Antioxidant activity of ethanolic extracts of amaranth seeds I. Klimczak, M. Małecka and B. Pachołek Antioxidant activity of ethanolic extracts obtained from two amaranth 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 practically applicable. The total content of phenolic compounds was estimated 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 identified by reversed-phase high-performance liquid chromatography (RPHPLC). The technique involved gave a good separation of the free phenolic acids in the amaranth seeds. Significant differences in phenolic acids profiles of both amaranth species were observed.
1 Introduction
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 quantification of free phenolic acids in amaranth seeds.
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 processing and storage. Compounds with antioxidant activity are widely spread in raw sources of plants origin. They exist in different 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 compounds 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 mechanisms. Thus, plant extract, not single compounds, are of practical 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 amaranth, known in many countries as decorative plant or weed. The interest of its cultivation stems from many valuable properties 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, tocotrienols and squalene [3, 4, 7, 16]. Raw materials rich in unsaturated fatty acids usually contain compounds which are protective against oxidation and can be used as a source of antioxidants [10, 12]. There is scant information in the literature concerning the antioxidant properties of extracts of amaranth seeds, although several phenolic compounds in herbs of amaranth species were identified, among other phenolic acids [14], rutin and quercetin [6]. The objectives of this study were to Correspondence: Dr. I. Klimczak, University of Economics, Faculty of Commodity Sciences, Al. Niepodleglosci 10, PL-60-967 Poznan, Poland E-mail:
[email protected] Fax: +4861-853-3993 Abbreviations: BHA, butylated hydroxyanisole; RP, reversed phase; SPE, solid-phase extraction Keywords: Antioxidant activity / Amaranth / Phenolic acids / Solidphase extraction / Reversed-phase high-performance liquid chromatography 184
2 Materials and methods Locally cultivated Amaranthus caudatus (A.c.) and A. paniculatus (A.p.) seeds were obtained from grain distributor PPHU “Szarłat” (Łomz˙a, Poland). Seeds were dried, comminuted, defatted with hexane and extracted threefold using 80% aqueous ethanol. Solutions were collected and solvent evaporated 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]. Comparative samples with the commercial antioxidant butylated hydroxyanisole (BHA) and controls with any additives were included to the experiment. All experiments were run in triplicate 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 purified and phenolic acids isolated on quaternary amine Bakerbond SPE columns [5]. The HPLC analyses were performed on Waters HPLC apparatus equipped with a Nova Pak C18 column (150 6 3.9 mm, 5 lm; Waters, Milford, MA, USA) and lBondapack C18 as a guard column. A mobile phase – acetonitrile (solvent A)/demineralised water containing 20 mL of acetic acid per litre (solvent B) – was used to develop the solvent 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 antioxidant activity of ethanolic extracts of amaranth seeds were statistically 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 analysis (StatSoft, 2000).
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 amaranth seeds Table 1.
Antioxidant activity of ethanolic extracts from amaranth seeds in model emulsion system (b-carotene-linoleic acid)
Sample
Time (min) 0
20
40
60
80
100
120
Amaranthus paniculatus Control +0.01% A.p. +0.02% A.p. +0.05% A.p. +0.1% A.p. +0.02% BHA
100 100 100 100 100 100
63.12a 91.00b 92.28bc 96.74c 97.00c 97.00c
36.29a 82.00b 85.33b 93.65c 96.30c 96.60c
19.41a 74.55b 80.50c 90.70d 94.15d 96.20d
11.76a 67.75b 75.90c 88.45d 92.05de 95.90e
7.49a 62.40b 72.48c 86.43d 90.20de 95.60e
4.53a 57.45b 69.40c 84.70d 88.60d 95.60e
Amaranthus caudatus Control +0.01% A.c. +0.02% A.c. +0.05% A.c. +0.1% A.c. +0.02% BHA
100 100 100 100 100 100
63.12a 92.00b 95.00b 96.20b 97.66b 97.00b
36.29a 83.00b 89.95bc 94.30c 96.69c 96.60c
19.41a 75.15b 86.10c 93.30cd 96.02d 96.20d
11.76a 67.55b 82.35c 91.85d 95.71d 95.90d
7.49a 61.70b 79.00c 90.45d 94.65d 95.60d
4.53a 56.05b 75.85c 88.91d 94.12d 95.60d
a, b, c, d, e: values within each column followed by the same letter are not significantly different (P a 0.05). Antioxidant activity measured by changes in absorbance values at 470 nm. Absorbances of the samples at beginning of the experiments were set at 100%.
