Free radical scavenging ability of Ximenia americana L . stem bark and leaf extracts

1 Chemistry Department, University State Piauí, Teresina – Piauí, Brazil. 2 Chemistry Department, Institute Federal of Pernambuco, Pesqueira – Pernambuco, Brazil. Chemistry Department, Institute Federal of Maranhão, Codó Maranhão, Brazil. Institute of Chemistry and Biotechnology, University Federal of Alagoas, Maceió – Alagoas, Brazil. 5 Chemistry Department, University Federal of Piauí, Teresina – Piauí, Brazil.


INTRODUCTION
Irreversible damage to proteins, carbohydrates, lipids and nucleic acids are some of the factors responsible for the development of numerous degenerative diseases, such as atherosclerosis, Parkinson's disease, Alzheimer's disease, cardiovascular diseases, cancer and diabetes (Lodyato et al., 2004).Fruits and vegetables are important in human diet because they contain antioxidative components that protect against free radicals and that have been associated with reduced risk of these degenerative diseases (Zhang andHamauzu, 2004, Sharma, et al., 2015).The compounds responsible for antioxidant activity are of endogenous origin, such as enzymes and proteins, and of exogenous origin, such as low-molecular-mass substances like vitamins, carotenoids, flavonoids, anthocyanins and other phenolic compounds commonly present in plants (Argolo et al., 2004;Zhang and Hamauzu, 2004;Yin et al., 2015).Several methods have been developed to determine the antioxidant capacity of pure substances and extracts of plants.One of the most reliable methods involves measuring the sequestration of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), or other color radicals (Roginsky andLissi, 2005, Fu et al., 2010).DPPH is a stable free radical at room temperature, with a typical violet color in methanol solution, and its use is indicated as a reagent for testing the radical scavenging activity of small molecules (Roginsky andLissi, 2005, Fu et al., 2010).In this type of assay, scavenging of the DPPH radical is verified by monitoring the decrease of absorbance at 516 nm resulting from the antioxidant activity (AH) (DPPH + AH  DPPH-H + A) (Argolo et al., 2004;Sánchez-Moreno et al, 1998).Ximenia americana is a thorny bush belonging to the family Olacaceae, found in tropical regions of America and Africa (Pott and Pott, 1994).In Brazil it is known by several popular names: ameixa-da-baia, ameixa-da-terra, ameixa- de-espinho and sândalo-do-brasil (Braga, 1960).All the parts of this plant are used in folk medicine (James et al., 2008, Braga, 1960).
The roots have antiseptic properties and are used in the treatment of fever, jaundice and headaches.The leaves have a laxative action and are indicated in the treatment of measles (Omer and Elnima, 2003).The powdered stem is recommended for healing ulcers, as a depurative, as a menstrual regulator and for gastric upsets.The infusion of its flowers serves to reduce bloody diarrhea (Braga, 1960).This plant is also active against rheumatism, cancer and many infections (Mevy et al., 2006).A pharmacological study of X. americana indicates that this species presents anticancer and antineoplastic (Voss, et al, 2006), antimicrobial (Geyid et al., 2005, Omer and Elimina, 2003), antibacterial (Koné et al., 2004, Kawo et al., 2011, Da Silva et al., 2015), activity antipyretic (Soro et al., 2009), radical scavenging (Maikai et al., 2010, Le et al., 2012) and pesticidal activity (Fatope et al., 2000).
In this work, the DPPH radical scavenging activity was evaluated and the total phenolic content of ethanol extracts (EtOH) of the stem bark and leaves of X. americana was determined, as well as the fractions obtained from the liquid-liquid partitioning of the extracts.In addition, biomonitored fractionation of the ethyl acetate fraction of the stem bark and leaves was performed, resulting in the isolation of the flavonoids, epicatechin and quercetin as active compounds.This is the first report on the isolation and characterization of epicatechin in this specie, as well of the DPPH radical scavenging activity and total phenolic content of ethanol extract (EtOH) and fractions of the stem bark of X. americana.

Vegetable material
The stem and leaves of the species Ximenia americana L. were collected in April 2010 in the municipality of Campo Maior, state of Piauí, Brazil, and identified by Dr. Maria Edilene Alencar.An exsiccatum was deposited in the Grasiela Barroso herbarium at the Federal University of Piauí under number TEPB 14407.

