Research Article Received: 19 September 2012
Revised: 22 October 2013
Accepted article published: 19 November 2013
Published online in Wiley Online Library: 23 December 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6484
Comparison of dietary tocotrienols from barley and palm oils in hen’s egg yolk: transfer efficiency, influence of emulsification, and effect on egg cholesterol Constanze M Walde, Astrid M Drotleff and Waldemar Ternes∗ Abstract BACKGROUND: The main component in tocotrienols (T3) from barley (Hordeum vulgare L.) is α-T3, the vitamer with the highest bioavailability, while palm oil T3 is particularly rich in γ -T3. Unlike tocopherols, T3 are known for their cholesterogenesisinhibiting, neuroprotective and anticarcinogenic properties. In this study the oral bioavailabilities of T3 from barley oil (3.98 mg day−1 ) and T3 from palm oil (3.36 mg day−1 ) in nanoemulsified formulations (NE) and self-emulsifying systems (SES) were compared using hen’s eggs as a bioindicator. In addition, the transfer efficiencies of barley oil T3 and palm oil T3 into egg yolk were compared, as well as their effects on egg cholesterol levels. RESULTS: Nanoemulsification led to T3 levels (132.9 µg per egg) higher than with non-emulsified barley oil (112.8 µg per egg) and barley oil SES (116.7 µg per egg) owing to the high proportions of α-T3 (99–117 µg per egg), which has a particularly high transfer efficiency (4.32–6.75%). T3 contents of eggs from hens fed barley oil supplements (112–132 µg per egg) were significantly higher than those of eggs from hens fed palm oil supplements (70–78 µg per egg). Addition of barley and palm oils to laying hen feed decreased egg yolk cholesterol by 4 and 6% respectively. CONCLUSION: Results from this animal study may help to establish T3 from barley as a dietary supplement and to develop nutritionally improved hen’s eggs. c 2013 Society of Chemical Industry Keywords: barley; tocotrienol; tocopherol; oral absorption; hen’s egg yolk
INTRODUCTION
810
Barley (Hordeum vulgare L.) is the richest cereal source of the vitamin E subclass tocotrienols (T3), and oil from barley is thought to have the highest T3 content of any natural oil.1 Moreover, barley is distinguished by being a natural source of all eight tocochromanol vitamers (Fig. 1), with T3 comprising about 76% of the total tocochromanols, including 47% α-T3, the most biologically available vitamer.2 As determined in rats, the oral bioavailability of α-T3 is highest (27.7 ± 9.2%), followed by γ -T3 (9.1 ± 2.4%) and δ-T3 (8.5 ± 3.5%).3 This makes T3 from barley oil superior to T3 from palm oil, the most prevalent commercial source of T3. Crude palm oil is particularly rich in γ -T3 (39% of the average total tocochromanol content of 587 mg kg−1 ).4 World palm oil production increased enormously during recent years, reaching 38.5 million tons in 2007.5 However, oil palm (Elaeis guineensis) cultivation in tropical regions of Asia, Africa and Latin America is subject to negative criticism owing to its environmental impact. Brewer’s spent grain (BSG) is a barley by-product that is generated in large amounts in beer production. In 2008, European breweries produced a total of 427 million hectolitres of beer and an estimated more than 8 million tons of residual wet BSG.6 Previously, we discovered that the sieving fraction 97% purity) were provided by Davos Life Science (Singapore). All solvents were of high-performance liquid chromatography (HPLC) grade. n-Hexane, tert-butyl methyl ether (TBME), ethyl acetate, 2-propanol, 1-trimethylsilylimidazole (98% purity) and 1,1,2-trichlorotrifluoroethane were obtained from Sigma Aldrich (Steinheim, Germany), 1,4-dioxane (not stabilized) from Carl Roth (Karlsruhe, Germany), absolute ethanol (>99% purity) from
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be due to the post-transcriptional inhibition of β-hydroxy-βmethylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), a rate-limiting enzyme involved in cholesterol synthesis.11 More than 30 years ago, Qureshi et al.9 reported that substances in barley lowered hepatic cholesterol synthesis in chickens. In 1986, extraction of barley flour yielded an oily fraction with a cholesterol biosynthesis-inhibiting compound, identified as αT3.10 The hypocholesterolaemic effect of T3 in oil made from BSG was first observed by Weber et al.12 Human subjects with average serum total cholesterol of 266 mg dL−1 were given daily doses of 3 g of oil from BSG (T3 content not stated). Total cholesterol and low-density lipoprotein (LDL) cholesterol were decreased by 34–40 and 32–37 mg dL−1 respectively. High-density lipoprotein (HDL) cholesterol was not affected. In order to maximize the health-promoting effects of T3, efforts have been made to optimize its bioavailability. Ingestion of T3 in emulsified formulations such as self-emulsifying systems or nanoemulsions (droplet sizes 20–500 nm) is reported to enhance the oral bioavailability. Yap and Yuen13 increased the bioavailability of T3 from Malaysian palm oil (148.7 mg of Tocomin 50% in a single dose) in self-emulsifying formulations with droplet sizes of 1–10 µm by two to three times that of a non-self-emulsifying oily solution given to fasting subjects. Recently, we conducted a study on humans to test the bioavailability of three different preparations: T3 from palm oil, T3 from a palm oil nanoemulsion (droplet size 40 nm) and T3 isolated from barley (DBSG); each preparation contained 450 mg of total T3. According to preliminary results, the nanoemulsified formula led to an increase in the AUC0–24 h (area under plasma concentration–time curve) value for total T3 in blood plasma (49.6 µg h mL−1 ) compared with the palm oil extract (35.1 µg h mL−1 ). The oral bioavailability of total T3 from barley was highest, even though the preparation was not emulsified; the AUC0–24 h was 64.6 µg h mL−1 , and α-T3 levels in blood plasma were particularly high.14 However, the oral bioavailability of barley T3 in emulsified formulations has not been investigated. Thus the first aim of this study was to determine the oral bioavailability of dietary T3 from non-emulsified barley oil, in nanoemulsified formulations (NE) and in self-emulsifying systems (SES) and to compare the findings with those for T3 from palm oil. Laying hens were chosen for this study because hen’s eggs can be collected non-invasively and
