Effects of broccoli extract and various essential oils on intestinal and faecal microflora and on xenobiotic enzymes and the antioxidant system of piglets

Kristin Mueller, Nicole M. Blum, Holger Kluge, Rolf Bauerfeind, Julia Froehlich, Anneluise Mader, Karola R. Wendler, Andreas S. Mueller

Delacon Biotechnik Ges.m.b.H., Steyregg, Austria.
Institute of Agricultural and Nutritional Sciences, Preventive Nutrition Group, Martin Luther University of Halle-Wittenberg, Halle, Germany.
Institute of Animal Nutrition, Free University of Berlin, Berlin, Germany.
Institute of Veterinary Hygiene and Infectious Diseases of Animals, Justus Liebig University Giessen, Giessen, Germany.

DOI: 10.4236/ojas.2012.22012                

Abstract

Objective: Since the ban of antibiotics as growth promoting feed additives in the EU in 2006 research in alternatives has gained importance. Phytogenic feed additives represent a heterogenous class of different plant derived substances that are discussed to improve the health of farm animals by direct and indirect antioxidant effects and by influencing microbial eubiosis in the gastrointestinal tract. Consequently our study aimed to investigate the influence of broccoli extract and the essential oils of tur- meric, oregano, thyme and rosemary, as selected individual additives, on intestinal and faecal microflora, on xenobiotic enzymes, and on the antioxidant system of piglets. Methods: 48 four weeks old male weaned piglets were assigned to 6 groups of 8. The piglets were housed individually in stainless steel pens with slatted floor. The control group (Con) was fed a diet without an additive for 4 weeks. The diet of group BE contained 0.15 g/kg sulforaphane in form of a broccoli extract. 535, 282, 373 and 476 mg/kg of the essential oils of turmeric (Cuo), oregano (Oo), thyme (To) and rosemary (Ro) were added to the diets of the remaining 4 groups to stan-dardise supplementation to 150 mg/kg of the oils’ key terpene compounds ar-turmerone, carvacrol, thymol and 1,8-cineole. The composition of bacterial microflora was examined by cultivating samples of jejeunal and colonic mucosa and of faeces under specific conditions. The mRNA expression of xenobiotic and antioxidant enzymes was determined by reversing transcrip- tase real time detection PCR (RT-PCR). Total antioxidant status was assayed using the Trolox Equivalent Antioxidant Capacity (TEAC), and lipid peroxidation was determined by measuring thiobarbioturic acid reactive substances (TBA- RS). Results: Compared to Con piglets all additives positively influenced weight gain and feed conversion in week 1. Over the whole trial period no significant differences in performance parameters existed between the experimental groups. Compared to group Con performance of Ro piglets was, however, slightly impaired. Com- pared to Con piglets Cuo, Oo and To increased the ratio of Lactobacilli:E. coli attached to the jejunal mucosa, whereas BE and Ro impaired this ratio slightly. In contrast in colonic mucosa Ro improved Lactobacilli:E. coli ratio. In faecal samples an improvement of Lactobacilli:E. coli ratio could be analysed for To and Ro. Ro was the only additive that reduced the incidence rate of piglets tested positive for enterotoxic E. coli (ETEC). All additives significantly increased jejunal TEAC and reduced TBA-RS. In the liver BE, Cuo, Oo and To increased TEAC in tendency and Ro significantly. Liver TBA-RS were slightly reduced by all additives compared to Con piglets. Whereas the influence of BE, To and Ro on jejunal TEAC mainly was derived from the induction of xenobiotic and antioxidant enzymes (indirect antioxidant effects), Cuo and Oo influenced TEAC by direct antioxidant effects. Discussion and Conclusions: Our results have shown: That within the labiatae oils Oo and To have the potential to improve performance slightly. That phytogenic substances have a small but not sig- nificant influence on intestinal microflora. That phytogenic feed additives up-regulate the anti- oxidant system of piglets either by direct or by indirect antioxidant effects and that they may thereby improve health status. That within the labiatae oils Oo has a high direct antioxidant potential whereas Ro potently induces xenobiotic and antioxidant enzymes. That broccoli extract is an attractive new phytogenic additive, improving antioxidant status by indirect antioxidant effects. That defined combinations of selected phytogenic substances may produce additive effects. That health promoting effects of phytogenic additives in the future should be studied systematically under the challenge with pathogenic microorganisms or food derived to-xins.

