Phenolic extracts from Crataegus × mordenensis and Prunus virginiana: Composition, antioxidant activity and performance in sunflower oil

Phenolic extracts from Crataegus × mordenensis and Prunus virginiana: Composition, antioxidant activity and performance in sunflower oil

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LWT - Food Science and Technology 59 (2014) 308e319

Contents lists available at ScienceDirect

LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt

Phenolic extracts from Crataegus  mordenensis and Prunus virginiana: Composition, antioxidant activity and performance in sunflower oil Felix Aladedunye a, *, Roman Przybylski b, Karsten Niehaus c, Hanna Bednarz c, €us a Bertrand Mattha a

Max Rubner-Institut (MRI), Federal Research Institute for Nutrition and Food, Department for Safety and Quality of Cereals, Working Group for Lipid Research, Schützenberg 12, D-32756 Detmold, Germany Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4 c Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 August 2013 Received in revised form 12 December 2013 Accepted 2 June 2014 Available online 12 June 2014

The prevailing stigmatization of synthetic antioxidants and the inefficiency of endogenous antioxidants like tocopherols during high temperature processing of edible oils necessitate the search for effective natural antioxidants. Here, polyphenolic extracts from chokecherry and hawthorn fruits were screened for possible antioxidative application in fats/oils. Extracts were successively partitioned on sephadex columns and fractions were screened for radical scavenging activity using DPPH and b-carotene assays. Furthermore, sunflower oil fortified with extracts was assessed for stability using accelerated storage at 65  C, Rancimat at 120  C, and frying at 180  C. Phenolic extracts showed significantly high radical scavenging and antioxidant activity in the oil. At the end of storage, hydroperoxide formation in sunflower oil was reduced by up to 50%, and the induction period significantly increased in the presence of extracts. Similarly, a significantly high frying stability was observed for the fortified samples, suggesting that the phenolic extracts can offer effective natural alternative to synthetic antioxidants during frying. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Polyphenols Edible wild fruits Vegetable oils Thermo-oxidative stability Natural antioxidants

1. Introduction Oxidation of vegetable oil is a major challenge in the food industry, resulting both in huge economic losses and diminishing nutritional quality of lipid-containing food. The resistance of an edible oil/fat to oxidative deterioration is a function of both its inherent quality and the applied conditions. In general, oils that are more unsaturated oxidize more readily than less unsaturated one, prompting several modifications of the fatty acid composition of conventional oils to obtain counterparts with reduced polyunsaturated fatty acid content (Matth€ aus, 2006). However, a number of studies has demonstrated that the minor components (also known as unsaponifiables), representing less than 5% of the oil composition, can also exert profound influence on their oxidative stability (Abuzaytoun & Shahidi, 2006; Normand, Eskin, & Przybylski, 2001). Unfortunately, many of the oil's endogenous minor components offer only limited performance under stringent conditions applied during frying. Fortification of endogenous minor

* Corresponding author. Tel.: þ49 (0) 5231 741 381; fax: þ49 (0) 5231 741 200. E-mail addresses: [email protected], [email protected] (F. Aladedunye). http://dx.doi.org/10.1016/j.lwt.2014.06.002 0023-6438/© 2014 Elsevier Ltd. All rights reserved.

components of edible oils is an alternative way of enhancing their oxidative stability without compromising their level of essential fatty acids. Furthermore, in addition to enhancing oxidative stability, appropriate fortification may also improve the nutritional and functional quality of the oil. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are often added to processed oils to retard oxidative degradation during storage and frying. However, like most endogenous antioxidants, they offer little or no protection during frying of food; they are known to evaporate or decompose, and thus do not remain in the frying oil long enough to provide sufficient protection (Augustin & Berry, 1983; Kochhar & Gertz, 2004). Beside the poor performance under frying conditions, consumers' acceptance of synthetic antioxidants remains negative due to their perceived detrimental effect on human health. Consequently, there is a growing interest in the search for effective natural antioxidants. A recent trend in the search for natural antioxidants is the application of phenolic extracts from various parts of plants. Majority of the studies in this direction are usually limited to common spices and herbs (Achat et al., 2012; Al-Bandak & Oreopoulou, 2011; Aranha & Jorge, 2012; Che Man & Jaswir, 2000; Houhoula,