Extraction and fractionation of the plant
The collected stems and leaves of X. americana were separated and dried at room temperature (26 ºC1) away from light.Dried and powdered, stem bark (3.3 kg), stem wood (2.5 kg) and leaves (1.5 kg) were extracted three consecutive times with 95% ethanol in a stainless steel extractor at a temperature of 26 ºC1.
Ethanol extracts of the stem bark (620 g, 18%), stem wood (250 g, 10%) and leaves (230 g, 15%) were obtained after concentration in a low-pressure rotary evaporator and elimination of the residual humidity by lyophilization.
The ethanol extracts of stem bark (500 g) and leaves (210 g) were suspended separately in a MeOH/H 2 O mixture (2:3) and subjected successively to the liquid-liquid partitioning process with C 6 H 14 (3 x 700 ml), CHCl 3 (3 x 700 ml) and EtOAc (3 x 700 ml).After removal of the solvent at low pressure in the rotary evaporator and lyophilization of the hydromethanol fraction, four fractions were obtained of the ethanol extract of the leaves: in hexane (15.5 g; 7.4%), in chloroform (7.5 g; 3.2%), in ethyl acetate (80.9 g; 38.5%) and hydromethanol (101 g, 48%), and four fractions of the ethanol extract of the stem bark: in hexane (10.75 g; 1.7%), in chloroform (7.2 g; 1.2%), in ethyl acetate (220 g; 45%) and hydromethanol (240.4 g; 48%).The raw extracts in ethanol and fractions resulting from partitioning were subjected to in vitro antioxidant activity assays.
An aliquot of subfraction C1-EtOAc (40.0 g) was subjected to silica gel column chromatography (300 g).Seven fractions were collected with a volume of 500 mL each, using CHCl 3 , MeOH and mixtures of these as eluants.
The EtOAc (3 g) fraction was subjected to silica gel column chromatography (30 g) and eluted with a mixture of CHCl 3 and MeOH.Thirty-five fractions of 30 mL each were collected, analyzed by silica-gel thin-layer chromatography (CCD), and grouped.The 17-24 (1.5 g, 50%) fraction was purified in a Sephadex LH-20 column using MeOH as solvent.This procedure was repeated twice and led to the isolation of compound 2 (1 g, 66.6%).

Statistical analysis
The results presented in this study correspond to the mean value of three repetitions (n=3) ± standard deviation of the mean.The results of antioxidant activity that presented a probability of occurrence of the null hypothesis of less than 5% (P < 0,05), applying ANOVA followed by Tukey's multiple comparison test, were considered statistically different.All the analyses were performed using Microcal Origin 8.0 software.

Structural identification of the isolated substances
Compound 1 and 2 (Figure 1) was identified to be (-) epicatequin e quercetin, respectively for comparison of the spectroscopic data (IR and the one-and two-dimensional NMR) with those reported in the literature Lopes et al., 2012, Wei et al., 2013.

