HPLC Separation of Vitamin E and Its Oxidation ... - ACS Publications

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HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF‑7 Breast Cancer Cells in Vitro Astrid M. Drotleff,*,† Anne Büsing,† Ina Willenberg,‡ Michael T. Empl,‡ Pablo Steinberg,‡ and Waldemar Ternes† †

Department of Analytical Chemistry and ‡Department of Food Toxicology, Center for Food Sciences, Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany S Supporting Information *

ABSTRACT: Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, β-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normalphase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γtocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0−100 μM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 μM) than that of nonoxidized γtocotrienol (134 μM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro. KEYWORDS: tocotrienols, tocopherols, vitamin E, dimeric oxidation products, HPLC-DAD, human MCF-7 breast cancer cells, alamarBlue assay, viability

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INTRODUCTION Vitamin E comprises eight vitamers: four tocopherols (α-, β-, γ-, and δ-T, 1−4) and four tocotrienols (α-, β-, γ-, and δ-T3, 5− 8) (Figure 1). Tocotrienols are available from several sources: primarily, from crude palm (Elaeis guineensis) oil (total tocotrienols = 364 mg/kg),1 and rice bran oil (total tocotrienols = 466 mg/kg), both of which are particularly rich in γ-T3; annatto seeds (total tocotrienols = 1400 mg/kg), which contain predominantly δ-T3;2,3 and barley oil extracted from brewers’ spent grain (total T3 = 516.8−850.2 mg/kg), predominantly αT3,4 the tocotrienol vitamer with the highest bioavailability.5 Tocotrienols differ from tocopherols only by three isolated double bonds in the isoprenoid side chains (Figure 1). All vitamin E forms (tocochromanols) are potent antioxidants, but tocotrienols are distinguished by exhibiting health benefits not attributed to tocopherols. For example, tocotrienols, but not tocopherols, are reported to lower blood cholesterol by inhibiting the HMG-CoA reductase. Furthermore, α-T3, but not α-T, is reported to exert a neuroprotective effect at the nanomolar level, and γ-T3 has been shown to possess a higher antitumor activity than tocopherols in breast cancer cells in vitro and, in some studies, in vivo at the micromolar level.3,6 The positive physiological effects of tocotrienols are principally related to their molecular structure. Tocotrienol vitamers are easily accessible for oxidation, for example, during storage and processing of tocotrienol-rich foods or pharmaceuticals, and this may lead to the degradation of tocotrienols during the formation of tocotrienol oxidation products. However, there are as yet no studies available on the biological © 2015 American Chemical Society

activity of tocotrienol-rich preparations that have undergone controlled oxidation. All vitamers have potent antioxidant activities, as they scavenge other radicals by donating hydrogen from the phenolic group on the chromanol ring. Among the vitamers, the α-forms are most prone to oxidation: they have the highest hydrogen-donating power, as they possess the highest methylation degree at the chromanol ring, which favors resonance stabilization of the tocochromanoxyl radical. This is why the α-vitamers are known to have the highest absolute antioxidant activity and degrade most rapdily in lipid matrices. However, at a high temperature, in high concentration, and in the presence of oxygen, the α-forms of tocochromanols can easily act as pro-oxidants by undergoing side reactions, leading to a poor relative antioxidant activity against lipid peroxidation.7−10 In fats and oils, tocotrienols were found to be better antioxidants than their corresponding tocopherols. Although γtocotrienol was found to be more effective in oils and fats, only the α-form of tocotrienol has considerable antioxidant activity in biological systems.11 Oxidations of tocochromanols in polar protic solvents such as ethanol typically involve different pathways and lead to different products than do oxidations in inert, apolar, aprotic solvents such as n-hexane.12 We recently separated seven different oxidation products resulting from thermal oxidation (3 Received: Revised: Accepted: Published: 8930

September September September September

8, 2015 22, 2015 25, 2015 25, 2015 DOI: 10.1021/acs.jafc.5b04388 J. Agric. Food Chem. 2015, 63, 8930−8939

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Journal of Agricultural and Food Chemistry