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Oreopoulou, & Tzia, 2004; Kalantzakis & Blekas, 2006), however, edible wild fruits can present a renewable source for natural and novel phenolic antioxidant mixture. Hawthorn is the common name of all plant species (~280) in the genus Crataegus of the Rosaceae family. Hawthorn extract has been used extensively in traditional Chinese and European medicines in acknowledgment of their bioactive constituents and antioxidant activity (Chang, Zuo, Harrison, & Chow, 2002). Morden Hawthorn (Crataegus  mordenensis Boom.) is a hybrid from Crataegus laevigata and Crataegus secculenta and was first raised at the Agricultural Canada Plant Breeding Station in Morden, Manitoba in 1935 (Wyman, 1969). Whereas the polyphenolic profiles, antioxidant activity and other biological effects of some hawthorn species including Crataegus Monogyne, Crataegus Pinnafidita, Crataegus laevigata, and Crataegus Oxyacantha have been well studied (Bernatoniene et al., 2009; Gazdik et al., 2008; Liu, Kallio, & Yang, €  ł-Łętowska, Oszmian  ski, & 2011; Oztürk & Tunçel, 2011; Soko  ski, 2007; Ying et al., 2009), Wojdyło, 2007; Wojdyło & Oszmlan similar investigations on Crataegus  mordenensis are rather scarce. The fatty acid, tocopherol and sterol compositions of Crataegus  mordenensis seed oil was reported by Anwar, Przybylski, Rudzinska, Gruczynska, and Bain (2008). To the best of our knowledge, there is no report on the potential of Crataegus  mordenensis polyphenolics as antioxidant for edible oil. Chokecherry (Prunus virginiana), also a member of the Rosaceae family, is one of the most widely distributed native tall shrubs in North America (St. Pierre, Zatylny, & Tulloch, 2005). Its habitats include moist sites in open areas, along fencerows, roadsides, and borders of woods as well as sandy or rocky hillsides and ravines (Stephen, 1973, p. 530). Native Americans and early settlers used chokecherries as fruit and in juice, wine, jellies, syrups and beverages (St. Pierre et al., 2005). Like Crataegus  mordenensis, investigations into the phenolic profile and antioxidant activity of P. virginiana are scarce (Acuna, Athan, Ma, Nee, & Kennelly, 2002; Hosseinian & Beta, 2007; Hosseinian et al., 2007), and there is no data on their effect on the oxidative stability of fats/oils. Thus, the purpose of the present study is to assess, for the first time, the potential of natural phenolic mixtures from Canadian hawthorn and chokecherry fruits as antioxidants for improving thermooxidative stability of sunflower oil.

309

extraction solvent (acetone/water/acetic acid; 700 mL/295 mL/ 5 mL) for 2 min using a T 25 Ultra Turrax (IKA Labortechnik, Staufen, Germany) operating at 13,500 rpm followed by sonication at 50  C in an ultrasound bath for 30 min. After filtration, the residue was re-extracted with fresh solvent following the same process. Acetone was removed from the combined filtrate under vacuum at 30  C using an RV 10C rotary evaporator (IKA Labortechnik, Staufen, Germany). Subsequently, the concentrated filtrate was defatted with hexane (3  500 mL) and successively extracted with ethyl acetate and n-butanol (3  500 mL each). The hexane extract was discarded while the ethyl acetate and n-butanol extracts were evaporated under vacuum at 40  C, flushed with nitrogen and kept at 18  C for further analysis. 2.3. Fractionation on C18 cartridge The ethyl acetate and butanol extracts were further purified using a C18 SPE cartridge as follows: Extract (1 g) was dissolved in 50 mL methanol:water (35 mL:15 mL) and applied to a C18 SPE cartridge that had been preconditioned with 10 mL methanol. Eluates from separate C18 cartridges were combined and evaporated at 40  C under vacuum using a rotary evaporator. 2.4. Fractionation on Sephadex The C18 SPE purified ethyl acetate extract was further fractionated on a Sephadex column as follows: Sephadex LH-20 powder (20 g) was swollen for 24 h in water and the suspension was poured into a glass column (20  350 mm). The extract (2 g) was suspended in water and applied onto the top of the column. The reservoir was filled with water and the flow rate was adjusted to about 1 mL/min. Subsequently, the extract was successively eluted with water (200 mL), methanol:water (50 mL:150 mL), methanol:water (100 mL:100 mL), methanol:water (150 mL:50 mL), methanol (200 mL), and finally, methanol:acetone (100 mL:100 mL). Fractions were monitored by HPLC-PDA, pooled together based on the similarity of their HPLC chromatograms, and evaporated to dryness under vacuum at 40  C using a rotary evaporator. After the removal of residual solvent under a gentle stream of nitrogen, the residue was re-dissolved in 5 mL of methanol and kept at 4  C until used.

2. Materials and methods 2.1. Materials Fully ripened fruits from wildly grown chokecherry and cultivated hawthorn plants were collected in the Oldman River Valley, Alberta, Canada during the months of August and October. Samples for each of the two fruits were randomly collected from several plants grown in three different locations. Sunflower oil and frozen par-fried French fries in institutional pack were obtained from a local food store. All solvents were of HPLC grade (Merck, Germany). C18 SPE 1000-mg cartridges with 6 mL reservoir were obtained from J.T Baker (Deventer, Netherlands). Lipophilic Sephadex LH20 was obtained from SigmaeAldrich (Steinheim, Germany). The water used was either doubly distilled or of HPLC grade. All chemicals, including phenolic standards, were obtained from SigmaeAldrich (Steinheim, Germany). 2.2. Extraction and partitioning of phenolic extracts After de-stemming and removal of damaged fruits, the fresh fruits were air dried at ambient temperature and pulverized with a Grindomix GM 200 (Retsch, Haan, Germany) at 10,000 rpm for 30 s. The ground samples (100 g) were homogenized with 1 L of

