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Journal of Integrative Agriculture  2013, Vol. 12 Issue (5): 803-814    DOI: 10.1016/S2095-3119(13)60301-9
Physiology & Biochentry · Tillage · Cultivation Advanced Online Publication | Current Issue | Archive | Adv Search |
Detection of Tocopherol in Oilseed Rape (Brassica napus L.) Using Gas Chromatography with Flame Ionization Detector
 Nazim Hussain, Zahra Jabeen, LI Yuan-long, CHEN Ming-xun, LI Zhi-lan, GUO Wan-li, Imran Haider Shamsi, CHEN Xiao-yang , JIANG Li-xi
Key Laboratory of Crop Germplasm Resources of Zhejiang Province, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China
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摘要  The variation among Chinese genotypes of Brassica napus L. for seed tocopherols content and their analysis using gas chromatography has not been comprehensively reported till to date. In the present study, the tocopherol contents of four Chinese genotypes of Brassica napus L., namely, Gaoyou 605, Zhejiang 619, Zheshuang 758, and Zheshuang 72, were evaluated using three modified sample preparation protocols (P1, P2, and P3) for tocopherol extraction. These methods were distinguished as follows. Protocol one (P1) included the evaporation of solvent after extraction without silylation. Protocol two (P2) followed the direct supernatant collection after overnight extraction without drying and silylation. Protocol three (P3) included trimethylsilylation with N,O-bis(trimethylsilyl) trifluoroacetamide. Genotypic comparison of tocopherol and its isoforms revealed that Gaoyou 605 was dominant over the other genotypes with (140.5±10.5), (316.2± 9.2), and (559.1± 24.3) μg g-1 of seed meal α-, γ-, and total (T-) tocopherol, respectively, and a 0.44±0.04 α- to γ-tocopherol ratio. The comparison of the sample preparation protocols, on the other hand, suggests that P3 is the most suitable method for the tocopherol extraction from Brassica oilseeds and for the analysis of tocopherols using gas chromatography flame ionization detector (GC-FID). Trimethylsilylation is the key step differentiating P3 from P1 and P2. Variations detected in tocopherol contents among the Chinese rapeseed (B. napus) genotypes signify the need to quantify a wide range of rapeseed germplasm for seed tocopherol dynamics in short and crop improvement in long.

Abstract  The variation among Chinese genotypes of Brassica napus L. for seed tocopherols content and their analysis using gas chromatography has not been comprehensively reported till to date. In the present study, the tocopherol contents of four Chinese genotypes of Brassica napus L., namely, Gaoyou 605, Zhejiang 619, Zheshuang 758, and Zheshuang 72, were evaluated using three modified sample preparation protocols (P1, P2, and P3) for tocopherol extraction. These methods were distinguished as follows. Protocol one (P1) included the evaporation of solvent after extraction without silylation. Protocol two (P2) followed the direct supernatant collection after overnight extraction without drying and silylation. Protocol three (P3) included trimethylsilylation with N,O-bis(trimethylsilyl) trifluoroacetamide. Genotypic comparison of tocopherol and its isoforms revealed that Gaoyou 605 was dominant over the other genotypes with (140.5±10.5), (316.2± 9.2), and (559.1± 24.3) μg g-1 of seed meal α-, γ-, and total (T-) tocopherol, respectively, and a 0.44±0.04 α- to γ-tocopherol ratio. The comparison of the sample preparation protocols, on the other hand, suggests that P3 is the most suitable method for the tocopherol extraction from Brassica oilseeds and for the analysis of tocopherols using gas chromatography flame ionization detector (GC-FID). Trimethylsilylation is the key step differentiating P3 from P1 and P2. Variations detected in tocopherol contents among the Chinese rapeseed (B. napus) genotypes signify the need to quantify a wide range of rapeseed germplasm for seed tocopherol dynamics in short and crop improvement in long.
Keywords:  Brassica napus L.       genotypic variation       tocopherol analysis       gas chromatography       flame ionization detector  
Received: 11 January 2012   Accepted:
Fund: 

The work was supported by the National Natural Science Foundation of China (30971700 and 31171463) and Natural Science Foundation of Zhejiang Province (Z3100130). We thank Ms. Zhang Dongqing of Zhejiang Academy of Agricultural Sciences and Prof. Zhou Weijun of Zhejiang University, China, for providing us the rapeseed cultivars.

