Chemical characterization of pressed and refined licuri ( Syagrus coronata ) oils

Knowing the composition of foods is fundamental to assess their potentialities and the availability of nutrients to consuming populations. The objective of this work was to increase knowledge about the licuri fruit (Syagrus coronata) by quantifying the fatty acids and verifying the antioxidant potential in the kernel ́s fruit and in the pressed oil ́s fruit. The lipid profile data shows a predominance of saturated fats, high levels of medium-chain fatty acids such as lauric and myristic fatty acids, a composition very similar to the coconut oil. Antioxidant activity was observed using methods of DPPH radical sequestration, ferric reduction and total level of phenolic compounds. The oil characterization in the Licuri fruit brings the prospect of using the same in cosmetic and pharmaceutical industry, as well as a healthy alternative for consumption by the population living in the semi-arid region in Brazil.


Introduction
Due to its continental size, Brazil has an abundance of foods, especially from vegetal source that are either yet to be nutritionally characterized or need better characterization.Knowing the nutritional composition of foods is fundamental to assess the availability of nutrients, so they can be adequately consumed by the population.Furthermore, it is also important for verification of the nutritional adequacy of diets, for the development of studies about the relationships between diets and diseases, for agricultural planning and its use by the food, chemical and pharmaceutical industry.
Licuri or ouricuri (Syagrus coronata) is a poorly studied palm tree commonly found in the Brazilian Cerrado, and thus it is part of the group of foods that need better characterization.The species has clear preference for the dry, arid regions of Brazil's Caatingas.Its distribution includes the north of Minas Gerais, the whole eastern and central portion of Bahia and the south of Pernambuco, including the states of Sergipe and Alagoas.However it blossoms and yields fruit throughout the year, it is in the months of March, June and July that it bears more fruit, which characterizes its harvest period (BONDAR, 1938).
Licuri has been studied mainly as a source of animal nutrition, especially ruminants, and more specifically, caprines.Numerous studies demonstrate that supplementing animal nutrition with licuri oil has positive aspects such as improved sensorial acceptance of goat's milk and its fatty content, and an improvement in the milk's stability (QUEIROGA et al., 2010).
The licuri fruit is commonly found as part of the diets of many small communities in the Brazilian Northeast, where its extract that is a kind of milk and its kernel are consumed also as sweets, liqueurs and ice creams.
Its kernel is also used for oil extraction because of the high oil content (54%) (PINTO et al., 2012).Licuri oil is similar to the coconut palm (Cocus nucifera, Lin) oil (BONDAR, 1938), with 83.2% short-and medium-chain fatty acid content (QUEIROGA et al., 2010).It is used in diets as a source of vegetable oil, and in the production of high quality soaps, given that licuri oil is considered the best Brazilian oil for this type of production.
Due to its similarity with coconut oil, currently linked to a number of health benefits such as weight loss, and in an attempt to expand consumption and research, the objective in carrying out this study was to improve knowledge about this native Brazilian specie by quantifying the fatty acids and lipids present in kernel oil and licuri pressed oil, as well as studying its antioxidant potential.

Sampling
Samples of licuri fruit kernel were obtained from markets at Chapada Diamantina, Bahia.Samples of cold-pressed licuri fruit oil were obtained from a producer's cooperative in the Piemonte da Diamantina region, Bahia.

Fatty acid assay
The assays were carried out at the Chromatographic Assay Center (CEACROM) of Universidade Estadual do Sudoeste da Bahia.
The methodology proposed by Bligh and Dyer (1959) was used to get the oil from the kernels of the licuri fruit finely processed.These extractions were carried in triplicate.
Both lipids obtained from kernel extraction (n = 3) and pressed oil samples (n = 3) were transesterified according to the methodology proposed by Bannon et al. (1982), with modifications according to Simionato et al. (2010).The chromatographic assay was carried out using a Thermo-Finnigan Trace-GC-Ultra gas chromatographer equipped with a flame ionization detector (FID) and a BPX-70 fused-silica capillary column (120 m, 0.25 mm i.d.).The operation parameters established after verification of the best resolution conditions were: injector and detector temperatures, 250 and 280°C, respectively.The column temperature was programmed at 140°C for 10 minutes, proceeding through a first ramp of 15°C min. - until it reached 200°C for 1 minute.The second ramp was 10°C min. - until reaching 230°C for 1 minute.
The third ramp was 0.4°C min. - until it reached 233°C for 3 minutes.The fourth ramp was 0.5°C min. - until it reached 238°C for 2 minutes.Total assay time amounted to 41.50 minutes.Gas (White Martins) flows were 30 mL min. - for hydrogen, 30 mL min. - for nitrogen, and 250 mL min. - for synthetic air.
Injections (1.2 μL) were carried out in duplicate and the fatty acids methyl ester peak areas were determined with software ChromQuest 4.1.
Fatty acid (FA) identification was carried out after verification of the peak equivalent chain length and comparison of sample retention times with a standard sample containing a mixture of fatty acid methyl esters (189-19 Sigma, EUA), as described by Simionato et al. (2010).
Quantification of FA in mg g -1 of total lipids was carried out in relation to the internal standard, methyl tricosanoate (23:0), Sigma, EUA.The calculation of FA concentrations contained in the samples were carried out according to Joseph and Ackman (1992), using the following equation 1: where: A X = fatty acid methyl esters area; A 23:0 = internal standard area; M 23:0 = mass of internal standard added to the sample (in milligrams); M A = sample mass (in grams); F RT = theoretical response factor of fatty acid methyl esters; F CT = conversion factor to express results in mg of fatty acids per g of total lipids (LT).
Verification of agreement between theoretical response and experimental factors was carried out as described by Simionato et al. (2010) andCosta et al. (2011).
The detection limit was visually estimated (RIBANI et al., 2004) from successive dilutions of 10 times each of a methyl tridecanoate standard 2.0 mg mL -1 until peak disappearance.The last dilution where the characteristic peak could be identified was determined as the detection limit.The quantification limit was stipulated as being 10 times larger than the detection limit.The extracts were obtained according to Seneviratne et al. ( 2009), using 20 ml of Methanol/Water (80:20, v v -1 ) and 1.0 g of the sample.