Keywords

Pigs; Phytogenic Feed Additives; Broccoli Extract; Essential Oils; Xenobiotic Enzymes; Antioxidant System

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Kyriakis, S.C., Sarris, K., Lekkas, S., Tsinas, C., Gianna- kopoulos, C., Alexopoulos, C. and Saoulidism, K. (1998) Control of post weaning diarroea syndrome of piglets by in-feed application of origanum essential oils. Proceed- ings of the 15th IPVS Congress 1998, Birmingham, Eng- land, 218.
[2]	Tsinas, A.C., Giannakopoulos, C.G., Papasteriades, A., Alexopoulos, C., Mavromatis, J. and Kyriakis, S.C. (1998) Use of Origanum Essential Oils as Growth Promoter in Pigs. Proceeding 15th IPVS Congress 1998, Birmingham, England, 221.
[3]	Zitterl-Eglseer, K., Wetscherek, W., Stoni, A., Kroismayr, A. and Windisch, W. (2008) Bioverfügbarkeit der ?the- rischen ?le eines phytobiotischen Futterzusatzes und der Einfluss auf die Leistung bzw. N?hrstoffverdaulichkeit bei Absetzferkeln. Die Bodenkultur, 59, 121-129.
[4]	Mares, P., Novák, L., Zeman, L., Kratochvílová, P. and Vecerek, M. (2007) The Evaluation of Phytogenic Addi- tives Using in Pig Nutrition. 6th BOKU-Symposium Tierern?hrung, 149-151.
[5]	Oswald, J. and Wetscherek, W. (2007) Wirkung von Oregano auf die Aufzuchtleistung von Ferkeln. 6th BOKU- Symposium Tierern?hrung, 174-181.
[6]	Hashemi, S.R. and Davoodi, H. (2011) Herbal plants and their derivatives as growth and health promoters in animal nutrition. Veterinary Research Communications, 35, 169-180. doi:10.1007/s11259-010-9458-2
[7]	Schirmer, B.C. and Langsrud, S. (2010) Evaluation of natural antimicrobials on typical meat spoilage bacteria in vitro and in vacuum-packed pork meat. Journal of Food Science, 75, 98-102. doi:10.1111/j.1750-3841.2009.01485.x
[8]	Kong, B., Wang, J. and Xiong, Y.L. (2007) Antimicrobial activity of several herb and spice extracts in culture me- dium and in vacuum-packaged pork. Journal of Food Protection, 70, 641-647.
[9]	Busatta, C., Vidal, R.S., Popiolski, A.S., Mossi, A.J., Da- riva, C., Rodrigues, M.R., Corazza, F.C., Corazza, M.L., Vladimir Oliveira, J. and Cansian, R.L. (2008) Applica- tion of Origanum majorana L. essential oil as an antim- icrobial agent in sausage. Food Microbiology, 25, 207- 211.doi:10.1016/j.fm.2007.07.003
[10]	Maenner, K., Vahjen, W. and Simon, O. (2011) Studies on the effects of essential-oil-based feed additives on per- formance, ileal nutrient digestibility, and selected bacte- rial groups in the gastrointestinal tract of piglets. Journal of Animal Science, 89, 2106-2112. doi:10.2527/jas.2010-2950
[11]	Castillo, M., Martín-Orúe, S.M., Roca, M., Manzanilla, E.G., Badiola, I., Perez, J.F. and Gasa, J. (2006) The re- sponse of gastrointestinal microbiota to avilamycin, bu- tyrate, and plant extracts in early-weaned pigs. Journal of Animal Science, 84, 2725-2734. doi:10.2527/jas.2004-556
[12]	Hagemüller, W., Jugl-Chizzola, M., Zitterl-Eglseer, K., Gab- ler, C., Spergser, J., Chizzola, R. and Franz, C. (2006) The use of Thymi Herba as feed additive (0.1%, 0.5%, 1.0%) in weanling piglets with assessment of the shed- ding of haemolysing E.coli and the detection of thymol in the blood plasma. Berliner und Munchener Tierarztliche Wochenschrift, 119, 50-54.
