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 R_f values from 0,04 to 0,85. Radical scavenging activity of the one with R_f 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.

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.

In this paper, we introduce a TLC-DPPH method for simultaneous separation and activity measurement of antioxidative compounds based on TLC and video scan techniques. The method was successfully applied to rapeseed meal extract.

EXPERIMENTAL

Materials and apparatus

Rapeseed meal and reagents 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 F_254s 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.

Apparatus Camag Video Documentation System including a HV-C20 3 x 1/2" CCD video camera (Hitachi, Japan) was connected with Reprostar 3 transilluminator cabinet and used under Video Store 2 (version 2.23) and Video Scan (version 1.01) software ( Camag, Muttenz, Switzerland) for TLC plates imaging. Perkin-Elmer Lambda 11 UV/VIS spectrophotometer (Uberlinger, Germany) for cuvette assay, Camag Chromatogram Immersion Device III and Linomat IV applicator were applied.

Methods

Extraction 0,5 g rapeseed meal was extracted with 2 x 10 ml methanol / H₂O (80:20 v/v) at ambient temperature using Ultra-Turrax T-25 homogenizer (IKA, Germany). The supernatant was evaporated to dryness after 15 min centrifugation at 4 °C, 6800 rpm. Dry extracts was redissolved in 0,5 ml methanol and used as crude extract. Diluted solution (10 x) of the crude extract was prepared for cuvette assay.

Cuvette assay of DPPH radical scavenging activity 50 μl sample was added to 2,95 ml DPPH solution (4,5 mg DPPH in 100 ml methanol) in a disposable cuvette (Plastibrand^® 12,5x12,5x45 mm). The absorbance at 517 nm was then monitored at 15 seconds interval from 0 to 5 min. Methanol was used as the blank solution and pyrogallol (24,8 mg in 50 ml DMSO) as a positive control representing 100% radical scavenging activity in each experiment.

TLC-DPPH separation and determination of radical scavenging activity Rapeseed extracts and all the standards were applied as 9 mm wide band, 10 mm apart on 10 x 20 cm RP-18 aluminium plate by Linomat IV at the rate of 15 s/μl. The starting position was 12 mm from both sides and 10 mm from the bottom of the plates. The solvent system optimized according to PRISMA model (Nyiredy et al., 1988) for rapeseed extract was methanol and H₂O (55:45 v/v) with 1,1% o-phosphoric acid as modifier. The plates were developed in an unsaturated twin trough chamber (10 x 20 cm, Camag, Muttenz , Switzerland) to the distance of 75 mm. After 15 min air-drying, the plates were dipped in 0,04% DPPH solution for 5 seconds and images were captured under visible light at exactly 2 min after dipping. Radical scavenging activity was then determined quantitatively by measuring the area of bright yellow bands against the purple background as proposed by Glavind (1967) with video scan software.

RESULTS AND DISCUSSIONS

Radical scavenging activity by cuvette assay

Reaction kinetics The 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 (Fig 1a). 2,3-Dihydroxybenzoic acid, ellagic acid, ferulic acid and protocatechuic acid followed a rather slow reaction kinetics (Fig.1b). Radical scavenging species in rapeseed extracts showed a relatively quick reaction kinetics reaching a steady state in about 3 min. L-ascorbic acid was the only compound reaching a steady state in less than one minute, which was in accordance with previous results (Brand-Williams et al., 1995). Sinapic acid, syringic acid, n-propyl gallate and α-tocopherol (Kurechi et al., 1980) were found to reach the steady state within 5 min.

Fig. 1 Reaction kinetics of DPPH with tested compounds and rapeseed extracts

Fig.2 DPPH scavenging activity by cuvette assay (2μmol/ml) after 2 min reaction

DPPH radical scavenging activity The two most active compounds were n-propyl gallate and syringic acid (Fig 2). Vanillic acid, 3-coumaric acid, p-coumaric acid and 3-hydroxybenzoic acid were poor radical scavengers. L-ascorbic acid and all the other phenolic compounds formed another group possessing rather high radical scavenging activities from 25% to 70% of pyrogallol inhibition value at 2μmol/ml level.

Fig.3 DPPH scavenging activity by TLC-DPPH method expressed as α-tocopherol indices

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. L-ascorbic acid, gallic acid and 2,3-dihydroxybenzoic acid were the most active scavengers based on α-tocopherol indices. Vanillic acid, 3-coumaric acid, p-coumaric acid and 3-hydroxybenzoic acid showed no activity by the TLC-DPPH method. Detection limits of TLC-DPPH method were determined according to signal / noise ratio value of 3 as showed in Table 1.

Correlation between the results of cuvette assay and TLC-DPPH method

Pearson correlation coefficients ranged from 0,661 to 0,809 and Spearman coefficients for ranks from 0,744 to 0,853 depending on the sample amount applied to plates and measurement time in cuvette assay. The results for syringic acid, n-propyl gallate and L-ascorbic acid showed the lowest correlation between the two methods.

Table 1. Detection limits of L-ascorbic acid and phenolic compounds by TLC-DPPH method

Compounds	Detection limits (ng)	Compounds	Detection limits (ng)	Compounds	Detection limits (ng)
n-propyl gallate	40	Gallic acid	100	Protocatechuic acid	310
Syringic acid	40	Caffeic acid	110	L-ascorbic acid	350
Sinapic acid	50	Catechin	170	Ferulic acid	390
Ellagic acid	60	Quercetin	180	alpha-tocopherol	690
2,3-OH-benzoic acid	90	Chlorogenic acid	210

Free radical scavenging activity of rapeseed extract

2μl rapeseed crude extract was directly applied on RP-18 plates. Ten DPPH radical scavenging bands were separated and detected with R_f values ranging from 0,04 to 0,85 (Fig. 4). DPPH scavenging activity of the one with R_f value 0,41 was highest, possessing 38% of the total scavenging activity. Shahidi et al. (1995) suggested that sinapate have the highest antioxidant activity potential in rapeseed meal.

The two rapeseed meal extracts showed a slight difference in their radical scavenging activity. Meal no.1 possessed approximately 10% higher total activity than meal no.2. Similar results were also obtained by cuvette assay (Fig. 2). Processing method, rapeseed varieties and meal storage time could be responsible for these differences. Moreover, a very unpolar band (Rf = 0,0) with 3 % of the total scavenging activity was detected in both rapeseed extracts. A major part (68-75 %) of compounds in rapeseed extract was found to be active based on DPPH scavenging area and the total bands area visualized by 10% H₂SO₄ (v/v) in methanol followed by heating at 120 °C for 15 min.

Fig.4 DPPH scavenging activity of rapeseed extract fractions by TLC-DPPH method

CONCLUSION

TLC-DPPH combined with video scanning technique provided a simultaneous separation and radical scavenging activity measurement of antioxidative compounds in rapeseed meal. Correlation between this TLC method and the cuvette assay for most of the standards studied was good. The method suitable for L-ascorbic acid and most phenolic compounds including phenolic acids, flavonoids and tocopherols was shown to be a simple, fast and efficient radical scavenging activity assay.

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