2.5. Separation and identification of phenolic compounds by HPLCMS High performance liquid chromatography was carried out using a LaChrom Elite® HPLC system equipped with an L-2130 Hitachi gradient pump and an L-2200 autosampler (Merck, Germany). The sample was separated at ambient temperature on a Lichrosphere 100 RP-18e column (5 mm; 250  4 mm; Merck, Germany) using a mobile phase consisting of solvent A (0.1mL/100mL formic acid in water) and solvent B (0.1mL/100mL formic acid in acetonitrile) at a flow rate of 1 mL/min using the following gradient: 100% A at time 0 min; 95% A, 5% B (5 min); 65% A, 35% B (35 min); 45% A, 55% B (45 min); 20% A, 80% B (55 min); 20% A, 80% B (60 min); 100% A (63 min); 100% A (70 min). Injection volume was 10 mL and the analytes were monitored at 280, 360, and 520 nm with an L-2455 PDA detector (Merck, Germany). Phenolic compounds were identified with an ultrahigh resolution maXis impact q-TOF mass spectrometer (Bruker, Bremen, Germany) and by comparison of retention time and UV spectra with pure standards. The mass spectrometer was equipped with an ESI ion source operated in both positive and negative modes, acquiring MS and auto MS/MS data at 3 Hz acquisition speed and m/ z 80e1300 scan range. MS source settings were as follows: dry gas

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temperature, 200  C; dry gas flow, 8 L/min; nebulizer gas pressure 2 bars; capillary voltage 4500 V; broadband collision-induced dissociation (bbCID), 25 eV; in-source CID, 60 eV. Molecular formula determination was carried out by combined evaluation of mass accuracy, isotopic patterns, adduct and fragment information using SmartFormula3D. TargetAnalysis software (Bruker, Germany) was used to screen extracts for phenolic compounds previously reported in the literature. 2.6. Determination of Total Phenolic Content (TP) The total phenolics (TP) in the extracts and fractions were determined using the method described by Singleton and Rossi (1965) with some modifications. In brief, 100 mL of FolineCiocalteu reagent, and 300 mL of Na2CO3 (20%) were added to 20 mL of appropriately diluted samples. Then the volume was completed to 2000 mL with distilled water. After a 30 min incubation period, absorbance was read at 765 nm. The concentration was calculated using gallic acid as standard, and the results were expressed as milligrams gallic acid equivalents (GAE) per gram extract. 2.7. DPPH radical scavenging assay The DPPH assay was performed according to a method described by Sanchez-Moreno, Larrauri & Saura-Calixto (1998) with some modifications. Briefly, an aliquot of methanolic (0.1 mL) containing different concentrations of extracts/fractions was added to 3.9 mL of DPPH (0.025 g/L in methanol) and the absorbance at 515 nm was measured at 1 min interval for 30 min. The percentage of remaining DPPH was calculated as follows:

 % DPPH remaining ¼

½DPPHT¼30 ½DPPHT¼0

  100

where: [DPPH]T¼0 and [DPPH]T¼30 are the initial DPPH concentration and the concentration at 30 min, respectively. The percentage of remaining DPPH was plotted against the antioxidant concentration to obtain the amount of antioxidant required to decrease the initial DPPH concentration by 50% (EC50). BHT and a-tocopherol were used as references. 2.8. b-Carotene bleaching assay Determination of the antioxidant activity using a b-carotene/ linoleic acid system was as described by Miraliakbari and Shahidi (2008) with some modifications. In brief, 40 mg of linoleic acid and 400 mg of Tween 20 were transferred into a flask, and 1 mL of a solution of b-carotene (2 mg/mL) in chloroform was added. Chloroform was removed by rotary evaporation at 40  C. Then 100 mL of distilled water was added slowly to the residue and the solution was vigorously agitated to form a stable emulsion. An aliquot of 4.8 mL of this emulsion was transferred into a test tube containing 0.2 mL of sample or methanol, and the absorbance was measured at 470 nm, immediately, against a blank consisting of the emulsion without b-carotene. The tubes were then incubated at 50  C in the dark and the absorbance was read after 2 h. BHT and a-tocopherol at 45 mg/g concentration were used as reference antioxidants. Each test was performed in triplicate. The antioxidant activity (AA) was calculated in terms of percentage inhibition relative to the control using the following equation:

 AAð%Þ ¼ 1 

 As0  As2  100 Ac0  Ac2

where: As0 and As2 are the absorbance of the test samples measured at 0 and 2 h, respectively, while Ac0 and Ac2 denote the absorbance of the control (b-carotene-containing emulsion and methanol instead of sample), measured at 0 and 2 h, respectively. 2.9. Addition of phenolic extracts to oil A solution of the extract in methanol was added to sunflower oil in a flask to deliver the desired amount of phenolic content in gallic acid equivalent (GAE) per gram of oil. The solvent was evaporated under vacuum at 50  C using a rotary evaporator followed by sonication at 50  C in an ultrasound bath for 30 min. This procedure offers effective dissolution and dispersal of phenolic extracts in the oil. Residual solvent was removed by gentle stream of nitrogen. 2.10. Accelerated storage e Schaal oven test The ability of the polyphenolic extracts to inhibit oxidative deterioration of oil during storage was determined using the Schaal oven test. Sunflower oil (1.0 g), fortified with phenolic extracts at 200 mg GAE/g of oil, were introduced in the vials (2 mL, 12  32 mm). The uncapped vials were stored in darkness at 65  C for up to 7 days. Samples were examined at 24 h intervals by collecting individual vials at the particular period. The oxidative stability of the samples was evaluated by peroxide value (PV). The effectiveness of the phenolic extracts was compared with BHT, as synthetic antioxidant. Experiments were set up in two repetitions for each tested antioxidant, and samples from each repetition were analyzed in duplicate. 2.11. Thermal oxidation by Rancimat Sunflower oil fortified with extracts at two different concentrations of phenolic extracts (200 and 500 mg GAE/g of oil) were submitted to thermo-oxidation under Rancimat conditions using a 743 Rancimat (Metrohm, Filderstadt, Germany). In brief, 3.6 g oil was weighed into the reaction vessel, which was placed into the heating block kept at 120  C. The air flow was set at 20 L/h for all determinations. Volatile compounds released during the degradation process were collected in a receiving flask filled with 60 mL distilled water. The conductivity of this solution was measured and recorded. The software of the Rancimat automatically evaluated the resulting curves. BHT was used as the reference antioxidant. Experiments were set up in three repetitions at each concentration for each tested antioxidant. 2.12. Frying Sunflower oil (100 g) fortified with phenolic extracts at 500 mg GAE/g of oil was weighed into 250 mL glass crystallizing dishes (9.5 cm diameter  5.5 cm height; Schott Duran, Wertheim/Main, Germany). The oil was placed on a hot plate with a probe to control temperature at 180 ± 2  C and heated for 8 h with an hourly frying of 10 g of frozen French fries (approximately 4.5  1  1 cm) for 5 min. Oils were heated without replenishing and samples (2.0 g) were collected at the 4th and 8th hour and immediately frozen at 18  C until analyzed. Experiments were set up in two repetitions for each tested antioxidant and samples for each repetition were analyzed in duplicate (n ¼ 4). 2.13. Determination of Peroxide Value (PV) PV was assessed according to procedure described by Szterk,  ska, Derewiaka, & Lewicki, 2010. Briefly, 200 mg of Roszko, Sosin oil was dissolved in 5 mL of hexane. Two hundred microliters of the

