Free radical‐scavening activity of phenolics by reversed‐phase TLC

Abstract

A method was developed to measure the radicalscavenging activity of compounds separated by reversed-phase TLC (RP-TLC) using phenolic acids as model analytes. TLC separation was followed by dipping the plate in a 0.04% (wt/vol) solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in methanol. The compounds possessing radical-scavenging activity were detected as bright yellow bands against a purple background. A video documentation system based on a CCD video camera was used for the detection and quantification of the activity. The developed RP-TLC-DPPH method was compared to the widely used spectrophotometric DPPH assay. The results obtained by the two methods correlated well, apart from syringic acid, ascorbic acid, and n-propyl gallate, which proved to be outliers in the regression analyses. The correlation coefficient, after excluding outliers, was r 2 = 0.923. The RP-TLC-DPPH method was applied for the measurement of free radical-scavenging activity of rapeseed meal fractions. A total of 10 separated zones with free radical-scavenging activity were detected, with R f values ranging from 0.04 to 0.85. The results show that the method can be used for the effective fractionation and analysis of potential antioxidative compounds in natural extracts.Paper no. J10222 in JAOCS 80, 9-14 (January 2003).KEY WORDS: 1,1-Diphenyl-2-picrylhydrazyl, free radical scavenging activity, phenolic compounds, reversed-phase thinlayer chromatography.The oxidation of lipids is the main mechanism responsible for the deterioration of food (1). The oxidation process can be prevented effectively by using free radical-scavenging antioxidants such as BHA, BHT, and propyl gallate. Furthermore, berries, fruits, and vegetables naturally contain many antioxidative compounds, for example, simple phenolics, flavonoids, and tannins. In addition to the conjugated diene method, which measures the rate of hydroperoxide formation and decomposition in different lipid systems and emulsions (2,3), radical-scavenging assays such as measurement of hydroxyl radical-scavenging activity, superoxide anion radical-scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (4) have also been used.DPPH is a stable, water-soluble free radical with an absorption maximum at 517 nm (5). This strong absorption is due to the unpaired electron of the DPPH radical (4). When this radical compound accepts an electron or a hydrogen radical from a radical scavenger, the absorption vanishes and the resulting decolorization is stoichiometric with respect to the number of electrons taken up.Many variations of the spectrophotometric DPPH method, first developed by Blois (4), are currently used, and this method has attained the status of a routine procedure in the screening of antioxidants (6-10). These methods measure the total DPPH radical-scavenging activity of the extracts or fractions of interest but lack the ability to isolate the activity caused by only one or a few components of the mixture.TLC combined with DPPH radical detection of antioxida...

Antioxidant capacity of rapeseed meal and rapeseed oils enriched with meal extract

“…These differences can be related to the different varieties of rapeseed, technological method of meal production (conventional, supercritical CO 2 ), conditions of phenolic extract preparation (hydrolysis with sodium hydroxide or enzymes, polarity of solvent, temperature, etc.). Moreover, the AC of canola meals were analyzed by the b-carotene bleaching test (3.50-95.3%) [9,24], DPPH (38%, 2.30 mg/mL, 0.28-0.61 mg/mL) [1,23,24], and TEAC methods (45-131 mmol TE/100 g) [24], whereas FRAP method was not applied for AC determination of the rapeseed meal extracts.…”

“…Also, UV spectra of methanolic extracts and the partial least squares (PLS) regression method were used to predict the total content of phenolic acids in meal [4]. However, for separation, identification, and quantification of the individual phenolic compounds, chromatographic techniques are required [3,9,18,[20][21][22][23]. Many bioactive compounds extracted from rapeseed meal have health beneficial characteristics and antioxidative properties, which were determined by 2,2 0 -diphenyl-1-picrylhydrazyl radical (DPPH ) [1,3,23,24], b-carotene-linoleic acid (linoleate) [9,22,24] the reducing power [24], and Trolox equivalent antioxidant capacity (TEAC) methods [24].…”

Separation and evaluation of free radical‐scavenging activity of phenol components of Emblica officinalis extract by using an HPTLC–DPPH• method

“…A similar method both on normal phase [12] and on RP [13] TLC was used for screening antioxidants present in plant extracts.…”

Application of metabolite profiling and antioxidant activity in assessing the quality of processed and unprocessed stingless bee’s propolis

“…described in [28] with slightly modification. The TLC plates were incubated in the dark for 30 minutes and were then visualized under white light using CAMAG TLC Visualizer (DXA252 digital camera).…”