Total phenolic content

To determine the total phenolic content, Folin-Ciocalteu technique was used (19). Phenolic compounds were extracted from the oil with a solution of water/methanol 60:40 (by volume). Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to suitable aliquots of the extracts. After 3 min, a sodium carbonate solution (35%) was added to the mixture, which was diluted with water to a final volume of 1000 μL. Absorbance was measured after 2 h at a wavelength of 725 nm on a Shimadzu spectrophotometer UV-1800 (Kyoto, Japan). The blank was composed of all the constituents of the reaction except that the phenolic solution was substituted by distilled water. The course of the except was accompanied by a calibration curve with standard gallic acid (Sigma-Aldrich). Total phenolic content was expressed in mg of gallic acid equivalents (GAE) per kg.

Total carotenoid content

Total carotenoid content in oil and fat samples was determined by a spectrophotometric method (20) adapted by the Laboratory of Oils and Fats at FEA, Unicamp, Brazil. Samples were diluted in 100% hexane (Synth, Diadema, SP, Brazil) at a concentration of 0.004 g/mL. The absorbance readings were performed at 453 nm using a Shimadzu spectrophotometer UV-1800. Mass of the tested oil was enough to obtain absorbance values between 0.5 and 0.8. Total carotenoid content was calculated and expressed as μg of β-carotene per g of oil and the specific absorption coefficient of 1% hexane was ε=2592∙10² mL/(g∙cm).

Tocopherol determination

The mass fractions of tocopherols were determined using the methodology described in AOCS (21). Samples,
diluted in hexane at a mass concentration of 0.01 g/mL, were analyzed by UHPLC (Series 200a; PerkinElmer, Waltham, MA, USA), with a LiChrosorb® Si 60 Hibar® HPLC column (250 mm×4 mm, 5 μm i.d.; Sigma-Aldrich) and a fluorescence detector (Series 200a; PerkinElmer). The mobile phase used was hexane (99%) and isopropanol (1%) (J.T.Baker, Phillisburg, NJ, USA) and the detection was performed at 290 nm excitation and 330 nm emission. Compounds were identified by chromatographic comparisons with authentic standards. Quantification was based on the fluorescence signal response, using the internal standard method, and the results were expressed in mg per g of oil samples.

DPPH radical scavenging activity

Radical scavenging activity in the samples was studied by reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich) radicals (22). Samples were prepared in ethyl acetate (Synth) at concentrations of 5 to 100 µg/mL. An ethyl acetate solution of DPPH radicals of 0.2 mM was freshly prepared. Then, 50 µL of oil solution and 150 µL of DPPH solution were mixed. The decrease in absorption at 520 nm was measured for 16 min using a NovoStar microplate reader (BMG LABTECH, Ortenberg, Germany). Measurements were performed in triplicate. Radical scavenging activity (in %) was estimated from the differences between the absorbance of DPPH solution with or without control sample and was calculated according to the following equation:

ORAC assay

Oxygen radical absorbance capacity (ORAC) assays were performed in a 96-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland), using fluorescein (Sigma-Aldrich) as a fluorescent probe (23). The measurements were performed in triplicate using a NovoStar Microplate reader (BMG LABTECH) with fluorescence filters at 485 nm excitation and 520 nm emission. The reaction, performed at 37 °C, was started by thermal decomposition of 2,2’-azobis(2-methylpropionamidine) (AAPH; Sigma-Aldrich) in a 75 mM-phosphate buffer (pH=7.4). ORAC values were defined as the difference between the area under the fluorescein decay curve and the blank (net area under curve, AUC). The course of the reaction was accompanied by standard curve with 6-hydroxy-2,5,7,8-
-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-
-Aldrich). Regression equations between net AUC and antioxidant concentration were calculated. ORAC-fluorescein values were expressed in μmol of Trolox equivalents (TE) per mg of samples (23, 24).

Sample preparation

Oil samples were mixed in dimethyl sulfoxide (DMSO)/Tween 80 (9:1) (Sigma-Aldrich) at an initial concentration of 80 mg/mL. Then, Eagle’s minimum essential medium (EMEM; Sigma-Aldrich) was added to reach the final oil concentrations from 0.5 to 20 mg/mL. Emulsified systems were used for all tests with mammalian cells and they were processed on ULTRA-TURRAX tube drive workstation (IKA, Staufen, Germany) for 5 min at 4000 rpm.

Cytotoxicity assay

Ninety-six well plates (TPP Techno Plastic Products AG) were inoculated with HepG2 cells at a density of 2^∙10⁶ cell/mL. After incubation for 24 h, the adherent cells were washed with phosphate-buffered saline (PBS; Sigma-Aldrich) and then incubated for 5 h with EMEM (Sigma-Aldrich) containing different concentrations of oil emulsions (0.5-4 mg/mL). Untreated cells were used as positive controls, and the emulsified system without the sample was taken as control. The average result of positive controls was considered as 100% HepG2 viability. After removing the medium, cells were treated with 10 µL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich) and 90 µL of PBS, and the plate was incubated at 37 °C for 3 h (25). The formed formazan crystals were dissolved by adding 100 µL of 10% sodium dodecyl sulfate (Synth) in 0.01 M HCl (Synth) to each well, and the plate was incubated for 18 h. Absorbance measurements (at λ=540 nm) were performed using a NovoStar microplate reader (BMG LABTECH).

HepG2 endogenous catalase activity

HepG2 cells were seeded in 60-mm Petri dishes at 8∙10⁵ cell/dish. The cells were incubated in EMEM with 10% FBS, changing the medium every 2 days until they reached 80% of confluence. They were washed twice with PBS, treated with 3 mL of the samples (0.5 and 1 mg/mL of oil) and the emulsion control (without the sample) and incubated for 5 h. After that, they were once again washed with PBS at 4 °C, scrapped and centrifuged for 10 min at 4 °C and 266^×g (Sorval Legend XT; Thermo Fisher Scientific, Waltham, MA, USA). Supernatant was discharged, the pellet was re-suspended in 200 µL of PBS at 4 °C and placed in the ultrasonic cold bath (Unique Ultrasonic Cleaner; Indaiatuba, SP, Brazil) for 5 min. The protein content of the cell extract was evaluated by Bradford assay (26). The enzymatic activity of the extracts was analyzed using the catalase assay kit CAT100 (Sigma-Aldrich), according to manufacturer’s instructions.

Tocopherols

Among the natural antioxidants, the most widely used in edible vegetable oil are the tocopherols. Antioxidant activity of these compounds is given by their ability to provide hydrogen of the phenolic group to oil free radicals, interrupting the chain of lipid peroxidation (27). In this study, α- and γ-tocopherols were the major tocopherols detected in buriti oil (Table 1). The β- and δ-tocopherol fractions are also present in this oil, although in lower mass fractions. Contrarily, tocopherols were not detected in murumuru fat sample. The non-interesterified blend, mostly produced with buriti oil, is also rich in α- and γ-tocopherols, with low concentrations of β- and δ-tocopherol.

Regardless of the used enzymes, the restructured lipids had similar mass fraction of α-tocopherol as the non-interesterified blend. The γ-tocopherol fraction was significantly increased and the δ-tocopherol fraction was reduced after the interesterification. Commercial lipase, when used alone, caused a more expressive decrease in β-tocopherol fraction. These results may indicate the occurrence of modifications of tocopherol isomers in the restructured lipids, revealing or reducing the antioxidant effect of the molecules. However, further studies need to be conducted to confirm these results.

Overall, the enzymatic interesterification had little effect on the mass fraction of tocopherols. The restructured lipids had significant amounts of α- and γ-tocopherol fractions. The α-tocopherol is considered a potent lipid-soluble antioxidant, playing a role in the in vivo mechanisms of action. The importance of this function is to maintain the integrity of long-chain polyunsaturated fatty acids present in the cell membranes (28). The γ-tocopherol is recognized as the most active in the removal of reactive nitrogen species with anti-inflammatory activity and possible action in inhibiting carcinogenesis mechanism (29).

Total carotenoids

Carotenoids are considered natural antioxidants, like tocopherols. When present in the oil, these compounds act to protect the lipid peroxidation, by scavenging free radicals. In addition to this protection, these natural antioxidants are also known to play a role in chronic disease prevention. Among different forms of carotenes, the β-carotene is the most important to the human body, being an important precursor of vitamin A (30, 31).

Table 1 shows that buriti oil is a source of total carotenoids determined as β-carotene (781.6 μg/g). Other studies with buriti oil also confirm that it is an important source of β-carotene (6, 7, 31). Murumuru fat had lower mass fraction of total carotenoids than buriti oil and interesterified samples. The effect of interesterification of buriti oil and murumuru fat on carotenoid content is presented in Table 1. Enzymatic interesterification did not significantly alter the final mass fraction of carotenoids. All produced restructured lipids kept similarly high content of carotenoids from the buriti oil. This result is important because one of the most interesting and appealing characteristic of the buriti oil is its high mass fraction of carotenes, and the studied process maintained this attribute in the new lipid samples. Other studies using Lipozyme TLIM treatment in different types of oil showed similar results, reducing the carotenoid content after interesterification (32, 33). This type of enzymatic interesterification normally occurs under mild conditions, maximizing the retention of minor compounds, contrarily to chemical interesterification (7, 34).

Total polyphenols

Phenolic compounds are considered the main antioxidant metabolites in plants. They have the ability to donate hydrogen or electrons to form stable radicals (35). In the present study, the enzymatic interesterification produced restructured lipids in which the antioxidant phenolic compounds have been preserved. Buriti oil showed to be a valuable source of phenolic compounds (Table 1). Although the mass fraction of total phenols in buriti is lower than in olive oil (170-210 mg/kg), it is larger than in other vegetable oils (36). The non-
-interesterified blend and the restructured lipid samples had the same mass fraction of total phenolics (p<0.05), indicating that the interesterification had no effect on the concentration of these compounds. Total phenolics were also determined in the study of Nagaraju and Belur (37), where coconut and olive oil were treated with the commercial lipase IM-60 (Novo Nordisk, Copenhagen, Denmark), showing no significant differences between blends and restructured lipids (approx. 115 mg GAE/kg).