ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 7, pp. 800–805. © Pleiades Publishing, Ltd., 2014. Original Russian Text © N.V. Sizova, 2013, published in Khimiya Rastitel’nogo Syr’ya, 2013, No. 1, pp. 157–163.

LOWMOLECULARWEIGHT COMPOUNDS

Determination of Tocopherols as Lipid Antioxidants in Vegetable Oils and Animal Fats N. V. Sizova1 Institute of Petroleum Chemistry, Siberian Branch, Russian Academy of Sciences, Akademicheskii pr. 3, Tomsk, 634021 Russia Received January 18, 2012

Abstract—The content of vitamin E (tocopherols) in natural vegetable oils and fats has been determined by microcalorimetry. This technique belongs to kinetic methods for determining vitamin E, which are based on the capacity of tocopherol to inhibit liquidphase radical oxidation reactions. It has been shown on a model reaction of initiated oxidation of cumene that fatty oils inhibit the radical reaction with an induction period proportional to the content of tocopherols in oil. From experimental curves, the content of tocopherols in oils obtained by different technological methods has been estimated. It has been shown that the amount of tocopherols is an indicator of the oil quality; therefore, the method proposed can be used to control the way of oil manufacture, oil quality, and the presence of synthetic antioxidants. Keywords: tocopherol, antioxidants, vegetable oils, determination, liquidphase radical oxidation DOI: 10.1134/S1068162014070164 1

INTRODUCTION Vitamin E (tocopherol) is called the vitamin of youth and life extension, which is reflected in its name (Greek tokos offspring + Latin ferre bear). This name is connected with the history of its discovery. In 1922 Evants and Dishop studied the causes of infertility in rats maintained on an artificial diet. The introduction of orange juice into the ration of rats did not cure them of infertility, whereas the addition of lettuce leaves restored the reproductive functions. Further it was found that the new vitamin is contained in great amounts in cereal germs and vegetable oils, is fatsol uble, and has many chemical analogues. At present, the term vitamin E involves a large group of natural substances possessing a similar bio logical activity. Along with the most widely distributed and most active αtocopherol, its 11 homologues and stereoisomers are known; they all have been currently isolated from vegetable oils or synthesized. At room temperature, tocopherols are an oily light yellow liq uid insoluble in water, soluble in chloroform, ethyl ether, and petroleum ether, and slightly soluble in ethyl alcohol and acetone [1, 2]. The biological value of vegetable oils is determined by a set of fatty acids; polyunsaturated acids and fat soluble vitamins are considered to be most valuable. Table 1 lists the data on the content of vitamin E in foodstuffs. The main depot of vitamin E is seeds, ker nels, and nuts and, respectively, all fatty oils; a maxi 1 Corresponding

author; phone: (3822) 492551; (3822) 491457; email: [email protected].

fax:

mum amount of vitamin E is contained in wheat germ oil. Vitamin E is thermally rather stable; when heated to 170°C in air and to 220°C in vacuo, it retains the biological activity. Therefore, the preparation of food in boiling water does not lead to the loss of toco pherols, but the roasting in heated vegetable oil should be avoided. Tocopherols can be identified in oils from specific electron absorption spectra with a maximum at 292 nm for αtocopherol, 297 nm for βtocopherol, and 298 nm for δ and γtocopherols. In factory laboratories, vita min E is determined by chromatography according to the AllUnion State Standard [3]. There are methods for determining tocopherol as an antioxidant since it behaves in model reactions as a classical inhibitor of radical processes [4]. There is evidence that toco pherols perform the function of a natural antioxidant [5]; on this basis, physicians recommend to increase the intake of vitamin E for the normalization of enhanced lipid oxidation, a process accompanying many diseases. The data on the content of vitamin E in many oils are out of date; sometimes, the results of measure ments are obviously untrue. Thus, in [6], the content of tocopherols in wheat germ oil was reported to be 1200 mg %, whereas according to numerous measure ments, it is 280–350 mg %. The content of tocopherols in camelina seed oil was reported in the same paper to be 785–821 mg %, whereas our measurements by two inde pendent methods gave 100–110 mg % [7].

