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Revista Brasileira de Farmacognosia

Print version ISSN 0102-695XOn-line version ISSN 1981-528X

Rev. bras. farmacogn. vol.25 no.6 Curitiba Nov./Dec. 2015 

Original Articles

Evaluation of acetylcholinesterase inhibitory activity of Brazilian red macroalgae organic extracts

Levi P. Machadoa 

Luciana R. Carvalhoa 

Maria Cláudia M. Youngb 

Elaine M. Cardoso-Lopesc 

Danilo C. Centenod 

Leonardo Zambotti-Villelae 

Pio Colepicoloe 

Nair S. Yokoyaa  * 

aNúcleo de Pesquisa em Ficologia, Instituto de Botânica, São Paulo, SP, Brazil

bNúcleo de Pesquisa em Fisiologia e Bioquímica, Instituto de Botânica, São Paulo, SP, Brazil

cDepartamento de Farmácia, Universidade Nove de Julho, São Paulo, SP, Brazil

dCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Paul, SP, Brazil

eInstituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil


Alzheimer's disease affects nearly 36.5 million people worldwide, and acetylcholinesterase inhibition is currently considered the main therapeutic strategy against it. Seaweed biodiversity in Brazil represents one of the most important sources of biologically active compounds for applications in phytotherapy. Accordingly, this study aimed to carry out a quantitative and qualitative assessment of Hypnea musciformis (Wulfen) J.V. Lamouroux, Ochtodes secundiramea (Montagne) M.A. Howe, and Pterocladiella capillacea (S.G. Gmelin) Santelices & Hommersand (Rhodophyta) in order to determine the AChE effects from their extracts. As a matter of fact, the O. secundiramea extract showed 48% acetylcholinesterase inhibition at 400 μg/ml. The chemical composition of the bioactive fraction was determined by gas chromatography–mass spectrometry (GC–MS); this fraction is solely composed of halogenated monoterpenes, therefore allowing assignment of acetylcholinesterase inhibition activity to them.

Keywords Acetylcholinesterase inhibition; Halogenated monoterpenes; Hypnea; Ochtodes; Pterocladiella; Rhodophyta


Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a loss of basal forebrain cholinergic neurons and reduced level of the neurotransmitter acetylcholine (ACh) (Alzheimer's Association, 2014). AD affects up to 5% of people over 65 years of age, rising to 20% of those aged over 80 years, suggesting the importance of treating this disease based upon increased life expectancy, particularly in developed countries worldwide. Specifically, estimates show that 27.7 up to 35 million people were affected by this disease from 2005 through 2010, respectively. In this same survey, it was found that treatment costs increased from 156 USD billion to 604 billion (Wimoa et al., 2007, 2013).

Control over AD is achieved by extending the action of acetylcholine (ACh) via acetylcholinesterase inhibition (AChEI). Current drugs exhibit two action mechanisms, either prosthetic or acid-transferring. Prosthetic inhibitors have an affinity for the anionic site of acetylcholinesterase and prevent acetylcholine from accessing it (competitive inhibitors). Acid-transferring inhibitors react with the enzyme and form an intermediate compound. Depending on the stability of this product, the effects could be short-term and reversible or long-acting and irreversible (Nair and Hunter, 2004).

The current drugs used against AD are plant-derived alkaloids: rivastigmine and galantamine. These drugs are AChEIs and can be used to treat early and moderate stages of AD by increasing the endogenous levels of acetylcholine to boost cholinergic neurotransmission (McGleenon et al., 1999). Although these drugs display some undesirable side effects, such as hepatotoxicity and gastrointestinal disorder, there is a growing interest in finding new AChE inhibitors from natural sources, but with few off-target effects (Rhee et al., 1997).

Marine organisms have a high chemical diversity of ecologically active substances with protective functions against predators, biofoulers and epiphytes, as well as against constant changes in abiotic conditions tolerated by algae in the marine coastal environment (Amsler, 2008; Ferreira et al., 2012; Mesko et al., 2015). Such diversity provides a useful resource for the discovery of novel bioactive carbon compounds, many of them with potential applications in the pharmaceutical, nutraceutical, and agricultural fields (Smit, 2004; Cardozo et al., 2006; Gressler et al., 2011a, 2011b; Torres et al., 2014).

Seaweed extracts have been tested against ACh enzyme with successful results (Natarajan et al., 2009). As such, seaweeds represent a promising group of species for developing new marine natural products based on the ease of obtaining biomass by performing cultures for the exploitation of bioactive compounds (Baweja et al., 2009 and therefore contributing to the conservation of natural populations (Yokoya and Yoneshigue-Valentin, 2011).

