Article pubs.acs.org/JAFC
Oxidative Stability of Pomegranate (Punica granatum L.) Seed Oil to Simulated Gastric Conditions and Thermal Stress Francesco Siano,*,† Francesco Addeo,†,§ Maria Grazia Volpe,† Marina Paolucci,# and Gianluca Picariello*,† †
Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche (CNR), Via Roma 64, I-83100 Avellino, Italy Dipartimento di Agraria, Università di Napoli “Federico II”, Parco Gussone, I-80055 Portici (Napoli), Italy # Dipartimento di Scienze e Tecnologie, Università degli Studi del Sannio, via Port’Arsa 11, I-82100 Benevento, Italy §
ABSTRACT: The fatty acid composition of pomegranate (Punica granatum L.) seed oil (PSO) is dominated by punicic acid, a conjugated linolenic acid (18:3ω-5). As a free fatty acid, punicic acid is rapidly oxidized in air and extensively isomerizes upon acid-catalyzed methylation at 90 °C. In contrast, triacylglycerol-bound punicic acid in PSO was unchanged by simulated gastric conditions and was degraded by 5−7% by severe heating (up to 170 °C for 4 h), as herein assessed by gas chromatography, attenuated total reflectance−Fourier transform infrared spectroscopy, 1H and 13C NMR, and high-resolution electrospray ionization mass spectrometry. Total polar compounds of PSO were slightly affected by thermal stress, accounting for 5.71, 6.35, and 9.53% (w/w) in the unheated, heated at mild temperature (50 °C, 2 h), and heated at frying temperature (170 °C, 4 h) PSO, respectively. These findings support from a structural standpoint the potential use of PSO as a health-promoting edible oil. KEYWORDS: pomegranate seed oil, punicic acid, conjugated linolenic acids, oxidative stability, isomerization of fatty acids
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INTRODUCTION Pomegranate (Punica granatum L.) contains 37−143 g of seeds/kg of fruits, resulting as a byproduct of the juice industry. Variable amounts (14−27% in weight of dried seeds) of an edible oil with characteristic composition can be extracted from pomegranate seeds through conventional physical or chemical methods.1,2 The components of pomegranate seed oil (PSO) including fatty acids (FA), phospholipids, tocopherols, and triterpenoids have been characterized in detail. 3−5 An increasing number of in vitro and in vivo investigations are attributing a number of potential health-promoting properties to PSO. The bioactive effects of PSO have been correlated primarily to the high amount of punicic acid (44.5−86.1% FA),6 an ω-5 conjugated linolenic fatty acid with potent antioxidant, anticarcinogenic, antiarteriosclerotic, immunomodulatory, and lipid metabolism regulation effects.7−10 Conjugated linolenic fatty acids (CLnA) include a family of positional and geometrical isomers of the octatrienoic acid, among which three 8,10,12−18:3 and four 9,11,13−18:3 isomers naturally occur in triacylglycerols (TAG) of a variety of plant seeds.11,12 Punicic acid, also named trichosanic acid, is the cis(c)9,trans(t)11,cis13−18:3 octatrienoic acid (18:3ω-5). Due to the reactivity of the conjugated double bonds, conjugated linoleic acids (CLA) and structurally related isomers of CLnA are chemically unstable and can easily undergo oxidative deterioration, especially when exposed to oxygen, moisture, light, and heat. The oxidative degradation of FA impairs the shelf stability, sensory properties, and nutritional quality of the oil. CLA are extremely susceptible to oxidation as free FA, although the degradation rate varies with the geometrical configuration of double bonds.13 CLnA are expected to be more unstable than CLA owing to the extended electron conjugation, which provides additional stabilization to © 2016 American Chemical Society
reactive free radical or carbocation intermediates. Differently from the nonconjugated α-linolenic acid, punicic acid isomerizes into positional and geometrical congeners during acidic methylation at 90 °C, yielding a series of isomers with prevalent trans configuration of double bonds.14 Similarly, free punicic acid is completely oxidized within 30 min upon air exposure at 50 °C.13 It has been reported that the oxidation rate of conjugated FA is significantly slower when they are esterified in TAG.15 However, the stability of triacylglycerol-bound punicic acid in PSO to mild or severe thermal stress has not been exhaustively investigated. Furthermore, after ingestion, PSO is exposed to gastric acid and ions, which could catalyze the chemical interconversion or the rearrangement of double bonds along the fatty acid acyl chains, so affecting the bioaccessibility of bioactive lipids. Considering that the conjugated linolenic acid isomers have different metabolism and bioactive properties,7,10 the stability of punicic acid in TAG has key implications to substantiate the dietary and nutraceutical use of PSO. In this work, the oxidative stability of TAG-bound punicic acid of PSO to simulated gastric conditions as well as to heating at mild (50 °C) and frying (170 °C) temperature was assessed using gas chromatography−flame ionization detector (GCFID) of fatty acid methyl ester derivatives (FAME), attenuated total reflectance−Fourier transform infrared (ATR-FTIR), and high-resolution electrospray ionization (ESI) mass spectrometry (MS). Received: Revised: Accepted: Published: 8369
