Food Chemistry 173 (2015) 966–971

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Food Chemistry

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Stabilisation of phytosterols by natural and synthetic antioxidantsin high temperature conditions

http://dx.doi.org/10.1016/j.foodchem.2014.10.0740308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +48 61 848 73 07; fax: +48 61 848 73 26.E-mail address: dominik.kmiecik@up.poznan.pl (D. Kmiecik).

Dominik Kmiecik ⇑, Józef Korczak, Magdalena Rudzinska, Anna Gramza-Michałowska, Marzanna Hes,Joanna Kobus-CisowskaFaculty of Food Science and Nutrition, Poznan University of Life Sciences, Wojska Polskiego 31, 60-634 Poznan, Poland

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 June 2013Received in revised form 6 October 2014Accepted 14 October 2014Available online 30 October 2014

Chemical compounds studied in the article:Phytosterols (PubChem CID: 12303662)Tocopherols (PubChem CID: 14986)b-Sitosterol (PubChem CID: 222284)Campesterol (PubChem CID: 173183)Brassicasterol (PubChem CID: 5281327)Sinapic acid (PubChem CID: 637775)

Keywords:PhytosterolsOxyphytosterolsHeating TAGsPhenolic antioxidantsTocopherol

The aim of the study was to assess the potential applicability of natural antioxidants in the stabilisation ofphytosterols. A mixture of b-sitosterol and campesterol was incorporated into triacylglycerols (TAGs).The following antioxidants were added to the prepared matrix: green tea extract, rosemary extract, amix of tocopherols from rapeseed oil, a mix of synthetic tocopherols, phenolic compounds extracted fromrapeseed meal, sinapic acid and butylated hydroxytoluene (BHT). Samples were heated at a temperatureof 180 �C for 4 h. After the completion of heating, the losses of phytosterols were analysed, as well as thecontents of b-sitosterol and campesterol oxidation products. The total content of phytosterol oxidationproducts in samples ranged from 96.69 to 268.35 lg/g of oil. The effectiveness of antioxidants decreasedin the following order: phenolic compounds from rapeseed meal > rosemary extract > mix of tocopherolsfrom rapeseed oil > mix of synthetic tocopherols > green tea extract > sinapic acid > BHT.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The capacity of phytosterols to reduce blood cholesterol levels,observed as early as the 1950s (Pollak, 1953), is at present the pri-mary property with which these compounds are associated.Numerous findings have confirmed the positive effect of the dailyconsumption of 2–3 g phytosterols and phytostanols (saturatedforms of sterols) on the reduction of the total contents of choles-terol and its LDL fraction in human blood by several to around adozen percentage points. Previous investigations resulted in thelaunch of the first margarine with an addition of plant sterols,Benecol�, in 1995 by Raiso, Finland. At present, there are numerousproducts available on the food market (milk, cheeses, yoghurts,mayonnaises, chocolate products) enriched with plant sterols andstanols and aimed at a reduction of blood cholesterol levels.

However, phytosterols undergo oxidation. The presence ofdouble bonds in phytosterol molecules (Fig. 1) makes them

sensitive to the effect of light, metal ions, pigments, enzymes andelevated temperature. Under the influence of these factors, phytos-terol oxidation occurs, most frequently as a result of autoxidationreactions, leading to the formation of phytosterol oxidation prod-ucts (POPs), such as 7-hydroxy, 7-keto, epoxy, 25-hydroxy and tri-ols of sterols (Soupas, 2006). The reaction scheme of the formationof POPs during autooxidation is shown in Fig. 2. Since vegetableoils are the primary natural source of phytosterols in the humandiet and they contain 70–1100 mg sterols per 100 g oil, they mayalso be a source of POPs formed during oxidation which have anadverse effect on the human organism. The content of phytosteroloxidation products in oils may depend, e.g., on the manner of theirproduction, storage method or the type of oil (Dutta, 2004;Rudzinska, Kazus, & Wasowicz, 2001). A much higher increase inthe levels of derivatives of oxidised phytosterols is observed duringthe storage of oil at elevated temperature (Cercaci, Rodriguez-Estrada, Lercker, & Decker, 2007), as well as during the heating ofvegetable oils (Johnsson & Dutta, 2006; Kmiecik et al., 2011).