Hence, motivated by the economic and social impact of AD, this study was aimed at testing the qualitative and quantitative AChEI activity of organic extracts obtained from three Brazilian red algae. The chemical composition of the bioactive fractions was investigated by gas chromatography-mass spectrometry (GC-MS).

Materials and methods

Algal material and extraction

Two hundred grams (fresh weight; equivalent to thirty individuals of each species) of Hypnea musciformis (Wulfen) in J.V. Lamouroux, Ochtodes secundiramea (Montagne) M.A. Howe and Pterocladiella capillacea (S.G. Gmelin) Santelices & Hommersand were collected from a lateritic reef at Manguinhos Beach (20°11′12″ S, 40°11′24″ W) in the municipality of Serra, Espírito Santo State, Brazil. After collection, seaweed biomass was cleaned and immediately transported to the laboratory in dark plastic flasks containing filtered seawater maintained at 22 °C by means of thermal packaging. Furthermore, the seaweeds were properly identified and deposited in the Herbarium VIES, Universidade Federal do Espírito Santo, under voucher numbers 18.853, 18.854 and 18.855, corresponding to H. musciformis,O. secundiramea, and P. capillacea, respectively.

The freshly cleaned seaweeds were weighed and then immediately extracted by using 10 ml of dichloromethane/methanol (DCM/MeOH) (2:1, v/v) per 1 g of seaweed (Machado et al., 2014a). The material was maintained in a dark room at 20 °C for one week, and then the extracts were filtered through a Whatman No. 5 filter paper and concentrated by under low pressure. The extraction yield was calculated for each algal sample. All chemicals used were of analytical grade (Merck, Darmstadt, Germany).

Inhibitory activity of acetylcholinesterase (AChEI)

Qualitative evaluation by autographic assay

The AChEI activity of DCM/MeOH crude extract was detected by using a thin-layer chromatography (TLC) autographic assay as previously described (Marston et al., 2002). Aliquots of 100 μg of each dried seaweed extract and 0.3 μg of physostigmine (Sigma, used as positive control) were dissolved, spotted on TLC layers (Silica gel 60 F254, 10 × 10 cm, layer thickness 0.2 mm, E. Merck, Germany), which were developed with mobile phase hexane:ethyl acetate:methanol (2:7:1 v/v/v), and then dried. Next, the plates were sprayed with the enzyme solution (6.66 U/ml) (Electric eel AChE type V, product no. C 2888, 1000 U - Sigma-Aldrich), thoroughly dried, and incubated in a humid atmosphere at 37 °C for 20 min. Afterwards, the plates were sprayed with 0.25% of 1-naphthylacetate in ethanol (5 ml) plus 0.25% of aqueous Fast Blue B salt solution (20 ml). The spots corresponding to potential acetylcholinesterase inhibitors were identified as clear zones against a purple background.

Retention factor values (Rf) of bioactive compounds were determined and employed for their preparative scale isolation by thin-layer chromatography (20 cm × 20 cm, layer thickness 1.5 mm, 60 F 254, Sigma-Aldrich). Extract samples of 80 mg were applied on preparative plates which were developed under identical conditions to those used at analytical scale essays. After elution, the AChE-positive regions were removed from the plates and extracted with DCM/MeOH (90:10 v/v).

Quantitative evaluation by microplate assay

AChE activity was measured using a modified 96-well microplate assay based on Ellman's method (Ellman et al., 1961, as modified by Rhee et al., 2001). Such extremely sensitive method is based on measuring thiocholine production when acetylthiocholine is hydrolyzed. This is accomplished by the continuous reaction of thiol with 5,5′-dithiobis(2-nitrobenzoic acid). Solutions: A. Tris/HCl 50 mM, pH 8; B. Tris/HCl 50 mM, pH 8, with 0.1% bovine albumin fraction V; C. Tris/HCl 50 mM, pH 8, with NaCl (0.1 M) and MgCl2-6H2O (0.02 M).

To each well of a 96-well microplate, 25 μl acetylthiocholine iodide (15 μM), 125 μl of 5,5′-dithiobis (2-nitrobenzoic acid) in solution C (3 μM DTNB or Ellman's reagent), 50 μl of solution B, 25 μl of seaweed extract dissolved in MeOH and diluted in solution A at concentrations of 1.56, 12.5, 25, 50, 100, 200 and 400 μg/ml were added. The absorbance was measured at 405 nm for 30 sec. After 25 μl of the enzyme AChE (0.22 U/ml) was added, the absorbance was again read every 5 min of incubation for four times. The percentage of AChE inhibition was calculated by comparing the reaction rates of samples to the negative control (10% MeOH in solution A, considering 100% as the total activity of AChE). Physostigmine was used as standard at concentrations ranging from 1.56 to 400 μg/ml. A BioTek ELISA reader (PowerWave(tm) HT Microplate Reader and KC4(tm) software) was used to determine reaction rates. This analysis was carried out in triplicate (n = 3) to calculate the mean and standard deviation.

CG-MS analysis of bioactive fraction obtained from O. secundiramea extract

Currently, different strategies have been reported in the literature in order to estimate algal compounds applying CGMS (Gressler et al., 2009). The bioactive fraction obtained from O. secundiramea was analyzed by a gas chromatograph-mass spectrometer (GCMS-QP2010 Plus, Shimadzu, Japan) provided with a HP-5MS column (30 m × 0.25 mm × 0.1 μm). The samples were injected in split mode at 220 °C, and the transfer line was set to 240 °C. Helium (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min. A linear oven temperature program was employed. The temperature was ramped from 60 to 260 °C at a rate of 3 °C/min and then held at 260 °C for 40 min. Detection was performed in full scan mode, mass-to-charge ratio (m/z) 50-650. Electron impact ionization was employed (collision energy = 70 eV), and the mass spectrometer ion source was maintained at 240 °C. Compounds were identified by their GC retention times and mass spectra (NIST08 Mass Spectral Library and literature data). The area of each peak in the total ion chromatogram was integrated.

Results and discussion

Fig. 1 shows the qualitative results of the autographic assay of acetylcholinesterase inhibition activity (AChEI), and Table 1 displays the fresh seaweed biomass weights, dried extract and extraction yield along with AChEI data of the autographic assay: Rf and bioactive compound intensity.

Fig. 1 TLC qualitative acetylcholinesterase inhibition (AChEI) assay. PC: positive control, physostigmine (0.03 μg). DCM/MeOH extracts (100 μg) of (1) Hypnea musciformis, (2) Pterocladiella capillacea and (3) Ochtodes secundiramea. TLC elution system: hexane:ethyl acetate:methanol (2:7:1 v/v/v). 

Table 1 Seaweed biomass, extract dry weights, extraction yields and autographic acetylcholinesterase inhibition (AChEI) assay results. 

Seaweed Fresh weight (g) Dried extracts (g) Yield (%) Rf of AChEI compounds Intensity of AChEI activity
Hypnea musciformis 200 1.82 0.91 0.01 Low
Pterocladiella capillacea 200 0.4 0.2 0.65 Low
Ochtodes secundiramea 200 2.82 1.41 0.62–0.71 Strong

The AChEI activity of plant extracts is classified according to Vinutha et al. (2007) as potent inhibitors (greater than 50% inhibition), moderate inhibitors (30-50% inhibition) and weak inhibitors (below 30% inhibition). According to this classification, the extracts of O. secundiramea have moderate activity (48.59 ± 0.8%), while H. musciformis and P. capillacea extracts have weak activity (7.21 and 5.38%, respectively), at a higher concentration (Fig. 2).

Fig. 2 Quantitative evaluation of acetylcholinesterase activity (AChE) of the DCM/MeOH algal extracts (1.56, 12.5, 25, 50, 100, 200 and 400 μg/ml), compared to the standard physostigmine. Mean and standard deviation (n = 3). 

As described in the literature, seaweed extracts exhibit poor anti-AChE activity, with IC50 ranging from 2.6 to 10 mg/ml, far smaller than that showed by O. secundiramea extract. The Ecklonia stolonifera ethanolic extract (IC50 100 μg/ml) and Ishige okamurae methanolic and ethanolic extracts (IC50 163.07 μM and IC50 137.25 μM, respectively) are the only exceptions (Stirk et al., 2007; Yoon et al., 2008; Kartal et al., 2009; Suganthy et al., 2010; Pangestuti and Kim, 2011).

DCM/MeOH extract (15 mg) were employed in the fractionation carried out under the same conditions as those described for the analytical TLC experiments. The bioactive fraction (2.45 mg; Rf 0.62-0.71), which corresponded to 16.3% of the crude extract, was analyzed by GC-MS; the resulting data are shown in Table 2 and Fig. 3.

Table 2 GC–MS data from bioactive fraction (Rf 0.62–0.71) obtained from preparative TLC carried out with Ochtodes secundiramea extract. 