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311

Fig. 1. representative chromatograms of purified ethyl acetate extract (HTx) and most active fractions (HT2 and HT3) from hawthorn fruit at 280 nm. A ¼ HTx; B ¼ HT2; C ¼ HT3. See Table 1 for peak identifications. See text for HPLC conditions.

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Fig. 2. representative chromatograms of purified ethyl acetate extract and most active fractions (CC2 and CC4) from chokecherry fruit at 280 nm. A ¼ CCx; B ¼ CC4; C ¼ CC2. See Table 1 for peak identifications.

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313

Table 1 Identification of phenolic constituents of hawthorn and chokecherry fruit extract. Peak

A B C D E F G H I J K L M N O P Q R S T a b c

RT (min)

9.3 12.4 12.7 16.8 18.9 19.5 20.0 24.1 25.0 27.1 27.6 28.6 29.0 30.3 30.7 31.2 31.8 33.7 40.9 43.3

UV lmax (nm)

225, 216, 218, 218, 260, 218, 215, 234, 268, 268, 256, 255, 254, 254, 254, 266, 254, 227, 255, 253,

266 226, 238, 240, 292 237, 225, 279 339 337 353 351 349 350 351 349 349 283 369 371

293 325 325 320 278

[MH] Found

Expected

169.0143 341.0869 353.0881 353.0877 167.0347 179.0351 289.0731 577.1369 577.1563 431.0985 609.1467 463.0883 463.0879 433.0777 433.0781 447.0947 447.0947 435.1310 301.0350 317.0302

169.0142 341.0873 353.0878 353.0878 167.0344 179.0350 289.0718 577.1351 577.1557 431.0978 609.1461 463.0882 463.0882 433.0776 433.0776 447.0933 447.0933 435.1297 301.0354 317.0297

MS2

mSigmac

Identity

125.0243 179.0347 191.0560 191.0562, 123.0451 135.0448 245.0798, 407.0763, 413.0877 311.0560 301.0337 301.0334 301.0333 301.0324 301.0323 285.0391 301.0357 273.0758, 178.9987, 178.9983

7.3 24.2 12.9 3.0 2.3 2.3 16.3 15.4 4.8 12.9 15.8 12.1 10.8 3.1 24.1 19.3 21.0 6.8 4.1 8.9

Gallic acida Caffeoyl hexosideb Neochlorogenic acidb Chlorogenic acida Vanillic acida Caffeic acida Epicatechina B-type Procyanidinb Vitexin rhamnosidea Vitexina Rutina Quercetin glucosidea Quercetin galactosidea Quercetin arabinosidea Quercetin pentosideb Kaempferol glucosideb Quercetin rhamnosidea Phloridzina Quercetina Myricetina

179.0351

203.0694 289.0720

167.0337 151.1038

Components conclusively identified by comparison with authentic standards. Component tentatively identified based on published (Chen et al., 2012; Cui et al., 2006; Liu et al., 2011) UV spectra, MS and MS/MS data. mSigma describes the isotopic pattern quality; values ˂ 20 are considered excellent and values ˂ 30, good.

