Contents of constituents and antioxidant activity of seed and pulp extracts of Annona coriacea and Annona sylvatica

The antioxidant potential of fruit pulp and seeds of extracts of the Annona coriacea, and A. sylvatica (Annonaceae) were investigated, as well contents total phenolics, flavonoids, condensed tannins and ascorbic acid. Was used to determine the antioxidant activity the 1,1-diphenyl-1-picrylhydrazyl free radical (DPPH), β-carotene bleaching and ABTS radical cation method. The total phenol, total flavonoid, condensed tannin, and ascorbic acid contents were measured spectrophotometrically. In this study, the pulp and seeds of the fruits were extracted using methanol/water (8:2) for maceration. The seed extracts of A. coriacea demonstrated a moderate antioxidant effect with free radical scavenging activity of 31.53%, by the DPPH test, 51.59% by the β-carotene bleaching test and 159.50 μM trolx/g of extract in the ABTS assay. We found that the hydromethanolic seed extract of A. coriacea had high total phenol (147.08 ± 4.20 mg of GAE/g of extract) and flavonoid (131.18 ± 2.31 mg of QE/g of extract) content. This indicated that the antioxidant activity of the extracts was related to the contents of these constituents.


Introduction
Free radicals and other oxidants have been associated with being responsible for the development of a number of chronic and degenerative diseases such as cancer, cardiovascular diseases, cataract, Alzheimer's, aging, immune system decline, and cerebral disorders (Atoui et al., 2005;Barreiros et al., 2006;Sian, 2003).The production of free radicals is controlled in living things by several antioxidant compounds, which can come from the diet and other sources (Alasalvar et al., 2005;Atoui et al., 2005).These fruits have attracted considerable attention as a source of natural antioxidants.
The genus Annona (Annonaceae) consists of 250 species distributed across Brazil, and has a great variety of of fleshy, odorous and tathy fruits, known as "araticum" or "marolo", widely consumed "in natura" and used by the population to prepare juice, ice-cream or jelly (Silva and Tassara, 2001).The other parts of the fruit are also widely used in folk medicine for antiparasitic or antitumoral treatment of intestinal diseases.The infusion of leaves and powdered seeds is used to combat diarrhea and induce menstruation (Almeida et al., 1994;Silva et al., 1994).In Brazil, A. coriacea, "marolo", is popular used against chronic diarrhea (Rodrigues and Carvalho, 2001), antimalarial (Mesquita et al., 2007), anti-helmintic (Santos andSant'Ana, 2000, 2001), and leishmaniasis (Akendengue et al., 1999).The leaves of the A. sylvatica, "araticum da mata", are used to treat malaria or as a febrifuge (Balbach, 1986).On consulting the literature, no reports on the comparative studies of the antioxidant activities of A. coriacea or A. sylvatica were found.
Methods were developed for a quick, simple, and reliable quantification of the antioxidant capacity.In general, the methods were divided into two major groups: Assays based on a single electron transfer (SET) reaction, with a change in color as the oxidant is reduced, and assays based on a hydrogen atom transfer (HAT) (Huang et al., 2005), which measures the activity of the antioxidant to scavenge the peroxyl radicals, such as, the total radical trapping antioxidant parameter (TRAP) assay, the oxygen radical absorbance capacity (ORAC) assay, and the luminol-chemiluminescence-based peroxyl radical scavenging capacity (LPSC) assay (Alho and Leinonen, 1999;Huang et al., 2005;Ou et al., 2001).The assays electron transfer includes the reaction of ferric reduction (FRAP), the α-tocopherol/Trolox equivalent antioxidant capacity (TEAC), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Benzie and Strain, 1996;Brand-Williams et al., 1995;Huang et al., 2005;Re et al., 1999).
The beneficial effects of the plant food are attributed to the variety of their phytochemistry including the polyphenols, which have revealed a remarkable spectrum of biochemical and pharmacological actions, thought to be due to their antioxidative and free-radical scavenging properties (Noroozi et al., 1998).
In the present study, we evaluated the antioxidant activity of Annona coriacea and A. sylvatica fruits (pulp and seeds), measured by DPPH, β-carotene bleaching, and the ABTS radical cation method, in our search for natural antioxidants.In addition, a number of parameters were determined to characterize this activity, among which are: Total polyphenols (TPP), total flavonoids (TF), condensed tannins (CT), and ascorbic acid content (TAA).

