Antioxidant activity of ethanolic extracts of amaranth seeds
I. Klimczak, M. Małecka and B. Pachołek
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
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
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
184 Nahrung/Food 46 (2002) No. 3, pp. 184 – 186 i WILEY-VCH Verlag GmbH, 69469 Weinheim 2002
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.
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” (Łomża, 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 C18 col-
umn (150 6 3.9 mm, 5 lm; Waters, Milford, MA, USA) and
lBondapack C18 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).
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 100 63.12a 36.29a 19.41a 11.76a 7.49a 4.53a
+0.01% A.p. 100 91.00b 82.00b 74.55b 67.75b 62.40b 57.45b
+0.02% A.p. 100 92.28bc 85.33b 80.50c 75.90c 72.48c 69.40c
+0.05% A.p. 100 96.74c 93.65c 90.70d 88.45d 86.43d 84.70d
+0.1% A.p. 100 97.00c 96.30c 94.15d 92.05de 90.20de 88.60d
+0.02% BHA 100 97.00c 96.60c 96.20d 95.90e 95.60e 95.60e

Amaranthus caudatus
Control 100 63.12a 36.29a 19.41a 11.76a 7.49a 4.53a
+0.01% A.c. 100 92.00b 83.00b 75.15b 67.55b 61.70b 56.05b
+0.02% A.c. 100 95.00b 89.95bc 86.10c 82.35c 79.00c 75.85c
+0.05% A.c. 100 96.20b 94.30c 93.30cd 91.85d 90.45d 88.91d
+0.1% A.c. 100 97.66b 96.69c 96.02d 95.71d 94.65d 94.12d
+0.02% BHA 100 97.00b 96.60c 96.20d 95.90d 95.60d 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 pre-
pared 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 sys-
tem 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 incu-
bation. BHA (0.02%) was significantly more active in restrain-
ing 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 Figure 1. Effect of different concentrations of A.p. and A.c. ethanolic
extracts, the antioxidant index was calculated by subtracting extracts on the antioxidant index. a, b, c: values with the same letter
the absorbance value of the sample from the control after are not significantly different (P a 0.05).
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 acids composition in the ethanol extracts of A.p. and A.c. In
extracts led to increase of the antioxidant activity; 0.05% addi- general, significant differences in profiles of phenolic acids of
tion of amaranth seeds extract was proposed as practically both amaranth species were observed. Protocatechuic acid was
applicable. Further increase of extract concentration in the the predominant one and covered 34% of the total amount of
model system gave no statistically significant increase of its phenolic acids in A.p. Caffeic acid was the main component of
antioxidant activity. Although the antioxidant activity of A.p. the phenolic acids in A.c. (52%). The gallic and sinapic ones
and A.c. at 0.05% concentration statistically did not differ sig- were observed only in A.p. Phenolic acids, such as ferulic, caf-
nificantly from each other, significant differences were feic, p-hydroxybenzoic, p-coumaric, protocatechuic, vanillic
noticed for A.p. in relation to BHA activity, whereas the activ- and syringic acid, have been found in other cereals [15, 20].
ity of A.c. was similar to that of BHA. The antioxidant index Ferulic acid constitutes more than 90% of the total phenolic
of BHA (91.07) was significantly stronger than that of A.p. acids in durum wheat grains and flour [11, 15]. Ferulic, vanil-
and A.c. at the same concentration (P a 0.05). lic and p-coumaric acids occur in the highest amount in durum
The total content of phenolics in A.p. and c. were 56.22 l wheat bran [17]. In barley both ferulic and p-coumaric acid are
0.8 and 39.17 l 0.9 mg/100 g of seeds, respectively. The con- the predominant [9]. The content of free phenolic acids in ana-
centration of total phenolic compounds in both amaranth spe- lysed samples covered about 53% of the total amount of phe-
cies analysed is lower than in other vegetables and fruits, but it nolic compounds in A.p., whereas 27% in A.c. (Table 2).
can be compared to total phenolic content in other cereals [8]. Therefore, the observed antioxidant activity of the amaranth
Table 2 shows the results of quantitative analysis of phenolic seeds can be on account of the free phenolic acids occurrence.

Nahrung/Food 46 (2002) No. 3, pp. 184 – 186 185

Klimczak et al.

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186 Nahrung/Food 46 (2002) No. 3, pp. 184 – 186