The samples of babassu oil were obtained directly from producers in the states of Tocantins and Maranhão, Brazil. Extra-Virgin Babassu Oil (EVBO) was obtained by cold pressing whole, healthy almonds without yellowing or superficial physical damage, followed by decantation, filtration and packaging. The Virgin Babassu Oil (VBO) was obtained by crushing previously selected and toasted almonds, followed by baking the mass to obtain the oil, which was separated by decanting, filtered and reheated for total removal of the water resulting from the cooking step and then packaged.

During the experiments, the following solvents and reagents were used: methanol with purity > 99% (Ecibra, Santo Amaro, São Paulo, Brazil) hexane with purity 98.5% (Synth, Diadema, São Paulo, Brazil), ferric chloride hexahydrate with purity 97% (Sigma-Aldrich, Sant Louis, USA) and ferrous sulfate heptahydrate with purity ≥ 99% (Sigma-Aldrich, Sant Louis, USA). Acetonitrile, methanol, hexane, isopropanol and acetic acid (Sigma-Aldrich, Sant Louis, USA) (both of whit purity ≥ 99.9%). Deionized water (deionizer Marte Científica, Santa Rita do Sapucaí, Minas Gerais, Brazil) and ultrapure water (Ultra water purifier MS2000, Gehaka, São Paulo, Bazil).

Folin-Ciocalteau reagents, DPPH radical solution (2,2_-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris (2-pyridyl) -s-triazine), all from Sigma-Aldrich (Sant Louis, USA) were used for the analysis of antioxidant activity.

The analytical standards for the chromatographic analyzes were: gallic whit purity ≥ 99.9%, caffeic acid whit purity ≥ 98%, catechin whit purity ≥ 99%, quercetin with purity ≥ 99%, rutin with purity ≥ 96%, α-tocopherol with purity ≥ 96%, β-tocopherol, γ-tocopherol with purity ≥ 96% and δ-tocopherol with purity ≥ 96%, lycopene with purity ≥ 95%, β-carotene with purity ≥ 97% and α-carotene with purity ≥ 95%, all from Sigma-Aldrich (Sant Louis, USA).

The determination of the antioxidant capacity is based mainly on two mechanisms of reaction, the transfer of a hydrogen atom and / or the transfer of an electron. In addition to the mechanism, the objective is to determine the protective effect of the material against free radicals, which differ in the initiator radical, reaction kinetics and side reactions [55Apak R, Özyürek M, Güçlü K, Çapanoğlu E. Antioxidant activity/capacity measurement. 1. Classification, physicochemical principles, mechanisms, and Electron Transfer (ET)- Based assays. J Agric Food Chem 2016; 64(5): 997-1027.
[http://dx.doi.org/10.1021/acs.jafc.5b04739] [PMID: 26728425] ]. Thus in investigating the total antioxidant capacity of a substance, it is important that at least one test of each mechanism is used. Thus, the samples were tested for the DPPH radioactivity and ferric reducing antioxidant potential assay (FRAP). In the DPPH assay both mechanisms are involved and in the FRAP assay the transfer of a hydrogen atom is involved [56Antolovich M, Prenzler PD, Patsalides E, McDonald S, Robards K. Methods for testing antioxidant activity. Analyst (Lond) 2002; 127(1): 183-98.
[http://dx.doi.org/10.1039/b009171p] [PMID: 11827390] -58Alves CQ, David JM, David JP, Bahia MV, Aguiar RM. Métodos para determinação de atividade antioxidante in vitro em substratos orgânicos. Quim Nova 2010; 33(10): 2202-10.
[http://dx.doi.org/10.1590/S0100-40422010001000033] ].

The results for the determinations of the antioxidant activity of the different babassu oils studied (EVBO and VBO) can be observed in Table 1. Regardless of the test used, the antioxidant activity was higher for the oil extracted by cooking the babassu mass, ranging from ≈ 9.3 times higher up to 19.7 times higher than for the antioxidant activity of the extra virgin babassu oil.