Antioxidant activity by sequestration of DPPH radical
The antioxidant activities of licuri refined and pressed oils were measured through the reaction with radical DPPH (2,2-difenil-1-picrilhidrazil), according to Brand-Williams et al. (1995), with a few modifications.Different methanol extract aliquots from the samples -200, 350 and 500 μLwere mixed with 2.0 mL of DPPH methanol solution (4.70%).The mixture was left in the dark for 30 minutes.Absorbance was measured at 517 mm, where methanol was used as blank.The inhibition percentage of radical DPPH was determined according to equation 2. where: Abs DPPH• = DPPH• absorbance Abs amostra = sample absorbance The results of % inhibition DPPH• were plotted into a graph as a function of the extract concentration and IC 50 , a value that estimates the necessary antioxidant concentration to inhibit 50% of the DPPH radical, was found with linear regression.
Antioxidant activity using the ferric reduction antioxidant power method (FRAP) Ferric reduction capacity in the licuri kernelextracted oil and in the pressed oil obtained from the cooperative was measured according to the methodology proposed by Rufino et al. (2006).After obtaining the methanol extracts, three dilutions were prepared, 0.125, 0.25 and 0.5 mg mL -1 , for the refined oil and pressed oil extracts.
Linear analytical curves were obtained using ferrous sulphate solutions in concentrations between 2000 and 20 M.The result was expressed by the quantity of ferrous sulphate in M produced per gram of licuri refined oil and/or pressed oil.

Total phenolic compounds
The levels of total phenolic compounds in the samples were determined from the preparation of three dilutions -0.125, 0.25 and 0.5 mg mL -1 -of licuri refined oil and pressed oil extracts, according to the procedure proposed by Wettasinghe and Shahidi (1999), using the Folin-Ciocalteau reagent (FCR).
Linear analytical curves were obtained using a gallic acid stock solution in a 1 mg mL -1 concentration, which was diluted so as to obtain concentrations between 0.5 and 0.01 mg of gallic acid equivalent mL -1 .
Total phenolic compounds present in the extracts obtained were expressed in M of ferrous sulphate per gram of licuri refined oil and/or pressed oil.

