aspect, the invention relates to the use of the algal extract of the invention as a medicament, i.e., to the algal extract of the invention for use in medicine.

In another aspect, the invention relates to a composition, hereinafter "composition of the invention", comprising an algal extract of the invention, and a cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical acceptable vehicle. The particulars of the algal extract of the invention have already been defined above and are incorporated herein by reference. In a particular embodiment, the composition of the invention is a cosmetic or personal care composition comprising an algal extract of the invention together with a cosmetically acceptable vehicle. As used herein, the term "cosmetic composition" or "personal care composition" refers to a composition suitable for use in personal hygiene of human beings or animals, or in order to enhance the natural beauty or change the body appearance without affecting the structure or functions of the human or animal body, comprising one or more products providing such effects. If desired, the cosmetic composition provided by the invention can contain, in addition to the algal extract of the invention, one or more cosmetic products, i.e., substances or mixtures intended to be placed in contact with the external parts of the human or animal body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the buccal mucosa, for the exclusive or main purpose of cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odors. Illustrative examples of cosmetic products include the products contained in the INCI (International Nomenclature of Cosmetic Ingredients) list. Cosmetic or personal care compositions include products such as balms, pads, pomades, creams, etc.

In a particular embodiment, the cosmetic or personal care composition provided by the present invention comprises an algal extract of the invention and an acceptable oral or topical carrier therefor.

In another particular embodiment, the cosmetic or personal care composition provided by the present invention comprises between 0.1% and 5% by weight of an algal extract of the invention.

In another particular embodiment, the cosmetic or personal care composition provided by the present invention comprises a sunscreen agent. Illustrative, non-limitative, examples of sunscreens include microfme titanium dioxide; microfme zinc oxide; boron nitride; p-aminobenzoic acids, esters and derivatives thereof; methoxycinnamate esters; benzophenones; 2-phenylbenzimidazole-5-sulfonic acid; disodium phenyl dibenzimidazole tetrasulfonate; terphthalylidene dicamphor sulfonic acid; alkyl-, diphenylacrylates; triazine sunscreens; benztriazolyl sunscreens; camphor sunscreens; organic pigment sunscreens; silicone sunscreens; and salicylate sunscreens.

In another particular embodiment, the algal extract of the invention in the cosmetic or personal care composition provided by the present invention is contained in an aqueous phase of said composition, i.e., the cosmetic or personal care composition provided by the present invention comprises an aqueous phase which comprises the algal extract of the invention. In another particular embodiment, the cosmetic or personal care composition provided by the present invention comprises an oil-in-water emulsion.

In another particular embodiment, the cosmetic or personal care composition provided by the present invention for application to the skin in the form of a cream emulsion, gel, milk, suspension, O/W emulsion, W/O emulsion, liposome foam, aqueous or emulsion lotion, spray or a waxy stick.

In another particular embodiment, the cosmetic or personal care composition provided by the present invention can be used in the prevention, amelioration or treatment of damage of mammalian skin. In another particular embodiment, the cosmetic or personal care composition provided by the present invention can be used for inhibiting one or more of the following skin conditions: inflammation, photo-ageing skin damage, uneven pigmentation, wrinkles and sagging skin. In another particular embodiment, the composition of the invention is a cosmeceutical composition comprising an algal extract of the invention together with a cosmeceutically acceptable vehicle. As used herein, the term "cosmeceutical composition" refers to a composition suitable for use in the body or animal body comprising one or more cosmeceutical products (functional cosmetics, dermoceuticals or active cosmetics), i.e., topical hybrid products with cosmetical-pharmaceutical characteristics containing active ingredients having effect on user's skin, hair and/or nails, at higher and more effective concentrations, therefore they are located in an intermediate level between cosmetic and drug. Illustrative examples of cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc. The person skilled in the art will understand that the algal extract of the invention or the compositions containing them can be part of a food or feed, or of a nutraceutical, pharmaceutical, or cosmeceutical product, which constitutes an additional aspect of the present invention. Said products can be in a liquid, semi-solid or solid form.

In another particular embodiment, the composition of the invention is a food or feed comprising an algal extract of the invention. As used herein, the term "food" is any substance or product of any nature, solid or liquid, natural or processed which due to its characteristics, applications, components, preparation and state of preservation, can usually or ideally be used for some of the following purposes: a) as normal nutrition for human beings or animals or as pleasurable foods; or b) as dietetic products, in especial cases of human or animal food. The term "feed" includes all the natural materials and finished products of any origin which, separately or conveniently mixed with one another, are suitable as animal food. A ready-to-eat food is that which does not need to be diluted by means of an aqueous solution suitable for consumption for example. In principle, the ingredients present in a ready-to-eat food are balanced and there is no need to add additional ingredients to the food to make it ready to eat, such considered by a person skilled in the art. A concentrated food is that in which one or more ingredients are present at a higher concentration than in a ready-to-eat food, therefore for use it is necessary to dilute it by means of an aqueous solution suitable for consumption for example. Non-limiting, illustrative examples of foods provided by this invention include both dairy products and derivatives, for example, fermented milks, yoghurt, kephir, curd, cheeses, butters, ice creams, milk-based desserts, etc., and non- dairy products, such as baked products, cakes and pastries, cereals, chocolates, jams, juices, other fruit derivatives, oils and margarines, prepared dishes, etc. In particular embodiment, the food comprises between 0.1% and 5% by weight of an algal extract of the invention.

In another particular embodiment, the composition of the invention is a nutraceutical composition comprising an algal extract of the invention together with a nutraceutically acceptable vehicle. As used herein, the term "nutraceutical composition" refers to a composition suitable for use in human beings or animals, comprising one or more natural products with therapeutic action which provide a health benefit or have been associated with disease prevention or reduction, for example, fucoxanthin, fucoxanthinol, etc., and it includes dietary supplements presented in a non-food matrix (e.g., capsules, powder, etc.) of a concentrated natural bioactive product usually present (or not) in the foods and which, when taken in a dose higher than that existing in those foods, exerts a favorable effect on health which is greater than effect which the normal food may have. Therefore, the term "nutraceutical composition" includes isolated or purified food products as well as additives or food supplements which are generally presented in dosage forms normally used orally, for example, capsules, tablets, sachets, drinkable phials, etc.; such products provide a physiological benefit or protection against diseases, generally against chronic diseases. If desired, the nutraceutical composition provided by the invention can contain, in addition to the algal extract of the invention, one or more nutraceuticals (products or substances associated with disease prevention or reduction), for example, flavonoids, omega-3 fatty acids, etc., and/or one or more prebiotics (non-digestible food ingredients which stimulate probiotic activity and/or growth), for example, oligofructose, pectin, inulin, galacto- oligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

In a particular embodiment, the nutraceutical composition provided by the present invention comprises an algal extract of the invention and an acceptable oral carrier therefor. In a more particular embodiment, the alga is a microalga, such as any of the microalgae mentioned in connection with the algal extract of the invention. In another particular embodiment, the nutraceutical composition provided by the present invention comprises between 0.1% and 5% by weight of an algal extract of the invention, for example, of an algal extract of the invention wherein the alga is a microalga. In another particular embodiment, the algal extract of the invention in the nutraceutical composition provided by the present invention is contained in an aqueous phase of said composition. In another particular embodiment, the nutraceutical composition provided by the present invention comprises an oil-in- water emulsion. In another particular embodiment, the composition of the invention is a pharmaceutical composition comprising an algal extract of the invention together with a pharmaceutically acceptable vehicle, especially suitable for oral, topical, rectal or vaginal administration; to that end, said composition comprises a pharmaceutically acceptable vehicle comprising one or more excipients suitable for oral administration, for example, in the form of capsule, powder, granulate, tablet (coated or non-coated), sachet, matrix, suspension, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for topical administration, for example, in the form of cream, ointment, salve, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for rectal administration, for example, in the form of suppository, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for vaginal administration, for example, in the form of bolus, suppository, etc. Information about excipients suitable for the formulation of pharmaceutical compositions intended for oral, topical, rectal or vaginal administration, as well as about the production of said pharmaceutical compositions can be found in the book "Tratado de Farmacia Galenica", by C. Fauli i Trillo, 10^th Edition, 1993, Luzan 5, S.A. de Ediciones.

As used herein, the term "subject" includes any mammal animal including human being.

In a particular embodiment, the pharmaceutical composition provided by the present invention comprises an algal extract of the invention and an acceptable oral carrier therefor. In a particular embodiment, the pharmaceutical composition provided by the present invention comprises between 0.1% and 5% by weight of an algal extract of the invention, for example, of an algal extract of the invention wherein the alga is a microalga. In another particular embodiment, the algal extract of the invention in the pharmaceutical composition provided by the present invention is contained in an aqueous phase of said composition, i.e., the pharmaceutical composition provided by the present invention comprises an aqueous phase which comprises the algal extract of the invention. In another particular embodiment, the pharmaceutical composition provided by the present invention comprises an oil-in- water emulsion. In another particular embodiment, the pharmaceutical composition provided by the present invention comprises a sunscreen agent such as any of the sunscreen agents mentioned on connection with the cosmetic composition provided by this invention.

In another particular embodiment, the algal extract of the invention in the pharmaceutical composition provided by the present invention is contained in an aqueous phase of said composition. In another particular embodiment, the pharmaceutical composition provided by the present invention comprises an oil-in- water emulsion. In another particular embodiment, the pharmaceutical composition provided by the present invention for application to the skin in the form of a cream emulsion, gel, milk, suspension, O/W emulsion, W/O emulsion, liposome foam, aqueous or emulsion lotion, spray or a waxy stick. In another particular embodiment, the pharmaceutical composition provided by the present invention can be used in the prevention, amelioration or treatment of damage of mammalian skin. In another particular embodiment, the pharmaceutical composition provided by the present invention can be used for inhibiting one or more of the following skin conditions: inflammation, photo-ageing skin damage, uneven pigmentation, wrinkles and sagging skin.