www.soci.org Fisher Chemical (Schwerte, Germany) and tetrahydrofuran from Merck (Darmstadt, Germany). The internal standard 2,2,5,7,8pentamethyl-6-chromanol (PMC), cholestanol, pyrogallol (>98% purity) and the emulsifying agents Tween 80 and Span 80 were purchased from Sigma Aldrich, 5α-cholestan-3β-ol from Merck, Labrasol from Gattefosse (Gennevilliers, France), sodium chloride from Carl Roth and potassium hydrate from Riedel-de ¨ (Seelze, Germany). The 960 kg L−1 ethanol used for extraction Haen was denatured with methyl ethyl ketone and came from Grussing ¨ GmbH Analytika (Filsum, Germany). Sample materials Barley oil Barley oil was extracted on laboratory scale from milled DBSG sieving fractions β-T3 > γ -T3 > δ-T3. Supplemental T3 (sum of vitamers) from palm oil caused a significantly higher (P ≤ 0.05) α-T3 transfer efficiency than did barley oil supplements. This suggests that, on the basis of the palm oil T3 vitamer pattern, α-T3 is preferentially transferred from feed to egg. The α-T3 transfer efficiency of T3 from barley oil supplements tended to be less effective than from palm oil preparations, but the higher α-T3 contents in barley resulted in significantly higher contents in eggs (Table 4). High amounts of α-T3 in supplements seemed to affect the γ -T3 transfer. The high α-T3 contents in the T3 from barley oil supplements resulted in a highly significantly (P ≤ 0.01) lower γ -T3 transfer efficiency than with palm oil preparations. These results indicate clearly that T3 vitamers influence each other in their effect on the transfer efficiency rate. In terms of the transfer efficiency of total T3, barley oil supplementation tended to result in higher T3 transfer efficiency values (2.85 ± 0.08 to 4.10 ± 0.61%) than did palm oil preparations (2.53 ± 0.30 to 2.75 ± 0.05%). These results suggest that the transfer efficiency of T3 from barley oil is superior to that from the palm oil preparations owing to the higher proportions of α-T3 in the barley oil preparations. Barley oil in emulsified formulations tended to improve the transfer efficiency of T3 and T vitamers, as reflected in higher tocochromanol contents in eggs. No differences were observed in the transfer efficiency of T3 from emulsified palm oil supplements. Among the T vitamers, α-T had the highest transfer efficiency; it was the major tocochromanol vitamer found in egg yolk, followed by γ -T, β-T and δ-T. The α-T transfer efficiency tended to be lower in eggs of hens fed barley oil supplements. This may be due to competition of the higher α-T3 contents with α-T for the α-T transfer protein (α-TTP). Tocopherols are selected preferentially by α-TTP located in the liver. Hosomi et al.21 reported that the relative affinities calculated from the degree of competition were 100% for α-T, 38% for β-T, 9% for γ -T, 2% for δ-T and 12% for α-T3, indicating that the methyl group at position 5 (R1 , Fig. 1) on the chromanol ring is important for recognition by α-TTP. Walker et al.16 evaluated egg transfer of T3 and T vitamers by adding different concentrations of Tocomin 50% (produced from palm oil) to the feed. Although they observed the same
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order of biodiscrimination between T3 vitamers as in our study, the transfer efficiency determined for α-T3 was only about 2%. Those investigators reported the highest transfer efficiency for α-T, ranging from 3.4 to 9.9%. In our study, the values for α-T were about 37–54% for barley oil supplements and as much as 62–69% for palm oil supplements. These values are similar to those of Galobart et al.,22 who reported a transfer efficiency of 41.8% for αT on feeding a diet containing an α-T concentration of 50 mg kg−1 , twice as much as our feed enrichment level. With supplementation of higher Tocomin 50% concentrations, Walker et al.16 observed a decrease in transfer efficiencies for T3 and T, assuming a feedback or competitive inhibition for tocochromanols. In our study, we determined much higher transfer efficiencies for T3 than did Walker et al.,16 probably because our daily T3 feed enrichment level was only half of the T3 concentrations in their lowest Tocomin 50% addition. Apart from that, those authors provide no information on the T3 and T contents in the premix and natural ingredients of the base diet, so that no reliable comparison with our results is possible. Despite their low affinity for α-TTP, T3 may be transferred into plasma in considerable amounts through lipoprotein exchanges.23 During chylomicron catabolism, T3 and T vitamers were transferred from chylomicron remnants to circulating HDL and further passed to other lipoproteins such as LDL and very low-density lipoproteins (VLDL). Fairus et al.24 proved the presence of α-, γ - and δT3 in circulating lipoprotein fractions in human plasma after supplementation with a palm oil tocotrienol-rich fraction (TRF). T3 concentrations were highest in HDL, intermediate in triglyceriderich particles and lowest in LDL. The α-T3 was the most abundant T3 vitamer circulated in plasma and lipoproteins. In addition, enterocytes secrete T3 in both chylomicrons with larger capacity and in HDL.25 After secretion, T3 in chylomicrons and HDL are transported from mesenteric lymph to the blood circulatory system. Small HDL can deliver T3 upon maturation to peripheral tissues independently of α-TTP action.26 In our study, we found that the transfer efficiency of α-T3 was four times higher than that of β-T3 and as much as 11 times higher than that of γ -T3. Preferential absorption of α-T3 is in agreement
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Dietary tocotrienols from barley and palm oils in hen’s egg yolk
200 Egg cholesterol (mg/egg)
*
*
Baseline
190
Day 28
* 180 * 170
160
150 Barley OIL Control
Barley OIL NE
Barley OIL SES
Palm OIL Control
Palm OIL NE
Palm OIL SES
Figure 2. Cholesterol levels in hen’s egg yolk. *Significant difference from baseline to end of trial: P ≤ 0.05.