transformation of δ-T3 to its quinone derivative, which possibly contributes to cytotoxicity and apoptotic effects through oxidative damage of mitochondria and functional destabilization in the course of redox-recycling of the quinone. Furthermore, it has been suggested that the anticancer effect of tocotrienols is at least partially caused by targeting multiple intracellular signaling pathways associated with tumor cell viability, 19 independent of the antioxidant activity of tocotrienols.20 Knowledge of the in vitro bioactivity may help to design and interpret results obtained in in vivo studies. The majority of in vitro studies on the anticancer effects of γ- and δ-T3 have in common that the effective extracellular concentrations applied in vitro exceed the physiological concentrations.21 With regard to the oral bioavailability of tocotrienol vitamers, α-T3 is preferentially transferred into the bloodstream when compared to γ- and δ-T3.5 Strategies are being developed to overcome the limited bioavailability, to enhance the efficacy of therapeutic protocols.21 Tocotrienols have a comparatively short elimination half-life in vivo due to their high metabolic rate and antioxidant activity. Redox inactivation should stabilize the tocotrienols in vivo. This hypothesis is supported by reports that redox-silent derivatives of tocotrienols such as esters, carbamates, or ethers, which mask the hydroxyl group at the C6 position critical for antioxidant activity, show potent or even enhanced anticancer properties.6,18,22 Numerous studies have shown that tocotrienols23−28 or palm TRF29−31 significantly inhibit the growth of estrogen receptorpositive MCF-7 human breast cancer cells in vitro. For example, concentrations that inhibited 50% of cell growth in comparison to the control (i.e., the IC50 value) over 72 h were in the range from 8 to 25 μM and decreased in the order α-T3 > δ-T3 ≥ γ-T3 > palm TRF > α-T-free palm TRF, which means that the antiproliferative activities of coexisting tocotrienols from palm TRF and α-T-free palm TRF were found to be higher than those of pure tocotrienols.32 The growth-inhibiting effect of tocotrienols on MCF-7 cells was shown to occur estrogen-independently.21 Oxidized tocotrienol preparations have not yet been investigated in regard to their activity against breast cancer cells despite the fact that oxidation products of tocotrienols often are unavoidable during food processing or storage of tocotrienol supplements. Consequently, it was of interest to study the effect of tocotrienol-rich preparations that have undergone controlled oxidation on the viability of human breast cancer cells, in particular, the MCF-7 cell line. The aims of this study were (i) to apply our previously described HPLC-DAD and HPLC-PBI-EIMS methods for the analysis of α-T3 oxidation products to the separation and evaluation of oxidation products generated from the other vitamers; (ii) to determine the composition of equimolar tocotrienol test compounds (α-T3, γ-T3, and palm TRF) before and after thermal oxidation in the model matrix nhexane; (iii) to compare the effect of the untreated and thermally oxidized tocotrienol test compounds exemplarily on the viability of human MCF-7 breast cancer cells; and (iv) to estimate in a first approach the potential anticancer activity of the tocotrienol oxidation products. This study did not attempt to elucidate cell toxicity mechanisms but to provide first evidence of the importance of tocotrienol oxidation products by testing the effects of nonoxidized and oxidized tocotrienols under the same cell culture conditions. The results are expected to be valuable in the search for tocotrienol derivatives with

Figure 1. Structures of the vitamin E subgroups tocopherol (T) and tocotrienol (T3) and the most important oxidation products of tocotrienols and tocopherols.

h, 100 °C) of α-T3 (model matrix n-hexane) from the eight tocochromanols by HPLC with DAD and fluorescence detection (HPLC-DAD-F), and identified these oxidation products spectroscopically (HPLC-PBI-EIMS, i.e., HPLCEIMS with particle beam interface (PBI)).8,13 The predominant oxidation products from α-T3 were α-tocotrienol dihydroxydimer (17), 7-formyl-β-tocotrienol (9), and 5-formyl-γtocotrienol (11) (Figure 1). Minor proportions of α-tocotrienol quinone and its dimers were also detected with this method. Data in the literature suggest that non-α-T3 vitamers should form dimers as major oxidation products,14−17 but there are as yet no reports of HPLC analysis of the oxidation products of β-, γ-, and δ-T3. The potential role of tocotrienols in the treatment and prevention of breast cancer by inducing growth inhibition and apoptosis was recently reviewed.6 Viola et al.18 investigated human SKBR3 breast carcinoma cells and found fast and sustained mitochondrial uptake of δ-T3 followed by oxidative 8931

DOI: 10.1021/acs.jafc.5b04388 J. Agric. Food Chem. 2015, 63, 8930−8939

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Figure 2. Normal-phase HPLC-UV chromatograms of the eight tocochromanol reference substances after thermal oxidation in n-hexane (3 h at 100 °C), recorded at 280 nm. The corresponding parent compounds are marked by underlined numbers. (A) Oxidized tocotrienols: 5, α-tocotrienol; 6, β-tocotrienol; 7, γ-tocotrienol; 8, δ-tocotrienol; 9, 7-formyl-β-tocotrienol; 11, 5-formyl-γ-tocotrienol; 13, 15, α-tocotrienol spirodimers/trimers; 17, α-tocotrienol dihydroxydimer; 19, 7-(β-tocotrienoxy)-β-tocotrienol; 21, 5-(γ-tocotrienoxy)-γ-tocotrienol; 23a, 23b, 5-(γ-tocotrienyl)-γ-tocotrienol, atropisomers; 25, 5-(δ-tocotrienoxy)-δ-tocotrienol. (B) Oxidized tocopherols: 1, α-tocopherol; 2, β-tocopherol; 3, γ-tocopherol; 4, δ-tocopherol; 10, 7-formyl-β-tocopherol; 12, 5-formyl-γ-tocopherol; 14, 16, α-tocopherol spirodimers/trimers; 18a, 18b, α-tocopherol dihydroxydimers; 20, 7-(βtocopheryl)-β-tocopherol; 22, 5-(γ-tocopheroxy)-γ-tocopherol; 24a, 24b, 5-(γ-tocopheryl)-γ-tocopherol, atropisomers; 26, 5-(δ-tocopheroxy)-δtocopherol. mL of n-hexane; 2 mL samples of the vitamer solutions (each 1000 μg/mL) were transferred into 4 mL brown glass vials and placed in a drying oven for 3 h at 100 °C. The vials were left open during heat treatment. Thereafter, the oxidized, dry samples were brought to room temperature, and the sample volume was adjusted to 500 μL with nhexane. The solutions were used for HPLC-UV and HPLC-PBI-EIMS analysis. For use in cell viability assays, α-T3, γ-T3, and palm TRF were each dissolved in n-hexane at a concentration of 150 mM. Five hundred microliter samples of vitamer solutions were transferred into 1.4 mL brown glass vials and placed in a drying oven at 110 °C for 10 h (α-T3 and palm TRF) or 16 h (γ-T3). The vials were left open during heat treatment, after which the oxidized, dry samples were brought to room temperature, and the volumes were adjusted to 500 μL with ethanol. The composition of these ethanolic stock solutions was determined by HPLC-DAD after appropriate dilution. HPLC Separation and Spectroscopic Analysis of Tocochromanol Oxidation Products. The separation and identification of the eight reference substances (individually oxidized α-, β-, γ-, and δ-T and α-, β-, γ-, and δ-T3) were performed using our previously established HPLC-PBI-MS method8 with an additional in-line detection at 280 nm on an 8700 UV detector from Knauer (Berlin, Germany). The HPLC-DAD analysis (200−400 nm) of the oxidized and nonoxidized test compounds α-T3, γ-T3, and palm TRF was performed using a 250 mm × 4 mm i.d., 3 μm, ProntoSil 120-3 Diol column (Knauer, Berlin, Germany) according to our previously established HPLC-DAD-F method8 with a smaller sample loop (20 μL) and a modified gradient program for accelerated elution as follows: 0−5 min, 100% A; 20 min, 70% A; 30 min, 100% B; 30−60 min, 100% B, where eluent A was n-hexane and eluent B was nhexane/dioxane/TBME (95:3:2, v/v/v).

