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Inhibition of Acetylcholinesterase and Fatty Acid Composition in Theobroma grandiflorum Seeds.

1. INTRODUCTION

Cupuacu (Theobroma grandiflorum) from the

Sterculiaceae family is an arboreal fruit species, that reaches a height of 15-20 m in the forest, but remains below 8 m when cultivated [1]. This species is still found wild in the eastern subregion of Brazilian Amazonia, especially in the states of Amazonas, Para, Maranhao, Rondonia and Acre, while commercial crops can be found in other states, like in Bahia. Other tropical countries such as Costa Rica, Colombia, Ecuador, French Guyana, Guyana, and Surinam also cultivate this species [2].

The potential use of the agro-industrial byproducts of cupuacu has only slightly been addressed by the scientific community [3]. Fat extracted from T. grandiflorum seeds is rich in saturated fatty acids [4] and may be used in food products, being extensively as cocoa butter substitute in chocolate-like derivatives [5] and as important ingredient in a variety of cosmetics [6].

The aim of this study was to verify the fatty acid profile present in cupuacu seeds collected in Boa Vista, Roraima state, Brazil, as well as analyzing their acetylcholinesterase enzyme inhibition.

2. MATERIAL AND METHODS

Fruit obtaining and extract preparation

Cupuacu fruit was obtained in Boa Vista city (Roraima state, Brazil). The sample was 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.

Pulp and seeds were manually separated. Seeds were dried, ground to a fine powder and extracted with hexane solvent in a Soxhlet apparatus. The solvent was then removed in a rotoevaporator and the residue was dried slowly under nitrogen stream to furnish an extract containing the fatty material [7].

Fatty acid composition

An aliquot of the extract (12 mg) was placed in a 2.0 mL cryogenic tube and 100 [micro]L of a solution of ethanol (95%) / potassium hydroxide mol [L.sup.-1] (5%) were added. After vortexing for 10 s, fat was hydrolyzed using a microwave oven (Panasonic Piccolo), at a power of 80W for 5 minutes. After cooling, 400 [micro]L of hydrochloric acid 20% were added together with 20 mg of NaCl and 600 [micro]L of ethyl acetate. After vortexing for 10 s, sample was left to 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 [8]. 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 analyses were performed on a HP-7820A chromatograph (Agilent) equipped with a gas flame ionization detector (GC-FID). As data acquisition program EZChrom Elite Compact (Agilent) was used. BP-20 column 15 m x 0.25 mm x 0.20 [micro]m with temperature gradient was used at 80 [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 was 1 [micro]L. Identification of the peaks was performed by comparison with standards of methylated fatty acids C14-C22 FAME (Supelco 18917) [8].

Acetylcholinesterase inhibition assay (in vitro)

Acetylcholinesterase inhibition assay was carried out as per the method of Ellman [9,10] modified [11]. 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 in Tris/HCl at pH 8.0 was added. Then, 25 [micro]L of acetylthiocholine iodide (Electrophorus electricus, Sigma Aldrich) 1000 U [mL.sup.-1]; 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 (0.1% m/v, pH=8) containing bovine serum albumin was added to each well. Absorbance was measured at 405 nm every 1 min for 10 times.

3. RESULTS AND DISCUSSION

Fatty acid composition of the seed fat of Cupuacu

The GC-FID analysis provided nine fatty acids (96.7%), as shown below in the chromatogram of Figure 1 and in Table 1.

Note that among the saturated fatty acids predominating in cupuacu seeds fat are stearic, arachidic e palmitic acids (Table 1). Oleic acid was found in greater proportion in the studied sample (40.0%). The same applies to the unsaturated rate (43.4%). There is a slight difference in the the [omega]-9, [omega]-6 and [omega]-3 contents relating to the literature.

Comparison among chemical composition of fatty acids of fat from cupuacu seeds and butter cocoa were qualitatively similar. However cocoa butter presented higher palmitic acid concentration (23.3%) which seems a disadvantage compared to cupuacu fat since this saturated fatty acid is known to induce atherogenesis [12], weight gain, insulin resistance, inflammation, and is also capable of increasing production of reactive oxygen species [3]. On the other hand, the major fatty acid found in cupuacu fat was oleic acid (40%), a monounsaturated fatty acid (MUFA) also detected in cocoa (36.8%) [13] and inversely correlated with insulin resistance in individuals dyslipidaemia [14].