October October October October
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2016 2016 2016 2016 DOI: 10.1021/acs.jafc.6b04611 J. Agric. Food Chem. 2016, 64, 8369−8378
Article
Journal of Agricultural and Food Chemistry
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were recorded and processed using ChromQuest 5.0 software (Thermo). ATR-FTIR. ATR-FTIR analyses were performed using a Spectrum 400 spectrophotometer (PerkinElmer, Waltham, MA USA), equipped with a DTGS detector. Overall, 32 scans/spectrum were acquired in the 4000−650 cm−1 range with a resolution of 4 cm−1. Samples were analyzed without any previous treatment. To test repeatability, analyses were performed in triplicate and average spectra were used. Spectra were elaborated using PE Spectrum software version 10.5.1, purchased with the instrument. Flow Injection Analysis−High-Resolution ESI-MS. Prior to ESI-MS analysis, glycerolipids were cationized as Na+-adducts. To this end, 5 μL of oil sample was dissolved in 1 mL of CHCl3 and vigorously shaken with 1 mL of aqueous 0.5 M sodium acetate. The chloroform layer was 20-fold diluted in CHCl3/MeOH 2/1 (v/v) and infused at a flow rate of 3 μL/min by a micro syringe into the H-ESI source of a high-resolution Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) operated in the positive ion mode. The m/z 400−3000 range was scanned at a resolution of 140,000 (m/z 200, fwhm). Source temperature was 320 °C, whereas capillary and skimmer voltages were 3.5 kV and 15 V, respectively. The sheath gas was N2 at 5 units. The TAG structure was assigned or confirmed by MS/MS fragmentation using collision-induced decay at 27−30 units. The instrument was calibrated externally using the Calmix (mixture of ultramark, MRFA peptide and caffeine) purchased from Thermo, achieving an accuracy better than 1.5 ppm over the entire m/z range. Spectra were the sum of at least 2 min of acquisition and were elaborated using Xcalibur 3.1 software (Thermo). Repeatability and reproducibility, determined as intra- and interday relative standard deviations, were assessed using the ion intensity of PSO TAG in five replicate analyses over three different days. Postanalysis calculations were carried out using the Microsoft Excel 2013 software. 1 H and 13C NMR Analyses. High-resolution 1H and natural abundance 13C NMR spectra of unheated and heated (170 °C, 4 h) PSO were acquired on a Bruker DRX-600 spectrometer, equipped with an inverse TCI CryoProbe (Bruker BioSpin, Rheinstetten, Germany), fitted with a gradient along the Z-axis at a probe temperature of 27 °C. Oil samples (20 mg) were dissolved in 0.75 mL of CDCl3 and transferred to 5 mm NMR tubes. Chemical shifts were compared to CDCl3 as the internal standard, which was assumed to resonate at δH 7.26 (CHCl3 impurity) and δC 77.00 ppm. Resonances were assigned on the basis of chemical shifts recorded on standard TAG (Fluka, Switzerland) and on the basis of literature data.22,23 Minor PSO Components. PSO (5 g) was saponified according to the method of Caligiani et al.24 The unsaponifiable fraction was extracted in diethyl ether and washed with water until neutral reaction. Ether phase was dehydrated with anhydrous sodium sulfate, filtered on paper, and dried under vacuum. The residue (100 μL) was dissolved in 50 mL of hexane; 1 μL was analyzed by GC-FID using the same chromatograph as above, equipped with an RTX-5 30 m × 0.25 mm × 0.25 μm column (Restek, Bellefonte, PA, USA). Samples were introduced through the autosampler in 1:10 split mode at 250 °C. The oven temperature program started at 200 °C (held for 2 min) and linearly increased to 300 °C (20 °C min−1) to the end of the analysis. FID temperature was 260 °C. Plant sterol mix (Matreya, State College, PA, USA), squalene, and α- and γ-tocopherols (Sigma) were used as external standards for qualitative and quantitative determinations. Total polyphenols were determined according to the Folin− Ciocalteu method, as previously described,16 using gallic acid in the 5− 50 μg/mL range to construct the standard curve. Average results from triplicate determinations were expressed as milligrams of gallic acid equivalents per 100 g of dry oil (mg GAE/100 g oil).