Results presented as means ± standard deviations of the analyzes of each type of babassu oil. Results followed by different letters in the same line are significantly different by the f test at 5% probability.

Chromatographic conditions were suitable for separating the different phenolic compounds, gallic acid, caffeic acid, catechin, quercetin and rutin (Fig. 1) and tocopherols (α, β, γ, and δ-tocopherol) (Fig. 2) present in the babassu oil samples.

The results for the quantification of the bioactive compounds, phenolics, tocopherols and carotenoids in the different babassu oils (EVBO and VBO) can be observed in Table 2. For most of the compounds, there was no difference between the types of oil. In cases where the oils differed, VBO presented content about three times higher than EVBO, except for gallic acid, where this ratio was reversed. With regard to catechin, it can be observed that for the EVBO samples the presented values are low and with high variability. Thus, the presence of this compound can be confirmed, but it is not possible to determine in what quantities.

Results presented as means ± standard deviations of the analyzes of each type of babassu oil. Results followed by different letters in the same line are significantly different by the test f at 5% probability.

Table 1
Antioxidant activity of babassu crude oils obtained by different extraction methods.

Fig. (1) Chromatogram characteristic of babassu oil samples. Separation of different phenolic compounds: 1) Gallic acid; 2) Catechin; 3) Caffeic acid; 4) Routine; 5) Quercitina.

Fig. (2) Chromatogram characteristic of babassu oil samples. Separation of the different tocopherols: 1) α-tocopherol, 2) β-tocopherol and 3) γ-tocopherol.

Table 2
The content of the bioactive compounds identified in the different types of crude babassu oils.

Analyzes for concentrations of total phenolic compounds showed a significant difference between the types of babassu oil studied. Phenolic compounds are substances that have structures with aromatic rings and double conjugated bonds from which they exert their antioxidant action, besides being the most abundant antioxidants in the diet [59Shahidi F, Ambigaipalan P. Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects – A review. J Funct Foods 2015; 18: 820-97.
[http://dx.doi.org/10.1016/j.jff.2015.06.018] ]. They are generally determined via reaction with the Folin-Ciocalteau reagent. However, this reagent is not only sensitive to phenolic compounds [60Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 2005; 53(10): 4290-302.
[http://dx.doi.org/10.1021/jf0502698] [PMID: 15884874] ]. Other structures with reducing power may also influence the results, for example of conjugated aldehydes, ketones, dienes and trienes, in addition to the melanoidins.

The process of obtaining VBO has two important stages of heating. At first, the mass of crushed kernel is cooked with a little water for oil scoring, which by being less dense floats on the cake and can be separated. In the second stage, this oil collected, still has a certain amount of water and therefore goes through an evaporation process. Thus, this exposure to heat may have favored oxidation and darkening reactions and consequently raised the total phenolic values of virgin babassu oil.

The oxidation of fatty acids leads to the formation of compounds such as aldehydes, ketones, dienes and conjugated trienes, among others [61McClemenets DJ, Decker EA. Lipids Fenema’s food chemistry 2007; 155-216.]. And melanoidins are heterogeneous brown pigments that have aromatic rings, are formed by reactions of non-enzymatic browning [62Mesias M, Delgado-Andrade C. Melanoidins as potential functional food ingradient. Curr Opin Food Sci 2017; 14: 37-42.
[http://dx.doi.org/10.1016/j.cofs.2017.01.007] , 63D. J., & Huber, E. A.Carbohydrates Fenema’s food chemistry 2007; 75-130.]. These pigments are produced during the Maillard reaction. This reaction occurs between the carbonyl groups of reducing sugars and the amino groups of amino acids, peptides or proteins, and is catalyzed by heating [64Ramonaityte DT, Kersiene M, Adams A, Tehrani KA, Kimpe ND. Binding capacity of brown pigments present in special Spanish sweet wines. Food Sci Technol 2009; 42: 1729-37., 65Wang HY, Qian H, Yao WR. Melanoidins produced by the Maillard reaction: Structure and biological activity. Food Chem 2011; 128: 573-84.
[http://dx.doi.org/10.1016/j.foodchem.2011.03.075] ]. The presence of proteins and carbohydrates in the babassu cake [66Castro AM, Castilho LR, Freire DMG. Characterization of babassu, canola, castor seed and sunflower residual cakes for use as raw materials for fermentation processes. Ind Crops Prod 2016; 83: 140-8.
[http://dx.doi.org/10.1016/j.indcrop.2015.12.050] ] together with the heating during the VBO extraction process contribute to the formation of these compounds.

