Effects of α- and γ-tocopherols on formation of hydroperoxides and two decomposition products from methyl linoleate

The antioxidant effects of α-and γ-tocopherols (at 0, 10, 100, 500, and 1000 ppm) were evaluated in a model system based on the autoxidation of methyl linoleate in bulk for 4 d at 40°C. Samples were collected every 24 h and analyzed for the 9 cis,trans, 9 trans,trans, 13 cis,trans, and 13 trans,trans isomers of hydroperoxide, hydroxy, and ketodiene oxidation products by high-performance liquid chromatography. Results showed that both α- and γ-tocopherols are effective hydrogen donors as evidenced by their abilities to inhibit the formation of hydroperoxides, hydroxy compounds, and ketodienes and the cis,trans to trans,trans isomerization of hydroperoxides. Compared with γ-tocopherol, α-tocopherol was a more efficient antioxidant at very low concentrations (10 ppm) but a less efficient antioxidant at the high concentrations (100–1000 ppm). This paradoxical behavior is explained on the basis of differences in ease of hydrogen donation between the two tocopherol homologs. Although α-tocopherol shows some loss of efficiency with increasing concentration, it is not a prooxidant when compared to the control void of antioxidants.     

Behavior of alpha,gamma, and delta tocopherols with linoleic acid in aqueous media

Tocopherols can exhibit opposite effects in aqueous media on linoleic acid autoxidation rate. The effect which was observed depended on tocopherol concentration and on the tocopherol itself. At 0.05 mole of tocopherol per mole of linoleic acid, α-tocopherol was prooxidant while in similar conditions, δ-tocopherol was anti-oxidant as well as γ-tocopherol. However, this latter one exhibited a slight antioxidant activity. When tocopherol concentration decreased (twice as weak), α-tocopherol still exhibited the same pro-oxidant activity, while the antioxidant effect of γ and δ tocopherols was increased. The study of tocopherol stability by HPLC has shown that tocopherol oxidation increased in order δ < γ < α. There was a relationship between the ability for a tocopherol to be easily oxidized by air and its prooxidant activity. Tocopherol oxidation would enhance the formation of a perhydroxyl radical ([·OOH] or one of this type [O₂0=, ·OH]) which was responsible for the prooxidant effect.     

Effects of α- and γ-tocopherols on the autooxidation of purified sunflower triacylglycerols

The antioxidant effects of α- and γ-tocopherols were evaluated in a model system based on the autooxidation of purified sunflower oil (p-SFO) triacylglycerols at 55°C for 7 d. Both tocopherols were found to cause more than 90% reduction in peroxide value when present at concentrations >20 ppm. α-Tocopherol was a better antioxidant than γ-tocopherol at concentrations ≤40 ppm but a worse antioxidant at concentrations >200 ppm. Neither α- nor γ-tocopherol showed a prooxidant effect at concentrations as high as 2000 ppm. The amount of tocopherols consumed during the course of oxidation was positively correlated to the initial concentration of tocopherols, and the correlation was stronger for α- than for γ-tocopherol. This correlation suggested that, besides reactions with peroxyl radicals, destruction of tocopherols may be attributed to unknown side reactions. Addition of FeSO₄, as a prooxidant, caused a 12% increase in the peroxide value of p-SFO in the absence of tocopherols. When tocopherols were added together with FeSO₄, some increase in peroxide value was observed for samples containing 200, 600 or 1000 ppm of α- but not γ-tocopherol. The addition of FeSO₄, however, caused an increase in the amount of α- and γ-tocopherols destroyed and led to stronger positive correlations between the amount of tocopherols destroyed during oxidation and initial concentration of tocopherols. No synergistic or antagonistic interaction was observed when α- and γ-tocopherols were added together to autooxidizing p-SFO.     