The rate of thermo-oxidative changes may be reduced due tothe application of substances with antioxidant properties. These

D. Kmiecik et al. / Food Chemistry 173 (2015) 966–971 967

substances may be synthetic (butylated hydroxytoluene – BHT,butylated hydroxyanisole – BHA, tert-Butylhydroquinone –TBHQ)or come from natural sources. Natural antioxidants, which are atpresent used by many researchers to stabilise fats, are a much big-ger group of compounds and originate mainly from two sources:vegetable oils and tissues of other plants, vegetables and herbs.Native antioxidants found in oils include tocopherols, tocotrienols,sesamol, sesaminol and their isomers, oryzanol, squalene and cer-tain phytosterols (Warner, 2005). Another group of natural antiox-idants comprises extracts produced from tea leaves, rosemary,sage, as well as other plants such as thyme, oregano, marjoram,oat or peanut husks. The antioxidant properties of these extractsresult from the presence of the flavonoids, phenolic acids andselected sterols they contain (Amarowicz et al., 2008; Kobuset al., 2009).

The potential applicability of natural antioxidants in the stabili-sation of oil and its components subjected to heating has beenconfirmed, e.g., by Khan and Shahidi (2001) and Besmira, Jiang,Nsabimana, and Jian (2007). Applied natural antioxidants and theirmixtures frequently exhibit a similar or higher antioxidant activitythan the applied synthetic antioxidants.

The aim of this study was to assess the potential applicability ofantioxidants obtained from natural sources (rapeseed meal, rose-mary, green tea and crude oil) in the stabilisation of phytosterolsduring the heating of the triacylglycerols of oil in model systems.

2. Materials and methods

2.1. Materials

Triacylglycerols (TAGs) were collected from rapeseed oil pur-chased in a local market using column chromatography accordingto Chimi, Cillard, and Cillard (1994). Rapeseed oil was dissolved inhexanes (1:3, v/v) and run through a glass column packed withactivated carbon, aluminium oxide (activated at 300 �C), and anhy-drous sodium sulphate. During the refining process, the columnand receiver system were shielded from external light. Next, therecovered hexanes were removed under a vacuum at <40 �C, afterwhich the triacylglycerols free of endogenous antioxidants weresealed in an amber vial under a nitrogen headspace. TAGs werestripped from the oil directly before conducting the plannedexperiments.

Fig. 1. Chemical structures of ph

Rosemary extract was prepared from rosemary leaves (Rosmari-nus officinalis L.) via ethanolic extraction according to Gramza-Michałowska, Abramowski, and Jovel (2008). Green tea extractwas prepared from China Lung Ching leaves (Camellia sinensis L.)via ethanolic extraction according to Gramza-Michałowska,Hes, and Korczak (2008). Natural tocopherols were obtained fromcrude rapeseed oil via a three-step crystallisation process accord-ing to Szulczewska-Remi, Kałucka-Nogala, Kwiatkowski, Lampart-Szczapa, and Rudzinska (2005). Phenolic compounds from rape-seed meal were obtained according to Wanasundara, Amarowicz,and Shahidi (1994). Briefly, rapeseed was ground and defeatedwith petroleum for 12 h using a Soxlet apparatus and dried.Phenolic compounds were obtained via double ethanolic extrac-tion with 95% ethanol for 20 min at 80 �C. After extraction, alcoholwas evaporated under a vacuum at 40 �C.

