Article pubs.acs.org/JAFC

Effect of Cyclolinopeptides on the Oxidative Stability of Flaxseed Oil Oyunchimeg Sharav,† Youn Young Shim,*,‡ Denis P. Okinyo-Owiti,‡ Ramaswami Sammynaiken,§ and Martin J. T. Reaney*,†,‡,⊥ †

Department of Food and Bioproduct Sciences, and ‡Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada § Saskatchewan Structural Sciences Centre, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada ⊥ Department of Food Science and Engineering, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China ABSTRACT: Polar compounds present in flaxseed oil increase its oxidative stability. Flaxseed oil becomes less stable to oxidation when filtered with silica. This observation may be linked to antioxidant compounds present in flaxseed oil. Flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. The polar fractions from flaxseed oil were then added back to silica-treated flaxseed oil to determine the impact of fractions containing polar compounds on oxidative stability (induction time) at 100 °C. A polar fraction containing mainly cyclolinopeptide A (CLA, 1), but also containing β/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.36 ± 0.28 to 3.20 ± 0.41 h. When oxidative stability was determined immediately after addition of the polar fractions other flaxseed fractions and solvent controls did not affect oil stability. However, when the oxidative stability index (OSI) test was delayed for three days and oil samples were held at room temperature after the addition of the polar fractions to the flaxseed oil, it was observed that the control oil treated with silica had become highly sensitive to oxidation. A polar fraction containing a mixture of CLs (1, 5, 7, 9, 11), improved the oxidative stability of peptide-free oil with respect to the control when the OSI measurement was made three days after adding the fraction. In addition, effects of 1 on the oxidative stability of peptide-free oil containing divalent metal cations was investigated. KEYWORDS: Linum usitatissimum L., cyclolinopeptide, flaxseed, antioxidant, oxidative stability

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shelf life than refined flaxseed oil.11 On the other hand, coldpressing procedures do not involve heat or chemical treatments and the oil is extracted under mild conditions.12 Siger et al.11 reported several antioxidant compounds in cold-pressed flaxseed oil, including flavonoids (12.7−25.6 mg/100 g), phenolic acids (76.8−307.3 mg/100 g), α-tocopherols (0.55− 9.11 mg/100 g), γ-tocopherols (10.56−15.00 mg/100 g), and plastochromanol-8 (3.37−5.53 mg/100 g). However, tocopherols are considered the main antioxidants in flaxseed oil. Some authors report weak13 or no relationship between flaxseed oil oxidative stability and the content of tocopherols or phenol compounds in the oil.14 Therefore, it is possible that other minor oil soluble compounds play a role in slowing the oxidation of flaxseed oil. Cyclolinopeptides (CLs) are hydrophobic compounds present in flaxseed oil with polarity comparable to known natural antioxidants (Table 1 and Figure 1), but the antioxidant activity of these compounds has not been studied. The oxidative stability of CLs in vegetable oil was reported by Brühl et al.6 while the stability of CLs in partially defatted flaxseed products was reported by Aladedunye et al.7 The first discovered CL, CLA (1), was characterized in 1959.15 Its unique structure induced researchers to investigate its

INTRODUCTION Flaxseed (Linum usitatissimum L.) is an oilseed adapted to cool northern climates. Whole flaxseed contains 41% fat, 28% dietary fiber, and 21% protein having an amino acid profile comparable to that of soybean meal.1 Flaxseed is one of the richest dietary sources of α-linolenic acid (ALA), having high levels of polyunsaturated fatty acids (73%).2 Food and Agriculture Organization statistics show that global flaxseed production has been stable at ∼2 000 000 tonnes between 2008 and 2012 with the majority of the crop being produced in North America, Asia, and Europe.3 Flaxseed oil is used predominantly as drying oil but it is increasingly being used as a source of dietary omega-3 fatty acids.4 Whereas the high content of unsaturated fatty acid present in flaxseed is important to its purported health benefits, its chemical structure makes it vulnerable to oxidation. Oxidation of flaxseed oil during improper processing and storage reduces its nutritional value and causes the development of undesirable flavors.5,6 Despite its distinctive unstable oil content, flaxseed produces antioxidants that protect the meal in storage.7−9 Flaxseed contains several known bioactive compounds, including lignans, phenolic acids, anthocyanin pigments, flavonols, flavones, and phytic acid, all of which may contribute to the seed’s antioxidant capacity.10 The concentration of these antioxidants in flaxseed oil depends on the extraction procedure and the treatment of the oil. Moreover, refining may also reduce minor compounds that have an effect on oil shelf life. The unrefined oil may have higher nutritional value and longer © 2013 American Chemical Society

Received: Revised: Accepted: Published: 88

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Table 1. Amino Acid Sequences and Chemical Formulas of CLs CL (code) CLA (1) 1-Met-CLB (2) 1-MetO-CLB (3) 1-Met-CLD (4) 1-MetO-CLD (5) 1-Met-CLE (6) 1-MetO-CLE (7) 1-Met-CLF (8) 1-MetO,3-MetO-CLF (9) 1-Met-CLG (10) 1-MetO,3-MetO-CLG (11) 1-MetO,3-Met-CLG (12) 1-Met,3-MetO-CLF (13)

namea 15

CLA CLB47 CLC47 CLD′48 CLD47 CLE′48 CLE47 CLF′48 CLF49 CLG′48 CLG49 CLH49 CLI49

amino acid sequence (NαC-)b

molecular formula

protonated ion mass (m/z)c

Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile Met-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile MetO-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile Met-Leu-Leu-Pro-Phe-Phe-Trp-Ile MetO-Leu-Leu-Pro-Phe-Phe-Trp-Ile Met-Leu-Val-Phe-Pro-Leu-Phe-Ile MetO-Leu-Val-Phe-Pro-Leu-Phe-Ile Met-Leu-Met-Pro-Phe-Phe-Trp-Val MetO-Leu-MetO-Pro-Phe-Phe-Trp-Val Met-Leu-Met-Pro-Phe-Phe-Trp-Ile MetO-Leu-MetO-Pro-Phe-Phe-Trp-Ile MetO-Leu-Met-Pro-Phe-Phe-Trp-Ile Met-Leu-MetO-Pro-Phe-Phe-Trp-Val

C57H85N9O9 C56H83N9O9S C56H83N9O10S C57H83N9O8S C57H77N9O8S C51H76N8O8S C51H76N8O9S C55H73N9O8S2 C55H73N9O10S2 C56H75N9O8S2 C56H75N9O10S2 C56H75N9O9S2 C55H73N9O9S2

1040.65 1058.61 1074.56 1048.60 1064.54 961.59 977.52 1052.51 1084.47 1066.56 1098.50 1082.38 1068.35

Peptides are named according to references where they are first described. bThe first and third positions of amino acid sequences are highlighted in Figure 1. Abbreviations are Met for methionine and MetO for methionine S-oxide. cFrom reference and ESI-MS data obtained as described in supplemental file by Gui et al.27 a

tested for their effects on the oxidative stability of peptide-free flaxseed oil. Subsequently the effects of enriched CLs on the oxidative stability of flaxseed oil in the presence of metal cations were determined. Analysis of the compounds present in fractions that contribute to oxidative stability may reveal compounds contributing to the antioxidant mechanism in flaxseed oil. There are no previous reports of the antioxidant activity of CLs in flaxseed oils.

bioactivity. Recent studies show that peptides possess a wide range of biological activities and can act as anticancer16 and antimicrobial17 agents. Peptides have also demonstrated immunomodulatory and antioxidant activities. Moreover, some peptides produced in living cells act as antioxidants. Well-known examples of such antioxidants include hormones (melatonin,18 angiotensin, oxytocin, and enkephalin19) and skeletal muscle peptides carnosine and anserine.20 The antioxidative mechanism of peptides is not well understood. Some authors suggest that peptides that contain aromatic residues (tyrosine, histidine, tryptophan, and phenylalanine) are able to stabilize reactive oxygen species (ROS) through electron transfer.21 Peptides are also well-known metal chelators and this property may contribute to antioxidant properties. The antioxidant activity of peptides produced by protein digestion is often greater than that of the intact proteins and the amino acid order and structure affect antioxidant activity.22 In accelerated oxidation tests of lipids, the oxidation rate is normally low until natural antioxidants are consumed and then the rate of oxidation increases. The time required for the increase in oxidation rate is called the induction time. The active oxygen method (AOM) accelerates lipid oxidation by applying both elevated temperatures and oxygen to an oil. The oxidative stability index (OSI) is an accelerated oxidation assay that determines production of volatile organic compounds produced during oil oxidation. The main criticism of this assay is that the oxidation mechanism at high temperatures is not necessarily similar to the mechanism encountered under normal storage conditions. Nevertheless, it is reported that the shelf life of the sample at ambient conditions can be predicted based on OSI at elevated temperature.23,24 In general, lipid oxidation is the result of many complex chemical processes and interactions. The reaction between unsaturated fatty acid groups in lipids and ROS created in different ways is important to the stability of lipids containing unsaturated fats.25 The presence of CLs in flaxseed oil has led us to hypothesize that these compounds may contribute to the stability of crude flaxseed oil. The purpose of this study is to isolate polar fractions present in flaxseed oil, including CLs, to determine if these fractions increased the oxidative stability of flaxseed oil that has been treated to remove polar compounds. The effect of CL-containing fractions obtained from crude flaxseed oil were

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MATERIALS AND METHODS

General. Cold-pressed crude flaxseed oil was donated by Biorginal Food and Science, Inc. (Saskatoon, SK, Canada), and stored at 4 °C. Canola oil was purchased from a retail supplier (Saskatoon, SK, Canada). Silica gel 60 (particle size 0.040−0.063 mm, 230−400 mesh) was purchased from EMD Chemicals Inc. (Gibbstown, NJ, USA). High-pressure liquid chromatography (HPLC) and column chromatography solvents were HPLC grade and purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. A Milli-Q system (Millipore, Bedford, MA, USA) was used to prepare deionized water for all mobile phases. Metal stearates (Zn2+, Ni2+, and Co2+) were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). CL 1 was prepared by reverse phase chromatography of a silica gel solid phase extraction of flaxseed oil according to Reaney et al.26 and Gui et al.27 At the time of this study highly enriched CL 1 was available in relatively large quantities while other peptides were not available in quantities sufficient to conduct similar studies. Silica Gel Flash Column Chromatography. A cotton ball was placed in the bottom of the glass column (2.5-cm i.d.) which was then packed with silica gel (80 cm3). A sand layer (1-cm; 50−70 mesh) was placed on top.27 Flaxseed oil (400 mL) was introduced into the column at a ratio of 5:1 (v/v, oil to silica gel) and was allowed to elute through the column under gravity to yield peptide-free oil.27 The column was eluted with 400 mL of 100% hexane (A), 250 mL of 20% (v/v) ethyl acetate (EtOAc) in hexane (B), 250 mL of 50% (v/v) EtOAc in hexane (C), 250 mL of 100% EtOAc, and (D) finally 250 mL of 10% (v/v) methanol (MeOH) in dichloromethane (DCM, E) (Figure 2). Each of the fractions B−E (∼230 mL each) were collected in volumetric flasks and diluted to a final volume of 250 mL. Samples of each fraction (B−E; 20 mL each) were taken and concentrated under vacuum pressure at 40 °C using a Buchi Rotavapor R-200 evaporator (9000 Pa, Brinkman Instruments, Inc., Westbury, NY, USA) and the resulting residue was subjected to HPLC and HPLCMS analysis. HPLC Analysis. HPLC analysis was conducted using an Agilent 1200 series HPLC systems (Agilent Technologies Canada Inc., Mississauga, ON, Canada) equipped with a quaternary pump, an autosampler, and a variable-wavelength diode array detector (DAD, 89

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Figure 1. Structures of CLs. Met and MetO in the first and third positions of amino acid sequences are highlighted in CLs. to 60:40 in 0.5 min, to equilibration for 5.5 min) and a flow rate of 0.40 mL/min. OSI Analysis. Effect of Polar Fractions on Oxidation of SilicaTreated Flaxseed Oil. Eluate from each fraction (B−E; 30 mL) was added to 30 mL of silica-treated flaxseed oil, and the mixture was concentrated under vacuum at 40 °C using a Buchi Rotavapor R-200 evaporator (9000 Pa) to remove organic solvent. Control samples were prepared by adding the same solvent used to elute the column to silica-treated flaxseed oil and concentrating under vacuum pressure as described above. Crude and silica-treated flaxseed oil samples were kept under vacuum pressure in a desiccator for 24 h prior to OSI analysis (as described below). Effect of the Concentration of Polar Fraction on the OSI of SilicaTreated Flaxseed Oil. Fractions D and E (200 mL) were collected from a silica gel flash column and concentrated as described above. The concentrate was added to 30 mL of silica-treated flaxseed oil and stored in a desiccator that was sealed and connected with a vacuum system for 24 h. The OSI of these samples was determined as described below.

190−300 nm). The samples were separated on a ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm i.d., 5 μm, Agilent Technologies Canada Inc.) with an in-line filter. The mobile phase consisted of a linear gradient of water−acetonitrile and a flow rate of 2 mL/min.27 The injections of silica extracts onto the HPLC column were conducted to show if compounds were present and were not meant to be quantitative. Chromatograms of peptide extracts had peaks that exceeded an absorbance of 2.5 AU. With this strong absorbance quantative analysis of peptides was not possible. Mass Spectrometry. Tocopherols in fraction D were separated using a Chromolith FastGradient RP-18e column (50 × 2.0 mm i.d., 3 μm, Merck KGaA, Darmstadt, Germany). An Agilent HPLC (1200 series) equipped with a quaternary pump, an autosampler, DAD (190−300 nm), and a degasser directly connected to a Bruker micrOTOF-Q II Mass Spectrometer (Hybrid Quadrupole-TOF MS/ MS; Bruker, Bremen, Germany) with Apollo II ESI ion source (operated with Nebulizer gas at 4.0 bar and dry gas temperature held at 200 °C) was used in HPLC MS analysis. The mobile phase consisted of a linear gradient of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic in acetonitrile (60:40 for 2 min, to 10:90 in 8 min, 90

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obtained by silica gel flash chromatography are presented in units of g/100 mL of flaxseed oil (Table 2). Silica-treated Table 2. Yield of Flaxseed Oil Extracts in Different Solvents fraction

yield g/100 mLa

Bb Cb D

24.96 ± 0.12a 2.29 ± 0.28b 1.11 ± 0.98b

E

1.09 ± 0.34b

details of content NDc NDc CLs (1−8, 10, 12) and tocopherols (β-, γ-, and δ-) CLs (1−13)

Values are mean ± standard deviation (SD) (n = 4). Means followed by the same letter are not significantly different at p < 0.05. bOil was a major component of these fractions. cND: not detected. a

flaxseed oil, with a clear yellow color, was obtained by passing fresh crude flaxseed oil through a silica gel column. Using an approach analogous to that applied in this study, Abuzaytoun and Shahidi29 analyzed fractions obtained by passing coldpressed hempseed and flaxseed oil through a column packed with two adsorbents (activated silicic acid and activated charcoal) and eluting the compounds with a series of organic solvents. They observed that after absorption on silica and carbon, the oil was essentially free of tocopherols and lacked light absorbance at 550−710 nm, which would indicate a loss of chlorophyll molecules as well. CLs were identified by their standard HPLC retention times as described by Gui et al.27 The crude flaxseed oil was rich in 1−13 (Table 3 and Figure 3). HPLC analyses of fractions B−E revealed the presence of CLs in fractions D and E. Fraction D contained CLs (1−8, 10, 12), whereas fraction E contained a mixture of CLs (1−13) (Figure 4C and D). Peptides containing methionine (Met) group (2, 4, 6, 8, and 10) were decreased, and CLs (9 and 11) were not apparent in the HPLC chromatograms of polar compounds isolated from flaxseed oil. Oxidation of the Met group of CLs into methionine S-oxide (MetO) is observed regularly with the exposure of flaxseed oil to oxygen during oil extraction.30 Most of the peptides were observed in fractions D and E. In addition, mass spectrometric analysis of fraction D indicated the presence of γ-tocopherol (Figure 5), β- and δ-tocopherols (data not shown). Oxidative Stability Test. To determine the presence of antioxidant substances, the OSI values were determined at 100 °C on crude pressed flaxseed oil, silica-treated flaxseed oil, and silica-treated flaxseed oil with polar fractions (B−E) added (Table 4). Adding solvents to silica-treated flaxseed oil and subsequently evaporating them was conducted to act as a control. The OSIs of solvent-treated control oils, ranging from 2.03 to 2.65 h, were not significantly different from that of silica-treated flaxseed oil (Table 4). An effect of the addition and removal of solvents was observed with solvent D treated oils having lower stability (Table 4). This was not explored further. Crude flaxseed oil had higher oxidative stability than silica-treated flaxseed oil, indicating that some of the chemical constituents that were removed by silica gel play a significant role in reducing the rate of flaxseed oil oxidation. OSI times of crude and silica-treated flaxseed oils varied from 3.00 to 4.30 h and 1.15 to 2.75 h, respectively (Table 4). Abuzaytoun and Shahidi26 observed that passing flaxseed oil over a silicic acid/ charcoal column reduced markers of oxidation, thiobarbituric acid-reactive substances and conjugated dienes of cold-pressed flaxseed oil from 6.01 to 4.54 μM/g and from 1.65 to 1.09 μM/ g, respectively. However, the oils were more susceptible to

Figure 2. Flowchart for isolation of CL-containing fractions and sample preparation for OSI. Effect of CL 1 and Divalent Metal Cations on the OSI of Flaxseed Oil. Stock solutions of metal stearates (Ni2+ and Zn2+; 1.25 mM) and 1 (2.5 mM) were prepared in silica-treated flaxseed oil. The stock solutions were then added to silica-treated flaxseed oil to achieve the desired final concentrations of metal stearate (0.125 mM) and 1 (0.25 mM). The OSI of these samples was determined as described below. In a separate study, stock solutions of zinc stearate (1.0 mM) and 1 (1.0 mM) were prepared in canola oil. Subsequently, 1 (1 mL, 2 mL, 3 mL) and zinc stearate (4 mL) stock solutions were added to 23 mL of silica-treated flaxseed oil. The mixture volume was increased to 30 mL by the addition of canola oil. The final concentration of 1 was 0.03− 0.09 mM and that of Zn2+ was 0.12 mM. The OSI of these samples was determined as described below. In a third study, stock solutions of 1 (0.625 mM) and cobalt stearate (0.625 mM) were prepared in canola oil. The canola oil along with metal-stearate-treated canola oil were added to 25 mL of silica-treated flaxseed oil until the volume of the oil was 32 mL. The addition of treated canola oil produced mixed oils with final concentrations of 0.04 or 0.12 mM 1 and/or 0.02 mM cobalt stearate. The OSI of these samples was determined as described below. Measurement of OSI. OSI was measured with an OSI instrument (Omnion, ADM, Rockville, MD, USA) using AOCS official method Cd 12b-92.28 Samples (5.0 ± 0.05 g) were taken in a glass test tube and then placed in the preheated (100 °C) OSI instrument. Air was bubbled through each sample (130 mL/min flow rate) during heating. Technical replicate samples (n = 4) were analyzed in each experiment. Crude oil and silica-treated flaxseed oil were taken as reference samples. OSI values were calculated using the Omnion OSI v 8.18 software, which detects the end point mathematically by determining the maximum of the second derivative of conductivity plotted against time. Statistical Analysis. All analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) and data are presented as mean ± standard deviation (SD) (n = 4). Differences between mean values were evaluated using one-way analysis of variance (ANOVA) followed by a posthoc least significant difference (LSD) test, or an unpaired Student’s t test. Statistical significance was accepted at p < 0.05.

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RESULTS AND DISCUSSION CL Isolation and Identification. UV absorbance indicated the presence of carotenoids in the oil. In the present study, CLs (2, 3, 5, and 7) containing polar fractions from silica were prepared by flash column chromatography of cold-pressed flaxseed oil, and the solvent was removed from the fractions by concentrating under vacuum. The yields of the extracts 91

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Table 3. HPLC Profile of Crude Flaxseed Oil and its Fractions crude flaxseed oil CL 1 2 3 4 5 6 7 8 9 10 11 12 13 a

retention time (min)a retention time (min)b 24.05 21.55 13.61 NDe 17.22 NDe 15.72 NDe NDe NDe 12.83 NDe NDe

fraction D

fraction E

area (mAU × s)c retention time (min)b area (mAU × s)c retention time (min)b area (mAU × s)c

24.24d 21.59 13.74 25.92 17.80 24.24d 15.70 23.22 12.35 25.20 12.83 18.42 16.58

2434d 654 5147 896 3950 2434d 9679 204 736 629 2467 5696 1786

24.20d 21.73 13.77 26.08 17.90 24.20d 15.72 23.33 NDe 25.28 NDe 18.53 16.66

30743d 24744 5718 17842 2458 30743d 5884 5980 NDe 17650 NDe 3282 1027

24.17d 21.77 13.78 26.01 17.93 24.17d 15.77 23.39 12.36 25.32 12.85 18.56 16.68

55747d 15135 39923 2063 25429 55747d 74445 1022 4550 1822 16993 41303 11886

Retention time assigned by Gui et al.27 bMean of retention time for 4 runs. cMean of peak area for 4 runs. dCoeluted. eND: not detected.

Figure 3. HPLC profile of fresh crude flaxseed oil.

Figure 4. HPLC profile of polar fractions obtained by silica absorption of flaxseed oil: (A) fraction B; (B) fraction C; (C) fraction D; (D) fraction E.

oxidation with continued storage. Several natural antioxidant compounds have been determined in flaxseed oil and the quantity of these compounds depends on cultivar, growing conditions, and processing method. According to the study of Choo et al.,12 cold-pressed flaxseed oil purchased in New

Zealand, including locally produced and imported oil, contained 76.8−307.3 mg/100 g phenolic acid, 12.7−25.6 mg/100 g flavonoids, 3.4−5.5 mg/100 g plastochromanol-8, and 11.1−24.5 mg/100 g tocopherols. In addition, cold-pressed flaxseed oil may contain up to 150 mg/100 g CLs.27 Therefore, 92

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Figure 5. Mass spectrum of fraction D containing tocopherols.

Table 4. Oxidative Stabilitya of CLs-Containing Fractions and Flaxseed Oils OSI (h) 0 month fraction B C D E crude flaxseed oil silica-treated flaxseed oil

OSI (h) 10 months

1st day 2.78 2.41 3.20 2.77 4.30 2.36

± ± ± ± ± ±

0.57c 0.50c 0.41b 0.48c 0.56a 0.28c

1st day NDb NDb 3.56 ± 3.02 ± 3.00 ± 2.75 ±

0.68a 0.45ab 0.10ab 0.20b

3rd day NDb NDb 2.86 ± 2.35 ± 3.07 ± 1.15 ±

0.21b 0.27b 0.47a 0.07c

reference (solvent) 2.65 ± 2.54 ± 2.03 ± 2.34 ± NDb NDb

0.41a 0.26a 0.03a 0.30a

a Values are mean ± SD (n = 4). Means in the same vertical column followed by the same letter are not significantly different at p < 0.05. bND: not detected.

reduced oxidative stability observed with silica treatment may be related to the removal of any or all of these polar compounds. The presence of other minor compounds and synergistic interaction among substances should be considered.12,29 Addition of fraction D to silica-treated flaxseed oil increased its stability to oxidation when compared to silicatreated flaxseed oil and solvent treated control oil (Figure 6, Table 4). However, oils treated with fractions B, C, or E were not significantly different from solvent-treated oil controls (Table 4). Fraction D contained 1 and trace amounts of γ-, β-, and δ-tocopherols. These compounds could potentially contribute to the observed increase in oxidative stability.

Fractions B and C did not affect oxidative stability of silicatreated flaxseed oil when compared with appropriate controls. Fraction E contained more polar CLs than Fraction D (Table 4). The effect of silica treatment on other compounds such as phenolics and flavonoids with known antioxidant properties31,32 was not investigated, but these compounds were not evident in extracts and may remain bound to the silica column. Reports of the presence of phenolic compounds in flaxseed11,12,33 are based on the use of colorimetric methods that employ low specificity reagents. For instance, the Folin− Ciocalteu reagent was used to determine the flavonoid content of cold-pressed flaxseed oil,12 but this reagent may also react with a broad range of reducing substances including nitrogencontaining compounds such as indole derivatives.34 D/Ltryptophan produces a strong absorbance at 745 nm when reacted with Folin−Ciocalteu reagent.34 As indicated in Figure 4, fraction E contains 5, 9, and 11. Each of these peptides contains tryptophan and MetO residues. It is possible that these substances reduce the Folin−Ciocalteu reagent, giving a false indication of the presence of phenolic compounds in flaxseed oil. Many studies have revealed that polar fractions obtained from various vegetable oils contain phenolic compounds that contribute to antioxidant activity. For instance, Zhang et al.35 contacted parsley essential oil with silica gel and conducted a stepwise solvent elution of the silica with EtOAc, EtOAc/ MeOH (1:1, v/v), and MeOH. This study revealed that the antioxidant activity of the fractions increased with the polarity of the solvent used to elute that fraction from the silica. Phenolic compounds are also reported in fractions exhibiting

Figure 6. OSI of flaxseed oil and CLs-containing fractions. 93

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oxidation. Also, if 1 binds to metals in flaxseed oil it may affect the rate of oil oxidation. Metal binding with 1 has been reported. Balasubramanian et al.40 reported that 1 binds weakly to Ca2+ ions in MeOH. Moreover, Chatterji et al.41 detected 1: Tb3+ complexes stabilized by the f orbital of the lanthanide. Although binding of both ions with 1 was stabilized by Phe residues, the interaction with Ca2+ did not involve charge. Silica gel adsorption readily removes divalent metal ions with high efficiency from different substrates.42−44 Hence, silicatreated flaxseed oil is likely greatly reduced in, or free from. divalent metal ions. We observed that transition metal ions Zn2+ and Ni2+ accelerated the oxidation rate of silica-treated flaxseed oil (Table 5). Induction times of silica-treated flaxseed oil in the presence of these metals at 0.25 mM concentration were reduced by over 0.8 h when compared with controls without added metal. Whereas 1 had no effect on the oxidation of oil when added alone, 1 slowed oxidation induction by Ni2+ and Zn2+ by 17 and 37%, respectively (Table 5). The is the first study describing the impact of a CL on the rate of oil oxidation in the presence of a transition metal (Zn2+, Ni2+, and Co2+). The observed results may be attributed to 1 binding with metal soaps, making them unavailable for oil oxidation. In addition, zinc ion is abundant in flaxseed (4 mg/100 g) and this study revealed that 1 could potentially interact with zinc. Additional experiments were focused on the dose-dependent interaction of zinc with 1. The effects of 1 and zinc ion on oxidation of silica-treated flaxseed oil at mole ratios of 1:4, 1:2, and 1:1.3 of 1 to Zn2+ were explored (Table 6). Canola oil was used as a more stable oil to dissolve metal stearates and 1 as dissolving these compounds in flaxseed oil led to unacceptably rapid oxidation (not shown). The addition of canola oil increased overall oil stability (from 2.35 h up to 3.5−8 h) and, hence, OSI times are not comparable to those in other studies. This agrees with other studies where blends of flaxseed oil with sesame, milk thistle,36 and olive oil37 have superior oxidative stability when compared to unblended flaxseed oil. This phenomenon occurs with other oils. For example, Isbell et al.45 reported that adding just 5% of crude meadowfoam oil to triolein enhanced its stability by 35%. The addition of canola oil modified the impact of zinc on oil OSI. Zinc soaps did not lower the OSI of blends of canola and flaxseed oil. It is possible that antioxidant compounds are present in canola oil that inhibit zinc oxidation pathways. Canola oil is rich in tocopherols; commercial canola oil contains up to 700 mg/kg tocopherols. Moreover, during refining processes, especially after bleaching and deodorizing, metal cations would have been almost cleared from the commercial oil used in this study.46 CL 1 improved the oxidative stability of canola/flax blends when added alone to the oil. However, the presence of zinc reduced the antioxidant effect of 1 (Table 6). At 0.12 mM concentration, 1 enhanced the induction time by 11.4 and 6.7% when canola oil was present at 26.0 and 16.6%, respectively. An interaction of 1 with zinc on oxidative stability was also observed in this study. As shown in Table 6, the OSI times of a

the strongest antioxidant activity. In comparison to fraction D and the solvent control, fraction E has little or no effect on flaxseed oil oxidation in spite of its higher polarity (Table 3). It is possible that fraction E does not contains phenolic compounds. Elevated induction time may be linked to specific CLs abundant in fraction D but are absent or less abundant in fraction E, or synergic activity of the peptides with tocopherols detected in fraction D. The dose−response and time-dependent activity of fractions D and E were studied after 10 months storage at 4 °C in a refrigerator (Table 4 and Figure 7). The

Figure 7. OSI of flaxseed oil and CL 1 + M2+.

induction time of crude flaxseed oil decreased from 4.30 to 3.07 h during the 10-month storage period. Guseva et al.36 observed that the peroxide value of cold-pressed flaxseed oil increased from 1.8 to 2.9 after six months storage at 10 °C, while Yildirim34 reported that over 126 days storage at 4 °C, the peroxide value increased from 3.01 to 6.73. In addition, the fatty acid and antioxidant contents in crude flaxseed oil changed significantly over the first seven months of storage, after which no further changes were observed.37 In this study the OSI times were measured over three days (Table 4) after removal from storage at 4 °C and maintaing the sample at room temperature. The oxidative stability of silica-treated flaxseed oil decreased significantly, by over 50%, after the temperature was increased, while oil subjected to other treatments was more stable. Silicatreated flaxseed oil with added fractions D and E decreased in induction period by 20% after three days removal from storage. Therefore, both fractions D and E have antioxidant properties that may be effective for long storage periods at low temperatures. It is important to note that the largest impact of fractions D and E was observed only after long storage followed by removal from storage for three days. Influence of CL 1 on Metal Cation Induced Oxidation of Flaxseed Oil. Crude cold-pressed vegetable oil contains small amounts of metals. The quantity of trace metals varies depending on genetics, environmental conditions during plant growth, and processing methods used for oil extraction.38,39 These metals potentially act as lipid oxidation catalysts. In this study, transition metal ions (Zn2+, Co2+, and Ni2+) were added to flaxseed oil to determine their effects on the rate of oil Table 5. OSIa of Flaxseed Oil with 1, Zn2+, and Ni2+ Stearates

a

silica-treated flaxseed oil

1 (0.125 mM)