The peroxidizing effect of α-tocopherol on autoxidation of methyl linoleate in bulk phase

In order to understand the effect of α-tocopherol on the autoxidation mechanism of edible oil under storage conditions, methyl linoleate was allowed to autoxidize at 50 C in bulk phase without any radical initiator. The reaction was monitored by determining the production of four isomeric hydroperoxides (13-cis,trans; 13-trans,trans; 9-cis,trans; 9-trans,trans) by high performance liquid chromatographic analysis after reduction. In the absence of α-tocopherol, the rate of autoxidation depended on the sample size, and the duration of the induction period was affected by the initial level of hydroperoxides. However, the distribution of c-t and t-t hydroperoxide isomers remained constant during the propagation period regardless of the sample size. The addition of α-tocopherol at 0.1 and 1.0% caused a linear increase in the amount of hydroperoxides and elevated the distribution of the c-t isomers. The rate of hydroperoxidation appeared to be governed by the initial concentration of α-tocopherol rather than the sample size or the initial hydroperoxide level. This peroxidizing effect of α-tocopherol was suppressed by the presence of ascorbyl palmitate. A mechanism in which chromanoxy radical participates is proposed for the effect of α-tocopherol on lipid autoxidation in bulk phase. It is therefore suggested that α-tocopherol at high concentrations influences the mechanism of autoxidation of edible oil.     

Effect of experimental factors on the prooxidant behavior of α-tocopherol

We have investigated the effect of experimental factors on the prooxidant effect of α-tocopherol during the autoxidation of linoleic acid. The prooxidant effect depended on two factors: the concentration of α-tocopherol (≥ 5 x 10⁻³ mol α-tocopherol/1 mol linoleic acid) and the solvent, an aqueous system in which the prooxidant effect occurred more easily. On the other hand, the prooxidant behavior of α-tocopherol was unaffected by the type of surfactant used in water as well as by the presence of different salts. The initial content of hydroperoxides affected the intensity of the prooxidant effect which varied in an inverse ratio to the initial hydroperoxide level.     

Effect of α-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides

α-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180°C were analyzed by capillary gas chromatography. In the presence of either α-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans andtrans, trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of α-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 9-oxononanoate to increase. A similar effect of α-tocopherol was observed on the distribution of volatiles formed from a mixture of thetrans,trans 9- and 13-hydroperoxides. This effect of α-tocopherol was, however, insignificant with purecis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, α-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic β-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect thetrans,trans isomers of linoleate hydroperoxides.     

Antioxidant activities of major components of γ-oryzanol from rice bran using a linoleic acid model

The change in hydroperoxides of linoleic acid incubated with constant micro air flow at 37°C was used to evaluate the antioxidant activities of three major components of γ-oryzanol from rice bran (cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, and campesteryl ferulate) compared with α-tocopherol and ferulic acid. The four hydroperoxide isomers of linoleic acid, 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid [9HPODE(t,c)], 9-hydroperoxy-10-trans, 12-trans-octadecadienoic acid, 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid [13HPODE(c,t)], and 13-hydroperoxy-9-trans, 11-trans-octadecadienoic acid, were measured using normal-phase high-performance liquid chromatography with an ultraviolet detector. The three components of γ-oryzanol evidenced significant antioxidant activity when they were mixed with linoleic acid in a molar ratio of 1∶100 and 1∶250 but not in a molar ratio of 1∶500 (P<0.05). α-Tocopherol and ferulic acid also demonstrated significant antioxidant activity at all three molar ratios (P<0.05). The highest molar ratio (1∶100) of α-tocopherol, however, caused greater levels of 9HPODE(t,c) and 13HPODE(c,t) than the other two less concentrated treatments.     