Corresponding Authors:  Correspondence JIANG Li-xi, Tel: +86-571-88982905, Fax: +86-571-88982130, E-mail: jianglx@zju.edu.cn     E-mail:  jianglx@zju.edu.cn

Cite this article: 

Nazim Hussain, Zahra Jabeen, LI Yuan-long, CHEN Ming-xun, LI Zhi-lan, GUO Wan-li, Imran Haider Shamsi, CHEN Xiao-yang , JIANG Li-xi. 2013. Detection of Tocopherol in Oilseed Rape (Brassica napus L.) Using Gas Chromatography with Flame Ionization Detector. Journal of Integrative Agriculture, 12(5): 803-814.

[1]Anonymous. 1982. Nomenclature of tocopherols andrelated compounds - Recommendations 1981. EuropeanJournal of Biochemistry, 123, 473-475

[2]Appelqvist L Å. 1972. Other lipids. In: Rapeseed,Cultivation, Composition, Processing and Utilization.Elsevier Publishing Co., Amsterdam, UK. pp. 145-147

[3]AOAC. 1980. Official Methods of Analysis of theAssociation of Official Analytical Chemists. 13thedition. Association of Official Analytical Chemists,Washington D.C.Azuma K, Ippoushi K, Ito H, Higasho H, Terao J. 1999.Evaluation of the antioxidants activity of vegetableextracts in linoleic emulsion and phospholipids bilayers.Journal of the Science of Food and Agriculture, 79,2010.

[4]Baysal T, Ersus S, Swmans D A J. 2000. Supercritical CO2extraction of ?-carotene and lycopene from tomatopaste waste. Journal of Agriculture and FoodChemistry, 48, 5507.

[5]Blau K, King G. 1979. Handbook of Derivatives forChromatography. Heyden & Sons Ltd., London, UK.Boem F H G, Lavado R S, Porcelli C A. 1996. Note on theeffects of winter and spring waterlogging on growth,chemical composition and yield of rapeseed. FieldCrops Research, 47, 175-179

[6]Bourgeosis C. 1992. Determination of Vitamin E:Tocopherols and Tocotrienols. Elsevier, New York,USA.Bramley P M, Elmadfa I, Kafatos A, Kelly F J, Manios Y,Roxborough H E, Schuch W, Sheehy P J A, Wagner KH. 2000. Vitamin E. Journal of the Science of Food andAgriculture, 80, 913-938

[7]Burton G W, Ingold K U. 1986. Vitamin E-application of theprinciples of physical organic chemistry to theexploration of its structure and function. Accounts ofChemical Research, 19, 194-201

[8]Candish J K. 1983. Tocopherol content of some South-EastAsian foods. Journal of Agricultural and FoodChemistry, 31, 166-170

[9]DellaPenna D, Pogson B J. 2006. Vitamin synthesis in plants:Tocopherols and carotenoids. Annual Review in PlantBiology, 57, 711-738

[10]Demurin Y, Skoric D, Karlovic D. 1996. Genetic variabilityof tocopherol composition in sunflower seeds as a basisof breeding for improved oil quality. Plant Breeding,115, 33-36

[11]Drotleff A M, Ternes W. 2001. Determination of RS, E/Ztocotrienolsby HPLC. Journal of Chromatography (A),909, 215-223

[12]Drozd J. 1976. Chemical derivatization in gaschromatography.Chemicke Listy, 70, 268-312

[13]Drissi A, Girona J, Cherki M, Godas G, Derouiche A, ElMessal M, Saile R, Kettani A, Sola R, Masana L, et al.2004. Evidence of hypolipemiant and antioxidantproperties of argan oil derived from the argan tree(Arganial spinosa). Clinical Nutrition, 23, 1159-1166

[14]Esterbauer H, Puhl H, Dieberrotheneder M, Waeg G, RablH. 1991. Effect of antioxidants on oxidative modificationof Ldl. Annals of Medicine, 23, 573-581

[15]Eitenmiller R R. 1997. Vitamin E content of fats and oils -Nutritional implications. Food Technology, 51, 78-81

[16]FAOSTAT. 2010. Searchable online statistical database fromFood and Agriculture Division of the United Nations.[2012-01-05] http://faostat.fao.org/site/339/default.aspx

[17]Fernie A R, Trethewey R N, Krotzky A J, Willmitzer L. 2004.Innovation - Metabolite profiling: from diagnostics tosystems biology. Nature Reviews Molecular CellBiology, 5, 763-769

[18]Fiehn O. 2002. Metabolomics - The link between genotypesand phenotypes. Plant Molecular Biology, 48, 155-171

[19]Goffman F D, Becker H C. 1998. Phänotypische variabilitätdesGehalts und musters der Tocopherole in den Samenvon Winterraps (Brassica napus L.). Vorträge fürPflanzenzüchtung, 42, 105-106

[20](in German)Goffman F D, Becker H C. 2001. Genetic analysis oftocopherol content and composition in winter rapeseed.Plant Breeding, 120, 182-184

[21]Goffman F D, Becker H C. 2002. Genetic variation oftocopherol content in a germplasm collection ofBrassica napus L. Euphytica, 125, 189-196

[22]Goffman F D, Velasco L, Becker H C. 1999. Tocopherolsaccumulation in developing seeds and pods of rapeseed(Brassica napus L.). Fett-Lipid, 101, 400-403

[23]Grela E R, Günter K D. 1995. Fatty-acid composition andtocopherol content of some legume seeds. Animal FeedScience and Technology, 52, 325-331

[24]Grob R L, Barry E F. 2004. Modern Practice of GasChromatography. Wiley, UK. p. 1045.Gül M K, Amar S. 2006. Sterols and the phytosterol contentin oilseed rape (Brassica napus L.). Journal of CellMolecular Biology, 5, 71-79

[25]Hamama A A, Bhardwaj H L, Starner D E. 2003. Genotypeand growing location effects on phytosterols in canolaoil. Journal of the American Oil Chemists Society, 80,1121-1126

[26]Hao Z G, Parker B, Knapp M, Yu L L. 2005. Simultaneousquantification of alpha-tocopherol and four majorcarotenoids in botanical materials by normal phase liquidchromatography-atmospheric pressure chemicalionization-tandem mass spectrometry. Journal ofChromatography (A), 1094, 83-90

[27]Herrera E. 2006. Metabolismo de la vitamina E. Nutrición yObesidad, 3, 16. (in Spanish)Ingold K, Hughes L, Slaby M, Burton G. 1987. Synthesis of2R,49R,89R-?-tocopherols selectively labelled withdeuterium. Journal of Labelled Compounds andRadiopharmaceuticals, 24, 817-831

[28]Jensen C R, Mogensen V O, Mortensen G, Fieldsend J K,Milford G F J, Andersen M N, Thage J H. 1996. Seedglucosinolate, oil and protein contents of field-grownrape (Brassica napus L.) affected by soil drying andevaporative demand. Field Crops Research, 47, 93-105

[29]Kamal-Eldin A, Görgen S, Pettersson J, Lampi A M. 2000.Normal phase high performance liquid chromatographyof tocopherols and tocotrienols. Comparison ofdifferent chromatographic columns. Journal ofChromatography (A), 881, 217-227

[30]Kataya H A H, Hamza A E. 2008. Red cabbage (Brassicaoleracea) ameliorates diabetic nephropathy in rats.Evidence-Based Complementary and AlternativeMedicine, 5, 281-287

[31]Knapp D R. 1979. Handbook of Analytical DerivatizationReactions. John Wiley and Sons, New York, USA.Liebler D C, Burr J A, Ham A J L. 1999. Gas chromatographymass spectrometry analysis of vitamin E and itsoxidation products. Oxidants and Antioxidants (A),299, 309-318

[32]Liu R H, Meng J L. 2006. RFLP and AFLP analysis of interandintraspecific variation of Brassica rapa and B. napusshows that B. rapa is an important genetic resource forB. napus improvement. Acta Genetica Sinica, 33, 814-823

[33]Lon G L, Winefordner J D. 1983. Limit of detection.Analytical Chemistry, 55, A712-A719.

[34]Machlin L J. 1980. Vitamin E: A Comprehensive Treatise.Marcel Dekker, New York, USA. p. 660.Machlin L J. 1984. Handbook of Vitamins: Nutritional,Biochemical and Clinical Aspects. Marcel Dekker, NewYork.Majors R E. 1995. Trends in sample preparation anautomation - what the experts are saying. Lc Gc-Magazine of Separation Science, 13, 742-749

[35]Majors R E. 1999. An overview of sample preparationmethods for solids. Lc Gc North America, 17, S8-S13.Marwede V, Schierholt A, Mollers C, Becker H C. 2004.Genotype X environment interactions and heritabilityof tocopherol contents in canola. Crop Science, 44,728-731