In another aspect, the invention relates to an algal extract of the invention for use as an adiponectin production accelerator, an antiangiogenic agent, an antiallergic agent, an antiangiogenic agent, an antibacterial agent, an anticancer agent, an antidiabetic agent, an antifungal agent, an antiinflamatory agent, an antimalarial agent, an antineoplastic agent, an antiobesity agent, an antioxidant agent, an antihypertensive agent, a bone protective agent, a cerebrovascular protective agent, a cholesterol lowering agent, an hepatoprotective agent, an ocular protective agent, a neovascularization inhibitor, or as a skin-protective agent; or, expressed in an alternative way, the invention also relates to the use of an algal extract of the invention as an adiponectin production accelerator, an antiangiogenic agent, an antiallergic agent, an antiangiogenic agent, an antibacterial agent, an anticancer agent, an antidiabetic agent, an antifungal agent, an antiinflamatory agent, an antimalarial agent, an antineoplastic agent, an antiobesity agent, an antioxidant agent, an antihypertensive agent, a bone protective agent, a cerebrovascular protective agent, a cholesterol lowering agent, an hepatoprotective agent, an ocular protective agent, a neovascularization inhibitor, or as a skin-protective agent (especially to protect skin against UV-B radiation photodamage and UV-B radiation induced skin pigmentation).

In another aspect, the invention relates to an algal extract of the invention for use in the prevention and/or treatment of Alzheimer Disease, arthritis, cancer, cirrhosis, coronary arterial disease, depression, diabetes, hypertension, hyperuricemia, an inflammatory disease (e.g., a gastrointestinal inflammatory disease (Crohn's Disease, etc.), gingivitis, joint/muscle recovery from overexertion, osteoarthritis, psoriasis, rheumatoid arthritis, etc.), metabolic syndrome, obesity, osteoporosis, Parkinson, skin pigmentation, virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma; or for the prevention, amelioration or treatment of damage of mammalian skin, particularly, for inhibiting a skin condition selected from the group consisting of inflammation, photo- ageing skin damage, uneven pigmentation, wrinkles, sagging skin and combinations thereof.

Alternatively, the invention also relates to the use of an algal extract of the invention in the manufacture of a pharmaceutical composition for the prevention and/or treatment of Alzheimer Disease, arthritis, cancer, cirrhosis, coronary arterial disease, depression, diabetes, hypertension, hyperuricemia, an inflammatory disease (e.g., a gastrointestinal inflammatory disease (Crohn's Disease, etc.), gingivitis, joint/muscle recovery from overexertion, osteoarthritis, psoriasis, rheumatoid arthritis, etc.), metabolic syndrome, obesity, osteoporosis, Parkinson, skin pigmentation, virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma; or for the prevention, amelioration or treatment of damage of mammalian skin, particularly, for inhibiting a skin condition selected from the group consisting of inflammation, photo-ageing skin damage, uneven pigmentation, wrinkles, sagging skin and combinations thereof. The invention also relates to a method for the treatment of a disease which comprises administering to a subject in need thereof a therapeutically effective amount of an algal extract of the invention, wherein said disease is Alzheimer Disease, arthritis, cancer, cirrhosis, coronary arterial disease, depression, diabetes, hypertension, hyperuricemia, an inflammatory disease (e.g., a gastrointestinal inflammatory disease (Crohn's Disease, etc.), gingivitis, joint/muscle recovery from overexertion, osteoarthritis, psoriasis, rheumatoid arthritis, etc.), metabolic syndrome, obesity, osteoporosis, Parkinson, skin pigmentation, virus associated malignancy such as adult T-cell leukemia and Burkitt lymphoma.

The invention also relates to a method for the treatment of damage of mammalian skin, particularly, for inhibiting a skin condition selected from the group consisting of inflammation, photo-ageing skin damage, uneven pigmentation, wrinkles, sagging skin and combinations thereof, which comprises administering to a subject in need thereof of an effective amount of an algal extract of the invention.

The particulars of the algal extract of the invention have already been defined above and are incorporated herein by reference. The following examples illustrate the invention and must not be considered as limiting same.

EXAMPLE 1

Purified fucoxanthin chemical and enzymatic derivatization (derivatization process controls)

In the chemical derivatization process, 5 mg of pure fucoxanthin (purity > 95%, Sigma- Aldrich) were dissolved in diethyl ether. One volume of methanol containing 10 g Γ¹ of potassium hydroxide was added. Saponification reaction was carried out under nitrogen atmosphere, in the dark, and at 0°C, during 60 minutes. To stop the reaction, a volume, equivalent to that of the diethyl ether extract, of water containing 100 g Γ¹ of sodium chloride was added to the mixture. Two phases were obtained. The upper phase (containing the carotenoids dissolved in diethyl ether) was recovered by decantation. This organic extract was neutralized twice by addition of two more volumes of water containing 100 g Γ¹ of sodium chloride and agitation, in order to remove traces of methanol and potassium hydroxide. Finally, diethyl ether was eliminated by rotoevaporation. This chemical derivatization process, when applied to algae extracts, was named "C-MEDPA Derivatization Process", wherein MEDPA means "(Micro)algae Extracts Derivatization Platform to Actives".

In the enzymatic derivatization approach, 5 mg of purified fucoxanthin were suspended with the same weight of taurocholic acid in aqueous phosphate buffer (PBS) pH 7 containing 50 mg of pig liver lipase per gram of pigment (100-400 units/g, Sigma- Aldrich). Alternatively, 10 units of cholesterol esterase from Pseudomonas fluorescens (Sigma- Aldrich) per gram of biomass were used. The extract was incubated at 37°C for 24 hours. Carotenoids were extracted with subsequent 1 : 1 volumes of diethyl ether, repeating the liquid-liquid extraction step until the organic phase was colorless. Extracts were combined, and diethyl ether was eliminated by rotoevaporation. This derivatization process, when applied to algae extracts, was named "E-MEDPA Derivatization Process". All extracts (both chemically and enzymatically obtained) were independently analyzed by high performance liquid chromatography (HPLC) in a device equipped with a photodiode array detector and a CI 8 column (250 mm x 4.6 mm x 5 μιη). The system was previously equilibrated with methanol (solvent A) and water (solvent B) at 1 ml/min 86% A constant flow. 20 μΐ samples were injected at 86% A during 10 min, raised to 100% A during 10 min, held 40 min. Detection wavelength was set at 450 nm.

The remaining extract from the chemical derivatization process contained up to 281 mg g^"1 of fucoxanthin, 132 mg g^"1 of fucoxanthinol, and other carotenoids derived from the saponification reaction such as amaurociaxanthin A (137 mg g^"1) or isofucoxanthinol (72 mg g^"1), with a final recovery up to 70% of the initial fucoxanthin. Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β-carotene. Results and experimental conditions are summarized in Tables 1 and 10. The remaining extract from the enzymatic (pig liver lipase) derivatization process contained up to 722 mg g^"1 of fucoxanthin and 242 mg g^"1 of fucoxanthinol, with a final recovery up to 97% of the initial fucoxanthin. Results and experimental conditions are summarized in Tables 1 and 10.

Table 1

Results and experimental conditions of chemical and enzymatic derivatization of pure

fucoxanthin

Reaction Amarouciaxanthin

Hydrolysis Solvent Fucoxanthin Fucoxanthinol Isofucoxanthinol time A

o hydrolysis - - 1000 mg g^"1 0 mg g^"1 0 mg g^"1 0 mg g ¹

Chemical Methanol 60 min 281 mg g-¹ 132 mg g ¹ 137 mg g ¹ 72 mg g ¹

(KOH)

Enzymatic PBS 24 h 722 mg g-¹ 242 mg g^"1 0 mg g^"1 0 mg g ¹

(Lipase)

Figure 1 shows an overlaid HPLC chromatogram of the initial fucoxanthin, together with the HPLC chromatograms of the reaction products resulting from the chemical derivatization process (C-MEDPA) and from the enzymatic derivatization process (E- MEDPA).

EXAMPLE 2

Haptophyte and Heterokontophyte Microalgae Extracts Derivatization A group of haptophyte and heterokontophyte strains were selected to evaluate the efficacy of the chemical derivatization process (C-MEDPA) on extracts of such microalgae genus rich in xantophylls, and specifically in fucoxanthin.

Cultures of the Isochrysis off. galbana UTEX LB2307 strain belonging to the class Prymnesiophyceae; Thalassiosira pseudonana UTEX LBFD2 and Navicula incerta UTEX 2044 belonging both to the class Bacillariophyceae; Pavlova lutheri UTEX LB1293 belonging to the class Pavlophyceae; and Ochromonas sp. UTEX LB275 strain belonging to the class Crysophyceae, were sourced from The Culture Collection of Algae at the University of Texas at Austin.

Once the cultures entered in the growth stationary phase, biomass was centrifuged and lyophilized. Initial extracts were obtained by ethanol addition to the dry biomass in 3 : 1 ratio (three parts liters of ethanol for each kg of dry biomass), repeating this step until the extract was colorless. Ethanolic extracts were filtered, in order to eliminate possible residual dead cells. Obtained extracts were rotovapored to dryness. The soft extract obtained was dissolved in the minimum possible amount of diethyl ether and processed as described in Example 1 following the C-MEDPA derivatization process but replacing the derivatization agent (10 g Γ¹ of potassium hydroxide) with 6 g Γ¹ of potassium hydroxide.

All extracts, before and after the C-MEDPA derivatization process, were analyzed by HPLC as described in Example 1. Concentration of fucoxanthin and fucoxanthin derivatives in the remaining extracts are shown in Table 2 (in mg g^"1) and Table 3 (in % of reaction products). Experimental conditions are summarized also in Table 4. As illustrative, Figure 2 shows an overlaid HPLC chromatogram of the P. lutheri extract before and after the C-MEDPA derivatization process.