with other reports. In a human study, Yap et al.27 reported that a γ T3 content twice as high as α-T3 resulted in similar concentrations of α- and γ -T3 in plasma. In 2010, Abuasal et al.28 discovered a saturable carrier-mediated process for γ -T3 present in the intestinal lumen. As the exposure to increasingly higher doses of γ -T3 in the lumen would likely reduce the percentage of γ -T3 transported into the enterocytes, it is difficult to obtain elevated levels of γ -T3 in blood after oral administration. This might explain the low γ -T3 concentrations in egg yolk following barley oil and γ -T3-rich palm oil supplementation in our study. In conclusion, we found with supplementation of similar total T3 amounts that the bioavailability of T3 from barley was superior to that of T3 from palm oil preparations owing to barley’s particularly high proportions of α-T3, which has a particularly high transfer efficiency rate.
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were not as great as determined in vitro. However, supplemental TRF (total T3 in feed, 500 mg kg−1 ) from palm oil (α-T, 16.8%; α-T3, 26.5%; γ -T3, 42.0%; δ-T3, 14.1%) decreased serum total cholesterol only by about 17%, although the percentages of γ -T3 and δ-T3 were higher. This can probably be attributed to the differences in oral bioavailability of single T3 vitamers, with α-T3 being absorbed preferentially. Among these positive results, supplementation of up to 200 mg kg−1 α-T3 in feed failed to alter egg cholesterol contents of laying hens.31 In our study, supplementation of barley oil and palm oil successfully decreased total cholesterol in egg yolk by up to 4 and 6% respectively. Although the percentage reduction reported by Yu et al.30 was higher, the amounts of T3 in our supplements (∼3 mg day−1 ) were considerably lower. In our study, T3 from palm oil supplements showed a slightly greater cholesterol-lowering potency than barley oil, but only to a small degree. This could be due the higher γ -T3 contents of the hen feed.
CONCLUSION T3 from barley oil supplements led to significantly higher T3 levels in egg yolk than did T3 from palm oil preparations owing to the predominantly high α-T3 contents of the former. Addition of barley oil as well as palm oil to laying hen feed decreased egg yolk cholesterol contents. These results may help to establish T3 from barley as a dietary supplement for the prevention of health risks such as hypercholesterolaemia or as a pharmaceutical effective against lifestyle diseases. T3-rich formulations from barley would provide a profitable alternative to the palm oil preparations that now dominate the market. In addition, the transfer efficiency of T3 from barley into egg yolk has now been described for the first time, and these results may also be valuable in developing hen’s eggs that are nutritionally enriched in specific, health-promoting T3 vitamers.
ACKNOWLEDGEMENTS This work is part of the Food Network project funded by the Ministry for Science and Culture of Lower Saxony (Germany) via the Research Association of Agricultural and Nutritional Science of Lower Saxony (Forschungsverbund Agrar- und Ern¨ahrungswissenschaften Niedersachsen (FAEN)). The authors wish to gratefully acknowledge Leiber GmbH (Bramsche, Germany), the German Institute of Food Technologie (Quakenbruck, Germany), Bruno Fehse u. Sohn GmbH & Co. KG ¨ (Estorf-Leeseringen, Germany) and Davos Life Science (Singapore) for their support.
REFERENCES 1 Moreau RA, Wayns KE, Flores RA and Hicks KB, Tocopherols and tocotrienols in barley oil prepared from germ and other fractions from scarification and sieving of hulless barley. Cereal Chem 84:587–592 (2007). 2 Andersson AAM, Lampi AM, Nystrom L, Piironen V, Li L, Ward JL, et al., Phytochemical and dietary fiber components in barley varieties in the HEALTHGRAIN diversity screen. J Agric Food Chem 56:9767–9776 (2008). 3 Yap SP, Yuen KH and Lim AB, Influence of route of administration on the absorption and disposition of α-, γ - and δ-tocotrienols in rats. J Pharm Pharmacol 55:53–58 (2003). 4 McLaughlin PJ and Weihrauch JL, Vitamin E content of foods. J Am Diet Assoc 75:647–665 (1979). 5 Lam MK, Tan KT, Lee KT and Mohamed AR, Malaysian palm oil: surviving the food versus fuel dispute for a sustainable future. Renew Sustain Energ Rev 13:1456–1464 (2009).