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Table 1. Content of tocopherols in foodstuffs (mg%, amount of mg in 100 g) [1, 2] Product

Content, mg%

Product

Content, mg%

Wheat grain

0.9

Wheat germ oil

Nonpareil wheat meal

0.03

Cotton oil

70–100

Firstrate wheat meal

1.5

Sunflower oil

50–75

Smallsized bran

3.2

Corn oil

10–23

Largesized bran

0.3

Soybean oil

75–170

Wheat germs

15.8

200–300

Peanut oil

14

Coconut oil

At present, the technologies for oil production are being improved; on the market, there are oils of differ ent purity grade obtained by cold and hot pressing. Therefore, the choice of an oil for the diet or as an ingredient for biologically active food additives, cos metic creams, and oily dosage forms is of importance, among other things, from the viewpoint of the content of tocopherol since it is a marker of the quality and freshness of oil [7]. We have earlier shown that the microcalorimetric method makes it possible to reliably determine toco pherol as an antioxidant [8–10]. These data agree well with the results obtained by kinetic and chromato graphic methods [8]. We have also shown that toco pherol, like any other antioxidant, is expended on heating and storage of oil; therefore, its high concen tration is indicative of the freshness of oil [10]. The rate constant of the tocopherol–peroxyl radical reac tion, which characterizes the activity of an antioxi dant, compares well with the constant for tertbutyl phenols (butyloxyanizol, butyloxytoluene) [8]; how ever, tocopherol is unable to protect oil from burning through. In the present work, we measured the content of the antioxidant tocopherol in oils of one type obtained from different manufacturers using different technol ogies and in animal fats. In addition, the method enables one to detect the presence of exogenous anti oxidants. Objects of Investigation Oils purchased in stores and pharmacy networks or kindly provided by manufacturers were used. Several oil samples were produced by cold pressing in the Tomsk plant OOO Laboratoriya Ruzaeva and are sam ples of rawsqueezed oils that contain no synthetic contaminants and were not subjected to refinement and deodorization. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

3–8

Camelina seed oil (Camelina sativa) was provided by OOO PKP Provansal (Tomsk, Russia). It is distin guished by a large amount of polyunsaturated fatty acids and requires additional protection against oxida tion after deodorization, which is accomplished by the addition of a complex antioxidant. Therefore, we examined three oil samples: unrefined, refined deodorized, and refined deodorized supplemented with the antioxidant Antrantsin 33. Samples of grape seed oil (Vitis vinifera L.) obtained by extraction and pressing were provided by the Institute of Grape and Wine “Magarach” (Yalta). Melted animal fats were obtained from hunters occupied with the catching of animals in Tomsk region; another sample of badger fat was purchased in a pharmacy (OOO Bagira, Orenburg, Russia). Coconut oil of two manufacturers was brought from Thailand: one sample was purchased in a phar macy network and the other, in a “Nature Product” store. METHODS Measurements were carried out on an MKDP2 microcalorimeter of unique construction, manufac tured in Institute of Petroleum Chemistry, Siberian Branch, Russian Academy of Sciences; the method of measurements is described in [8]. The method of microcalorimetry belongs to kinetic methods and is based on the registration of the heat of a model reac tion of initiated cumene oxidation in the presence of additives of natural oils. With an active antioxidant, this method enables one to estimate the activity of inhibitors (constant k7) by the formula (1) presented below and the amount of antioxidants in a mixture of complicated composition by formula (2). Wt = ΔHVk2/k7[RH][1/(τ – t), (1) where ΔH is the enthalpy of the process under study equal to 111 ± 2 kJ/mol [4], and V is the volume of the Vol. 40

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SIZOVA q × 10–3, J/s 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 –1.0 –1.2 –1.4 –1.6 –1.8 –2.0 –2.2 –2.4 –2.6 –2.8 0:05:00

