Studies of the correlation between antioxidant properties and the total phenolic content of different oil cake extracts

Industrial Crops and Products 39 (2012) 210–217

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Studies of the correlation between antioxidant properties and the total phenolic content of different oil cake extracts ˇ b , Nataˇsa Poklar Ulrih a , Helena Abramoviˇc a,∗ Petra Terpinc a , Barbara Ceh a b

Biotechnical Faculty, University of Ljubljana, SI-1111 Ljubljana, Slovenia Slovenian Institute of Hop Research and Brewing, SI-3310 Zˇ alec, Slovenia

a r t i c l e

i n f o

Article history: Received 27 January 2011 Received in revised form 10 February 2012 Accepted 18 February 2012 Keywords: Oil cakes Extraction solvent Antioxidant activity Oxidative stability Correlation analysis

a b s t r a c t In this work, quantitative correlations between the antioxidant properties and the total phenolic content (TPC) of different oil cake extracts were studied. The oil cakes from camelina (Camelina sativa), linseed (Linum usitatissimum), rapeseed (Brassica napus), and two varieties of white mustard (Sinapis alba) were analysed as potential sources of antioxidant compounds. Two solvents of different polarity were used to obtain the extracts, resulting in great variation in antioxidant activity. The highest phenolic content was observed for white mustard, followed by camelina, rapeseed and linseed. The antioxidant properties were evaluated by determination of their reducing capacity, free radical scavenging activity and metal chelating ability, by the ␤-carotene bleaching method and as the effectiveness of inhibition of conjugate diene and triene formation in the bulk oil. The methanolic extracts exhibited higher reducing power (max. rapeseed), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging activity (max. rapeseed) and chelating ability (max. linseed), while the ethanolic extracts were more efficient in the ␤-carotene bleaching test (max. camelina). White mustard extracts inhibited conjugate diene and triene formation the most. These various antioxidant activities were compared to the synthetic antioxidant butylated hydroxytoluene (BHT). A lack of positive correlations among the different antioxidant activity assays and total phenolic contents was observed. © 2012 Published by Elsevier B.V.

1. Introduction Plants rich in antioxidants, including polyphenolic compounds, tocopherols, vitamin C and carotenoids, are attracting to the food industry as replacements for synthetic ones, which use is being restricted due to safety concerns. The synthetic antioxidants have been widely used to control lipid oxidative rancidity in foods, which is a major cause of quality deterioration, nutritional losses, offflavour development and discolouration. Besides prolonging the shelf-life of food products, these compounds are able to retard the progress of many oxidative stress-related chronic diseases in man. Therefore, dietary antioxidants also have an important role as nutraceuticals due to their role in protecting the body from free radicals, reactive oxygen species and reactive nitrogen species, which are derived either from normal metabolic processes or from external sources (Kohen and Nyska, 2002; RiceEvans et al., 1997; Bera et al., 2006). This protection is likely to involve several mechanisms of action, including inhibition of the generation of free radicals, enhancement of the scavenging capacity against free radicals, reducing capacity and metal chelating

∗ Corresponding author. Tel.: +386 1 320 37 82; fax: +386 1 256 62 96. E-mail address: [email protected] (H. Abramoviˇc). 0926-6690/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.indcrop.2012.02.023

ability. The antioxidant activity assays usually involve these reactions (Prior et al., 2005; Huang et al., 2005). Based on the antioxidant activity assays and the lipidic system used as substrate, a wide range of activities can be determined (Moure et al., 2000; Foti, 2007). Phenolic compounds may either occur naturally in the original plant or be formed during processing. The seeds of oil crops, particularly those containing a high percentage of polyunsaturated fatty acids are thought to be rich in antioxidants. The phenolic content is closely dependent on the oil extraction process as this determines the partitioning behaviour of the phenols and hence their distribution between the oil and waste fraction (Obied et al., 2005; Zhao et al., 2006). As is well known, a higher amount of phenolic compounds is released from the seeds when the oil is extracted at higher temperatures and higher pressure. In addition to the processing technique, the occurrence of specific phenols depends on the cultivar and maturity of the plant, climatic conditions, and storage time. In oilseed products, phenolic compounds occur as the hydroxylated derivatives of benzoic and cinnamic acids, coumarins, flavonoid compounds and lignins (Oomah et al., 1995). However, many of the antioxidants in oilseeds are not necessarily fat soluble (they occur in conjugation with sugar molecules), or their extraction into the oil is low (Terpinc et al., 2012).