enhanced anticancer properties and in future studies on the nature of the observed biological effects. Although it is not possible to draw direct conclusions from tocochromanol oxidation products generated in model systems on the relevance of these oxidation products in food systems, the expected results should nevertheless help in future studies dealing with unwanted oxidative degradation during storage and processing of tocotrienol-rich foods or pharmaceuticals.

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MATERIALS AND METHODS

Chemicals and Test Compounds. The following tocotrienols were obtained from Davos Life Science (Singapore): α-, β-, γ-, and δT; α-, β-, γ-, and δ-T3 (each >97%); and palm tocotrienol-rich fraction (TRF) (>97%). The HPLC solvents used were ethanol, n-hexane, and acetonitrile, from Sigma-Aldrich (Steinheim, Germany); 1,4-dioxane (not stabilized), from Carl Roth (Karlsruhe, Germany); and tert-butyl methyl ether (TBME) and chloroform (stabilized), from Acros Organics (Geel, Belgium). The cell culture medium used was Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% 10000 U/mL penicillin, 1% 10000 μg/mL streptomycin, and 1% 200 mM L-glutamine (all from Biochrom, Berlin, Germany). MCF-7 human breast cancer cells were obtained from LGC Standards (Wesel, Germany). The alamarBlue reagent was purchased from AbD Serotec (Puchheim, Germany). Thermal Oxidation of Vitamers in Model Matrix n-Hexane and Preparation of Ethanolic Stock Solutions. To generate tocotrienol oxidation products for use as HPLC reference substances, our previously described method8 for the thermal oxidation of α-T3 was applied to the test compounds studied here: 100 mg of each vitamer (α-, β-, γ-, and δ-T; α-, β-, γ-, and δ-T3) was dissolved in 100 8932


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Table 1. UV and EIMS Spectroscopic Characteristics of the Most Important Tocochromanol Oxidation Products Formed during Individual Thermal Oxidation in the Model Matrix n-Hexane structure no. of oxidation product, chemical name educt: α-form 9, 7-formyl-β-tocotrienol 10, 7-formyl-β-tocopherol 11, 5-formyl-γ-tocotrienol 12, 5-formyl-γ-tocopherol 13, α-tocotrienol spirodimers/ 15, α-tocotrienol spirotrimers 14, α-tocopherol spirodimers/ 16, α-tocopherol spirotrimers 17, α-tocotrienol dihydroxydimer 18, α-tocopherol dihydroxydimers educt: β-form 19, 7-(β-tocotrienoxy)-β-tocotrienol 20, 7-(β-tocopheroxy)-β-tocopherol educt: γ-form 21, 5-(γ-tocotrienoxy)-γ-tocotrienol 22, 5-(γ-tocopheroxy)-γ-tocopherol 23, 5-(γ-Tocotrienyl)-γ-tocotrienol, atropisomers 24, 5-(γ-tocopheryl)-γ-tocopherol, atropisomers educt: δ-form 25, 5-(δ-tocotrienoxy)-δ-tocotrienol 26, 5-(δ-tocopheroxy)-δ-tocopherol a

retention time (min)

M+ (main EIMS fragments)

UV max (nm) 277, 290, 287, 290,

244 28333 279, 244 282, 27533

m/z m/z m/z m/z m/z

6.2 3.6 7.6 4.2 13.0−13.6

385, 395, 380, 386, 300

438 (219, 191, 179, 151) 444 (219, 205, 203, 179, 151) 438 (219, 191, 179, 151) 444 (219, 205, 203, 179, 151) 845/nd (847, 845, 625, 424, 422, 205, 203, 165)

7.0−7.5

339, 301, 290/292, 29033

m/z 857/nd (859, 857, 630, 430, 205, 203, 165)

57 49.9, 50.8

297 300, 290, 27533

m/z 847 (424, 422, 205, 203, 165) m/z 859 (430, 205, 203, 165)

11.1 6.9

295 29514

m/z 818 (410, 203, 151) m/z 830 (416, 191, 151)

7 4.6 11.8, 20.2

296 29715 304

m/z 818 (410, 191, 151) m/z 830 (416, 191, 151) m/z 818 (410, 191, 151)

5.9, 11.5

304, 30515

m/z 830 (416, 191, 151)

34.9 15.0

297 29515

m/za 790 (396, 191, 161) m/z 802 (402, 191, 161)