Table 2

Concentration of carotenoids in microalgae extracts and in C-MEDPA derivatized microalgae extracts (mg g^"1)

Before

After derivatization

derivatization

Fuco- Amarou Iso

Species Fucoxanthin Fucoxanthin

xanthinol ciaxanthin A fucoxanthinol

I. off. galbana 13.3 2.46 2.89 3.29 1.80

T. pseudonana 9.34 0.91 1.71 2.53 5.25

P. lutheri 9.50 6.18 2.55 0.56 3.36

N. incerta 2.94 0.59 1.85 2.53 0.88

Ochromonas sp. 4.17 0.41 0.77 1.02 1.62 Table 3

Concentration of carotenoids in microalgae extracts (% of reaction products)

Before

After derivatization

derivatization

Species Fucoxanthin Fucoxanthin Fucoxanthinol Amarouciaxanthin A Isofucoxanthinol

I. off. galbana 100 23.6 27.7 31.5 17.2

T. pseudonana 100 8.71 16.4 24.3 50.5

P. lutheri 100 48.9 20.2 4.40 26.5

N. incerta 100 10.1 31.5 43.2 15.1

Ochromonas sp. 100 10.8 20.1 26.7 42.4

Table 4

Experimental conditions of the chemical derivatization process (C-MEDPA)

applied to Haptophyte and Heterokontophyte Microalgae Extracts

Species Culture Hydrolysis Solvent Reaction time

2 1 [ Erlenmeyer

I. off. galbana 6 g r¹ KOH Methanol 60 min

flask

2 1 [ Erlenmeyer

T. pseudonana 6 g r¹ KOH Methanol 60 min

flask

2 1 [ Erlenmeyer

P. lutheri 6 g r¹ KOH Methanol 60 min

flask

2 1 [ Erlenmeyer

N. incerta 6 g r¹ KOH Methanol 60 min

flask

2 1 [ Erlenmeyer

Ochromonas sp. 6 g r¹ KOH Methanol 60 min

flask

EXAMPLE 3

Scale-up of the C-MEDPA derivatization process for obtaining novel bioactive extracts from I. salbana

For the scale-up of the C-MEDPA derivatization process applied to /. galbana cultures, a specific mutant of /. galbana UTEX LB2307 strain, named GAT-6007.00.002, was selected. /. galbana GAT-6007.00.002 was obtained through a directed evolution process to high exposure to UV light of UTEX LB2307 strain cultures (as described by Bougaran et al., 2012. Enhancement of Neutral Lipid Productivity in the Microalgae Isochrysis Affinis Galbana (T-ISO) by a Mutation- Selection procedure. Biotechnol. Bioengin. 109(11):2737:45) and mutants were sorted by flow cytometry for survival and significant high xanthophills contents (as described by Toepel et al., 2004. Cytometry, 4th Edition. New Developments. Methods in Cell Biology Vol. 15. Chapter 15). GAT-6007.00.002 is a selected variation of UTEX LB2307 strain that behaves mainly as the wild-type with a significant enhancement (around 10% increase) of fucoxanthin accumulation at the tested growth conditions. Cultures of /. galbana GAT-6007.00.002 were carried out in f/2 medium (Guillard and Ryther, 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239) in 50 1 air-lift column photobioreactors, with a light intensity of 100 μιηοΐ photons m^"2 s^"1, temperature of 25°C and bubbling C0₂ enriched air at 1 1 min ¹. Once the culture entered in the stationary phase of growth, biomass was centrifuged and lyophilized. Initial extracts of fucoxanthin and C-MEDPA derivatized extracts were obtained as described in Example 2.

Both extracts, before and after the C-MEDPA derivatization process, were characterized by HPLC as described in Example 1 and chromatograms showed in Figure 3. The C-MEDPA derivatized extract contained up to 50 mg g^"1 of fucoxanthin, 15 mg g^"1 of fucoxanthinol, among other carotenoids derived from the saponification reaction such as amaurociaxanthin A (20 mg g^"1) or isofucoxanthinol (10 mg g^"1), with a final recovery up to 95% of the initial fucoxanthin. Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β- carotene. Results and experimental conditions are summarized in Table 5.

Table 5

Results and experimental conditions of the C-MEDPA derivatization process applied to 7. galbana extracts Amarou

Reaction Isofuco- Species Culture Hydrolysis Solvent Fucoxanthin Fucoxanthinol ciaxanthin

time xanthinol

A

^~L sol O ; ; ; ^~

Methanol - 100 mg g 0 mg g 0 mg g 0 mg g galbana PBR hydrolysis

· 50 1 6 g l ¹ _{l l l} lO mg g-

Methanol 60 min 50 mg g 15 mg g 20 mg g galbana PBR KOH ¹

Figure 3 shows the HPLC chromatogram of the /. galbana extract before (A) and after (B) the C-MEDPA derivatization process.

EXAMPLE 4

Scale-up of the C-MEDPA derivatization process for obtaining novel bioactive extracts from Thalassiosira pseudonana UTEX LBFD2

For the scale-up of the C-MEDPA derivatization process applied to Thalassiosira pseudonana cultures, the strain of Example 2 was chosen, i.e., T. pseudonana UTEX LBFD2. The production of microalgal biomass was carried as described in Example 3 and the C-MEDPA derivatized extract as in Example 2 but replacing the derivatization agent (6 g Γ¹ of potassium hydroxide) with 2 g Γ¹ of potassium hydroxide. Characterization of the obtained extracts was performed following the HPLC protocol described in Example 1.

The C-MEDPA derivatized extract contained 80 mg g^"1 of fucoxanthin, 7 mg g^"1 of fucoxanthinol, among other carotenoids derived from the saponification reaction such as amaurociaxanthin A (7 mg g^"1) or isofucoxanthinol (1 mg g^"1). Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β-carotene. Results and experimental conditions are summarized in Table 6.

Table 6

Results and experimental conditions of the C-MEDPA derivatization process applied to T. pseudonana extracts Amarou

Reaction Fuco- Fuco- Iso

Species Culture Hydrolysis Solvent ciaxanthin

time xanthin xanthinol fucoxanthinol

A

ί 5οϊ NO ; ; ; ^~~

Methanol - 93 mg g 0 mg g 0 mg g 0 mg g pseudonana PBR hydrolysis

T- 50 1 2 g ¹

Methanol 60 min 80 mg g 7 mg g 7 mg g 1 mg g pseudonana PBR KOH

EXAMPLE 5

Scale-up of the E-MEDPA derivatization process for obtaining novel bioactive extracts from I. salbana

A second fraction of the lyophilized biomass from /. galbana GAT-6007.00.002 obtained as described in Example 3, is processed to fucoxanthin enriched extracts by ethanol extraction according to the protocols described in Example 2 (in 3 : 1 proportion ethanol: lyophilized biomass). This first fucoxanthin enriched extract was then derivatized following the enzymatic strategy tested in Example 1 and identified as E- MEDPA derivatization process. Thus, 100 mg of taurocholic acid per gram of extract were added to the fucoxanthin enriched extract and rotoevaporated to dryness. The remaining dried extract was dissolved in aqueous phosphate buffer (PBS) pH 7 containing 6 grams of pig liver lipase (100-400 units/mg, Sigma- Aldrich) per gram of biomass, or alternatively, in another approach, in aqueous phosphate buffer (PBS) pH 7 containing 50 units of cholesterol esterase from Pseudomonas fluorescens per gram of biomass (Sigma-Aldrich), and incubated at 37°C for 24 hours. Carotenoids were extracted with subsequent 1 : 1 volumes of diethyl ether, repeating the liquid-liquid extraction step until the organic phase was colorless. Extracts were combined, and diethyl ether was eliminated by rotoevaporation. The obtained E-MEDPA derivatized extracts contained 65 mg g^"1 of fucoxanthin/35 mg g^"1 of fucoxanthinol and 13 mg g^"1 of fucoxanthin/86 mg g^"1 of fucoxanthinol, respectively, from pig liver lipase and cholesterol esterase. Other naturally occurring carotenoids, such as diadinoxanthin, diazoxanthin or β-carotene were also present in the composition at lower amounts. Results and experimental conditions are summarized in Table 7. Table 7

Results and experimental conditions of the E-MEDPA derivatization process applied to 7. galbana extracts

Amarou-

Reaction FucoFuco- Iso-

Species Culture Hydrolysis Solvent ciaxanthin

time xanthm xanthinol fucoxanthinol

A

I. 50 1 No 100 mg g-

Methanol - galbana PBR 1 0 mg g^"1 0 mg g^"1 0 mg g^"1 hydrolysis

I. 50 1

Lipase PBS 24 h 65 mg g^"1 35 mg g^"1 0 mg g^"1 0 mg g^"1 galbana PBR

I. 50 1

Esterase PBS 24 h 13 mg g^"1 86 mg g^"1 0 mg g^"1 0 mg g^"1 galbana PBR

Figure 4 shows the profiles of a cholesterol esterase enzymatically hydrolyzed fucoxanthin-fucoxanthinol extract obtained compared to an initial fucoxanthin enriched extract. EXAMPLE 6

Scale-up of the C-MEDPA derivatization process for obtaining novel bioactive extracts from I. salbana - variation (1)

A third fraction of the lyophilized biomass from /. galbana GAT-6007.00.002 (Example 5) was processed to render fucoxanthin enriched extracts by ethanol extraction according to the protocols described in Example 2 (in 3: 1 proportion ethanol: lyophilized biomass). This first fucoxanthin enriched extract was then derivatized following a variation of the chemical derivatization process tested in Example 1 and identified as the C-MEDPA derivatization process; thus, once the solid residue was dissolved in the minimum possible amount of solvent, the same volume of ethanol containing 4 g Γ¹ of potassium hydroxide was added to the ether diethylic extract. The C-MEDPA derivatization process was carried out under nitrogen atmosphere, in the dark and at 0°C, during 10 minutes. To stop the reaction, the same protocol described in Example 3 was used. Finally, diethyl ether was eliminated by rotoevaporation. The remaining derivatized extract contained 30 mg g^"1 of fucoxanthin, 3 mg g^"1 of fucoxanthinol, among other carotenoids derived from the saponification reaction such as amaurociaxanthin A (13 mg g^"1) or isofucoxanthinol (1 mg g^"1). Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β-carotene. Results and experimental conditions are summarized in Table 8.

