Fatty acid composition and biological ac

J Appl Phycol DOI 10.1007/s10811-013-0098-0 Fatty acid composition and biological activities of Isochrysis galbana T-ISO, Tetraselmis sp and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries Luísa Custódio & Fernando Soares & Hugo Pereira & Lsa Barreira & Catarina Vizetto-Duarte & Maria João Rodrigues & Amélia Pilar Rauter & Fernando Alberício & Jỗo Varela Received: 16 May 2013 / Revised and accepted: 18 July 2013 # Springer Science+Business Media Dordrecht 2013 Abstract Organic and water extracts of Isochrysis galbana TISO (=Tisochrysis lutea), Tetraselmis sp and Scenedesmus sp were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC) I galbana T-ISO had the highest TPC (3.18 mg GAE g−1) and radical scavenging activity, with an IC50 value of 1.9 mg mL−1 on the acetone extract The extracts exhibited a higher ability to chelate Fe2+ than Cu2+, and the maximum Fe2+ chelating L Custódio (*) : F Soares : H Pereira : L Barreira : C Vizetto-Duarte : M J Rodrigues : J Varela (*) Centre of Marine Sciences, University of Algarve, Faculty of Sciences and Technology, Ed 7, Campus of Gambelas, 8005-139 Faro, Portugal e-mail: lcustodio@ualg.pt e-mail: jvarela@ualg.pt A P Rauter Faculty of Sciences, Center of Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Lisbon, Campo Grande, Ed C8, Piso 5, 1749-016 Lisbon, Portugal F Alberício Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain F Alberício CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain F Alberício School of Chemistry, University of KwaZulu-Natal, 4001 Durban, South Africa F Alberício Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, 08028 Barcelona, Spain capacity was observed in the hexane extract of Scenedesmus sp (IC =0.73 mg mL − ) and Scenedesmus sp (IC50 =0.73 mg mL−1) The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC50 values of 0.11 and 0.15 mg mL−1, respectively, and in the water extract of I galbana (IC50 =0.16 mg mL−1) The acetone extract of I galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC50 =81.3 μg mL−1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL−1) All species presented a highly unsaturated fatty acids profile Results suggest that these microalgae, particularly I galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae Keywords AChE inhibitors Antioxidants Functional foods Marine natural products Microalgae Polyunsaturated fatty acids Introduction In recent years, healthcare costs have steadily increased due to factors such as longer life spans, higher incidence of chronic illnesses and the existence of more expensive and safer medical treatments This trend has raised the interest in the development of functional foods and nutraceuticals due to the beneficial health effects associated with their intake, namely risk reduction, relief and/or treatment of common health problems or ailments such as cancer and Alzheimer’s disease (AD; Chacón-Lee and González-Maríđo 2010) J Appl Phycol Microalgae display a high degree of biodiversity and are considered as one of the most promising sources for new products and applications (Pulz and Gross 2004) The elevated contents and balanced composition of several important biomolecules, namely fatty acids (FA), carotenoids, phycobilins, vitamins, sterols and polysaccharides (Plaza et al 2009; Guedes et al 2011), confer microalgae the potential to improve the nutritional value of foods and animal feed In aquatic ecosystems, microalgae are the most important primary producers of biomass These organisms display efficient oxygenic photosynthesis and simple nutritional requirements, coupled with important biotechnological features namely, fast growth in liquid medium and the ability to accumulate or secrete metabolites On the other hand, microalgae can be grown under different conditions, including large photobioreactors, making it possible to rapidly produce biomass and biomolecules at a large scale (Sánchez et al 2008) Moreover, algal biomass can often be enriched in a desired particular bioactive compound upon exposure to abiotic stresses (Coesel et al 2008) In the early 1950s, microalgae were mainly viewed as an alternative protein source in the context of impending insufficient protein supply caused by the rapid increase of the world population (Spolaore et al 2006) Since then, microalgae have gained increasing importance in the food industry due to their unique biochemistry, being considered as