3 Results and discussion For the antioxidant activity evaluation the samples were prepared by mixing the emulsion of b-carotene and linoleate with the amaranth extract at four different concentrations (0.01, 0.02, 0.05 and 0.1% w/w). Samples with BHA added at 0.02% w/w were studied for comparison. Data in Table 1 show that the addition of amaranth extracts in the range of 0.01–0.1% restrained degradation of b-carotene in a model emulsion system during the 120 min of incubation. No distinct differences in antioxidant activity were observed at concentrations between 0.05 and 0.1% of A.p. during the whole time of incubation. BHA (0.02%) was significantly more active in restraining of degradation of b-carotene than A.p. 0.05 and 0.1% only after 120 min of incubation. Antioxidant activities of A.c. 0.05, 0.1% and BHA were similar (P A 0.05) during the whole time of incubation. In order to compare the antioxidant activity of analysed extracts, the antioxidant index was calculated by subtracting the absorbance value of the sample from the control after 120 min of incubation. The effect of different concentrations of analysed extracts of A.p. and A.c. on the antioxidant index is shown in Fig. 1. The increase of the concentration of the extracts led to increase of the antioxidant activity; 0.05% addition of amaranth seeds extract was proposed as practically applicable. Further increase of extract concentration in the model system gave no statistically significant increase of its antioxidant activity. Although the antioxidant activity of A.p. and A.c. at 0.05% concentration statistically did not differ significantly from each other, significant differences were noticed for A.p. in relation to BHA activity, whereas the activity of A.c. was similar to that of BHA. The antioxidant index of BHA (91.07) was significantly stronger than that of A.p. and A.c. at the same concentration (P a 0.05). The total content of phenolics in A.p. and c. were 56.22 l 0.8 and 39.17 l 0.9 mg/100 g of seeds, respectively. The concentration of total phenolic compounds in both amaranth species analysed is lower than in other vegetables and fruits, but it can be compared to total phenolic content in other cereals [8]. Table 2 shows the results of quantitative analysis of phenolic Nahrung/Food 46 (2002) No. 3, pp. 184 – 186
Figure 1. Effect of different concentrations of A.p. and A.c. ethanolic extracts on the antioxidant index. a, b, c: values with the same letter are not significantly different (P a 0.05).
acids composition in the ethanol extracts of A.p. and A.c. In general, significant differences in profiles of phenolic acids of both amaranth species were observed. Protocatechuic acid was the predominant one and covered 34% of the total amount of phenolic acids in A.p. Caffeic acid was the main component of the phenolic acids in A.c. (52%). The gallic and sinapic ones were observed only in A.p. Phenolic acids, such as ferulic, caffeic, p-hydroxybenzoic, p-coumaric, protocatechuic, vanillic and syringic acid, have been found in other cereals [15, 20]. Ferulic acid constitutes more than 90% of the total phenolic acids in durum wheat grains and flour [11, 15]. Ferulic, vanillic and p-coumaric acids occur in the highest amount in durum wheat bran [17]. In barley both ferulic and p-coumaric acid are the predominant [9]. The content of free phenolic acids in analysed samples covered about 53% of the total amount of phenolic compounds in A.p., whereas 27% in A.c. (Table 2). Therefore, the observed antioxidant activity of the amaranth seeds can be on account of the free phenolic acids occurrence. 185
Klimczak et al. Table 2. Content of free phenolic acids in amaranth seeds obtained by application of combined SPE and RP-HPLC method Phenolic acids
A.paniculatus (lg/g seeds)
A. caudatus (lg/g seeds)
Gallic acid Protocatechuic acid p-Hydroxybenzoic acid Caffeic acid p-Coumaric acid (8) Ferulic acid (9) Sinapic acid (10) Salicylic acid (11) Total
40.64 l 1.1 100.92 l 8.7a 15.62 l 1.3a 51.67 l 0.45a 43.57 l 0.9a 40.05 l 1.3a 0.48 l 0.1 2.65 l 0.2a 295.50
– 4.65 l 0.4b 20.89 l 0.8b 55.79 l 0.96b 5.20 l 0.5b 18.41 l 0.8b – 1.92 l 0.2a 106.86
a, b: values in rows column with the same letter are not significantly different (P a 0.05)
Further analyses of the bound phenolic compounds fractions in amaranth seeds are necessary. In conclusion, the present work indicated that ethanolic extracts of the amaranth seeds showed appreciable antioxidant activity in our model system b-carotene and linoleate; 0.05% addition of amaranth seeds extract was proposed as practically applicable. The extracts showed great variation in their HPLC phenolic acid profiles. Based on the results obtained, amaranth seeds could be a potential source of natural antioxidants.
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