0:15:00 0:10:00

0:25:00 0:20:00

0:35:00 0:30:00

0:45:00 0:40:00

0:55:00 0:50:00

1:00:00

Fig. 1. Heat release in a model reaction of cumene oxidation [oxygen, t = 60°C, the initiation rate wi = 6.8 × 10–8 L/(mol s)] in the presence of deodorized camelina seed oil, C = 28.55 g/L. Azobisisobutyronitrile 4.6 mg.

reaction mixture (L). By substituting the known values of cumene concentration [RH]60 = 6.9 mol/L, the vol ume of cumene (V = 4 mL), and k2 = 1.75 L/mol s [9] into the above expression, k7 is calculated. The inhibition rate constant k7 refers to the interac tion of peroxyl radical with tocopherol (reaction II): •

k2

•

k

RH + RO 2

ROOH + R•,

(I)

7 (II) InH + RO 2 ROOH + In•. In the case of natural vegetable oils, the inhibition rate constants for tocopherols vary in the range of 1.4– 6.8 × 104 mol/L s [9]. It was shown with camelina and rape oils as examples that the inhibition rate constant for unrefined oil (6.2 × 104 L/mol s) is higher than for refined deodorized oil (1.4 × 104 L/mol s [9]. If the inhibitor is effective, the induction period τ (s) is observed, which is related to the rate of initiation wi (L/mol s) and the initial concentration of antioxi dants [AO]0 (mol/L) by the formula: [ InH ] τ = fn 0 . (2) wi From the period of induction of cumene oxidation, it is possible to estimate the concentration of antioxi dants for complex mixtures of the natural origin. A limitation of the method is the solubility in cumene of

fractions under study; among objects isolated from vegetable raw materials are fatty and volatile oils, lipid fractions of plants, and carbon dioxide extracts. Figure 1 shows an experimental curve of heat release in the presence of camelina seed oil; it is shown that, in the presence of a strong tocopherol antioxidant, an induc tion period occurs. It is known that volatile oils con tain no tocopherol; therefore, the oxidation rate of a model reaction in the presence of the volatile milfoil oil decreases (Fig. 2). We showed using carbon dioxide extracts that, with the technology of extraction being the same, the objects vary significantly in the degree of antioxidant activity depending on the starting raw material. In our calculations, we consider tocopherol to be a potent inhibitor with one functional group (n = 1) and the stoichiometric coefficient of inhibition f = 2 and calculate the tocopherol concentration from the induction period. Because 11 tocopherol homologues are present in oils, we averaged the weights of four main homologues (α, β, γ, and σ) and obtained an average weight of 416 relative units, which differs by 3% from the weight of the most abundant and active homologue αtocopherol. We have previously shown that the rate constants of the inhibition of oxidation for all oils (camelina, wintercress, rape seed, cedar) differ insignificantly, k7 = (1.4–6.8) × 104 mol/L s [9].

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q × 10–3, J/s 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 –1.0 –1.2 –1.4 –1.6 –1.8 –2.0 –2.2 –2.4 –2.6 0:05:00 0:10:00 0:15:00 0:20:00 0:25:00 0:30:00 0:35:00 0:40:00 0:45:00 0:50:00

Fig. 2. Heat release of (in) a model reaction of cumene oxidation [oxygen, t = 60°C, the initiation rate wi = 6.8 × 10–8 L/(mol s)] in the presence of the volatile milfoil oil. Tomsk. C = 0.9 g/L, the oxidation rate 1.48 × 10–6 mol/L s, 4.2 mg of azobisisobuty ronitrile, 4 mL of cumene, and oxygen.