Antioxidant activities of α- and γ-tocopherols in the oxidation of rapeseed oil triacylglycerols

Antioxidant properties of 5 to 500 μg/g levels of α-and γ-tocopherols, in the oxidation of rapeseed oil triacylglycerols (RO TAG), were studied at 40°C in the dark. Each tocopherol alone and in a mixture was studied for its stability in oxidizing RO TAG. Also the effects of tocopherols on the formation of primary and secondary oxidation products of RO TAG were investigated. Both tocopherols significantly retarded the oxidation of RO TAG. At low levels (≤50 μg/g), α-tocopherol was more stable and was a more effective antioxidant than γ-tocopherol. At higher α-tocopherol levels (>100 μg/g), there was a relative increase in hydroperoxide formation parallel to consumption of α-tocopherol, which was not found with γ-tocopherol. Therefore, γ-tocopherol was a more effective antioxidant than α-tocopherol at levels above 100 μg/g. As long as there were tocopherols present, the hydroperoxides were quite stable and no volatile aldehydes were formed. In a mixture, α-tocopherol protected γ-tocopherol from being oxidized at the addition levels of 5+5 and 10+10 μg/g but no synergism between the tocopherols was found. α-Tocopherol was less stable in the 500+500 μg/g mixture than when added alone to the RO TAG. No prooxidant activity of either tocopherol or their mixture was found.     

An investigation of the basic conditions for tocopherol determination in vegetable oils and fats by differential pulse polarography

The polarographic behavior of α-, γ-, and δ-tocopherols was studied according to the proposed official IUPAC method for tocopherol determination in vegetable oils and fats. Each of the tocopherols had a different polarographic response; however, the tocotrienols had the same half-wave potentials and probably also the same polarographic response as the corresponding tocopherols. Additives previously investigated plus several others were examined for possible interference. The results by polarography and a new high performance liquid chromatography (HPLC) method were compared. The analysis by t-test at 99% significance level showed no differences for the determinations of α-tocopherol, but the results for three of the γ-tocopherol results were less consistent under the same conditions. The results for the determination of δ-tocopherol were below the detection limit for polarography and could not be statistically evaluated. The polarographic method investigated was found to be uncomplicated and therefore suitable for routine work. However, when using the method, one has to take into account possible interference by additives and the limitations due to the lack of separation of β- from γ-tocopherol and/or the interference of tocotrienols with the corresponding tocopherol peaks. From this aspect the HPLC method gives better resolution.     

Relative autoxidative and photolytic stabilities of tocols and tocotrienols

The relative stabilities of selected individual tocols and tocotrienols and of equimolar mixtures of either α- plus γ- or α- plus δ- tocopherols were determined in methyl myristate and methyl linoleate during autoxidation and photolysis. Solutions containing 0.05% of the appropriate tocopherol(s) or tocotrienols were subjected to UV light (254 nm) or to a flow of 4.3 ml/min of oxygen, both at 70 C. Tocopherols (T) and tocotrienols (T−3) were determined by gas chromatography without preliminary separation or purification. Under photolytic conditions, stabilities in increasing order in methyl myristate were γ-T−3<α-T−3<δ-T<α-T <γ-T<5,7-T<β-T and in methyl linoleate were α-T<α-T−3≤γ-T−3≤β-T≤5,7-T <γ-T<δ-T. A solvent effect on the initial rate of photolysis was observed for 5-methyl substituted tocols but not for the tocols with an unsubstituted 5-position or for the tocotrienols. Under autoxidative conditions, stabilities in increasing order in methyl myristate were α-T=α-T−3 <β-T−3<γ-T−3<δ-T−3<γ-T<δ-T=β-T and in methyl linoleate were α-T<α-T−3 <γ-T−3<β-T<γ-T<δ-T. Tocopherols were much more stable during autoxidation in methyl myristate than they were in methyl linoleate. In mixtures, there was no significant protection of α-tocopherol by either γ- or δ-tocopherol under any of the conditions used. However, α-tocopherol was highly effective in protecting γ- and δ-tocopherols in methyl myristate during both photolysis and autoxidation and in methyl linoleate during photolysis. During autoxidation in methyl linoleate, α-tocopherol protection of γ- and δ- tocopherols after 24 hr was slight tough measurable.     