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Diet effects on cholesterol contents in hen’s eggs Figure 2 shows the cholesterol contents at baseline and at the end of the treatment for each group. Barley OIL Control, Palm OIL Control and Palm OIL SES caused a significant (P < 0.05) drop in yolk cholesterol in comparison with the baseline values of each: 172.23 ± 7.87, 188.89 ± 8.77 and 188.33 ± 14.50 mg per egg respectively. At the end of the experiment, cholesterol levels in yolks were 165.06 ± 5.70, 183.45 ± 7.00 and 176.68 ± 12.36 mg per egg for the respective groups. Moreover, Barley OIL SES led to a decrease in cholesterol levels, but this was not statistically significant (P > 0.05). Barley oil or palm oil in nanoemulsified formulations (Barley OIL NE or Palm OIL NE) failed to lower cholesterol levels in egg yolk. The supplementation of Palm OIL NE led to a cholesterol level (177.14 ± 3.22 mg per egg) that was significantly higher (P > 0.05) than the baseline value (166.37 ± 4.00 mg per egg). This may be related to the reduced laying hen performance and egg quality parameters during the trial in comparison with the other supplements. Furthermore, Weiss et al.29 observed that the dietary inclusion of surface-active agents such as Tween 80 in hen feed (50 g kg−1 ) led to an increase in cholesterol levels in egg yolk, probably caused by the improved cholesterol absorption in the gastrointestinal tract. In 1992, Pearce et al.11 discovered the cholesterol-lowering potency of both α- and γ -T3 in vitro using cultured cells, but found that γ -T3 was 30 times more HMG-CoA reductase efficient than α-T3. As Yu et al.30 observed invivo in chickens, α-T3 (17%) was indeed less effective than γ -T3 (20%) and δ-T3 (27%) in lowering serum total cholesterol after supplementing feed with isolated T3 vitamers (each 500 mg kg−1 ), but differences between vitamers
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www.soci.org 6 Bohnsack C, Ternes W, Busing A and Drotleff A, Tocotrienol levels in ¨ sieving fraction extracts of brewer’s spent grain. Eur Food Res Technol 232:563–573 (2010). 7 Sen CK, Khanna S and Roy S, Tocotrienols: vitamin E beyond tocopherols. Life Sci 78:2088–2098 (2006). 8 Serbinova E, Kagan V, Han D and Packer L, Free radical recycling and intramembrane mobility in the antioxidant properties of alphatocopherol and alpha-tocotrienol. Free Radic Biol Med 10:263–275 (1991). 9 Qureshi AA, Burger WC, Prentice N, Bird HR and Sunde ML, Suppression of cholesterol and stimulation of fatty acid biosynthesis in chicken livers by dietary cereals supplemented with culture filtrate of Trichoderma viride. J Nutr 110:1014–1022 (1980). 10 Qureshi AA, Burger WC, Peterson DM and Elson CE, The structure of an inhibitor of cholesterol biosynthesis isolated from barley. J Biol Chem 261:544–550 (1986). 11 Pearce BC, Parker RA, Deason ME, Qureshi AA and Wright JJK, Hypocholesterolemic activity of synthetic and natural tocotrienols. J Med Chem 35:3595–3606 (1992). 12 Weber FE, Chaudhary V and Qureshi AA, Suppression of cholesterol biosynthesis in hypercholesterolemic subjects by tocotrienol of barley ingredients made from brewers grain. Cereal Foods World 36: p. 680 (1991). 13 Yap SP and Yuen KH, Influence of lipolysis and droplet size on tocotrienol absorption from self-emulsifying formulations. Int J Pharmaceut 281:67–78 (2004). 14 Ternes W, Drotleff AM, Bohnsack C, Schneider I and Hahn A, Tocotrienol extracts from barley by-products: unique health benefits of the better Vitamin E, in Biomedical Engineering Acta, Vol. 4, ed. by Podbielska H and Trziszka T. Indygo Zahir Media, Wrocław, pp. 43–51 (2011). 15 Sookwong P, Nakagawa K, Nakajima SI, Amano Y, Toyomizu M and Miyazawa T, Tocotrienol content in hen eggs: its fortification by supplementing the feed with rice bran scum oil. Biosci Biotechnol Biochem 72:3044–3047 (2008). 16 Walker LA, Wang T, Xin HW and Dolde D, Supplementation of layinghen feed with palm tocos and algae astaxanthin for egg yolk nutrient enrichment. J Agric Food Chem 60:1989–1999 (2012). 17 Littmann-Nienstedt S, Neuere Beobachtungen zum Cholesterin- und Trockenmassengehalt von Schaleneiern. Lebensmittelchemie 50: p. 64 (1996). 18 Leong TSH, Wooster TJ, Kentish SE and Ashokkumar M, Minimising oil droplet size using ultrasonic emulsification. Ultrason Sonochem 16:721–727 (2009).
CM Walde, AM Drotleff, W Ternes 19 Busing A and Ternes W, Separation of α-tocotrienol oxidation products ¨ and eight tocochromanols by HPLC with DAD and fluorescence detection and identification of unknown peaks by DAD, PBI-EIMS, FTIR, and NMR. Anal Bioanal Chem 401:2843–2854 (2011). 20 Sookwong P, Nakagawa K, Murata K, Kojima Y and Miyazawa T, Quantitation of tocotrienol and tocopherol in various rice brans. J Agric Food Chem 55:461–466 (2007). 21 Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, et al., Affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett 409:105–108 (1997). 22 Galobart J, Barroeta AC, Baucells MD, Cortinas L and Guardiola F, Alpha-tocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with omega3-polyunsaturated fatty acids. Poultry Sci 80:1496–1505 (2001). 23 Kayden HJ and Traber MG, Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res 34:343–358 (1993). 24 Fairus S, Nor RM, Cheng HM and Sundram K, Postprandial metabolic fate of tocotrienol-rich vitamin E differs significantly from that of α-tocopherol. Am J Clin Nutr 84:835–842 (2006). 25 Anwar K, Iqbal J and Hussain MM, Mechanisms involved in vitamin E transport by primary enterocytes and in vivo absorption. J Lipid Res 48:2028–2038 (2007). 26 Gee PT, Unleashing the untold and misunderstood observations on vitamin E. Genes Nutr 6:5–16 (2011). 27 Yap SP, Yuen KH and Wong JW, Pharmacokinetics and bioavailability of α-, γ - and δ-tocotrienols under different food status. J Pharm Pharmacol 53:67–71 (2001). 28 Abuasal B, Sylvester PW and Kaddoumi A, Intestinal absorption of γ -tocotrienol is mediated by Niemann-Pick C1-like 1: in situ rat intestinal perfusion studies. Drug Metab Dispos 38:939–945 (2010). 29 Weiss JF, Johnson RM and Naber EC, Effect of some dietary factors and drugs on cholesterol concentration in egg and plasma of hen. J Nutr 91:119–128 (1967). 30 Yu SG, Thomas AM, Gapor A, Tan B, Qureshi N and Qureshi AA, Dose–response impact of various tocotrienols on serum lipid parameters in 5-week-old female chickens. Lipids 41:453–461 (2006). 31 Beyer RS and Jensen LS, Tissue and egg cholesterol concentrations of laying hens fed high-protein barley flour, α-tocotrienol, and cholesterol. Poultry Sci 72:1339–1348 (1993).
