Fatty acid composition and biological ac

and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries Luísa Custódio&Fernando Soares&Hugo Pereira&Luísa Barreira& Catarina Vizetto-Duarte&Maria J

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&Luísa Barreira&

Catarina Vizetto-Duarte&Maria João Rodrigues&Amélia Pilar Rauter&

Fernando Alberício&João 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

T-ISO (=Tisochrysis lutea), Tetraselmis sp and Scenedesmus sp

were evaluated for their antioxidant activity,

acetylcholines-terase (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 IC50value 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

capacity was observed in the hexane extract of Scenedesmus

s p ( IC5 0= 0 7 3 m g m L− 1) a n d S c e ne d e s m u s s p (IC50=0.73 mg mL−1) The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC50values 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=81.3μg 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 pharmaceu-tical industry and the production of functional food ingredi-ents 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 med-ical treatments This trend has raised the interest in the devel-opment 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 prob-lems or ailments such as cancer and Alzheimer’s disease (AD; Chacón-Lee and González-Maríño2010)

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

DOI 10.1007/s10811-013-0098-0

Microalgae display a high degree of biodiversity and are

considered as one of the most promising sources for new

prod-ucts and applications (Pulz and Gross2004) The elevated

con-tents and balanced composition of several important

biomole-cules, namely fatty acids (FA), carotenoids, phycobilins,

vita-mins, 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,

micro-algae are the most important primary producers of biomass

These organisms display efficient oxygenic photosynthesis and

simple nutritional requirements, coupled with important

biotech-nological 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

insuffi-cient 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-Maríño 2010) In spite of the high number of

microalgae species described until now (more than 50,000;

Bhakuni and Rawat2005), 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

(Olaizola2003) 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

supple-ments for humans and/or as animal feed additives

(Chacón-Lee and González-Maríño2010) Having this in mind, this

work evaluated the potential of three different species of

microalgae, namely Isochrysis galbana T-ISO (=Tisochrysis

lutea, Bendif et al2013, Tetraselmis sp and Scenedesmus sp.,

to be used as functional foods and/or as sources of

biomole-cules 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),

potassi-um 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 culti-vation system, in closed ‘Flat Panel Flow Through’ and

‘Tubular’ photobioreactors Sterility was ensured by mechan-ical and physmechan-ical 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 superna-tants recovered The extraction was repeated three times and the supernatants combined and filtered (Whatman no 4) The res-idue 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 solu-tions, 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 1,000μg 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 (IC , milligram per milliliter)

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 1 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

1,000μg 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

maintained in RPMI-1640 culture media supplemented with glucose (1,000 mg mL−1), 10 % fetal bovine serum (FBS),

L-glutamine (2 mM), penicillin (50 U mL−1) and streptomycin (50 μg mL−1) S17 cell line was grown in DMEM culture media supplemented with glucose (1,000 mg mL−1), 10 % FBS,L-glutamine (2 mM), penicillin (50 U mL−1) and strep-tomycin (50μg mL−1) Both lines were grown at 37 °C and 5.0 % CO2in humidified atmosphere

Cell viability and selectivity Exponentially growing cells were seeded at a density of 5 × 103 cells/well on 96-well plates and incubated for 24 h at 37 °C in 5.0 %

CO2 The extracts (100 μL) were then applied at con-centrations ranging from 3.9 to 125 μg mL−1 for 72 h Positive control cells were treated with etoposide at the same concentrations and during the same incubation period as the extracts, while negative control cells were treated with DMSO at the highest concentration used in test wells (0.5 % v/v) The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (Mosmann 1983) was used to assess the effect of the extracts on mitochondrial metabolic activity, as an indi-cator of cell viability Results were expressed in terms

of cell viability (percentage) and IC50 values (micro-gram per milliliter) The selectivity index (SI) of the extracts was estimated using the following equation:

SI = CT/CNT, where CT and CNT indicate the extract-induced cytotoxicity on tumour cells (HepG2) and on non-tumour cells (S17), respectively (Oh et al 2011)

Table 1 Extract yields

(percent-age), total phenolic content (TPC,

milligrams GAE per gram of

microalgae), and radical

scaveng-ing (RSA) and metal chelatscaveng-ing

activities (IC 50 , milligrams per

milliliter) on Fe 2+ and Cu 2+ ions

of different microalgae extracts

Values represent mean±standard

error (n=6) Different letters in

the same column indicate

signifi-cant differences between extracts

for the same species by the Tukey

HSD test at p<0.05

nd not detected

a

Positive control

Species Extract Yield TPC RSA Metal chelating activity

Fe2+ Cu2+

I galbana T-ISO Hexane 11.19 0.94±0.06a >10 1.29±0.03cd 0.90±0.09d

Diethyl ether 2.90 0.64±0.03b 3.09±0.01b 2.67±0.07b 1.81±0.05c Acetone 3.37 1.13±0.03a 1.90±0.04c 1.86±0.15c 3.53±0.02a Water 4.03 0.37±0.02c 9.47±1.03a 3.61±0.19a 2.42±0.14b Total 21.49 3.08

Tetraselmis sp Hexane 1.05 0.07±0.01b 2.41±0.40b 1.71±0.15ab 0.99±0.07c

Diethyl ether 0.82 0.08±0.00b 2.52±0.15b 1.73±0.18ab 2.52±0.15a Acetone 1.61 0.19±0.01a 4.35±0.21a 1.41±0.17b 2.10±0.04ab Water 6.17 n.d 4.46±0.01a 1.91±0.18a 2.96±0.10a Total 9.65 0.34

Scenedesmus sp Hexane 0.62 0.05±0.00c 6.53±0.19a 0.73±0.02d 0.91±0.12c

Diethyl ether 1.56 0.36±0.01b >10 2.84±0.2b 2.06±0.04b Acetone 1.64 0.51±0.02a 3.56±0.14b 4.11±0.14a >10 Water 1.26 0.17±0.02c >10 1.64±0.02c 5.49±0.14a Total 5.08 1.10

Statistical analysis

Results are expressed as mean±standard error of the mean, and

experiments were conducted at least four times Significant

differ-ences 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 IC50values 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 (Table1) 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; Table1) Scenedesmus sp

had the lowest content in weakly and non-polar (hexane-extractable) compounds (Table1) The TPC values, consider-ing the sum of all the extracts, were 0.34, 1.10 and 3.18 mg GAE g−1in Tetraselmis sp., Scenedesmus sp and I galbana T-ISO, respectively (Table1), 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 quan-tified FAME (Table2) 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 (Table2) 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

Table 2 FAME profile of I.

galbana T-ISO, Tetraselmis sp.

and Scenedesmus sp in

percent-age 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 Capric acid nd 0.51±0.02 nd

C12:0 Lauric acid nd n.d nd C14:0 Myristic acid 18.26±0.07 0.70±0.02 1.28±0.06 C15:0 Pentadecanoic acid 0.93±0.01 nd 0.33±0.01 C16:0 Palmitic acid 14.41±0.03 24.85±0.32 20.45±0.80 C17:0 Margaric acid nd nd 0.59±0.02 C18:0 Stearic acid 0.25±0.04 0.40±0.07 0.77±0.10 C20:0 Arachidic acid 0.32±0.03 nd nd C22:0 Behenic acid 0.51±0.09 nd 0.85±0.05 C24:0 Lignoceric acid nd nd 0.92±0.06

∑ SFA 34.68±0.26 26.43±0.42 25.19±1.09 C16:1 Palmitoleic acid 20.48±0.13 7.86±0.09 18.99±2.97 C18:1 Oleic acid 15.17±0.16 27.08±0.73 0.87±0.04 C20:1 Eicosenoic acid nd 1.49±0.01 nd C22:1 Docosenoic acid 0.43±0.01 nd nd