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Therefore, after oil extraction, the remaining meal, also referred to as oil cake, are underutilized and provides an excellent source of raw material with potential applications as nutraceuticals and functional food ingredients (Shahidi, 2000; Moure et al., 2001; Balasundram et al., 2006). The antioxidant activity of several oilseed cakes has been reported, including camelina, rapeseed, mustard, linseed, sesame and evening primrose (Peschel et al., 2007; Matthäus, 2002; Naczk et al., 1998, 2005; Terpinc et al., 2012; Thiyam et al., 2004, 2006; Salminen et al., 2006; Vuorela et al., 2004; Shahidi et al., 2006; Wettasinghe and Shahidi, 1999; Wanasundara et al., 1996). The present research work focuses on the antioxidant potential of pressing residues from camelina, linseed, rapeseed, and white mustard. Linseed belongs to the Linaceae family and is rich in ␣-linolenic acid. It is grown around the world for oil and fibre production. Camelina, rapeseed, and white mustard are all members of the Brassicaceae family and contain ␻−6 and ␻−3 fatty acids. They have been cultivated for many years for the production of animal feed, vegetable oils and edible seeds for human consumption and recently even for biodiesel production. The aim of the current study was to compare the antioxidant activity of extracts obtained from fat-free residues of five different oilseeds. The influence of the extraction solvent (methanol (70%, v/v) and ethanol (96%, v/v)) on the total phenolic content (TPC) in the extracts and on their antioxidant activity was investigated. When exploring the antioxidant potential of different extracts, the use of a single test is insufficient to identify the different mechanisms involved. Therefore, four antioxidant assays, namely the reducing power assay, DPPH· radical scavenging activity, ferrous ion-chelating ability and alkylperoxyl radical scavenging capacity in the ␤-carotene-linoleic acid emulsion system were performed. In addition, the efficiency of the added extracts in promoting the oxidative stability of oil samples incubated at elevated temperature was also investigated. Finally, the correlations among different antioxidant assays and their applicability to oil cakes were evaluated.

2. Materials and methods 2.1. Plant materials Flax (Linum usitatissimum L.), rapeseed (Brassica napus L. var. napus) and two varieties of white mustard (Sinapis alba L.) were supplied by Slovenian Institute of Hop Research and Brewing (IHPS) from the field experiment in 2007. Camelina, with its other common names of false flax or gold of pleasure (Camelina sativa (L.) Crantz) was obtained from a local farm in the Koroˇska region, Slovenia. Seed cakes were obtained after cold-pressing of the seeds. Seeds (15 kg) were crushed and pressed by screw press machine within an hour and the temperature of the system was around 35 ◦ C.

2.2. Reagents and solvents Chloroform, ethanol (96%), methanol (99.9%) trichloroacetic acid, sodium carbonate, iron(II) chloride, potassium hexacyanoferrate(III), hexane and cyclohexane were obtained from Merck (Darmstadt, Germany). ␤-Carotene, DPPH· reagent, linoleic acid (95%), Tween 20, Folin–Ciocalteu reagent, ferrozine, and gallic acid were purchased from Sigma (Sigma–Aldrich GmbH, Steinheim, Germany). Potassium dihydrogen phosphate and di-sodium hydrogen phosphate were obtained from Kemika (Zagreb, Croatia). Iron(III) chloride was purchased from Carlo Erba (Milano, Italy). All the reagents were of analytical quality. For preparation of solutions ultrapure water (MiliQ, Millipore) was used.

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2.3. Extraction of phenolics The five different seedcakes obtained were ground and defatted with hexane (1:10, w/v) on a Vibromix 314 EVT shaker (Tehtnica Zˇ elezniki) and left to dry overnight at room temperature. An aliquot of 5 g of each sample was then extracted three times in 50 ml of methanol (70%, v/v) or ethanol (96%, v/v). The first extraction process involved stirring for 12 h at ambient temperature. The solid was recovered by filtration and subjected to two additional 45 min long extraction steps using ultrasound instead of a shaker. The solvent from the combined extract was evaporated under reduced pressure using a rotary vacuum-evaporator at 40 ◦ C. 2.4. Determination of total phenolic content (TPC) TPC in the tested cakes was evaluated using the Folin–Ciocalteu assay, as adapted by Gutfinger (1981), with some modifications. Briefly, 200 ␮l of diluted cake extract, a gallic acid (as a calibration standard), or Milli-Q water (as blank) was mixed with 125 ␮l of freshly prepared Folin–Ciocalteu reagent diluted with water (1:2, v/v) and 125 ␮l of sodium carbonate solution (20%, w/v). The final mixture was supplemented to 1 ml with Milli-Q water. After 40 min, the absorbance was measured in 1 cm cuvette at 765 nm (A765 nm ) on a model 8453 Hewlett Packard UV-Visible spectrophotometer (Hewlett Packard, Waldbronn, Germany) at room temperature. Results in triplicate were expressed in mg of gallic acid (GA) per 1 g of defatted cake. 2.5. Reducing power The reducing power of the cake extracts was determined by the modified (Terpinc et al., 2012) method of Juntachote et al. (2006). Different concentrations of methanolic or ethanolic extract (0.5 ml) were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.8) and potassium hexacyanoferrate(III) (2.5 ml, 1%, w/v). Then, trichloroacetic acid solution (2.5 ml, 20%, w/v) was added. After centrifugation at 13,000 min−1 for 15 min on a model 5415C Centrifuge (Eppendorf, Hamburg, Germany), the supernatant (2.5 ml) was mixed with MilliQ water (2.5 ml) and iron(III) chloride (1.0 ml, 1%, w/v). After 25 min, the absorbance was measured at 740 nm (A740 nm ). The reducing power of the extracts was quantitatively expressed as the slope of the lines representing the dependence of A740 nm on the concentration of total extractable phenolics in the reaction mixture (not shown) and denoted as the coefficient of reducing power (CR ). 2.6. DPPH· radical scavenging activity The DPPH· radical scavenging activity of the cake extracts was measured by the following procedure described by Brand-Williams et al. (1995). Briefly, a solution of DPPH· (2.9 ml, 0.1 mM) in 96% ethanol was added to 0.1 ml of extract solution at different concentrations. After 30 min, the absorbance was measured at 517 nm (A517 nm ). Ethanol (96%) was used as a blank. The control solution consisted of 0.1 ml of 96% ethanol and 2.9 ml of DPPH· solution. Triplicate analyses were run for each extract. The capability of scavenging the DPPH· radical was calculated as the percentage of DPPH· remaining in the resulting solution, using the following equation: DPPHremaining =