Optimal tocopherol concentrations to inhibit soybean oil oxidation

The optimal concentration for tocopherols to inhibit soybean oil oxidation was determined for individual tocopherols (α-, γ-, and δ-tocopherol) and for the natural soybean oil tocopherol mixture (tocopherol ratio of 1∶13∶5 for α-, γ-, and δ-tocopherol, respectively). The concentration of the individual tocopherols influenced oil oxidation rates, and the optimal concentrations were unique for each tocopherol. For example, the optimal concentrations for α-tocopherol and γ-tocopherol were ∼100 and ∼300 ppm, respectively, whereas δ-tocopherol did not exhibit a distinct concentration optimum at the levels studied (P<0.05). The optimal concentration for the natural tocopherol mixture ranged between 340 and 660 ppm tocopherols (P<0.05). The antioxidant activity of the tocopherols diminished when the tocopherol levels exceeded their optimal concentrations. Above their optimal concentrations, the individual tocopherols and the tocopherol mixture exhibited prooxidation behavior that was more pronounced with increasing temperature from 40 to 60°C (P<0.05). A comparison of the antioxidant activity of the individual tocopherols at their optimal concentrations revealed that α-tocopherol (∼100 ppm) was 3–5 times more potent than γ-tocopherol (∼300 ppm) and 16–32 times more potent than δ-tocopherol (∼1900 ppm).     

A multivariate study of the correlation between tocopherol content and fatty acid composition in vegetable oils

The main biochemical function of the tocopherols is believed to be the protection of polyunsaturated fatty acids (PUFA) against peroxidation. A critical question that must be asked in reference to this is whether there is a biochemical link between the tocopherol levels and the degree of unsaturation in vegetable oils, the main source of dietary PUFA and vitamin E. We used a mathematical approach in an effort to highlight some facts that might help address this question. Literature data on the relative composition of fatty acids (16:0, 16:1, 18:0, 18:1, 18:2, and 18:3) and the contents of tocopherols (α-, β-, δ-, and γ-tocopherol) in 101 oil samples, including 14 different botanical species, were analyzed by principal-component analysis and linear regression. There was a negative correlation between α- and γ-tocopherols (r=0.633, P<0.05). Results also showed a positive correlation between linoleic acid (18:2) and α-tocopherol (r=0.549, P<0.05) and suggested a positive correlation between linolenic acid (18:3) and γ-tocopherol.     

High performance liquid chromatography of the tocols in corn grain

A sensitive and selective method was developed for analyzing the tocol isomers in corn grain by high performance liquid chromatography (HPLC) with fluorescence detection. The relative proportions and the total amounts of the tocol isomers (α-tocopherol, α-tocotrienol, γ-tocopherol and γ-tocortrienol) varied greatly among the 15 corn inbreds that were examined. Although γ-tocopherol has traditionally been considered to be the predominant vitamin E isomer in corn, inbreds with equal or higher levels of α-tocopherol have been discovered. No tocotrienols were found in corn germ oil, only α-and γ-tocopherols. Analysis of the tocopherols of the germ oils of inbreds and their reciprocal crosses indicated that the proportions of the α- and γ-isomers and the total amount of the tocopherols are heritable. Presented at the 74th AOCS annual meeting, Chicago, 1983.     

Tocopherol Content and Profile of Soybean: Genotypic Variability and Correlation Studies