Table 8

Results and experimental conditions of C-MEDPA derivatization process applied to 7. galbana extracts - Variation (I)

Reaction Fuco- Fuco- Amarou- Iso-

Species Culture Hydrolysis Solvent

time xanthin xanthinol ciaxanthin A fucoxanthinol

I. 50 1 No 100 mg g-

Methanol 0 mg g^"1 0 mg g^"1 0 mg g^" galbana PBR hydrolysis

I. 50 1

KOH 4 g l^_1 Ethanol 60 min 30 mg g^_1 3 mg g^"1 13 mg g^_1 1 mg g^"1 galbana PBR

EXAMPLE 7

Scale-up of the C-MEDPA derivatization process for obtaining novel bioactive extracts from I. salbana - variation (II)

A fourth fraction of the lyophilized biomass from /. galbana GAT-6007.00.002 (Example 5) was processed to fucoxanthin enriched extracts by ethanol extraction according to the protocols described in Example 2 (in 3: 1 proportion ethanol: lyophilized biomass). This first fucoxanthin enriched extract was then derivatized following a variation of the chemical derivatization process tested firstly in Example 1 and identified as the C-MEDPA derivatization process; thus, once the solid residue was dissolved in the minimum possible amount of solvent, 0.1 volumes of 0.1M lithium tert-butoxide in THF was added. The C-MEDPA derivatization process was carried out under nitrogen atmosphere, in the dark and at 0°C, during 10 minutes. To stop the reaction, the same protocol described in Example 3 was used. Finally, diethyl ether was eliminated by rotoevaporation. The remaining derivatized extract contained 20 mg g^"1 of fucoxanthin, 10 mg g^"1 of fucoxanthinol, among other carotenoids derived from the saponification reaction such as amaurociaxanthin A (10 mg g^"1) or isofucoxanthinol (5 mg g^"1). Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β-carotene. Results and experimental conditions are summarized in Table 9.

Table 9

Results and experimental conditions of C-MEDPA derivatization process applied to 7. galbana extracts - Variation (II)

Reaction Fuco- Fuco- Amarou- Iso-

Species Culture Hydrolysis Solvent

time xanthin xanthinol ciaxanthin A fucoxanthinol

1. 50 1 No 100 mg 0 mg g^" 0 mg g^"

MeOH - O mg g-¹ galbana PBR hydrolysis g

I. 50 1 Li tert- 20 mg g^" 10 mg 10 mg

THF lO min 5 mg g ¹ galbana PBR ButO ¹ g^"1 g^"1

EXAMPLE 8

Scale-up of the C-MEDPA derivatization process for obtaining novel bioactive extracts from I. salbana - variation fill)

A fifth fraction of the lyophilized biomass from /, galbana GAT-6007.00.002 (Example 5) was processed to fucoxanthin enriched extracts by ethanol extraction according to the protocols described in Example 2 (in 3: 1 proportion ethanol: lyophilized biomass). This fucoxanthin enriched extract was then derivatized following a variation of the chemical derivatization process tested in Example 1 and identified as the C-MEDPA derivatization process; thus, once the solid residue was dissolved in the minimum possible amount of solvent, 0.5 volumes of 30% aqueous NH₄OH was added. The C- MEDPA derivatization process was carried out under nitrogen atmosphere, in the dark and at 0°C, during 60 minutes. To stop the reaction, the same protocol described in Example 3 was used. Finally, diethyl ether was eliminated by rotoevaporation. The remaining derivatized extract contained 80 mg g^"1 of fucoxanthin, 3 mg g^"1 of fucoxanthinol, among other carotenoids derived from the saponification reaction such as amaurociaxanthin A (3 mg g^"1). Other naturally occurring carotenoids can also be in the extract, such as diadinoxanthin, diazoxanthin or β-carotene. Results and experimental conditions are summarized in Table 10.

Table 10

Results and experimental conditions of C-MEDPA derivatization process of 7. galbana extracts - Variation (III)

Reaction Fuco- Amarou- Iso- Species Culture Hydrolysis Solvent Fucoxanthin

time xanthinol ciaxanthin A fucoxanthinol

I. 50 1 No

MeOH 100 mg g-¹ O mg g-¹ 0 mg g^"1 0 mg g^" galbana PBR hydrolysis

I. 50 1 60

NH₄OH MeOH 80 mg g^"1 3 mg g^"1 3 mg g^"1 0 mg g^" galbana PBR

Table 11

Summary of the experimental conditions for C-MEDPA and E-MEDPA derivatization processes

% of pigment in extract

Exampl Derivatization Rection Fucoxanthi Fucoxanthino Amarouciaxanthi

Species Culture Solvent Isofucoxanthinol e Agent time n 1 n A

1 Pure fucoxanthin - 10 g/l KOH Methanol 60 min 28.1 13.2 13.7 7.2

1 Pure fucoxanthin - Lipase PBS 24 hours 72.2 24.2 0.00 0.00

2 1 Erlenmeyer

2 /. galbana 6 g/1 KOH Methanol 60 min 0.25 0.29 0.33 0.18 flask

2 1 Erlenmeyer

2 T. pseudonana 6 g/1 KOH Methanol 60 min 0.09 0.17 0.25 0.53 flask

2 1 Erlenmeyer

2 P. lutheri 6 g/1 KOH Methanol 60 min 0.62 0.26 0.06 0.34 flask

2 1 Erlenmeyer 0.09

2 N. incerta 6 g/1 KOH Methanol 60 min 0.06 0.19 0.25

flask

2 1 Erlenmeyer

2 Ochromonas sp. 6 g/1 KOH Methanol 60 min 0.04 0.08 0.10 0.16 flask

50 1

3 I. galbana No - - 9.80 0.00 0.00 0.00 photobioreactor

50 1

3 I. galbana 6 g/1 KOH Methanol 60 min 5.00 1.50 2.00 1.00 photobioreactor

50 1

4 T. pseudonana 2 g/1 KOH Methanol 60 min 8.00 0.70 0.70 0.10 photobioreactor

50 1

5 I. galbana Lipase PBS 24 hours 6.50 3.50 0.00 0.00 photobioreactor

I. galbana 50 1

5 Esterase PBS 24 hours 1.30 8.60 0.00 0.00 photobioreactor

I. galbana 50 1

6 4 g/1 KOH Ethanol 10 min 3.00 0.30 1.30 1.00 photobioreactor

I. galbana 50 1 0.1M lithium tert- 7 THF 10 min 2.00 1.00 1.00 0.50 photobioreactor butoxide

I. galbana 50 1 30% aqueous

8 Methanol 60 min 8.00 0.30 0.30 0.00 photobioreactor NH₄OH

EXAMPLE 9

Antiinflammatory effect of the C-MEDPA and E-MEDPA derivatized extracts compared to fucoxanthin enriched extracts of I. salbana

This Example was performed in order to evaluate the anti-inflammatory effect of some representative algal extracts obtained by treatment with the C-MEDPA or with the E- MEDPA derivatization processes. To that end, the /. galbana C-MEDPA and E- MEDPA extracts of Examples 3, 5 and 6 were tested.

The /. galbana C-MEDPA and E-MEDPA extracts of Examples 3, 5 and 6 have the particulars shown in Table 12.

Table 12

/. galbana C-MEDPA and E-MEDPA extracts

% of pigment in extract

Derivatization FucoFuco- Amaroucia Isofuco-

Example Species

Agent xanthin xanthinol xanthin A xanthinol

3 /. galbana No 98.0 0.00 0.00 0.00

3 I. galbana 6 g/1 KOH 50.0 15.0 20.0 10.0

6 I. galbana 4 g/1 KOH 30.0 3.00 13.0 10.0

5 I. galbana Lipase 65.0 35.0 0.00 0.00

The extracts of Table 12 were diluted in DMSO in order to normalize the total content of fucoxanthins (presented in any of the following forms: fucoxanthin, fucoxanthinol, amarouciaxanthin A and iso fucoxanthinol) to 100 mg total fucoxanthins/ml.

Normal human epidermal keratinocyte progenitor cells (HPEKp, Cellntec) were grown in a defined keratinocytes medium, CnT-07 (Cellntec), at 37°C with 5% C0₂ in the humidified incubator. After one passage of expansion, cells were used for the experiment.

Keratinocytes were seeded at a density of 10,000 cells/well in a 96 well plate. After two days of culture, cells were treated with the different /. galbana extracts (concentrations as described below in Table 13). 24 hours later cells were challenged with PMA phorbol 12-myristate- 13 -acetate for 5 hours. Plates were then centrifuged, and the cell culture supernatant harvested. Cytokine [interleukin 8 (IL-8)] expression was then determined by ELISA (Human IL-8 Platinum ELISA Kit; Affymetrix eBioscience). All the conditions were performed in triplicates. Expression levels in the cell culture supernatants were normalized to the cell numbers using Janus B green staining to determine cell number. Results were expressed as percentage using untreated control as 100%.

Table 13

/. galbana C-MEDPA and E-MEDPA extracts

The results are shown in Figure 5 wherein it can be appreciated that the production of IL-8 is considerably repressed in keratinocytes treated with the extracts comprising fucoxanthin and fucoxanthinol obtained in Examples 3 and 6 after chemical treatment with the C-MEDPA derivatization process or in Example 5 after enzymatic treatment with the E-MEDPA derivatization process.

