ABSTRACT

Phenolics are common constituents of many oilseeds. There is a great diversity in the natural occurrence and distribution of these compounds, reflecting differences in their role in plant metabolism and physiology. Cruciferous plants accumulate sinapic acid esters in their tissues; they are especially rich in sinapoylcholine (sinapine). It has been demonstrated that sinapine, during seed germination, can be a source of choline for membrane lipid synthesis. However, the presence of relatively high levels of sinapine may compromise the use of rapeseed for animal feed. Better analytical techniques are required to facilitate advancements in the understanding of the role of phenolics in oilseeds. These techniques are also crucial to the development of germplasm, rich in beneficial or low in adverse components.

INTRODUCTION

The success of edible rapeseed: canola is an effect of selective breeding techniques, with its primary goal to lower the amount of erucic acid and glucosinolates in seed.

The second stage in rapeseed development involved the use of biotechnology methods for production of seed containing modified fatty acid profiles, both for human consumption and industrial use. A wide range of designer rapeseed has been developed including low oleic, high oleic, high lauric, high erucic, and high GLA (gamma linolenic acid) rapeseed. Novel rapeseed varieties resistant to environmental stress are in common use; this includes herbicide- resistant canola.

The third stage, which we are now witnessing, is the production of desired substances in canola seed. It includes substances that are ‘foreign’ to rapeseed, like carotenoids, peptides, and enzymes.

With the advent of biotechnology applied to manipulation of rapeseed genes, a variety of novel rapeseed cultivars designed for specific purposes are appearing on the market. More attention has been paid recently to components existing in rapeseed, and having important (positive or negative) effect on its nutritional value, especially when used as animal feed.

These components include phenolics and their metabolites (sinapine, tannins) which apart from their nutritional function are important factors in plant physiology, protect plants from diseases and predators, and in addition play an important role as antioxidants during oil storage in seed compartments.

This paper describes some findings on the determination of endogenous sinapine concentrations in canola, and discusses the relevance of relying on analytical methods that are best suited for this purpose.

MATERIALS AND METHODS

Canola samples from 1997 check sample cultivar tests consisting of the following varieties: Parkland, Reward, Goldrush, Tobin, Excel, Legend, Cyclone, Innovator, and Quantum. All canola samples were grown in Canadian Prairie Provinces. Seeds were de-fatted with hexane using the Swedish tube method. Dry, defatted canola meal was used for determination of sinapine level.

All chemicals were purchased from Sigma (St. Louis, MO). Sinapine was isolated and purified as sinapine bisulfate in POS laboratories according to known procedures (Clandinin, 1961). The UV spectra were recorded with the HP – 8453 spectrophotometer.

Capillary electrophoresis. The buffer used for electrophoresis was 150 mM boric acid, 15mM dimethyl β-cyclodextrin, 75mM SDS, pH-8.5. Electrophoresis was carried out using a HP ^3DCE instrument with UV detection. An uncoated fused-silica capillary of i.d. 50 μm and effective length 72cm was used. Separations were performed at 30kV. The capillary temperature was 35^oC. The photodiode detector and monitoring at 230 nm was used.

Spectrophotometric determination of sinapine in canola meal.

From the stock solution of sinapine (concentration equal to 200 μg/mL), a series of methanol solutions were prepared. The absorbance of each sample was measured at 330 nm. From the data collected, the following linear regression formula was calculated from collected data:

Sinapine concentration [μg/mL] = 21.838 x Absorbance.

For sinapine determination, 1.5 g of dry, defatted meal was extracted three times with 35 mL of methanol under reflux for 30 minutes. Methanol extracts were combined, evaporated at reduced pressure with a rotavapor, transferred to a 100 mL volumetric flask and the final volume was adjusted to 100 mL with methanol. For measurement, 100 μL of solution was diluted to 10 mL with methanol and the absorbance was recorded at 330 nm. Sinapine content in the meal was calculated from the formula:

% Sinapine= (2.184 x Absorbance x 10)/ Sample Wt. [g]

Analysis of sinapine by capillary electrophoresis (CE).

Calibration curve. Tryptophan was used as an internal standard (IS) for this determination. The calibration curve for concentration of sinapine vs. tryptophan and related area under corresponding peaks was constructed.

For the CE determination, 20 mL of solution (I) – (see above), was evaporated to dryness under reduced pressure at 40^o C. The residue was dissolved in water, 1 mL of aqueous tryptophan solution (2 mg/mL) solution was added as an internal standard.