Predicted m/z values from 5-(δ-tocopheroxy)-δ-tocopherol EIMS results. M+, molecular ion; nd, not detected. Statistics. All cell viability experiments were performed four times with independent cell cultures. Data were tested for outliers and homoscedasticity. An analysis of variance (ANOVA) was performed to determine statistically significant differences between the different experimental groups. The selected significance level was α = 0.001. All statistical parameters were calculated using Valoo 2.3.34

Molar contents of dimeric oxidation products in oxidized test compounds were approximatively calculated from the corresponding peak area (UV detection), assuming that a dimeric tocotrienol oxidation product has the same molar extinction coefficient (ε, 1 g−1 cm−1) as the monomeric parent compound, which results in half the molar concentration of a dimeric compound compared to its monomeric parent compound with the same peak area. This approach is supported by literature data, according to which α-T and its dihydroxydimer have very similar ε values.33 Cell Viability Assay. The six ethanolic stock solutions containing either 150 mM nonoxidized α-T3, γ-T3, or palm TRF or 150 mM oxidized α-T3, γ-T3, or palm TRF were diluted with ethanol to prepare 0, 20, 25, 50, and 100 mM solutions. To facilitate the comparison between oxidized and nonoxidized test compounds, the concentration values of dilutions of oxidized test compounds refer to the vitamer concentration prior to oxidation. The ethanolic dilutions were further diluted 1:1000 with complete medium to yield concentrations of 20, 25, 50, and 100 μM for use in the cell viability assays. MCF-7 cells were grown as monolayer cultures in medium in a humidified atmosphere of 5% CO2 at 37 °C and passaged twice a week by bringing them in suspension with 0.05% trypsin/0.02% EDTA (w/ v) in PBS (Biochrom, Berlin, Germany). For the experiment, cells at passages 7−16 were seeded in 96-well plates at a density of 5 × 103 cells/well and were allowed to adhere for 24 h. Then, the medium was removed and cells were treated with 200 μL of either α-T3 (0−100 μM), γ-T3 (0−100 μM), or TRF (0−100 μM) for 72 h. Ethanol (0.1%) was used as negative control. Culture medium in all wells was then replaced by medium containing 5% (v/v) alamarBlue reagent solution. After a 3 h incubation at 25 °C, fluorescence F (λex 560 nm, λem 590 nm) was measured in a plate reader. Experimental results were expressed as viability in percent relative to control (100%). The absolute half-maximal inhibitory concentration (IC50) and the 95% confidence interval (CI) were determined by nonlinear regression curve fit analysis using GraphPad Prism version 6.04 (GraphPad Software, La Jolla, CA, USA).

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RESULTS AND DISCUSSION HPLC-UV Analysis of the Eight Reference Substances (Individually Oxidized α-, β-, γ-, and δ-T3 and -T). The chromatograms resulting from HPLC-UV of each of the eight individually oxidized α-, β-, γ-, and δ-T3 and -T are shown in Figure 2. The molecular structures, main EIMS fragments, and UV spectroscopic data of the most important tocotrienol oxidation products are summarized in Figure 1 and Table 1. In all chromatograms (Figure 2), additional peaks appeared along with the recovered tocochromanols. Under the conditions applied in this experiment, both α-vitamers (1, 5) showed the same types of oxidation products, the dihydroxydimer (17, 18) being the most striking for each α-vitamer (Figure 2). There were no indications that quinone or epoxide derivatives were present. Chromatograms of oxidized non-α-tocotrienols (i.e., β-, γ-, and δ-T3, 6−8) (Figure 2A) showed the same peak pattern as the corresponding oxidized non-α-tocopherols (2− 4) (Figure 2B). By means of HPLC-PBI-MS analysis of the tocotrienol and tocopherol oxidation products, we found the same types of oxidation products for the corresponding pairs of vitamers (β-T3 and β-T, etc.) and determined that the non-αT3 vitamers formed dimers as major oxidation products: β-T3 (6) formed 7-(β-tocotrienoxy)-β-tocotrienol (19); γ-T3 (7) formed 5-(γ-tocotrienoxy)-γ-tocotrienol (21) and two mutual atropisomers (peaks 23a and 23b) of 5-(γ-tocotrienyl)-γtocotrienol (23);12 and δ-T3 (8) formed 5-(δ-tocotrienoxy)-δ8933


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Journal of Agricultural and Food Chemistry Table 2. Composition of Ethanolic Stock Solutions of T3 Test Compounds Determined by Means of HPLC-DAD

without thermal oxidation test compound α-tocotrienol 5, α-tocotrienol (α-T3) 17, α-tocotrienol dihydroxydimer 9, 7-formyl-β-tocotrienol 11, 5-formyl-γ-tocotrienol 13, 15, α-tocotrienol spirodimers/trimers test compound γ-tocotrienol 7, γ-tocotrienol (γ-T3) 21, 5-(γ-tocotrienoxy)-γ-tocotrienol 23a, 5-(γ-tocotrienyl)-γ-tocotrienol, atropisomer 23b, 5-(γ-tocotrienyl)-γ-tocotrienol, atropisomer test compound palm tocotrienol-rich fraction (TRF) 1, α-tocopherol (α-T) 5, α-tocotrienol (α-T3) 6, β-tocotrienol (β-T3) 7, γ-tocotrienol (γ-T3) 8, δ-tocotrienol (δ-T3) sum 21, 5-(γ-tocotrienoxy)-γ-tocotrienol 23a, 5-(γ-tocotrienyl)-γ-tocotrienol, atropisomer 23b, 5-(γ-tocotrienyl)-γ-tocotrienol, atropisomer 9, 7-formyl-β-tocotrienol 11, 5-formyl-γ-tocotrienol

mM

percentage of total tocochromanol

150

100

150

100

10.7 38.1 4.8 69.9 26.6 150

7.1 25.4 3.2 46.6 17.7 100

after thermal oxidation of 150 mM test compound (110 °C, n-hexane solution) and redissolution in ethanol mM

