Oil in Inaja Pulp (Maximiliano, maripa): Fatty Acid Profile and Anti-acetylcholinesterase Activity.
The inaja palm (Maximiliana maripa) is from Arecaceae family. It is considered a large plant (can reach up to 20 meters in height) and its distribution occurs in flooded areas . This palm is very hardy, regenerates quickly in the environments where it is burned [2, 3].
Geographically, the inaja palm is widely distributed throughout the Amazon to west-central South America. It is easily in the central and northern regions of Brazil . In the state of Roraima, Brazil, which borders the north of Venezuela, there is a significant population of this palm in these areas of savannah.
Oil is extracted from M. maripa seed and pulp for human consumption [3 ,4]. The inaja fruit is rich in fatty acids and minerals (phosphorus and magnesium) , and has interest as biofuel in the process of obtaining biodiesel .
The aims of this study is to verify the fatty acid profile in inaja pulp collected in Roraima state, Brazil, as well as analyzing their acetylcholinesterase enzyme inhibition.
2. MATERIAL AND METHODS
2.1 Obtaining Fruit and Extracting Oil from Inaja Pulp
The fruits were obtained in Mucajai (Roraima, Brazil), in Tantinho region, at 451 miles on the BR 174 highway (2[degrees]27'44" N, 60[degrees]55'10" W). Samples were taken to the Laboratory of Environmental Chemistry in the Center for Research and Post-Graduate in Science and Technology (NPPGCT) of the Federal University of Roraima, Boa Vista city, Roraima.
The pulp was removed, washed and dried for 24 hours at 50[degrees]C in an oven with air circulation. Pulp was grounded and sieved on a 20 to 40 mesh fabric to obtain a homogeneous granulation. Lipid extraction gave Soxhlet apparatus with hexane solvent, any procedure effected in triplicate .
2.2 Inaja Oil Analysis by GC-FID
Were dissolved in 2.0 mL cryogenic tube approximately 12 mg of the oil sample in 100 [micro]L of a solution of ethanol (95%) / potassium hydroxide mol L-1 (5%). After vortexing for 10 s, oil was hydrolyzed in a domestic microwave oven (Panasonic Piccolo), at a power of 80W for 5 minutes. After cooling, 400 [micro]L of hydrochloric acid 20% was added a spatula tip of NaCl (about 20 mg) and 600 [micro]L of ethyl acetate. After vortexing for 10 s rest for 5 minutes. An aliquot of 300 [micro]L of the organic layer was removed, placed into micro centrifuge tubes, dried by evaporation, thus obtaining free fatty acids . Subsequently, the free fatty acids were methylated with 100 [micro]L of [BF.sub.3] / methanol (14%), by heating for 10 minutes in water bath at 60[degrees]C. These samples were diluted in 400 [micro]L of methanol and analyzed by gas chromatography.
The analyzes were performed on a HP7820A chromatograph (Agilent) equipped with a gas flame ionization detector. As data acquisition program was used, EZChrom Elite Compact (Agilent). HP-INNOWax column 15 m x 0.25 mm x 0.20 [micro]m with temperature gradient was used: 120[degrees]C, 0 min, 7 [degrees]C [min.sup.-1] to 240 [degrees]C; injector (Split 1/50) detector at 250[degrees]C and 260 [degrees]C. Hydrogen was used as carrier gas (3.0 mL [min.sup.-1]), Injection volume 1[micro]L. Identification of the peaks was performed by comparison with standards of methylated fatty acids C14-C22 FAME (Supelcocat no 18917) .