[36]MSTATC. 1989. MS-DOS, ver. 2.10. Michigan StateUniversity, East Lansing, USA.Onay O, Kockar O M. 2006. Pyrolysis of rapeseed in a freefall reactor for production of bio-oil. Fuel, 85, 1921-1928

[37]Piironen V, Lindsay D G, Miettinen T A, Toivo J, Lampi AM. 2000. Review plant sterols: biosynthesis, biologicalfunction and their importance to human nutrition.Journal of the Science of Food and Agriculture, 80,939-966

[38]Regis. 1998. Regis Technologies ChromatographyCatalog. Regis Technologies Inc., Morton Grove,Illinois, USA. pp. 30-32

[39]Robertson D G. 2005. Metabonomics in toxicology: Areview. Toxicological Sciences, 85, 809-822

[40]Ruperez F J, Martin D, Herrera E, Barbas C. 2001.Chromatographic analysis of alpha-tocopherol andrelated compounds in various matrices. Journal ofChromatography (A), 935, 45-69

[41]Scialabba A, Salvini L, Faqi A S, Bellani L M. 2010.Tocopherol, fatty acid and phytosterol content in seedsof nine wild taxa of Sicilian Brassica (Cruciferae). PlantBiosystems, 144, 626-633

[42]Shewmaker C K, Sheehy J A, Daley M, Colburn S, Ke D Y.1999. Seed-specific overexpression of phytoenesynthase: increase in carotenoids and other metaboliceffects. The Plant Journal, 20, 401-412

[43]Steel R G D, Torrie J H. 1980. Principles and Procedures ofStatistics: A Biometrical Approach. 2nd ed. McGraw-Hill, New York.Sumner L W, Mendes P, Dixon R A. 2003. Plantmetabolomics: large-scale phytochemistry in thefunctional genomics era. Phytochemistry, 62, 817-836

[44]Tan B. 1989. Palm carotenoids, tocopherols andtocotrienols. Journal of the American Oil ChemistsSociety, 66, 770-776

[45]Taylor P, Barnes P. 1981. Analysis of vitamin E in edibleoils by high performance liquid chromatography.Chemistry & Industry, 20, 722-726

[46]Thies W. 1997. Quantitative Bestimmung der tocopherole durch HPLC. Angewandte Botanik, 71, 62.Traber M G, Sies H. 1996. Vitamin E in humans: Demandand delivery. Annual Review of Nutrition, 16, 321-347

[47]Vatassery G T, Lai J C K, Smith W E, Quach H T. 1998.Aging is associated with a decrease in synaptosomalglutamate uptake and an increase in the susceptibilityof synaptosomal vitamin E to oxidative stress.Neurochemical Research, 23, 121-125

[48]Wanasundara U N, Shahidi F. 1994. Canola extract as analternative natural antioxidant for canola oil. Journalof the American Oil Chemists Society, 71, 817-822

[49]Waters R P. 1976. Method for determining the α-tocopherolcontent of citrus essential oils. Journal of Food Science,41, 370.Wu J G, Shi C H, Zhang H Z. 2006. Partitioning geneticeffects due to embryo, cytoplasm and maternal parentfor oil content in oilseed rape (Brassica napus L.).Genetics and Molecular Biology, 29, 533-538

[50]Zielinski H, Ciska E, Kozlowska H. 2001. The cereal grains:focus on vitamin E. Czech Journal of Food Sciences,19, 182.Zhang G Q, Zhou W J, Gu H H, Song W J, Momoh E J J.2003. Plant regeneration from the hybridization ofBrassica juncea and B-napus through embryo culture.Journal of Agronomy and Crop Science, 189, 347-350

[51]Zhou W J. 2001. Oilseed rape. In: Zhang G P, Zhou W J.eds., Crop Cultivation. Zhejiang University Press,Hangzhou, China. pp. 153-178

[52](in Chinese)Zhou W J, Zhang G Q, Tuvesson S, Dayteg C, Gertsson B.2006. Genetic survey of Chinese and Swedish oilseedrape (Brassica napus L.) by simple sequence repeats(SSRs). Genetic Resources and Crop Evolution, 53,443-447

[53]Zum Felde T, Baumert A, Strack D, Becker H C, Moellers C.2007. Genetic variation for sinapate ester content inwinter rapeseed (Brassica napus L.) and developmentof NIRS calibration equations. Plant Breeding, 126,291-296.
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