%a

after 10 h of thermal oxidation 45.6 31 7.4 10 0.3 0.2 0.9 0.6 ndc n.d. after 16 h of thermal oxidation 29.8 20 8.2 11 7.5 10 9.7 13 after 10 h of thermal oxidation 0.3 2.5 3.4 9 1.5 32 9.1 13 ≤6.7b ≤25b ≤21.0 6.3 9 9.8 14 5.6 8 0.5 1.3 1.2 3.1

Percentage of peak area of parent vitamer not having been thermally oxidized. bThis value most probably includes a minor α-T3 oxidation product (α-tocotrienol dihydroxydimer). cnd, not detected. a

the monomer-connecting ethyl bond; in the case of the α-T3dihydroxydimer (17), there were no indications of the presence of atropisomers (peak 17) (Figure 2B), thus suggesting that the specific steric structures of the tocopherol or tocotrienol side chains play a role in this phenomenon. The data on oxidized non-α-tocotrienols (Figure 2) are in agreement with previous work on tocotrienol oxidation products.14−17 The dimeric oxidation product resulting from the β-vitamers was assigned to the ether dimer 19 and not the diphenol dimer by comparison with chromatographic and UV spectroscopic characteristics reported by Nilsson et al.14 In accordance with data from the literature,14,15,17 only one type of dimer was found for the δvitamers, that is, the ether dimer 25. This is the first report on the HPLC-UV and -PBI-EIMS characteristics of the oxidation products of all eight tocochromanols resulting from their individual thermal oxidation (100 °C, 3 h) in n-hexane. However, it should be kept in mind that all of these oxidation products may decompose further to form decomposition products that cannot be detected with this method.13 Composition of Test Compounds before and after Thermal Oxidation. The T3 test compounds, that is, individual α-T3, individual γ-T3, and mixed tocochromanols from palm TRF, were dissolved in n-hexane and thermally oxidized at 110 °C for 10−16 h. Table 2 shows the composition and the total content (sum of all vitamers) of tocochromanols of the test compounds before and after thermal oxidation. Figure 3 shows the HPLC-DAD chromatograms of palm TRF before (Figure 3A) and after thermal oxidation (Figure 3B,C). The amounts of tocochromanols and oxidation products in the

tocotrienol (25) (Figures 1 and 2A; Table 1). As the oxidation of tocochromanols takes place at the phenolic chromanol “head” and not at the side chain, the oxidation products of tocochromanols that share the same chromanol “head” are expected to be the same, differing only in the side chain. For αT3, this has previously been described by us in detail and confirmed by spectroscopic analysis.8 Tocochromanoxyl radicals primarily undergo radical−radical coupling reactions in apolar, aprotic solvents such as n-hexane, as used in the present study. If other potentially unstable radicals are absent, the tocochromanoxyl radicals react by selfcoupling, forming dimers.12,35 A major product of the αtocotrienoxyl radical is the α-T3-dihydroxydimer (17), which is formed by dimerization through the ortho methyl groups at the initial position C-5. The β-, γ-, or δ-tocotrienoxyl radicals, which have free (nonmethylated) ortho positions, primarily form dimers through these positions, thereby leading to tocotrienol (diphenol) dimers due to ortho−ortho couplings, for example, 5-(γ-tocotrienyl)-γ-tocotrienol (23);, and to tocotrienol−tocotrienyl ether dimers due to coupling of the tocotrienoxyl oxygen of one tocotrienol with the ortho position of the other, for example, 5-(γ-tocotrienoxy)-γ-tocotrienol) (21) (Figure 1). Remarkably, the HPLC-UV chromatogram of oxidized α-T showed the α-T dihydroxydimer (18) as a double peak (peak 18a and 18b) (Figure 2B). This is in agreement with prior findings.33,36 The double peak is probably due to the presence of two α-T dihydroxydimer atropisomers (18), which differ in the positions of the chromanol rings caused by the rotation of 8934


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recovered was 20% (Table 2); assuming linear degradation, the calculated recovery after 10 h is 75%. In the palm TRF sample, the vitamers degraded to different degrees: thermal oxidation led to an extensive loss of α-T (only 2.5% was recovered) followed by α-T3, γ-T3, δ-T3, and β-T3, with residual percentages of 9, 13, ≤25, and 32%, respectively, so that the oxidized palm TRF contained strikingly less total tocochromanols (21.0 mM) than oxidized α-T3 (45.6 mM) and γ-T3 (29.8 mM), even though the γ-T3 test compound was oxidized for a longer time (for 16 h) than the other test compounds (10 h). The chemistry of oxidation of the tocochromanols is complex and depends largely on the oxidation conditions,35 for example, concentration, type of solvent or lipid matrix, temperature, and presence of light or oxygen. Because of the complex test systems and the resulting difficulties in evaluating the relative antioxidant activity of tocochromanols in vitro, there is a widespread controversy regarding this topic,9 and it is difficult to draw any general conclusions.37 In our hexanoic palm TRF sample, tocochromanols did not compete with lipids or model substances as substrates for oxidation, but different tocochromanols competed with themselves. Clearly, the α-vitamers in the palm TRF degraded most intensely, but apparently without simultaneously protecting γ-T3 from degradation (Table 2). The results suggest that the above-mentioned pro-oxidant property of the α-vitamers may cause a higher overall degradation of the vitamers in the oxidized palm TRF sample than the samples with individual tocotrienols. There are as yet no reports in the literature on the composition of highly concentrated hexanoic solutions of T3-rich mixed tocochromanols after thermal oxidation. The oxidation products detected in α- and γ-T3 test compounds (Table 2) were found to be the same types of oxidation products as was expected from the results from the corresponding reference substances (Table 1). The oxidation products detected in the test compounds were predominantly dimers (Table 2). All of these dimers (17, 21, 23) are phenols or diphenols and can still act as antioxidants.12 It should be noted that HPLC-DAD analysis showed no indications of dimeric oxidation products other than those composed of two γ-T3 or two α-T3 and that at most a minor proportion of an αT3-dimer was formed, as indicated in the footnote of Table 2. This corresponds to the findings of Ha and Igarashi,15 who autoxidized α- and γ-T mixtures in methyl linoleate (10 h, 97.8 °C) and detected no mixed dimers, composed of different types of vitamers, among the isolated oxidation products by means of spectroscopic structure determination and found that dimers of γ-T predominated over those of α-T. Only small amounts of the aldehydic α-T3 oxidation products 5-formyl-γ-tocotrienol (11) and 7-formyl-β-tocotrienol (9) were also present in the α-T3 and TRF sample after thermal oxidation (Table 2; Figure 3B). These compounds, analogously to the aldehydes of tocopherols,38 should have negligible antioxidant properties because of the formation of strong intramolecular hydrogen bonds between the hydroxyl group and the corresponding carbonyl group in the ortho position. The conditions of thermal oxidation applied in our study led to an intensive decrease in all tocotrienol vitamers and necessarily to the generation of oxidation products. As suggested by the material balance (Table 2), the tocotrienol oxidation products newly separated, identified, and quantitated in the present study probably coexist with small-molecule products, which have lost the characteristic molecular moieties

Figure 3. Diol-modified normal-phase HPLC-DAD chromatograms of palm TRF (A) before and (B, C) after thermal oxidation in the model matrix n-hexane (10 h at 110 °C), recorded at (A, C) 297 nm or (B) 269 nm. 1, α-tocopherol (T); 5−8, tocotrienols (T3); 9, 7-formyl-βtocotrienol; 11, 5-formyl-γ-tocotrienol; 23a, 23b, 5-(γ-tocotrienyl)-γtocotrienol, atropisomers.

test compounds were assessed using a HPLC-DAD method similar to the HPLC-UV method for the oxidized reference substances (Figure 2), but with application of a steeper elution gradient, which is the reason for their overall shorter retention times (Figure 3) in comparison to those of the reference substances. Thermal oxidation of α-T3, γ-T3, and palm TRF, each containing 150 mM total T3, resulted in a decrease in tocochromanol compounds and the emergence of oxidation products. After a 10 h thermal oxidation of the α-T3 test compound, the percentage of α-T3 recovered was 31%. On the basis of preliminary tests and in analogy with tocopherols, where the redox potential of γ-T is higher (+0.348 V) than that of α-T (+0.273 V),12 γ-T3 was expected to be a weaker hydrogen donor and to degrade less intensely than α-T3. Therefore, the γ-T3 test compound was treated for a longer time (for 16 h instead of 10 h). The percentage of γ-T3 8935


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Journal of Agricultural and Food Chemistry of tocotrienols in the course of the oxidation progress and which were not identified by any of the analytical methods applied by us. In summary, the experimental design provided distinctly composed test compounds before and after thermal oxidation and, therefore, allowed the assessment of the importance of tocotrienol oxidation products with respect to anticancer effects of test compounds in an initial in vitro approach. Effect of Nonoxidized Tocotrienols on the Viability of Human MCF-7 Breast Cancer Cells. Treatment of human MCF-7 breast cancer cells for 72 h with α-T3, γ-T3, and palm TRF reduced the cell viability in a dose-dependent manner in comparison to the control, with no tocochromanols (Figure 4). After treatment with 100 μM α-T3, γ-T3, or palm TRF, significantly (p < 0.001) fewer living cells were determined by means of the alamarBlue assay: 58 ± 2, 68 ± 4, and 29 ± 5%, respectively. In accordance with Loganathan et al.,32 we found individual α- and γ-T3 vitamers to be less potent in reducing the viability of MCF-7 cells than equimolar total concentrations of mixed tocochromanols from palm TRF (Figure 4). This also agrees with the findings of Yu et al.,39 who found that MCF-7 cells were most sensitive to the δ-T3 vitamer, which was a component of our palm TRF. The IC50 values of α-T3, γ-T3, and palm TRF were greater than 100 μM in the cases of α-T3 and γ-T3 and 59 μM (95% CI = 51−68 μM) for palm TRF (Figure 4); when the MCF-7 breast cancer cells were treated with the γ-T3 vitamer at the lowest concentration (20 μM), there was a slight increase in cell viability (115 ± 4%) (Figure 4B). The IC50 values determined in our study are higher than data in the literature on anticancer effects of tocotrienols in MCF-7 cells.23−27,29−32 One explanation for this difference is that the other research groups conducted the viability tests with MCF-7 cells in media containing inactivated FBS, charcoal-stripped FBS, or a lower percentage of FBS: because FBS contains growth factors, stimulating endogenous hormones,40 it might attenuate the effects of tocotrienols on cell viability.24 Moreover, MCF-7 cells from different laboratories have been reported to have different biological properties, most probably due to an inherent genetic variability when cultured and passaged under different conditions.41 Furthermore, our observation that nonoxidized γ-T3 (IC50 = 134 μM, 95% CI = 92−196 μM) possesses a lower potency than nonoxidized α-T3 (IC50 = 125 μM, 95% CI = 107−145 μM) is in contrast to some study results reported in the literature.21,27 Our observation was made after a treatment period of 72 h. However, after a treatment period of only 24 h, the IC50 values of α-T3 (93 μM, 95% CI = 72−121 μM) and γT3 (20 μM, 95% CI = 16−25 μM) were much lower than after 72 h, and the IC50 value of γ-T3 was lower than that of α-T3, which is in accordance with literature data.27 After a treatment period of 48 h, the MCF-7 cell viability was considerably stimulated when compared to the 24 h results by nearly every one of the applied concentrations of nonoxidized and oxidized test compounds, but most intensely at the lowest concentrations of each test compound. These obvious hormetic effects were attenuated after a treatment period of 72 h and turned into a dose-dependent decrease of cell viability, except for oxidized palm TRF. Therefore, and to achieve the most conclusive comparison between the effects of nonoxidized and oxidized test compounds, we decided to evaluate the data from the 72 h experiments, as displayed in Figure 4.