The mix of phytosterols (b-sitosterol – 75%, campesterol – 14.5%,b-sitostanol – 10.5%) and sinapic acid were purchased fromSigma-Aldrich (Sigma–Aldrich St. Louis, MO, USA). a-tocopherol(synthetic, 95% grade) was purchased from Sigma (Germany). b-,d-, c-tocopherols (synthetic) were purchased from Eisai (Japan).Butylated hydroxytoluene (BHT) was purchased from Merck(Germany). Standards for the identification of phytosterols(b-sitosterol, campesterol, stigmasterol, and brassicasterol) andoxyphytosterols (5a-cholestane, cholestane-3b,5a,6b-triol, 5a,6a-epoxy-cholestane-3b-ol, 5-cholestane-3b,19-diol, 5-choles-tane-3b,7b-diol, 5b,6b-epoxy-cholestane-3b-ol, 5-cholestane-3b-ol-7-one) were purchased from both Sigma–Aldrich (Sigma–AldrichSt. Louis, MO, USA) and Steraloids (Steraloids, Newport, RI, USA).

2.2. Sample preparation

In the first step, a mix of phytosterols (2%) was dissolved inchloroform, added to triacylglycerols and then the solvent wasremoved under a vacuum at <40 �C. In the second step, the follow-ing were added to a mix of TAGs and phytosterols (5 g): ethanolextract of rosemary (0.02%), ethanol extract of green tea (0.1%), amix of tocopherols extracted from oil (natural tocopherols)(0.02%), a mix of synthetic tocopherols (0.02%), phenolic com-pounds extracted from rapeseed meal (0.02%), sinapic acid(0.02%) and BHT (0.02%). Then, the solvent was evaporated. Themix of synthetic tocopherols consisted of a-, b-, d- and c-tocopoh-erol. The concentrations of these components were 24, 0.1, 45 and

ytosterols and phytostanols.

Fig. 2. Autooxidation scheme of phytosterols.

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1.5 mg/100 g of oil, respectively. Samples of a mix of TAGs andphytosterols with antioxidants were heated at 180 �C for 4 h in aRancimat (Metrohm, Switzerland). During the heating of the TAGsamples in the Rancimat, 2 l of air per hour were passed throughthe sample. The Rancimat was chosen to ensure constant condi-tions during the heating of all samples. The control sampleconsisted of a mix of TAGs and phytosterols heated withoutantioxidants.

2.3. Total polar compounds analysis

Total polar compounds (TPCs) were determined using theOfficial Method ISO 8420 (1999). The analysis was performed ina glass column (0.45 m � 20 mm) packed with silica gel condi-tioned with a mixture of light petroleum ether and diethyl ether(87:13 v:v). The sample was dissolved in an eluant solvent andseparated in a column. Non-polar fractions are eluted by themobile phase, while polar substances were absorbed on silica gel.The amounts of polar and non-polar fractions were determinedby weight after evaporation of solvents.

2.4. Analysis of phytosterols

Phytosterols were analysed using the procedure described byRudzinska et al. (2001). Oil samples (200 ll) with the internal stan-dard – 5a-cholestane (500 lg) were saponified with 1 M KOH inmethanol at room temperature for 18 h. The unsaponifiable fractionwas extracted with diethyl ether and the solvent was evaporatedunder nitrogen. Sterols were silylated with bis(trimethylsilyl)triflu-oroacetamide (BSTFA) with 1% trimethylchlorosilane (TCMS)(Sigma–Aldrich St. Louis, MO) and analysed in a Hewlett Packard6890 GC (Agilent, Wilmington, DE, USA) equipped with aDB5 (J&W Scientific, Folsom, CA, USA) capillary column(30 m � 0.32 mm � 0.25 lm). Analysis parameters were as fol-lows: oven temperature 290 �C; injector and detector (FID) temper-atures 310 �C; and carrier gas, helium at 1.6 mL/min. Phytosterols

were identified by comparing their retention times (relative to5a-cholestane) with those of commercially available standards.