solution was mixed with 5 mL of methanol/chloroform/HCl solution (1:1:0.012, v/v). Thereafter, 100 mL of FeCl2 (0.4 g/100 mL water) and 100 mL of NH4SCN (30 g/100 mL water) were added. The reaction was kept at room temperature for 5 min, and the absorbance was measured at 480 nm using all reagents for the blank sample. 2.14. Determination of polar materials, polymerized triacylglycerols, anisidine and iodine values The total polar components (TPC), dimerized and polymerized triacylglycerols (DPTG), anisidine value (AnV), and iodine value (IV) of frying oils were determined by Fourier-Transformed Near Infrared Spectroscopy (FT-NIR) following the DGF standard method C-VI 21 (2013). An MPA multipurpose FT-NIR analyzer equipped with an OPUS LAB spectroscopy software interface (Bruker Optik GmbH, Ettlingen, Germany) was used for data acquisition and analysis. 2.15. Statistical analysis Data are presented as means ± standard deviation (SD). Data were analyzed by single factor analyses of variance (ANOVA) using SPSS package (version 10.0). Statistically significant differences between means were determined by Duncan's multiple range tests for P < 0.05. 3. Results and discussions 3.1. Extraction, fractionation and phenolic compositions The extraction and fractionation protocol employed in the current study is depicted in Supplementary Fig. 1S. Aqueous acetone has been reported to offer a higher polyphenolic content than €nen, Hopia, & aqueous methanol (Buendia et al., 2010; K€ ahko Heinonen, 2001), informing the choice of extraction solvent in the present study. It is also well-known that ultrasound assistance considerably improves both the kinetic and yields of phenolic compounds from vegetal sources (Achat et al., 2012; d'Alessandro,

Kriaa, Nikov, & Dimitrov, 2012). Under the conditions used in the present study, the yields from the ethyl acetate and butanol partitioning of the hawthorn fruit aqueous acetone extract were 2.0 and 7.9%, respectively. The corresponding yields for chokecherry were 1.3 and 6.6%. Successive fractionation with Sephadex columns afforded a total of 8 and 11 fractions for chokecherry and hawthorn, respectively. Fractions were bulked based on the similarity of their phenolic compositions; representative HPLC chromatograms for hawthorn and chokecherry extracts are presented in Figs. 1 and 2, respectively. Phenolic constituents of hawthorn and chokecherry ethyl acetate extracts are rather similar (Figs. 1A and 2A). Based on their characteristic UV spectra, MS and auto MS/MS data, and comparison with authentic standards and published data (Chen, Inbaraj, & Chen, 2012; Cui, Nakamura, Tian, Kayahara, & Tian, 2006; Liu et al., 2011), the following flavonoids were identified in the extracts: epicatechin, B-type procyanidin dimer, quercetin, myricetin, rutin, quercetin glucoside, quercetin galactoside, quercetin arabinoside, quercetin rhamnoside, kaempferol glucoside, vitexin rhamnoside, and phloridzin (Table 1). For instance, peaks L, M, N, O and Q had UV absorption maxima at about 350 nm and a characteristic MS2 fragment at m/z 301 in negative mode following the loss of hexose (162 amu; L, M), pentose (132 amu; N, O) or rhamnose (146 amu; Q) indicating the presence of quercetin glycosides (Cui et al., 2006; Liu et al., 2011). Peak P was identified as kaempferol glucoside in accordance with a molecular ion at m/z 447 [MH] and the MS2 fragment at m/z 285 after elimination of a glucose unit (162 amu). Hydroxycinnamic acid derivatives including: gallic acid; caffeic acid; vanillic acid; caffeoylquinic acid; and caffeoyl hexoside, with caffeic acid derivatives predominating in both extracts were also identified based on their characteristic UV spectra, MS and auto MS/ MS data, and comparison with authentic standards and published data (Table 1). For instance, peak B was identified as caffeoyl hexoside based on the parent ion at m/z 341 in the negative mode, the fragment ion at m/z 179 for caffeic acid, corresponding to the loss of a hexose moiety (162 amu), and matching absorption maxima with those reported by Chen et al. (2012). As indicated by HPLC and total phenolic content (TP) analysis, the bulk of the phenolic compounds resided in the ethyl acetate

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extracts despite the higher extract yield from butanol. For instance, the TP for the purified ethyl acetate extract for chokecherry and hawthorn were 97 and 132 mg GAE/g, respectively while the respective values for the purified butanol extracts were 51 and 76 mg GAE/g. Depending on their purity and phenolic constituents, the TP, based on gallic acid equivalent (GAE), for the various phenolic fractions isolated from chokecherry and hawthorn fruits ranged from 51 to 407 mg/g and 76e698 mg/g, respectively (Fig. 3). Activity of fractions were further screened by a number of established tests in order to derive a natural polyphenolic mixture that can significantly improve the stability of vegetable oils during high temperature processing and storage. 3.2. Radical scavenging activity of phenolic extracts The DPPH radical scavenging activity of the different phenolic fractions examined in this study is presented in Fig. 4A. All the fractions from both fruits showed good DPPH radical scavenging activity, with a number of them exhibiting significantly higher (P < 0.05) activity than tocopherol and BHT. The EC50 of the extracts ranges from 6 to 360 g/kg DPPH and 8e290 g/kg DPPH for chokecherry and hawthorn, respectively (Fig. 4A). Under the same DPPH assay experimental conditions, the EC50 for BHT and a-tocopherol were 221 and 257 g/kg DPPH, respectively. In accordance with the TP content, ethyl acetate extracts from hawthorn exhibited significantly higher radical scavenging activity than the corresponding chokecherry extract. A previous study by Socha, Juszczak, Pietrzyk, and Fortuna (2009) also found similar results, with the ethyl acetate extracts of commercial hawthorn fruits possessing a higher TP content and DPPH radical scavenging activity than the chokecherry counterpart. As expected, a positive relationship was observed between the amounts of phenolic compounds in a particular extract and its DPPH radical scavenging activity (Figs. 3 and 4A). In other words, an extract with a higher TP generally have lower EC50 value, thus, informing our decision to screen out those fractions with very low TP values.