A

s 517 nm

Ac

517 nm



× 100%

(1)

where As 517 nm is the absorbance of the sample solution after 30 min and Ac 517 nm is the absorbance of the control solution with no antioxidant added. The percentage of DPPH· remaining against the sample concentration was plotted to obtain the amount of

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antioxidant necessary to decrease the initial concentration of DPPH· by 50% (EC50 ). 2.7. Iron-chelating capacity The iron-chelating capacity of the cake extracts was determined according to the method used by Decker and Welch (1990). An aliquot of the extract of the oil cakes (1.0 ml) was mixed with 3.7 ml of methanol (or ethanol for ethanolic extracts), 0.1 ml of iron(II) chloride (2 mM) and 0.2 ml of ferrozine (5 mM). The mixture was allowed to stand for 15 min, before the absorbance was measured at 562 nm. The absorbance of the control was determined by replacing the sample with methanol or ethanol. Antioxidant activity was expressed as the coefficient of chelating ability (CCA ) and calculated using the following equation: CCA =



1−

As Ac

562 nm



× 100%

(2)

2.10. Conjugated dienes, conjugated trienes Absorbance measurements at 232 nm and 268 nm of oil samples diluted with cyclohexane were performed according to the standard IUPAC method (IUPAC, 1987) to evaluate the amounts of conjugated dienes and conjugated trienes formed during storage at elevated temperature. The coefficient of antioxidant ability in prevention of formation of CD and CT in oil (CCD , CCT ) was expressed according to the formula:



CCD = 1 − and



CCT = 1 −

As Ac

232 nm /s

As Ac

268 nm /s



232 nm /c

× 100%

(4)

× 100%

(5)



268 nm /c

where Ac 562 nm is the absorbance of the control and As 562 nm is the absorbance in the presence of the sample. Triplicate analyses were run for each sample.

respectively, where Ac 232 nm is the absorbance of the control and As 232 nm is the absorbance of the sample at 232 nm; Ac 268 nm is the absorbance of the control and As 268 nm is the absorbance of the sample at 268 nm;  s and  c are the concentrations of an oil with and without antioxidant, respectively, in cyclohexane (g/100 ml).

2.8. Antioxidant activity by the ˇ-carotene bleaching test

2.11. Statistical analysis

The antioxidant activity of the cake extracts in an aqueous emulsion system of linoleic acid and ␤-carotene was determined according to a slightly modified method of Moure et al. (2000). ␤Carotene solution in chloroform (3.0 ml, 0.2 mg/ml) was mixed with 60 mg of linoleic acid and 600 mg of Tween 20. After evaporation of chloroform, 150 ml of Milli-Q water was added slowly and mixed well to form an emulsion. Aliquots of this emulsion (5.0 ml) and 0.2 ml of cake extracts (or methanol for the control) were mixed, thus the final concentration of antioxidant in the reaction mixture amounted to 10 ppm. The absorbance was measured at 470 nm against the blank, immediately after preparation (tY´ = 0 min) and after 120 min of incubation at 50 ◦ C. The blank was prepared by adding 0.2 ml of methanol to an emulsion consisting of linoleic acid, Tween 20 and Milli-Q water. All determinations were carried out in triplicate. The coefficient of antioxidant activity (CAA ) of the cake extracts in the emulsion was evaluated in terms of bleaching of the ␤-carotene using the following formula:

All the experiments were carried out in triplicate. Data were reported as means ± standard deviation (SD) for triplicate determinations. The Pearson correlation test was employed to determine the correlation coefficients among means.



CAA = 1 −

562 nm



As

470 nm (t=0)

− As

470 nm (t=120)

Ac

470 nm (t=0)

− Ac

470 nm (t=120)

× 100%

(3)

where As 470 nm (t=0) and Ac 470 nm (t=0) are the absorbances measured at zero time of incubation for the test sample and control, respectively, and As 470 nm (t=120) and Ac 470 nm (t=120) are the absorbances measured in the test sample and control, respectively, after incubation for 120 min. 2.9. Stabilization of oil The methanolic extracts of oil cakes were added to safflower oil at a concentration of 50 ppm. Safflower oil without antioxidant was used as a control. The oil samples were stirred on a Vibromix 314 EVT shaker (Tehtnica Zˇ elezniki) for 10 min to a uniform dispersion. After that, a stream of nitrogen was passed for 10 min to remove methanol from the added extracts. Synthetic antioxidant (BHT) was also employed at a concentration of 50 ppm, to directly compare the efficacy of the extracts. Oil samples with added antioxidant and control oil were placed in a laboratory dryer at 50 ◦ C for 14 days. All oil samples were prepared in triplicate. The oxidative deterioration level was assessed by measurement of conjugated dienes (CD) and conjugated trienes (CT).

3. Results and discussion 3.1. Total phenolic content The data produced on the antioxidant activity and TPC of the five oilseed cakes tested in this study are presented in Table 1, and major differences can be observed among them. The TPC of oil cakes were expressed as mg GA per g of defatted cake. In general, the highest phenol content was observed for white mustard, followed by camelina and rapeseed. Linseed exhibited a significantly lower phenolic content than the other plants considered. These results are partly in accordance with Matthäus (2002), who reported that the 70% methanol extract from white mustard had the highest phenolic content, followed by rapeseed and camelina. In the current study we determined slightly lower absolute values. It is hard to compare the results obtained in different investigations. The differences may arise for various reasons. As seen from Table 1, there are some differences already between two varieties of the same plant. We have to take into consideration the different extraction procedures and variety of total phenolic determinations. From the data in Table 1, it can be concluded that extraction with 70% methanol was more effective than with 96% ethanol. These results are in agreement with Matthäus (2002) who reported that different solvents used for the extraction of the fat-free residues have different capacities for extracting substances from these residues. In general, as reported before, the amounts of extractable phenolic substances decreased with decreasing polarity of the solvent. According to Zhao et al. (2006), the extraction solvent determines not only the total phenolic content, but also the amounts of individual compounds. The results of their investigation indicated that 80% methanol has a higher extraction capacity for catechin, syringic acid, ferulic acid, protocatechuic acid, caffeic acid, vanillic acid, gallic acid and p-coumaric acid than 80% ethanol. Another important aspect is the selection of appropriate reference substances with chemical and physicochemical properties similar to those of the samples to be studied, but this is often difficult (Parejo et al., 2002).

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Table 1 Total phenolic content and total antioxidant capacities of oil cake extracts from camelina, linseed, rapeseed and white mustard in comparison to BHT. Extr. solvent

TPCa (mg GA/g)

CR b (␮g/ml)−1

EC50 c (␮g/ml)

CCA d (%)

CAA e (%)

CCD f (%)

CCT g (%)

Camelina

Ethanol Methanol

4.5 ± 0.3 7.7 ± 0.4

0.189 ± 0.002 0.277 ± 0.002

5.7 ± 0.2 4.0 ± 0.1

19 ± 2 55 ± 2

73 ± 2 66 ± 1

nd 14 ± 2

nd 16 ± 3

Linseed

Ethanol Methanol

0.12 ± 0.03 0.9 ± 0.1

0.501 ± 0.039 0.636 ± 0.015

3.6 ± 0.3 3.0 ± 0.2

69 ± 1 95 ± 1

nd 53 ± 8

nd nd

nd nd

Rapeseed

Ethanol Methanol

3.4 ± 0.5 4.9 ± 0.4

0.713 ± 0.096 0.751 ± 0.025

2.8 ± 0.3 2.6 ± 0.1

22 ± 1 41 ± 1

nd 64 ± 1

nd 16 ± 1

nd 15 ± 2

White mustard Variety 1 Variety 2

Methanol Methanol

14 ± 1 13 ± 1

0.183 ± 0.002 0.250 ± 0.006

15.1 ± 0.9 12.5 ± 0.5

2.4 ± 0.1 1.8 ± 0.1

22 ± 2 32 ± 4

19 ± 1 23 ± 3

23 ± 1 22 ± 4

BHT

/

0.191 ± 0.010

13.6 ± 0.2

x

92 ± 1

27 ± 1

20 ± 2

/

x: no activity; nd: not determined. a Total phenolic content expressed in mg of gallic acid (GA) per 1 g of defatted cake. b Coefficient of reducing power. c Concentration of test compound required to produce 50% inhibition of DPPH· radical. d Coefficient of chelating ability. e Coefficient of antioxidant activity determined with ␤-carotene bleaching test. f Coefficient of antioxidant ability for prevention of formation of conjugated dienes. g Coefficient of antioxidant ability for prevention of formation of conjugated trienes.

Rapeseed meal contains several classes of phenolic compounds including phenolic acids, flavones and flavonols (Wanasundara et al., 1996). The most active antioxidant components of rapeseed meal were identified as glucopyranosyl sinapate, 4-vinylsyringol and sinapine (the choline ester of sinapic acid) (Vuorela et al., 2004; Thiyam et al., 2004, 2006). 4-Vinylsyringol, also referred to as canolol is a decarboxylation product of sinapic acid. Besides sinapic acid as the predominant compound, the presence of other phenolic acids has been reported in rapeseed meal (Table 2). Similarly, sinapic acid in free form and as its derivative sinapine also occurred in considerable quantities in camelina cake. According to Salminen and Heinonen (2008) beside hydroxycinnamic acids, flavanols and flavonols such as quercetin glucoside also contribute to the antioxidant activity of camelina cake. On the other hand, white mustard contains considerable more p-hydroxybenzoic acid than sinapic acid, while most of the other phenolic acids (Table 2) occurred in low concentrations (Kozlowska et al., 1983a,b). Linseed is a rich source of the lignan secoisolariciresinol diglucoside, meanwhile other phenolic compounds (Table 2) have also been found in linseed meal, e.g. the hydroxycinnamic acid derivatives, p-coumaric acid glucoside and ferulic acid glucoside (Johnsson et al., 2002) and the flavonoids herbacetin diglucoside and kaempferol diglucoside (Qiu et al., 1999). 3.2. Reducing power The reducing power is often used as an indicator of electrondonating activity, which is an important mechanism for testing the antioxidative action of phenolics. For the measurement of reductive ability, the transformation of ferric ion (Fe3+ ) to the ferrous form (Fe2+ ) was investigated using the potassium ferricyanide reduction method. The presence of reductants such as antioxidant substances in the tested sample causes the aforementioned reduction of Fe3+ , therefore the reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The yellow colour of the test solution changes into various shades of green and blue colours in a dose-dependent manner. Table 1 presents the CR values for the investigated extracts. A higher CR value indicates a higher reduction capacity. Using this assay, methanolic extracts were found to have better reducing capacity than ethanolic ones. This observation is in agreement with Zhao et al. (2006) who reported that various solvent extracts from the same barley variety showed significant differences in their reducing power, ethanolic being less effective than methanolic.

The reducing power of the oilseeds investigated exhibited the following order: rapeseed > linseed > camelina > white mustard. The quite significant differences in electron donating ability for different white mustard varieties suggested that variety already might have a substantial influence on antioxidant activity when the latter was estimated by reducing power assay. 3.3. DPPH· radical scavenging activity The model using scavenging of the stable DPPH· radical is a widely used assay to evaluate antioxidant activity in a relatively short time compared with other methods. The decrease in absorbance occurs when the DPPH· radical accepts an electron or hydrogen from an antioxidant. The radical become a stable molecule what is visually noticeable as a colour change from purple to yellow. The EC50 values of 70% methanol and 96% ethanol extracts in comparison to that of BHT are shown in Table 1. A lower EC50 value indicates a higher free radical scavenging activity. Generally, methanolic extracts demonstrated greater antiradical power. These results are in accordance with the report that 70% methanol is the preferred solvent for extracting DPPH· scavenging agents from plant material as compared to ethanol (Zhou and Yu, 2004). Among the methanolic extracts, rapeseed exhibited the most effective scavenging ability for the DPPH· radical, followed by linseed and camelina. The extract from white mustard showed significantly the weakest scavenging potential, irrespective of its variety. 3.4. Iron-chelating capacity Transition metal ions can accelerate lipid oxidation reactions by hydrogen abstraction and peroxide decomposition leading to the deterioration of flavour and taste in food. Chelating agents that form ␴-bonds with metal are effective as secondary antioxidants because they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion (Gordon, 1990). The chelating capacities for ferrous ions by five different oilseed extracts were estimated by the ferrozine assay. The latter can quantitatively form complexes with Fe2+ . In the presence of co-existing chelating agents, complex formation is inhibited and the red colour of the complex fades. Measurement of the colour reduction therefore allows an estimate of the metal chelating activity of the co-existing chelator (phenolic compounds) (Gülc¸in, 2006). In this assay, all the tested extracts interfered with the formation of the ferrous-ferrozine

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Table 2 List of phenolic acids, their derivatives and flavonoids, found in oil cakes of camelina, linseed, rapeseed, and white mustard. Phenolic compound Camelinaa

Protocatechuic acid p-Hydroxybenzoic acid Salicylic acid Sinapic acid Ellagic acid Sinapine 4-Vinylphenol 4-Vinylsyringol 4-Vinylguaiacol 4-Vinylcatechol Rutin Catechin Quercetin

Linseedb

Protocatechuic acid Gallic acid Vanillic acid Gentistic acid p-Hydroxybenzoic acid Syringic acid p-Coumaric acid o-Coumaric acid Ferulic acid Sinapic acid Caffeic acid Chlorogenic acid Herbacetin Kaempferol

Rapeseedc

p-Hydroxybenzoic acid Vanillic acid Gentistic acid Protocatechuic acid Syringic acid Gallic acid Sinapic acid p-Coumaric acid o-Coumaric acid Ferulic acid Caffeic acid Chlorogenic acid Sinapine 4-Vinylsyringol Apigenin Kaempferol Naringenin

White mustardd

Salicylic acid p-Hydroxybenzoic acid Vanillic acid Gentistic acid Protocatechuic acid Syringic acid p-Coumaric acid o-Coumaric acid Ferulic acid Caffeic acid Sinapic acid Sinapine

a

Refs: Terpinc et al. (2011a, 2012) and Salminen and Heinonen (2008). Refs: Dabrowski and Sosulski (1984), Hall et al. (2006), Kozlowska et al. (1983a), Kozlowska et al. (1983b) and Qiu et al. (1999). c Refs: Oskoueian et al. (2011), Shahidi and Naczk (1992), Naczk et al. (1998), Kozlowska et al. (1983a,b), Szydlowska-Czerniak et al. (2010), and Salminen and Heinonen (2008). d Refs: Kozlowska et al. (1983a,b). b

chelating power. As shown, all tested oilseed cake extracts exhibited metal chelating activity at the tested concentration. The results reveal that phenolic compounds extracted with methanol have a greater capacity for iron binding compared to those extracted with ethanol. Similar observation was published by Zhao et al. (2006). However, these results are not consistent with the research study of Jung et al. (2006), where exactly the opposite was found. The methanolic extract from linseed cake showed excellent chelating ability. The chelating effects of camelina and rapeseed were notably lower, but still not negligible. However, the iron chelating capacity of white mustard was found to be very low. There were some differences among different white mustard varieties, suggested that variety might have some influences on the chelating effectiveness. On the other hand, at the same concentration, the well-known commercial antioxidant BHT was not effective as a chelator at all. 3.5. ˇ-Carotene bleaching test The mechanism of bleaching of ␤-carotene is a freeradical-mediated phenomenon. ␤-Carotene in this model system undergoes rapid discoloration in the absence of an antioxidant. The linoleic acid free radical formed upon abstraction of a hydrogen atom from one of its methylene groups attacks the highly unsaturated ␤-carotene molecule. As ␤-carotene molecules oxidizes, the compound loses its characteristic orange colour, which can be monitored spectrophotometrically. The presence of different extracts can hinder the extent of ␤-carotene bleaching by scavenging the linoleic acid free radical and other free radicals formed in the system (Jayaprakasha et al., 2001). The antioxidant activity of the cake extracts investigated and BHT at 10 ppm concentration is presented by CAA in Table 1. A higher CAA value indicates a higher antioxidant activity in the emulsified system. As shown, adding camelina and rapeseed cake extracts to the emulsion at relatively low concentration considerably reduced ␤-carotene bleaching, indicating appreciable antioxidative activity. Further, also contribution of linseed extract was not insignificant. On the other hand, white mustard, regardless of the variety, exhibited no substantial antioxidant capacity in this assay. A similar tendency was observed by Matthäus (2002) who reported that methanolic extracts of rapeseed and camelina had a strong effect, whereas the extract obtained from mustard possessed only a relatively small antioxidant activity. However, the antioxidant activity determined by the ␤-carotene bleaching method was very different according to the plant material analysed. As mentioned before (Jayaprakasha et al., 2001), extracts prepared by different solvents exhibited varying degrees of antioxidant activity. This was observed with camelina extracts, where higher antioxidant activity was found for the ethanolic than the methanolic extract. Extracts prepared by 96% ethanol generally are expected to contain less polar phenolic compounds compared to 70% methanolic extracts. The highest CAA among all the tested samples was observed for the nonpolar BHT. These results together confirm the well-known phenomenon that the polar antioxidants remaining in the aqueous phase of the emulsion are more diluted in the lipid phase and thus are, less effective in protecting the emulsified linoleic acid, whereas lipophilic antioxidants due to higher partition into lipid phase reveal greater activity in the emulsion (Moure et al., 2001; Terpinc et al., 2011b). 3.6. Conjugated dienes, conjugated trienes

complex. The metal chelating activities of the tested extracts were concentration-dependent only at lower concentrations, while afterwards a plateau was achieved (data not shown). The percentages of metal chelating capacity, denoted as CCA , of the tested samples at a concentration of 0.025 mg/ml in the reaction mixture are summarized in Table 1. A higher CCA value indicates a higher

Besides the role of natural antioxidants as protective compounds against disease, they also prevent oxidative deterioration of lipid food during processing, distribution, and storage (Vági et al., 2005). The formation of conjugated dienes (primary oxidation products) and trienes (secondary oxidation products) in