Regarding antioxidant capacity, both types of babassu oils can be considered low antioxidant power. Ferreira et al. (2011) evaluating the antioxidant capacity of different oils, including babassu oil, obtained comparable results. Essential oils such as oregano and thyme, which have high antioxidant capacity, have values between IC50 3.9 and 1.1 mg/mL [67Viuda-Martos M, Navajas YR, Zapata ES, et al. Antioxidant activity of essential oils of five spice plants widely used in a Mediterranean diet. Flavour Fragrance J 2010; 25: 13-9.
[http://dx.doi.org/10.1002/ffj.1951] ]. When compared with excellent antioxidant substances, such as ascorbic acid and BHT [68Cansian RL, Mossi AJ, Oliveira D, et al. Atividade antimicrobiana e antioxidante do óleo essencial de ho-sho (Cinnamomum camphora Ness e Eberm Var. Linaloolifera fujita). Food Sci Technol (Campinas) 2010; 30(2): 378-84.
[http://dx.doi.org/10.1590/S0101-20612010000200014] , 69Ferreira BS, de Almeida CG, Faza LP, et al. Comparative properties of Amazonian oils obtained by different extraction methods. Molecules 2011; 16(7): 5875-85.
[http://dx.doi.org/10.3390/molecules16075875] [PMID: 21750480] ], babassu oil presents activity up to 11.11 x 10⁵ times lower. This may be related to the plant's own anatomy. The babassu kernel is protected within the fruit by at least three layers, epicarp, mesocarp, and endocarp [32Teixeira MA. Babassu–A new approach for an ancient Brazilian biomass. Biomass Bioenergy 2008; 32: 857-64.
[http://dx.doi.org/10.1016/j.biombioe.2007.12.016] , 34Carrazza LR, Ávila JCC, Silva ML. Manual Tecnológico de Aproveitamento Integral do Fruto e da Folha do Babaçu (Attalea spp) 2^nd ed. 2012; 68., 70Lorenzi H. Flora brasileira Lorenzi: Arecaceae (palmeiras) 2010; 367.] that form a protective physical barrier for the constituent substances of this almond, so the plant need not produce other compounds for this purpose.

The differences presented by EVBO and VBO oils, both for the antioxidant activity discussed above and the content of phenolic compounds may be related to the higher release of these bioactive substances from the food matrix when it is heated. The presence of water in the cooking step increases the solubility of flavonoids and phenolic acids, consequently increasing their extraction during the process. With the evaporation of water these components remain in the extracted oil. Equivalent results were demonstrated by Serevinatne et al. (2008) in a study about the influence of the method of coconut oil extraction on the content of phenolic compounds of this product [71Seneviratne KN, Dissanayake MSD. Variation of phenolic content in coconut oil extracted by two conventional methods. Int J Food Sci Technol 2008; 43: 597-602.
[http://dx.doi.org/10.1111/j.1365-2621.2006.01493.x] ].

Generally flavonoids, among them, catechin, quercetin and rutin, are the predominant class among the phenolics present in plant foods. However, the presence of phenolic acids, such as gallic and caffeic acids, is considered beneficial since these compounds are considered multipurpose bioactive. These acids, in addition to the known antioxidant effect, may still have antimutagenic, anticarcinogenic, anti-inflammatory and antimicrobial action [59Shahidi F, Ambigaipalan P. Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects – A review. J Funct Foods 2015; 18: 820-97.
[http://dx.doi.org/10.1016/j.jff.2015.06.018] , 72Xu G, Ye X, Liu D, Ma Y, Chen J. Composition and distribution of phenolic acids in Ponkan (Citrus poonensis Hort. ex Tanaka) and Huyou (Citrus paradisi Macf. Changshanhuyou) during maturity. J Food Compos Anal 2008; 21: 382-9.
[http://dx.doi.org/10.1016/j.jfca.2008.03.003] ].