[13]	Shan, B., Cai, Y.Z., Sun, M. and Corke, H. (2005) Anti- oxidant capacity of 26 spice extracts and characterization of their phenolic constituents. Journal of Agricultural and Food Chemistry, 53, 7749-7759. doi:10.1021/jf051513y
[14]	Negi, P.S., Jayaprakasha, G.K., Rao, J.M. and Sakariah, K.K. (1999) Antibacterial activity of tumeric oil: A by- product from curcumin manufacture. Journal of Agricul- tural and Food Chemistry, 47, 4297-4300. doi:10.1021/jf990308d
[15]	Anonymous (2012) Evonik Industries product informa- tion (no authors listed). TEGO? Turmerone: the distilled fraction of tumeric oil extracted from the roots of Cur- cuma longa by supercritical carbon dioxide. http://www.cenerchem.com/PDFs/TEGO%20Turmerone%20Tech%20Lit%200208.pdf
[16]	Mueller, K., Blum, N.M., Kluge, H. and Mueller, A.S. (2011) Influence of broccoli extract and various essential oils on performance and expression of xenobiotic- and antioxidant enzymes in broiler chickens. British Journal of Nutrition, 1-15. [Epub ahead of print] doi:10.1017/S0007114511005873
[17]	Clarke, J.D., Dashwood, R.H. and Ho, E. (2008) Multi- targeted prevention of cancer by sulforaphane. Cancer Letters, 269, 291-304. doi:10.1016/j.canlet.2008.04.018
[18]	Aires, A., Mota, V.R., Saavedra, M.J., Rosa, E.A.S. and Bennett, R.N. (2009) The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from human intestinal tract. Journal of Applied Microbiology, 106, 2086-2095.
[19]	Iqbal, M., Sharma, S.D., Okazaki, Y., Fujisawa, M. and Okada, S. (2003) Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: Possible role in protection against chemical carcinogenesis and toxicity. Pharmacology & Toxicology, 92, 33-38. doi:10.1034/j.1600-0773.2003.920106.x
[20]	Petri, N., Tannergren, C., Holst, B., Mellon, F.A., Bao, Y., Plumb, G.W., Bacon, J., O’Leary, K.A., Kroon, P.A., Knutson, L., Forsell, P., Eriksson, T., Lennernas, H. and Williamson, G. (2003) Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo. Drug Metabolism and Disposition, 31, 805-813. doi:10.1034/j.1600-0773.2003.920106.x
[21]	Miyakoshi, M., Yamaguchi, Y., Takagaki, R., Mizutani, K., Kambara, T., Ikeda, T., Zaman, M.S., Kakihara, H., Takenaka, A. and Igarashi, K. (2004) Hepatoprotective effect of sesquiterpenes in turmeric. Biofactors, 21, 167- 170. doi:10.1002/biof.552210134
[22]	Yoxall, V., Kentish, P., Coldham, N., Kuhnert, N., Sauer, M.J. and Ioannides, C. (2005) Modulation of hepatic cy- tochromes P450 and phase II enzymes by dietary doses of sulforaphane in rats: Implications for ist chemopreventive activity. International Journal of Cancer, 117, 356-362. doi:10.1002/ijc.21191
[23]	Hu, R., Xu, C., Shen, G., Jain, M.R., Khor, T.O., Gopal- krishnan, A., Lin, W., Reddy, B., Chan, J.Y. and Kong, A.N.T. (2006) Gene expression profiles induced by can- cer chemopreventive isothiocyanate sulforaphane in liver of C57BL/6J mice and C57BL/6J/Nrf2 (-/-) mice. Cancer Letters, 243, 170-192. doi:10.1016/j.canlet.2005.11.050
[24]	Gowda, N.K.S, Ledoux, D.R., Rottinghaus, G.E., Ber- mudez, A.J. and Chen, Y.C. (2008) Efficacy of tumeric (Curcuma longa), containing a known level of curcumin, and a hydrated sodium calcium aluminosilicate to ame- liorate the adverse effects of aflatoxin in broiler chicks. Poultry Science, 87, 1125-1130. doi:10.3382/ps.2007-00313
[25]	Hong, F., Freeman, M.L. and Liebler, D.C. (2005) Identi- fication of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane. Chemi- cal Research in Toxicology, 18, 1917-1926. doi:10.1021/tx0502138
[26]	Abdull-Razis, A.F., Baratta, M., De Nicola, G.R., Iori, R. and Ioannides, C. (2010) Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation active- ties and may contribute directly to the chemopreventive activity of cruciferous vegetables. Toxicology, 277, 74-85. doi:10.1016/j.tox.2010.08.080
[27]	Abdull-Razis, A.F., Bagatta, M., De Nicola, G.R., Iori, R. and Ioannides, C. (2011) Up-regulation of cytochrome P450 and phase II enzyme systems in rat precision-cut rat lung slices by the intact glucosinolates, glucoraphanin and glucoerucin. Lung Cancer, 71, 298-305. doi:10.1016/j.lungcan.2010.06.015
[28]	Gao, S.S., Chen, X.Y., Zhu, R.Z., Choi, B.M. and Kim, B.R. (2010) Sulforaphane induces glutathione S-trans- ferase isozymes which detoxify aflatoxin B(1)-8,9-epox- ide in AML 12 cells. BioFactors, 36, 289-296. doi:10.1002/biof.98
[29]	Gesellschaft für Ern?hrungsphysiologie (2006) Empfeh- lungen zur Energie und N?hrstoffversorgung bei Schwei- nen. DLG-Verlag, ISBN-Nr.: 978-3-7690-0683-4.