MATERIALS AND METHODS
Pomegranate seeds, representative of several local varieties, were the byproducts of the juice-manufacturing process of a local farm (Giovomel, Aiello Del Sabato, Avellino, Italy). Seeds were representative of several local varieties of pomegranate. Raw PSO was obtained by cold pressing using a press oil machine (E-Bassi Co., Mantova, Italy). In the laboratory, PSO was dehydrated with anhydrous sodium sulfate (1% w/v) and cleaned by centrifugation (3500g, 15 min). A standard sample of punicic acid was extracted from PSO with methanol and purified by reversed phase HPLC as previously reported.16 Chemicals and HPLC grade solvents were from Sigma-Aldrich (St. Louis, MO, USA). In Vitro Simulated Gastric Conditions. PSO (5.0 g) was suspended 1:1 (v/v) in a buffer simulating gastric fluid and incubated for 2 h at 37 °C under gentle magnetic stirring. Simulated gastric fluid (SGF) was prepared according to the method of Minekus et al.17 and adjusted at pH 2.5 with 1 N HCl. Gastric liposomes, prepared with egg phosphatidylcholine (Sigma-Aldrich), were included at the final concentration 0.17 mM in the digestion mixture.17 PSO aliquots were sampled at t = 0, 30, 60, 90, and 120 min, extracted with nhexane, evaporated under N2, and subjected to further analysis. Simulated gastric digestion was carried out in duplicate. Heating of PSO. PSO (10 g aliquots) was transferred into a 100 mL glass Pyrex beaker and incubated in a thermo-ventilated oven at a preset temperature of 50 °C for 2 h or 170 °C for up to 4 h. Aliquots (1 g) were sampled at regular time intervals of 30 min. Fractionation of Oil Samples. “Native” and heated PSO was fractionated by silica gel liquid chromatography into polar and nonpolar components, according to the gel mini column method18 based on the IUPAC procedure.19 Briefly, a 500 mg oil sample was dissolved in 2 mL of petroleum ether/diethyl ether (90:10, v/v) and fractionated using glass columns (150 mm × 10 mm i.d.) packed with 15 g of silica gel (Merck grade 60, 70−230 mesh). The nonpolar fraction was eluted with 60 mL of petroleum ether/diethyl ether (90:10, v/v), whereas the polar fraction was eluted with 50 mL of diethyl ether. The solvent was evaporated to dryness by rotary evaporator, and lipids were finally flushed under N2 stream. Separation efficiency was assessed by TLC using precoated silica gel plates (Merck KGaA, Darmstadt, Germany), which were developed with hexane/ diethyl ether/acetic acid 75:25:1 (v/v/v). Lipid spots were visualized by exposure to iodine vapors or, alternatively, by spraying the TLC plates with 10% (w/v) phosphomolybdic acid (Sigma) in ethanol and heating at 150 °C for 5 min. Total polar compounds (TPC) were determined gravimetrically on the chromatographic eluates and are reported as the mean of three independent determinations. Preparation of FAME. Analysis of the FAME was performed according to AOAC Official Method 996.0.20 The oil samples (0.200 g) were transferred into Pyrex test tubes with screw caps, and 2 mL of 1.25 N HCl−methanol solution was added. Samples were incubated in a water bath at 50 or 90 °C for 60 min. FAME were extracted with nhexane, after the addition of 2 mL of distilled water. The organic phase was filtered using Millex 0.45 μm PVDF disposable syringe filters (EMD Millipore Corp., Billerica MA, USA), and 1 μL was directly injected into the gas chromatograph for analysis. GC-FID. FAME were analyzed with a Trace GC gas chromatographer (Thermo Scientific, Inc., San Jose, CA, USA) equipped with a FID, using an SP-2560 100 m × 0.25 mm × 0.20 μm capillary column (Supelco-Sigma-Aldrich). Samples were introduced through a split− splitless injection system of an AS 3000 autosampler in split mode (ratio 1:100) at 260 °C. The oven temperature program started at 140 °C (held for 5 min) and linearly increased to 260 °C (4 °C min−1) up to the end of the analysis, according to previously described operating conditions.21 FID temperature was 260 °C. Fatty acid composition of PSO was obtained by comparison with the retention times of the standard mixture FAME 37 components (Sigma) and was expressed as a percentage area. Punicic acid was assigned by separate analysis of the standard FA, previously identified by HPLC, UV spectrum, and mass spectrometry.16 Each sample was analyzed in triplicate at least. Data
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RESULTS
GC-FID Analysis. The GC-FID chromatogram of FAME of PSO methylated by the conventional method using 1.25 M HCl in anhydrous methanol at 50 °C for 1 h is shown in Figure 1A. As expected, punicic acid was dominant in PSO, 8370
DOI: 10.1021/acs.jafc.6b04611 J. Agric. Food Chem. 2016, 64, 8369−8378
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at 90 °C (Figure 1B), producing both positional and geometrical isomers of punicic acid (Table 1). Differently from CLnA, saturated, monounsaturated, and nonconjugated polyunsaturated FA were only faintly affected by methylation temperature. The possible isomerization of conjugated polyunsaturated FA in acidic and ionic conditions simulating the gastric physiological environment has not been investigated so far. The GC profile of FAME (obtained by methylation at 50 °C) was unmodified when PSO was exposed to in vitro simulated gastric conditions (Figure 1C), indicating that CLnA are stable to the simulated gastric fluid at pH 2.5 and 37 °C. In addition, punicic acid still was 46.3% of total FA (83.8% of the content in unheated PSO) even after deep thermal stress of PSO (4 h, 170 °C), whereas the positional and geometrical isomers weakly increased (Figure 1D). In particular, positional octatrienoic isomers of punicic acid overall represented 5.2% of FA in severely heated oil (Table 1). Minor PSO Components. Minor components in the unsaponifiable fraction of PSO have been already extensively characterized.5,24−26 To complete the characterization of the PSO sample used in this study, phytosterols and tocopherols were determined by GC-FID after saponification of TAG (Table 2). In agreement with other studies, β-sitosterol, campesterol, and stigmasterol (in order of decreasing abundance) were the main phytosterols of PSO.5,24 Consistent with previous determinations, PSO contained a significant amount of squalene, accounting for 78.1 mg/100 g oil, and a number of aliphatic alcohols,24 which were not quantified in this study. Data available about tocopherols in PSO are conflicting,5,24,26 apart from a certain variability depending on the pomegranate biotype. Comparison with standards demonstrated that the most abundant component of the class was γtocopherol (Table 2) accounting for 194.0 ± 8.7 mg/100 mL oil, in close agreement with some of the previous papers.5,25,27 The total polyphenol content of PSO was 23.07 ± 1.44 mg GAE/100 g oil, expressed as gallic acid equivalent. The HPLC analysis of the 80% methanol of PSO, as already reported,16 indicated the presence of a multitude of low-abundance phenolic compounds, each occurring in low quantities (ppm and sub-ppm level) in the oil, in part identified by other authors.26 However, the most abundant component in the polar extract of PSO was free punicic acid. Although dedicated investigations indicated that PSO does not contain carotenoids,5,26 pigments of PSO still await identification. Tocopherols, phenolic compounds, and pigments are expected to prevent at least in part the oxidative degradation of PSO, although high levels of tocopherols could increase the peroxidation rate during the induction period.28 ATR-FTIR. FTIR is a powerful analytical tool to investigate the chemical modifications of edible oils, including the formation of geometrical isomers of unsaturated FA.29 ATRFTIR overcomes the drawbacks due to interferences in the detection of characteristic bands of cis/trans double bonds, enabling the assessment of isomerization events.30 The official methods for the determination of total isolated trans FA in fats and oils relies on the detection of the diagnostic C−H out-ofplane deformation band observed at 966 cm−1.31 The effects of the cis/trans interconversion on the IR absorption pattern of CLA have been well characterized as well. The CLA fatty acid isomers exhibit typical patterns in the mid-infrared spectrum, with the trans,trans, and cis,trans CLA isomers producing a single absorption band at 988 cm−1 or a doublet at 981 and 947
Figure 1. GC-FID chromatograms of FAME from PSO. Unheated PSO was methylated in acidic conditions at (A) 50 °C for 1 h or (B) 90 °C for 1 h. The methylation at 90 °C clearly induces isomerization of punicic acid. (C) PSO exposed to simulated gastric fluid at 37 °C for 2 h. (D) PSO thermally stressed at frying temperature (170 °C for 4 h).
representing 55.3% of the fatty acid content in the analyzed sample (Table 1). The content of punicic acid in our PSO sample was lower than previously reported by other authors.9 However, our value was in close agreement with recent determinations8,25 and consistent with fluctuations of the FA composition of PSO, depending on the pomegranate cultivar as well as on several abiotic and biotic conditions (geographical origin, soil, sun exposure, growing conditions, harvesting stage, etc.). Saturated FA, that is, palmitic and stearic acids, were minor components accounting for 5.87 and 1.97%, respectively, although slightly higher amounts have been reported in some specific pomegranate biotypes.3 PSO contained only trace amounts of other CLnA (overall 4.85%), including catalpic (t9,t11,c13-18:3), α-eleostearic (c9,t11,t13-18:3), and β-eleostearic (t9,t11,t13-18:3) acids.23 GC peaks of CLnA were tentatively assigned according to the reported elution order.6 The GC-FID profile of FAME confirmed that methylation at 50 °C did not affect the stability of punicic acid.14 In contrast, the combined effect of acid and higher temperature had a drastic impact on the stability of punicic acid, in that conjugated double bonds extensively isomerized during transesterification 8371
DOI: 10.1021/acs.jafc.6b04611 J. Agric. Food Chem. 2016, 64, 8369−8378
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Journal of Agricultural and Food Chemistry Table 1. Fatty Acid Composition of PSO Determined by GC Analysis of FAME (Area % ± SD) fatty acid miristic (C14:0) palmitic (C16:0) palmitoleic (C16:1) stearic (C18:0) oleic (C18:1ω-9 c) linoleic (C18:2ω-6 c) arachidic (C20:0) γ-linolenic (C18:3ω-6) cis-11-eicosenoic (C20:1) α-linolenic (C18:3ω-3) punicic (C18:3ω-5 ctc) positional CLnA isomers α-eleostearic (C18:3ω-5 ctt) catalpic (C18:3ω-5 ttc) β-eleostearic (C18:3ω-5 ttt) ∑ SFA ∑ MUFA ∑ PUFA ω-6 ∑ CLnA ∑ PUFA ω-3 a
PSO (methylated 50 °C) 0.10 5.87 0.17 1.97 9.67 14.03 0.23 0.09 0.38 0.82 55.27
± ± ± ± ± ± ± ± ± ± ±
0.01 0.35 0.03 0.22 0.55 1.02 0.05 0.02 0.12 0.18 2.40
2.50 1.61 0.74 8.17 9.84 14.12 59.38 0.82
± ± ± ± ± ± ± ±
0.75 0.42 0.12 0.16 0.29 0.52 1.80 0.18
PSO (methylated 90 °C) 0.08 6.81 0.11 2.62 8.57 10.81 0.34 0.08 0.41 0.20 8.66 19.47 9.86 12.72 11.58 9.85 9.09 10.89 62.29 0.20
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.25 0.02 0.31 0.62 1.22 0.07 0.01 0.10 0.08 1.05 3.52a 1.88 1.55 2.05 0.66 0.70 1.18 3.02 0.08
PSO (SGF, 2 h) 0.07 5.26 0.18 2.13 6.97 10.94 0.47 0.10 0.44 0.27 58.01
± ± ± ± ± ± ± ± ± ± ±
0.01 0.34 0.05 0.56 0.86 1.45 0.09 0.01 0.22 0.12 4.05
2.16 1.69 0.79 7.93 7.59 11.04 62.65 0.27
± ± ± ± ± ± ± ±
0.95 0.62 0.21 0.42 0.88 1.46 3.90 0.12
heated PSO (170 °C, 4 h) 0.15 4.46 0.13 1.98 6.49 10.33 0.39 0.10 0.42 0.21 46.33 5.23 4.27 2.70 0.99 6.98 7.04 10.43 59.52 0.21
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.24 0.05 0.44 0.82 1.56 0.10 0.02 0.28 0.07 3.05 1.25a 0.96 0.84 0.36 0.38 0.50 1.57 3.28 0.07
Total positional isomers of punicic acid (e.g., 8,10,12- and 10,12,14-octatrienoic acids).
Figure 2. ATR-FTIR spectra of (A) unheated PSO, (B) pure punicic acid, (C) PSO methylated in acidic conditions at 50 °C for 1 h, (D) PSO methylated in acidic conditions at 90 °C for 1 h, (E) PSO exposed to simulated gastric fluid at 37 °C for 2 h, and (F) PSO thermally stressed at frying temperature (170 °C for 4 h).
cm−1, respectively.32 To the best of our knowledge, the IR spectra of edible oils containing conjugated trienoic FA have not been described previously. PSO is a complex mixture of TAG, containing a high amount of punicic acid and minor levels of its geometrical isomers, for example, catalpic and αeleostearic acids. Tripunicin is the most abundant triacylglycerol, and the predominant bands of the ATR-FTIR spectrum of PSO arise from the structural features of punicic acid. The doublet of bands centered at 987 and 936 cm−1 in the ATRFTIR fingerprint region of PSO (Figure 2A) characteristically
distinguishes the geometry of the double bonds of punicic acid, as confirmed by comparison with the ATR-FTIR spectrum of the pure FA (Figure 2B). No appreciable variation in the ATRFTIR, especially at the level of these two diagnostic peaks, was observed when PSO was converted into FAME with 1.25 N HCl−methanol at 50 °C for 1 h. Indeed, the ATR-FTIR spectra of PSO and relative FAME obtained at 50 °C (Figure 2C) were strictly correlated, confirming that the cis/trans double bond geometry can be assigned by FTIR either directly on oils or on the corresponding FAME.33 In contrast, the 8372
DOI: 10.1021/acs.jafc.6b04611 J. Agric. Food Chem. 2016, 64, 8369−8378
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at 723 cm−1 for nonconjugated olefins, shifted at higher wavenumbers, most likely due to the extended conjugation.36 In line with the preservation of the structural traits of punicic acid, no additional ATR-FTIR absorption bands due to neoformed oxidation components were evident after treatment of PSO in simulated gastric fluid and after protracted heating at frying temperature (Figure 2E,F). 1 H and 13C NMR. Unheated and heated (170 °C, 4 h) PSO samples were compared by 1H and 13C NMR spectroscopy (Figures 3 and 4, respectively). 1H and 13C resonances of conjugated linolenic acid isomers have been assigned in detail by Cao et al.22 Because of the peculiar cis/trans geometry of conjugated double bonds, members of the CLnA class have distinct proton and carbon chemical shifts as well as different proton−proton coupling constants. In particular, punicic acid can be distinguished by α-eleostearic, β-eleostearic, and catalpic acids on the basis of characteristic signal patterns in the olefinic region of both 1H and 13C spectra. NMR spectra were consistent with the predominance of punicic acid in PSO and confirmed the substantial identity of unheated and heated oils. Very weak 1H and 13C signals (Figures 3 and 4, respectively) were indicative of a minor process of heat-induced cis/trans isomerization, which has been estimated to induce interconversion of nearly 5−7% of the original punicic acid, in line with the results of other techniques. The gross assessment of oil deterioration has been obtained by the integrated intensity of neo-formed olefinic (proton and carbon) signals compared to those of punicic acid in PSO. Oxygenated compounds such as hydroxyls, epoxides, or aldehydes in heated PSO escaped detection by NMR, most likely due to their very low relative abundance. Flow Injection Analysis−High-Resolution ESI MS. Because of the peculiar FA composition, the ESI MS spectrum of TAG (Figure 5A) clearly distinguishes PSO from the most common edible oils. The signal m/z 895.7 of tripunicin (PuPuPu), that is C54:9 TAG (as Na+ adduct), dominated the MS spectrum of PSO, as it represents >30% of total TAG.37
Table 2. Determination of Minor Components of PSO component squalene phytosterols campesterol stigmasterol β-sitosterol sitostanola others tocopherols α-tocopherol γ-tocopherol δ-tocopherol a
mg/100 g oil 78.1 ± 4.1 36.4 17.5 296.8 16.2 50.3
± ± ± ± ±
2.0 1.8 13 2.9 3.8
15.6 ± 3.5 194.0 ± 8.7 9.3 ± 1.1
Tentatively assigned according to Fernandes et al.5
transesterification of PSO with 1.25 N HCl−methanol at 90 °C for 1 h resulted in the appearance of a strong absorption peak at 992 cm−1 (Figure 2D), consistent with the extensive isomerization of the double bonds, which are prevalently converted into the thermodynamically more stable trans configuration. Compared to CLA, the conjugation of an additional double bond in CLnA produces a shift of the absorption bands at slightly higher wavenumber (966 vs 992 cm−1). The ATR-FTIR pattern of PSO was preserved either in conditions of simulated gastric digestion (Figure 2E) or after heating at frying temperature (170 °C) for 4 h (Figure 2F). The faint absorption band at 966 cm−1 of the isolated trans double bonds, as well as the small peak at 3016 cm−1 corresponding to the cis double bond stretching, did not change upon treatments under examination and were most likely due to FA other than punicic acid, such as oleic and linoleic acid.34 These findings are consistent with the weak absorption bands at 1419 and 759 cm−1 produced by bending/rocking and out-of-plane C−H vibration of cis olefins, respectively, which disappeared only after acidic methylation at 90 °C,35 whereas they persisted after treatment with gastric fluid and in thermally stressed PSO. Also in this case, the out-of-plane C−H vibration, which is observed
Figure 3. 1H NMR comparison of unheated (upper panel) and heated (170 °C, 4 h) PSO. Proton signals are labeled according to their position on the carbon chain of punicic acid. Signals due to interconversion of punicic into α- and β-eleostearic acids are evidenced. 8373
DOI: 10.1021/acs.jafc.6b04611 J. Agric. Food Chem. 2016, 64, 8369−8378
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Figure 4. 13C NMR comparison of unheated (upper panel) and heated (170 °C, 4 h) PSO. Carbon signals are labeled according to their position in the punicic acid chain.
Na+ adducts are 911.80 and 911.66, respectively. Thus, the experimental value corresponds to oxidized tripunicin, which is also consistent with its detection in the polar fraction. However, the signal at m/z 911.65 was also intense in the nonpolar fraction, in this case probably corresponding to the epoxy form of tripunicin,40 which already occurred in the unheated oil (Figure 5A). MS cannot distinguish the epoxy and hydroxy forms of unsaturated TAG as they have identical molecular formulas. The relative reactivity of the epoxy-tripunicin would also explain the decrease of the m/z 911.65 signal observed after 2 h at 50 °C, resulting in relatively large amounts of the hydroxyl derivative distributed over a series of signals at different m/z values after simulated frying (Figure 5C). Triacylglycerol dimers and trimers gave more intense signals in the spectra of the nonpolar fraction, indicating that oligomers produced via carbon−carbon cross-links of the acyl chains are more abundant than those later-eluting polar compounds containing bridging oxygen or oxygenated functional groups.41 Except for isomeric compounds, such as regioisomers, the high accuracy and resolution of the Orbitrap-based measurement would allow singling out the components within the lipid classes, also including triacylglycerol dimers and trimers (inset of Figure 5C). However, providing a comprehensive inventory of the lipids in thermo-oxidized PSO was beyond the purpose of the current work. Total Polar Compounds (TPC). The percentages of TPC in unheated PSO and PSO heated at 50 °C for 2 h and at 170 °C for 4 h were 5.71 ± 0.50, 6.35 ± 0.38, and 9.53 ± 0.41 (w/ w), respectively. Heat treatment at 50 °C had only a slight impact on PSO oxidation. However, after 4 h at frying temperatures, TPC were at levels comparable with those of other vegetable oils, such as sunflower, high-oleic acid sunflower, soybean, and olive oils.42−44 TPC increased in a relatively smooth fashion during the first hours at frying temperature (not shown). Changes at longer heating time,
The signal at m/z 873.70 corresponds to C52:6 TAG including PuPuP (P, palmitic acid) and its isobaric regioisomers. The group of signals in the m/z 1740−1790 range are Na+ adducts of triacylglycerol dimers, which are diagnostic compounds of triacylglycerol autoxidation, already detected in unheated oil. The ESI MS spectrum of PSO did not change by heating at 50 °C for 2 h (Figure 5B), aside from a slight increase of the intensity of triacylglycerol dimers (7.8 vs 6.1% with respect to the base peak) and a decrease of the signal at m/z 911.65. Similarly, the MS spectrum of PSO incubated for up to 2 h with simulated gastric fluid was unmodified if compared with the untreated control (data not shown). Thermally stressed PSO at frying temperature (170 °C, 4 h) still contained tripunicin as the dominant component and grossly retained its compositional traits (Figure 5C). However, in line with the changes observed for other edible oils, several classes of neo-formed species produced by thermo-oxidation were detected, including (i) diacylglycerols (DAG), which were generated by hydrolytic oxidation (m/z 613.48, 615.50, 635.46); (ii) β-scission products and core aldehydes (m/z 760−820); (iii) products of TAG-free FA (m/z 1140−1260) and TAG−DAG (m/z 1460−1540) coupling; (iv) increased amounts of oxidized TAG with progressive increment of the molecular mass by 16 Da of tripunicin, that is m/z 911.67, 927.67, 943.66, 959.66; and (v) increased triacylglycerol dimers (relative intensity 9.8%) and triacylglycerol trimers in the m/z 2620−2660 range. The nonpolar and polar fractions of heated PSO (170 °C, 4 h) were analyzed by ESI MS separately (Figure 6) to improve the detection and enable a more straightforward assignment of the neo-formed compounds.38 The signal m/z 911.65 dominating the polar fraction of PSO has been previously assigned to SSPu (S, stearic acid).39 However, the occurrence of SSPu seems quite unlikely, because S occurs at very low amount in PSO. The high accuracy of the Orbitrap-based MS measurement allowed discrimination between the two options. The expected m/z values of SSPu and oxidized tripunicin as 8374
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Figure 5. High-resolution ESI Q-Orbitrap MS of (A) unheated PSO, (B) PSO heated at 50 °C for 2 h, and (C) PSO heated at 170 °C for 4 h. TAG are detected as Na+ adducts. Base peak (m/z 895.68) is tripunicin. Insets demonstrate the capability of high-resolution mass spectrometry to single out the triacylglycerol dimer (A) and triacylglycerol trimer (B) components.
during which TPC might increase exponentially,45 were not investigated in this study because PSO is not commonly employed for deep-frying.
oxidative injury. The homolytic decomposition of hydroperoxides partially produces reactive peroxy and alkoxy radicals as the thermal treatment progresses. Radicals quickly degrade into a heterogeneous series of neo-formation compounds that can be monitored as specific markers of the oxidative damage. The high content of punicic acid of PSO justifies the iodine value of 217−221 g I2/100 g oil,4 much higher than those of other edible oils such as sunflower oil (128−150 g I2/100 g oil) and soybean oil (124−139 g I2/100 g oil). Thus, PSO is particularly susceptible to a rapid oxidative deterioration. Free punicic acid completely isomerizes after exposure to air for 30 min even at mild temperature (50 °C).14 In contrast, the results of our work confirm that PSO is practically unchanged and that triacylglycerol-bound punicic acid did not appreciably isomerize after exposure to air at 50 °C for up to 120 min. Similarly, we observed that PSO was not modified under in vitro simulated gastric conditions. Punicic acid in PSO TAG
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DISCUSSION Because of the presence of double bonds, nonenzymatic oxidation mainly affects polyunsaturated fats during industrial and domestic heating of oils, producing a number of oxidation products. Because the formation of oxygenated functional groups enhances the polarity of the lipid molecules, the level of TPC can be monitored as a valuable parameter to assess the deterioration of oil. Positional and geometrical isomerization of double bonds in unsaturated FA also reflects the formation of reactive intermediates. The oxidative factors lead to the formation of hydroperoxides mainly located in the allyl position with respect to the double bonds as primary oxidation products. The level of hydroperoxides is correlated to the severity of the 8375
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Figure 6. High-resolution ESI Q-Orbitrap MS of (A) nonpolar and (B) polar fractions purified by silica gel liquid chromatography from thermally stressed (170 °C, 4 h) PSO. Insets in panel B are magnified views of signals due to (I) progressively oxidized TAG and (II) oxidized triacylglycerol trimers.
exert locally its potential bioactivity without substantial isomerization or structural degradation. In this regard, free punicic acid has been found absorbed, although with low absorption rates, and incorporated into human plasma and red cell plasma membranes.49 In Caco-2 cells and in rats, CLnA are also partly converted into CLA,50−52 which have enhanced intestinal uptake. Thus, CLnA represent dietary precursors of bioactive CLA, besides being themselves potential health-promoting components. This aspect is particularly relevant for the dietary use of PSO, which supplies a relatively high amount of punicic acid, compared to other food matrices normally containing CLA at very low levels. In other terms, a small supplement of PSO supplies punicic acid and can significantly increase the daily intake of CLA, which for an adult person on an ordinary diet does not overcome 0.5−1 g,53 whereas the recommended intake for delivering health-promoting effect ranges from 1.5 to 3.5 g/day.54 In conclusion, our data indicate a high bioaccessibility and an interesting potential uptake of punicic acid as either free FA or 2-monoacylglycerol following assumption of PSO, thus supporting the use of PSO as dietary and nutritional supplement. The oxidative stability of triacylglycerol-bound punicic acid also should rule out possible health hazards related to the ingestion of conjugated FA, which might promote oxidative stress in the human body.48 However, the research in this field is still in its early stages, and many nutritional and physiological aspects, especially those related to the bioconversion of CLnA, await elucidation.
isomerized at a very low rate, even after protracted thermal stress at 170 °C. Interestingly, our analyses confirm the general capability and potentiality of ATR-FTIR to monitor the modifications of edible oils and to assess the severity of thermally induced oxidative damages. The classes of compounds generated by thermo-oxidation at frying temperature were the same as described in the cases of other edible oils, such as sunflower, linseed, soybean, and olive oils,36,40,46 thereby confirming that no additional oxidative mechanisms are operating in the presence of CLnA. Differently from what one could expect on the basis of the unsaturation level of PSO, TPC did not increase significantly, indicating that upon heating PSO undergoes oxidation reactions similarly to other edible oils with a lower iodine value upon heating. In effect, the development of polar compounds is not strictly correlated with the unsaturation level, and other factors could contribute to or prevent the oil degradation.40 In the current case, triacylglycerol-bound punicic acid exhibited increased stability to oxidation with respect to free FA due to a series of concomitant factors, such as (i) higher (>10-fold) intrinsic stability of esterified conjugated FA;15,47,48 (ii) limited susceptibility of TAG to oxidative and hydrolytic degradation, because of the much lower water solubility; (iii) the presence in PSO of tocopherols, phenolic compounds, and pigments5,15 which act as radical scavengers, preventing in part the oxidative damage of acyl chains; and (iv) protection exerted by other lipid components of PSO.47 On the basis of our results, it can be reasonably expected that TAG-bound punicic acid preserves its structure in the relatively soft chemical conditions of the duodenal compartments. Thus, after lipolytic release as either 2-monoacylglycerol or free FA, punicic acid can be adsorbed or 8376
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AUTHOR INFORMATION
Corresponding Authors
*(F.S.) E-mail:
[email protected]. Phone: +39 0825 299301. *(G.P.) E-mail:
[email protected]. Phone: +39 0825 299521. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully thank Adele Cutignano and Dominique Melck of the Institute of Biomolecular Chemistry (ICB)−CNR, Pozzuoli (Naples, Italy), for the acquisition of NMR spectra.
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