Figure 4. Effect of test compounds before and after thermal oxidation on the viability of MCF-7 human breast cancer cells in medium containing 10% fetal bovine serum after a treatment period of 72 h: (A) α-tocotrienol (α-T3); (B) γ-tocotrienol (γ-T3); (C) palm tocotrienol-rich fraction (palm TRF). Pairs of bars labeled with an asterisk (∗) indicate significantly different viabilities caused by equimolar test compounds (p < 0.001). Data are representative of four independent experiments. 8936


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oxidized γ-T3, the viability-inhibiting effect was greater than that of equimolar nonoxidized γ-T3; consequently, the calculated IC50 (85 μM, 95% CI = 65−111 μM) was lower than for nonoxidized γ-T3 (Figure 4B). These data suggest that, above a minimum concentration of 25−50 μM γ-T3, the effect on cell viability of the test products containing oxidation products generated from any amount of γ-T3 given, was significantly stronger than that of the equimolar nonoxidized γT3 test products. However, it remains unknown whether this enhanced activity was caused by the oxidation products per se or whether the γ-T3 molecule acted more effectively through an interaction with the oxidation products. Nevertheless, our observation that the cell viability decreasing activity of the oxidized γ-T3 test compound was greater than that of equimolar, nonoxidized γ-T3 may be of value in the abovementioned search for redox-silent tocotrienol derivatives to identify substances with higher effectivity and bioavailability and deserves further investigation. As the in vitro bioactivity greatly depends on the intracellular concentration of tocotrienol forms,18,21 assessing the cellular uptake of the specific γ-T3 oxidation products should help in the evaluation if these can be associated with the improved activity or if other and, so far, unknown characteristics of oxidized γ-T3 or the tested MCF-7 cell line could explain these results. Whereas the nonoxidized palm TRF test compound decreased the viability of cancer cells more extensively than the individual α- and γ-T3, the oxidized palm TRF surprisingly did not reduce cell viability; on the contrary, a stimulating effect was observed at all concentrations of oxidized palm TRF (Figure 4C), and oxidation products generated from equimolar palm TRF test compounds clearly did not affect cell viability. This surprising result was predictable neither from the results obtained with the tested oxidized α- or γ-T3 test compounds nor from the decrease of the highly anticancerous δ-T3 vitamer. However, it is likely that the oxidation of palm TRF led to the formation of much less bioactive or bioavailable products, which prevented cell viability from decreasing as was observed for oxidized α- and γ-T3. It is obvious that, particularly in systems containing mixed tocochromanols, the mechanisms leading to changes in tumor cell viability, the interactions between different compounds, and the relationship between structure and effectiveness are complex and merit further investigation. This conclusion is supported by the results of a preliminary experiment in our laboratory, in which oxidized palm TRF (0−100 μM) clearly decreased the cell number of human colon adenocarcinoma HT-29 cells after a treatment period of 96 h in a dose-dependent manner as determined by performing the sulforhodamine B (SRB) assay. There are few studies available on the activity of oxidation products of tocochromanols in biological systems, and where such products are discussed, in most cases these are quinones, oxidation products primarily formed in polar, protic solvents. Shrader et al.45 reported that α-tocopherol quinone, an oxidative metabolite of α-T, is a potent cellular protectant against oxidative stress, whereby its biological activity depends upon its ability to undergo reversible two-electron redox cycling. Dolfi et al.46 observed that γ- and δ-tocopherol quinones had much higher inhibitory effects on human colon cancer HCT-116 and HT-29 cells compared to their parent compounds after a 72 h incubation; however, α-T and its quinone derivative were rather ineffective. Jones et al.47 found that γ-tocopherol quinone, but not α-tocopherol quinone, induced apoptosis in human ZR-75-1 breast cancer cells. Goh