RESULTS AND DISCUSSION The content of tocopherol homologues in oils and fats is an indicator of the quality of oil and the method of its production and purification. Thus, as it is evident from Table 2, the content of vitamin E in sunflower seed and cedar oils from OOO ”Laboratoriya Ruza eva” obtained by cold pressing is 10–20% higher than in oils of largetonnage industrial manufacture, par ticularly in refined oils. It was shown on samples of sunflower, camelina, and rape seed oils that up to 10% of vitamin E is lost during refinement and deodoriza tion. According to reference data, the content of toco pherols in wheat germ oil should be 200–300 mg % [1, 2]. However, the content of vitamin E in oils of one manufacturer (Novosibirsk) corresponds to the refer ence data, whereas in oils of another manufacturer (Moscow) it is almost three times less, which indicates a falsification. Two samples of apricot and linseed oils from two different manufacturers are nearly equal in quality. Grape seed oil obtained by pressing is richer in tocopherol than oil obtained by extraction. Animal oils, which have been long used in folk medicine as external and internal agents for the treat ment of catarrhal diseases, are of special interest. In the last years, animal oils have been included in the formula of medicinal and massage creams. Because fat is a depot of nutrient substances and fatsoluble vita mins for animals, it is logical to assume that vitamin E RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

also accumulates in the subcutaneous fat layer. Vita min E comes into the animal fat with food from nuts and berries, and its content in the body in the years of the highest nut yields is increased. As is seen from Table 2, mink, badger, and bear fats contain about 10– 15 mg % of vitamin E. One sample of badger fat con tains an antioxidant; therefore, the calculation for tocopherols yields a higher tocopherol content. To prevent oxidation, the antioxidant Antrantsin 33 was added to a sample of camelina seed oil accord ing to the manufacturer’s technology; in this case, our method reveals an increase in the antioxidant concen tration by 15%. Therefore, the method described can be applied for determining the additions of synthetic antioxidants. Vitamin E can act as an antioxidant; however, its efficacy is insufficient to completely stop the oxidation process. We have previously shown that the concentra tion of tocopherols during intensive oxidation of fatty oils at an elevated temperature (50°C) over a period of 30 days decreases to zero [10]. In this case, oils lose fluidity and convert to viscous films. As any other anti oxidant, vitamin E is expended during the storage of fatty oil. On storage of oils in domestic refrigerators, their oxidation proceeds slowly, and during the useful life ensured by the manufacturer, the oxidation prod ucts are within the limits of the norm. However, after the emergence of organoleptic signs of oxidation Vol. 40

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SIZOVA

Table 2. Content of tocopherols in vegetable oils and animal fats (mg %, mg in 100 g). The content of the antioxidant tocopherol in oils was determined in a model reaction of cumene oxidation [oxygen, t = 60°C, the initiation rate wi = 6.8 × 10–8 L/(mol s)] Grade of oil, manufacturer

[AO] × 10–3, mol/kg

Content of tocopherol, mg %

1

2

3

Cedar oil (Pinus sibirica) OOO Dary prirody, Tomsk OOO Sibekoprodukt, Tomsk PKP Lazurin, Novosibirsk OOO Laboratoriya Ruzaeva, Tomsk Sunflower seed oil (Helianthus) OAO Gubinskoe maslo Altai, Barnaul Polden, OOO PKP Provansal, Tomsk OOO Laboratoriya Ruzaeva, Tomsk Wheat germ oil (Triticum) OOO SibTar, Novosibirsk OOO Biask, Moscow Cameline seed oil (Camelina sativa) OOO PKP Provansal, Tomsk Unrefined oil Refined deodorized oil Refined deodorized oil supplemented with the antioxidant Antratsin 33 Rape seed oil (Brassica napus oleifera) OOO PKP Provansal, Tomsk Unrefined oil Refined oil Grape seed oil (Vitis vinifera L.) (Institute of Grape and Wine Magarach, Yalta) Obtained by extraction Obtained by pressing Walnut oil (Juglans regia L.) OOO Laboratoriya Ruzaeva, Tomsk Hemp oil (Cannabis sativa) OOO Laboratoriya Ruzaeva, Tomsk Flax oil (Linum usitatissimum L.), Novosibirsk Flax oil (Linum usitatissimum L.), OOO Laboratoriya Ruzaeva, Tomsk Coconut oil, “Nature product”, Tailand Coconut oil (pharmacy network) OOO Dary prirody, Tomsk OOO Biask, Moscow Peach oil (Persica mill.) OAO Pharmaceutical Factory, St. Petersburg Almond oil (Amygdalus communis) OOO Biask, Moscow Bear fat, melted; Tomsk Mink fat, melted; Tomsk Badger fat, melted; Tomsk OOO Bagira, Orenburg