The tocopherols are part of the compounds with vitamin E activity and are the main antioxidants naturally present in vegetable oils [51Flakelar CL, Prenzler PD, Luckett DJ, Howitt JÁ, Doran G. A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography. Food Chem 2017; 214: 147-55.
[http://dx.doi.org/10.1016/j.foodchem.2016.07.059] [PMID: 2750 7459] ]. The biological activity varies among the isomers, with an increased vitamin activity associated with greater methylation and heightened activity associated with methylation at the fifth carbon, vitamin activity: α > β > γ > δ [6Kerrihard AL, Pegg RB. Utilizing the bioactive contents of specialty oils and fats. Specialty Oils and Fats in Food and Nutrition - Properties, Processing and Applications 2015; 317-48.
[http://dx.doi.org/10.1016/B978-1-78242-376-8.00013-2] , 73Alimentarius C. Codex standard for named vegetable oils (revised, 2015). Codex Standards 2015; 210: 1-14., 74Frank J. Dietary phenolic compounds and vitamin E bioavailability. Model studies in rats and humans. PhD thesis, Swedish University of Agricultural Sciences 2004.]. According to the Specified Oil Standard [73Alimentarius C. Codex standard for named vegetable oils (revised, 2015). Codex Standards 2015; 210: 1-14.] baba ssu oil has low or undetectable levels of these compounds. The results presented in this study show low levels of total tocopherols ( 6.5 mg/100 g of olive oil), and not all of them are found. This fact may be related to the fatty acid composition of babassu oil. Since babassu oil is mostly composed of saturated fatty acids and is therefore more stable, a high concentration of these natural antioxidants is not expected. Other highly saturated vegetable oils, such as coconut oil and palm kernel oil, also have low or undetectable levels of tocopherols [73Alimentarius C. Codex standard for named vegetable oils (revised, 2015). Codex Standards 2015; 210: 1-14.]. On the other hand, in more unsaturated vegetable oils have highly levels of tocopherols, such as palm ( 19 mg/100 g oil) [75Kua YL, Gan S, Morris A, Ng HK. A validated, rapid, simple and economical high-performance liquid-chromatography method to quantify palm tocopherol and tocotrienols. J Food Compos Anal 2016; 53: 22-9.
[http://dx.doi.org/10.1016/j.jfca.2016.09.003] ], soybean oil ( 40 mg/100g oil) [76Khan S, Lisa AS, Obaid M, Chowdhury K. Tocopherol content of vegetable oils/ fats and their oxidative deterioration during storage. World J Pharm Pharm Sci 2015; 4(04): 1537-48.], canola ( 65 mg/100g oil) [77Flakelar CL, Luckett DJ, Howitt JA, Doran G, Prenzler PD. Canola (Brassica napus) oil from Australian cultivars shows promising levels of tocopherols and carotenoids, along with good oxidative stability. J Food Compos Anal 2015; 42: 179-86.
[http://dx.doi.org/10.1016/j.jfca.2015.03.010] ], sunflower ( 80 mg/100g oil) [51Flakelar CL, Prenzler PD, Luckett DJ, Howitt JÁ, Doran G. A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography. Food Chem 2017; 214: 147-55.
[http://dx.doi.org/10.1016/j.foodchem.2016.07.059] [PMID: 2750 7459] ] and olive oil (200-450 mg/100g olive oil) [78Chen H, Angiuli M, Ferrari C, Tombari E, Salvetti G, Bramanti E. Tocopherol speciation as first screening for the assessment of extra virgem olive oil quality by reverse-phase high-performance liquid chromatography/fluorescence detector. Food Chem 2011; 125: 1423-9.
[http://dx.doi.org/10.1016/j.foodchem.2010.10.026] ].