2.5. Analysis of phytosterol oxidation products (POPs)

Phytosterol oxidation products (oxyphytosterols) were ana-lysed using the procedure described by Rudzinska et al. (2001).Oil samples (200 ll) with an internal standard, i.e. 19-hydroxy-C(500 lg), were transesterified with a mixture of sodium methylateand methyl tetr-butyl ether (MTBE) (4:6 v:v) and left for 1 h. Theoxyphytosterol fraction was extracted with chloroform and rinsedtwice with water and 1% citric acid. After the removal of the waterlayer, samples were dried under nitrogen and transferred by chlo-roform to an SEP-PAK NH2 cartridge (Waters, Milford, MA, USA)conditioned with 2 � 5 mL of hexane. The column was washedwith 2 � 5 mL of hexane, 5 mL of hexane:MTBE (5:1 v:v) and5 mL of hexane:MTBE (3:1 v:v). Then, the fraction of oxyphytoster-ols was eluted with 7 mL of acetone and dried under nitrogen. Afterthe evaporation of the acetone, samples were silylated with bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlo-rosilane (TCMS) (Sigma-Aldrich Poland), left for 4 h, and analysedin a Hewlett Packard 6890 GC (Agilent, Wilmington, DE, USA)equipped with an HP-5MS (J&W Scientific, Folsom, CA, USA) capil-lary column (50 m � 0.2 mm � 0.33 lm) and FID. The initial oventemperature was 260 �C for 20 min, and then it was increased at0.5 �C/min to 275 �C and this was followed by 3 �C/min to 290 �C.The injector temperature was 300 �C, splitless and the detectortemperature was 310 �C. The carrier gas was helium at 1 mL/min.POPs were identified by comparing their retention times withthose of commercially available standards of oxycholesterols andliterature data.

2.6. Statistical analysis

All assays were performed in three replications. The values ofmeans and standard deviations were calculated with the use of

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Microsoft Office Excel 2007 (Microsoft, Redmond, WA). The signif-icance of differences between means was determined at p < 0.05using the analysis of variance (ANOVA) followed by Tukey’s multi-ple range test with STATISTICA PL 8.0 (StatSoft, Inc., Tulsa, OK). Astatistical analysis between means was also performed using Pear-son’s correlation with STATISTICA PL 9.0 (StatSoft, Inc., Tulsa, OK).

3. Results and discussion

In all heated samples, an increase in total polar compounds(TPCs) was observed. The lowest TPC content was observed inthose samples with an addition of a rosemary extract (12.7%) andan addition of a mix of synthetic tocopherols (24.6%). The highestcontent of polar compounds was found in samples with a syntheticantioxidant BHT (39.2%) and a mixture of tocopherols obtainedfrom rapeseed oil (37.6%). No statistically significant differenceswere found between these two sets of samples at p < 0.05. More-over, the content of the polar fraction in these samples was signif-icantly higher than in a mixture of TAGs and phytosterols, whichwas heated without any antioxidants. The other applied antioxi-dants inhibited the processes of triacylglycerol degradation eitheronly to a slight degree or not at all. The content of the polar fractionwas 33.1% in a sample without any antioxidants and with green teaextract, 32.7% when phenolic compounds extracted from canolameal were applied and 35.0% for sinapic acid (Table 1). The highactivity of rosemary extract and a mix of tocopherols during theheating of TAGs from rapeseed oil at a temperature 180 �C was alsoobserved by Nogala-Kałucka et al. (2005). During the acceleratedRancimat� test, rosemary extract turned out to be almost twiceas effective as the BHT. In turn, the application of rosemary extractand an extract from green tea leaves, as well as tocopherols,resulted in the protection of both sunflower oil TAGs in bulk andemulsion stored at an elevated temperature (40 �C); however, theantioxidant efficiency of the additives was lower than that ofBHT. Authors indicated different mechanisms of action fortocopherols, green tea extract and rosemary extract, dependingon the temperature and the fat matrix adopted. A lack of BHT activ-ity at a high temperature may also be explained by its rapiddecomposition and evaporation in the air from samples (Hamana& Nawar, 1991).

The total phytosterol content in the unheated TAG sample was17.95 mg/g of oil (Table 2). Heating of samples resulted in losses ofphytosterols added to TAGs. The lowest losses were observed insamples with an addition of green tea extract, a mix of tocopherolsfrom rapeseed oil, a mix of synthetic tocopherols or phenolic com-pounds extracted from rapeseed meal. The content of phytosterolsin these samples was 16.69, 17.70, 17.10 and 17.16 mg/g of oil,respectively. Lower contents of phytosterols were recorded insamples with an addition of sinapic acid (16.22) and rosemary

Table 1Total polar compounds in tag heated for 4 h at 180 �C.