Zn2+ (0.25 mM)

1 + Zn2+ (1:2)

Ni2+ (0.25 mM)

1 + Ni2+ (1:2)

2.36 ± 0.20a

2.21 ± 0.40a

1.50 ± 0.20b

2.05 ± 0.25ab

1.45 ± 0.15b

1.71 ± 0.13ab

Values are mean ± SD (n = 4). Means in a horizontal row followed by the same letter are not significantly different at p < 0.05. 94

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Table 6. OSIa of Flaxseed/Canola Oil Blends with 1 and Zn2+ Stearates 1: Zn2+ flaxseed/canola oil blends

1 (0.12 mM)

3.50 ± 0.33 c 8.07 ± 0.33cc

3.90 ± 0.30a 8.61 ± 0.41a

b

Zn

2+

(0.12 mM)

3.50 ± 0.31c 8.25 ± 0.38b

1:4

1:2

1:1.3

3.58 ± 0.17c 8.08 ± 0.02c

3.65 ± 0.28bc 8.20 ± 0.10b

3.75 ± 0.13b 8.77 ± 0.23a

Values are mean ± SD (n = 4). Means in the same horizontal row followed by the same letter are not significantly different at p < 0.05. bContent of canola oil is 26.0% by volume of blend. cContent of canola oil is 16.6% by volume of blend.

a

Table 7. OSIa of Flaxseed/Canola Oil Blends with 1 and Co2+ Stearates flaxseed/canola oil blends

1 (0.08 mM)

3.41 ± 0.13 a

3.55 ± 0.16a

b

Co

2+

(0.02 mM)

3.05 ± 0.15c

1: Co2+

1: Co2+

2:1

6:1

3.10 ± 0.14b

3.11 ± 0.15b

Values are mean ± SD (n = 4). Means in a horizontal row followed by the same letter are not significantly different at p < 0.05. bContent of canola oil is 28.5% for volume canola oil. a

the canola/flax blend with 1 is similar to that of the blend with highest ratio of 1 to Zn2+ studied (1:1.3). Addition of smaller amounts of 1 did not counteract the effect of Zn2+ on OSI. It is possible that CLA almost completely binds Zn2+ at this ratio. In contrast, oil with 1 and Zn2+ added at a molar ratio of 1:4 oxidizes at the same rate as oil in the presence of metal soaps with no 1 added. This likely indicates that the presence of excess amounts of metal ion exceeds the metal binding capacity of 1. In addition, the effect of Co2+ ions on the oxidation of silica-treated flaxseed oil was studied. However, 1 had no effect on the rate of oil the oxidation in the presence of cobalt as there was no influence of 1 on OSI in the presence of cobalt stearate. Because cobalt has a strong redox potential it may be able to initiate oxidation. It cannot be determined if binding to 1 has occurred (Table 7). In conclusion, crude flaxseed oil has higher oxidative stability than silica-treated flaxseed oil, indicating that some of the chemical constituents removed upon treatment with silica gel play a significant role in protecting flaxseed oil from oxidation. Fractions containing 1 and tocopherols (fraction D), and a mixture of CLs (1−13) (fraction E), improved the oxidative stability of peptide-free oil, suggesting the significant role played by these compounds in protecting oil from oxidation. CLs have their own role in the strong antioxidant system of flaxseed oil. They may behave as antioxidants or pro-oxidants depending on concentration and environment. Dose- and timedependent antioxidant properties of these peptides were observed. CL 1 is able to interact with metals selectively. Further studies are required to determine if 1 inhibits oil oxidation processes by binding metals or other mechanisms. Although, the OSI test was able to illuminate an approximate picture of oxidation in the presence of CLs, possible trace compounds in the studied fractions prevented the conclusive demonstration of the direct role of CLs as antioxidants. Further research should be conducted with simplified model systems to definitively show the interaction of pure CLs with antioxidants and radicles.

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Funding

This research was funded by the Saskatchewan Ministry of Agriculture’s Agriculture Development Fund (20080205) and Genome Canada support of Total Utilization of Flax GENomics (TUFGEN). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Dr. P. G. Arnison (Prairie Plant Systems Inc.) for helpful comments. ABBREVIATIONS USED HPLC, high-performance liquid chromatography; MeOH, methanol; EtOAc, ethyl acetate; AOM, active oxygen method; OSI, oxidative stability index; ROS, reactive oxygen species; Met, methionine; MetO, methionine S-oxide

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REFERENCES

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