EXAMPLE 10

Antiinflammatory effect of C-MEDPA and E-MEDPA derivatized extracts compared to pure fucoxanthin and fucoxanthinol

This Example was performed in order to evaluate the anti-inflammatory effect of some representative unicellular algal extracts obtained by treatment with the C-MEDPA or with the E-MEDPA derivatization processes. To that end, some /. galbana C-MEDPA and E-MEDPA derivatized extracts from Examples 3 and 5 were tested.

Materials & Methods Compositions and Extracts

Compositions A, B, C, D and E, and Extracts 1, 2 and 3, at a concentration of 500% in dimethylsulfoxide (DMSO). The particulars of said Compositions and Extracts are shown below in Experimental Conditions.

PMA (Phorbol 12-Myristate 13-Acetate, Sigma, P1585).

Control without stimulus + DMSO (0.25%)

Control with stimulus (PMA) (50 ng/ml) + DMSO (0.25%)

Cells and specific culture media

Primary human epidermal keratinocytes (pooled) from Cellntec (HPEKp).

PCT Epidermal Keratinocyte Medium (CnT-07) from Cellntec.

Cell culture

Normal primary human epidermal keratinocyte cells (HPEKp, Cellntec) were grown in PCT Epidermal Keratinocyte Medium, CnT-07 (Cellntec), at 37°C with 5% C0₂ in a humidified incubator with 90% humidity, controlling viability in each culture by staining with trypan blue (Merck Millipore). All the assays were performed with cells at 80-90% confluence and viability higher than 90%.

Stimulus and ELISA

The effect of the different extracts on IL-8 secretion by human keratinocytes was analyzed by the following protocol.

Keratinocytes were seeded at a density of 10,000 cells/ well in a 96 well plate. After two days of culture, cells were treated with the actives (Compositions A-E and Extracts 1-3) at 1%. 24 hours later cells were challenged with PMA (50 ng/ml) for 5 hours. Plates were then centrifuged, and cell culture supernatant harvested. Cytokine expression (IL-8) was then determined by ELISA. All the conditions were performed in triplicates. Expression levels in the cell culture supernatants were normalized to the cell numbers using hexosaminidase activity to determine cell number. Results were expressed as percentage using untreated control as 100%.

Supernatants from triplicates of each Experimental Condition were grouped in order to diminish differences among wells under the same conditions and thus to guarantee a higher homogeneity intra-conditions.

In order to determine IL-8 present in cells supernatant, a sandwich ELISA was used. Briefly, an ELISA plate (Nunc™ MaxiSorp™) was coated with 50 μΐ/well of a mouse monoclonal antibody anti-human IL-8 (Sigma) at 4 μg/ml, overnight, at 4°C. After washing the plate twice with PBS-HT (NaCl 280 mM, KC1 2.7 mM, Na₂HP0₄ 10 mM, KH₂PO₄ 2 mM and 0.1% Tween-20, pH 7.4), the plate was blocked with 150 μΐ/well of PBS with 1% de skimmed milk powder for 1 h at 37°C. Subsequently, the recombinant IL-8 (BD Pharmigen) pattern curve, diluted with keratinocytes medium having 0.25% DMSO (dilutions 1/2, from 25 ng/ml), was incubated, and the samples to be measured without dilution, for 2 h at 37°C. A total of 20 samples (see Experimentals Conditions below) were analyzed. The samples were analyzed by duplicate. After washing 5 times, a rabbit polyclonal antibody anti-IL-8 (Biosource) was incubated at 1 μg/ml in blocking buffer, for 1 h at 37°C. The plate was washed then 5 times and incubated with a goat secondary antibody anti-rabbit (Jackson) at a dilution of 1/10,000 in blocking buffer for 30 min at 37°C. Finally, plates were washed 5 times and TMB substrate was added. After stopping the reaction with HCl 1 M, absorbance was read at 450 nm with a spectrophotometer Multiskan Ascent (Thermo).

In order to normalize the secretion values of IL-8 along the number of cells in each well, the number of adherent cells was calculated by using a colorimetric assay based on the hexosaminidase enzyme. Namely, supernatant was removed and cells were washed 2 times with PBS-Ca/Mg before adding 60 μΐ/well of substrate buffer (7.5 mM p- nitrofenol-N-acetyl-P-D-glucosaminide (Sigma), 0.1 M sodium citrate and 0.25% Triton X-100 in ddH₂0, pH 5). After incubating the plate 4 h at 37°C and overnight at 4°C, the reaction was stopped with 90 μΐ/well of developing buffer (Glycine 50 mM and EDTA (ethylendiaminetetraacetic acid) 5 mM in ddH₂0, pH 10.4). Absorbance in each well was read at 405 nm with a spectrophotometer Multiskan Ascent (Thermo).

Experimental Conditions

1) Control without stimulus + DMSO (0.25%)

2) Control with stimulus PMA (50 ng/ml) + DMSO (0.25%)

3) PMA (50 ng/ml) + 1% Composition A

4) PMA (50 ng/ml) + 1% Composition B

5) PMA (50 ng/ml) + 1% Composition C

6) PMA (50 ng/ml) + 1% Composition D

7) PMA (50 ng/ml) + 1% Composition E

8) PMA (50 ng/ml) + 1% Extract_l (Extract of Example 3. a)

9) PMA (50 ng/ml) + 1% Extract_2 (Extract of Example 3.b)

10) PMA (50 ng/ml) +1% Extract_3 (Extract of Example 5)

wherein all the compositions and extracts were at 0.25% DMSO that was used as sample dilution vehicle. Compositions and Extracts:

Composition A: 2.5 μΜ Fucoxanthin (97% purity, Sigma Aldrich)

Composition B: 2.5 μΜ Fucoxanthinol (97% purity, Sigma Aldrich)

Composition C: 2.5 μΜ Fucoxanthin + 2.5 μΜ Fucoxanthinol (1 : 1) (97% purity, Sigma Aldrich)

Composition D: 2.5 μΜ Fucoxanthin + 0.5 μΜ Fucoxanthinol (5: 1) (97% purity, Sigma Aldrich)

Composition E: 0.08 μΜ Fucoxanthin (97% purity, Sigma Aldrich)

Extract 1 : The extract of Example 3. a at 1%: Methanol extract from /. galbana obtained according to Example 3, having Fucoxanthin 0.08 μΜ.

Extract 2: The extract of Example 3.b at 1%: Extract from /. galbana obtained according to Example 3, by C-MEDPA, having Fucoxanthin 0.04 μΜ +

Fucoxanthinol 0.012 μΜ + Amauroxanthin A 0.016 μΜ + Iso fucoxanthinol

0.008 μΜ (i.e., 0.08 μΜ in "carotenoids").

Extract 3: The extract of Example 5 at 1%: Extract from /. galbana obtained according to Example 5, by E-MEDPA (lipase), having Fucoxanthin 0.052 μΜ + Fucoxanthinol 0.028 μΜ (i.e, 0.08 μΜ in "carotenoids"). Results

Results are shown in Figure 6, wherein it can be appreciated that:

IL-8 expression by keratinocytes induced to inflammation via PMA was reduced at similar levels by high concentrations (2.5 μΜ) of pure fucoxanthin and fucoxanthinol (Compositions A and B); therefore, both compositions at the same concentration (2.5 μΜ) appear to have the same efficacy in reducing inflammation;

combining pure fucoxanthin and fucoxanthinol until total 5 μΜ or 3 μΜ of both compositions (Compositions C and D), a linearity in the response to inflammation reduction was maintained;

- when pure fucoxanthin was reduced to 0.08 μΜ (Composition E), the antiinflammatory response was practically lost;

Extract 1 (a non-derivatized extract according to the present invention), having 0.08 μΜ fucoxanthin, practically maintains the same anti-inflammatory activity as that obtained with 0.08 μΜ pure fucoxanthin; and

however, Extracts 2 and 3, obtained by the derivatization processes C-MEDPA and E-MEDPA according to the invention, respectively, having 0.08 μΜ carotenoids [i.e., fucoxanthin, fucoxanthinol, amauroxanthin A and isofucoxanthinol (Extract 2) and fucoxanthin and fucoxanthinol (Extract 3)], have an anti-inflammatory activity similar to that achieved with 2.5 pure fucoxanthin or fucoxanthinol.

These results show that, against simple combinations of pure fucoxanthin and fucoxanthinol, the algal extracts provided by this invention show a synergistic effect provided by different anti-inflammatory active compounds derivatized by the C- MEDPA or E-MEDPA derivatization process, which increases about 30 times the antiinflammatory activity of pure fucoxanthin or fucoxanthinol or their pure mixtures, i.e., the anti-inflammatory activity obtained with Extracts 2 and 3 was quite similar to that obtained with Compositions A and B but the concentration of fucoxanthin and fucoxanthinol in Extracts 1 and 2 was about 30 times lower than in Compositions A and B. EXAMPLE 11

Antiinflammatory effect of C-MEDPA derivatized extracts

This Example was performed in order to evaluate the anti-inflammatory effect of some representative algal extracts obtained by treatment with the C-MEDPA derivatization processes provided by this invention. To that end, the /. galbana C-MEDPA derivatized extract of Example 3 was tested.

Method

HaCat keratinocytes were seeded at a density of 14,000 cells/well in a 24-well plate in DMEM supplemented with 10% Foetal Bovine Serum, 2mM Glutamine and 0,5% penicillin/streptomycin (from a stock of 10,000 IU/lOmg). After two days of culture, cells were treated with the extract from Example 3 C-MEDPA derivatized (hereinafter referred to as "EXT3"), whose concentration is described below. 24 hours later cells were challenged with LPS (2.5 ug/mL) (Sigma-Aldrich, ref L2630) for 5 hours. Plates were then centrifuged 10 min at 10,000 g, and cell culture supernatant harvested. Cytokine expression [interleukin-lbeta (IL-Ιβ) and tumor necrosis factor alpha (TNFa)] was then determined using a commercial ELISA kit (Quiagen). All the conditions were performed in triplicates. Cell viability was assessed using Trypan Blue and expression levels in the cell culture supernatants were normalized to the cell numbers. Results are expressed as percentage using untreated control as 100%).