Although DPPH assay is an excellent method to evaluate the radical scavenging activity of potential antioxidative compounds, the results are sometimes poorly correlated with performance in real food, principally because the nature and polarity of the radical encountered in food system is different from that of the DPPH radical. Further, DPPH radical scavenging reaction occurs in an organic solvent, thus the impact of antioxidant partitioning is excluded (Decker, Warner, Richards, & Shahidi, 2005). To overcome this disadvantage, as a complementary test, the radical scavenging activity of the phenolic extracts was further assessed with the bcarotene assay. However, fractions with DPPH radical scavenging activity weaker than that of a-tocopherol (EC50 257 g/kg DPPH) were screened out from further study with the b-carotene assay. Extracts were added at concentration that delivered phenolic compounds of approximately 50 mg/mL based on TP analysis. As shown in Fig. 4B, all the tested extracts showed good antioxidant activity and the efficiency of the different fractions at the equivalent phenolic concentration (~50 mg/mL) are rather similar, suggesting that the phenolic components of the fractions are the active principles. The relatively higher antioxidant activity of fractions; HT3 (AA ¼ 87.9%), HT2 (AA ¼ 84.7%) from hawthorn, and CC2 (AA ¼ 80.7%), CC3 (83.1%), and CC4 (AA ¼ 81.6%) from chokecherry may be related to their higher purity compared to other fractions. 3.3. Performance during storage While chemical tests like the DPPH radical scavenging and bcarotene bleaching assays can provide important information on the potential of an antioxidative compound, assessment in real food system is still imperative. Thus, ability of the polyphenolic fractions from hawthorn and chokecherry to protect fats/oils during ambient storage was evaluated in sunflower oil during accelerated storage at 65  C for 7 days. Formation of lipid hydroperoxides, the primary products of oxidative deterioration was monitored by peroxide value (PV). The increase in PV in the oil substrate over the storage period is depicted in Fig. 5. All the tested extracts offered significant

Fig. 3. Total phenolic content (TP, mg GAE/g of extract) of representative phenolic fractions from hawthorn (HT) and chokecherry (CC) fruits. HTx, CCx ¼ purified ethyl acetate extracts from hawthorn and chokecherry fruits, respectively. HT1 e HT8, CC1 e CC7 ¼ respective fractions by column chromatography. Values are means of triplicate analysis (n ¼ 3).

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315

Fig. 4. DPPH radical scavenging activity (A) and b-carotene antioxidant activity (B) of various phenolic fractions from hawthorn (HT) and chokecherry (CC) fruits. EC50 is the concentration of extract required to decrease DPPH amount by 50%. HTx, CCx ¼ purified ethyl acetate extracts from hawthorn and chokecherry fruits, respectively. See Fig. 3 for explanation. Values are means of triplicate analysis (n ¼ 3).

protection against oxidative deterioration of the sunflower oil. Formation of hydroperoxides in the oil was inhibited by up to 50% in the presence of polyphenolic fractions from hawthorn and chokecherry (Fig. 5). Fractions HT3 from hawthorn, and CC2, and CC4 from chokecherry were particularly effective, with activity significantly better than the respective ethyl acetate extracts. However, none of the fractions showed comparable efficiency to BHT during the accelerated storage test, presumably due to their poor lipophilicity or/and the influence of some unidentified prooxidant components in the phenolic fractions. The superior

performance of CC2 compared to other fractions from chokecherry fruits may be related to the relatively better lipophilic nature and/or higher radical scavenging activity of quercetin, the predominant polyphenol in this fraction (Fig. 2C). Polyphenol aglycones are generally recognized as better radical scavengers than their glycosides, and quercetin has indeed been shown to improve oxidative stability of bulk canola and fish oils (Chen, Chan, Ho, Fung, & Wang, 1996; Huber, Rupasinghe, & Shahidi, 2009). The same explanation could be cited for the better antioxidant activity of HT3 compared to other hawthorn extracts; however, since all the phenolic

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Fig. 5. Changes in peroxide values during accelerated storage of sunflower ( ) and chokecherry ( ytoluene (C BHT); Refined, bleached and deodorized sunflower oil without extract/fraction ( each tested antioxidants and samples from each repetition were analyzed in duplicate (n ¼ 4).

compounds in these fraction are also represented in the ethyl acetate extract (Fig. 1A and C), the influence of extract/fraction purity on their effectiveness under the storage condition may be significant.

oil

fortified

with

different

phenolic

fractions from hawthorn ) fruits. Butylated hydrox). See Fig. 3 for explanation. Experiments were set up in two repetitions for

3.4. Performance under Rancimat Although the oxidative conditions under the Rancimat are different from those during actual frying, the method can provide a

Fig. 6. Oxidative stability of sunflower oil fortified with different phenolic fractions from hawthorn (HT) at 500 mg/g ( ) or 200 mg/g ( ) and chokecherry (CC) at 500 mg/g ( ) or 200 mg/g ( ) fruits as measured by Rancimat induction period. BHT e butylated hydroxytoluene; Ctr e refined, bleached and deodorized (RBD) sunflower oil without extract/ fraction. See text for details. Experiments were set up in three repetitions at each concentration (n ¼ 3).