Fatty acid analysis
Fatty acids detection and quantification limits were estimated based on methyl tridecanoate, resulting in values 0.015 and 0.15 mg g -1 of fatty acids, respectively.The analytical curve went linear from 0.005 mg g -1 , R 2 = 0.9995, with the following equation: Y = 1.10 7 X + 11948 where Y is the chromatographic response and X is the concentration of fatty acid methyl ester.
The results obtained for the quantification of fatty acids in licuri refined and pressed oils are presented in Table 1.The levels of saturated fatty acids in the refined oil and pressed oil samples were approximately five times higher than unsaturated fatty acids.The most predominant saturated fatty acid on both samples was lauric acid, at 43.4 and 44.2% of total fatty acids present in the refined oil and the pressed oil, respectively.This was followed by myristic acid (13.89 and 14.45%) and oleic acid (12.83 and 12.08%) for both samples.Similar results were found by La Salles et al. (2010), in a study about fatty acid composition in refined licuri oil for biodiesel production.
Coconut oil samples used by Chandrashekar et al. (2010) while studying hypocholesterolemic effects, presented a predominance of the same fatty acids with values of 48.5, 21.2 and 5.2%, results that prove the similarities in the composition of both oils.
This lipid composition demonstrates that licuri oil is rich in Medium-Chain Fatty Acids (MCFA), some 60.02 and 60.50% of the composition for refined oil and pressed oil, respectively.These fatty acids have from 6 to 12 carbons and have unique properties with important nutritional and medical applications.
Among the nutritional applications is its use as supplement for athletes who exercise for long periods, as reported by Gomes and Aoki (2003).Differently from long-chain fatty acids, mediumchain acids are oxidized rapidly during rest periods and exercise, which makes them a quick source of energy to the organism, preventing it from consuming stocks of muscular glycogen, therefore delaying fatigue.MCFA demonstrate also to be beneficial in the treatment of pathologies caused by deficient lipid absorption, burns and generalized infections (GOMES; AOKI, 2003).
Population studies by Fife (2005) show the possible health benefits of MCFA in the treatment or prevention of diseases such as cancer and diabetes.Such studies state that contrary to what was thought, coconut oil does not influence cholesterol concentrations in blood, helps to decrease body fat accumulation and avoids blood clot formation, therefore it is not linked to cardiac disease.
A study by Silva et al. (2011) assessing the effects of extra virgin coconut oil supplement on the lipid and cardiovascular profile of hypercholesterolemic individuals concluded that consumption of 30 mL a day of oil can significantly reduce body weight, body mass index, triglycerides, very low density lipoprotein (VLDL) and shows a tendency to reduce total cholesterol, low density lipoprotein (LDL) and to slightly increase high density lipoprotein (HDL).Due to the similar compositions of coconut and licuri oils, we suggest that similar results can be achieved with consumption of licuri oil.
Furthermore, licuri refined and pressed oils presented respectively 6.87 and 6.92% of palmitic acid in their composition, lower than the levels found in coconut oil 7.83% (NEVIN; RAJAMOHAN, 2006), corn 12.00% and soy 12.66% (JORGE et al., 2005), cotton 23.91% and palm 48.86% (CORSINI et al., 2008).These can be considered positive results, since palmitic acid is one of the saturated fatty acids that have the most hypercholesterolemic or atherogenic effect (DRISKELL, 2006).

Antioxidant activity potential
The various analytical assays used to determine antioxidant capacity are mainly based on two reaction mechanisms, Hydrogen Atom Transfer (HAT), and Single Electron Transfer (SET).Both mechanisms have the objective of determining the protective effect of the sample against free radicals, however they are different as to the initiating radical, the kinetics of the reaction and the side reactions (CASTELO-BRANCO; TORRES, 2011).Thus, when investigating the total antioxidant capacity of a substance it is important to use at least one assay from each mechanism.In the DPPH radical assay both mechanisms are involved, and in the ferrous reduction assay (FRAP) only the transference of one hydrogen atom is involved.
The results from the investigation into the antioxidant potential of licuri refined and pressed oils, both for the DPPH radical assay and the ferrous ion reduction (FRAP) are presented on Table 2. IC 50 is defined as the concentration of substrate that can inhibit 50% of the activity of free radical DPPH (BRAND-WILLIS et al., 1995).This concentration can be considered high, if compared with antioxidants par excellence, such as ascorbic acid (IC 50 = 2.15 μg mL -1 ) and BHT (IC 50 = 5.37 μg mL -1 ) (CANSIAN et al., 2010).
A comparative study between the antioxidant activities of cold and hot extracted coconut oil carried out by Seneviratne et. al (2009), demonstrated that extraction at high temperatures can incorporate a higher quantity of antioxidants.Furthermore, there are no other studies in literature about the antioxidant potential of oils extracted from fruit kernels, however, articles related with plant extracts or essential oils prove the low antioxidant activity of licuri refined oil (ANDRADE et al., 2012;CANSIAN et al., 2010).
Phenolic compounds are substances that have structures with aromatic rings and conjugated double bonds from which they exert their antioxidant activity, and they are the most abundant antioxidants in diets.Phenolic compound concentrations were 189.93 ± 23.72 and 553.33 ± 89.62 mg equivalent of gallic acid per gram of licuri pressed oil and refined oil, respectively.In a study carried out by Gutfinger (1981), the phenolic content of olive oil varied according to the method of extraction.Oils extracted with solvents had higher quantities of phenols than virgin oils extracted by press.These results are similar to those found in the present study, and justify the increased antioxidant activity for licuri kernel oil in all assays (NEVIN; RAJAMOHAN, 2006;SENEVIRATNE et al., 2009).
There is growing interest in research for new sources of antioxidants, especially those that can prevent the pernicious effects of free radicals on the human body and on food fats and other compounds.Among them, natural sources of antioxidants are being emphasized, especially foods that are rich in these compounds and that can be consumed by the population as part of a balanced diet.

Conclusion
The results obtained demonstrate that just as with coconut oil, licuri is a good source of mediumchain fatty acids and also has a significant quantity of phenolic compounds and oxidant potential, which can open perspectives in order to improve consumption of this native fruit of the Brazilian Caatinga, since these substances can bring important health benefits.

Table 1 .
Fatty acid quantification (mg g -1 ) of licuri refined kernel oil and pressed oil.

Table 2 .
Antioxidant capacity of licuri refined and pressed oils as determined by DPPH• assay and ferrous reduction capacity (FRAP).