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the bulk lipid system was monitored during the period of incubation of safflower oil. During the early stages of polyunsaturated fatty acid oxidation, the double bond migrates from its position and the unconjugated system is converted into a conjugated system that absorbs light at 234 nm. With the progress of oxidation, hydroperoxides undergo decomposition reactions that result in an increase of conjugated trienoic structures (keto acids and aldehydes), which can be evaluated by absorbance measurements at 268 nm. The ability of the extracts to prevent the formation of CD and CT at a concentration of 50 ppm over an oxidation period of 14 days is shown in Table 1. The greater the values of CCD and CCT , the more effective is the extract in suppressing oxidation in the oil. The CCD and CCT values for linseed could not be determined because the concentration of phenolic compounds in the prepared extract was too low. At early stages of the incubation, the CCD values for the samples under study were very similar to the control (data not shown). After 14 days of incubation at 50 ◦ C, the rate of CD formation in the oil samples increased significantly less in the presence of extracts than in the control. Camelina and rapeseed were similarly effective against CD formation. Also Thiyam et al. (2004) reported that rapeseed cake extracts contain significant amounts of phenolic compounds, which effectively prevent lipid oxidation. Similarly, Salminen et al. (2006) found that rapeseed and camelina cake extracts were effective antioxidants toward both protein and lipid oxidation. Phenolic compounds extracted from white mustard showed the highest antioxidative activity of the cakes tested, but not higher than BHT, a well-known commercial antioxidant. A different trend was observed when measuring CT. The level of accumulated CT did not increase significantly during the experiment. It is noticeable from Table 1 that addition of white mustard methanolic extract at 50 ppm inhibited oxidation significantly in safflower oil compared to the control after 14 days of exposure at 50 ◦ C, even more than BHT at the same concentration, and therefore far below its legal limit as a food additive. The inhibitory effects of camelina and rapeseed methanolic extracts against CT formation and consequently oil deterioration were similar, but not as important as that of white mustard. 3.7. Correlations among oil cake antioxidant activity assays and total phenolic contents To add to understanding of the interrelationship between oil cake antioxidant activity evaluation indices and phenolic compound contents, all the prepared extracts were used in an analysis of the correlation between TPC and reducing power, free radical scavenging activity, metal chelating activity, ␤-carotene bleaching inhibition or conjugated diene and triene formation. With reference to Table 3, TPC gave a statistically significant negative correlation with EC50 , CCA and CAA , with Pearson’s correlation coefficients |r| > 0.75. The positive correlation for EC50 in fact means that TPC correlated negatively with DPPH· radical scavenging effectiveness, suggesting that the oilseed with the lowest phenolic content (linseed) exhibited the greatest antioxidant properties. All these results indicate that TPC might not be a good predictor of the antioxidant ability of oil cakes and therefore confirm the non-specificity of the Folin–Ciocalteu method. Polyphenols are the major plant compounds with antioxidant activity, although they are not the only ones (Moure et al., 2001). Some nonphenolic compounds which are known to react with Folin–Ciocalteu reagent but are not effective as free radical scavengers (citric acid, ferrous sulfate, d-glucose) may also interfere with the correlation between TPC and the antioxidant activity of some extracts (Magalhães et al., 2006). Further, the lack of a positive correlation could be also explained by differences in the phenolic composition among plant extracts. The genotype affects the presence and ratio of a range of phenolic compounds with alterations in their hydrophilic nature, number

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Table 3 Correlations among oil cake antioxidant activity parameters and total phenolic contents.

a

TPC CR b EC50 c CCA d CAA e CCD f CCT g

TPCa

CR b

EC50 c

CCA d

CAA e

CCD f

CCT g

1 −0.656 0.893 −0.764 −0.768 0.753 0.985

/ 1 −0.713 0.495 0.308 −0.385 −0.739

/ / 1 −0.701 −0.882 0.775 0.997

/ / / 1 0.469 −0.912 −0.946

/ / / / 1 −0.801 −0.987

/ / / / / 1 0.813

/ / / / / / 1

a

Total phenolic content. Coefficient of reducing power. c Concentration of test compound required to produce 50% inhibition of DPPH· radical. d Coefficient of chelating ability. e Coefficient of antioxidant activity determined with ␤-carotene bleaching test. f Coefficient of antioxidant ability for prevention of formation of conjugated dienes. g Coefficient of antioxidant ability for prevention of formation of conjugated trienes. b

