Małgorzata Nogala-Kałucka, Marek Gogolewski, Eleonora Lampart-Szczapa, Marcin Jaworek, Aleksander Siger, Aleksandra Szulczewska
Akademia Rolnicza w Poznaniu, Katedra Biochemii i Analizy Żywności
Determination of vitamin E active compounds
as biological antioxidants occurring
in rapeseeds of the selected varieties
Oznaczanie zawartości związków witamino-E aktywnych jako
biologicznych antyoksydantów w nasionach wybranych odmian rzepaku
Key words: rapeseed, tocopherols, HPLC, alpha-T equivalent Słowa kluczowe: rzepak, tokoferole, HPLC, ekwiwalent alfa-T The aim of the study was the determination of the
homologous tocopherol level in seeds of oilseed rape varieties cultivated in some plantations in Wielkopolska region during three years (2000– 2002). The following selected varieties of rapeseeds were investigated: Silvia (the year of harvesting 2000); Silvia, Lisek, Marita and Bermuda (the year of harvesting 2001) as well as Lisek, Lirajet, Buffalo, Wotan and Kaszub harvested in year 2002. The high performance liquid chromatograph (HPLC) Waters 600 Asc. Milford with Millenium 32 program was applied for a qualitative and quantitative identification of tocopherols. Separation was carried out on the LiChrosorb Si 60 column and mixture of n-hexane and 1.4-dioxane (97:3, v/v) with a flow rate of 1.5 ml/min was used as a mobile phase. The fluorimetric detector (Water 474) worked at excitation λ=290 nm and emission λ=330 nm. The biological activity of the rapeseeds was estimated. Statistical analysis of the results was performed using the computer program Statistica. Investigations showed presence of four tocopherol homologues in each of the studied varieties. Total tocopherol content was to 25 mg/100g dm. There was more gamma- than alpha- tocopherol in all of the varieties. Among the remaining tocopherols,
beta-Celem badań było oznaczenie poziomu zawar-tości homologicznych tokoferoli w nasionach odmian rzepaku uprawianych na terenie Wielko-polski w trzech kolejnych latach (2000–2002). Badano następujące wybrane odmiany rzepaku: Silvia (zbiór 2000), Silvia, Lisek, Marita i Ber-muda (zbiór 2001) oraz Lisek, Lirajet, Buffalo, Wotan i Kaszub ze zbioru w roku 2002. Do identyfikacji jakościowej i oznaczeń ilościowych tokoferoli stosowano chromatografię cieczową HPLC. Rozdziału tokoferoli dokonywano na kolumnie LiChrosorb Si 60 stosując mieszaninę
n-heksanu i 1,4-dioksanu (97:3, v/v) jako fazę
ruchomą o szybkości przepływu 1,5 ml/min. Detektor fluorymetryczny (Waters 474) pracował przy wzbudzeniu λ=290 nm i emisji λ=330 nm. Otrzymane wyniki analizowano statystycznie wykorzystując do obliczeń program Statistica. Badania wykazały obecność czterech homologów tokoferoli we wszystkich badanych odmianach. Ich sumaryczna zawartość kształtuje się na po-ziomie 25 mg/100 g s.m, przy czym w badanych odmianach występowało więcej homologu gamma-tokoferolu niż alfa. Z pozostałych form toko-feroli, w śladowych ilościach, w zależności od odmiany, występował beta-tokoferol, natomiast delta-tokoferol oznaczano w ilościach około 1% w zależności od odmiany i roku zbioru.
Naj-tocopherol was present in trace amounts depending on the variety, whereas quantity of delta-tocopherol was determined as ca. 1% depending on the variety and year of harvesting. The smallest tocopherol amount characterized the Buffalo variety with low fat content. Lisek and Silvia were the richest in tocopherols. Also the highest biological activity, expressed as the alpha-tocopherol equivalent, was stated in these varieties and the lowest one – in Buffalo. Evaluation of the biological activity of the rapeseeds seems to be a characteristic significantly differentiating individual varieties.
mniejszą zawartością tokoferoli charakteryzo-wała się odmiana Buffalo. Najlepszymi pod względem zawartości tokoferoli okazały się Lisek i Silvia. Najwyższą wartość biologiczną wyrażoną w ekwiwalentach alfa-tokoferolu stwier-dzono również dla tych odmian, natomiast naj-mniejszą dla odmiany Buffalo. Określanie war-tości biologicznej dla nasion rzepaku wydaje się być cechą charakterystyczną znacząco różniącą poszczególne odmiany.