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c 2013 Society of Chemical Industry
J Sci Food Agric 2014; 94: 810–818
Revised: 22 October 2013
Accepted article published: 19 November 2013
Published online in Wiley Online Library: 23 December 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6484
Comparison of dietary tocotrienols from barley and palm oils in hen’s egg yolk: transfer efficiency, influence of emulsification, and effect on egg cholesterol Constanze M Walde, Astrid M Drotleff and Waldemar Ternes∗ Abstract BACKGROUND: The main component in tocotrienols (T3) from barley (Hordeum vulgare L.) is α-T3, the vitamer with the highest bioavailability, while palm oil T3 is particularly rich in γ -T3. Unlike tocopherols, T3 are known for their cholesterogenesisinhibiting, neuroprotective and anticarcinogenic properties. In this study the oral bioavailabilities of T3 from barley oil (3.98 mg day−1 ) and T3 from palm oil (3.36 mg day−1 ) in nanoemulsified formulations (NE) and self-emulsifying systems (SES) were compared using hen’s eggs as a bioindicator. In addition, the transfer efficiencies of barley oil T3 and palm oil T3 into egg yolk were compared, as well as their effects on egg cholesterol levels. RESULTS: Nanoemulsification led to T3 levels (132.9 µg per egg) higher than with non-emulsified barley oil (112.8 µg per egg) and barley oil SES (116.7 µg per egg) owing to the high proportions of α-T3 (99–117 µg per egg), which has a particularly high transfer efficiency (4.32–6.75%). T3 contents of eggs from hens fed barley oil supplements (112–132 µg per egg) were significantly higher than those of eggs from hens fed palm oil supplements (70–78 µg per egg). Addition of barley and palm oils to laying hen feed decreased egg yolk cholesterol by 4 and 6% respectively. CONCLUSION: Results from this animal study may help to establish T3 from barley as a dietary supplement and to develop nutritionally improved hen’s eggs. c 2013 Society of Chemical Industry Keywords: barley; tocotrienol; tocopherol; oral absorption; hen’s egg yolk
INTRODUCTION
810
Barley (Hordeum vulgare L.) is the richest cereal source of the vitamin E subclass tocotrienols (T3), and oil from barley is thought to have the highest T3 content of any natural oil.1 Moreover, barley is distinguished by being a natural source of all eight tocochromanol vitamers (Fig. 1), with T3 comprising about 76% of the total tocochromanols, including 47% α-T3, the most biologically available vitamer.2 As determined in rats, the oral bioavailability of α-T3 is highest (27.7 ± 9.2%), followed by γ -T3 (9.1 ± 2.4%) and δ-T3 (8.5 ± 3.5%).3 This makes T3 from barley oil superior to T3 from palm oil, the most prevalent commercial source of T3. Crude palm oil is particularly rich in γ -T3 (39% of the average total tocochromanol content of 587 mg kg−1 ).4 World palm oil production increased enormously during recent years, reaching 38.5 million tons in 2007.5 However, oil palm (Elaeis guineensis) cultivation in tropical regions of Asia, Africa and Latin America is subject to negative criticism owing to its environmental impact. Brewer’s spent grain (BSG) is a barley by-product that is generated in large amounts in beer production. In 2008, European breweries produced a total of 427 million hectolitres of beer and an estimated more than 8 million tons of residual wet BSG.6 Previously, we discovered that the sieving fraction 97% purity) were provided by Davos Life Science (Singapore). All solvents were of high-performance liquid chromatography (HPLC) grade. n-Hexane, tert-butyl methyl ether (TBME), ethyl acetate, 2-propanol, 1-trimethylsilylimidazole (98% purity) and 1,1,2-trichlorotrifluoroethane were obtained from Sigma Aldrich (Steinheim, Germany), 1,4-dioxane (not stabilized) from Carl Roth (Karlsruhe, Germany), absolute ethanol (>99% purity) from
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be due to the post-transcriptional inhibition of β-hydroxy-βmethylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), a rate-limiting enzyme involved in cholesterol synthesis.11 More than 30 years ago, Qureshi et al.9 reported that substances in barley lowered hepatic cholesterol synthesis in chickens. In 1986, extraction of barley flour yielded an oily fraction with a cholesterol biosynthesis-inhibiting compound, identified as αT3.10 The hypocholesterolaemic effect of T3 in oil made from BSG was first observed by Weber et al.12 Human subjects with average serum total cholesterol of 266 mg dL−1 were given daily doses of 3 g of oil from BSG (T3 content not stated). Total cholesterol and low-density lipoprotein (LDL) cholesterol were decreased by 34–40 and 32–37 mg dL−1 respectively. High-density lipoprotein (HDL) cholesterol was not affected. In order to maximize the health-promoting effects of T3, efforts have been made to optimize its bioavailability. Ingestion of T3 in emulsified formulations such as self-emulsifying systems or nanoemulsions (droplet sizes 20–500 nm) is reported to enhance the oral bioavailability. Yap and Yuen13 increased the bioavailability of T3 from Malaysian palm oil (148.7 mg of Tocomin 50% in a single dose) in self-emulsifying formulations with droplet sizes of 1–10 µm by two to three times that of a non-self-emulsifying oily solution given to fasting subjects. Recently, we conducted a study on humans to test the bioavailability of three different preparations: T3 from palm oil, T3 from a palm oil nanoemulsion (droplet size 40 nm) and T3 isolated from barley (DBSG); each preparation contained 450 mg of total T3. According to preliminary results, the nanoemulsified formula led to an increase in the AUC0–24 h (area under plasma concentration–time curve) value for total T3 in blood plasma (49.6 µg h mL−1 ) compared with the palm oil extract (35.1 µg h mL−1 ). The oral bioavailability of total T3 from barley was highest, even though the preparation was not emulsified; the AUC0–24 h was 64.6 µg h mL−1 , and α-T3 levels in blood plasma were particularly high.14 However, the oral bioavailability of barley T3 in emulsified formulations has not been investigated. Thus the first aim of this study was to determine the oral bioavailability of dietary T3 from non-emulsified barley oil, in nanoemulsified formulations (NE) and in self-emulsifying systems (SES) and to compare the findings with those for T3 from palm oil. Laying hens were chosen for this study because hen’s eggs can be collected non-invasively and
www.soci.org Fisher Chemical (Schwerte, Germany) and tetrahydrofuran from Merck (Darmstadt, Germany). The internal standard 2,2,5,7,8pentamethyl-6-chromanol (PMC), cholestanol, pyrogallol (>98% purity) and the emulsifying agents Tween 80 and Span 80 were purchased from Sigma Aldrich, 5α-cholestan-3β-ol from Merck, Labrasol from Gattefosse (Gennevilliers, France), sodium chloride from Carl Roth and potassium hydrate from Riedel-de ¨ (Seelze, Germany). The 960 kg L−1 ethanol used for extraction Haen was denatured with methyl ethyl ketone and came from Grussing ¨ GmbH Analytika (Filsum, Germany). Sample materials Barley oil Barley oil was extracted on laboratory scale from milled DBSG sieving fractions β-T3 > γ -T3 > δ-T3. Supplemental T3 (sum of vitamers) from palm oil caused a significantly higher (P ≤ 0.05) α-T3 transfer efficiency than did barley oil supplements. This suggests that, on the basis of the palm oil T3 vitamer pattern, α-T3 is preferentially transferred from feed to egg. The α-T3 transfer efficiency of T3 from barley oil supplements tended to be less effective than from palm oil preparations, but the higher α-T3 contents in barley resulted in significantly higher contents in eggs (Table 4). High amounts of α-T3 in supplements seemed to affect the γ -T3 transfer. The high α-T3 contents in the T3 from barley oil supplements resulted in a highly significantly (P ≤ 0.01) lower γ -T3 transfer efficiency than with palm oil preparations. These results indicate clearly that T3 vitamers influence each other in their effect on the transfer efficiency rate. In terms of the transfer efficiency of total T3, barley oil supplementation tended to result in higher T3 transfer efficiency values (2.85 ± 0.08 to 4.10 ± 0.61%) than did palm oil preparations (2.53 ± 0.30 to 2.75 ± 0.05%). These results suggest that the transfer efficiency of T3 from barley oil is superior to that from the palm oil preparations owing to the higher proportions of α-T3 in the barley oil preparations. Barley oil in emulsified formulations tended to improve the transfer efficiency of T3 and T vitamers, as reflected in higher tocochromanol contents in eggs. No differences were observed in the transfer efficiency of T3 from emulsified palm oil supplements. Among the T vitamers, α-T had the highest transfer efficiency; it was the major tocochromanol vitamer found in egg yolk, followed by γ -T, β-T and δ-T. The α-T transfer efficiency tended to be lower in eggs of hens fed barley oil supplements. This may be due to competition of the higher α-T3 contents with α-T for the α-T transfer protein (α-TTP). Tocopherols are selected preferentially by α-TTP located in the liver. Hosomi et al.21 reported that the relative affinities calculated from the degree of competition were 100% for α-T, 38% for β-T, 9% for γ -T, 2% for δ-T and 12% for α-T3, indicating that the methyl group at position 5 (R1 , Fig. 1) on the chromanol ring is important for recognition by α-TTP. Walker et al.16 evaluated egg transfer of T3 and T vitamers by adding different concentrations of Tocomin 50% (produced from palm oil) to the feed. Although they observed the same
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order of biodiscrimination between T3 vitamers as in our study, the transfer efficiency determined for α-T3 was only about 2%. Those investigators reported the highest transfer efficiency for α-T, ranging from 3.4 to 9.9%. In our study, the values for α-T were about 37–54% for barley oil supplements and as much as 62–69% for palm oil supplements. These values are similar to those of Galobart et al.,22 who reported a transfer efficiency of 41.8% for αT on feeding a diet containing an α-T concentration of 50 mg kg−1 , twice as much as our feed enrichment level. With supplementation of higher Tocomin 50% concentrations, Walker et al.16 observed a decrease in transfer efficiencies for T3 and T, assuming a feedback or competitive inhibition for tocochromanols. In our study, we determined much higher transfer efficiencies for T3 than did Walker et al.,16 probably because our daily T3 feed enrichment level was only half of the T3 concentrations in their lowest Tocomin 50% addition. Apart from that, those authors provide no information on the T3 and T contents in the premix and natural ingredients of the base diet, so that no reliable comparison with our results is possible. Despite their low affinity for α-TTP, T3 may be transferred into plasma in considerable amounts through lipoprotein exchanges.23 During chylomicron catabolism, T3 and T vitamers were transferred from chylomicron remnants to circulating HDL and further passed to other lipoproteins such as LDL and very low-density lipoproteins (VLDL). Fairus et al.24 proved the presence of α-, γ - and δT3 in circulating lipoprotein fractions in human plasma after supplementation with a palm oil tocotrienol-rich fraction (TRF). T3 concentrations were highest in HDL, intermediate in triglyceriderich particles and lowest in LDL. The α-T3 was the most abundant T3 vitamer circulated in plasma and lipoproteins. In addition, enterocytes secrete T3 in both chylomicrons with larger capacity and in HDL.25 After secretion, T3 in chylomicrons and HDL are transported from mesenteric lymph to the blood circulatory system. Small HDL can deliver T3 upon maturation to peripheral tissues independently of α-TTP action.26 In our study, we found that the transfer efficiency of α-T3 was four times higher than that of β-T3 and as much as 11 times higher than that of γ -T3. Preferential absorption of α-T3 is in agreement
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Dietary tocotrienols from barley and palm oils in hen’s egg yolk
200 Egg cholesterol (mg/egg)
*
*
Baseline
190
Day 28
* 180 * 170
160
150 Barley OIL Control
Barley OIL NE
Barley OIL SES
Palm OIL Control
Palm OIL NE
Palm OIL SES
Figure 2. Cholesterol levels in hen’s egg yolk. *Significant difference from baseline to end of trial: P ≤ 0.05.
with other reports. In a human study, Yap et al.27 reported that a γ T3 content twice as high as α-T3 resulted in similar concentrations of α- and γ -T3 in plasma. In 2010, Abuasal et al.28 discovered a saturable carrier-mediated process for γ -T3 present in the intestinal lumen. As the exposure to increasingly higher doses of γ -T3 in the lumen would likely reduce the percentage of γ -T3 transported into the enterocytes, it is difficult to obtain elevated levels of γ -T3 in blood after oral administration. This might explain the low γ -T3 concentrations in egg yolk following barley oil and γ -T3-rich palm oil supplementation in our study. In conclusion, we found with supplementation of similar total T3 amounts that the bioavailability of T3 from barley was superior to that of T3 from palm oil preparations owing to barley’s particularly high proportions of α-T3, which has a particularly high transfer efficiency rate.
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were not as great as determined in vitro. However, supplemental TRF (total T3 in feed, 500 mg kg−1 ) from palm oil (α-T, 16.8%; α-T3, 26.5%; γ -T3, 42.0%; δ-T3, 14.1%) decreased serum total cholesterol only by about 17%, although the percentages of γ -T3 and δ-T3 were higher. This can probably be attributed to the differences in oral bioavailability of single T3 vitamers, with α-T3 being absorbed preferentially. Among these positive results, supplementation of up to 200 mg kg−1 α-T3 in feed failed to alter egg cholesterol contents of laying hens.31 In our study, supplementation of barley oil and palm oil successfully decreased total cholesterol in egg yolk by up to 4 and 6% respectively. Although the percentage reduction reported by Yu et al.30 was higher, the amounts of T3 in our supplements (∼3 mg day−1 ) were considerably lower. In our study, T3 from palm oil supplements showed a slightly greater cholesterol-lowering potency than barley oil, but only to a small degree. This could be due the higher γ -T3 contents of the hen feed.
CONCLUSION T3 from barley oil supplements led to significantly higher T3 levels in egg yolk than did T3 from palm oil preparations owing to the predominantly high α-T3 contents of the former. Addition of barley oil as well as palm oil to laying hen feed decreased egg yolk cholesterol contents. These results may help to establish T3 from barley as a dietary supplement for the prevention of health risks such as hypercholesterolaemia or as a pharmaceutical effective against lifestyle diseases. T3-rich formulations from barley would provide a profitable alternative to the palm oil preparations that now dominate the market. In addition, the transfer efficiency of T3 from barley into egg yolk has now been described for the first time, and these results may also be valuable in developing hen’s eggs that are nutritionally enriched in specific, health-promoting T3 vitamers.
ACKNOWLEDGEMENTS This work is part of the Food Network project funded by the Ministry for Science and Culture of Lower Saxony (Germany) via the Research Association of Agricultural and Nutritional Science of Lower Saxony (Forschungsverbund Agrar- und Ern¨ahrungswissenschaften Niedersachsen (FAEN)). The authors wish to gratefully acknowledge Leiber GmbH (Bramsche, Germany), the German Institute of Food Technologie (Quakenbruck, Germany), Bruno Fehse u. Sohn GmbH & Co. KG ¨ (Estorf-Leeseringen, Germany) and Davos Life Science (Singapore) for their support.
REFERENCES 1 Moreau RA, Wayns KE, Flores RA and Hicks KB, Tocopherols and tocotrienols in barley oil prepared from germ and other fractions from scarification and sieving of hulless barley. Cereal Chem 84:587–592 (2007). 2 Andersson AAM, Lampi AM, Nystrom L, Piironen V, Li L, Ward JL, et al., Phytochemical and dietary fiber components in barley varieties in the HEALTHGRAIN diversity screen. J Agric Food Chem 56:9767–9776 (2008). 3 Yap SP, Yuen KH and Lim AB, Influence of route of administration on the absorption and disposition of α-, γ - and δ-tocotrienols in rats. J Pharm Pharmacol 55:53–58 (2003). 4 McLaughlin PJ and Weihrauch JL, Vitamin E content of foods. J Am Diet Assoc 75:647–665 (1979). 5 Lam MK, Tan KT, Lee KT and Mohamed AR, Malaysian palm oil: surviving the food versus fuel dispute for a sustainable future. Renew Sustain Energ Rev 13:1456–1464 (2009).
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Diet effects on cholesterol contents in hen’s eggs Figure 2 shows the cholesterol contents at baseline and at the end of the treatment for each group. Barley OIL Control, Palm OIL Control and Palm OIL SES caused a significant (P < 0.05) drop in yolk cholesterol in comparison with the baseline values of each: 172.23 ± 7.87, 188.89 ± 8.77 and 188.33 ± 14.50 mg per egg respectively. At the end of the experiment, cholesterol levels in yolks were 165.06 ± 5.70, 183.45 ± 7.00 and 176.68 ± 12.36 mg per egg for the respective groups. Moreover, Barley OIL SES led to a decrease in cholesterol levels, but this was not statistically significant (P > 0.05). Barley oil or palm oil in nanoemulsified formulations (Barley OIL NE or Palm OIL NE) failed to lower cholesterol levels in egg yolk. The supplementation of Palm OIL NE led to a cholesterol level (177.14 ± 3.22 mg per egg) that was significantly higher (P > 0.05) than the baseline value (166.37 ± 4.00 mg per egg). This may be related to the reduced laying hen performance and egg quality parameters during the trial in comparison with the other supplements. Furthermore, Weiss et al.29 observed that the dietary inclusion of surface-active agents such as Tween 80 in hen feed (50 g kg−1 ) led to an increase in cholesterol levels in egg yolk, probably caused by the improved cholesterol absorption in the gastrointestinal tract. In 1992, Pearce et al.11 discovered the cholesterol-lowering potency of both α- and γ -T3 in vitro using cultured cells, but found that γ -T3 was 30 times more HMG-CoA reductase efficient than α-T3. As Yu et al.30 observed invivo in chickens, α-T3 (17%) was indeed less effective than γ -T3 (20%) and δ-T3 (27%) in lowering serum total cholesterol after supplementing feed with isolated T3 vitamers (each 500 mg kg−1 ), but differences between vitamers
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www.soci.org 6 Bohnsack C, Ternes W, Busing A and Drotleff A, Tocotrienol levels in ¨ sieving fraction extracts of brewer’s spent grain. Eur Food Res Technol 232:563–573 (2010). 7 Sen CK, Khanna S and Roy S, Tocotrienols: vitamin E beyond tocopherols. Life Sci 78:2088–2098 (2006). 8 Serbinova E, Kagan V, Han D and Packer L, Free radical recycling and intramembrane mobility in the antioxidant properties of alphatocopherol and alpha-tocotrienol. Free Radic Biol Med 10:263–275 (1991). 9 Qureshi AA, Burger WC, Prentice N, Bird HR and Sunde ML, Suppression of cholesterol and stimulation of fatty acid biosynthesis in chicken livers by dietary cereals supplemented with culture filtrate of Trichoderma viride. J Nutr 110:1014–1022 (1980). 10 Qureshi AA, Burger WC, Peterson DM and Elson CE, The structure of an inhibitor of cholesterol biosynthesis isolated from barley. J Biol Chem 261:544–550 (1986). 11 Pearce BC, Parker RA, Deason ME, Qureshi AA and Wright JJK, Hypocholesterolemic activity of synthetic and natural tocotrienols. J Med Chem 35:3595–3606 (1992). 12 Weber FE, Chaudhary V and Qureshi AA, Suppression of cholesterol biosynthesis in hypercholesterolemic subjects by tocotrienol of barley ingredients made from brewers grain. Cereal Foods World 36: p. 680 (1991). 13 Yap SP and Yuen KH, Influence of lipolysis and droplet size on tocotrienol absorption from self-emulsifying formulations. Int J Pharmaceut 281:67–78 (2004). 14 Ternes W, Drotleff AM, Bohnsack C, Schneider I and Hahn A, Tocotrienol extracts from barley by-products: unique health benefits of the better Vitamin E, in Biomedical Engineering Acta, Vol. 4, ed. by Podbielska H and Trziszka T. Indygo Zahir Media, Wrocław, pp. 43–51 (2011). 15 Sookwong P, Nakagawa K, Nakajima SI, Amano Y, Toyomizu M and Miyazawa T, Tocotrienol content in hen eggs: its fortification by supplementing the feed with rice bran scum oil. Biosci Biotechnol Biochem 72:3044–3047 (2008). 16 Walker LA, Wang T, Xin HW and Dolde D, Supplementation of layinghen feed with palm tocos and algae astaxanthin for egg yolk nutrient enrichment. J Agric Food Chem 60:1989–1999 (2012). 17 Littmann-Nienstedt S, Neuere Beobachtungen zum Cholesterin- und Trockenmassengehalt von Schaleneiern. Lebensmittelchemie 50: p. 64 (1996). 18 Leong TSH, Wooster TJ, Kentish SE and Ashokkumar M, Minimising oil droplet size using ultrasonic emulsification. Ultrason Sonochem 16:721–727 (2009).
CM Walde, AM Drotleff, W Ternes 19 Busing A and Ternes W, Separation of α-tocotrienol oxidation products ¨ and eight tocochromanols by HPLC with DAD and fluorescence detection and identification of unknown peaks by DAD, PBI-EIMS, FTIR, and NMR. Anal Bioanal Chem 401:2843–2854 (2011). 20 Sookwong P, Nakagawa K, Murata K, Kojima Y and Miyazawa T, Quantitation of tocotrienol and tocopherol in various rice brans. J Agric Food Chem 55:461–466 (2007). 21 Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, et al., Affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett 409:105–108 (1997). 22 Galobart J, Barroeta AC, Baucells MD, Cortinas L and Guardiola F, Alpha-tocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with omega3-polyunsaturated fatty acids. Poultry Sci 80:1496–1505 (2001). 23 Kayden HJ and Traber MG, Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res 34:343–358 (1993). 24 Fairus S, Nor RM, Cheng HM and Sundram K, Postprandial metabolic fate of tocotrienol-rich vitamin E differs significantly from that of α-tocopherol. Am J Clin Nutr 84:835–842 (2006). 25 Anwar K, Iqbal J and Hussain MM, Mechanisms involved in vitamin E transport by primary enterocytes and in vivo absorption. J Lipid Res 48:2028–2038 (2007). 26 Gee PT, Unleashing the untold and misunderstood observations on vitamin E. Genes Nutr 6:5–16 (2011). 27 Yap SP, Yuen KH and Wong JW, Pharmacokinetics and bioavailability of α-, γ - and δ-tocotrienols under different food status. J Pharm Pharmacol 53:67–71 (2001). 28 Abuasal B, Sylvester PW and Kaddoumi A, Intestinal absorption of γ -tocotrienol is mediated by Niemann-Pick C1-like 1: in situ rat intestinal perfusion studies. Drug Metab Dispos 38:939–945 (2010). 29 Weiss JF, Johnson RM and Naber EC, Effect of some dietary factors and drugs on cholesterol concentration in egg and plasma of hen. J Nutr 91:119–128 (1967). 30 Yu SG, Thomas AM, Gapor A, Tan B, Qureshi N and Qureshi AA, Dose–response impact of various tocotrienols on serum lipid parameters in 5-week-old female chickens. Lipids 41:453–461 (2006). 31 Beyer RS and Jensen LS, Tissue and egg cholesterol concentrations of laying hens fed high-protein barley flour, α-tocotrienol, and cholesterol. Poultry Sci 72:1339–1348 (1993).
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