Antioxidants Polar compounds (%)

Unheated TAG 2.8 ± 0.4 a*

Without antioxidant 33.1 ± 0.3 bBHT 39.2 ± 1.8 cGreen tea extract 33.1 ± 1.7 bRosemary extract 12.7 ± 0.1 dTocopherols (natural) 37.6 ± 0.6 cfTocopherols (synthetic) 24.6 ± 0.7 ePhenolic compounds from rapeseed meal 32.7 ± 0.6 bSinapic acid 35.0 ± 0.9 bf

Values are means of three determinations ± SD.* Means followed by different letters indicate significant differences (p < 0.05)

between samples.

extract (15.80). The lowest phytosterol content was found in theheated samples without antioxidants (14.15) or with BHT(15.51). When analysing losses of individual phytosterols (campes-terol and b-sitosterol), the highest activity was also observed forboth additions of tocopherols and the addition of phenolic com-pounds extracted from rapeseed meal. A considerable protectiveaction was also found in the case of b-sitosterol for an additionof green tea extract and sinapic acid. Losses of phytosterols duringheating may depend both on the conditions of the process (tem-perature and time), and the chemical composition of oil (the typeof oil), as well as the presence of antioxidants (Rudzinska,Korczak, Gramza, Wasowicz, & Duta, 2004; Winkler, Warner, &Glynn, 2007). The presence of synthetic antioxidants in oil, suchas BHT or BHA (butylated hydroxyanisole), does not always guar-antee the effective protection of its components, including phytos-terols. Investigations have shown that at 60 �C BHT exhibits ahigher antioxidant activity in relation to phytosterols than an addi-tion of either a-tocopherols, a rosemary extract or a green teaextract (Rudzinska et al., 2004). In turn, at 180 �C and as a resultof its decomposition under the influence of temperature, this activ-ity is slight, leading to similar losses of phytosterols as in samplesof oil heated without antioxidants (Kmiecik, Korczak, Rudzinska,Gramza-Michałowska, & Hes, 2009).

As a result of heating of TAGs with phytosterols, an increase wasalso observed in the content of phytosterol oxidation products inall samples; however, this varied and depended on the appliedantioxidant. The total content of b-sitosterol and campesterol oxi-dation products in samples ranged from 96.69 lg/g of oil for thesample with an addition of phenolic compounds from rapeseedmeal to 268.35 lg/g of oil for the sample heated without an antiox-idant. Samples with an addition of the other antioxidants wereordered as follows: rosemary extract < mix of tocopherols fromrapeseed oil < mix of synthetic tocopherols < green teaextract < sinapic acid < BHT. Contents of POPs in these sampleswere 106.70, 123.32, 126.29, 164.77, 176.01 and 191.33 lg/g ofoil, respectively. However, a detailed analysis of the contents ofb-sitosterol and campesterol oxidation products revealed certainirregularities in the activity of individual antioxidants in relationto individual phytosterols. The total content of b-sitosterol oxida-tion products, depending on the applied antioxidant, ranged from58.09 to 164.53 lg/g of oil (Table 3). The effectiveness of antioxi-dants decreased in the following order: phenolic compoundsextracted from rapeseed meal > mix of synthetic tocopherols > mixof tocopherols from rapeseed oil > rosemary extract > green teaextract > sinapic acid > BHT. The highest content of b-sitosteroloxidation products was observed in the sample heated withoutantioxidants. Most frequently, the dominant group of oxidisedderivatives comprised 5a,6a-epoxysterols (in samples with rose-mary extract and a mix of tocopherols from rapeseed oil) and5b,6b-epoxysterols (in samples with green tea extract, a mix ofsynthetic tocopherols and sinapic acid). The content of 5a,6a-epoxysterols was 17.04 and 20.60 lg/g of oil, while for 5b,6b-epox-ysterols it was 20.64, 20.21 and 30.38 lg/g of oil, respectively. Asimilar trend was observed by Rudzinska et al. (2004) during theheating of stigmasterols in sunflower TAGs with added BHT, a-tocopherol, rosemary extract and green tea extract. In the samplewith added phenolic compounds extracted from rapeseed meal,the dominant group consisted of 7b-hydroxysterols, the contentof which amounted to 15.04 lg/g of oil. In turn, triols of b-phytos-terols constituted a dominant group both in samples of TAGs sub-jected to heating without an addition of antioxidants and in thosewith BHT. The content of these compounds was 50.59 and27.78 lg/g of oil, respectively. A higher content of triols in TAGsamples may be explained by the high temperature, generatingconditions promoting a much faster conversion of epoxysterolsto triols.

