Journal of Medicinal Plants Research Vol. 6(27), pp.

4450-4455, 18 July, 2012

Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.822
ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Antioxidant and antimicrobial activities of Chowlai

(Amaranthus viridis L.) leaf and seed extracts
Muhammad Javid Iqbal1*, Sumaira Hanif1, Zahed Mahmood1, Farooq Anwar2 and Amer Jamil1
1
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan.
2
Department of Chemistry, University of Sargodha, Sargodha-40100, Pakistan.
Accepted 19 June, 2012

The present study was conducted to evaluate the phenolics, antioxidant and antimicrobial activities of
leaf and seed extracts from an edible herb namely Amaranthus viridis L. The extract yields of active
components, produced using pure and aqueous methanol, from the leaves and seeds ranged from 5.4
to 6.0 and 2.4 to 3.7%, respectively. The extracts contained appreciable levels of total phenolic contents
(1.03 to 3.64 GAE, g/100 g) and total flavonoid contents (18.4 – 5.42 QE, g/100 g) and also exhibited
good 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity as revealed by IC50 (14.25 - 83.43
µg/ml). Besides, the tested extracts showed considerable antimicrobial activity against selected
bacterial and fungal strains with minimum inhibitory concentrations (MIC) ranging from 179-645 µg/ml.
Of the parts tested, the seed extracts exhibited superior antioxidant and antimicrobial activity. It was
concluded that A. viridis leaf and seed can be explored as a potential source for isolation of antioxidant
and antimicrobial agents for uses in functional food and pharmaceuticals.

Key words: Amaranthus viridis, phytochemical constituents, minimum inhibitory concentrations (MIC), total
phenolics, total flavonoids, 1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavengers.

INTRODUCTION

Amaranthus viridis L. (Amaranthaceae), commonly past few years because of their preventive role in
known as “Chowlai”, is a fast growing herb mainly protecting from oxidative-stress related chronic diseases
cultivated in Asia, Africa and Latin America (Amin et al., (Halovrson et al., 2002).
2006). Being resistant to drought, hot climate and pests, Existence of microorganisms causes food spoilage and
and with little requirements for its cultivation, this pseudo- results in deterioration of the quality and quantity of
cereal has attracted much attention as an important food processed food products. Some plant-based biologically
commodity (Sexna et al., 2007). In the last decade, the active compounds isolated from herbs have been
use of amaranth has expanded not only in the common explored for the growth inhibition of pathogenic microbes
diet, but also in diet of people with celiac disease or because of their antimicrobial potential (Abubakar et al.,
allergies to typical cereals (Berti et al., 2005). Reactive 2008). The medicinal value and multiple biological
oxygen species (ROS) and reactive nitrogen species functionalities of several plants are defined by their
produced as a result of oxidation have been shown to be phytochemical constituents (Fallah et al., 2005). Many
linked with different degenerative disorders such as herbal species being a promising source of bioactive
aging, inflammation, cancer, cardiovascular compli- compounds such as phenolics, anthocyanins, flavonoids,
cations, and osteoporosis (Wilcox, 2004). Interest in and carotenoids, are usually used to impart flavor and
search for new natural antioxidants has grown over the enhance the shelf-life of dishes and processed food
products, recently reported work was (Nisar et al., 2010a,
b, 2011; Qayum et al., 2012; Zia-Ul-Haq et al., 2008,
2011a, b, 2012). Due to their high antioxidant potency,
*Corresponding author. E-mail: [email protected] or the consumption of many such plants species is
[email protected]. recommended (Ozsoy et al., 2009). Antioxidant
 Iqbal et al. 4451

properties of green leafy vegetables and herbs including were determined following the modified procedure of Edeoga et al.
different amaranth species have been preliminarily (2005), and quercetin was used as standard as quercetin
equivalent (QE).
studied (Ozbucak et al., 2007).
The main aim of the present study was to evaluate the
phenolic compounds, antioxidant and antimicrobial DPPH radical scavenging assay
activities of pure and aqueous methanol extracted
components from leaves and seeds of locally grown 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay was carried out
Amaranthus viridis plants to explore their potential spectrophotometrically (Miliauskas et al., 2004). The percent
inhibition was calculated as:
pharmaceutical and/or functional food uses.
I (%) = 100 × (Ablank __ Asample / Ablank)
MATERIALS AND METHODS Where Ablank is absorbance of the control reaction (containing all
reagents except the test sample), and Asample is the absorbance of
Collection and pretreatment of plant material test samples. Extract concentrations providing 50% inhibition (IC50)
values were calculated from the plot of percentage scavenging
The leaves and seeds of fully matured A. viridis L. were collected versus extracts concentration.
during June to July 2009, from the local fields of Faisalabad,
Pakistan, and identified by the Department of Botany, GC
University Faisalabad, Pakistan. Collected specimens were dried at Antimicrobial activity
room temperature and stored in polyethylene bags at 4°C.
Microbial strains

Chemical and reagents The A. viridis leaf and seed extracts were individually tested against
a panel of microorganisms (locally isolated), including two bacteria,
1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, Folin-Ciocalteu Staphylococcus aureus and Escherichia coli, and two pathogenic
reagent, sodium nitrite, butylated hydroxytoluene (BHT) were fungi, Fusarium solani and Rhizopus oligosporus. The selected
purchased from Sigma Chemical Co (St. Louis, USA) and strains have strong pathogenic activities against plant and animals
anhydrous sodium carbonate, methanol and ethanol used were that lead to a significant loss of lives and food. The pure bacterial
obtained from Merck (Darmstadt, Germany). All culture media and fungal strains were obtained from the Bioassay section, Protein
antibiotic, discs and sterile solution of 10% (v/v) DMSO in water Molecular Biochemistry laboratory, Department of Chemistry and
were purchased from Oxoid (Hampshire, UK). Biochemistry, University of Agriculture Faisalabad, Pakistan. The
bacterial strains were cultured at 37°C overnight, w hile fungal
strains were cultured overnight at 28°C in an incuba tor (Memmert,
Preparation of A. viridis extracts Germany).