F. Aladedunye et al. / LWT - Food Science and Technology 59 (2014) 308e319

fast assessment of the thermal stability and efficiency of an antioxidative compound under more challenging conditions than obtainable using the Schaal oven test. In contrast to the peroxide value, which provides a static measure for the assessment of fats and oils, the determination of oxidative stability by means of the Rancimat method is a dynamic measurement and the induction period (IP) measured by the Rancimat can be a useful “screening” €us, method for frying oils and the applied antioxidants (Mattha 2006). The IP for sunflower oils with or without exogenous phenolic compounds at 200 and 500 mg GAE/g from hawthorn and chokecherry is presented in Fig. 6. At the lower phenolic concentration, the activity of the extracts was marginal, but comparable to that of BHT. However, at the higher concentration, the efficiency of the phenolic extracts was markedly better than BHT. At 500 mg GAE/ g of oil, HTx, HT2, and HT3, from hawthorn, and CCx, CC3, and CC4 from chokecherry significantly extended the IP of the sunflower oil (Fig. 6). The observed effectiveness of CC3 and HT2 (both containing significant amount of chlorogenic acid) in the Rancimat test despite their relatively weaker activity under storage condition may be due to their higher thermal stability compared to quercetin glycosides. According to Van der Sluis, Dekker, and van Boekel (2005), the loss of quercetin rhamnoside at 100  C, for instance, was at least 4 times higher than that of chlorogenic acid. The higher temperature of the Rancimat condition may also have improved the solubility of the relatively polar phenolic constituents (chlorogenic acid and caffeic hexoside) in these fractions. 3.5. Performance during frying Prevailing conditions during frying are rather peculiar and may be difficult to simulate by accelerated tests like Rancimat. The high temperature (>160  C), presence of water, oxygen, metals and other pro/antioxidative components from food materials often result in an array of reactions with huge impact on the performance of applied antioxidants. Hence, the need to assess the antioxidative phenolic mixture under actual frying conditions. Based on their consistence in all the screening tests, the performance of fractions HT3 from hawthorn and CC4 from chokecherry and their respective ethyl acetate extracts were further evaluated during frying in sunflower oil. Results are presented in Table 2. Because the components measured are non-volatile and are representative of the major reactions occurring during frying, TPC is one of the most reliable parameters for assessing the frying stability of fats/oils. As shown in Table 2, the TPC increased significantly during the entire frying period, irrespective of oil samples, however, statistically significant differences (P < 0.05) were found among the treatments. At the end of the 8 h of frying, the increase in TPC of the control sunflower oil was 48.4%, compared to 36.7,

317

41.3, 38.0, 40.1, and 46.8% in samples fortified with HT3, HTx, CC4, CCx, and BHT, respectively. Thus, the applied extracts/fractions significantly inhibited thermo-oxidative degradation of the sunflower oil. Compared to BHT, the phenolic fractions/extracts also showed a statistically significant (P < 0.05) superior performance during the entire frying period (Table 2). As a consequence of prolonged oxidation, ether, peroxy, or carbon-bridged dimers and oligomers of triacylglycerols are formed. Because of their high molecular weight, these components remain in the oil and can offer reliable indication of the extent of polymerization reaction occurring in the oil during frying. As shown in Table 2, all the extracts/fractions examined exhibited significant anti-polymerization activity in the oil, with performance significantly higher than BHT, the synthetic antioxidant control. Although at the end of the frying period, the oil fortified with extracts from hawthorn contained lower amount of DPTG compared to the sample containing chockecherry extract, the difference was not statistically significant. Thermal decomposition of hydroperoxides during frying generates a number of secondary oxidation products, with carbonyl compounds being the most prominent. Anisidine value provides a reliable indication of the level of nonvolatile aldehydes in the oil, and by extension, the level of oxidative deterioration occurring in the frying oil. Changes in AnV of sunflower oil with and without fortification are presented in Table 2. As expected, a significant increase in AnV was observed over the entire frying period, regardless of sample treatments. However, in agreement with TPC and DPTG results, the oil supplemented with phenolic extracts/ fractions exhibited a significantly higher frying stability compared to the unfortified sunflower oil control, as assessed by the amount of carbonyl secondary oxidation products formed during the frying. At the end of the 4 h of frying, the accumulation of aldehydic secondary oxidation product was reduced by up to 30% in the presence of applied phenolic fractions. No significant difference was observed in the performance of extracts from hawthorn and chokecherry with regards to AnV. Polyunsaturated fatty acids such as linoleic and linolenic are the primary target of thermo-oxidation. The decrease in the level of unsaturation as a consequence of oxidation was also monitored by the iodine value (IV) of samples. Similar to other indices evaluated in this study, samples containing phenolic extracts showed higher IV compared to the control sunflower oil and samples fortified with BHT, indicating that polyunsaturated fatty acids were better protected in the presence of the applied natural phenolic extracts. The poor performance of BHT during frying contrasts sharply with its excellent performance during storage, underscoring significant differences in the nature and mechanisms of reaction occurring under the two conditions. Lipid oxidation under storage

Table 2 Frying performance of sunflower oils fortified with phenolic extracts/fractions. Sample