Table 2Influence of synthetic and natural antioxidants on contents of phytosterols in TAGs heated for 4 h at 180 �C.

Antioxidant Campesterol Sitosterol Sitostanol Total(mg/g oil)

Unheated TAG 2.60 ± 0.05 a* 13.38 ± 0.85 a 1.97 ± 0.04 a 17.95 ± 0.84 aeWithout antioxidant 1.79 ± 0.02 b 10.41 ± 0.21 b 1.96 ± 0.21 a 14.15 ± 0.39 bBHT 2.11 ± 0.06 c 11.49 ± 0.49 bc 1.91 ± 0.08 a 15.51 ± 0.49 bcGreen tea extract 1.82 ± 0.04 b 12.94 ± 0.19 a 1.94 ± 0.04 a 16.69 ± 0.22 adeRosemary extract 2.35 ± 0.02 d 11.50 ± 0.19 bc 1.95 ± 0.01 a 15.80 ± 0.18 bdTocopherols (natural) 2.46 ± 0.10 ad 13.30 ± 0.22 a 1.94 ± 0.01 a 17.70 ± 0.13 aTocopherols (synthetic) 2.59 ± 0.12 a 12.56 ± 0.17 ac 1.95 ± 0.04 a 17.10 ± 0.25 adePhenolic compounds from rapeseed meal 2.58 ± 0.01 a 12.64 ± 0.31 a 1.93 ± 0.03 a 17.16 ± 0.29 aSinapic acid 1.83 ± 0.02 b 12.46 ± 0.63 ac 1.93 ± 0.00 a 16.22 ± 0.65 cde

Values are means of three determinations ± SD.* Means in the same column followed by different letters indicate significant differences (p < 0.05) between samples.

Table 3Influence of synthetic and natural antioxidants on contents of b-sitosterol oxidation products in TAGs heated for 4 h at 180 �C.

Antioxidant 7a-OH 7b-OH 5a,6a-Epoxy 5b,6b-Epoxy Triol 7-Keto Total(lg/g oil)

Unheated TAG nd* nd nd nd nd nd ndWithout antioxidant 11.36 ± 0.17 a** 12.68 ± 0.14 a 29.58 ± 0.21 a 37.34 ± 1.56 a 50.59 ± 0.89 a 22.99 ± 0.64 a 164.53 ± 4.40 aBHT 20.96 ± 0.25 b 18.74 ± 0.12 b 13.95 ± 0.10 b 13.50 ± 0.03 b 27.78 ± 0.13 b 17.52 ± 0.02 b 112.44 ± 5.85 bGreen tea extract 20.48 ± 1.25 c 17.74 ± 0.78 c 18.39 ± 0.35 c 20.64 ± 0.95 c 5.69 ± 0.89 c 12.50 ± 1.94 c 95.45 ± 0.87 cRosemary extract 7.73 ± 1.03 b 8.28 ± 0.18 d 17.04 ± 0.24 c 11.58 ± 0.46 b 12.11 ± 0.05 d 11.12 ± 0.47 d 67.87 ± 0.38 dTocopherols (natural) 5.34 ± 0.55 d 18.19 ± 0.49 b 20.60 ± 0.15 d 6.69 ± 0.22 d 18.36 ± 0.14 e 9.46 ± 0.38 e 78.64 ± 0.46 eTocopherols (synthetic) 10.73 ± 0.68 a 7.25 ± 0.39 d 15.35 ± 0.70 e 20.21 ± 0.47c 13.61 ± 0.69 ce 7.36 ± 0.35 e 74.50 ± 4.92 ePhenolic compounds from rapeseed meal 6.66 ± 0.50 d 15.40 ± 0.56 a 9.47 ± 0.49 f 11.70 ± 0.92 b 8.99 ± 1.11 ce 5.87 ± 0.29 e 58.09 ± 0.05 eSinapic acid 9.59 ± 0.91 e 28.83 ± 0.12 e 5.70 ± 0.06 g 30.38 ± 1.84 e 12.30 ± 0.85 d 12.37 ± 0.56 c 99.18 ± 1.40 c

