Fatty acids profile and anticholinesterase activity of fish lipids from Brazilian Northeast

Acetylcholine deficiency is a neurochemical characteristic of patients with clinical diagnosis of Alzheimer ́s disease. Substances that inhibit the enzyme acetylcholinesterase, increasing levels of acetylcholine in the brain, are a promising form of treatment. Studies relate the use of omega-3 fatty acids in the treatment and prevention of Alzheimer's disease. The Northeast Region of Brazil has an enormous biological diversity and a wide variety of fish species. In this work, the oils of eleven species of marine fish found on the coast of Ceará, Brazil, were analyzed in relation to the fatty acid profile and the inhibitory activity of the enzyme acetylcholinesterase. Total lipids were extracted from fish samples by Folch metodology. The lipid extracts of the fish and industrialized fish oil, used for comparison, were esterified and fatty acid profiles were analyzed. The acetylcholinesterase inhibitory activity was measured quantitatively. The oils presented a high percentage of saturated fatty acids, which is a general characteristic of tropical fish. Oleic acid was the highest monounsaturated fatty acid. Oils of Scomberomorus cavalla, Lutjanus synagris and Haemulon plumieri presented expressive percentages of polyunsaturated fatty acids and the most potent anticholinesterase activities. This research showed the oils of S. cavalla, L. synagris and H. plumieri may be promising functional food products of active fatty acids as new therapies to treatment or prevention of Alzheimer's disease. The expressive concentration of unsaturated and polyunsaturated fatty acids together with their relevant anticholinesterase activity are characteristics of the importance of these fish oils. Research, Society and Development, v. 10, n. 10, e450101018968, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18968 2


Introduction
Alzheimer's disease (AD) is a neurodegenerative disease with a slow and continuous progression affecting mainly the elderly population of the world (over 65 years of age). It has become increasingly worrying in view of the increase in life expectancy. It is the most common cause of dementia. Its characteristics are difficulty in memory, language, problem solving and other cognitive abilities that affect one's ability to perform everyday activities (Alzheimer's Association, 2018).
Several observational epidemiological studies have pointed to the benefits of omega 3, especially at the early stages of AD (Barberger-Gateau et al., 2007;Huang et al., 2005;Wu et al., 2015).
According to the Alzheimer's Association (2018), several changes occur to the brain during the development of the disease. Such changes may result from different factors.
Among the changes observed, decreased quantities of DHA, one of the most abundant fatty acids in a healthy brain, are found in AD patients, especially in the most affected regions (Belkouch et al., 2016;Calviello, Serini & Piccioni, 2008).
Acetylcholine deficiency is also a neurochemical characteristic of clinically diagnosed patients. Acetylcholine is one of the major neurotransmitters by which electrical impulses are driven through nerve cells and transmitted to other nerve cells or to voluntary and involuntary muscles (Elufioye, Obuotor, Sennuga, Agbedahunsi & Adesanva, 2010).
Substances that inhibit the enzyme acetylcholinesterase by increasing the levels of acetylcholine in the brain are a promising form of AD treatment (Huang, Su & Li, 2013). Some drugs have been used for inhibiting this enzyme. On the other hand, natural products such as extracts and substances isolated from plants, algae and other marine organisms have been widely investigated (Bianco et al., 2015;Morais et al., 2017;Penido et al., 2017).
Substances or materials that have a positive effect on cognitive disorders, whether by anti-inflammatory, antioxidant or acetylcholinesterase inhibitory action or by two or more mechanisms, are of great interest for the treatment of AD (Belkouch et al., 2016;Penido et al., 2017).
The Brazilian Northeastern Region has an enormous biological diversity and a great variety of fish species. Fish oils can be investigated as for its pharmacological and biotechnological potential. Thus, the oils of eleven fish species commonly found in the state of Ceará, Northeastern Region of Brazil, were analyzed and an industrialized fish oil commercialized in pharmacies rich in omega 3 fatty acids, was used for comparison. The objective of this work is to evaluate which types of fish oils have the greatest inhibiting action of the enzyme acetylcholinesterase to value local fishes.

Methodology
The present study refers to an experimental qualitative research, with numerical data obtained in the laboratory (Pereira et al., 2018)

Collection and preparation of samples
Fish samples were obtained near the fish market in Fortaleza, Ceará, Northeast of Brazil, from local anglers. Three fresh specimens of each species of sea fishes were purchased. The samples were transported to the laboratory in iceboxes. The heads were removed and the fillets were separated and milled in a processor until a homogeneous mass was formed. This mass was used for analysis the species analyzed were Balistes capiscus (grey triggerfish), Cephalopholis fulva (coney), Industrialized fish oil was purchased from local stores, in a bottle containing 60 capsules, for comparison.

Lipid extraction
Total lipids were extracted from fish samples using the method of Folch (Folch, Lees & Stanley, 1957) in 100 g aliquots of samples and a mixture of chloroform/methanol (3:1).

Transmethylation of fish lipids
The lipid extracts were esterified according to the methodology of the International Union of Pure and Applied Chemistry (Internation Union of Pure and Applied Chemistry [IUPAC], 1987). The method comprises mixing 500 mg of lipids with hexane (5 mL) and KOH at 0.1M into methanol (5 mL) in a capped test tube heated in 50°C water bath for one hour with a subsequent separation of methyl esters.

