Phenolic Profile and Antioxidant Activity of Pulp and Peel from Peach and Nectarine Fruits

Peach (Prunus persica L.) is a fruit of high nutritional and economic value. Carbohydrates, dietary fibers, minerals and organic acids are among the major constituents of peach fruit, which contribute to the nutritional quality of both fresh fruits and juice. Polyphenolic compounds found in peach may play an important role in physiological functions related to human health. Different polyphenolics may have varied biological activities including antioxidant activity. In this study antioxidant characteristics between peel and pulp of different peach cultivars (‘Radmilovčanka’, ‘June Gold’, ‘Blake’, ‘Hale’, ‘Vesna’, ‘Adria’) and one of nectarine (‘Fantasia’) were investigated. The peel and pulp extracts showed a huge amount of total phenolics (TP), total flavonoids (TF), total hydroxycinnamates (TH) and total flavonols (TFL), ranging from 42.7-211.4, 11.1-128.5 mg GAE/100 g fresh weight (f.w.) (TP), 21.9-94.9, 5.0-58.9 mg CE/100 g f.w. (TF), 28.4-389.2, 8.5-165.8 mg kg f.w. (TH) and 17.3-54 mg kg f.w. (TFL). High contents of phenolic compounds were significantly correlated with high antioxidant capacities. Peach pulp and peel differ significantly in their phenolic profiles: the pulp contains mainly chlorogenic, neochlorogenic and p-coumaric acids, whereas the peel possesses chlorogenic, neochlorogenic and p-coumaric acids together with several flavonol glycosides in huge amounts. Our results indicate that cultivar and extraction solvent play important roles in phenolic compositions and antioxidant properties of peach and nectarine extracts, which was shown using statistical analysis (ANOVA). There are high correlations between extracted phenolic compounds and peach and nectarine cultivars, and used solvent and part of the fruit (peel and pulp).


Introduction
Traditional fruits and vegetables possess numerous healthy properties.The positive influence of these natural products is attributed to their bioactive compounds: dietary fiber and antioxidants, mainly phenolic compounds, flavonoids, phenolic acids (Gorinstein et al., 2002;Alothman et al., 2009).As it has been shown, diets rich in dietary fiber and other bioactive substances have decreased the risk of diseases such as coronary atherosclerosis, obesity and cancer (Rimm et al., 1996).It is well known that phenolic compounds possess antioxidant properties and prevent the oxidation of low density lipoprotein cholesterol (LDL-C) (Silva et al., 2002).
Phenolic compounds constitute a large and heterogeneous class of compounds.Within each plant species, the nature of those compounds can vary from organ to organ but is constant.These further characterized (Kajdžanoska et al., 2011).Various solvent systems have been used for the extraction of polyphenols from plant materials.Water and aqueous mixture of ethanol (Gorinstein et al., 2002), methanol (Orazem et al., 2011;Manzoor et al., 2012a) and acetone (Hamauzu et al., 2006) are commonly used.Alothman et al. (2009) reported that aqueous acetone was superior to methanol and ethanol in the extraction of phenols from fruits.
Recently, the investigation on peach from Serbia (Mitic et al., 2013) was performed using the combination of methanol and HCl for the extraction of polyphenolic compounds, and not separating pulp from peel.
The objective of this study was to determine the polyphenol profile and antioxidant capacity of peel and pulp of six different peach cultivars and one nectarine cultivar, and to examine the efficiency of different HCl concentrations in 80% acetone (v/v) for the extraction of polyphenolic compounds.

Fruit samples
Six different peach cultivars ('Radmilovčanka', 'June Gold', 'Blake', 'Hale', 'Vesna' and 'Adria') and one of nectarine ('Fantasia') were picked in the phase of commercial maturity during 2013 harvest season in southern Serbia, and stored at ˗20 °C.Prior to analysis, peaches were defrosted, pilled and mixed in the kitchen blender.