* nd – Not detected. Values are means of three determinations ± SD.** Means in the same column followed by different letters indicate significant differences (p < 0.05) between samples. 7a-OH – 7a-hydroxysitosterol; 7b-OH – 7b-

hydroxysitosterol; 5a,6a-epoxy – sitosterol-5a,6a-epoxide; 5b,6b-epoxy – sitosterol-5b,6b-epoxide; triol – sitostanetriol; 7-keto – 7-ketositosterol.

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The total content of campesterol oxidation products observed inthe samples was lower than in the case of b-sitosterol oxidationproducts and ranged from 38.60 to 103.82 lg/g of oil (Table 4).The lowest level of campesterol oxidation products, as before,was found in the sample of TAGs heated with an addition of phe-nolic compounds extracted from rapeseed meal, while the highestlevel was for the sample heated without an antioxidant. The effec-tiveness of individual antioxidants decreased in the followingorder: phenolic compounds extracted from rapeseed meal > rose-mary extract > mix of tocopherols from rapeseed oil > mix of syn-thetic tocopherols > green tea extract > sinapic acid > BHT. Thetotal content of campesterol oxidation products was 38.6, 38.83,44.68, 51.79, 69.32, 76.83 and 78.89 lg/g of oil, respectively. Sim-ilar to the analyses of b-sitosterol oxidation products, the majorgroup found in samples was 5b,6b-epoxysterols. Their levels insamples with an addition of green tea extract, rosemary extractand a mix of synthetic tocopherols were 18.71, 10.39 and

Table 4Influence of synthetic and natural antioxidants on contents of campesterol oxidation prod

Antioxidant 7a-OH 7b-OH 5a,6(lg/g oil)

Unheated TAG nd* nd ndWithout antioxidant 14.86 ± 0.09 a** 12.36 ± 0.33 a 24.15BHT 13.01 ± 0.10 b 11.04 ± 0.15 a 11.50Green tea extract 5.24 ± 0.56 c 15.83 ± 0.34 b 14.95Rosemary extract 5.67 ± 0.47 c 6.20 ± 0.08 c 6.70Tocopherols (natural) 2.86 ± 0.21 d 11.07 ± 0.12 a 3.55Tocopherols (synthetic) 6.22 ± 0.46 ce 9.94 ± 0.09 a 8.06Phenolic compounds from rapeseed meal 9.99 ± 0.97 f 6.52 ± 0.31 c 2.20Sinapic acid 7.93 ± 0.10 e 18.71 ± 0.25 d 18.90

* nd – not detected. Values are means of three determinations ± SD** Means in the same column followed by different letters indicate significant differe

hydroxycampesterol; 5a,6a-epoxy – campesterol-5a,6a-epoxide; 5b,6b-epoxy – campe

15.89 lg/g of oil, respectively. In samples with other antioxidants,the predominant groups were 7a-hydroxy (in samples with pheno-lic compounds extracted from rapeseed meal), 5a,6a-epoxy (insamples with sinapic acid) and 7-ketosterols (in samples withoutantioxidants or with a mix of tocopherols from rapeseed oil). Levelsof these compounds amounted to 9.99, 18.90, 25.53 and 13.40 lg/gof oil, respectively.