2.3 Acetylcholinesterase Inhibition Assay
Aliquots of a working solution (25 [micro]L) (sample in DMSO 10 mg [mL.sup.-1]) were added to microplate wells and positive and negative controls were also prepared. To the first five wells of a column (positive control) 25 [micro]L of an eserine solution prepared at 10 mg [mL.sup.-1] (31 mM; 2.7 mM in the whole reaction mixture 275 [micro]L) in Tris/HCl at pH 8.0) was added. Then, 25 [micro]L of acetylthiocholine iodide (ATChI, Sigma A5751) 15 mM; the reaction mixture, 125 [micro]L of 5',5-dithio-bis (2- nitrobenzoate) (DTNB, Sigma D8130) (3 mM) and 50 [micro]L of Tris/HCl (50 mM, pH 8) containing 0.1% (m/v) bovine serum albumin was added to each well. Absorbance was measured at 405 nm every 1 min for 8 times. Then 25 [micro]L (0.226 U [mL.sup.-1]) of Electric eel AChE (type VI-S) provided by Sigma (C3389-500UN) in Tris/HCl was added to each well. Absorbance was measured at 405 nm by 10 times [9,10].
3. RESULTS AND DISCUSSION
3.1 Fatty Acids Determination in Inaja Pulp
The GC-FID analysis provided 11 fatty acids (94.01%), as shown below in the chromatogram of Figure 1 and in Table 1.
Note that the unsaturated fatty acids, UFA, predominate in the inaja pulp oil studies of Roraima are oleic, linoleic and linolenic acids (Table 1). The oleic acid which is found in greater proportion in the studied sample (22.32%).
This acid is with values close to the inaja and other fruits of the Amazon region, as shown in Table 1. To saturated fatty acids, SFA (62.78%), the most abundant in this oil are palmitic (20.76%), myristic (20.48%) and lauric acids (17.42%). There is a difference of lauric and myristic sample studies compared with inaja of literature, but the values are close to as palmitic acid, even to other oleaginous species. The same applies to the unsaturated, UFA (22.56%), note the rn-9 and rn-6 sample and literature, while the [omega]-3 there is a slight difference. The amount of polyunsaturated fatty acids, PUFAs, differs from inaja of literature, but it is close to other species in Table 1. According to Crowley and Frohlich (1998)  differences may occur due biotic and abiotic factors that influence the composition and concentration of fatty acids.
Table 2 provides a comparison of the main constituent of olive oil, oleic acid . The amount of oleic acid inaja pulp lipid is similar to that found in cocoa butter and higher than coconut oil [16, 17].
Observed that the percentage of linoleic acid in the inaja pulp is 4.72% and 3.95% for linolenic acid, found in higher concentrations than in coconut oil .
Fatty acids when in equilibrium in the diet have benefits for human health by preventing heart disease, inflammation and even neurodegenerative diseases. Some fatty acids such as linoleic and linolenic acids, mammals do not synthesize and can only be obtained through diet [18, 19].
3.2 Inhibition of Acetylcholinesterase
The test results for acetylcholinesterase enzyme inhibition by inaja pulp oil is considered potent, as Table 3. This classification is given by Vinutha et al. (2007)  in which higher than 50% inhibitions are potent and inhibition values between 30-50% are moderate and below 30% are weak inhibitors.
This enzyme has biochemical importance in humans, but its sudden increase may develop neurodegenerative diseases, one of them is Alzheimer disease [20, 21].
The inaja pulp oil could be beneficial for society because according to the World Health Organization estimated about 115 million people develop Alzheimer's disease by 2050 . Other Amazonian plants or found in the Brazilian Amazon have equal importance, namely: Combretum laurifolium (crude extract) , Lantana camara (essential oil)  and Annona hypoglauca (oil) .
The oil from the inaja pulp has a fatty acid profile similar to other oils or fats, so its lipid content can bring many benefits to human health. Thus, we can see a potent inhibition of acetylcholinesterase, almost 64%. The good results obtained of the fatty acid profile from inaja suggests future studies of chemical and biological prospecting for this plant species with the intention of developing bio products for human health.
Article history: Received: 26 July 2015; revised: 17 December 2015; accepted: 23 January 2016. Available online: 31 March 2016.
We are grateful to CAPES and CNPq for their fellowship.