Inhibition of Acetylcholinesterase

The results for acetylcholinesterase enzyme inhibition by cupuacu seed fat was moderate, as shown in Table 2. This classification is given in the literature [15] in which values higher than 50% of inhibition were considered potent, inhibition values between 30-50% were rated as moderate and below 30% should be considered weak inhibitors.

This enzyme has biochemical importance in humans, but its sudden increase may develop neurodegenerative conditions, one of them is Alzheimer's disease [16-17]. Use of extracts from cupuacu seeds as additive in food could be beneficial for society to help in minimizing the effects of Alzheimer disease since according to the World Health Organization about 107 million people will develop Alzheimer's disease by 2050 [16].

4. CONCLUSION

The fatty acids from cupuacu seeds have profile similar to other fats of importance such as cocoa butter. Its lipid content can bring benefits to human health and may find applications if pharmaceutical, cosmetics and foods industry. The inhibition of acetylcholinesterase reached almost 40%, being of interest since the seeds are an agroindustrial residue. The good results obtained of the fatty acid profile from cupuacu seeds suggests future chemical and biotechnological applications of this fruit species with the intention of developing bioproducts for human health and cosmetic industry.

5. ACKNOWLEDMENTS

We are grateful to CAPES for fellowship and Federal University of Roraima, Brazil.

6. REFERENCES AND NOTES

[1] Quijano, C. E.; Pino, J. A. Food Chem. 2007, 104, 1123. [CrossRef]

[2] Pugliese, A. G. Compostos fenolicos do cupuacu (Theobroma grandiflorum) e do cupulate: Composicao e possiveis beneficios [Master's thesis.] Sao Paulo, Brazil: Programa de Pos-Graduacao em Ciencia dos Alimentos-Bromatologia, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, 2010.

[3] Salgado, J. M.; Rodrigues, B. S.; Donado-Pestana, C. M.; Dias, C. T. S.; Morzelle, M. C. Plant Foods Hum. Nutr. 2011, 66, 384. [CrossRef]

[4] Gilabert-Escriva, M. V.; Goncalves, L. A. G.; Silva, C. R. S.; Figueira, A. J. Sci. Food Agr. 2002, 82, 1425. [CrossRef]

[5] Genovese, M. I.; Tomas-Barberan, F. A.; Truchado, P.; Genovese, M. I. J. Agric. Food Chem. 2013, 61, 2720. [CrossRef]

[6] Alves, R. M.; Sebbenn, A. M.; Artero, A. S.; Clement, C.; Figueira, A. Tree Genet. Genomes 2007, 3, 289. [CrossRef]

[7] Santos, R. C.; Melo Filho, A. A.; Chagas, E. A.; Takahashi, J. A.; Ferraz, V. P.; Costa, A. K. P.; Melo, A. C. G. R.; Montero, I. F.; Ribeiro, P. R. E. African Journal Biotechnology 2015, 14, 2377. [CrossRef]

[8] Christie, W. W. Gas Chromatography and Lipids: A Practical Guide. Ayr: The Oil Press, 1989.

[9] Ellman, G. L.; Courtney, K. D.; Andres Jr., V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88. [CrossRef]

[10] Rhee, I. K.; van de Meent, M.; Ingkaninan, K.; Verpoorte, R. J. Chromatogr. A 2001, 915, 217. [CrossRef]

[11] Teles, A. P. C.; Takahashi, J. A. PAecilomide, a new acetylcholinesterase inhibitor from Paecilomyces lilacinus. Microbiological Res. 2013, 168, 204. [CrossRef]

[12] Luccas, V. Fracionamento termico e obtencao de gorduras de cupuacu alternativas a manteiga de cacau para uso na fabricacao de chocolate. [Doctoral dissertation.] Campinas, Brazil: Faculdade de Engenharia Quimica, Universidade Estadual de Campinas, 2001.

[13] Matos, T. M. Estudo do aproveitamento do residuo da gordura de cupuacu para biodiesel. [Master's thesis.] Manaus, Brazil: Programa de Pos-Graduacao em Quimica, Universidade Federal do Amazonas, 2013.