Concentration of the extract from Example 3 C-MEDPA derivatized used (EXT3) in this assay:

- 50 mg/g fucoxanthin;

15 mg/g fucoxanthinol;

20 mg/g amaurociaxanthin A; and

10 mg/g iso fucoxanthinol

Thus, the extract contains 95 mg/g of fucoxanthin or fucoxanthin-type products. Procedure: 50 mg of EXT3 were resuspended in 6 mL EtOH. The suspension was mixed with 1L of a 50 mg/ml liposome emulsion (99% phosphatidylcholine - PPC) until encapsulated using an orbital shaker.

The extract contained 95 mg/g of fucoxanthin and fucoxanthin-type product; thus the concentration of the extract to be encapsulated was about 4,8 mg/L. Since molecular weight of fucoxanthin is 658.91 g/mol, the concentration of encapsulated fucoxanthin and fucoxanthin-type products was 0.087 μΜ.

Results are shown in Figure 7. As it can be seen in said figure, treatment of keratinocytes with EXT3 results in a decrease of IL-Ιβ and TNFa pro-inflammatory cytokines under induced inflammation conditions using LPS. The resulting decrease of cytokine expression is of nearly 60% in the case of IL-Ιβ and of around 35% in the case of TNFa. As to TNFa, EXT3 is even able to reduce the basal expression of TNFa around a 30%>.

EXAMPLE 12

Lipolysis effect of the C-MEDPA derivatized extracts of Thalassiossira pseudonana

This Example was performed in order to preliminarily evaluate the lipolysis effect of a representative algal extract obtained by treatment with the C-MEDPA derivatization process. To that end, the Thalassiosira pseudonana C-MEDPA extract of Example 4 was tested [that extract was named in this assay as "Extract- 1 A"].

Briefly, human subcutaneous preadipocytes were seeded in preadipocyte medium into a 96-well plate at a density of 40.625 cells/cm² and were incubated in a humidified 37°C incubator with 5% C0₂. After 48 hours incubation, cells were induced to differentiate by exchanging with adipocyte differentiation medium and the plates were incubated for 7 days in an incubator. After 7 days, cells were fed with fresh adipocyte maintenance medium and were incubated for another 7 days. After differentiation, cells appeared rounded with large lipid droplets apparent in the cytoplasm indicating mature adipocytes. Cells were treated for 3 hours either in the presence of actives ("Extract- 1 A" at two concentrations in the final assay volume: 0,000048% and 0,000024%) or in buffer alone as untreated control. Isoproterenol treatment was used as a positive control.

After 3 hours of treatment, cell culture supematants were analysed for glycerol release using an absorbance-based method. A standard curve generated using glycerol standards were used to calculate glycerol released by each actives. Glycerol release/well was normalized to the cell number using Janus B green to determine cell number. All the conditions were performed in triplicates and the results were depicted in percentage release of glycerol normalized to untreated cell controls (100%).

The results (Figure 8) show that:

- at the highest concentration tested (0,000048%), the "Extract- 1 A" showed an increase in the lipolytic activity of about 45% when compared to the untreated control (i.e., about 50%> equivalent with respect to the positive control isoproterenol); and

- at the lowest lower concentration (0,000024%), the "Extract- 1 A" showed an increase in the lipolytic activity of about 27% when compared to the untreated controls (i.e., about 31 % when compared to the positive control isoproterenol).

EXAMPLE 13

Analysis of fatty acids in algal extracts of the invention

This Example was carried out in order to analyze the fatty acid (FA) content in some algal extracts provided by this invention obtained after treating an algal culture to the C- MEDPA derivatization process or to the E-MEDPA derivatization process.

Thus, analysis of FA content were performed for the /. galbana extracts obtained in Examples 2, 3 and 5 (i.e., subjected to C- or E-MEDPA derivatization process).

Briefly, around 25 mg of freeze-dried sample were weighed in 15 ml polypropylene tubes with screw cap. 0.5 mg of triundecanoin were added as internal standard. Extraction and transesterification of fatty acids was carried out by the addition of 1.25 ml of 0.5M potassium hydroxide in methanol, vigorously vortexed and reacting for 5 minutes at 75°C. Once the sample had cooled down, 2 ml of boron trifluoride were added, and samples were kept again at 75°C for 5 more minutes. Finally, 1 ml of a saturated sodium chloride aqueous solution and 1 ml of hexane were added, and samples were shaken vigorously. Upper phase (hexane) was transferred to chromatographic vials.

Instrumental analysis was carried out by gas chromatography (CG) with flame ionization detector, equipped with a SP-2330 (30 m x 0.25 mm x 0.20 μιη, Sigma Aldrich). Two μΐ were injected in split mode, with the injector set at 270°C, using helium as carrier gas at 1 ml/min and detection temperature of 270°C. Oven initial temperature was set at 150°C, increasing at 4°C/min to 220°C, held 2.5 min. Samples were quantified according to the internal standard method.

The results are shown in Figure 9 and in Tables 14-16.

1. The C-MEDPA derivatization process as well as the E-MEDPA derivatization process results in no significant effect on the natural fatty acids profiles of crude extracts.

2. The contents of total fatty acids (FAs) in the C-MEDPA extracts as well as in the E-MEDPA extract are higher than 225 mg/g (mg FA/g extract), i.e, higher than 22.5% by weight with respect to the extract.

3. Total PUFAs [including linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (CI 8:3 n3), cz^'s- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), cz^'s-5,8,l l,14,17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16- docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), and cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3)] in the C- MEDPA extracts are maintained higher than 125 mg/g (mg PUFAs/g extract), i.e, higher than 12.5% by weight with respect to the extract. Total PUFAs [including linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (CI 8:3 n3), cz^'s- 11,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14-eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), cz^'s-5,8,l l,14,17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16- docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), and cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3)] in the E- MEDPA extract is maintained about 90 mg/g (mg PUFAs/g extract), i.e, about 9.0% by weight with respect to the extract.

Further, the content of DHA in C-MEDPA extracts and E-MEDPA extracts is maintained between 1 5 and 60 mg DHA g extract, i.e, between 1 .5 and 6% by weight with respect to the extract.

Total MUFAs and PUFAs [including palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cz^'s-5,8,l l,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3)] in the C-MEDPA extracts are maintained higher than 175 mg/g (mg MUFAs+PUFAs/g extract), i.e, higher than 17.5% by weight with respect to the extract.

Total MUFAs and PUFAs [including palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8: 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), cz^'s-5,8,l l,14,17-eicosapentaenoic acid [EPA] (C20:5 n3) and cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3)] in the E-MEDPA extract are maintained higher than 140 mg/g (mg MUFAs+PUFAs/g extract), i.e, higher than 14.0% by weight with respect to the extract. Table 14

Fatty acid content in 7. galbana extracts (Example 3)

Capric acid C10:0 9.5 7.9

Laurie acid C12:0 0.5 0.7

Tridecanoic acid C13:0 0.0 0.0

Myristic acid C14:0 53.2 54.5

Myristoleic acid C14:l n5 0.9 1.0

Pentadecanoic acid C15:0 0.7 0.7 czs-lO-Pentadecenoic acid C15:l n5 0.0 16.9

Palmitic acid C16;0 28.1 26.7

Palmitoleic acid C16:l n7 26.4 25.2

Heptadeeanoic acid C17:0 1.5 2.4 cis-10-Heptadecanoic acid C17:l n7 0.0 0.0

Stearic acid CI 8:0 2.8 2.2

Elaidic acid C18: lt n9 0.3 0.4

Oleic acid C18:lc n9 35.8 29.7

Linolelaidic acid C18:2t n6 0.0 0.0

Linoleic acid (LA) C18:2c n6 61.2 58.0 γ-Linoleic acid (GLA) C18:3 n6 5.8 5.2

AracMdic acid C20:0 0.0 0.0 a-Linolenic acid (ALA) C18:3 n3 31.6 29.3 eis-l l-Eieosenoic acid C20:l n9 0.0 0.0

Heneicosanoic acid C21:0 0.0 0.0 cis-l 1,14-Eieosadienoic acid C20:2 n6 0.0 0.0

7CM-8,11,14-Eicosatrienoic acid C20:3 n6 0.0 0.0

Behenic acid C22:0 0.0 0.0

cis-l 3,1 -Docosadienoic acid

Lignoceric acid C24:0 0.0 0.0

Nervonie acid C:24:l n9 0.4 0.4

Table 15

Fatty acid content in I. galbana E-MEDPA (lipase) derivatized extract (Example 5)

Capric acid C10:0 8.1

Laurie acid C12:0 0.5

Tridecanoic acid C13:0 0.0

Myristie acid C14:0 37.1

Myristoleic acid C14: l n5 0.2

Pentadecanoic acid CI 5:0 0.4 czs-lO-Pentadecenoic acid C15: l n5 9.3

Palmitic acid C16:0 28.4

Palmitoleic acid C16: l n7 17.1

Heptadeeanoie acid C17:0 1.9 cis-10-Heptadecanoic acid C17: l n7 0.0

Stearic acid CI 8:0 9.3

Elaidic acid C18: lt n9 0.2

Oleic acid CI 8:1c n9 36.4

Linolelaidic acid C18:2t n6 0.0

Linoleic acid (LA) CI 8:2c n6 41.5 γ-Linoleic acid (GLA) C18:3 n6 3.5

AracMdic acid C20:0 0.0 a-Linolenic acid (ALA) C18:3 n3 20.5 cis-l l-Eieosenoic acid C20: l n9 0.0