RESULTS

A typical CE analysis of canola meal extract is presented in fig. 1A. Apart from sinapine exhibiting peak at time 18.39 min, several minor peaks corresponding to other phenolic components of canola meal are present: sinapic acid, ferulates and glucopyranosyl sinapate (GPS). The UV spectra of compounds present in the meal are given in fig. 1B.

It is evident that all of these compounds do contribute to the total absorption at 330 nm. Therefore, spectrophotometric determination of sinapine provides overestimated results.

Figure 1. A/. Capillary electrophoresis (CE) analysis of canola meal extract. B/. UV spectra of components present in canola meal and separated by CE. All spectra shows significant absorption or absorption peak at 330 nm. C/. Sinapine absorption in the condition of CE separation. (For experimental conditions refer to the text)

The comparison of data obtained from spectrometric determination of canola meal with those from CE determination, shows that CE results are approximately fifty percent lower (Table 1).

Table 1. Sinapine content [%] in canola meal.

Canola meal variety	‘Sinapine’ by spectrophotometry (%)	Sinapine by CE (%)
Goldrush	2.18	1.37
Parkland	2.69	1.32
Reward	2.36	1.41
Tobin	2.38	1.38
Excel	3.02	1.51
Legend	2.70	1.48
Cyclone	3.71	1.73
Quantum	3.26	1.23
Innovator	2.86	1.28

The presented results show that there is considerable variation concerning the types of phenolic components present in meal of different canola varieties. For example, in Goldrush, sinapine is a major phenolic component, while the Quantum variety contains large amounts of other phenolics, and sinapine represents only about one-third of phenolics exhibiting absorption at 330nm.

DISCUSSION AND CONCLUSIONS.

The accurate determination of sinapine levels in canola seed, as well as that of other phenolic compounds is an important aspect in the quality control and classification of canola varieties.

Phenolics are common constituents of many oilseeds. There is a great diversity in the natural occurrence and distribution of these compounds, reflecting differences in their role in plant metabolism and physiology. For instance, cruciferous plants accumulate sinapic acid esters in their tissues; they are especially rich in sinapoylcholine (sinapine). It has been demonstrated that sinapine, during seed germination, can be a source of choline for membrane lipid synthesis. However, the presence of relatively high levels of sinapine may compromise the use of rapeseed for animal feed (Butler et al.). The characteristic ‘fishy’ taint in eggs produced by hens fed diets containing rapeseed meal is due to trimethylamine. The source of the triethylamine in such eggs originates from the presence of sinapine in the meal.

Therefore, it is of great interest to rapeseed breeders, processors and nutritionists to attempt to eliminate, or at least lower the content of sinapine in rapeseed meal without affecting other phenolics important to plant physiology.

Today’s sophisticated technologies applied in molecular biology also require better analytical methods for analysis of seed components. Currently used methods for the analysis of sinapine in rapeseed are lacking such precision. The main reason for this deficiency is that those methods are based on determination of UV absorbance at the 320 - 330 nm range, which is characteristic for sinapine. However, there are many other phenolics exhibiting strong absorption in the same region. As a consequence, these methods tend to overestimate the sinapine level, and not differentiate from other ‘harmless’ or beneficial phenolics present in seeds. Other procedures include the titanium tetrachloride method (Ismail and Eskin, 1979), gas chromatography (Krygier et al., 1982) and high-performance liquid chromatography (Clausen et al., 1983). An improved method for sinapine determination combines purification of canola extract using cation-exchange columns followed by spectrophotometric determination (Wang et al., 1998); however, a considerable amount of laborious operations is required.

The application of capillary electrophoresis (CE) to the natural products, biotechnology and pharmaceutical industry has flourished in recent years. This technique provides several advantages such as low operating costs, short separation times, high separation efficiency, and requires reduced amounts of material for the analysis. Moreover, CE can be coupled with several mass spectrometry techniques (MALDI, FAB, electrospray) for a positive identification of the substance of interest.

Our data presents a novel approach to the analysis of sinapine in rapeseed and might provide a useful alternative for canola seed screening, given the reported shortfalls of the methods currently in use. The CE based method is very quick, precise and cost effective overall.

Although the initial cost of the system is significant, the fact that it represents an extensive range of very powerful analytical methodologies makes CE an important and versatile technique that has a great potential in the determination of sinapine and other phenolics in rapeseed. Reliable and precise analyses of these compounds can constitute a valuable tool available to breeders and biotechnologists for assessing genetic variation and/or environmental impact in canola varieties.

REFERENCES

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