Plant seed oils, including soybean seed oil, represent the major source of naturally derived tocopherols, the antioxidant molecules that act as free radical quenchers preventing lipid peroxidation in biological systems and vegetable oil products. All four isomers of tocopherols, i.e. α, β, γ, δ tocopherols that exist in nature are found in soybean seeds. The biological activity and the contribution of these isomers in improving the oxidative stability of vegetable oil are in reverse order. Because of the nutritive value and the importance for oil stability, enhancement of tocopherol content, through breeding programs, in soybean seeds has become a new and an important objective. Genotypic variability, which is the basis of every breeding program, is scarcely reported for tocopherol content and profile in soybean seeds. In the present investigation, the tocopherol content and profile in seed samples of 66 genotypes of Indian soybean were determined. The ratios observed between the lowest and the highest values for α, β, γ, δ, total tocopherol content were 1:13.6, 1:10.4, 1:7.5, 1:9.1, 1:7.9, respectively. The mean contents for α, β, γ, δ and total tocopherols were 269, 40, 855, 241 and 1,405 μg/g of oil, respectively. Total tocopherol content was the highest in ‘Co Soya2’ followed by ‘Ankur’. Concentration of α-tocopherol was the highest (27%) in ‘Ankur’ followed by ‘MACS124’ (26%) whereas gamma tocopherol concentration was the highest (69%) in ‘VLS1’ and ‘PK327’ followed by ‘MACS13’ (67%). In view of the fact that levels of unsaturated fatty acids, apart from tocopherols, also determine the oxidative stability of vegetable oils, the relationship of four isomers of tocopherols with each other as well as with different unsaturated fatty acids and oil content was also investigated in the present study. All the four isomers of tocopherols exhibited highly significant correlations with each other (p < 0.001) whereas γ-tocopherol and total tocopherol content showed a significant relationship with linoleic acid (p < 0.05).     

Properties of α-, γ-, and δ-tocopherol in purified fish oil triacylglycerols

The effect of α-tocopherol (αTOH) (50–2000 ppm), γ-tocopherol (γTOH) (100–2000 ppm), and δ-tocopherol (δTOH) (100–2000 ppm) on the formation and decomposition of hydroperoxides in purified fish oil triacylglycerols (TAG) was studied. The tests were conducted at 30°C in the dark. Purified fish oil TAG oxidized very rapidly with no apparent induction period. The relative ability of the tocopherols to retard the formation of hydroperoxides decreased in the order αTOH> γTOH>δTOH at a low level of addition (100 ppm), but a reverse order of activity was found when the initial tocopherol concentration was 1000 ppm. This dependence of relative antioxidant activity on tocopherol concentration was caused by the existence of concentrations for maximal antioxidant activity for αTOH and for γTOH. An inversion of activity, on the basis of hydroperoxide formation, was observed for αTOH at 100 ppm and for γTOH at 500 ppm, whereas the antioxidant activity of δTOH increased with level of addition up to 1500–2000 ppm. None of the tocopherols displayed any prooxidant activity. All three tocopherols strongly retarded the formation of volatile secondary oxidation products in a concentration-dependent manner. At concentrations above about 250 ppm there appeared to be a linear relationship between rate of consumption of αTOH and initial αTOH concentration, in accordance with the linear relationship observed between the initial rate of formation of hydroperoxides and the initial αTOH concentration. The rate of consumption of γTOH also increased with initial concentration, but to a lesser extent than for αTOH. At high levels of addition the rate of consumption of δTOH was independent of initial concentration, appearing to reflect the greater stability of this tocopherol homolog and participation in reactions with lipid peroxyl radicals only. Presented in part at the AOCS annual meeting in San Diego, California, April 2000.     

A dimeric oxidation product of γ-tocopherol in soybean oil

A dimeric oxidation product (5-γ-tocopheroxy-γ-tocopherol) has been isolated from soybean oil and identified. The dimer content in extracted oil was increased by elevating the moisture level in raw soybeans. With moisture increase, no change in the quantity of α-tocopherol was observed, but γ- and δ-tocopherol contents were greatly decreased and two kinds of dimer were formed from γ- and δ-tocopherols. When the moisture level in moistened beans was lowered, these dimers reverted to their corresponding original tocopherols. The same results were obtained by treating pulverized soybeans with various reducing agents. γ-Tocopherol added to autoxidizing soybean oil was oxidized more easily in the presence of oxidation products derived from tocopherols and turned into the dimeric product.     

Chromatographic separation of the stereoisomers of α-tocopherol

The diastereoisomers of2-ambo-α-tocopherol were completely separated as TMS ethers by gas chromatography on a 115 m × 0.25 mm glass capillary column coated with SP2340, at a column temperature of 195 C. In the same way,all-rac-α-tocopherol was separated into four peaks, corresponding to the four racemates present, and having the same retention ratios as the four diastereoisomers of 4′-ambo-8′-ambo-α-tocopherol (produced by the hydrogenation of natural α-tocotrienol). Retention data and relative peak areas for the diastereoisomers of the synthesized α-tocopherols and several commercial products were determined. Limited data on the isomers of other tocopherols also are reported.     

Effects of steeping conditions during wet-milling on the retentions of tocopherols and tocotrienols in corn

Vitamin E is a natural antioxidant that plays significant roles in food preservation and disease prevention. There are eight naturally occurring vitamin E isomers (tocols): α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols. Corn oil is a major source of vitamin E. Most of the corn oil produced in the United States is a co-product of corn wet-milling. There is limited knowledge about the effects of corn wet-milling on the retention of these vitamin E isomers. A high-performance liquid chromatography method was developed for simultaneous determinations of tocols in steeped corn samples. Effects of steeping conditions (steeping time and SO₂ concentration) on retention of tocols in corn were investigated. α-Tocopherol, γ-tocopherol, α-tocotrienol, and γ-tocotrienol are the predominant vitamin E isomers in the corn variety used in the study. Steeping conditions had little effect on the concentration of α-tocopherol and α-tocotrienol. However, a higher concentration of SO₂ and a shorter steeping time gave a slightly higher γ-tocotrienol content and lower γ-tocopherol content. Corn kernels steeped in a vitamin C solution had a much higher concentration of the tocols than those steeped in SO₂ solution.     

Effect of α- and γ-tocopherols on thermal polymerization of purified high-oleic sunflower triacylglycerols

The antipolymerization effects of α- and γ-tocopherols were compared in model systems composed of purified high-oleic sunflower triacylglycerols at 180°C. γ-Tocopherol was much more effective as an antipolymerization inhibitor than α-tocopherol, partly due to lower oxidizability/disappearance. Purified triacylglycerols of sunflower, rapeseed, and high-oleic sunflower oils were less stable than their nonpurified forms containing tocopherols. Results confirmed that tocopherols per se can act as antipolymerization agents in high-oleic oils at frying temperatures. No synergism was observed when α- and γ-tocopherols were present together although larger amounts of residuals were left for both tocols. Results suggested that high-oleic/high-γ-tocopherol oils (such as high-oleic canola and high-oleic soybean oils) may provide better frying oils than high-oleic/high-α-tocopherol oils (such as high-oleic sunflower oil).     

Effects of tocopherols, ascorbyl palmitate, and lecithin on autoxidation of fish oil

The effects of α-, γ/δ, and δ-tocopherol concentrates (0.2–2.0%) alone and in combination with ascorbyl palmitate (0.1%) and lecithin (0.5%) on oxidative stability and flavor of fish oil were studied. Stability was assessed on oil stored in air at 20°C by peroxide value (PV) and off-flavor formation. Polymer content, para-anisidine value, and conjugation were used to characterize selected samples. When used alone, the protective effect of the tocopherols, as measured by PV, was δ≫γ/δ≫α, especially at the 2% concentration. Binary systems of ascorbyl palmitate-lecithin and lecithin-γ/δ or-δ-tocopherol were strongly synergistic in delaying peroxidation. The ternary blends provided the greatest protection against autoxidation. Refined fish oil with 2% δ-tocopherol, 0.1% ascorbyl palmitate, and 0.5% lecithin showed no significant peroxidation at 20°C over a period of 6 mon. The original antioxidant effect noted for the ternary systems in delaying peroxidation was not reflected in improved flavor stability. Off-flavors developed within 3 wk, making the oils unsuitable for use at high concentrations in ambient products that are unprotected from air.