of available hydroxyl groups and complexity of reaction kinetics (Balogh et al., 2010). Moreover, even samples with similar concentrations of total phenolics may vary remarkably in their antioxidant activity, thanks to synergistic and antagonistic interactions among the antioxidants. The presence of antioxidants such as ascorbic acid, vitamin E, carotenoids, sodium sulfite and polysaccharides might also be responsible for enhancing the antioxidant activity. The results of the correlation studies of TPC with antioxidant activities could be further attributed to the fact that the antioxidant capacity in the present evaluation was estimated at the end of the observation period (when the steady state was reached). It has been confirmed that the antioxidant activity obtained at a fixed end point does not necessarily coincide with the evaluation by kinetic parameters (Terpinc et al., 2009; Terpinc and Abramoviˇc, 2010). It was reported earlier that no significant correlation could be found between the total phenolic content and antioxidant activity of various plant extracts (Kähkönen et al., 1999). On the contrary, a strong correlation of total phenolics with reducing power assay was found in canola cake extract in an investigation performed by Hassas-Roudsari et al. (2009). In our investigation both methods for determination of primary (CCD ) and secondary (CCT ) oxidation products formed in stored bulk oil correlated well with TPC. In general, different methods used to determine antioxidant activity are based on different reaction mechanism, thus they often give different results. To decide whether antioxidant capacities could be predicted from one assay to another, a correlation analysis was carried out. As shown in Table 3, all four methods were positively correlated with each other, regardless whether they are based on SET or HAT reaction mechanisms. The quite good linear relationship of DPPH· radical scavenging ability with other assays (|r| > 0.7), suggested that this method could provide some reliable information on the antioxidant properties of selected cake extracts. On the other hand, only moderate correlations (|r| > 0.5) among CCA , CR and CAA values were observed. Different assays produced different ranking orders. However, the non-significant correlation between individual methods suggested that the compounds which could scavenge the peroxyl radical from oxidized linoleic acid were mostly unable to reduce ferric ions or chelate metals. Moreover, as the reducing capacity determination is based upon reduction of ferric ion, antioxidants that act by radical quenching (H transfer), particularly thiols and carotenoids, will not be determined (Magalhães et al., 2008). On the other hand, the surprisingly strong correlation observed between CAA and EC50 indicated that, besides the polarity of the compound, also the ability to scavenge free radicals affects the behaviour of antioxidants in the emulsions. Regarding Table 3, CCD and CCT exhibited statistically significant

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negative correlations with results for DPPH· radical scavenging effectiveness, chelating ability and ␤-carotene bleaching inhibition. It is hardly to explain, why those antioxidant compounds extracted from the tested oilseeds, which were the most effective as scavengers of free radicals, metal chelators and reductants, at the same time made the weakest contribution against lipid oxidation in a homogeneous lipid system and vice versa. 4. Conclusion In conclusion, the present study has clearly demonstrated that both the plant material and the extraction solvent influenced the amount of total phenolic compounds and greatly affected their antioxidant activity. Interestingly, a negative correlation was found between TPC and the antioxidant efficiencies of the corresponding extracts, suggesting that phenolic compounds are not the only contributors to the antioxidant activities of the defatted oil cakes. Moreover, considering the insignificant correlation among CCA , CR , CAA , EC50 , CCD and CCT values, our samples contained antioxidants with varying reactivity in different methods. As the polarity of the solvent increases, a higher yield of total extractable polyphenolics was obtained. Moreover, 70% methanolic extracts offer better antioxidant activity than 96% ethanolic in terms of reducing capacity, DPPH· radical scavenging and chelating activity. However, ethanolic extracts exhibited a better response in alkylperoxyl radical scavenging in the ␤-carotene-linoleic acid emulsion system. In comparison to other investigated oil cakes, the extract from rapeseed exhibited the highest reducing power and was the most effective in scavenging DPPH· radicals. On the other hand, the extract from linseed was best in chelating the ferrous ion and the camelina extract was very efficient in preventing the oxidation of emulsified linoleic acid. The white mustard extracts yielded the highest total phenolic content, but exhibited the weakest antioxidant activity determined by the relevant assays. Nevertheless, the latter most successfully prevented conjugated diene and triene formation and also significantly enhanced the oxidative stability of the bulk lipid substrate. Therefore it is quite unreliable to use solely one method and highly crucial which measurements should be used to decide on or determine the antioxidant activity of a compound or an extract. In summary, it is difficult to decide which cake is the best potential source of natural antioxidants, because of their different mechanisms of action. However, some of the extracts showed an even higher antioxidant activity than that of the well-recognized synthetic antioxidant BHT. Therefore, the incorporation of tested oilseeds in food has great advantages. It would be interesting to perform further studies using the investigated oilseed cakes as food additives in order to increase the shelf life of foods by preventing lipid peroxidation. Moreover, the results obtained support the possibility that these byproducts of the oil-extracting process, normally used as fodder in animal nutrition or as a fuel in heating plants, could contribute a protective effect on human health. Acknowledgement The authors would like thank the Slovenian Research Agency for financial support through the research program P4-0121. References Balasundram, N., Sundram, K., Samman, S., 2006. Phenolic compounds in plant and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem. 99, 191–203. Balogh, E., Hegedüs, A., Stefanovits-Bányai, E., 2010. Application of and correlation among antioxidant and antiradical assays for characterizing antioxidant capacity of berries. Scientia Horticul. 125, 332–336.

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