6. REFERENCES AND NOTES
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Ismael M. Fernandez (a) *, Diana M. S. Mozombite (b), Ricardo C. Santos (a), Antonio A. Melo Filho (a,b), Pedro Romulo E. Ribeiro (b), Edvan A. Chagas (a,c), Jacqueline A. Takahashi (d), Vany P. Ferraz (e), Ana C. G. Reis de Melo (b), and Selvin A. S. Maldonado (f)
(a) Post-Graduate in Biodiversity and Biotechnology Program, Bionorte, State Coordination of Roraima, Federal University of Roraima, UFRR, Campus Cauame, BR 174, s/n, Km 12, District Monte Cristo, CEP 69310-250, Boa Vista-RR-Brazil.
(b) Post-Graduate in Chemistry Program, Center for Research and Post-Graduate Studies in Science and Technology, NPPGCT, UFRR, Av Capitao Ene Garcez, no. 2413, Campus Paricarana, CEP 69310-000, Boa Vista-RR-Brazil.
(c) Embrapa-Brazilian Agricultural Research Corporation. Rodovia 174, Km 8, Industrial District, CEP 69301970, Boa Vista-RR-Brazil.
(d) Institute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, UFMG, Av Antonio Carlos, no. 6627, Pampulha, CEP 31270-901, Belo Horizonte-MG-Brazil.
(e) Chromatography Laboratory, Institute of Exact Sciences, Department of Chemistry, UFMG.
(f) Post-Graduate in Agronomy Program, POSAGRO, Campus Cauame, UFRR.
* Corresponding author. E-mail: email@example.com
Captions: Figure 1. Inaja pulp oil chromatogram.
Table 1. Fatty acids in inaja pulp oil. Sample Retencion Amount Fatty acid Time (min) (%) Capric acid (C10:0) 0.761 0.38 Lauric acid (C12:0) 1.563 17.42 Myristic acid (C14:0) 2.862 20.48 Palmitic acid (C16:0) 4.742 20.76 Palmitoleic acid (C16:1) 4.903 0.24 Stearic acid (C18:0) 6.867 3.40 Oleic acid ([omega]-9) (C18:1) 7.071 22.32 Linoleic acid ([omega]-6) (C18:2) 7.562 4.72 Linolenic acid ([omega]-3) (C18:3) 8.197 3.95 Arachidic acid (C20:0) 8.977 0.34 Behenic acid (22:0) 11.040 - Saturated 62.78 Monounsaturated 22.56 Polyunsaturated 8.67 Compared with the literature Inaja Tucuma Palma Fatty acid   Yagua  Capric acid (C10:0) - 1.0 - Lauric acid (C12:0) 4.6 0.1 0.5 Myristic acid (C14:0) 10.7 0.2 0.3 Palmitic acid (C16:0) 25.1 22.6 22.2 Palmitoleic acid (C16:1) 0.3 0.4 - Stearic acid (C18:0) 1.6 3.0 2.6 Oleic acid ([omega]-9) (C18:1) 39.2 64.7 64.0 Linoleic acid ([omega]-6) (C18:2) 12.9 4.7 2.6 Linolenic acid ([omega]-3) (C18:3) 1.5 3.6 7.3 Arachidic acid (C20:0) 1.3 0.2 0.2 Behenic acid (22:0) - - - Saturated 43.3 27.1 25.8 Monounsaturated 39.5 65.1 64.0 Polyunsaturated 14.4 8.3 9.9 Table 2. Profile main fatty acids in edible oils and fat. % [omega]-9 [omega]-6 [omega]-3 Olive oil  67.0-81.0 3.5 -14.0 0.3-1.2 Cacao butter  26.3 41.0 0.34 Coconut oil  5.0-10.0 1.0-2.5 0.2 Table 3. Percentage inhibition of acetylcholinesterase and its classification. % Inhibition Classification Inaja 63.76 Potent
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|Author:||Fernandez, Ismael M.; Mozombite, Diana M.S.; Santos, Ricardo C.; Filho, Antonio A. Melo; Ribeiro, Pe|
|Publication:||Orbital: The Electronic Journal of Chemistry|
|Date:||Jan 1, 2016|
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