Phenolic Profile and Antioxidant Activity of Pulp and Peel from Peach ...

Available online: www.notulaebotanicae.ro Print ISSN 0255-965X; Electronic 1842-4309 Not Bot Horti Agrobo, 2016, 44(1):175-182. DOI:10.15835/nbha44110192

Original Article

Phenolic Profile and Antioxidant Activity of Pulp and Peel from Peach and Nectarine Fruits Branka T. STOJANOVIC1, Snezana S. MITIC1, Gordana S. STOJANOVIC1, Milan N. MITIC1, Danijela A. KOSTIC1, Dusan Dj. PAUNOVIC1, Biljana B. ARSIC2* 1

University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, P. O. Box 224, 18000 Niš, Republic of Serbia; brankastojanovic81@gmail.com; mitich_s@yahoo.com; stgocaus@yahoo.com; milanmitic83@yahoo.com; danijelaaakostic@yahoo.com; dpaunovic3@gmail.com 2 University of Niš, Faculty of Sciences and Mathematics, Department of Mathematics, Višegradska 33, P. O. Box 224, 18000 Niš, Republic of Serbia; ba432@ymail.com (*corresponding author)

Abstract 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-1 f.w. (TH) and 17.3-54 mg kg-1 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). Keywords: chlorogenic acid, extracts, glycosides, HPLC, statistics

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

factors have been used, in recent years, in the characterization of several food products of plant origin by their phenolic profile. Factors contributing to the variability in phenolic distribution include cultivar and genetic origin, maturity, climate, position of tree, and agricultural practices (Witzum and Steinberg, 1991). Moreover, contents of organic acids, carbohydrates and phenolics are not uniformly distributed within different parts of fruits, and most of them are concentrated in the epidermal and subepidermal layers of fruits (Mattila et al., 2006; Manzoor et al., 2012a). Peach (Prunus persica L.) fruit have high economic and nutritional value (Manzoor et al., 2012b). Carbohydrates, organic acids, minerals and dietary fibers are among the major constituents of peach fruit, which contribute to the nutritional quality of both fresh fruits and juice. The critical point in studying polyphenols in plant materials is the used extraction procedure since it dictates the nature and quantity of polyphenols that will be transferred into the extract and

Received: 17 Nov 2015. Received in revised form: 23 Apr 2016. Accepted: 25 Apr 2016. Published online: 14 June 2016.

Stojanovic BT et al. / Not Bot Horti Agrobo, 2016, 44(1):175-182 176

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. Materials and Methods

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. Chemicals Standards of gallic, p-coumaric, chlorogenic acid, quercetin and kaempferol-3-glucoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid), Iron(II)sulphate, 2,4,6tri(2-pyridyl)-s-triazine (TPTZ), 2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2’-diphenyl-1picrylhydrazil (DPPH), Folin-Ciocalteau’s reagent, and catechin were obtained from Fluka (United Kingdom). Deionized water was used for the preparation of all solutions, and it was produced using MicroMed high purity water systems (TKA Wasseraufberei tungssystem GmbH). 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 5080% 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 (r2