∑ MUFA 36.08±0.30 36.32±0.83 19.87±3.01 C16:2(n-6) Hexadecadienoic acid 0.70±0.01 1.66±0.01 1.88±0.08 C18:2(n-6) Linoleic acid 12.07±0.18 21.33±0.32 10.32±0.34 C16:3(n-3) Hexadecatrienoic acid nd 1.94±0.02 2.36±0.10 C16:3(n-6) Hexadecatrienoic acid nd nd 0.55±0.02 C18:3(n-3) α-Linolenic acid nd nd 39.25±1.39 C18:3(n-6) γ-Linolenic acid nd nd nd C20:4(n-6) Arachidonic acid acidaciacid 0.70±0.02 2.90±0.08 nd C20:5(n-3) Eicosapentaenoic acid 2.77±0.23 9.41±0.21 nd C22:6(n-3) Docosahexaenoic acid 12.68±0.22 nd nd

∑ PUFA 29.24±0.70 37.26±0.63 54.94±1.95

∑n-3 15.45±0.45 11.36±0.22 41.62±1.48

∑n-6 13.79±0.25 25.90±0.41 13.33±0.46

(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)

Table3compares 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

con-tent 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 (IC =0.73 mg mL−1) and I

galbana T-ISO (IC50=1.29 mg mL−1) (Table1) The lowest

IC50values 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; Table1)

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 differ-ences were found between these and the IC50value obtained with galanthamine (IC50=0.14 mg mL−1)

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 evalu-ated 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

Table 3 FAME profile of different commercial microalgae species

Species ∑ PUFA ALA AA EPA DHA

I galbana T-ISOa 29.24 nd 0.70 2.77 12.68

Tetraselmis sp.a 37.26 nd 2.90 9.41 nd

Scenedesmus sp.a 54.94 39.25 nd nd n.d.

Arthrospira platensisb 41.63 nd nd nd nd

Arthrospira maximab 40.36 nd nd nd nd

Chlorella pyrenoidosab 35.48 15.87 nd 0.40 nd

Chlorella vulgaris b 38.30 15.79 nd nd 0.30

Nannochloropsis oculata c 38.96 0.65 5.98 21.84 3.23

Crypthecodinium cohnii d 32.70 nd nd 0.10 32.60

Dunaliella salina e 60.05 14.79 4.17 nd 6.93

Haematococcus pluvialis f,g 45.80 16.18 0.89 0.57 nd

Values represent mean (n=4)

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

indi-cated values correspond to the percentage of each FA per total identified

FAME

Fig 1 AChE inhibition (IC50 values, milligrams per milliliter) of microalgae extracts For the same species columns with different letters are significantly different at p<0.05 (one-way ANOVA, Tukey HSD test) Solid bars and errors bars represent the mean and standard error values, respectively (n=6)

sp.(IC50= 93.17 μg mL−1) Etoposide had an IC50 value

of 1.85 μg mL−1 The acetone extract of I galbana

T-ISO displayed an SI of 3.1 at the highest concentration

tested

Discussion

The chemical nature and the polarity of the solvents used in the extraction process determines the extraction yield, the

Fig 2 a Effect of the application of different microalgae extracts

(125 μg mL −1 ) for 72 h on the viability of HePG 2 cells in comparison

to a control (DMSO, 0.5 %, v/v) *p<0.01, significant differences

com-pared to the control b Cytotoxic activity (IC 50 values, microgram per

milliliter) of different extracts of microalgae against a human tumoural

cell line (HepG 2 cells) and a non-tumoural cell line (S17 cells) Columns with different letters are significantly different at p<0.05 (one-way ANOVA, Tukey HSD test) Solid bars and errors bars represent the mean and standard error values, respectively (n=6)