Introduction
Tocopherols belong to a group of compounds generally present in oilseeds but their presence was also noticed in other parts of green plants such as stems, leaves, buds, flowers, pods, roots and tubers (Osuna-Garcia et al. 1998, Grusak and DellaPenna 1999, Nogala-Kałucka et al. 2002). Alpha-tocopherol (alpha-T) is a predominant homologue identified in different parts of a plant, whereas all four tocochromanols are present in seeds and fruits. Tocopherols as natural antioxidants protect lipids against free radicals, some type of reactive oxygen, hydroxylic group and peroxides (Fig. 1) (Stuchlik and Zak 2002). During reactions alpha-toco-pheroxyl radicals and other oxidation products, such as alpha-tocopherol hydro-peroxydienone, alpha-tocopherol quinone or alpha-tocopherol quinol, are formed (Munne-Bosch and Alegre 2002). During oxidation tocopherol radicals may be regenerated, for example, by procyanidins (Carini et al. 1998). The recent findings open a new field in which other roles of tocochromanols in plant cells are investigated.
The level of tocopherols in oilseeds, e.g. in oilseed rape, can be a quality determining factor. Oil obtained from double low oilseed rape presently cultivated is one of the most valuable edible oils (Trautwein and Erbersdobler 1998, Grusak and DellaPenna 1999). Together with tocopherols other components — polyun-saturated fatty acid (PUFA), sterols as well as glucosinolates — bring completely new meaning to the oilseeds rape healthy value. According to literature, glucosinolates, if present in appropriate quantities and qualities, like tocochromanols, can show anticancerogenic activity (Steinmetz and Potter 1993). Rapeseeds composition is important for its healthy effect, as well as in fat and food industries. Genotype as well as agroclimatic conditions influence composition of PUFA in triacylglycerols of oils obtained from double low rapeseed varieties.
Oxidation Alkyl
radical
Polyunsaturated fatty acid (PUFA)
Alkyl radical
Conjugated diene
Lipid peroxy radical
Hydroperoxide
Reduction
Other compounds (e.g., n-hexanal, jasmonic
acid, traumatic acid) Alcohols O CH3 O H C H3 CH3 CH3 O CH3 O C H3 CH3 CH3 3 3 α-TOCOPHEROL α-TOCOPHEROXYL RADICAL
Other oxidation products
Recycling 1 O2 R1 R2 R1 R2 H R1 R2 O2 R1 R2 O O PUFA R1 R2 O OH • •
Fig. 1. Lipid peroxidation, and scavenging of lipid peroxy radicals by alpha-tocopherol in plants
Utlenianie tłuszczów i zmiatanie rodników nadtlenkowych przez alfa-tokoferol u roślin (Munne-Bosch
i Alegre 2002)
Chemical composition of the oil is the main factor influencing the stability as well as nutritional and healthy values (Jerzewska and Ptasznik 1999). Increased intake of the essential unsaturated fatty acids (susceptible to oxidation) should be accompanied by the increased intake of vitamin E which prevents quick oxidation of double bonds in the unsaturated acids (Ziemiański, Budzyńska-Topolowska 1991).
In the present work we investigated quality and quantity changes in tocopherols content in seeds coming from selected plantations of rapeseed varieties cultivated in Wielkopolska region during the last tree years (2000–2002). Biological activity was expressed as alpha-tocopherol equivalent for individual rape varieties, as it is done for seeds of different species of the crucifereous
(Brassicaceae) plants seeds. The rapeseeds can be the potential material for the
production of sprouts, which are products with functional food properties, more and more widely accepted not only by vegetarians but also by the rest of consumers.
Materials and methods
Seeds from the selected plantations of rapeseed varieties were investigated: Silvia (year of harvesting 2000); Silvia, Lisek, Marita and Bermuda (year of harvesting 2001) as well as those of five varieties harvested in year 2002 — Lisek, Lirajet, Buffalo, Wotan and Kaszub. All studied varieties were cultivated in Wielkopolska region. Directly after harvesting the seeds were delivered for analyses.