Peak Retention time (min) (%) Area Molecular mass m / z M+−halogen Main mass fragments (intensity of signal) Molecular formula Reference
1 18.036 5.23 136 41(50) 69(100) 78, 81(20) 93(90) 107(25) 119(50) 135(80) 136 (trace) C10H16 Polzin and Rorrer (2003)
2 30.913 11.41 229; 230 149 (M+−Br) 41(40) 53(40) 79(70) 91(100) 105 (95) 122(85) 133(55) 149(80) 185(30) 229, 230(70) C10H15OBr Paul et al. (1980)
3 31.636 2.82 292; 294; 296 213(M+−Br) 133(M+ -Br2) 41(15) 65(15) 77(20) 93(50) 105(40) 118(50) 133(100) C10H14Br2 Barahona and Rorrer (2003)
4 36.215 3.09 248; 249; 251 214(M+−Cl) 167(M+−Br) 133 (M+−BrCl) 44(60) 51(10) 65(20) 77(30) 93(100) 105(40) 117(60) 133(60)167 169(20) 212 213(5) C10H14BrCl Burreson et al. (1975)
5 39.856 13.82 311 229(M+−Br) 149 (M+−Br2) 41(50) 55 (50) 65(40) 79(80) 91(100) 105(40) 118(60) 133(95) 149(25) 229(20) C10H12OBr2 Paul et al. (1980)
6 40.682 5.81 323;325 217 (M+−BrCl) 167 (M+−Br2) 133(M+−Br2Cl) 41(40) 65(30) 77(50) 93(95) 105(50) 117(60) 133(100) 167(40) 217(40) C10H13Br2Cl Polzin et al. (2003)
7 43.298 13.81 408; 410 375 (M+−Cl) 329 (M+−Br) 249 (M+−Br2) 167(M+−Br3) 133 (M+−Br3Cl) 41(10) 77(40) 91(50) 105(30) 117(50) 133(100) 167 197 213 249 294 329 375 (5) C10H14Br3Cl Gerwick, 1984
8 45.273 30.13 328; 330; 332 294 (M+−BrCl) 249 (M+−Br) 213 (M+−BrCl) 167 (M+−Br2) 133 (M+−Br2Cl) 69(15) 77(40) 93(50) 105(40) 133(100) 167(50) 213(5) 249(5) 294(3) C10H15Br2Cl Coll and Wright (1987)
9 46.900 2.52 344; 346; 348 310 (M+−Cl) 267 (M+−Br) 229 (M+−BrCl) 185(M+−Br2) 147 (M+−Br2Cl) 65(20) 77(30) 91(100) 105(40) 117(60) 133(60); 147(15) 185, 187 (20) 197, 199(5) 212, 213(5) 229(10); 267 269(5) 285 287 (5); 310 311(5) C10H15Br2ClO Naylor et al. (1983)
10 47.972 18.36 360; 362; 364 329 (M+−Cl) 294(M+−Cl2) 284 (M+−Br2) 247 (M+−BrCl) 213 (M+−BrCl2) 167 (M+−Br2Cl) 133 (M+−Br2Cl2) 77(40) 91(50) 105 (30) 117(50) 133(100) 167(80) 213(40) 247(15) 284(20) 294(15) 329 (5) C10H14Br2Cl2 Coll et al. (1988)

Fig. 3 Total ion chromatogram (TIC) from GC–MS analysis of AChEI fraction obtained by preparative TLC carried out with O. secundiramea extract. 

The active fraction compounds present the typical fragmentation losses related to chlorine and bromine losses (respectively, 35 and 79 m/z), as clearly observed on the mass spectra. The characteristic multiplicity of halogen isotope abundance (35Cl:37Cl - 75.2:24.8% and 79Br:81Br - 50:50%) was observed in their molecular ions. As reported in numerous structural studies, both magnetic resonance spectra (13C NMR) (McConnell and Fenical, 1978; Paul et al., 1980; Naylor et al., 1983) and mass fragmentation (Polzin and Rorrer, 2003; Barahona and Rorrer, 2003) have played a key role in the structural characterization of halogenated monoterpenes (Maliakal et al., 2001; Machado et al., 2014a).

However, it can also be observed in the total ion chromatogram in which the peak 8 corresponds to the majority compound that has m/z value 329. Its mass spectra showed fragment ions at m/z 294, indicative of chlorine loss (M+−Cl), and at m/z 213 and m/z 133, which are characteristic of bromine and chlorine losses (M+−BrCl and M+−Br2Cl), respectively. The ions at m/z 93 and at m/z 133 are observed in the fragmentation of acyclic monoterpenes. This compound (C10H15Br2Cl) has been identified as (E)-1,2-dibromo-3-chloromethylene-7-methyloct-6-ene, by comparison of its mass spectrum data with those found in the literature, and allowed its discrimination among isomeric compounds (Coll and Wright, 1987).