Fig 3 Effect of different concentrations of microalgae extracts on HepG2

(dark grey) and S17 cells (light grey) viability (bar chart) and selectivity

(scatter line) a Tetraselmis sp, hexane; b Scenedesmus sp., hexane; c I.

galbana T-ISO, ether; d I galbana T-ISO, acetone Solid bars and errors bars represent the mean and standard error values, respectively (n=6)

composition, and thus the biological activity of a given

ex-tract, and defines both qualitatively and quantitatively the

extracted compounds (Franco et al.2008) In this sense, in

order to gain knowledge on the bioactivities and

phytochem-ical profile of the microalgal species included in this study, a

four-stage sequential extraction process using hexane, ether,

acetone and water was used to obtain different bioactive

compounds In this way, more diverse extracts are obtained

and the probability of false negative results is minimized

I galbana T-ISO had the highest content of total phenolics

in all extracts However, the values reported in this work for

Tetraselmis sp and Scenedesmus sp are lower than the ones

described for ethanol/water extracts of species of the same

genera, while the TPC of I galbana T-ISO is higher than the

one reported in the same species (Goiris et al 2012) The

chemical composition of microalgae, including phenolics, is

highly influenced by culture conditions namely nutrient

avail-ability and light intensity; thus, it is possible that the differences

observed are the result of different cultivation conditions

(El-Baky et al.2009; Coesel et al.2008) From our results, it is

clear that, except for I galbana T-ISO, the phenolics present in

the species under study were poorly hexane-extractable, which

agrees with previous findings by others (Hajimahmoodi

et al 2010) In fact, in this work, phenolics were mainly

extracted with solvents of medium polarity (ether and

acetone), which are commonly used for the extraction of

phenolic compounds from higher plants (Stalikas 2007)

However, I galbana T-ISO is similar to other eukaryotic

microalgae species (e.g Chlorella sp., Crypthecodinium

cohnii and Schizochytrium sp.) and cyanobacteria (e.g

Anabaena flos-aquae and Nostoc ellipsosporum; Stalikas

2007) that have also been reported to contain

hexane-extractable phenolic compounds Bioactive molecules are

de-fined as a heterogeneous group of essential and non-essential

compounds commonly present in small concentrations in

plants and food products, with a proven effect on human

health Phenolics are one of the most common types of

bio-active compounds, and a large body of evidence emphasizes

their role in human health, especially the potential to prevent

oxidative stress-related diseases, such as cancer and

neurolog-ical disorders (Dai and Mumper2010) Although microalgae

are considered as valuable sources of bioactive

com-pounds (Plaza et al.2009; Guedes et al.2011), there have been

few reports on the phenolic content of these organisms (Duval

et al.2000; Li et al.2007; Klejdus et al.2009; Hajimahmoodi

et al.2010; Uma et al.2011; Custódio et al.2012)

Microalgae contain significant concentrations of

polyun-saturated fatty acids (PUFA) from the omega-3 (n-3) series,

namelyα-linolenic (ALA; 18:3n-3), eicosapentaenoic (EPA;

C20:5n-3) and docosahexaenoic (DHA; C22:6n-3) acids This

feature led to the commercial exploitation of selected

microalgal strains rich in PUFA (e.g I galbana, N oculata,

Scenedesmus sp., C vulgaris) particularly for the aquaculture

industry since omega-3 PUFA are required for proper devel-opment of fish larvae and bivalves (Pulz and Gross 2004) Currently, microalgae are being considered not only as sources of PUFA for aquaculture but also as important sources

of these micronutrients for human nutrition Presently, the main human dietary sources of omega-3 and omega-6 PUFA are fish and vegetable oils, respectively (Simopoulos 2008) However, the decline in fishing stocks has increased the demand for alternative sources, and microalgae can be a viable supplement of PUFA for human nutrition