sources of food and as reservoirs of functional molecules (Chacón-Lee and González-Maríđo 2010) In spite of the high number of microalgae species described until now (more than 50,000; Bhakuni and Rawat 2005), just a few thousands of strains are kept in collections, and only a small fraction of it has been investigated for biochemical contents and bioactivities (Olaizola 2003) Moreover, among these, only a few species are commercialized Examples are Chlorella vulgaris, Haematococcus pluvialis, Dunaliella salina and Arthrospira (Spirulina) maxima, which are used as nutritional supplements for humans and/or as animal feed additives (ChacónLee and González-Maríđo 2010) Having this in mind, this work evaluated the potential of three different species of microalgae, namely Isochrysis galbana T-ISO (=Tisochrysis lutea, Bendif et al 2013, Tetraselmis sp and Scenedesmus sp., to be used as functional foods and/or as sources of biomolecules To achieve this purpose, the radical scavenging activity (RSA), ability to chelate iron and copper ions, total content in phenolic compounds, FA profile, in vitro acetylcholinesterase (AChE) inhibitory and cytotoxic activities, present in different extracts of microalgal dried biomass, were determined Materials and methods All chemicals used in the experiments were of analytical grade The compounds 1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium persulphate, fatty acid methyl ester (FAME) standards (Supelco® 37 Component FAME Mix), AChE (EC.3.1.1.7) from electrical eel, acetylthiocholine iodide, 5,5-dithiobis(2-nitrobenzoic acid) and galanthamine were purchased from Sigma (Germany) Algal biomass Samples were provided by NECTON S.A (Olhão, Portugal) as a dark green solid frozen paste Microalgae were grown outdoors in a semi-continuous cultivation system, in closed ‘Flat Panel Flow Through’ and ‘Tubular’ photobioreactors Sterility was ensured by mechanical and physical pre-treatment of the water used for algal production and by weekly control of the presence of Vibrio and total marine bacteria Algal biomass was concentrated by centrifugation at controlled speed, packed and frozen at −20 °C Before the experiments, samples were freeze-dried, milled and stored in the dark at −20 °C for 2–3 months Preparation of the extracts Samples were mixed with hexane (1:10w/v) and the algae cell walls disrupted using a disperser IKA T10B Ultra-Turrax at room temperature (RT) Samples were then centrifuged (5,000×g, 10 min., RT) and the supernatants recovered The extraction was repeated three times and the supernatants combined and filtered (Whatman no 4) The residue was then sequentially extracted with ether, acetone and water in a similar manner The organic extracts were dried under reduced vacuum pressure at 40 °C, while the water extracts were freeze dried All extracts were dissolved in DMSO at the concentration of 50 mg mL−1, aliquoted and stored (4 °C) Phytochemical analysis Total phenolic content The total phenolic content (TPC) of the extracts was determined by the F-C assay according to Velioglu et al (1998) Results are expressed as gallic acid equivalents (GAE) using a calibration curve of gallic acid standard solutions, in milligram per gram of microalgae (dry weight) FA composition The extraction and analysis of FA were according to a modified Lepage and Roy (1984) procedure as described by Pereira et al (2012) Antioxidant activity Samples were prepared by diluting the stock solution (50 mg mL−1, dry extract) to obtain concentrations ranging from 125 to 1,000 μg of dried extract per milliliter of solution Absorbances were measured in a microplate reader (Biotek Synergy 4) Results were expressed as antioxidant activity (percentage), relative to a control containing DMSO in place of the extract, and as half maximal inhibitory concentration (IC50, milligram per milliliter) J Appl Phycol RSA against DPPH free radical The RSA against DPPH radical was determined according to the method described by Moreno et al (2006) Butylated hydroxytoluene (BHT, E320) was used as a positive control at the same concentration of the extracts Iron and copper chelating activity The iron (Fe2+) and copper (Cu2+) chelating activities were determined according to Megías et al (2009) EDTA was used as a standard at a concentration of mg mL−1 AChE inhibitory activity The AChE inhibitory activity was measured by the Ellman method (Ellman et al 1961) as described by Orhan et al (2007) on samples