Food Chemistry 131 (2012) 134–140

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Food Chemistry

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Microalgae of different phyla display antioxidant, metal chelatingand acetylcholinesterase inhibitory activities

Luísa Custódio a, Tiago Justo a, Laura Silvestre a, Ana Barradas a, Catarina Vizetto Duarte a, Hugo Pereira a,Luísa Barreira a, Amélia Pilar Rauter b, Fernando Alberício c,d,e, João Varela a,⇑a Center of Marine Sciences, University of Algarve, Faculty of Sciences and Technology, Ed. 7, Campus of Gambelas, 8005-139 Faro, Portugalb University of Lisbon, Faculty of Sciences, Center of Chemistry and Biochemistry, Department of Chemistry and Biochemistry, Campo Grande, Ed. C8, Piso 5, 1749-016 Lisbon, Portugalc Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spaind CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spaine University of Barcelona, Department of Organic Chemistry, Martí i Franqués 1-11, 08028 Barcelona, Spain

a r t i c l e i n f o

Article history:Received 16 May 2011Received in revised form 28 June 2011Accepted 18 August 2011Available online 24 August 2011

Keywords:AcetylcholinesteraseAlzheimer’sDementiaMarine productsNeurological disordersMetal chelators

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.08.047

⇑ Corresponding author. Tel.: +351 289 800900x73E-mail address: jvarela@ualg.pt (J. Varela).

a b s t r a c t

Methanol and hexane extracts from Tetraselmis chuii, Nannochloropsis oculata, Chlorella minutissima andRhodomonas salina were evaluated for total phenolic contents, radical scavenging activity (RSA), metalchelating potential against copper and iron ions and acetylcholinesterase (AChE) inhibition. Only themethanol extracts contained phenolic compounds. The hexane extracts had the highest RSA. The extractshad a higher capacity to chelate Fe2+ ions, more pronounced in the lowest concentration of the hexaneextracts with values ranging from 73.3 ± 3.3% (R. salina) to 97.5 ± 1.1% (N. oculata). The highest AChEinhibitory activity was found in the hexane extracts at 10 mg/ml of C. minutissima (79.3 ± 1.9%), T. chuii(85.7 ± 0.7%) and R. salina (81.5 ± 7.5%). GC–MS analysis indicated polyunsaturated fatty acids and ste-roids as the most abundant compounds in the hexane extracts. The species under study provide a valu-able source of antioxidants, metal chelators and AChE inhibitors.

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1. Introduction

Dementia is a group of symptoms that may accompany neurolog-ical disorders or conditions, and occurs mainly in the elderly popu-lation. It is characterised by the deterioration of multiple cognitivefunctions, such as memory, thinking, comprehension, calculationand language. It is estimated that 63 million people will suffer fromdementia in 2030, 65% of whom in less developed countries (Wimo,Winblad, Aguero-Torres, & von Strauss, 2003). The most commoncauses of dementia include degenerative neurological diseases, suchas Alzheimer’s (AD), dementia with Lewy bodies, Parkinson’s, andHuntington’s. AD is estimated to account for 50–60% of dementiacases in people over 65 years of age (Filho et al., 2006). This diseasehas no cure and it is terminal within 3–7 years of diagnosis. Thecharacteristic pathology of AD includes the extracellular depositsof plasma amyloid beta peptide (Ab) in senile plaques, intracellularformation of neurofibrillary tangles and the loss of neuronal syn-apses and pyramidal neurons (Weinreb, Mandel, Bar-Am, & Amit,2011). Although the initiating factors underpinning this pathologyremain to be elucidated, it is well established that AD is associatedwith a reduction of the levels of acetylcholine (ACh), which is the

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major neurotransmitter in the central nervous system (CNS) (Filhoet al., 2006). Acetylcholinesterase (AChE) is considered to be thechief enzyme involved in ACh hydrolysis, as well as in the develop-ment of AD. According to the cholinergic hypothesis, the restorationof ACh levels, which are progressively lost during the progression ofAD, delays the loss of cognitive function (Filho et al., 2006). Recentstudies have shown that AChE inhibitors alleviate neuropsychiatricsymptoms in AD patients, and provide beneficial effects on cognitiveability by increasing ACh levels within the synaptic region (Zarotsky,Sramek, & Cutler, 2003). Inhibition of AChE serves also as a strategyfor the treatment of other neurological disorders, such as seniledementia, ataxia, myasthenia gravis and Parkinson’s disease (Pulok,Venkatesan, Mainak, & Houghton, 2007).

Another factor considered to be pathologically important in var-ious neurodegenerative processes is oxidative stress, which mayplay a key pathogenic role in AD as an early event during the pro-gression of the disease (Qureshi & Parvez, 2007). It was shown thatthe absence of natural antioxidants such as vitamin D exacerbatedAD in a mouse model (Mhatre & Hensley, 2007). Moreover,increased levels of free radicals contribute to the inflammatory pro-cess, which is known to enhance the development of AD (Mhatre &Hensley, 2007).

Various metals have also been implicated in the development ofneurological disorders. For instance, changes in iron homeostasis

L. Custódio et al. / Food Chemistry 131 (2012) 134–140 135

have been noticed in AD patients, who might display altered levelsof iron, ferritin and transferrin receptors in the hippocampus andcerebral cortex (Weinreb et al., 2011). Iron may promote the depo-sition of Ab and, specifically, redox-active iron is known to be in-volved in the development of oxidative stress through thepromotion of the Haber–Weiss/Fenton reaction (Weinreb et al.,2011). Thus, therapies involving the chelation of iron and other re-dox active metals (e.g. copper) are presently being considered as avaluable strategy in AD (Weinreb et al., 2011).

Microalgae are the most important primary producer of bio-mass, and one of the most diverse ecological groups of organisms.They exhibit a unique combination of features typical of higherplants, such as efficient oxygenic photosynthesis and simple nutri-tional requirements, and yet they display biotechnological attri-butes of proper microbial cells, namely fast growth in liquidmedium and the ability to accumulate or secrete metabolites. Infact, microalgal biomass is a natural source of a number of biolog-ically active compounds (e.g. carotenoids, phycobilins, fatty acids,polysaccharides, vitamins, and sterols) (Plaza, Herrero, Cifuentes,& Ibáñez, 2009) and has a very wide range of applications, fromanimal feed and aquaculture to human nutrition and health prod-ucts. In addition, the taxonomical diversity of microalgae and thepossibility of growing and harvesting them under different envi-ronmental conditions render these aquatic photosynthetic organ-isms particularly attractive as bioreactors, which can be enrichedin a particular bioactive compound upon exposure to abiotic stres-ses (Coesel et al., 2008).

Knowledge on AChE inhibitors from marine photosyntheticorganisms is particularly scarce. Some research has been reportedfor the genera Sargassum and Gracilaria (Choi et al., 2007; Natarajan,Shanmugiahthevar, & Kasi, 2009), but to the best of our knowledgethere is no information on the AChE inhibitory potential or metalchelating activity of microalgae. In this context, this work evaluatedthe in vitro AChE inhibitory activities of polar (methanol) and non-polar (hexane) extracts from four species of microalgae, namely Tet-raselmis chuii, Nannochloropsis oculata, Chlorella minutissima andRhodomonas salina by the Ellman method. Additionally, the radicalscavenging of the extracts, their capability to chelate iron and cop-per and their total phenolic content were assessed. A preliminarychemical characterisation of the bioactive extracts was made byGas Chromatography coupled with Mass Spectrometry (GC/MS).

2. Materials and methods

2.1. Enzyme and chemicals

All chemicals used in the experiments were of analytical grade.Acetylcholinesterase (EC.3.1.1.7) from electrical eel with a specificactivity P1000 units/mg (1 unit hydrolyses 1.0 lmole of acetylcho-line to choline and acetate per minute at pH 8.0 and 37 �C), acetyl-thiocholine iodide (ATChI), 5,5-dithiobis-(2-nitrobenzoic acid)(DTNB), galanthamine, pyrocatechol violet and 1,1-diphenyl-2-pic-rylhydrazyl (DPPH) were purchased from Sigma (Steinheim, Ger-many). Ethylenediaminetetracetic acid (EDTA), and sodiumcarbonate (Na2CO3) were from Fluka (Steinheim, Germany). Merck(Darmstadt, Germany) supplied ferrozine, copper sulphate penta-hydrate and Folin–Ciocalteau (F–C), while methanol was fromFischer Scientific (Loughborough, UK). Additional reagents and sol-vents were purchased from VWR International (Leuven, Belgium).

2.2. Algal cell biomass

Biomass from N. oculata was provided by NECTON S.A (Portugal)as a solid dark green frozen paste. Microalgae were grownoutdoors in closed ‘Flat Panel Flow Through’ and ‘Tubular’

photobioreactors in a semi-continuous cultivation system, usingwater pre-treated by mechanical and physical methods in orderto ensure sterility and inorganic nutrients. Cultures were testedweekly for the presence of Vibrio and total marine bacteria. Micro-algae were concentrated by centrifugation at controlled speed,packed under chilled conditions and frozen at –20 �C. R. salina, T.chuii and C. minutissima were grown at the LEOA (ExperimentalLaboratory of Aquatic Organisms, University of Algarve), in f/2 cul-ture media using 100 L plastic bags. Cultures were maintained un-der artificial light (100 lmol/s/m2) with a 12:12 h light/darkphotoperiod. Microalgal cells were collected at late exponentialphase, centrifuged at 2000g and maintained at �20 �C until furtheranalysis.

