11
Radical scavenging activity of leafy amaranths as potential antioxidant sources
Muhammad Ikhsan Sulaiman
1,
, Rita Andini
1
1
Department of Agricultural Processing Technology, Faculty of Agriculture, Syiah Kuala University
Darussalam, Banda Aceh, 23111, Indonesia Corresponding author: misulaimanthp.unsyiah.ac.id
Abstract
Amaranths are popular leafy vegetables in the diet among the people in Asia particularly Indonesia. Previous study exhibited that the
leaves of amaranths were rich in quality protein particularly containing high lysine, which is the limiting essential amino acid in cereals. Fast growing
and high tolerance to drought, marginal land and extreme climate had made this nutritious plant as promising crop for human future. This research
reported the DPPH
radical scavenging activity of leaves of 16 amaranth’s accessions from personal collection and USDA that were grown in polybags.
The leaves were harvested before flowering. Total phenol and inhibition capacity of DPPH free radicals IC
50
as indication for radical scavenging activity were measured by using uv-vis spectrophotometer. The results
indicated that Amaranthus tricolor from USA and A. hybridus from Zimbabwe was the weakest and the strongest IC
50
with 13.83 mgml and 1.30 mgml, respectively. Correlation was not observed between IC
50
and total phenol content. This result highlighted the importance of leafy
amaranth as potential sources of antioxidant. Keywords
Amaranthus, radical scavenging activity, antioxidant
1. Introduction
Amaranths are popular leafy vegetables in the diet among the people in Asia particularly Indonesia. Amaranth is species that spreads widely in tropical
to subtropical regions and found up to 2000 m above sea level [1]. Around 70 species of amaranth have been recorded worldwide harvested as grain,
vegetable, forage, ornamental or grow wild as weed [2]. Increasing amount of amaranth grain has been used in food industries benefiting from its high
level of oil and protein with particularly its excellent amino acids composition [3]. The leaves of amaranths are also good sources of protein
and amino acid especially lysine [4]. Fast growing and high tolerance to drought, marginal land and extreme climate has made this nutritious plant
as promising crop for human future [5].
12 Recently, researches in amaranth tend to explore the compounds in grain
and leaves that have health benefit to human life. Oil extract of amaranth seed contained unsaturated fatty acids, lectins, tocopherols, tocotrienols,
phytosterols, squalene, isoprenoid compounds, aliphatic alcohols, terpene alcohols, and polyphenols, which have properties to improving the
immunity system, decrease pain and inflammation [3]. Grain, leave and flower of amaranths are reported having high antioxidant activity [6,7,8] in
correspondence to the content of phenolic and flavonoid compounds. Interestingly, amaranth leaves exhibited more antioxidant capacity than
those from seeds [9]. Different types of polyphenols in the grain [10] and the leaves of amaranths [11] were recognized as quinic acid, quercetin,
kaempferol, gallic acid, caffeic acid, rutin and ferulic acid. Out of 70 betalain structures 50 betacyanins and 20 betaxanthines, 16 betachyanins
and 3 betaxanthines are found in the family of Amaranthaceae [6]. Betalains are water-soluble pigments having antioxidant, anti-inflammatory,
antibacterial, anticarcinogenic and anti-ageing properties. This article reported DPPH radical scavenging activities of amaranth leaves
of 16 cultivars from USDA and Indonesian collection. The results were expected to provide information on the potential of amaranth leaves as
antioxidant source for human health benefit.
2. Materials and Methods
2.1. Plant materials Amaranth seeds from different types vegetable, grain and weedy and
species Amaranthus blitum, A. caudatus, A. dubius, A. graecizans, A. hybridus
, and A. tricolor were obtained from USDA and personal collection gathered from different part of Indonesia Table 1. The seeds
were cultivated in polybag from August to September 2013 at Syiah Kuala University. Each accession was grown in three polybags as replication
where each of them consisted of three plantlets. Leaves were harvested around 20 up to 50 days after sowing depending on its physiological
maturity.
Table 1. Accession number, species, type and origin of the plant materials
Acc No. Species
Origin Type Acc No.
Species Origin
Type 1
A. blitum Medan
WD 9
A. hybridus Zimbabwe GR
2 A. caudatus Jawa Barat VG
10 A. hybridus Yunani
GR 3
A. cruentus Zimbabwe GR
11 A. tricolor
Banda Aceh VG 4
A. cruentus Zimbabwe GR
12 A. tricolor
India VG
5 A. dubius
Jamaica VG
13 A. tricolor
Taiwan VG
13
Acc No. Species
Origin Type Acc No.
Species Origin
Type 6
A. dubius Takengon
WD 14
A. tricolor Medan
VG 7
A. dubius Takengon
WD 15
A. tricolor Jakarta
VG 8
A. graecizans India VG
16 A. tricolor
USA VG
WD = Weedy, VG = Vegetable, GR = Grain
2.2. Chemicals Methanol, Folin-Ciocalteu reagent, Na
2
CO
3
was obtained from Merck. Gallic acid and DPPH 2,2-diphenyl-1-picrylhydrazyl was from Sigma-
Aldrich Corp. 2.3. Extraction method
Ten grams of fresh leaves were mashed in a mortar directly after harvesting. The pulp of the leaves was poured with 100 ml methanol to an Erlenmeyer
flask for extraction. Extraction was performed by shaking the mixture for three hours using orbital shaker with a rate of 8 rpm. After extraction time,
filtration was done to obtain a filtrate and it was brought for analysis. 2.4. Radical scavenging capacity
Analyses of radical scavenging capacity were performed using the DPPH radical inhibition capacity by antioxidant compounds in the methanol
extracted filtrate [6]. Standard curve was constructed by the reaction between 0, 25, 50 and 100 mg fresh leaves per liter and 0.5 ml 1 mM DPPH
to get a final volume of 4.5 ml. During the reaction time of 3 hours at room temperature, a purple-color stable free radical of DPPH measured
spectrophotometrically at 517 nm would be reduced into a yellow color diphenylpicryl hydrazine as a result of the reaction between the radicals and
antioxidant compounds. DPPH radical scavenging activity was calculated by using the formula: Inhibition = A
– A
1
A x 100. Where A
was the absorbance of the control without filtrate and A
1
was the absorbance in the presence of the filtrate. IC
50
value was calculated from the standard curve indicating the capacity of the antioxidant in mgml to reduce fifty
percent of the DPPH radicals. Reaction and measurement was conducted in darkness.
2.5. Total phenol Total phenolic compounds was analyzed uing Folin-Ciocalteu assay [6].
Briefly, 1 ml Folin-Ciocalteu reagent a mixture of Folin Ciocalteu reagent and methanol, 1:9, 1 ml 20 Na
2
CO
3
and 0.95 ml aquadest was reacted with 50
μ
l methanol extracted amaranth leaves in a cuvette. After vortexing and incubation time of 90 minutes at room temperature, the absorbance was
measured using UV-Vis Spectrophotometer Shimadzu UV1700, Japan at