Extraction of phenolics
The polyphenolic compounds from fruits (both pulp and peel) samples were extracted using conventional solvent extraction procedure.Ten grams of homogenized samples were extracted in an ultrasound bath with 30 ml of 80% (v/v) acetone solution containing 0, 0.1, 1 or 2% HCl.The contact time was 60 min.After the extraction, the samples were filtered through Whatman No. 1 filter paper and the residual tissues were washed with 2×20 mL of solvent.The filtrates were combined in the total extract.Finally, the obtained peach extracts were collected in volumetric flasks (100 mL).The obtained extracts were used for spectrophotometric and HPLC measurements.The extractions were performed in triplicates for each peach variety.

Determination of total phenolic content
Total phenolic (TP) contents of the acetone extracts were determined using the Folin-Ciocalteau assay (Gougoulias and Mashev, 2006).0.15 mL of acetone extract were mixed with 2.0 mL of 20% aqueous sodium carbonate solution and 0.5 mL of Folin-Ciocalteau's reagent and made up to 10 mL with deionized water.The solutions were mixed and, after ageing for 120 min at 25 °C, absorbance was measured at 760 nm, using an Agilent 8453 UV-VIS spectrophotometer (Agilent Technologies, USA).Results were expressed as mg of gallic acid equivalents (GAE) per 100 g of the fresh weight (f.w.).

Determination of total flavonoid content
The total flavonoid (TF) contents of peach extracts were determined by a colorimetric method according to Malencic et al. (2002).Known volumes of the samples were mixed with 2 mL of distilled water and subsequently with 0.3 mL of 5 % sodium nitrite solution.After 5 min, 3 mL of 1% aluminum chloride solution were added and the solution left for 5 min at room temperature.Then, 2 mL of 1 M sodium hydroxide were added to the mixtures diluted with deionized water to the final volume of 10 mL.The mixtures were thoroughly mixed and absorbance was immediately measured at 510 nm.Results were expressed as mg of catechin equivalent (CE) per 100 g of fresh weight (f.w.).

Determination of antioxidant activity
The antioxidant capacities of peach acetone extracts (peel and pulp) were studied in four antioxidant assays: scavenging DPPH radical (Brand-Williams et al., 1995), scavenging ABTS radical (Lee et al., 2003), iron (III) to iron(II) reduction power assay (RP) (Dorman et al., 2003) and ferric-reducing antioxidant power assay (FRAP) (Benzil and Strain, 1999).The total antioxidant activities of peach extracts for the first and the second assays were expressed as mmol of Trolox equivalent (TE) per 100 g of f.w.RPs of the extracts were expressed as mg of gallic acid equivalent (GAE) per 100 g of f.w., while FRAP values were expressed as mmol of ferrous ion equivalents (FE) per 100 g of f.w.

HPLC-DAD determination of phenolics composition
The individual phenolics were analyzed by the direct injection of the extracts (previously filtered through a 0.45 μm pore size membrane filter) into Agilent 1200 chromatographic system equipped with a quaternary pump, and UV-VIS photodiode array detection (DAD) for multi wavelength detection and fluorescence detection for the acquisition of the emission response, an 8 μL flow cell, and automatic injector and ChemStation software.The column temperature was 30 °C.After injection of 5 μL of sample extract, the separation was performed in the Agilent/eclipse XDBC-18 4.6×150 mm column.Two solvents were used for the gradient elution: A-(H2O+5% HCOOH) and B-(80% ACN+5% HCOOH+H2O).The used elution program was as follows: from 0 to 10 min 0 % B, from 10 to 28 min gradually increased 0-25% B, from 28 to 30 min 25% B, from 30 to 35 min gradually increased 25-50% B, from 35 to 40 min gradually increased 50-80% B, and finally for the last 5 min gradually decreased 80-0% B. The runs were monitored at the following wavelengths: hydroxycinnamates at 320 nm and flavonol glycosides at 360 nm.Retention times and spectra were compared to pure standards.Calibration curves at concentrations ranging from 0.05 to 5 mg ml -1 (r 2 <0.99) were made from chlorogenic acid, pcoumaric acid, quercetin and kaempferol-3-glucoside as standards.Neohlorogenic acid quantity was expressed as quantity of quercetin and kaempferol-3-glucoside, respectively.The results were expressed as milligrams per kg of fresh weight (f.w.).