at concentrations ranging from 125 to 1,000 μg mL−1 The change in colour was read at 412 nm, using a 96-well microplate reader (Biotek Synergy 4) Results are expressed as AChE percentage inhibition relative to a control containing DMSO in place of the sample and as IC50 values (milligram per milliliter) Galanthamine was used as a positive control at the same concentrations of the extracts In vitro cytotoxic activity Cell culture The human hepatocellular carcinoma cell line (HepG2 cells) and murine bone marrow stromal S17 cells were provided by CBME, University of Algarve HepG2 cells were Table Extract yields (percentage), total phenolic content (TPC, milligrams GAE per gram of microalgae), and radical scavenging (RSA) and metal chelating activities (IC50, milligrams per milliliter) on Fe2+ and Cu2+ ions of different microalgae extracts Species I galbana T-ISO Tetraselmis sp Scenedesmus sp Values represent mean±standard error (n=6) Different letters in the same column indicate significant differences between extracts for the same species by the Tukey HSD test at p10 3.09±0.01b 1.90±0.04c 9.47±1.03a 1.29±0.03cd 2.67±0.07b 1.86±0.15c 3.61±0.19a 0.90±0.09d 1.81±0.05c 3.53±0.02a 2.42±0.14b 2.41±0.40b 2.52±0.15b 4.35±0.21a 4.46±0.01a 1.71±0.15ab 1.73±0.18ab 1.41±0.17b 1.91±0.18a 0.99±0.07c 2.52±0.15a 2.10±0.04ab 2.96±0.10a 6.53±0.19a >10 3.56±0.14b 0.73±0.02d 2.84±0.2b 4.11±0.14a 0.91±0.12c 2.06±0.04b >10 Water Total 1.26 5.08 0.17±0.02c 1.10 >10 1.64±0.02c 5.49±0.14a 0.10±0.00 0.28±0.01 0.07±0.00 J Appl Phycol Table FAME profile of I galbana T-ISO, Tetraselmis sp and Scenedesmus sp in percentage of total FAME Values represent mean±standard error (n=4) nd not detected Fatty acid Common name I galbana T-ISO Tetraselmis sp Scenedesmus sp C10:0 C12:0 C14:0 C15:0 C16:0 Capric acid Lauric acid Myristic acid Pentadecanoic acid Palmitic acid nd nd 18.26±0.07 0.93±0.01 14.41±0.03 0.51±0.02 n.d 0.70±0.02 nd 24.85±0.32 nd nd 1.28±0.06 0.33±0.01 20.45±0.80 C17:0 C18:0 C20:0 C22:0 C24:0 ∑ SFA C16:1 C18:1 C20:1 C22:1 ∑ MUFA C16:2(n-6) C18:2(n-6) C16:3(n-3) C16:3(n-6) C18:3(n-3) C18:3(n-6) C20:4(n-6) Margaric acid Stearic acid Arachidic acid Behenic acid Lignoceric acid nd 0.25±0.04 0.32±0.03 0.51±0.09 nd 34.68±0.26 20.48±0.13 15.17±0.16 nd 0.43±0.01 36.08±0.30 0.70±0.01 12.07±0.18 nd nd nd nd 0.70±0.02 nd 0.40±0.07 nd nd nd 26.43±0.42 7.86±0.09 27.08±0.73 1.49±0.01 nd 36.32±0.83 1.66±0.01 21.33±0.32 1.94±0.02 nd nd nd 2.90±0.08 0.59±0.02 0.77±0.10 nd 0.85±0.05 0.92±0.06 25.19±1.09 18.99±2.97 0.87±0.04 nd nd 19.87±3.01 1.88±0.08 10.32±0.34 2.36±0.10 0.55±0.02 39.25±1.39 nd nd 2.77±0.23 12.68±0.22 29.24±0.70 15.45±0.45 13.79±0.25 0.83 0.84 9.41±0.21 nd 37.26±0.63 11.36±0.22 25.90±0.41 2.95 1.41 nd nd 54.94±1.95 41.62±1.48 13.33±0.46 0.32 2.18 C20:5(n-3) C22:6(n-3) ∑ PUFA ∑n-3 ∑n-6 ∑n-6/∑n-3 PUFA/SFA Palmitoleic acid Oleic acid Eicosenoic acid Docosenoic acid Hexadecadienoic acid Linoleic acid Hexadecatrienoic acid Hexadecatrienoic acid α-Linolenic acid γ-Linolenic acid Arachidonic acid acidaciacid Eicosapentaenoic acid Docosahexaenoic acid Statistical analysis Results are expressed as mean±standard error of the mean, and experiments were conducted at least four times Significant differences were assessed by analysis of variance (ANOVA) or using Duncan’s New Multiple Range Test when parametricity of data did not prevail SPSS statistical package for Windows (release 15.0, SPSS Inc.) was used The IC50 values were calculated by sigmoidal fitting of the data in the GraphPad Prism v 5.0 program Results Fractions yield and TPC The analysed microalgae differ in their chemical composition (Table 1) Whereas Tetraselmis sp has a high content in polar compounds (water-extractable), I galbana T-ISO seems to be rich in weakly and non-polar compounds (hexane-extractable; Table 1) Scenedesmus sp had the lowest content in weakly and non-polar (hexaneextractable) compounds (Table 1) The TPC values, considering the sum of all the extracts, were 0.34, 1.10 and 3.18 mg GAE g−1 in Tetraselmis sp., Scenedesmus sp and I galbana T-ISO, respectively (Table 1), the latter being the species with the highest TPCs in all extracts FA profile All species presented a highly unsaturated FA profile in which the percentage of PUFA ranged from 29.2 % (I galbana T-ISO) to 54.9 % (Scenedesmus sp.) of the total quantified FAME (Table 2) Linoleic acid (LA; 18:2n-6) was detected in all studied strains, ranging from 10 to 21 % of the total FAME