[30]	National Research Council (1998) Nutrient Requirements of Swine. 10th Revised Edition, The National Academies Press, Washington, DC.
[31]	Livak, K.J. and Schmittgen, T.D. (2001) Analysis of re- lative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)). Methods, 25, 402-408. doi:10.1006/meth.2001.1262
[32]	Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Hug- gett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. and Wittwer, C.T. (2009) The MIQE guidelines: Minimum information for publi- cation of quantitative real-time PCR experiments. Clini- cal Chemistry, 55, 611-622. doi:10.1373/clinchem.2008.112797
[33]	Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, Research0034.
[34]	Hosseini, A., Sauerwein, H. and Mielenz, M. (2010) Pu- tative reference genes for gene expression studies in propionate and β-hydroxybutyrate treated bovine adipose tissue explants. Journal of Animal Physiology and Animal Nutrition, 94, 178-184. doi:10.1111/j.1439-0396.2010.01002.x
[35]	Vreeburg, R.A., Bastiaan-Net, S. and Mes. J.J. (2011) Normalization genes for quantitative RT-PCR in differen- tiated Caco-2 cells used for food exposure studies. Food & Function, 2, 124-129. doi:10.1039/c0fo00068j
[36]	Miller, N.J., Rice-Evans, C., Davies, M.J., Gopinathan, V. and Milner, A. (1993) A novel method for measuring an- tioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science, 84, 407-412.
[37]	Wang, C.C., Chu, C.Y., Chu, K.O., Choy, K.W., Khaw, K.S., Rogers, M.S. and Pang, C.P. (2004) Trolox-equi- valent antioxidant capacity assay versus oxygen radical absorbance capacity assay in plasma. Clinical Chemistry, 50, 952-954. doi:10.1373/clinchem.2004.031526
[38]	Wong, S.H., Knight, J.A., Hopfer, S.M., Zaharia, O., Leach, C.N. Jr. and Sunderman, F.W. Jr. (1987) Lipoperoxides in plasma as measured by liquid-chromatographic separa- tion of malondialdehyde-thiobarbituric acid adduct. Clin- ical Chemistry, 33, 214-220.
[39]	Bradford, M.M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Bioche- mistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3
[40]	Jugl-Chizzola, M., Ungerhofer, E., Gabler, C., Hagmüller, W., Chizzola, R., Zitterl-Eglseer, K. and Franz, C. (2006) Testing of the palatability of Thymus vulgaris L. and Ori- ganum vulgare L. as flavouring feed additive for weaner pigs on the basis of a choice experiment. Berliner und Munchener Tierarztliche Wochenschrift, 119, 238-243.
[41]	Ilsley, S.E., Miller, H.M. and Kamel, C. (2005) Effects of dietary quillaja saponin and curcumin on the performance and immune status of weaned piglets. Journal of Animal Science, 83, 82-88.
[42]	Wenk, C. (2005) Einsatz von Kr?utern und deren Ex- trakten in der Tierern?hrung: Erwartungen und M?glich- keiten. 4th BOKU-Symposium Tierern?hrung, 17-27.
[43]	Shapiro, T.A., Fahey, J.W., Dinkova-Kostova, A.T., Hol- tzclaw, W.D., Stephenson, K.K., Wade, K.L., Ye, L. and Talalay, P. (2006) Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: A clinical phase I study. Nutrition and Cancer, 55, 53-62. doi:10.1207/s15327914nc5501_7
[44]	McMillan, M., Spinks, E.A. and Fenwick, G.R. (1986) Preliminary observations on the effect of dietary brussels sprouts on thyroid function. Human Toxicology, 5, 15-19. doi:10.1177/096032718600500104
[45]	Papatsiros, V.G., Tzika, E.D., Papaioannou, D.S., Kyriakis, S.C., Tassis, P.D. and Kyriakis, C.S. (2009) Effect of Ori- ganum vulgaris and Allium sativum extracts fort the control of proliferative enteropathy in weaning pigs. Polish Journal of Veterinary Sciences, 12, 407-414.
[46]	Manzanilla, E.G., Perez, J.F., Martin, M., Kamel, C., Bau- cells, F. and Gasa, J. (2004) Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science, 82, 3210-3218.
[47]	McCue, P., Vattem, D. and Shetty, K. (2004) Inhibitory effect of clonal oregano extracts against porcine pancre- atic amylase in vitro. Asia Pacific Journal of Clinical Nu- trition, 13, 401-408.
[48]	Prathapkumar, S.H., Rao, V.S., Paramkishan, R.J. and Bhat, R.V. (1997) Disease outbreak in laying hens arising from the consumption of fumonisin-contaminated food. British Poultry Science, 38, 475-479. doi:10.1080/00071669708418024
[49]	Gowda, N.K.S, Ledoux, D.R., Rottinghaus, G.E., Ber- mudez, A.J. and Chen, Y.C. (2008) Efficacy of tumeric (Curcuma longa), containing a known level of curcumin, and a hydrated sodium calcium aluminosilicate to ame- liorate the adverse effects of aflatoxin in broiler chicks. Poultry Science, 87, 1125-1130. doi:10.3382/ps.2007-00313
[50]	Yarru, L.P., Settivari, R.S., Gowda, N.K.S., Antoniou, E., Ledoux, D.R. and Rottinghaus, G.E. (2009) Effects of tumeric (Curcuma longa) on the expression of hepatic genes associated with biotransformation, antioxidant, and immune systems in broiler chicks fed aflatoxin. Poultry Science, 88, 2620-2627. doi:10.3382/ps.2009-00204
[51]	Giannenas, I., Florou-Paneri, P., Papazahariadou, M., Chri- staki, E., Botsoglou, N.A. and Spais, A.B. (2003) Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archiv fur Tierernahrung, 57, 99-106. doi:10.1080/0003942031000107299
[52]	El-Nekeety, A.A., Mohamed, S.R., Hathout, A.S., Hassan, N.S., Aly, S.E. and Abdel-Wahhab, M.A. (2011) Antioxi- dant properties of Thymus vulgaris oil against aflatoxin- induce oxidative stress in male rats. Toxicon, 57, 984-991. doi:10.1016/j.toxicon.2011.03.021
[53]	Offord, E.A., Macé, K., Avanti, O. and Pfeifer, A.M. (1997) Mechanisms involved in the chemoprotective ef- fects of rosemary extract studied in human liver and bronchial cells. Cancer Letters, 114, 275-281. doi:10.1016/S0304-3835(97)04680-6
[54]	Costa, S., Utan, A., Speroni, E., Cervellati, R., Piva, G., Grandini, A. and Guerra, M.C. (2007) Carnosic acid from rosemary extracts: A potential chemoprotective agent against aflatoxin B1. An in vitro study. Journal of Applied Toxicology, 27, 152-159. doi:10.1002/jat.1186
[55]	Fiala, J.L., Egner, P.A., Wiriyachan, N., Ruchirawat, M., Kensler, K.H., Wogan, G.N., Groopman, J.D., Croy, R.G. and Essigmann J.M. (2011) Sulforaphane-mediated reduc- tion of aflatoxin B1-N7-guanine in rat liver DNA: Im- pacts of strain and sex. Toxico, 121, 57-62. doi:10.1093/toxsci/kfr026
[56]	Blomberg, L., Henriksson, A. and Conway, P.L. (1993) Inhibition of adhesion of Escherichia coli K88 to piglet ileal mucus by Lactobacillus spp. Applied and Environ- mental Microbiology, 59, 34-39.
[57]	Boddupalli, S., Mein, J.R., Lakkanna, S. and James, D.R. (2012) Induction of phase 2 antioxidant enzymes by bro- ccoli sulforaphane: Perspectives in maintaining the anti- oxidant activity of vitamins A, C, and E. Frontiers in Genetics, 3, 7.