To determine the tocopherol content, the samples (3 g) of rapeseeds were ground and then saponified using 60% KOH. Then the samples were extracted using the peroxide free diethyl ether (Dunphy et al. 1966). After extraction ether was distilled off and in the residue (unsaponifiable matter) tocopherols were qualitatively and quantitatively determined using HPLC (Gogolewski et al. 2000, Nogala-Kalucka et al. 2002). The HPLC (Waters 600 Asc. Milford) equipment consisted of the gradient pump Waters Model 600, column, fluorimetric detector and Waters Millenium 32 data acquisition system. The residue was dissolved in
n-hexane and 20 µl samples were injected on the LiChrosorb Si 60 column (200
mm, 5 µm, Merck), and the mixture of n-hexane and 1.4 dioxane (97 : 3 v/v) was used as a mobile phase. The flow rate of mohle phase was 1.5 ml/min. The fluorimetric detector (Waters 474) worked by excitation λ=290 nm and emission λ=330 nm. The concentrations were calculated from calibration curves made for individual tocopherols. Standards of alpha-, beta- gamma- and delta-tocopherol (99.9%, Merck–Darmstadt, Germany) were used.
Biological activity of all rapeseeds samples was calculated according to the formula:
1 mg equivalent α-T = α-T + 0.5β-T + 0.25γ-T + 0.01δ-T (Elmadfa and Bosse 1985)
The results were evaluated with statistical analysis. Chemical analyses were carried out in three replicates — chromatographic analysis of each of them was repeated twice. Statistical analysis was performed using the computer program Statistica, version 6, StatSoft Inc. (2001).
Results and discussion
Wielkopolska is a region where different varieties of winter rape are cultivated. The choice of varieties for cultivation requires considering the sowing soil and agroclimatic conditions (e.g. ability of plants to regenerate after winter damages) together with a term of maturation (organization and schedule of field works) as well as the crop of seeds obtained from hectare (Wałkowski et al. 2002). For this reason very often a few varieties are cultivated simultaneously after selecting from the Register those which are characterized by high fat content, medium — early
maturation and high crop of seeds. Only the Kaszub variety was the early one, then Bermuda, Buffalo, Lisek, Marita and Silvia were medium-early, Lirajet and Wotan belonged to medium-late.
During the last three years of harvesting (2000–2002) on the same plantations from Wielkopolska region, many varieties were cultivated alternatively. As a matter of fact, we were not able to investigate changes in homologues of the tocopherols content due to the diversity of varieties during three years of investigations.
Changes in tocopherols content present in investigated seeds samples are presented in figures 2 and 3. The total content of homologous tocopherols is between 20.8 (Buffalo 2002) and 27.6 mg/100 g dry mass (Lisek 2002). Lirajet and Wotan belong to the medium-late and average or lowered fat content varieties (Wałkowski et al. 2002) and that is why the amount of tocopherols is about 25.0 mg/100 g d.m. Wotan variety with the results of total tocopherols content 21,8 mg/100 g d.m. differs statistically, which can be explained by lower content of fat (ca 41%). The remaining varieties belong to medium early ones among which Silvia shows higher content of fat (above 42%), together with tocopherols amount of 27.0 mg/100 g d.m. in year 2000 and 25.5 mg/100 g d.m. in year 2001. Buffalo is the poorest variety in tocopherols (about 21.0 mg/100 g d.m.) also with lowered fat content (41%). Data concerning the fat content was presented in an earlier publication about determination of some indicators of rapeseeds quality e.g. fat content (Nogala-Kałucka et al. 2002a).
Among the studied seeds of different winter rapeseeds varieties statistically significant differences (p ≥ 0.05) of tocopherol homologues content were revealed (Fig. 2, 3). These varieties are characterized by the highest level of gamma-tocopherol, up to 56.4 %, and also that of alpha-tocopherol — up to 50.5% for Silvia (2001).