2.3. Preparation of the extracts

For extract preparation, 1 g of lyophilised biomass was added to40 ml of hexane or methanol, homogenised using a disperser IKAT10B Ultra-Turrax, extracted overnight at room temperature (RT)with stirring and filtered (Whatman no. 4). The extracts were driedunder reduced vacuum pressure, resuspended in methanol or di-methyl sulfoxide (DMSO) for the methanol or hexane extracts,respectively, and stored at �20 �C.

2.4. Determination of total phenolic content (TPC)

TPC of the extracts was determined using the F–C colorimetricmethod as described by Velioglu, Mazza, Gao, and Oomah (1998).Briefly, 5 ll of the extracts were mixed with 100 ll of 10-fold di-luted F–C reagent, incubated at RT for 5 min and mixed with100 ll of sodium carbonate (Na2CO3, 75 g/l, w/v). Absorbancewas measured at 725 nm after 90 min incubation at RT on a micro-plate reader (Biotek Synergy 4). TPC was calculated as gallic acidequivalents (GAE) from the calibration curve of gallic acid standardsolutions, and expressed as GAE in milligrams per gram of initialdry material.

2.5. Radical scavenging activity (RSA)

RSA was evaluated by the DPPH method (Moreno, Scheyer,Romano, & Vojnov, 2006). Samples (22 ll at the concentration of1 mg/ml) were mixed with 200 ll of methanolic DPPH solution(120 lM) in 96-well flat bottom microtitration plates, and incu-bated in darkness at RT for 30 min. The absorbance was measuredat 515 nm (Biotek Synergy 4) and RSA was calculated as the per-centage inhibition relative to a blank containing methanol orDMSO. Butylated hydroxytoluene (BHT, 1 mg/ml) was used as apositive control.

2.6. Iron (ICA) and copper (CCA) chelating activity

ICA was determined by measuring the formation of the Fe2+

-ferrozine complex according to the method of Megías et al.(2009). Samples (30 ll) were mixed in 96-well microplates with250 ll of 100 mM Na acetate buffer (pH 4.9) and 30 ll of an aque-ous FeCl2 solution (0.1 mg/ml, w/v). After 30 min, 12.5 ll of anaqueous 40 mM ferrozine solution was added. The change in col-our was measured using a microplate reader (Biotek Synergy 4)at 562 nm, and the results were expressed as the percentage inhi-bition, relative to a control containing methanol or DMSO in placeof the sample.

The CCA was determined using pyrocatechol violet (PV) as de-scribed by Megías et al. (2009). Samples (30 ll) were mixed in96-well microplates with 200 ll of 50 mM Na acetate buffer (pH6.0), 6 ll of 4 mM PV dissolved in the latter buffer and 100 ll ofCuSO4�5H20 (50 lg/ml, w/v). The change in colour of the solution

Table 1Total phenolic contents (TPC, mg GAE/g DW) and DPPH radical scavenging (RSA,%)activity of microalgal extracts. Values represent means ± SD (n = 3).

TPC RSA

1 mg/ml 5 mg/ml 10 mg/ml

N. oculata Methanol 4.1 ± 0.5 12.2 ± 1.1c 35.2 ± 0.7b 59.4 ± 0.7a

Hexane – 5.20 ± 0.4c 12.2 ± 0.3b 70.3 ± 0.6a

T. chuii Methanol 8.6 ± 0. 20.0 ± 1.2c 33.5 ± 0.6b 45.8 ± 2.5a

Hexane – 14.0 ± 0.9c 24.1 ± 0.8b 68.1 ± 2.3a

C. minutissima Methanol 3.1 ± 0.3 20.8 ± 1.1b 38.3 ± 1.3a 35.3 ± 2.5a

Hexane – 16.4 ± 1.4c 42.6 ± 1.7b 46.0 ± 5.0a

R. salina Methanol – 9.4 ± 0.8a 6.6 ± 1.1b 12.0 ± 0.9a

Hexane – ni 15.1 ± 1.2a 22.9 ± 1.4a

BHT* – 88.5 ± 1.4

ni, no inhibition.a, b, c Different letters in the same row indicate significant differences by Duncan’sNew Multiple Range Test at p < 0.05.* Positive control, 1 mg/ml.