Statistical analysis
The data were reported as mean±standard deviation (SD) with triplicate determinations.Statistical analysis was performed using the application available for Microsoft Excel (XLSTAT2016) (Addinsoft, 2016).Analysis of variance, analysis of the differences between the categories with a confidence interval of 95% (Tukey) and analysis of the differences between the control category and other categories with a confidence interval of 95% (Dunnett test) were performed.

Total flavonoids
Total flavonoids (TF) of pulps and peels of the six different varieties of peach and one nectarine also varied considerably (Table 1).In the peels extracts, total flavonoids ranged from 21.9-94.9mg CE/100 g f.w, whereas, these amounts decreased in the pulps extracts levels of 5.0-58.9mg CE/100 g f.w.The peel of 'Radmilovčanka' peach exhibited significantly (p<0.05)higher content of flavonoids (94.9 mg CE/100 g f.w.) compared to those of 'Adria' and 'Fantasia' (23.9 and 21.9 mg CE/100 g f.w., respectively).In case of the pulp extract of cv.'Radmilovčanka', higher contents of TF were observed (58.9 mg CE/100 g f.w.) whereas cv.Hale had smaller content of TF (5.0 mg CE/100 g f.w.).Also, the contents of TF determined in the peach peels were found to be higher than those of the corresponding pulps.Cevallos-Casals et al. (2006) also reported that contents of phenolic compounds vary within different tissues of the same fruits and are mostly concentrated in the epidermal and subepidermal layers of the fruits.Higher concentrations of TF compounds in peach peel compared to the pulp were also reported by other researchers (Tomas-Barberan et al., 2001;Scordino et al., 2012).The effect of acidified solvents on the extraction of the peach phenolics was evaluated using aqueous HCl in 80% acetone as the solvent.The optimal acidified conditions for the extractions of phenolic compounds were 1 % (v/v) HCl for the pulp, and 2% (v/v) HCl for peel.

Antioxidant capacity
There are huge varieties of antioxidants present in fruits.Therefore, measurement of the antioxidant capacity of each compound separately represents very difficult task.Several methods have been developed to determine the antioxidant potential of plant extracts.Each method provides an estimation of the capacity that depends on the time of reaction, and the complexity of the reaction kinetics.Therefore, the single antioxidant method can not give profiles of the antioxidant capacities of compounds completely.Antioxidant can reduce radicals primarily by two mechanisms: the single electron transfer and the hydrogen atom transfer.ABTS, DPPH, FRAP and RP are commonly used for the evaluation of the activity of plant extracts (Ozgen et al., 2006;Gong et al., 2012).
DPPH is a stable free radical with a deep violet colour and absorption maximum at a wavelength of 515 nm.In this test, the violet colour of DPPH is reduced to a pale yellow colour due to the abstraction of hydrogen atom from antioxidants (Lee et al., 2003).
FRAP assay is commonly used to study the antioxidant capacity of plant materials.The antioxidant capacity of fruits extracts is determined by the ability of the antioxidants to reduce ferric iron to ferrous in FRAP reagent, which consists of 2,4,6tris(1-pyridyl)-5-triazine (TPTZ) prepared in sodium acetate buffer, pH 3.6.The reduction of ferric iron in FRAP reagent results in the formation of a blue product (ferrous-TPTZ complex) which absorbance can be read at 593 nm.The antioxidant capacity of peel (0.4-1 mmol Fe/100 g f.w.) was higher than in pulp (0.3-0.7 mmol Fe/100 g f.w.).Similar results were reported by Guo et al. (2003) (0.4 for pulp and 0.9 mmol Fe/100 g f.w. for peel, respectively), (Stratil et al., 2006;Remorini et al., 2008).In general, cultivars with higher total phenolic content showed high antioxidant capacity (Table 1).We found that the highest antioxidant capacities were present in 'Radmilovčanka' and 'June Gold'.
The assessment of reducing power (RP) of a compound may act as a good indicator of its potential antioxidant activity.The presence of the reducing agents in a typical sample causes the reduction of Fe 3+ to Fe 2+ , and reductive capability can be monitored colorimetrically due to the formation of Perl's Prussian blue complex at 700 nm (Dorman et al., 2003;Lim and Quah, 2007).Table 1 gives the reductive capability of peel and pulp extracts of different cultivars of peach fruit.The reducing potentials of the tested extracts were recorded over a range of 3.8 to 70.5 mg GAE/100 g f.w.Generally, the extracts from peels showed significantly higher reductive capabilities than the pulp extracts.ABTS •+ , a protonated radical, has a characteristic absorbance at (Mohdaly et al., 2010).The extracts demonstrated a wide range of ABTS •+ scavenging activities from 0.2 to 0.7 mmol TE/100 g f.w.
(Table 1).Scavenging of the ABTS •+ radicals by extracting was found to be higher than DPPH radical.Factors such as the stereoselectivity of radicals and the solubility of extracts in different test systems have been reported to affect the capacity of extracts to react with and quench different radicals (Yu et al., 2012).Similar results were found by Montevecchi et al. (2012) for Sicilian white peach cultivar (0.7-2.3 mmol TE/100 g f.w. for pulp and 0.4-1.4mmol TE/100 g f.w. for peel).
In our study, all extracts exhibited significant antioxidant activities.Extracts with 1% for pulp and 2% (v/v) HCl for peel, respectively, contain the most efficient ABTS •+ scavengers.Acidity of solvent alters its ability to dissolve selected groups of antioxidant compounds.
Obtained R 2 values were 0.833 (Neochlorogenic acid), 0.779 (Chlorogenic acid), 0.828 (p-Coumaric acid), and 0.798 (Total acids); they show that few other factors beside peach and nectarine cultivar, concentration of HCl and part of the fruit (pulp and peel) must be taken into account for the explanation of the variance.The values of p were <0.0001 (Neochlorogenic acid), <0.0001 (Chlorogenic acid), <0.0001 (p-Coumaric acid), and <0.0001 (Total acids); all of them are less than 0.05, so all models are valid.
Only two residuals were outside the limit [-2, 2] (outliers).Tukey test with a confidence interval of 95% shows that in case of: Individual phenolics in peels and pulps of peach varieties The extracts of peels and pulps from six different peach cultivars and one nectarine were analyzed by HPLC-DAD.The compounds were identified by comparison with those of standards at 200-400 nm.The HPLC chromatograms of three acids, i.e. neochlorogenic acid (peak 1), chlorogenic acid (peak 2) and pcoumaric acid (peak 3) found in peel peach extract recorded at 320 nm with a diode detector, are shown in Fig. 1.In Table 2, we show phenolic compounds (neochlorogenic acid, chlorogenic acid, and p-coumaric acid) in pulp and peel, and in Table 3 flavonols (quercetin-3-rutinoside, quercetin-3-glucoside, quercetin-3rhamnoside and kaempferol-3-rutinoside) in the peel of peach fruit.

Conclusion
In both peel and pulp, the predominant group of polyphenolics was hydroxycinnamates.Hydroxycinnamic acids represent 18.41 % of the total polyphenolics in the peel and 9.91 % of the pulp.Three quercetin glycosides and one kaempferol glycoside were found in the peel.The results obtained in this study will help to further understand the polyphenolic composition in peach and the roles of these compounds in health promoting physiological functions.

Table 1 .
Total phenolics contents, total flavonoid contents and antioxidant activities of peel and pulp extracts obtained with two solvent mixtures (mean of three replicates ± standard deviation*) *Values are expressed as means±SD (n=5), a in acetone extracts with 2% HCl; b in acetone extracts with 1% HCl.

Table 2 .
Individual hydroxycinnamic acids contents (mg/kg) in peels and pulps of six different peach and one nectarine cultivars

Table 3 .
Individual flavanol acid contents (mg/kg) in peels of six different peach and one nectarine cultivars