In vitro radical scavenging activity
Evaluation of the antioxidant activity of extracts and molecules in assays with the DPPH radical is a widely used method because it is easy to perform and less time-consuming than other methods.The reaction can be observed visually by thin-layer chromatography (qualitative assay) and its intensity can be evaluated by chromometric or spectrophotometric assays (Sánchez-Moreno et al., 1998;Soler-Rivas et al., 2000).The DPPH radical is sequestered by antioxidants through the donation of hydrogen to form the stable molecule of DPPH in the reduced form (Argolo et al., 2004).
Figure 2 shows the radical scavenging activity, expressed in percent of inhibition (PI), exhibited by the ethanol extracts of the stem bark, stem wood and leaves of X. americana, in the concentrations of 250, 200, 150, 100, 50 and 25 g/ml.Based on these data, it is evident that the ethanol extracts of the leaves, stem bark and wood of X. americana contain substances that act as hydrogen donors to the DPPH radical, although they show a differentiated action.The ethanol extract of the stem bark showed the highest radical scavenging activity in all the concentrations, and was the most effective in sequestering the DPPH radical, reaching a percent of inhibition of more than 90% at the concentration of 100 g/ml.The ethanol extract of the stem wood showed a PI = 78.29%at the concentration of 250 g/ml, displaying a lesser capacity to sequester the DPPH radical.
Taking into consideration the statistical analysis of the data on radical scavenging activity at all the tested concentrations (25, 50, 100, 150, 200 and 250 μg/ml), and applying ANOVA and Tukey's test, it was found that the ethanol extract of stem bark showed a significant difference (P<0.05) in relation to the ethanol extracts of stem wood and leaves, but did not show a significant difference (P<0.05), as a source of free radical scavenging substances, in relation to the positive control rutin.
The leaf extract showed a higher radical scavenging activity than the stem wood extract and a lower activity than the stem bark extract at all the tested concentrations.A comparison of the percentages of inhibition of the extracts and that of the positive control compound rutin indicated a radical scavenging activity comparable to that of the control, as shown in Figure 2. At a concentration of 100 µg/ml, the ethanol extracts of stem bark, leaves and stem wood showed a percentage of inhibition of the DPPH radical of 94.75% ± 0.27; 57.27% ± 4.20 and 42.83% ± 2.43, respectively.Because the stem wood extract showed less activity, it was not fractionated.
The hexane, chloroform, ethyl acetate and hydromethanol fractions resulting from the liquid-liquid partitioning of the ethanol extracts of stem bark and leaves were subjected to quantitative antioxidant assays.
The hexane fractions of the ethanol extracts of stem bark and leaves presented a low percentage of inhibition of DPPH radical, proving to be poor in antioxidant compounds.The ethyl acetate fractions of stem bark and leaves at the concentration of 100 µg/ml reduced the DPPH by 93.49% ± 2.85 and 92.33% ± 3.12, respectively, indicating that these fractions were the most active, as shown in Figure 3.The flavonoids epicatechin (1) from stem bark and quercetin (2) from leaves were isolated from the ethyl acetate fractions.At the concentration of 50 µg/ml, the isolated compounds epicatechin (1) and quercetin (2) and the positive controls gallic acid and rutin presented a percentage of DPPH inhibition of 62.62% ±2.5; 93.84% ± 0.13; 83.59% ± 3.7 and 64.4% ± 3.73, respectively, as shown in Figure 4.
Figure 5 shows the kinetic behavior of the reaction of DPPH with each extract or control, at the concentration of 100 μg/ml, based on the dose-response curve of the decrease in percentage of remaining DPPH (DPPH REM ) as a function of time (min).As can be seen, almost all the samples reduced the initial concentration of DPPH before of five minute of analysis, a behavior comparable to that of the positive control (rutin), and can therefore be classified as an antioxidant of rapid kinetics (0 to 5 min) (Sousa et al., 2007).Only the samples of ethanol extract and hydromethanol fraction of the leaves were considered antioxidants of slow kinetics (after 30 min).The increasing order of speed of the scavenging action was found to be: rutin control < epicatechin < MeOH/H2O fraction (stem bark) < EtOAc fractions (leaves) < EtOH extract (stem bark) < EtOAc fractions (stem bark) < gallic acid < quercetin, as shown in Table 1.The EC 50 is a parameter widely used to determine the antioxidant power of extracts or substances, and it can be stated that the lower the EC 50 value the higher the free radical scavenging power (Sanchez-Moreno et al., 1998).
The EC50 values of the extracts, fractions and compounds of X. americana were calculated, and the results indicate that the values of the ethyl acetate fractions of stem bark (29.62 μg/mL ± 3.00) and of leaves (33.03 μg/ml ± 2.66) of X. americana are lower than the standard compound, the rutin (47.08 μg/ml ± 4.65) and higher than gallic acid (24.90 μg/ml ± 0.04).The high radical scavenging activity of these fractions may be justified by the presence of epicatechin (1) (34.64 µg/ml ± 3.00) and quercetin (2) (16.40 µg/ml ± 1.30).
Even though the extracts present compounds such as quercetin (leaves) and epicatechin (stem bark), it is not possible to infer unequivocally that these are the only compounds responsible for the observed action, since the raw extract of stem bark and the EtOAc fraction of leaves show a result similar to that of the epicatechin compound.The determination of total phenols is an indirect method of evaluation of antioxidant activity, providing important information that can characterize natural products as sources of free radical scavenging substances (Roginsky and Lissi, 2005).Among the various methods developed for the quantification of phenolic compounds, the Folin-Ciocalteu reagent method has been widely used (Atoui et al., 2005).
The results of the analysis of total phenols represent the behavior found in the analysis of the antioxidant action.The ethanol extract of stem bark and the ethyl acetate, hydromethanol and chloroform fractions showed a total phenol content of 678.66; 878.47; 736.84 and 243.51 mg of EAG, respectively.On the other hand, the ethanol extract of leaves and the ethyl acetate, hydromethanol and chloroform fractions showed a total phenol content of 665.37; 662.24; 625.26 and 449.76 mg of EAG, respectively, as indicated in Table 1.From these results, it can be observed that the evaluated samples presented a high percentage of phenolic compounds.
The highest total phenolic content for ethanol extracts of stem bark and leaves were found in the ethyl acetate fraction.The chloroform fractions of both the stem bark and leaves presented a lower contents of total phenols, which is consistent with the lower antioxidant activity observed in the DPPH assay.

CONCLUSION
In this work, leaves and stem bark of Ximenia americana presented high radical scavenging activity by the DPPH photocolorimetric method and high total phenol content.The stem bark showed better results than the leaves.The quercetin compound isolated from the leaves had a better free radical scavenging activity compared to the positive controls gallic acid and rutin.This is the first report of the occurrence of epicatechin in this specie, as well of the DPPH radical scavenging activity and total phenolic content of ethanol extract (EtOH) and fractions of the stem bark of X. americana.

Fig. 3 :
Fig. 3: Antioxidant activity of the fractions of ethanol extracts of leaves and stem bark of X. americana

Table 1 :
Total phenol (TP) content and antioxidant activity (EC50) of the EtOH extracts and fractions of Ximenia americana.