Determination of fatty acid profiles GC/MS
Fatty acid profiles were analyzed by gas chromatography coupled to a mass spectrometer -Shimadzu Q P-2010 instrument with a DB-5ms fused silica capillary column containing dimethylpolysiloxane (30 m x 0.25 mm x 0.25 μm), drag gas: He (1 mL/min) in constant linear velocity mode, injection temperature: 250°C, and detector temperature: 200°C. The temperature of the column was set from 35 to 180°C at 4°C/min, from 180 to 280°C at 17°C/min and at 280°C for 10 min. The mass spectrum was obtained by electron impact at 70 eV. The compounds were identified by retention times, comparison of the mass spectra obtained with spectra recorded in a database (National Institute of Standards and Technology database NIST: 147,198 compounds, USA), and by visual comparison with spectra published in the catalog of mass spectra (Adams, 2001).

Determination of acetylcholinesterase inhibitory activity,
The inhibitory activity of acetylcholinesterase was quantitatively measured according to the method described by Ellman, Courtney, Valentino and Featherstone (1961)  Absorbance was measured at 405 nm for 30 seconds. Then, 25 μL of acetylcholinesterase enzyme was added and absorbance was measured per minute up to 25 minutes of enzyme incubation. As a negative standard, all solutions were used, except for the sample. The dilutions of the samples and the positive standards used in the quantitative microplate assays, starting from the 20 mg/mL concentration stock solution, were 200µg.mL -1 , 100 µg.mL -1 , 50 µg.mL -1 , 25 µg.mL -1 , 12.5 µg.mL -1 , 6.25 µg.mL -1 , 3.12 µg.mL -1 , 1.56 µg.mL -1 , and 0.78 µg.mL -1 .
The standard used as a positive control was Physostigmine.
All samples were analyzed in triplicate. After data normalization, a non-linear regression curve test was performed using the statistical software GraphPad Prism version 5.01.

Statistical Analysis
Data were expressed as mean ± standard deviation. Differences between values were examined using analysis of variance (ANOVA). The results were compared by Tukey test at a 5% significance level.

Fatty acid profile
The percentage of lipids (g/100 g) of the meat of different fish species varied between 0.10 and 4.80%, according to Table 1. All species could be classified as lean fishes because they had a fat content below 5% (Penfield & Campbell, 1990).
Oils of fish captured on the coast of Ceará showed a high percentage of saturated fatty acids (SFA) ( Table 2). This is a characteristic of fish from warm waters of tropical zones like in the Brazilian Northeast seas (Dey, Buda, Wiik, Halver & Farkas, 1993). In the analysis of lipids of sea fish, other studies also mention palmitic acid as the main constituent among SFA (Andrade, Bispo & Druzian, 2009;Ozogul, Ozogul & Alagoz, 2007;Prato & Biantolino, 2012). The fatty acid profile of commercially available industrialized fish oil is shown in Table 3. In relation to commercially available fish oil, palmitic acid and oleic acid are also presented as the main constituents of SFA and MUFA, but at lower concentrations than most other fish oils, especially in relation to saturated acids. With regard to PUFA, the content was expected since it is a fish oil with a high content of omega 3 fatty acids.

Inhibitory activity of the acetylcholinesterase enzyme
The results obtained in the inhibition of AChE activity were compared to that of the alkaloid physostigmine, which was the first discovered natural inhibitor. Santos et al. (2018), determined the anticholinesterase activity of extracts and fractions from 54 plants and classified the action according to the IC50 values as: high potency when IC50 <20 μg. mL -1 .
Evaluating the results obtained in the AChE inhibition test, it is observed that all fish oils presented results below 20 μg. mL -1 , therefore with a high inhibition power. Then, all oils extracted from regional fish showed good results for acetylcholinesterase Research, Society andDevelopment, v. 10, n. 10, e450101018968, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18968 8 inhibitory activity (AChEI) (Table 4), with emphasis on the oils of the species S. cavalla, L. synagris and H. plumieri, whose values of IC50 (the concentration that inhibited 50% of the activity of the enzyme) were the lowest, corresponding to a greater bioactivity. Oils with high concentrations of PUFA had the best anticholinesterase activity, including the oil used therapeutically.
The oils of L. synagris and S. cavalla, which had the best AChE inhibitory -IC50 values, contain the highest concentrations of DHA among the oils with the best activity.
Some studies on plant oils and plant pulps with high percentages of unsaturated fatty acids (UFA), with emphasis on oleic and linoleic acids, also show results, ranging from moderate to potent in relation to percent inhibition of the AChE enzyme (Fernández et al., 2016;Itriago et al., 2017;Santos et al., 2015;Vinutha et al., 2007).
Polyunsaturated fatty acids, especially EPA and DHA, are among the most studied nutrients with regard to neuroprotective effects. They promote improvement of cognitive abilities and acts on brain neurodegenerative diseases.
The efficient inhibitory activity of the acetylcholinesterase enzyme corroborates the potential of omega 3 fatty acids as they may increase acetylcholine concentration in the brain.
Moreover, a synergistic action between the PUFA anti-inflammatory process, cited by several authors, and inhibition of the enzyme should be evaluated in relation to the importance of PUFA and UFA for the treatment of AD-induced dysfunctions (Calder & Grimble, 2002;Mesquita et al., 2011).

Conclusion
In conclusion, this research indicated that the oils of S. cavalla, L. synagris and H. plumieri as functional food as new therapies to treat or prevent AD. They are promising due to an expressive concentration of UFA and PUFA together with a relevant anticholinesterase activity.
For future works, it is suggested to select fish samples with high levels of fatty acids for further analyses, such as carrying out tests with zebrafish and other models. It is also suggested to carry out new surveys with other fish species from the Northeast Coast to find new study targets. Besides, it is important to encourage the population to consume the fish species that had the best results.