TLC Method for Evaluation of Free Radical Scavenging Activity of Rapeseed Meal by Video Scanning Technology

Abstract

A reversed phase thin layer chromatography (TLC) method combined with video scanning detection for quantitative evaluation of free radical scavenging activity of antioxidative fractions from rapeseed meal by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) is reported. The activity was evaluated by measuring the area of bright yellow bands against the purple background by a CCD video camera after dipping the plate in 0,04% (w/v) DPPH solution. DPPH scavenging activity of L-ascorbic acid and 17 well-known phenolic compounds including α-tocopherol, phenolic acids and flavonoids was determined by this TLC-DPPH method. Correlation coefficients (R2) between activity and sample amount applied were 0,947--0,996 and the detection limits were 40--690 ng depending on compounds. Comparison of the results based on α-tocopherol index showed a good correlation between the activities measured by TLC-DPPH and by the conventional cuvette assay. The method was used to measure free radical scavenging activity of rapeseed meal fractions separated with methanol - H2O - H3PO4 (55:45:1,1) as mobile phase. Ten free radical scavenging bands were detected with Rf values from 0,04 to 0,85. Radical scavenging activity of the one with Rf value 0,41 was highest, possessing 38% of the total scavenging activity. The method was shown to be a simple, fast and efficient analysis for free radical scavenging activity of antioxidative compounds in rapeseed meal. No sample purification is needed. Further, both separation and the activity measurement can be done in the same TLC-DPPH plate simultaneously.

Keywords

TLC-DPPH, video-scan, free radical scavenging activity, antioxidative compounds, separation, rapeseed meal

Introduction

Lipid autoxidation via free radical chain reaction not only lowers the nutritional value of food and deteriorates the flavor and taste but also may be a cause of numerous diseases such as atherosclerosis, ischemia, inflammation, carcinogenesis and aging (Aruoma and Halliwell, 1991). To ensure nutritious and safe food, harmful effects of lipid oxidation should be prevented. In food industry addition of free radical scavenging antioxidants has been one of the effective ways to retard fat oxidation of food. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), n-propyl gallate (PG), monoglyceride citrate (MGC) and tert-butylhydroquinone (TBHQ) are widely used. However, consumers' demands for natural food ingredients have continuously increased and there has been a general desire to replace synthetic food additives with natural alternatives (Howell, 1986). Unfortunately, known natural antioxidants including tocopherols are less effective than synthetic ones. This leads to the interest and need to identify new natural antioxidants used as safe and effective additives in food industry (Aruoma, 1994, Wanasundara et al., 1994, Namiki, 1990). In consequence, efficient methods for screening natural antioxidative compounds are required.

Low erucic acid rapeseed is one of the world's most important oilseed crops. Relatively high content and quality of rapeseed protein make the seed a valuable raw material not only for oil industry but also for feed industry. The meal contains 1-2 % phenolic compounds which is about ten times higher than those in soybean meal (Shahidi and Naczk, 1992). However, the presence of phenolic compounds in rapeseed meal is undesirable because of their antinutritional and sensory properties e.g. dark color, bitter taste and astringency. Therefore, their removal would improve meal quality. Furthermore, phenolic compounds in rapeseed meal would provide a new source for natural food antioxidants.

ESR, chemiluminescence, oxygen radical absorbance capacity and enhanced chemiluminescence assay have been often used for measuring radical scavenging activity and antioxidative potentials of phenolic compounds (Nanjo et al., 1996; Robinson, 1997). Blois' DPPH method and its variations, methyl linoleate and β-carotene / linoleate models are often adopted for antioxidant activity evaluation (Cuvelier et al., 1992; Heinonen et al., 1997; Hopia et al., 1996; Huang et al., 1996; Miller, 1971). A DPPH-HPLC method was developed recently for DPPH radical scavenging activity of colored foods (Tomoko, 1998). However, all these methods are time consuming and can only measure total radical scavenging activity of extracts.

Experimental

Materials and apparatus

Rapeseed meals studied were from Raisio Group. Ltd (Finland) and Mildola Ltd (Finland). Sinapic acid, ferulic acid, vanillic acid, caffeic acid, 2,3-dihydroxybenzoic acid, 3-coumaric acid and 3-hydroxybenzoic acid were purchased from Extrasynthese (Genay, France). Syringic acid, p-coumaric acid, chlorogenic acid, ellagic acid, gallic acid, n-propyl gallate, protocatechuic acid, quercetin, catechin, pyrogallol and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were from Sigma Chemical Co. (St. Louis, USA). HPLC grade methanol from Rathburn Chemicals Ltd (Walkerburn, Scotland), L-ascorbic acid from Aldrich Chemical Co., dimethylsulphoxide (DMSO) from Acros Organics (New Jersey, USA), analytical grade o-phosphoric acid from Riedel-de Haen AG (Seelze, Germany), TLC RP-18 F254s aluminium sheets (Art. 1.05559) and analytical grade α-tocopherol as well as sulphuric acid from Merck KGaA (Darmstadt, Germany) were used. Water was purified with an Alpha Q water purification system (Millipore Co., USA). Methanol solution (20mM) was made for all the standards except ellagic acid which was dissolved in DMSO with the same molar concentration. 0,04% (w/v) DPPH dipping solution was prepared by dissolving 104,6 mg DPPH in 250,0 ml methanol.

Methods

The extraction method involved the use of a 0.5 g rapeseed meal, extracted with methanol / H2O (80:20 v/v) at ambient temperature. After 15 minutes of centrifugation, the supernatant was evaporated to dryness, and the extract was redissolved in methanol.

Results and Discussions

Radical Scavenging Activity by Cuvette Assay

The reaction kinetics of DPPH scavenging reaction of tested compounds and rapeseed extracts is presented in Figure 1. L-ascorbic acid reacted with DPPH immediately and reached a steady state in 15 seconds. Other fast-reacting compounds were sinapic acid, n-propyl gallate, and syringic acid.

Radical Scavenging Activity and Detection Limits by TLC-DPPH Method

There was a polynomial correlation with R2 value 0.947-0.996 between DPPH scavenged band area and sample amount applied to plates. In Figure 3, an example is shown with sample application amount of 20 nmol/band.

Free Radical Scavenging Activity of Rapeseed Extract

Ten DPPH radical scavenging bands were separated and detected with Rf values ranging from 0.04 to 0.85. The band with Rf value 0.41 was highest, possessing 38% of the total scavenging activity.

Conclusion

The TLC-DPPH method combined with video scanning technique provides a simple, fast, and efficient analysis for free radical scavenging activity of antioxidative compounds in rapeseed meal. The correlation between the TLC-DPPH method and cuvette assay for most of the standards studied was good.

References

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