[14] Sala-Vila, A.; Cofan, M.; Mateo-Gallego, R.; Cenarro, A.; Civeira, F.; Ortega, E.; Ros, E. Clin. Nutr. 2011, 5, 590. [CrossRef]

[15] Vinutha B.; Prashanth D.; Salma K.; Sreeja S. L.; Pratiti D.; Padmaja R.; Radhika S.; Amit A.; Venkateshwarlu K.; Deepak M. J. Ethnopharmacol. 2007, 109, 359 [CrossRef]

[16] Trevisan, M. T. S.; Macedo, F. V. V.; Meent, M. V.; Rhee, I. K.; Verpoorte, R. Quim. Nova 2003, 26, 301 [CrossRef]

[17] WHO, World Health Organization (2012). World Health Organization and Alzheimer's Disease International. Dementia: a public health priority. 2012, pp. 112 [Link]

Casandra Valentina Itriago Yanes (a) *, Antonio Alves de Melo Filho (a), Pedro Romulo Estevam Ribeiro (a), Ana Cristina Goncalves Reis Melo (a), Jacqueline Aparecida Takahashi (b), Vany Perpetua Ferraz (c), Diana Maruja Sagama Mozombite (d), and Ricardo Carvalho dos Santos (e)

(a) 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.

(b) 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.

(c) Chromatography Laboratory, Institute of Exact Sciences, Department of Chemistry, UFMG. Av. Antonio Carlos, 6627-31270-901--Belo Horizonte-MG, Brazil.

(d) Multi-Institutional Post-graduate program in Biotechnology, PPGBIOTEC of the University Federal of Amazonas, UFAM, Av. Gral. Rodrigo Otavio Jordao Ramos, no. 3000, CEP 69077-00, Manaus, AM, Brazil. eNational Postdoctoral Program of CAPES, PNPD/CAPES, associated to the Postgraduate Program in Agronomy of Universidade Federal de Roraima, POSAGRO/UFRR, Campus Cauame, BR 174, s/n, Km 12, District Monte Cristo, CEP 69310-250, Boa Vista-RR, and to the Brazilian Agricultural Research Corporation, Embrapa, Rodovia 174, Km 8, Industrial District, CEP 69301970, Boa Vista-RR-Brazil

* Corresponding author. E-mail: casandraitriago@hotmail.com

Article history: Received: 08 October 2016; revised: 17 April 2017; accepted: 25 May 2017. Available online: 19 June 2017. DOI: http://dx.doi.org/10.17807/orbital.v0i0.894

Caption: Figure 1. Cupuacu seeds fat gas chromatogram.
Table 1. Fatty acids in cupuacu seeds fat.

Fatty acid                          Retention    Amount   Cupuacu 1
                                    Time (min)    (%)       [11]

Lauric acid (C12:0)                    5.1        0.2        --
Myristic acid (C14:0)                  7.8        0.6        --
Palmitic acid (C16:0)                  10.4       8.0        7.0
Stearic acid (C18:0)                   12.8       32.7      34.2
Oleic acid ([omega]-9) (C18:1)         13.0       40.0      41.9
Linoleic acid ([omega]-6) (C18:2)      13.5       3.1        3.2
Linolenic acid ([omega]-3(C18:3)       14.2       0.3        --
Arachidic acid (C20:0)                 15.1       10.4      11.2
Behenic acid (22:0)                    17.2       1.4        1.7
Saturated                               --        53.3      54.3
Monounsaturated                         --        40.0      42.3
Polyunsaturated                         --        3.4        3.4

Fatty acid                          Cupuacu 2   Cupuacu 3   cocoa
                                       [2]        [12]       butter
                                                               [11]

Lauric acid (C12:0)                    --          --            --
Myristic acid (C14:0)                  --          --           0.1
Palmitic acid (C16:0)                 7.54        7.68          23.3
Stearic acid (C18:0)                  32.6        31.52         34.0
Oleic acid ([omega]-9) (C18:1)        41.31       42.55         36.8
Linoleic acid ([omega]-6) (C18:2)      4.9        3.76          2.7
Linolenic acid ([omega]-3(C18:3)       0.2         --            --
Arachidic acid (C20:0)                10.51       9.49          0.7
Behenic acid (22:0)                   1.72         1.4           --
Saturated                             52.81       50.09         58.1
Monounsaturated                       42.12       42.55         36.8
Polyunsaturated                       5.07        3.76          2.7

Cupuacu 1, 2, 3 and cocoa butter to data from the literature.

Table 2. Percentage inhibition of acetylcholinesterase
in cupuacu seed fat.

Cupuacu seed     % Inhibition   Classification
                 40.48          moderate
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Title Annotation:Full Paper
Author:Yanes, Casandra Valentina Itriago; de Melo Filho, Antonio Alves; Ribeiro, Pedro Romulo Estevam; Melo
Publication:Orbital: The Electronic Journal of Chemistry
Article Type:Report
Date:Apr 1, 2017
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