136 L. Custódio et al. / Food Chemistry 131 (2012) 134–140

was measured at 632 nm using a microplate reader (Biotek Syn-ergy 4). The extracts were assayed at the concentrations of 1, 5and 10 mg/ml, and the results were calculated as the percentageinhibition relative to a blank containing methanol or DMSO. Thesynthetic metal chelator EDTA was used as a standard at a concen-tration of 1 mg/ml.

2.7. AChE inhibitory activity

Inhibition of AChE activity by microalgal extracts was measuredby the colorimetric method described by Orhan et al. (2007).Briefly, 140 ll of 0.1 mM sodium phosphate buffer (pH 8.0), 20 llof the extracts at the concentrations of 1, 5 and 10 mg/ml and20 ll of AChE (0.28 U/ml) solution were dispensed in a 96-wellmicroplate and incubated for 15 min at RT. Afterwards, 10 ll ofATChI (4 mg/ml) and 20 ll of DTNB (1.2 mg/ml) were added. ATChIhydrolysis, catalysed by AChE, was monitored by the formation ofthe 5-thio-2-nitrobenzoate anion as a result of the reaction ofDTNB with thiocholines. The latter yellow-coloured anion was de-tected at a wavelength of 412 nm using a 96-well micro-platereader (Biotek Synergy 4). Results were expressed as the AChE per-centage inhibition relative to a control containing methanol orDMSO in place of the sample. Galanthamine was used as the posi-tive control at the concentration of 1 mg/ml.

2.8. Gas Chromatography and Mass Spectrometry (GC/MS) analysis

For component identification, the hexane extracts were submit-ted to GC/MS analysis, performed using an Agilent GC (6890 Series)– quadrupole MS system (5973), equipped with a fused silica cap-illary column (30 m � 0.25 mm � 0.25 lm, coated with DB-5),with the EI operating at 70 eV. Injector and detector temperatureswere set at 250 �C. The oven temperature program was 40 �C for1 min, 40–240 �C at 3 �C/min and helium was employed as carriergas (1 ml/min). The compound identification was performed bycomparing mass spectra with those contained in the National Insti-tute of Standards and Technology (NIST) library.

2.9. Statistical analysis

The experiments were conducted in triplicate and results areexpressed as mean ± standard error of mean (SEM). The data weresubjected to variance analysis (ANOVA) to assess treatment differ-ences and interactions, using the SPSS statistical package for Win-dows (release 15.0, SPSS Inc.). Significant differences betweenmeans were assessed by the Duncan’s New Multiple Range Test.Differences at p < 0.05 were considered to be significant.

3. Results and discussion

3.1. Total phenolic content (TPC)

As can be observed in Table 1, only the polar extracts containeddetectable levels of phenolic compounds. Indeed, the hexane ex-tracts had no phenolics, and low contents (<5 mg GAE/g) were de-tected in methanol extracts of C. minutissima and N. oculata, withvalues of 3.1 and 4.1 mg GAE/g, respectively (Table 1). The metha-nol extract of T. chuii exhibited the highest TPC (8.6 mg GAE/g)(Table 1). No phenolic compounds were detected in the crypto-phyte R. salina (Table 1). This is in agreement with the results ob-tained by Hajimahmoodi et al. (2010), where the polar extracts ofmicroalgae of the genera Anabaena, Chlorella, Chrocococcus, Fische-rella, Microchaete, Nostoc and Tolypothrix had the highest levels ofsuch compounds when compared with less polar extracts. How-ever, Li et al. (2007) have described that it is possible for hexane

fractions to exhibit the highest total phenolic levels in differentmicroalgae species such as Anabaena flos-aquae, Chlorella sp., Cryp-thecodinium cohnii, Nostoc ellipsosporum, Schizochytrium sp. andThraustochytrium sp. Phytophenolic compounds act as scavengersof singlet oxygen and free radicals by donating hydrogen fromthe phenolic hydroxyl groups, thereby forming a stable end prod-uct that does not initiate or propagate lipid oxidation (Soobrattee,Neergheen, Luximon-Ramma, Aruoma, & Bahorun, 2005). Much ofthe work conducted until now has emphasised the role of pheno-lics present in higher plants in relation to human diseases, suchas cancer and neurodegenerative disorders like AD (Dillard & Ger-man, 2000). Although algae are considered as a potential source ofnatural antioxidants, compared to higher plants, few studies haveevaluated the phenolic contents in (micro)algae. To the best ofour knowledge, this is the first report on the phenolic contents ofT. chuii, N. oculata, C. minutissima and R. salina.

3.2. Radical scavenging activity

In C. minutissima, T. chuii and N. oculata, the highest RSA was ob-tained with the application of methanol extracts at the concentra-tions of 1 and 5 mg/ml (Table 1). However, the application of thehexane extracts at the concentration of 10 mg/ml resulted in thehighest RSA, with values ranging from 46% to 70%, the highest va-lue obtained with samples of N. oculata (Table 1). Conversely, theextracts from R. salina had the lowest capacity to scavenge theDPPH radical (Table 1). These results indicate that the antioxidantcompounds of microalgae could have different polarities(Hajimahmoodi et al., 2010; Li et al., 2007). In the polar (methanol)extracts, the antioxidant activity can be due to the presence of phe-nolic compounds, in accordance with the findings by other authors(Hajimahmoodi et al., 2010). However, in the non-polar (hexane)extracts, the RSA detected cannot be attributed to the presenceof phenolic compounds, which differs greatly from other plant spe-cies like fruits, vegetables and medicinal plants (Li et al., 2007).

ROS are constantly produced in the brain due to its high con-sumption of oxygen for energy metabolism and also metabolic pro-cess of neurotransmission. Thus free radical-induced neuronaldamage is implicated in the pathogenesis and possible aetiologyof AD (Mhatre & Hensley, 2007). Our results indicate that the spe-cies under study could be a source of dietary antioxidants usefulfor the treatment or prophylaxis of oxidative stress-relateddiseases like AD and other pathologic conditions of the CNS.

3.3. Metal chelating activity

As can be seen in Tables 2 and 3, the extracts had a highercapacity to chelate Fe2+ ions than Cu2+, and the hexane extracts

Table 2Iron chelating activity (ICA,%) of microalgal extracts.

1 mg/ml 5 mg/ml 10 mg/ml

N. oculata Methanol 19.3 ± 5.9c 42.2 ± 5.6b 68.5 ± 6.6a

Hexane 97.5 ± 1.1 – –C. minutissima Methanol 15.5 ± 5.7c 25.8 ± 4.8b 38.4 ± 8.3a

Hexane 95.6 ± 1.3 – –T. chuii Methanol 11.9 ± 2.3c 23.0 ± 3.7b 32.2 ± 5.0a

Hexane 77.8 ± 6.2 – –R. salina Methanol 12.3 ± 2.2 23.0 ± 3.4a 24.6 ± 1.4a

Hexane 73.3 ± 3.3 – –EDTA* 94.9 ± 0.9 – –

– Not determined.a, b, c Different letters in the same row indicate significant differences by Duncan’sNew Multiple Range Test at p < 0.05.* Positive control, 1 mg/ml.

Table 3Copper chelating activity (CCA,%) of microalgal extracts.

1 mg/ml 5 mg/ml 10 mg/ml

N. oculata Methanol 21.7 ± 1.6c 53.0 ± 1.2b 69.0 ± 2.2a

Hexane 14.2 ± 1.8 24.3 ± 4.2 38.8 ± 2.1C. minutissima Methanol 13.6 ± 0.7c 35.3 ± 2.5b 53.5 ± 2.2a

Hexane 15.3 ± 2.0 37.4 ± 1.9 56.1 ± 5.4T. chuii Methanol 12.0 ± 0.8c 28.1 ± 2.3b 43.7 ± 2.4a

Hexane 15.4 ± 2.5 34.2 ± 2.1 39.9 ± 3.5R. salina Methanol 14.8 ± 1.1c 31.2 ± 1.2b 44.6 ± 1.0a

Hexane 19.1 ± 3.6c 55.8 ± 2.8b 69.6 ± 1.5a

EDTA* – 74.4 ± 2.8 – –

– Not determined.a, b, c Different letters in the same row indicate significant differences by Duncan’sNew Multiple Range Test at p < 0.05.* Positive control, 1 mg/ml.

L. Custódio et al. / Food Chemistry 131 (2012) 134–140 137

exhibited the highest Fe2+-chelating activity at the lowest concen-tration (1 mg/ml), with values ranging from 73% to 98%, the highestvalue being found in N. oculata extracts. Interestingly, the latterchelating activity was higher than that of the synthetic chelatorEDTA used as a positive control (Table 2). The values for theFe2+-chelating activity of methanol extracts ranged from 25% to69% in R. salina and N. oculata, respectively, at the concentrationof 10 mg/ml (Table 2). Oxidative damage due to reactions catalysedby divalent metals has been implicated in the development of neu-rological disorders. The disruption in the homeostasis of Fe2+ andCu2+ is of particular significance, in light of the increase in oxida-tive stress parameters, namely lipid peroxidation and the oxidativedamage to senile plaques (Gaeta & Hider, 2005). Thus, the delete-rious effects of Fe2+ and Cu2+ can be attenuated by agents that re-move them from solution. Our results suggest that the hexaneextracts are endowed with compounds with Fe2+-chelating activ-ity, and that these compounds do not have a phenolic nature. How-ever, the Fe2+-chelating activity detected in the methanol extractscan be due to the presence of phenolic compounds. Phenolic com-pounds possess diverse mechanisms of action for their antioxidantactivity, including metal chelation (Megías et al., 2009), and havebeen proposed for the treatment of neurodegenerative diseasesin which oxidative damage, caused by reactions catalysed by diva-lent metals, may be involved (Weinreb et al., 2011).

The Cu2+-chelating activity varied greatly among extracts andspecies (Table 3). In N. oculata, the best results were obtained withthe application of the methanol extract, and the highest value(69.0%) was achieved with the concentration of 10 mg/ml. In C.minutissima, no significant differences were observed between ex-tracts, and the best results were 53.5% and 56.1% for hexane andmethanol extracts, respectively (Table 3). The hexane extracts fromT. chuii generally exhibited a higher activity, although the best re-

sult (43.7%) was observed after the application of 10 mg/ml of themethanol extract (Table 3). The hexane extracts of R. salina had thehighest capacity to chelate Cu2+ ions, with values ranging from19.1% to 69.6% depending upon the extract concentration (Table3). Our results indicate the presence of Cu2+ binders in the micro-algae extracts under study, which may be used in the design of no-vel AD therapeutics. In fact, although the role of Cu2+ on AD hasbeen disputed by a recent nutritional study (Klevay, 2008), thereis direct evidence that an increase in the concentration of Cu ionsin AD patients is linked to ROS generation and neuronal cell death(Gaeta & Hider, 2005). In addition, the Cu chelating compoundsprobably have different chemical natures in the different species,since the metal chelating activity was detected in both polar andnon-polar extracts. In the polar extracts, those compounds mayhave a phenolic nature (Megías et al., 2009).

Taken together, our results indicate the possible beneficial roleof microalgae extracts as metal chelators, which provides a new in-sight into their use as a source of therapeutic compounds for AD.Histochemical studies indicate that the direct detection of redoxactivity in AD lesions is inhibited by the exposure of the tissue sec-tions to both Cu2+ and Fe2+ selective chelators (Sayre et al., 1997).Moreover, re-exposing the chelator-treated sections to either Cu2+

or Fe2+ salts can reinstate activity, suggesting that redox imbalancein AD is dependent on these metals (Perry, Cash, & Smith, 2002).These findings indicate that the accumulation of both Fe2+ andCu2+ has a significant impact on ROS generation and may beresponsible for the increase in global oxidative stress parametersobserved in AD.

3.4. AChE inhibitory activity

The AChE inhibitory activity (%) of the extracts was classified aspotent (>50% inhibition), moderate (30–50% inhibition), low (<30%inhibition) or nil (<5% inhibition) as suggested by Vinutha et al.(2007). The hexane extracts exhibited the highest AChE inhibitoryactivity, and except for N. oculata, potent inhibitions were obtainedupon treatment at concentrations of 5 and 10 mg/ml (Fig. 1). Thebest results were obtained with the application of the highest hex-ane extract concentration of C. minutissima, T. chuii and R. salina,with values of 79.3%, 85.7% and 81.5%, respectively (Fig. 1). Galan-thamine had an AChE inhibitory activity of 93.3%. Inhibition ofAChE is considered as a promising approach for the treatment ofAD and for possible therapeutic applications in the treatment ofParkinson’s disease, ageing, and myasthenia gravis (Pulok,Venkatesan, Mainak, and Houghton, 2007). Moreover, cholinester-ase inhibitors (ChEi) activate a-secretase, which acts on the b-amy-loid precursor protein preventing the formation of b-amyloidprotein (Natarajan et al., 2009). Therefore, ChEi not only increasethe level of Ach, but also prevent the formation of b-amyloidal pla-ques, thereby having a key role in the prevention of neuronal deathdue to inflammation in AD (Giacobini, 2004). The search for AChEiamong marine natural products is scarce: methanol extracts of G.gracilis and Sargassum had the highest ChE inhibitory potential(Natarajan et al., 2009), and plastoquinones isolated from S. sag-amianum have already been found to be potent ChEi (Choi et al.,2007). In this work, the results obtained with hexane extracts fromC. minutissima, T. chuii and R. salina point to a possible therapeuticvalue of bioactive compounds as ChEi, with application in the man-agement of AD and other neurological disorders.

3.5. GC/MS analysis

Since the hexane extracts exhibited the highest antioxidant andAChE inhibitory activities, they were analysed by GC/MS in order toidentify their main compounds, and the results are presented inFig. 2 and Table 4 Seven main chemical compound classes were

Fig. 1. AChE inhibition of hexane and methanol extracts of microalgae species, applied at the concentration of 1 mg/ml (A), 5 mg/ml (B) and 10 mg/ml (C). Values followed bydifferent letters are significantly different at p < 0.05 (one-way ANOVA, Duncan’s New Multiple Range Test).

Fig. 2. Main chemical compound classes identified in the hexane microalgal extracts by GC/MS. (A) T. chuii; (B) C. minutissima; (C) R. salina and (D) N. oculata. St, steroids;PUFA, polyunsaturated fatty acids; AA, alkanes and alkenes; PC, phenolic compounds; SFA + MUFA, saturated fatty acids + monounsaturated fatty acids; LCAlc, long-chainaliphatic hydrocarbons; Uni, unidentified compound.

138 L. Custódio et al. / Food Chemistry 131 (2012) 134–140

Table 4GC/MS analysis of the microalgal hexane extracts.

Compound Relative Abundance (%)

T. chuii C. minutissima R. salina N. oculata

5,8,11,14,17-Eicosapentaenoic acid 9.24 4.24 – –5,8,11,14,17-Eicosapentaenoic acid methyl ester 6.04 4.53 18.3 13.05,8,11,14,17-Eicosapentaenoic acid ethyl ester – – 1.21 –Oleic acid 1.60 14.7 1.82 1.73Stearic acid 1.59 – – –Palmitic acid 2.87 9.81 5.21 2.21Linoleic acid – 16.9 3.62 1.23Palmitoleic acid – 4.14 9.60 7.46Myristic acid – 3.23 5.57 2.54Stearic acid – 1.65 – –Pyrulic acid methyl ester – 1.16 – –4,7,10,13,16,19-Docosahexaenoic acid methyl ester – 1.11 – –1-Palmitoylglycerol – 1.04 – –2-Palmitoylglycerol 1.78 – – –2-hydroxy-1-(hydroxymethyl)ethyl (9Z)-9-(2-hex-1-en-1-ylidenecyclopropylidene)nonanoate 3.16 – –Campesterol 13.5 5.63 – –Brassicasterol – 1.10 2.09 –Cholesterol – – 3.88 8.55Fucosterol – – – 1.28Eicosane 1.30 – 3.89 –Nonadecane 1.24 – – –Octadecane 1.2 – 0.98 –Hexadecane 1.09 – –Heptadecane – – – 3.29Tetracosane 0.84 – 1.70 –Docosane – – 1.79 –Heptacosane – – 1.25Octacosane – – 1.10 –8-Heptadecene – – – 2.211,4-Eicosadiene – – – 1.881,19-Eicosadiene – 2.52 – –Octadecenyl aldehyde 2.40 – – –E-15-Heptadecenal 0.96 – – –8-(2-Octylcyclopropyl)octanal 0.78 – – –Vitamin E 0.35 1.03 0.22 2.32Phenol, 2,4-bis(1,1-dimethylethyl) – 1.81 – 3.13Linolenyl alcohol 8.38 – – –Phytol 4.05 2.7 1.37 1.619,12,15-Octadecatrien-1-ol – – 4.13 –Dihydroactinidiolide 1.7 – – –Di-tert-butylbenzene – 1.26 – 3.01Pyrrylmethyl(triethyl)stannane – – – 4.316,10-Methano-19-norandrost-4-ene-3,17-dione, 6-methoxy – – – 1.69

L. Custódio et al. / Food Chemistry 131 (2012) 134–140 139

identified: steroids, polyunsaturated fatty acids, other long-chainfatty acids; phenolic compounds, long-chain aliphatic hydrocar-bons (alkanes and alkenes), long-chain aldehydes and long-chainalcohols (Fig. 2). These compounds varied greatly in the analysedspecies, but polyunsaturated fatty acids (PUFA) were always themost abundant compounds in the hexane extracts of these micro-algae (Fig. 2, Table 4). The most abundant fatty acids ranged from14 to 22 carbons and varied in the degree of unsaturation, with T.chuii presenting the most unsaturated profile. Microalgae areknown to be an important source of long-chain fatty acids, fromwhich a large part is PUFA (Plaza et al., 2009). These compoundsare probably the major ones responsible for the high antioxidantactivity of the hexane extracts, since lipids and especially unsatu-rated lipids are good radical scavengers (Plaza et al., 2009). Thoughan unequivocal relationship between PUFA, or other long-chainfatty acids, and the inhibition of AChE has not been reported inthe literature, PUFA have been described to ameliorate AD diseasesymptoms improving cholinergic neurotransmission (Willis,Shukitt-Hale, & Joseph, 2009). These algae may therefore constitutean important nutritional supplement for AD patients. Steroidswere generally the second most abundant compounds in all spe-cies except R. salina (Table 4). Steroids are a type of organic com-pounds that contain a specific arrangement of four fused

cycloalkane rings. Belonging also to this chemical group, sterolscontain an additional hydroxyl group. Sterols occur naturally inplants, but sterol composition may vary widely from species tospecies. In most algae, campesterol, stigmasterol, ergosterol andfucosterol are generally the most abundant sterols, though choles-terol may also exist abundantly in some photosynthetic organismslike the Eustigmatophyceae (Patterson et al., 1994).

In fact, in this work, campesterol was the most abundant sterolin T. chuii and C. minutissima, but cholesterol was the principal ste-rol in R. salina and N. oculata (Table 4). The health benefits of sterolsfrom photosynthetic organisms include anti-inflammatory action(Micallef & Garg, 2009), protection against oxygen free radicals(Shao, Hernandez, Kramer, Rinker, & Tsao, 2010), and also preven-tion of b-amyloid-induced neurotoxicity (Lecanu et al., 2004). Thislink between phytosterols and ROS scavenging capacity (Shaoet al., 2010) may help to explain the high antioxidant activity ofthese algal hexane extracts. Some of the analysed algal extractsalso had significant amounts of long-chain aliphatic hydrocarbons,mostly under the form of n-alkanes (Table 4). These ranged from16 to 24 carbons in T. chuii, 18 to 28 carbons in R. salina (the heavi-est profile) and from 17 to 20 carbons in N. oculata (Table 4). In C.minutissima only eicosane (20 carbon atoms) was detected (Table4). Most of the algal extracts also had small amounts of long-chain

140 L. Custódio et al. / Food Chemistry 131 (2012) 134–140

aldehydes and alcohols. Interestingly, some of these compoundslike octadecenyl aldehyde and dihydroactinidiolide, detected in T.chuii, are used as pheromones by some higher plants and insects.Others like 2-palmitoylglycerol found also in T. chuii have nocicep-tive properties (Walker, Krey, Chu, & Huang, 2002) and 1-palmi-toylglycerol found in C. minutissima may act as an acetyl-coenzyme A acetyltransferase (ACAT) inhibitor, which can be use-ful in the stabilization of atheroschlerosis-induced damages (USPatent 5607970). Minor amounts of phenolic compounds wereidentified in the hexane extracts by GC–MS analysis, namely butyl-ated hydroxytoluene (BHT) and a-tocopherol (vitamin E). As al-ready stated, phytophenols are, in general, known by theirantioxidant activity (Megías et al., 2009).

4. Conclusions

Due to the increasing life expectancy, the number of people suf-fering from dementia will increase rapidly in both developed anddeveloping countries. AD is the most common cause of dementiaand affects 7.3 million European citizens. Contemplating the com-plexity of AD, novel therapeutic approaches for the treatment ofthis disease and also other neurological disorders comprise thesearch for bioactive compounds that act on multiple targets. Thiswork shows, for the first time, that the hexane extracts of T. chuii,N. oculata, C. minutissima and R. salina have DPPH radical scaveng-ing and metal chelating activity, coupled with cholinesteraseinhibitory activity towards AChE. In particular, T. chuii, C. minutiss-ima and R. salina may be considered for further studies in the man-agement of AD, due to their high metal chelating potential andAChE inhibitory activities. These bioactivities may be related tothe high abundance of PUFAs in these extracts. However, the pos-sibility of the observed bioactivities being related to the presenceof other minor compounds present in the samples cannot be ex-cluded. Though less abundant, several compounds were identifiedin these microalgae that may be linked to the observed bioactivi-ties and/or have other effects not studied here. These possibilitiesare now under investigation and shall be addressed in future work.

Acknowledgements

This work was supported by the SEABIOMED project (PTDC/MAR/103957/2008), funded by the Foundation for Science andTechnology (FCT) and the Portuguese National Budget. Luisa Custó-dio is an FCT post-doctoral research fellow (SFRH/BPD/65116/2009). NECTON S.A (Portugal) provided biomass from N. oculata.

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