The increased proliferation caused by tocotrienols described in this study has also been observed by other research groups applying experimental conditions similar to ours in regard to culture medium composition and duration of incubation. For example, Nesaretnam et al.42 observed that the lowest tested concentration of palm TRF (about 75 μM total tocochromanols) stimulated the proliferation of human MDA-MB-435 breast cancer cells when the cells were incubated in culture medium containing 10% FBS and palm TRF for 48 h, whereas higher concentrations of TRF caused an increasing inhibitory effect. Furthermore, Burdeos et al.43 recently found an increased cell proliferation rate of about 70% compared to the control after treating mouse hepatocellular carcinoma cells (Hepa 1-6) with culture medium containing 10% FBS and 1− 15 μM γ-T3 for 24 h; however, at higher concentrations, γ-T3 inhibited the proliferation of tumor cells. The reason for the increase in viability at lower tocochromanol concentration is still unknown, but it has been speculated that the antioxidative effect of γ-T3 could be responsible for this phenomenon.43 Explaining the differences in anticancer activity between the α- and γ-tocotrienols over time, the following literature may be relevant. The in vitro anticancer activity was shown to be a function of the cellular uptake rate and cell content of the different vitamers, as recently summarized by Viola et al.21 Tocotrienols are more potent in suppressing mouse mammary tumor cell growth and viability than tocopherols due to their preferential uptake, which rapidly provides levels leading to cytotoxic effects, and in this context γ- and δ-tocotrienols show higher uptake rates than α-T3.21,44 However, once the same intracellular concentration is reached, the difference in the anticancer activity between the vitamers decreases.21 In accordance with this observation, the testing of a series of tocotrienols in human SKBR3 breast carcinoma cells revealed that δ-T3 clearly was the most potent compound capable of inhibiting cell viability after a 24 h treatment, followed by γ- and α-T3. However, after 48 h, α-T3 showed the highest inhibitory effect, and this effect was also observed in murine TUBO breast carcinoma cells.18 Effect of Oxidized Tocotrienols on the Viability of Human MCF-7 Breast Cancer Cells. The α- and γ-T3 test compounds that underwent thermal oxidation affected the viability of MCF-7 cells in a dose-dependent manner (Figure 4A,B), as did the nonoxidized α- and γ-T3 test compounds. The calculated IC50 (145 μM, 95% CI = 101−209 μM) of the oxidized α-T3 was higher than that of nonoxidized α-T3, and the viability of cancer cells was in all cases significantly higher than when treated with equimolar nonoxidized α-T3 solutions (p < 0.001). This is plausible in consideration of the fact that only 31% of the initial α-T3 content was recovered in the oxidized α-T3 (Table 2). However, oxidized α-T3 decreased the viability of cancer cells to a greater extent than would be expected from the recovered α-T3 content alone, indicating that the oxidation products present in the test compounds also possess some antitumor activity in vitro. The oxidized γ-T3 compound enhanced cancer cell viability at the lowest tested concentrations (20 and 25 μM). In consideration of the fact that only 20% of the initial γ-T3 content in the oxidized γ-T3 was recovered (Table 2) and that, as mentioned above, low concentrations of tocotrienols in particular were found to stimulate cell viability, it is plausible that the cell viability increased significantly (p < 0.001) compared to treatment with the equimolar nonoxidized γ-T3 test compound. However, with higher concentrations of 8937


Journal of Agricultural and Food Chemistry et al.48 reported that no oxidative dimeric compounds from γT3 exhibited activity against tumor promotion by inhibition of the expression of Epstein−Barr virus early antigen in Raji cells, wheras monomeric γ-T3 did. In conclusion, this study has shown that individually thermally oxidized non-α-tocotrienols formed certain dimeric oxidation products, as did the corresponding non-α-tocopherols treated in the same way. Furthermore, it was shown that our HPLC-UV method is well applicable for separating the oxidation products from the parent compound, as was the HPLC-DAD method for determining the most important components of test products, even those containing mixed tocotrienols (i.e., palm TRF) that have undergone thermal oxidation in the model matrix n-hexane. Moreover, it was shown that equimolar concentrations of tocotrienol test compounds subjected to thermal oxidation had significantly different effects on the viability of human MCF-7 breast cancer cells compared to nonoxidized tocotrienol test compounds after a treatment period of 72 h in culture medium containing 10% FBS. Our findings also indicate that oxidation products of γ-T3 can contribute to the decrease in viability of human MCF-7 breast cancer cells and even enhance the antitumor activity in vitro, but that oxidized tocotrienol mixtures did not behave as expected from the results obtained with the individual vitamers. This is the first approach toward estimating the biological activity of tocotrienol oxidation products against breast cancer cells. These findings should be taken into account not only in the search for tocotrienol derivatives with enhanced anticancer properties but also in dealing with unwanted oxidative degradation during storage and processing of tocotrienol-rich foods or pharmaceuticals. Over all, these results should stimulate further investigations to elucidate the mechanisms responsible for enhancing or attenuating the anticancer properties of tocotrienols through oxidation.

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ACKNOWLEDGMENTS

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ABBREVIATIONS USED

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REFERENCES

We gratefully acknowledge Nicole Brauer for conducting the cell culture work.

CI, confidence interval; F, fluorescence; IC50, half-maximal inhibitory concentration; PBI, particle beam interface; T, tocopherol; T3, tocotrienol; TBME, tert-butyl methyl ether; TRF, tocotrienol-rich fraction

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04388. Figure showing the thermal oxidation of tocotrienols over time; figure and tables showing the effects of test compounds on the viability of MCF-7 breast cancer cells after treatment periods of 24, 48, and 72 h, the corresponding IC50data, and statistical evaluation; figure showing the effects of test compounds on the growth of human colon adenocarcinoma HT-29 cells after a treatment period of 96 h (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.M.D.) Phone: +49 511 856 7365. Fax: +49 511 856 827365. E-mail: astrid.drotleff@tiho-hannover.de. Funding

This work is part of the “Food Network” project funded by the German Ministry for Science and Culture of Lower Saxony through the Research Association of Agricultural and Nutritional Science of Lower Saxony (Forschungsverbund Agrarund Ernährungswissenschaften Niedersachsen, FAEN). Notes

The authors declare no competing financial interest. 8938


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