The data collected in Tables 3 and 4 show that the most effec-tive natural phenolic antioxidant was phenolic compoundsextracted from rapeseed meal. The rosemary extract limited theoxidation of phytosterols less than phenolic compounds from rape-seed meal and the least effective was the green tea extract. The dif-ferences in the activity of individual additives were probably dueto their composition and antioxidant capacity. The main phenoliccompound of rapeseed is sinapic acid and its choline ester calledsinapine. Sinapic acid constitutes over 73% of free phenolic acidsand 80–90% of the total phenolic acids, mainly occurring as esters

ucts in TAGs heated for 4 h at 180 �C.

a-epoxy 5b,6b-epoxy Triol 7-keto Total

nd nd nd nd± 0.66 a 23.81 ± 0.43 a 3.13 ± 0.18 a 25.53 ± 0.13 a 103.82 ± 1.01 a± 0.42 b 12.94 ± 0.34 b 15.30 ± 0.13 b 15.11 ± 0.43 b 78.89 ± 0.70 b± 0.25 c 18.71 ± 0.43 c 8.68 ± 0.43 c 5.92 ± 0.51 c 69.32 ± 0.53 c

± 0.37 d 10.39 ± 0.31 de nd 9.87 ± 0.11 d 38.83 ± 0.23 d± 0.42 e 12.26 ± 0.39 be 1.53 ± 0.46 d 13.40 ± 0.15 b 44.68 ± 0.71 e± 0.68 d 15.89 ± 0.92 f 1.11 ± 0.20 e 10.57 ± 0.11 d 51.79 ± 1.45 f± 0.31 e 8.79 ± 0.80 d 3.17 ± 0.08 d 7.93 ± 0.67 e 38.60 ± 0.31 d

± 0.45 f 15.73 ± 0.69 f 6.35 ± 0.64 f 9.22 ± 0.47 de 76.83 ± 0.56 b

nces (p < 0.05) between samples. 7a-OH – 7a-hydroxycampesterol; 7b-OH – 7b-sterol-5b,6b-epoxide; triol – campestanetriol; 7-keto – 7-ketocampesterol.

D. Kmiecik et al. / Food Chemistry 173 (2015) 966–971 971

and glucosides (Thiyam, Stöckmann, & Schwarz, 2006). Com-pounds responsible for antioxidant activity in rosemary weremainly the phenolic diterpenes, such as carnosic acid, carnosol,rosmanol, methyl carnosate, and rosmaric and caffeic acids. More-over, carnosic acid and carnosol are chiefly responsible for the anti-oxidant properties of rosemary extract (Vicente et al., 2013). Greentea is a rich source of polyphenols, including flavanols, flavandiols,flavonoids and phenolic acids. The most abundant group of pheno-lic compounds in green tea are catechins (flavonoids) (Hilal &Engelhardt, 2007). The highest activity of phenolic compoundsextracted from rapeseed meal is also due to the high total phenoliccontent and their activity (Wanasundara, Amarowicz, & Shahidi,1996). The total phenolic content of seven antioxidant fractionsfound by the author in canola meal has been calculated at about550 mg sinapic acid/g of canola meal. The total phenolic contentof rosemary extract and green tea extract are 219 and 144 mg gal-lic acid/g of extract, respectively (Gallego, Gordon, Segovia,Skowyra, & Almajano, 2013, Oh, Jo, Cho, Kim, & Han, 2013). Thelower content of phenolic compounds in rosemary extract com-pared to rapeseed meal is probably the cause of the higher levelsof phytosterol oxidation products in the sample heated with rose-mary extract. The highest level of oxyphytosterols was observed insamples with green tea extract, despite the higher addition for theoil sample (0.1% compared to 0.02%). This may mean that catechinsare not as effective in protecting phytosterols against oxidation asphenolic acid. Single antioxidants, such as BHT or sinapic acid,added to oil samples were less active than extracts obtained fromnatural sources. This means that not only the amount and type ofantioxidant is important but also the interactions between thedifferent compounds and the composition of mixture.

Acknowledgement

This study was financed by the Ministry of Education andScience, Poland under project No. 2 P06T 033 30.

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