profile ALA was only detected in Scenedesmus sp., where it accounted for almost 40 % of the total FAME (Table 2) EPA was detected in Tetraselmis sp (10 %) and I galbana T-ISO (3 %), while DHA was only detected in I galbana T-ISO representing almost 15 % of total FAME (Table 2) AA was detected at low percentages in I galbana T-ISO J Appl Phycol Table FAME profile of different commercial microalgae species Species ∑ PUFA ALA AA EPA DHA I galbana T-ISOa Tetraselmis sp.a Scenedesmus sp.a Arthrospira platensisb Arthrospira maximab Chlorella pyrenoidosab Chlorella vulgarisb Nannochloropsis oculatac Crypthecodinium cohniid Dunaliella salinae Haematococcus pluvialisf,g 29.24 37.26 54.94 41.63 40.36 35.48 38.30 38.96 32.70 60.05 45.80 nd nd 39.25 nd nd 15.87 15.79 0.65 nd 14.79 16.18 0.70 2.90 nd nd nd nd nd 5.98 nd 4.17 0.89 2.77 9.41 nd nd nd 0.40 nd 21.84 0.10 nd 0.57 12.68 nd n.d nd nd nd 0.30 3.23 32.60 6.93 nd Values represent mean (n=4) galbana T-ISO (IC50 =1.29 mg mL−1) (Table 1) The lowest IC50 values for Cu2+ chelating activity were obtained with the hexane extracts of I galbana T-ISO (IC50 =0.90 mg mL−1), Tetraselmis sp (IC50 =0.99 mg mL−1) and Scenedesmus sp (IC50 =0.91 mg mL−1; Table 1) AChE inhibitory activity The results obtained in this work indicate the presence of acetylcholinesterase inhibitors (AChEI) belonging to different classes of compounds, generally exhibiting medium to high polarity (Fig 1) In I galbana T-ISO, the AChEI were mainly water-extractable, with an IC50 value of 0.16 mg mL−1, while in Scenedesmus sp they were present in the ether (IC50 =0.15 mg mL−1), acetone (IC50 =0.15 mg mL−1) and water extracts (IC50 =0.11 mg mL−1) No significant differences were found between these and the IC50 value obtained with galanthamine (IC50 =0.14 mg mL−1) nd not detected a Current work b Otleş and Pire (2001) c Roncarati et al (2007) d Mendes et al (2007) e El-Baky et al (2004) f Damiani et al (2010) g Glycolipids and phospholipids were also taken into account The indicated values correspond to the percentage of each FA per total identified FAME (0.7 %) and Tetraselmis sp (2.9 %; Table 2) The lipid profile of microalgae presented particularly low ∑n-6/∑n-3 ratios (Table 2), especially I galbana T-ISO (0.84) and Scenedesmus sp (0.32) Table compares the percentage of the main PUFA present in the microalgae species included in this study, with some currently commercially produced microalgae, namely Arthrospira (former Spirulina), Chlorella, Nannochloropsis, Dunaliella and Haematococcus species Scenedesmus has a higher PUFA content than all the commercial species, except when compared with the slow growing species D salina, and is also a better source of ALA The commercial species Nannochloropsis oculata exhibits a higher AA and EPA content than the species under study However, it is a weaker source of DHA Only I galbana T-ISO, one of the species included in this work, can simultaneously provide AA, EPA and DHA Antioxidant activity In this work, the highest RSA was observed in the acetone extract of I galbana T-ISO (IC50 =1.9 mg mL−1), and in the hexane and ether extracts of Tetraselmis sp., with IC50 values of 2.4 and 2.5 mg mL−1, respectively (Table 1) Generally, the analysed extracts exhibited a higher ability to chelate Fe2+ than Cu2+, and the highest Fe2+ chelating capacity was observed in the hexane extracts of Scenedesmus sp (IC50=0.73 mg mL−1) and I In vitro cytotoxic activity The highest reduction in HepG2 cells viability was observed after application of extracts of I galbana T-ISO (ether and acetone), Tetraselmis sp (hexane) and Scenedesmus sp (hexane; Fig 2) The best result was achieved with the hexane extract of Tetraselmis sp., which reduced cell viability down to 10.8 %, similar to the result observed for etoposide (Fig 2a) The extracts responsible for a decrease in cell viability of 50 % or more, i.e ether and acetone extracts of I galbana T-ISO and hexane extracts of Tetraselmis sp and Scenedesmus sp., were further evaluated at different concentrations on HepG2 and S17 cells in order to determine IC50 values (Fig 2b) and selectivity (SI; Fig 3) The highest cytotoxic activity towards HepG2 cells was obtained after application of the hexane extracts of Tetraselmis sp (IC50 =58.25 μg mL−1) and Scenedesmus Fig AChE inhibition (IC50 values, milligrams per milliliter) of microalgae extracts For the same species columns with different letters are significantly different at p