[58]	Li, F., Hullar, M.A., Beresford, S.A. and Lampe, J.W. (2011) Variation of glucoraphanin metabolism in vivo and ex vivo by human gut bacteria. British Journal of Nutri- tion, 106, 408-416. doi:10.1017/S0007114511000274
[59]	Campbell, F.C. and Collett, G.P. (2005) Chemopreventive properties of curcumin. Future Oncology, 1, 405-414. doi:10.1517/14796694.1.3.405
[60]	Garg, R., Gupta, S. and Maru, G.B. (2008) Dietary curcu- min modulates transcriptional regulators of phase I and phase II enzymes in benzo[a]pyrene-treated mice: Me- chanism of its anti-initiating action. Carcinogenesis, 29, 1022-1032. doi:10.1093/carcin/bgn064
[61]	Joshi, J., Ghaisas, S., Vaidya, A., Vaidya, R., Kamat, D.V., Bhagwat, A.N. and Bhide, S. (2003) Early human safety study of turmeric oil (Curcuma longa oil) administered orally in healthy volunteers. Journal of the Association of Physicians of India, 51, 1055-1060.
[62]	Miyakoshi, M., Yamaguchi, Y. and Takagaki, R. (2004) Hepatoprotective effect of sesquiterpenes in turmeric. Biofactors, 21, 167-170. doi:10.1002/biof.552210134
[63]	J?ger, S., Scheffler, A. and Schmellenkamp, H. (2011) Pharmakologie ausgew?hlter Terpene. Pharmazeutische Zeitung online. http://www.pharmazeutische-zeitung.de/index.php?id=1354
[64]	Keum, Y.S. (2011) Regulation of the Keap1/Nrf2 system by chemopreventive sulforaphane: Implications of post- translational modifications. Annals of the New York Aca- demy of Sciences, 1229, 184-189. doi:10.1111/j.1749-6632.2011.06092.x
[65]	Meesters, R.J., Duisken, M. and Hollender, J. (2009) Cy- tochrome P450-catalysed arene-epoxidation of the bioac- tive tea tree oil ingredient p-cymene: indication for the formation of a reactive allergenic intermediate? Xenobi- otica, 39, 663-671. doi:10.1080/00498250902989094
[66]	Wang, X., Tomso, D.J., Chorley, B.N., Cho, H.Y., Cheung, V.G., Kleeberger, S.R. and Bell, D.A. (2007) Identifica- tion of polymorphic antioxidant response elements in the human genome. Human Molecular Genetics, 16, 1188- 1200. doi:10.1093/hmg/ddm228
[67]	Zhao, J., Zhang, J.S., Yang, B., Lv. G.P. and Li, S.P. (2010) Free radical scavenging activity and characterization of sesquiterpenoids in four species of Curcuma using a TLC bioautography assay and GC-MS analysis. Molecules, 15, 7547-7557. doi:10.3390/molecules15117547
[68]	Wingler, K., Müller, C. and Brigelius-Flohé, R. (2001) Stability of gastrointestinal glutathione peroxidase mRNA in selenium deficiency depends on its 3’UTR. Biofactors, 14, 43-50. doi:10.1002/biof.5520140107
[69]	Wingler, K., Müller, C., Schmehl, K., Florian, S. and Bri- gelius-Flohé, R. (2000) Gastrointestinal glutathione per- oxidase prevents transport of lipid hydroperoxides in CaCo-2 cells. Gastroenterology, 119, 420-430. doi:10.1053/gast.2000.9521
[70]	Simitzis, P.E., Symeon, G.K., Charismiadou, M.A., Bize- lis, J.A. and Deligeorgis, S.G. (2010) The effects of die- tary oregano oil supplementation on pig meat characteris- tics. Meat Science, 84, 670-676. doi:10.1016/j.meatsci.2009.11.001
[71]	Janz, J.A., Morel, P.C., Wilkinson, B.H. and Purchas, R.W. (2007) Preliminary investigation of the effects of low-level dietary inclusion of fragrant essential oils and oleoresins on pig performance and pork quality. Meat Science, 75, 350-355. doi:10.1016/j.meatsci.2006.06.027
[72]	Lahucky, R., Nuernberg, K., Kovac, L., Bucko, O. and Nuernberg, G. (2010) Assessment of the antioxidant po- tential of selected plant extracts—In vitro and in vivo ex- periments on pork. Meat Science, 85, 779-784. doi:10.1016/j.meatsci.2010.04.004