Plant material and extract preparation
The fruits of A. coriacea Mart. and A. sylvatica A. St. -Hil.were collected in December 2011, in Dourados, in the state of Mato Grosso do Sul, Brazil, which is located at an average altitude of 452 m; at 23°17'6" S latitude and 54°43'28" W longitude.The plants were identified by Dr. Zefa Valdevina Pereira, who is a Professor at the University Federal of the Grande Dourados, and a voucher specimen was deposited at the Herbarium of this University, A. coriacea (DDMS 186) and A. sylvatica (DDMS 4600).For preparing the extracts, the fruit pulp and air-dried and powdered seeds of each species were separately and successively extracted by maceration with methanol/water (8:2), at room temperature.The extract was filtered, concentrated under pressure in a rotaevaporator at 50 °C, and lyophilized.The extraction yield of the crude extracts was determined from the mass of the material prior to extraction and the mass of extract obtained after removal of methanol/water.The extraction yield was calculated in percentage after weighing.

Total phenol content
The total phenol content of the samples was determined using the Folin-Ciocalteau reagent (Djeridane et al., 2006).Briefly, 100 μL of extracts in methanol (1 g/L) were mixed with 1.0 mL of distilled water and 0.5 mL of Folin-Ciocaleu's (1:10 v/v) reagent.After mixing, 1.5 ml of 2% sodium bicarbonate was added, and the mixture was allowed to stand for 30 min with intermittent shaking.The absorbance was measured at 765 nm using a spectrophotometer.The total phenolic content was expressed as a gallic acid equivalent (GAE) in milligrams per gram (mg/g) of extract.The methanol solution was used as a blank.

Total flavonoid content
The amount of total flavonoids in the extracts was measured spectrophotometrically, as reported (Lin and Tang, 2007).Briefly, 500 μL of each extract was mixed with 1.50 mL of 95% ethanol, 0.10 mL of 10% aluminum chloride (AlCl 3 .6H 2 O), 0.10 mL of acetate sodium (NaC 2 H 3 O 2 .3H 2 O) (1 M), and 2.80 mL of distilled water.After incubation for 40 min, the absorbance was measured at 415 nm, using a spectrophotometer.To calculate the concentration of flavonoids, we prepared a calibration curve by using quercetin as the standard.The flavonoid content was expressed as quercetin equivalents (QE) in milligrams per gram (mg/g) of extract.

Condensed tannin content
Condensed tannin concentrations were determined by a modified version of the method developed by Maxson and Rooney (1972).The samples were mixed with 5 mL of the vanillin-HCl reagent (8% concentrated HCl in methanol and 4% vanillin in methanol).The absorbance at 500 nm was read after 20 min.Catechin was used as reference.The condensed tannin content was expressed as catechin equivalents (CE) in milligrams per gram (mg/g) of extract.

Ascorbic acid content
The determination of TAA was adapted from the spectrophotometric method developed by Roe and Kuether (1943), for the estimation of ascorbic acid content.A sample of 0.1 mL (100 mg extract/10 mL methanol HPLC) was added to 2, 4 dinitrophenylhydrazine reagent (2,4-DNPH).It was allowed to stand for 30 minutes and the absorbance was read in triplicate at 515 nm, using distilled water as blank.The result was expressed in milligrams of ascorbic acid per gram of extract.

DPPH free radical scavenging assay
Free radical scavenging activities of the test samples and of the positive control butylhydroxytoluene (BHT) were determined using the DPPH free radical method (Blois, 1958).Various concentrations of the samples were added to 3 mL of methanol DPPH solution (0.1 mM) prepared daily.The mixture was shaken and left to stand at room temperature, in the dark.After 30 min, the absorbance was measured at 517 nm against a blank (containing all reagents except the test samples).The assays were carried out in triplicate.The concentrations of the samples for 50% inhibition of DPPH (IC 50 ) were obtained from the graph of I% (inhibition percentage) versus a concentration of the sample in microgram per milliliter (µg/mL).I% was calculated using the equation: I% = (A blank -A sample /A blank ) × 100, where A blank is the absorbance of the blank solution and A sample is the absorbance of the test sample.

β-Carotene bleaching test
The β-carotene solution was prepared by dissolving 2 mg of β-carotene in 10 mL of chloroform. 1 mL of β-carotene-chloroform solution was mixed with 20 mg linoleic acid and 0.2 g Tween 40.Subsequently, the chloroform was removed by a rotary evaporator at 45°C.Distilled water (50 mL) was slowly added with vigorous agitation to form an emulsion.Emulsion aliquots (5 mL) were transferred with 0.2 mL of sample extracts.The control samples were prepared with 0.2 mL methanol instead of extracts (Jayaprakasha et al., 2001;Kaur and Kapoor 2002;Shahidi et al., 2001).As soon as the emulsion was added to each tube, zero time absorbance was read at 470 nm against the blank.The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was monitored by the subsequent reading of absorbance at 15 min intervals, until the color of the β-carotene in the control sample had disappeared (105 min).BHT was used as reference.Analyses were performed in triplicate.The antioxidant activity (AA) was calculated as a percent of inhibition, relative to the control, using the following equation: AA = [1 -(Ai -At) / (A'i -A't)] × 100, where Ai = absorbance of the sample at zero time; At = absorbance of the sample after incubation (105 min) at 50°C; A'i = absorbance of the control at zero time; A't = absorbance of control after incubation (105 min) at 50°C.

ABTS assay
The total antioxidant activity was measured by the improved azino-bis (ethylbenzothiazoline6-sulfonicacid) radical scavenging (ABTS) method (Rufino et al., 2007), with minor modifications.Briefly, 7.0 mM ABTS and 140 mM potassium persulfate were mixed for the production of the ABTS cation (ABTS) and kept in dark for 16 hours, at ambient temperature.The ABTS solution was diluted with ethanol (P.A.) till the absorbance obtained 0.700(±0.05),at 734 nm.For sample analysis, 3 mL of diluted ABTS solution was added to 30 µl of five different dilutions of the methanolic extract and mixed thoroughly.The reaction mixture was allowed to sit (6 min) in the dark, at an ambient temperature, and absorbance was recorded at 734 nm, using ethanol (P.A.) to the blank.A standard curve of various concentrations was prepared of ethanolic solution in trolox, in concentrations of: 100; 500; 1000, and 2000 µM.The results were expressed in (µM) trolox equivalent per gram of extract.

Statistical analysis
All of the experiments were conducted in triplicate.The data shown represent the mean ± standard deviation (SD) of three determinations.The IC 50 value was determined by linear regression, using Origin 5.0.

Results
The seed and pulp extracts of fruits A. coriacea (14.5 and 20.5%) and A. sylvatica (8.7 and 5.2%) showed great extraction yield (% w/w).
The seed extracts of A. coriacea demonstrated a moderate antioxidant effect and exhibited a free radical scavenging activity of 31.53%, by the DPPH test.The seeds also afforded antioxidant activity (51.59%) by the β-carotene bleaching test and 159.50 µM trolox/g of extract in the ABTS assay (Table 1).
The total phenolic content, flavonoids, condensed tannins, and ascorbic acid of the extracts are shown in Table 2.The results show that the hydromethanolic seed extract of A. coriacea has the highest total phenolic content (147.08 ± 4.20 mg GAE/g extract) and the highest flavonoid content (131.18 ± 2.31 mg QE/g extract).In comparison, the evaluated condensed tannin and ascorbic acid contents of the extracts are low (Table 2).
According to Pereira et al. (2013), the pulp of A. sylvatica has 8.84 g of sugar content, 17.89 g of carbohydrates, 1.82 g of protein, and 8.74 g of total fiber.The A. coriacea pulp content has 11.91 g of carbohydrates, 1.07 g of protein, and 5.62 g of total fiber (Hiane et al., 1992).
The DPPH free radical scavenging assay is based on the ability of certain substances to donate a hydrogen atom to the radical, reducing it to hydrazine, provoking a change in coloration, from pale yellow to violet.This change is accompanied by the fall in coloring of the 517 nm absorbance (Alves et al., 2010).The test of inhibition of the autoxidation of β-carotene/linoleic acid is based on the ability of certain substances to protect the beta-carotene from oxidation.This oxidation is caused by the free radicals formed during the peroxidation of linoleic acid, which attack the chromophore of the β-carotene emulsion resulting in a whitening reaction (Alves et al., 2010;Damasceno et al., 2011).
The antioxidant activity of vegetable extracts depends on the type and polarity of the extracting solvent, the isolation procedures, and the purity of the active compounds, as well as the assay techniques and substrate used (Meyer et al., 1998).Generally polar solvents provide slightly more active extracts than mixtures with less polar solvents.This factor may also have affected the results when using the methanol/water solvent, providing greater extraction of components.
It has been suggested that the phenolic content of plant materials is correlated to their antioxidant activity (Velioglu et al., 1998).In this study, we found that the hydromethanolic seed extracts of A. coriacea had the best radical scavenging activity, with high total phenol and flavonoid content.This indicates that the antioxidant activity of the extracts is related to the contents of these constituents.
Phenolic compounds are considered to be secondary metabolites that are synthesized by plants during normal development, in response to stress conditions, and the compounds occur ubiquitously in plants as a diversified group of phytochemicals derived from phenylalanine and tyrosine.In food, phenolics may contribute to the bitterness, astringency, color, flavor, odor, and oxidative stability of the products.In addition, they have numerous beneficial effects, such as, free radical scavenging oxygen species, modulate the activity of some specific enzymes, inhibit cell proliferation, and have antimicrobial, anti-inflammatory, and anti-allergic potential (Manach et al., 2004).2007), the phenolic compounds are distributed in the following categories: Simple phenolics, phenolic acids (benzoic and cinnamic acid derivatives), coumarins, flavonoids, hydrolysable and condensed tannins, stilbenes, lignans, and lignins.They have the ability to inhibit lipid peroxidation and lipoxygenase in vitro.Consumption of flavonoid containing fruits and vegetables has been linked to protection against cancer and heart disease (Hertog et al., 1992;Atoui et al, 2005).
The tannins condensed are polymers of flavonoids, whose structure is formed by connecting the series of monomers of units flavan-3-ol, or a derivative of this.This binding occurs usually between the 4 carbons of a structure and another 8. Variations may occur for different numbers monomers attached, for the occurrence of links for oxygenation pattern in the rings A and B of flavan-3-ol unit and the stereochemistry of substituent the C ring.The hydrolysable tannins are esters of gallic acid and hexahydroxydiphenic acid and glucose, as well as other polyols.Are soluble in water, potent antioxidants and responsible for the astringency of many fruits, through complexation of tannins and proteins.Our values have been relatively equivalent compared to many other fruits of different families present in Cerrado ecosystem (Rocha et al., 2011).
In conclusion, our study has demonstrated the moderate antioxidant properties of the seed extracts of Annona coriacea and shown that this effect can be attributed to the total phenols and flavonoid content.Further studies on isolation and structure elucidation of active components from the extract, as well as, investigations of their inhibitory mechanism are needed.In addition, the results reported in this paper may contribute to the appreciation of the nutrimental and functional value of A. sylvatica and A. coriacea fruits.

Table 1 .
Antioxidant activity of the four extracts of Annona fruits by DPPH, β-carotene bleaching, and ABTS test.Values are expressed as mean ± SD (n = 3); n.d.= not determined.IC 50 = corresponds to the concentration of 50% inhibition of DPPH and is calculated from the graph of I% (inhibition percentage) versus extract concentration in μg/mL.* %FRS = free-radical scavenging percentage (*antioxidant activity evaluated by the method of DPPH free-radical scavenging, with the final concentration equivalent to 250 µg/mL of extract).

Table 2 .
Contents of constituents of four extracts of Annona fruits.