DTPC (%)c 4h

HTx CCx HT3 CC4 Controla BHT

25.2 26.0 23.3 24.7 32.9 29.1

DDPTG (%)c 8h

± ± ± ± ± ±

2.0a 1.6a 1.4a 1.3a 2.4b 1.9b

41.3 40.1 36.7 38.0 48.4 46.8

4h ± ± ± ± ± ±

2.1a 1.8a 2.4b 1.9ba 2.1c 2.3c

16.1 15.8 14.2 13.5 20.8 19.3

DAnVc 8h

± ± ± ± ± ±

1.3a 1.1a 0.9ab 1.0b 1.2c 0.7c

26.1 28.3 24.7 26.2 33.8 34.1

IV (127.4)b

4h ± ± ± ± ± ±

1.6a 2.4a 1.8a 2.0a 1.9b 2.3b

123.2 120.4 110.8 112.5 160.7 149.2

8h ± ± ± ± ± ±

4.8a 6.1ab 4.5b 5.3b 4.6c 5.2d

175.2 180.9 173.2 180.3 224.1 217.3

4h ± ± ± ± ± ±

6.9a 8.2a 10.8a 9.6a 14.3b 10.1b

117.7 117.2 117.9 117.7 112.1 115.1

8h ± ± ± ± ± ±

1.4a 1.6a 1.2a 1.3a 1.4b 1.3ab

112.1 112.3 113.1 112.4 109.5 109.9

± ± ± ± ± ±

1.1a 0.9a 1.2a 1.0a 0.9b 1.1b

Values with the same online within the same column are not significantly different. Experiments were set up in two repetitions and samples for each repetition were analyzed in duplicate (n ¼ 4). a Control ¼ unfortified sunflower oil; b IV (127.4) ¼ Iodine value of fresh sunflower oil; DTPC ¼ change in Total Polar Compounds during frying; DDPTG ¼ change in Dimeric and Polymeric Triacyglycerides during frying; DAnV ¼ change in Anisidine Value during frying; c Initial TPC ¼ 3.8 ± 0.2%; initial DPTG ¼ 2.4 ± 0.3%; initial AnV ¼ 6.9 ± 0.5.

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condition is principally radical mediated; thus, the good radical scavenging activity of BHT coupled with its strong lipophilicity and small molecular size presumably enhanced its activity under the static storage condition. On the contrary, reactions during frying are dynamic and much more complex, involving both radical and nonradical reactions (Kochhar & Gertz, 2004). During deep-frying at elevated temperature with limited supply of oxygen and presence of food components, an acid-catalyzed, non-radical mechanism for the formation of CeC linked dimeric, polymeric or cyclic triacylglycerols predominates (Kochhar & Gertz, 2004). Thus, synthetic antioxidants such as BHT, which operates almost exclusively by radical scavenging mechanism, tend to offer poor protection during frying (Catel, Aladedunye, & Przybylski, 2012; Kochhar & Gertz, 2004). On the other hand, natural components which can undergo acid-catalyzed decomposition reaction with activation energy lower than that of triacylglycerol dimerization may inhibit polymerization during frying even though they exhibit negligible radical scavenging activity (Kochhar & Gertz, 2004). Under the prevailing frying conditions, the antioxidant mechanism of the phenolic extracts evaluated in this study is evidently beyond radical scavenging. Thus, besides the well-known metal chelating potentials of their aglycones, it is possible that the polyphenols acted as polymerization inhibitors through acid-catalyzed hydrolysis of their glycosides. This competitive reaction for acid catalysts available in the frying medium may have spared the triacylglycerols from acid-catalyzed thermal polymerization (Kochhar & Gertz, 2004). Whereas the acid-catalyzed hydrolysis of polyphenolic glycosides may be too slow or improbable at storage temperature, the activation energy is expected to significantly reduce at frying temperature and in the presence of acid catalysts from foods (Timell, 1964; Wolfenden, Lu, & Young, 1998). Procyanidins present in the extracts/fractions can also be activated by acid-catalysis and high temperatures during frying. Further, unlike BHT which becomes inactive at high temperature due to evaporative losses and thermal inactivation, natural polyphenolic compounds are much less volatile and their degradation products can also act as secondary antioxidants. For instance, one of the major components of the thermal degradation of quercetin glycoside at 180  C was quercetin (Rohn, Buchner, Driemel, Rauser, & Kroh, 2007), and thermal degradation of quercetin yielded protocatechuic acid as a major component (Buchner, Krumbein, Rohn, & Kroh, 2006). This release of quercetin aglycone through thermal degradation of the glycosides could also be adduced for the better performance of CC4 (containing predominantly quercetin glycosides) compared to CC2 (composed mainly of quercetin) during Rancimat, despite the superior performance of the later under storage conditions (Fig. 2B and C, Table 1). Furthermore, the possibility of synergistic interactions among the phenolic constituents in the extracts cannot be disregarded. 4. Conclusion The present study showed that phenolic extracts from Canadian hawthorn and chokecherry fruits can be employed as natural antioxidative and anti-polymerization additives in vegetable oils for improved frying and storage stability. Evidently, thermal degradation of frying oils/fats by reactions that are not radicalmediated is also happening during frying. As such, antioxidants like BHT which operates exclusively by radical scavenging will offer poor performance during frying, compared to additives that can impede both radical and non-radical complex chemical reactions occurring under the stringent frying conditions. Results indicated that quercetin glycosides are better antioxidants under frying conditions whereas the aglycone, quercetin, offered better protection during storage. Due to their unique and yet diverse structural

features, plant polyphenols offer promising alternatives to synthetic antioxidants for frying applications. However, further studies on the effect of applied phenolic extracts on the sensory attributes of frying fats/oils and the prepared food are required.

Acknowledgment The financial support of the Alexander von Humboldt-Stiftung is highly appreciated. We thank Dr Christian Gertz (Maxfry, Hagen, Germany) for assistance with FT-NIR analyses.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2014.06.002.

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