The compounds 1, 2, 7, 9, and 10 could also be identified by comparing the data collected from their mass spectra (Table 2) to the literature ones. Compounds 1 (C10H16), 2 (C10H15OBr), 7 (C10H14Br3Cl), 9 (C10H15Br2ClO), and 10 (C10H14Br2Cl2) are named as 7-methyl-3-methyleneocta-1,6-diene (myrcene) (Polzin and Rorrer, 2003); 6(S*)-bromoocta-1,3(8)-dien-4(R*)-ol (Paul et al., 1980); 2-chloro-1,6(S*),8-tribromo-3-(8)(Z)-ochtodene (Gerwick, 1984); 7-chloro-4,8,dibromo-1-ethyl-3,3-dimethyl-6-hidroxi-7,8-cyclohexene (Naylor et al., 1983); (3R*,4S*,5E)-4,8-dibromo-3,7-dichloro-3,7-dimethylocta-1,5-diene (Coll et al., 1988), respectively.

The compound 2 (C10H15OBr) [6(S*) bromoocto-1,3(8) dien-4(R)-ol] was prepared by synthesis and the remaining ones were not properly identified due to insufficient data: the compounds 3 (C10H14Br2), 4 (C10H14BrCl, and 6 (C10H13Br2Cl) are known as 3,7-dibromo-myrcene (Barahona and Rorrer, 2003); (Z)-10-bromo-7-chloro myrcene (Burreson et al., 1975); and (Z)-10-chloro-3,7-dibromo-myrcene (Gerwick, 1984), respectively.

Mass spectral fragmentation of compound 5 suggests an aromatic structure due to a sequence of peaks at m/z 91, 79, 65, 41; however the great number of isomers having the same structural formula did not allow the compound identification.

Currently, monoterpenes are the second major class of natural metabolites with AChEI activity, surpassed only by alkaloids (Barbosa-Filho et al., 2006), and extracts containing mixtures of monoterpenes generally display high AChEI. They are reversible competitive inhibitors of acetylcholinenesterase (Keane and Ryan, 1999) and such effect is attributed to the combined actions of these multifunctional compounds which can exert their activities through different mechanisms (Koroch et al., 2007). These observations are in accordance with the present study documenting the potential AChEI of an O. secundiramea extract containing halogenated monoterpenes.

Brominated monoterpenes are formed via reactions catalyzed by enzymes capable of generating nucleophilic halide anions, electrophilic hypohalite species, or halogen radicals, promoting the substitution of hydrogen atoms for halogens in aliphatic hydrocarbons (Moore, 2006; Hillwig and Liu, 2014). Their AChEI can result from the easy displacement of bromine by neutral or anionic nucleophiles which could then be removed by elimination reactions (Dagani et al., 2014).

Whereas the toxicity, or side effects, of alkaloids, widely used as a source of potential acetycholinesterase inhibitors, is a drawback in Alzheimer's disease treatment, O. secundiramea extract showed, in preliminary trials, no cytotoxicity or mutagenic activities at concentrations twice as much those tested in the present study (Machado et al., 2014b).

The results presented herewith provided evidence to corroborate the therapeutic potential of O. secundiramea monoterpenes based upon their moderate AChEI activity in combination with minimal off-target effects.


The authors thank financial supports from CNPq and FAPESP, scholarship from CAPES to the first author, and grants from CNPq to MCMY, PC, and NSY. This study is part of thesis presented by the first author to the Graduate Programme in Plant Biodiversity and Environment, Institute of Botany, São Paulo, Brazil. The authors would also like to thank Prof. O. Vieira for proof-reading and editing the manuscript in English.

Appendix A

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2015.09.003.


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Received: April 14, 2015; Accepted: September 7, 2015

* Corresponding author. (N.S. Yokoya).

Authors’ contributions

LPM collected and identified seaweed samples, as well as carried out lab experiments, analyzed the data, and wrote the manuscript. LRC performed chemical and chromatographic analyses and provided critical reading of the manuscript. MCMY and EMCL performed laboratory experiments involving biological studies. DCC and LZV carried out chemical studies. PC and NSY designed the study, supervised the laboratory work and contributed to critical reading of the manuscript. All authors have read the final manuscript and approved the submission.

Conflicts of interest

Authors have no conflicts of interest to declare.

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