Evaluation of the antioxidant activity of the aqueous and methanolic extracts of
seeds of Persea Americana Mill, variety hass, from the state Aragua in Venezuela
Evaluación de la actividad antioxidante de los extractos acuoso y metanólico de semillas de
Persea Americana Mill, variedad hass, provenientes del estado Aragua en Venezuela
]]> Franklin Pacheco-Coello*, Desirée Seijas-Perdomo
The high content of bioactive compounds in Persea americana seed has generated great interest worldwide due to its various potentialities, in which its antioxidant capacity stands out. The objective of the study was to evalúate the antioxidant activity of the aqueous and methanolic extracts of Persea americana Mili seeds, variety Hass, sold in supermarkets in the city of Maracay, Venezuela. The total phenol content of the extract was determined through the Folin-Ciocalteau method to then evalúate the antioxidant activity by three chemical methods (DPPH, FRAP and ABTS). A higher concentration of total phenolic compounds was obtained in the methanolic extract, with a statistical difference with respect to the aqueous extract. The antioxidant activity was higher in the methanolic extract in the three methods used (p <0.05). These results show the high antioxidant power of the avocado seed, considered as a waste product, which allows proposing strategies that allow its use for medicinal purposes.
Keywords. Avocado, Antioxidant, Total phenols, FRAP, DPPH, ABTS.
Resumen
El alto contenido de compuestos bioactivos en la semilla de Persea americana, ha originado un gran interés a nivel mundial por sus diversas potencialidades, en la que destaca su capacidad antioxidante. El estudio tuvo como objetivo evaluar la actividad antioxidante de los extractos acuoso y metanólico de semillas de Persea americana Mili, variedad Hass, comercializadas en supermercados de la ciudad de Maracay, Venezuela. Se determinó el contenido de fenoles totales del extracto a través del método Folin-Ciocalteau para luego evaluar la actividad antioxidante por tres métodos químicos (DPPH, FRAP y ABTS). Se obtuvo una mayor concentración de compuestos fenólieos totales en el extracto metanólico, con diferencia estadística respecto al extracto acuoso. La actividad antioxidante fue superior en el extracto metanólico en los tres métodos empleados (p<0.05). Estos resultados muestran el alto poder antioxidante de la semilla de aguacate, considerada como producto de desecho, lo que permite proponer estrategias que permitan su uso con fines medicinales.
Palabras clave. Palta, Antioxidante, Fenoles totales, FRAP, DPPH, ABTS.
INTRODUCCIÓN
Avocado is an evergreen tree belonging to the order Ranales, family Lauraceae and genus Persea, native to Central America and México 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 infoods, specifically in avocado waste, not only ulereases the possibility of increasing the number of phytochemicals that canimprove the quality of life of people, but also the alternative of using waste by-produets (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 effeets [8]. Therefore, the purpose of the present study was to evalúate the antioxidant capacity of the aqueous and methanolic extraets of avocado seed (Persea americana Mili, variety Hass) by three chemical methods.
EXPERIMENTAL
Origin ofplant 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 aleo ho lie extract, 10 g ofplant 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 extraets were filtered, using Whatman filter paper number 4. The extract was leñ to macérate 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 aleo ho lie extract were mixed with 250 μL of the Folin-Ciocalteu 1 N reagent (Analytical grade, Merck). It was leñ 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 Génesis 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 infiisions [11]: 100 μL of infusión were mixed with 3 mL of FRAP reagent consisting of a mixture of 300 mM sodium acétate and acetic acid buffer, 10 mM TPTZ solution (2,4,6-tri(2-pyridyl)-s-triazine), and 20 mM FeCl3 solution, inavolume ratio of 10:1:1.
The reaction was carried out at room temperatura for 4 min, and absorbance was recorded at 593 nm using a Génesis 20 UV/VIS spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). FeSÜ4 was used as standard, and the results were expressed as umol 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 (Génesis 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 radicáis 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 catión 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 ABT.+ solution was diluted with ethanol to obtain an absorbance of 0.70±0.02 at 734 nm, using a Génesis 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 inmg/mL-1.
Statistical analysis
All determinations were made fivefold and the valúes 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 triplícate, 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 Mili, which indicate that the extraction of phenolic compounds is influenced by the type of solvent used [14,15]
Antioxidant activity
The antioxidant capacity was determínate as recommended by several studies, which indicate that at least two methods should be used to evalúate 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 conclusión 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 radicáis, the ABTS radical is one of the most applied, as it is considered highly sensitive, practica!, fast and very stable [21]. The ABTS assay showed higher inhibition in the methanolic extract with statistical difference fromthe aqueous extract (Figure 2). These results coincide with what was reported in Bogotá 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 Mili 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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