The remaining homologues of beta-tocopherol are present in trace amounts while delta-tocopherol up to some percentage — 1.84% (Lisek 2001). These results confirm literature data referring to percentage of individual tocopherol homologues content in rapeseed where alpha-tocopherol amounted to 42%, gamma-tocopherol — 57% and delta-tocopherol — 0.9% (Goffman et al. 1999). Leckband and co-workers (2002) determined the tocopherol content in different rapeseed varieties in the project NAPUS 2000 quoting from 35.9 to 38.2% for alpha-tocopherol, and 59.3–62% for gamma-tocopherol. Similar results were obtained also during biosynthesis of tocochromanols studied in seeds of double low variety of spring oilseed rape Star (Nogala-Kałucka et al. 2002). Buffalo and Wotan contained alpha-tocopherol statistically not differing from year 2001, which is also observed in the case of gamma-tocopherol. Differences in tocopherol quantities in individual varieties may be due to climatic and soil conditions, agrotechnical processing as well as technological maturation of rapeseeds.
2000 2001
alpha-T gamma-T delta-T
Oilseed rape varieties
Fig. 2. Tocopherol content in rapeseed varieties cultivated in Wielkopolska region (crop 2000/2001)
Zawartość tokoferoli w odmianach rzepaku uprawianych na terenie Wielkopolski (zbiór 2000/2001)
alpha-T gamma-T delta-T
Oilseed rape varieties
Fig. 3. Tocopherol content in rapeseed varieties cultivated in Wielkopolska region (crop 2002)
Alpha- and gamma-tocopherol, predominant homologues in rapeseeds, show essential biological functions not only in plant world. Alpha-tocopherol possessing the highest biological activity is named vitamin E and it is an indispensable exogenic compound necessary for proper metabolism in humans and animals (Brigelius-Flohe and Traber 1999). Hence determination of the biological activity refers to calculation of the content of individual homologues with different vitamin activity, and their effectiveness is defined as alpha-tocopherol equivalents (Elmadfa and Muskat 1999). The demand depends on the amount of the consumed PUFA. In view of increasing popularity of sprouts rich in phyto compounds (Kozłowska et al. 2002) alpha-tocopherol equivalents in the investigated varieties were determined. (Fig. 4). Alpha-tocopherol equivalents in rapeseeds of all studied varieties differ from 12 to almost 17 mg. In the investigated rapeseed varieties we noticed statistically significant differences in alpha-tocopherol equivalent depending on a variety but also on a year of harvesting e.g. Lisek and Silvia. It is only possible to compare this value with wheat sprouts, where higher alpha-tocopherol activity, reaching 28 mg, was two times higher (Piironen et al. 1986).
Conclusions
• In all investigated winter rapeseed varieties alpha- and gamma-tocopherol were predominant homologues, followed by delta-tocopherol, while beta-tocopherol was present in trace amounts.
• In the investigated rapeseeds varieties we noticed statistically significant differences in alpha-tocopherol equivalent depending on a variety but also on a year of harvesting.
• Considering biological activity of vitamin E the determining of alpha-tocopherol equivalent can be a significant criterion in assessing the quality of rapeseeds.
Wnioski
• Dominującymi homologami we wszystkich badanych odmianach rzepaku ozimego był alfa- i gamma-tokoferol, w dalszej kolejności delta-tokoferol, podczas gdy beta-tokoferol występował w ilościach śladowych.
• Stwierdzono statystycznie istotne różnice dotyczące zawartości ekwiwalentu alfa-tokoferolu pomiędzy poszczególnymi odmianami, także w kolejnych latach zbioru.
• Z uwagi na aktywność biologiczną witaminy E, określenie ekwiwalentu alfa-tokoferolu może być istotnym wyróżnikiem jakości nasion rzepaku.
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2000 2001 2002 C D C B G F F E C A
Oilseed rape varieties
Data presents mean values from three replicates; mean values followed by different letters are statistically significant at p ≤ 0.05
Dane przedstawiają średnią z trzech powtórzeń; średnie oznaczone różnymi literami różnią się statystycznie istotnie przy p ≤ 0,05
Fig. 4. Biological activity calculated as mg alpha-T equivalent — Aktywność biologiczna przeliczona na ekwiwalent mg alfa-T Biological activity was calculated according to the formula — Aktywność biologiczną wyliczono według wzoru:
