INTRODUCCIÓN

Avocado is an evergreen tree belonging to the order Ranales, family Lauraceae and genus Persea, native to Central America and Mexico that can now be found in most tropical and subtropical regions of the world, such as in Colombia, Perú and Venezuela [1,2]. Among the species is the Persea americana classified by Miller, which developed several subspecies due to its geographical isolation, which originated different botanical types [3,4]. Avocado consumption has increased because it is increasingly valued by consumers, not only for its unique taste and texture but also for its health benefits [5,6].

The separation, isolation and characterization of phenolic compounds in foods, specifically in avocado waste, not only increases the possibility of increasing the number of phytochemicals that can improve the quality of life of people, but also the alternative of using waste by-products (epicarp and seed) at industrial level and thus achieve a comprehensive use of this fruit [7]. Seeds have been found to possess insecticidal, fungicidal and antimicrobial activities with a high content of phenolic compounds, which play an important role in potential health effects [8]. Therefore, the purpose of the present study was to evaluate the antioxidant capacity of the aqueous and methanolic extracts of avocado seed (Persea americana Mill, variety Hass) by three chemical methods.

EXPERIMENTAL

Origin of plant material (PM)

The seeds were obtained from avocados sold in a super market in the city of Maracay, Aragua state, Venezuela.

Sample preparation for extraction

To obtain the alcoholic extract, 10 g of plant material were weighed and previously crushed, placing them in a 400 mL beaker with 300 mL of 70% v/v methanol/water. It was macerated and left to rest for 24 h, and then it was subjected to microwave radiation for 15 s with a power of 125 MHz. The extracts were filtered, using Whatman filter paper number 4. The extract was left to macerate for 24 h, filtering with Whatman filter paper number 4[9].

Determination of total phenolics

For the determination of total phenolics, 50 μL of the alcoholic extract were mixed with 250 μL of the Folin-Ciocalteu 1 N reagent (Analytical grade, Merck). It was left to stand for 8 minutes and then 750 µL of 20% Na2CO3 and 950 µL of distilled water were added. Was incubated for 30 min at room temperature and the absorbance was read on a Genesis 20 UV/VIS spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). A calibration curve for Gallic Acid (Sigma-Aldrich, Germany) was prepared with concentrations of 50, 100, 200, 300, 400, 500 and 1000 ppm. The results were expressed in mg of Gallic Acid Equivalents (GAE) / g of PM [10].

Ferric-Reducing Antioxidant Power (FRAP) Assay

The FRAP test was used to determine the reducing capacity of infusions [11]: 100 μL of infusion were mixed with 3 mL of FRAP reagent consisting of a mixture of 300 mM sodium acetate and acetic acid buffer, 10 mM TPTZ solution (2,4,6-tri(2-pyridyl)-s-triazine), and 20 mM FeCl3 solution, in a volume ratio of 10:1:1.

The reaction was carried out at room temperature for 4 min, and absorbance was recorded at 593 nm using a Genesis 20 UV/VIS spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). FeSO₄ was used as standard, and the results were expressed as µmol Fe ²⁺/g PM.

Free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay

100 µL of sample and 2.9 mL DPPH (100 mM solution of DPPH in 80 % methanol) (Sigma Aldrich) were placed together in a quartz cell. Absorbance (Genesis 20 UV/VIS spectrophotometer) was monitored every 5 min for 30 min at a wavelength of 515 nm. The reference absorbance (A0) was obtained by substituting the sample volume for 80 % methanol. The percentage of DPPH reduction was obtained with the equation DPPH (%) = (A0-An) 100/ A0, where A0 and An were the reference and sample absorbance, respectively [12]. The data were used to determine the IC50 parameter, which represents the concentration (µg.mL-1) of phenolic compounds required to reduce the DPPH free radicals by 50 % [5].

2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid Assay (ABTS)

This test is based on the ability of antioxidants to sequester the cation radical ABTS ^•+(7 mM with K₂S₂O₈ at 2.45 mM)[13]. The reaction mixture was kept in the dark at room temperature for 14 h. After time, the ABTS^•+ solution was diluted with ethanol to obtain an absorbance of 0.70±0.02 at 734 nm, using a Genesis 20 UV/VIS spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). Subsequently, 250 µL of ABTS+ solution diluted with methanol was added to 10 µL of sample. Radical reduction was monitored for 5 min at 734 nm, using ascorbic acid (Vitamin C) as standard. The results were expressed in mg/mL^-1.

Statistical analysis

All determinations were made fivefold and the values ​​were expressed as the means ± the standard deviation. Statistical differences were determined by analysis of variance (ANOVA) by using the Statistic 9.0 program for Windows.

RESULTS AND DISCUSSION

Total Phenolics of the extracts

Prior to the evaluation of the antioxidant activity of the extracts, the concentration of total Phenolics was determined in triplicate, obtaining a concentration of 26.32±2.45 mg GAE/g PM in the aqueous extract and 57, 32 ±2.45 mg GAE/g PM for the methanolic extract, observing a statistical difference (p=0.035). The difference observed is similar to that reported in several studies carried out with Persea americana Mill, which indicate that the extraction of phenolic compounds is influenced by the type of solvent used [14,15]

Antioxidant activity

The antioxidant capacity was determinate as recommended by several studies, which indicate that at least two methods should be used to evaluate the antioxidant activity of an extract [16-18]. The antioxidant effect by the DPPH and FRAP method is shown in Table 1. The antioxidant capacity observed by the DPPH and FRAP methods shows that the extracts possess an important source of antioxidant compounds, a conclusion specially valid when compared to other studies worldwide, which indicate that avocado seeds can be used as a functional food ingredient or as an antioxidant additive [19,20].

Among the methods used to determine the capacity of an antioxidant to capture free radicals, the ABTS radical is one of the most applied, as it is considered highly sensitive, practical, fast and very stable [21]. The ABTS assay showed higher inhibition in the methanolic extract with statistical difference from the aqueous extract (Figure 2). These results coincide with what was reported in Bogota Colombia, whose study consisted in determining the phenolic profile of avocado byproducts, among them the seed, indicating that the extracts showed an antioxidant capacity (ABTS method) related to their content of bioactive compounds, considering these byproducts for pharmaceutical and industrial use [15].

CONCLUSIONS

We may conclude that the seed of Persea americana Mill variety Hass, constitutes an excellent source of phenolic compounds with high antioxidant power, conditioned to the way in which these are extracted from the plant material. Finally, this study supports and extends what has been found in several countries where avocado is a daily food, with high projection of exportation from South America to the Asian continent.

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