The FAME profiles obtained throughout this study corrobo-rate results published previously (Pratoomyot et al 2005; Huerlimann et al.2010) LA (18:2n-6) was detected in all studied strains, and ALA was only detected in Scenedesmus sp The essential FA, ALA and LA must be provided in the diet as mammals are unable to synthetize them Upon dietary intake, both essential FA are vital precursors in the synthesis of other important omega-3 and omega-6 PUFA, such as EPA, DHA and arachidonic (AA; C20:4n-6) acids Nevertheless, previous re-ports showed that the conversion into other PUFA is limited (Burdge et al 2003) and that the intake of EPA and DHA promotes health benefits EPA was present in Tetraselmis sp and I galbana T-ISO, and DHA was only detected in I galbana T-ISO EPA and DHA are known bioactive molecules, with beneficial effects on the cardiovascular system (Givens and Gibbs 2008), cognitive function and dementia (van Gelder et al.2007) and foetal and neonatal brain development (Ruxton et al.2005)

There is increasing evidence that the ingestion of products with a high omega-6/omega-3 ratio promotes the occurrence of medical conditions, such as cardiovascular and autoimmune diseases, cancer and atherosclerosis, whereas a lower omega-6/omega-3 ratio could prevent them (Simopoulos 2008; Komprda2012) In this work, as previously reported, the lipid profile of microalgae has low ∑n-6/∑n-3 ratios, especially I galbana T-ISO and Scenedesmus sp Interestingly, I galbana consumption has been shown to be associated with favourable effects in diabetic rat models (Nuño et al.2013), which might

be explained by the high PUFA content in this microalga and the beneficial effects of PUFA in counteracting diabetes (Suresh and Das2003)

Considering the previously reported total lipid content of I galbana [(24 to 28 % of the biomass dry weight (BDW); Kaplan et al 1986], the EPA and DHA contents of the I galbana T-ISO, included in this study, suggest that 1 g of the latter strain may contain 40 mg of EPA+DHA Although obtained by gross estimation, these values agree with a recent work that reports an n-3 PUFA content of 43.6 mg/g of BDW for a different strain of the Isochrysis genus (O’Neil et al 2012) As the European Food Safety Agency recommends

250 mg of EPA+DHA per day (EFSA2010), 6 g of I galbana T-ISO biomass could supplement or almost fulfil the recom-mended n-3 PUFA daily intake This amount can be even

lower if the final yield of lipids in this microalgae strain is

improved, since previous reports have shown that a correct

manipulation of the culture conditions can enhance the

con-centrations of PUFA in this species (Fidalgo et al 1998)

Taken together, these results suggest that I galbana T-ISO

and Scenedesmus sp can be interesting sources of PUFA for

the nutraceutical and functional food industries

When comparing the percentage of the main PUFA present in

the microalgae species under study with some currently

com-mercially produced microalgae, such as Arthrospira, Chlorella,

Nannochloropsis, Dunaliella and Haematococcus species, it can

be observed that 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 N oculata exhibits a higher AA and

EPA content than the species under study However, it is a

weaker source of DHA The species C cohnii is an exceptional

source of DHA, but it is a well-known heterotrophic species,

while the species considered in this work are autotrophic, are not

grown with carbon sources other than CO2, and thus, do not

incur in operational costs, such as substrate acquisition for

fermentation Moreover, C cohnii has low or undetectable levels

of EPA and AA Only I galbana T-ISO can simultaneously

provide AA, EPA and DHA

The generation of highly reactive free radicals such as

reactive oxygen species (ROS) occurs during normal cellular

function, namely the mitochondrial respiratory chain and

phagocytosis However, the imbalance between ROS

production and the detoxification capacity of biological

sys-tems results in oxidative stress, which has been implicated in

the development of cancer and neurological diseases such as

AD (Dai and Mumper2010) and Parkinson’s disease (PD;

Danielson and Andersen2008) The RSA of I galbana

T-ISO, Scenedesmus sp and Tetraselmis sp is higher than those

reported for I galbana, Scenedesmus quadricauda and

Tetraselmis tetrathele, respectively (Natrah et al 2007)

However, the results obtained in this work were obtained

using different methodologies, which can partly account for

the differences observed on the genera Isochrysis and

Tetraselmis (Goiris et al.2012; Ulloa et al.2012)

Our results suggest that different types of antioxidant

com-pounds are present in the microalgae under study (Li et al

2007; Hajimahmoodi et al 2010) In the polar extracts

(acetone), the antioxidant activity can be ascribed to the

pres-ence of phenolics, in accordance with the findings of other

authors (Hajimahmoodi et al 2010) However, in the

non-polar extracts (hexane), the RSA detected cannot be attributed

to the presence of phenolics since, except for I galbana T-ISO,

the TPC of the hexane fraction was generally low Instead, the

antioxidant activity of the less polar extracts could be due to

their FA contents Oleic acid, the major FA of Tetraselmis sp.,

has antioxidant properties against the DPPH radical (Cerón

et al.2007), while palmitic acid has a high activity against the

peroxynitrite anion (ONOO-; Fang et al.2010) It can thus be said that I galbana T-ISO and Tetraselmis sp contain dietary antioxidants with potential application in the prophylaxis and/or treatment of oxidative stress-related diseases like AD and other neurological disorders

Although high levels of iron are essential for brain devel-opment and function, this metal can also catalyse reactions that generate highly toxic free radicals, resulting in oxidative damage responsible for the development of neurological dis-orders (Gaeta and Hider 2005) Moreover, it has been ob-served in AD patients a link between the increase in the concentration of copper ions, ROS generation and neuronal cell death (Gaeta and Hider2005) In this sense, the use of metal chelating compounds is considered a valuable therapy approach for AD treatment (Gaeta and Hider 2005) Generally, the analysed extracts exhibited a higher ability to chelate Fe2+than Cu2+, and the Fe2+-chelating activity was observed in both polar and non-polar extracts, suggesting that the microalgae under study possess chelating compounds of different chemical natures In the more polar extracts, metal chelation could be partly explained by the presence of pheno-lics, since it is known that these compounds can bind to transition metals (Megías et al.2009) However, Cu2+ chelat-ing activity seemed to be mainly due to weakly polar/non-polar compounds, as the lowest IC50 values were obtained with the hexane extracts of I galbana T-ISO, Tetraselmis

sp and Scenedesmus sp Hence, these results as a whole indicate the presence of compounds in microalgae extracts able to bind to both Fe2+and Cu2+, pointing to a possible beneficial role of those extracts as metal chelators To the best of our knowledge, this is the first report on the metal chelating potential of I galbana T-ISO, Tetraselmis sp and Scenedesmus sp

One of the hallmarks of AD is the reduction of acetylcho-line levels (ACh), which is the major neurotransmitter in the central nervous system (Filho et al.2006) AChE is a

relative-ly selective enzyme responsible for the hydrorelative-lysis of ACh, which results in the cessation of cholinergic neurotransmis-sion (Ciro et al.2012) Thus, AChEI are considered one of the main treatments for AD (Pulok et al 2007) Inhibition of AChE provides symptomatic relief also on other neurological disorders, such as senile dementia, ataxia, myasthenia gravis and PD (Pulok et al 2007) All drugs presently used for

AD therapy namely tacrine, donepezil, rivastigmine and galanthamine are (acetyl)cholinesterase inhibitors ([A]ChEI); however, they exhibit adverse side effects such as gastro-intestinal disturbance and bioavailability problems Thus, the search for new and safer natural ChEI is still a rather important area of research

The results obtained in this work indicate the presence of AChEI belonging to different classes of compounds, generally exhibiting medium to high polarity Noteworthy is the fact that

in I galbana T-ISO and Scenedesmus sp the IC values were

similar to those of galanthamine, a pure compound isolated

from the bulbs of snowdrop (Galanthus sp.) that acts as an

anticholinesterase alkaloid-type drug In the ether and acetone

fractions, the inhibition of AChE could be at least partially

ascribed to the presence of phenolics, since these compounds

can also be ChEI (Williams et al.2011) There are more than

180 natural compounds with a direct relevance to AD

thera-peutics, mostly from plant origin (Williams et al 2011)

However, there is little information on the neuroprotective

activity of compounds from marine origin, either from

macroalgae (reviewed by Pangestuti and Kim 2011) or

microalgae (Custódio et al.2012) The results obtained with

the water fractions of I galbana T-ISO and with the ether,

acetone and water fractions of Scenedesmus sp indicate that

they are potential sources of natural AChEI

Hepatocellular carcinoma (HCC) accounts for 70–90 % of

primary liver cancers and is considered as one of the most

dominant human diseases worldwide, especially in East Asia

and South Africa (El-Serag and Rudolph2007) Moreover,

HCC is highly recalcitrant to orthodox chemotherapy and

radiation treatments and has a high rate of postsurgical

reappearance (50–70 % at 5 years; Gish et al.2007) In this

work, the microalgae extracts were evaluated for cytotoxic

activity against a hepatocellular carcinoma cell line (HepG2),

and also on non-tumoural cells (S17) Natural extracts are

considered interesting for in vitro cytotoxic activity when

IC50values are lower than 100μg mL−1(Boyd1997) In this

sense, all the extracts tested, except for the I galbana ether

extract, showed interesting in vitro cytotoxic activity Extracts

are considered selective when the selectivity index (SI) is

higher than 1 According to this, all the tested extracts were

selective, at diverse concentrations Moreover, according to

Mahavorasirikul et al (2010), samples with SI values superior

to 3 are considered highly selective This was the case of the

acetone extract of I galbana T-ISO, which displayed an SI of

3.1 at the highest concentration tested

Though the tested extracts exhibited IC50values higher than

etoposide in HepG2 cells, they were significantly more

selec-tive than this chemotherapy drug Moreover, the analysed

extracts are still a mixture of a reasonably large number of

compounds, and thus, a higher IC50was expected Although

microalgae have long been recognised as sources of important

biomolecules with potential medical uses (Guedes et al.2011),

there have been few reports on their cytotoxicity against

hu-man tumoural cells Potential antitumoral activity has been

ascribed for compounds present in H pluvialis, Tetraselmis

suecica, Arthrospira (Spirulina) platensis and Chlorella sp

(reviewed in Plaza et al.2009), but there are no reports of the

potential in vitro antitumoural activity of the species included

in this study

There is a growing trend to consume products that offer

balanced nutritional requirements and additional health

bene-fits, such as the reduction of the risk associated with specific

diseases This trend boosted the need to find natural sources for such products, and raised the interest in microalgae as a source of food ingredients and bioactives (Lordan et al.2011) Our results suggest that the microalgae used in this work could

be used as functional food and/or source of nutraceuticals due

to their contents in FA and bioactivities In particular I galbana T-ISO extracts, which displayed high phenolic con-tents and antioxidant activity, were able to chelate iron and copper ions and exhibited an outstanding selectivity against a human hepatocellular carcinoma cell line Moreover, this spe-cies is a good source of AA, EPA and DHA, and can be considered as an advantageous alternative to several currently produced microalgae

Acknowledgments This work was supported by the SEABIOMED project (PTDC/MAR/103957/2008), funded by the Foundation for Sci-ence and Technology (FCT) and the Portuguese National Budget LC is

an FCT post-doctoral research fellow (SFRH/BPD/65116/2009) CVD is

an FCT doctoral research student (SFRH/BD/81425/2011) All the algal species used in this study were provided by NECTON S.A (Portugal) The authors would like to dedicate this article to the memory of Fernando Soares, whose untimely passing remind us all of the pressing need for novel medical treatments for cancer.

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