Heneicosanoic acid C21 :0 0.0

cis -11,14-Eicosadieiioic acid C20:2 n6 0.0

CM-8,11,14-Eicosatrienoic acid C20:3 n6 0.0

Lignoceric acid C24:0 0.0

Table 16

Fatty acid content in I. galbana extracts (Example 2)

Capric acid C10:0 9.2 19.4

Laurie acid C12:0 1.5 0.7

Trideeanoic acid C13:0 0.1 0.0

Myristie acid C14:0 91.4 88.1

Myristoleic acid C14:l n5 2.0 1.3

Pentadeearioic acid CI 5:0 1.2 1.2 czs-lO-Pentadecenoic acid C15:l n5 0.0 0.0

Palmitic acid C16:0 53.1 59.4

Palmitoleic acid C16:l n7 45.3 27.6

Heptadecanoie acid C17:0 4.8 3.5 cis-10-Heptadecanoic acid C17:l n7 0.0 0.0

Stearic acid C I 8:0 4.9 6.2

Elaidic acid C18: lt n9 0.6 0.7

Oleic acid C18:lc n9 75.2 76.5

Linolelaidic acid C18:2t n6 0.0 0.0

Linoleic acid (LA) C18:2c n6 110.0 53.8 γ-Linoleic acid (GLA) C18:3 n6 9.5 2.8

Araehidie acid C20:0 0.0 0.0 a-Linolenic acid (ALA) C18:3 n3 55.0 54.5 eis-11-Eicosenoie acid C20:l n9 0.0 0.0

Heneicosanoic acid C21:0 0.0 0.0 cis-l 1,14-Eicosadienoic acid C20:2 n6 0.0 0.0

CM-8,11,14-Eicosatrienoic acid C20:3 n6 0.0 0.3

Behenic acid C22:0 0.0 0.7

Aracmdonic acid (ARA) C20:4 n6;

2.2 1.3 cis-l l,14,17-Eicosatrienoic acid C20:3 n3

Euricic acid C22:l n9 0.0 0.4

Tricosanoic acid C23:0 0.0 0.0

Lignoceric acid C24:0 3.3 0.0

Neramic acid C:24:l n9 0.0 0.0

EXAMPLE 14

Compositions comprising an algal extract of the invention

Different compositions for application to the skin in the form of cream emulsions, gels, milks, suspensions, O/W or W/O emulsions, or liposome foams, aqueous or emulsion lotions, sprays or waxy sticks where prepared by using the different algal extracts of the invention, namely, the algal extracts obtained by C-MEDPA or E-MEDPA derivatization processes, listed in Table 11 (identified below as "algal extract").

1) Composition 1 : Enriched Oil - cosmetics/cosmeceutics/nutraceutical a. Algal extract 1 % w/w

b. Vegetal oil (q.s. ad)

2) Composition : a. Triglyceride 86.5% w/w

b. Algal extract 12.5% w/w

c. Natural Tocopherols 1.0 % w/w

3) Composition 2: Liposomated Extract - cosmetics/cosmeceutics/nutraceutical a. Algal extract 1 % w/w

b. Lecithins (20 mg/ml) 9% w/w

c. Water 90% w/w

4) Composition 3 : Emulsion. Cosmetics/Cosmeceutics/Nutraceutical

a. Algal extract 1 % w/w.

b. Vegetal oil 8,9% w/w

c. Water 90% w/w

d. Surfactant (Tween 80®) 0, 1 % w/w

5) Composition 4: Cream. Cosmetics-cosmeceutics a. Algal extract 5% w/w

b. Vegetal oil 5% w/w

c. Water 26,5% w/w

d. Excipient (xanthan, gelam, chitosan...) 56,4%₀ w/w e. Surfactant (Tween 80®) 0, 1 % w/w ) Composition 5 : Gel. Cosmeceutic, medical device, wound dressing a. Algal extract 5% w/w

b. Chitosan or alginate or hyaluronic acid (q.s. ad) ) Composition 6: Capsules. Nutraceuticals a. Algal extract 1% w/w

b. Fucoidan 10%> w/w

c. Denaturated starch 89%) w/w ) Composition 7: Granulates. Nutraceuticals

a. Triglyceride 10.0%) w/w

b . Algal extract 2.5% w/w

c. Cyclodextrin 85.0% w/w

d. Natural Tocopherols 2.5% w/w

Claims

1. An algal extract comprising fucoxanthin and fucoxanthinol, together with other algal components, wherein said algal extract is obtained by a process comprising: a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; and

b) reacting the fucoxanthin previously obtained with:

2. Algal extract according to claim 1, which comprises between about 0.001% and about 90% of fucoxanthin by weight with respect to the total weight of the algal extract, preferably between 0.01% and 75% by weight, more preferably between 0.1 % and 45%) by weight, still more preferably between 1% and 15% by weight.

3. Algal extract according to any one of claims 1 or 2, which comprises between about 0.001% and about 90% by weight of fucoxanthinol with respect to the total weight of the algal extract of the invention, preferably between 0.01% and 75% by weight, more preferably between 0.1% and 45% by weight, still more preferably between 1% and 15% by weight.

4. Algal extract according to any one of claims 1 to 3, which comprises between 1%) and 99.998%) by weight of other algal components.

5. Algal extract according to any one of claims 1 to 4, which comprises:

- 0.001 % to 90% by weight of fucoxanthin;

- 0.001 % to 90% by weight of fucoxanthinol;

1%) to 99.998%) by weight of other algal components.

6. Algal extract according to any one of claims claim 1 to 5, wherein the other algal components comprise one or more fatty acids.

7. Algal extract according to any of claims 1 to 6, wherein said fatty acids are selected from the group consisting of caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:0), myristoleic acid (C14: l n5), pentadecanoic acid (CI 5:0), cz^'s- 10-pentadecenoic acid (C 15 : 1 n5), palmitic acid (C16:0), palmitoleic acid (C16: l n7), heptadecanoic acid (C17:0), cz^'s- 10-heptadecanoic acid (C17: l n7), stearic acid (C18:0), elaidic acid (C18: lt n9), oleic acid (C18: lc n9), linolelaidic acid (C18:2t n6), linoleic acid [LA] (CI 8 :2c n6), gamma- linoleic acid [GLA] (C18:3 n6), arachidic acid (C20:0), alpha-linolenic acid [ALA] (C18:3 n3), cis- 11-eicosenoic acid (C20: l n9), heneicosanoic acid (C21 :0), cis-l 1,14-eicosadienoic acid (C20:2 n6), cz^'s-8,l l,14-eicosatrienoic acid (C20:3 n6), behenic acid (C22:0), arachidonic acid [APvA] (C20:4 n6), cis-l 1,14,17-eicosatrienoic acid (C20:3 n3), euricic acid (C22: l n9), tricosanoic acid (C23:0), cis-5, 8,11,14, 17-eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-13,16-docosadienoic acid (C22:2 n6), lignoceric acid (C24:0), nervonic acid (C:24: l n9), cis-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cis- 4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3), and any combination thereof.

8. Algal extract according to any of claims 1 to 7, wherein said fatty acids comprise a PUFA selected from the group consisting of linolelaidic acid (C18:2t n6), linoleic acid [LA] (C18:2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha-linolenic acid [ALA] (C18:3 n3), cis-l 1,14-eicosadienoic acid (C20:2 n6), cz^'s-8,11,14- eicosatrienoic acid (C20:3 n6), arachidonic acid [ARA] (C20:4 n6), cis-l 1,14,17- eicosatrienoic acid (C20:3 n3), cis-5, 8, 11, 14, 17-eicosapentaenoic acid [EPA] (C20:5 n3),cz^'s-13,16-docosadienoic acid (C22:2 n6), cz^'s-4,7,10,13,16-docosapentaenoic acid (C22:5 n6), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof.

9. Algal extract according to any of claims 1 to 8, wherein said fatty acids are selected from the group consisting of palmitoleic acid (CI 6: 1 n7), oleic acid (CI 8 : 1c n9), linoleic acid [LA] (CI 8 :2c n6), gamma-linoleic acid [GLA] (CI 8:3 n6), alpha- linolenic acid [ALA] (C18:3 n3), arachidonic acid [ARA] (C20:4 n6), czs-5,8,11,14,17- eicosapentaenoic acid [EPA] (C20:5 n3), cz^'s-4,7,10,13,16,19-docosahexanoic acid [DHA] (C22:6 n3) and any combination thereof.

10. Algal extract according to any of claims 1 to 9, which comprises about 1% and about 50% by weight of fatty acids with respect to the total weight of the algal extract, preferably between 2% and 40% by weight, more preferably between 10% and 40%) by weight, still more preferably between 15%> and 40%> by weight, even more preferably between 15%> and 35%> by weight.

11. Algal extract according to any of claims 1 to 10, which comprises at least 1% by weight, usually 5%> by weight, normally 10%> by weight, preferably at least 15%> by weight, more preferably at least 20%> by weight, still more preferably, at least 25 %> by weight of free fatty acids.

12. Algal extract according to any of claims 1 to 11, which comprises at least 1% by weight, usually 5%> by weight, normally 10%> by weight, preferably at least 15%> by weight, more preferably at least 20% by weight, still more preferably at least 25% by weight of polyunsaturated fatty acids (PUFAs).

13. Algal extract according to any of claims 1 to 12, which comprises between 1%) and 50%) by weight, typically between 5%> and 40%> by weight, usually between 10% and 35% by weight, of a combination of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) with respect to the algal extract.

14. Algal extract according to any of claims 1 to 12, which comprises:

l%o to 10%o by weight of fucoxanthin;

- 0.05%) to l%o by weight of fucoxanthinol; and

- 89%o to 98.95%o by weight of other algal components,

wherein said other algal components comprise at least one fatty acid at a concentration between about 1%> and about 50%> by weight with respect to the total weight of the algal extract of the invention, preferably between 2% and 45% by weight, more preferably between 3% and 40% by weight, still more preferably between 4% and 35% by weight, even more preferably between 5% and 30%> by weight.

15. Algal extract according to any of claims 1 to 14, which comprises fucoxanthin and fucoxanthinol, together with other algal components, wherein said other algal components comprise between about 1% and about 50% by weight of fatty acids with respect to the total weight of the algal extract, and wherein said algal extract is obtained by a process comprising:

b) reacting the fucoxanthin previously obtained with:

16. Algal extract according to claim 15, wherein said fatty acids comprise one or more polyunsaturated fatty acids (PUFAs).

17. Algal extract according to any of claims 1 to 16, wherein said fucoxanthin producing alga is a microalga belonging to a class selected from the group consisting of Rapidophyceae, Bacillariophyceae (Diatomeas), Crysophyceae, Pavlophyceae, and Prymnesiophyceae.

18. Algal extract according to any of claims 1 to 17, wherein said fucoxanthin producing microalga is a microalga that belongs to a genus selected from the group consisting of Isochrysis, Thalassiosira, Navicula, Pavlova, Ochromonas, Phaeodactylum, Odontella, Skeletonema, Chaetoceros, Prymnesium, Nitzschia, Dinobryon, Synura, Chrysochromulina, Ochrosphaera, Cylindrotheca, Chromulina, Mallomonas, and Emiliania.

19. Algal extract according to any of claims 1 to 18, wherein said fucoxanthin producing microalga is selected from the group consisting of Isochrysis aff. galbana, Thalassiosira pseudonana, Navicula incerta, Pavlova lutheri, Ochromonas sp., Phaeodactylum tricornutum, Odontella aurita, Isochrysis galbana, Isochrysis sp., Pavlova gyrans, Skeletonema costatus, Chaetoceros gracilis, Chaetoceros calcitrans, Prymnesium parvum, Nitzschia heufleriana, Nitzschia sp., Dinobryon sp., Synura uvella, Synura petersenii, Chrysochromulina brevifikum, Ochrosphaera neapolitana, Cylindrotheca fusiformis, Chromulina neblosa, Mallomonas asmundae, Ochromonas danica, Ochromonas spherocystis, and Emiliania huxleyi, preferably, /. aff. galbana, T. pseudonana, N. incerta, P. lutheri, and Ochromonas sp.

20. Algal extract according to any one of claim 1 to 19, wherein the base is an inorganic base or an organic base.

21. Algal extract according to any one of claim 1 to 20, wherein the enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol is an hydrolase, preferably a lipase or a cholesterol esterase.

22. A process for producing an algal extract comprising fucoxanthin and fucoxanthinol, together with other algal components, according to any of claims 1 to 21, which comprises: a) culturing fucoxanthin producing microalgae biomass under conditions that allow the production of fucoxanthin; and b) contacting fucoxanthin with: b.l) a base under conditions for the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol; or, alternatively, with b.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting at least a portion of fucoxanthin to fucoxanthinol.

23. Process according to claim 22, wherein said fucoxanthin producing alga is a microalga belonging to a class selected from the group consisting of Rapidophyceae, Bacillariophyceae (Diatomeas), Crysophyceae, Pavlophyceae, and Prymnesiophyceae.

24. Process according to any of claims 22 or 23, wherein said fucoxanthin producing microalga is a microalga that belongs to a genus selected from the group consisting of Isochrysis, Thalassiosira, Navicula, Pavlova, Ochromonas, Phaeodactylum, Odontella, Skeletonema, Chaetoceros, Prymnesium, Nitzschia, Dinobryon, Synura, Chrysochromulina, Ochrosphaera, Cylindrotheca, Chromulina, Mallomonas, and Emiliania.

25. Algal extract according to any of claims 22 to 24, wherein said fucoxanthin producing microalga is selected from the group consisting of Isochrysis aff. galbana, Thalassiosira pseudonana, Navicula incerta, Pavlova lutheri, Ochromonas sp., Phaeodactylum tricornutum, Odontella aurita, Isochrysis galbana, Isochrysis sp., Pavlova gyrans, Skeletonema costatus, Chaetoceros gracilis, Chaetoceros calcitrans, Prymnesium parvum, Nitzschia heufleriana, Nitzschia sp., Dinobryon sp., Synura uvella, Synura petersenii, Chrysochromulina brevifikum, Ochrosphaera neapolitana, Cylindrotheca fusiformis, Chromulina neblosa, Mallomonas asmundae, Ochromonas danica, Ochromonas spherocystis, and Emiliania huxleyi, preferably, /. aff. galbana, T. pseudonana, N. incerta, P. lutheri, and Ochromonas sp.

26. Process according to any one of claims 22 to 25, wherein the base is an inorganic base or an organic base.

27. Process according to any one of claims 22 to 25, wherein the enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol is an hydrolase, preferably a lipase or a cholesterol esterase.

28. Process according to any one of claims 22 to 27, wherein the product resulting from step a) is removed from the culture medium, optionally dried, and subjected to a treatment, comprising: i) contacting said removed algal biomass containing fucoxanthin with an organic solvent in order to obtain an organic extract comprising fucoxanthin; and, if desired, ii) drying said organic extract comprising fucoxanthin from step i) to obtain a residue comprising fucoxanthin; and iii) dispersing said residue comprising fucoxanthin from step ii) in an organic solvent.

29. A process for producing an algal extract comprising fucoxanthin and fucoxanthinol, together with other algal components, according to any of claims 1 to 21, which comprises: a) culturing fucoxanthin producing microalgae biomass under conditions for producing fucoxanthin; b) removing the algal biomass comprising fucoxanthin resulting from step a); c) contacting the algal biomass containing fucoxanthin resulting from step a) with an organic solvent under conditions for obtaining an organic extract comprising fucoxanthin; and, if desired, drying said organic extract comprising fucoxanthin to obtain a residue comprising fucoxanthin; and dispersing said residue comprising fucoxanthin in an organic solvent; d) contacting the product resulting from step c) with fucoxanthin with: d.l) a base under conditions for the hydrolysis of at least a portion of fucoxanthin to fucoxanthinol; o, alternatively, with d.2) an enzyme that catalyzes the conversion of fucoxanthin to fucoxanthinol under conditions for converting at least a portion of fucoxanthin to fucoxanthinol; e) adding an aqueous saline solution to the product resulting from step d) under conditions for forming an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol; and f) removing and, if desired, drying the organic phase comprising fucoxanthin and fucoxanthinol, to render an extract comprising fucoxanthin and fucoxanthinol.

30. A process for producing an algal extract comprising at least 37% by weight of fucoxanthin and 25% by weight of fucoxanthinol, together with other algal components, which comprises: a) culturing a fucoxanthin producing microalgal under conditions for the production of fucoxanthin; b) removing and drying said microalgal biomass comprising fucoxanthin; c) contacting said dry microalgal biomass with ethanol under conditions for obtaining an ethanol extract comprising fucoxanthin; d) drying said ethanol extract comprising fucoxanthin to obtain a solid residue comprising fucoxanthin; e) dispersing said solid residue comprising fucoxanthin from step d) in diethyl ether; f) contacting the suspension resulting from step e) with a base in a medium comprising an alcohol; g) adding an aqueous sodium chloride solution to the product resulting from step f) under conditions for forming an aqueous phase and an organic phase, wherein the organic phase comprises fucoxanthin and fucoxanthinol; h) removing the organic phase comprising fucoxanthin and fucoxanthinol; i) washing said removed organic phase comprising fucoxanthin and fucoxanthinol; and j) drying said organic phase comprising fucoxanthin and fucoxanthinol to render an extract comprising at least 37% by weight of fucoxanthin and 25% by weight of fucoxanthinol.

31. A composition comprising an algal extract according to any one of claims 1 to 21, or obtainable by a process according to any of claims 22 to 30, and a cosmetic, cosmeceutical, food, nutraceutical, or pharmaceutical acceptable vehicle.

32. Use of an algal extract according to any one of claims 1 to 21, or obtainable by a process according to any of claims 22 to 30, as an adiponectin production accelerator, an antiangiogenic agent, an antiallergic agent, an antibacterial agent, an anticancer agent, an antidiabetic agent, an antifungal agent, an antiinflamatory agent, an antimalarial agent, an antineoplastic agent, an antiobesity agent, an antioxidant agent, an antihypertensive agent, a bone protective agent, a cerebrovascular protective agent, a cholesterol lowering agent, an hepatoprotective agent, an ocular protective agent, a neovascularization inhibitor, a skin-protective agent.

33. An algal extract according to any one of claims 1 to 21, or obtainable by a process according to any of claims 22 to 30, for use in medicine.

34. An algal extract according to any one of claims 1 to 21, or obtainable by a process according to any of claims 22 to 30, for use in the prevention and/or treatment of Alzheimer Disease, arthritis, cancer, cirrhosis, coronary arterial disease, depression, diabetes, hypertension, hyperuricemia, an inflammatory disease, metabolic syndrome, obesity, osteoporosis, Parkinson, skin pigmentation, virus associated malignancy, adult T-cell leukemia and Burkitt lymphoma; or for the prevention, amelioration or treatment of damage of mammalian skin, particularly, for inhibiting a skin condition selected from the group consisting of inflammation, photo-ageing skin damage, uneven pigmentation, wrinkles, sagging skin and combinations thereof.

35. A cosmetic, pharmaceutical, nutraceutical or food product comprising an algal extract according to any one of claims 1 to 21, or obtainable by a process according to any of claims 22 to 30, comprising between 0.1% and 5% by weight of said algal extract.

36. Cosmetic, pharmaceutical or nutraceutical product according to claim 35, which further comprises a sunscreen agent.