Antioxidant activity of ethanolic extracts of amaranth seeds I. Klimczak, M. Malecka and B. Pacholek 1 Introduction Oxidation causes many undesirable changes in food and leads both to the deterioration of sensory characteristics and to the lowering of its nutritive value. For this reason, antioxidants, which inhibit oxidation play an important role in food process- ing and storage. Compounds with antioxidant activity are widely spread in raw sources of plants origin. They exist in dif- ferent parts of plants – fruits, seeds, leaves, flowers, barks [8, 18, 20]. Good sources of antioxidants are many seasoning plants (rosemary, sage, oregano, thyme, mustard), as well as soya, oat, rice, barley, wheat germs, nut and cocoa shells, and many others [1, 2, 8, 19, 20]. Antioxidant activity of plant extracts mainly depends on the presence of phenolic com- pounds and among them derivatives and isomers of flavones, isoflavones, flavonols, catechins, phenolic acids, and the best known tocopherols are of the greatest importance. Antioxidant activity of plant extracts is often the effect of involvement of two or more compounds acting according to different mechan- isms. Thus, plant extract, not single compounds, are of practi- cal interest as food additives [10, 12]. The interest of some plants forgotten long ago and not used for nutritional purposes has lately increased. To these plants, called the alternatives, can be numbered among others amar- anth, known in many countries as decorative plant or weed. The interest of its cultivation stems from many valuable prop- erties such as high protein content (rich in exogenic amino acids), high fat content in relation to other cereals (rich in unsaturated fatty acids), the presence of tocopherols, tocotrie- nols and squalene [3, 4, 7, 16]. Raw materials rich in unsatu- rated fatty acids usually contain compounds which are protec- tive against oxidation and can be used as a source of antioxi- dants [10, 12]. There is scant information in the literature con- cerning the antioxidant properties of extracts of amaranth seeds, although several phenolic compounds in herbs of amar- anth species were identified, among other phenolic acids [14], rutin and quercetin [6]. The objectives of this study were to investigate the antioxidant activity of ethanolic extracts of amaranth seeds and to develop a combined SPE and RP-HPLC analytical method for separation, identification and quantifica- tion of free phenolic acids in amaranth seeds. 2 Materials and methods Locally cultivated Amaranthus caudatus (A.c.) and A. pani- culatus (A.p.) seeds were obtained from grain distributor PPHU “Szarłat” (Łomz ˙a, Poland). Seeds were dried, commin- uted, defatted with hexane and extracted threefold using 80% aqueous ethanol. Solutions were collected and solvent evapo- rated under reduced pressure at a temperature of 45 8C. The dry residue was dissolved in ethanol (96%). The antioxidant activity of the ethanolic extracts was evaluated by a procedure involving colorimetric measurement of b-carotene bleaching at 470 nm in aqueous emulsion of linoleic acid [18]. Compara- tive samples with the commercial antioxidant butylated hydro- xyanisole (BHA) and controls with any additives were included to the experiment. All experiments were run in tripli- cate and the presented results are the average of two trials. The amount of total phenolic compounds in crude extracts was determined according to the Folin-Ciocalteu procedure using caffeic acid as a calibration standard [13]. The Folin-Ciocalteu reagent was obtained from Sigma Aldrich (St. Louis, MO, USA). Analysis of phenolic acids present in ethanolic extract was performed by RP-HPLC. Crude seeds extracts were puri- fied and phenolic acids isolated on quaternary amine Baker- bond SPE columns [5]. The HPLC analyses were performed on Waters HPLC apparatus equipped with a Nova Pak C 18 col- umn (150 6 3.9 mm, 5 lm; Waters, Milford, MA, USA) and lBondapack C 18 as a guard column. A mobile phase – acetoni- trile (solvent A)/demineralised water containing 20 mL of acetic acid per litre (solvent B) – was used to develop the sol- vent gradient. Gradient of phase A run: 5–15% in 20 min, 15– 20% in 8 min, 20–100% in 2 min, 100% in 5 min; flow rate, 0.6 mL/min. Phenolic acids were identified by comparing their UV spectra and retention times with that of corresponding standards. Quantification of phenolic acids was done at 280 nm. Standard phenolic acids were purchased from Sigma. HPLC analyses were run in triplicate. The results of antioxi- dant activity of ethanolic extracts of amaranth seeds were sta- tistically analysed by two-way analysis of variance (ANOVA). The Tuckey test was used for multicomparison. To compare differences between mean values of phenolic acids, the paired t-test was employed. Significance was declared at P a 0.05. Statistica 5.5 software computer was used for statistical analy- sis (StatSoft, 2000). 184 Nahrung/Food 46 (2002) No. 3, pp. 184 – 186 i WILEY-VCH Verlag GmbH, 69469 Weinheim 2002 0027-769X/2002/0305-0184$17.50+.50/0 Antioxidant activity of ethanolic extracts obtained from two amar- anth species was evaluated in a b-carotene-linoleic acid model system. The addition of amaranth extracts in the range of 0.01–0.1% inhibited degradation of a b-carotene in a model emulsion during incubation at 60 8C; 0.05% addition of amaranth seeds extract was proposed as practi- cally applicable. The total content of phenolic compounds was esti- mated by the Folin-Ciocalteu method and ranged from 39.17 mg/100 g of Amaranthus caudatus to 56.22 mg/100 g of A. paniculatus seeds. Free phenolic acids contained in ethanolic extracts of amaranth seeds were purified and isolated by solid-phase extraction (SPE) and identi- fied by reversed-phase high-performance liquid chromatography (RP- HPLC). The technique involved gave a good separation of the free phe- nolic acids in the amaranth seeds. Significant differences in phenolic acids profiles of both amaranth species were observed. Correspondence: Dr. I. Klimczak, University of Economics, Faculty of Commodity Sciences, Al. Niepodleglosci 10, PL-60-967 Poznan, Poland E-mail: klimczak@novcil.ae.poznan.pl Fax: +4861-853-3993 Abbreviations: BHA, butylated hydroxyanisole; RP, reversed phase; SPE, solid-phase extraction Keywords: Antioxidant activity / Amaranth / Phenolic acids / Solid- phase extraction / Reversed-phase high-performance liquid chromato- graphy