1.00 1.26 1.30 1.44

43 54 55 61

1.55 1.64 2.00 2.18

67 71 86 90

6.49–6.99 2.31

300–350 100–105

2.73 2.39 2.77

117 102 Equivalent 119

1.97 1.77

85 77

2.01 2.56

88 106

1.24

58–53

1.99 2.01 1.60 0.65 0.11 1.81 1.84

85 70–86 73–77 25–28 5–7 78 79

2.31 1.15

102 49

0.33 0.30 0.21 1.3

14.0 13.0 9.0 Equivalent to 55 (addition of antioxidant)

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(unpleasant odor, bitter taste, greater viscosity), the oil cannot be used for food. CONCLUSIONS The content of vitamin E in natural oils and fats used in food, cosmetic, and pharmaceutical industries was determined. It was found that the content of toco pherols in oils obtained from one type of raw material differs by 25–30% depending on the method of pro duction. To summarize, it can be concluded that the high content of vitamin E in oils of one type may be an indicator of a careful technology of oil manufacture, which increases the food value of oil. The microcalo rimetric method used in the study makes it possible to determine quickly and precisely the quality and fresh ness of oil by the total antioxidant concentration from which the content of vitamin E is calculated as well as to determine the presence of exogenous antioxidants. ACKNOWLEDGMENTS The authors would like to thank L.N. Ruzaev for providing cedar, hemp seed, flax, and walnut oils, I.V. Chernousova (Institute of Grape and Wine “Magarach”, Yalta) for grape seed oil, and OOO PKP Provansal for the samples of unrefined and refined deodorized camelina seed, rape seed, and sunflower oils.

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REFERENCES 1. Vitaminy (Vitamins), Smirnov, V.V., Ed., Moscow, 1974. 2. Kalinin, F.L., Lobov, V.P., and Zhidkov, V.A., Sprav ochnik po biokhimii (A Handbook in Biochemistry), Kiev, 2013. 3. Masla rastitel’nye. Metody opredeleniya massovykh dolei vitaminov A i E. GOST 3041796 (Vegetable Oils. Meth ods for Determination of Mass Fractions of Vitamins A and E. GOST 3041796), Moscow, 1997. 4. Radchenko, L.M., Tsepalov, V.F., Konchalovskaya, M.E., Schmidt, A.A., Pastukhova, V.I., and Kozlova, Z.G., USSR Inventor’s Certificate No. 761902, Byull. Izo bret., 1980, no. 33. 5. Burlakova, E.B. and Khrapova, N.G., Usp. Khim., 1985, vol. 54. 6. Shikov, A.N., Makarov, V.G., and Ryzhenkov, V.E., Rastitel’nye masla i maslyanye ekstrakty: tekhnologiya, standartizatsiya, svoistva (Vegetable Oils and Oil Extracts: Technology, Standardization, and Proper ties), Moscow, 2004. 7. Sizova, N.V., Pikuleva, I.V., and Chikunova, T.M., Khim. Rastit. Syr’ya, 2003, no. 2, pp. 27–31. 8. Velikov, A.A. and Sizova, N.V., RF Patent No. 224205, Byull. Izobret., 2005, no. 9. 9. Sizova, N.V. and Andreeva, N.Yu., Khim.Farm. Zh., 2007, vol. 41, no. 6, pp. 49–52. 10. Sizova, N.V., Khim. Rastit. Syr’ya, 2009, no. 1, pp. 117– 119. 11. Sizova, N.V. and Popova, I.Yu., Khim.Farm. Zh., 2006, vol. 40, no. 4, pp. 29–33.

Translated by S. Sidorova

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