Ground (80 mesh) leaf and seed samples (100 g each) were
extracted separately with 1000 ml absolute methanol and 80% Disc diffusion method
methanol (80:20, methanol: water, v/v) using an orbital shaker
(Gallenkamp, UK) for 12 h at room temperature. The extracts were The antimicrobial activity of the leaf and seed extracts was
separated from solids by filtering through Whatman No. 1 filter determined by using disc diffusion method (CLSI, 2007). The discs
paper. The residues were extracted thrice and the extracts were (6 mm in diameter) were impregnated with 20 µg/ml sample
collected. The solvent was removed under vacuum at 45°C, using a extracts (20 µg/disc) and placed on inoculated agar. Rifampicin (20
rotary vacuum evaporator (N-N Series, Eyela, Rikakikai Co. Ltd. µg/disc) (Oxoid) and fulconazole (20 µg/disc) (Oxoid) were used as
Tokyo, Japan) and stored at 4°C till further analysis. positive reference for bacteria and fungi, respectively. Antimicrobial
activity was evaluated by measuring the inhibition zones (in mm) by
zone reader.
Phytochemical screening

The methanol extracts of the tested plant material were screened Minimum inhibitory concentration (MIC) assay for
for the presence of various phytoconstituents such as determination of antimicrobial activity
phlobatannins, tannins, alkaloids, terpenoids, glycosides,
flavonoids, and phenolic compounds (Abubakar et al., 2008). Incubator at 35 and 37°C; pipettes of various sizes (G ilson); sterile
tips, 100, 200, 500 and 1000 µL; 5 ml multi-channel pipette;
centrifuge tubes; vortex mixer; centrifuge (Fisons); Petri-dishes,
Antioxidant activity sterile universal bottles; UV-spectrophotometer (Shimadzu) and
sterile resazurin tablets (BDH Laboratory Supplies) were used.
Determination of total phenolic contents (TPC) Isosensitest medium was used throughout this assay, as it is pH
buffered. Although the use of Mueller Hinton medium was
Total phenolic contents (TPC) were determined using the Folin- recommended for susceptibility testing (NCCLS, 2000), the
Ciocalteu reagent method and gallic acid was used as gallic acid isosensitest medium had comparable results for most of the tested
equivalent (GAE) (Amin et al., 2006). bacterial strains.

Determination of total flavonoid contents (TFC) Use of standardized bacterial colony numbers

The total flavonoid contents (TFC) in the leaf and seed extracts The method wherein turbidity is compared to McFarland standards
4452 J. Med. Plants Res.

usually 0.5, is not able to give a standardized number of CFU for measurement. Where appropriate, the data were tested by one-way
bacterial strains only because this is operator-driven and is thus ANOVA using Minitab 15. Pearson correlation coefficients and p-
subjective. It also makes it difficult to compare different bacterial values were used to show correlations and their significance.
species as they have differing optical densities. A final Differences of P<0.05 were considered significant (Steel et al.,
concentration of 5 × 105 CFU/ml was adopted for this assay. Thus, 1997).
different strains and different bacterial species could be compared.

Preparation of microbial culture RESULTS AND DISCUSSION

Using aseptic techniques, a single colony was transferred into a
Extract yields
100 ml bottle of isosensitest broth capped and placed in incubator
overnight at 35°C. After 12 to 18 h of incubation, u sing aseptic
preparation and the aid of a centrifuge, clean samples of bacteria Yields (g/100 g) of A. viridis leaf and seed extracts
and fungi were prepared. The broth was spun down using a produced using different extraction solvents are given in
centrifuge at 4000 rpm for 5 min with appropriate aseptic Table 1. Maximum extract yields of antioxidant
precautions. The supernatant was discarded into an appropriately
components from leaves and seed were obtained with
labeled contaminated waste beaker. The pellet was re-suspended
using 20 ml of sterile normal saline and centrifuged again at 4000 80% methanol (6.0 g/100 g) and 100% methanol (3.7
rpm for 5 min. This step was repeated until the supernatant was g/100 g), respectively. The present results demonstrated
clear. The pellet was then suspended in 20 ml of sterile normal a significant (P<0.05) variations for the seed extract
saline and was labeled as Bs. The optical density of the Bs was yields, but non-significant (P>0.05) for the leaves extracts
recorded at 500 nm, and serial dilutions were carried out with yields between the solvents used. The present extraction
appropriate aseptic techniques until the optical density was in the
range of 0.5 to 1.0. The actual number of colony forming units was
yields are lower than those reported by Ozsoy et al.
calculated from the viability graph. The dilution factor needed was (2009) in water, methanol and ethyl-acetate extracts of
calculated and the dilution was carried out to obtain a concentration Amaranthus species (13.09 to 20.46 g/100 g). Kyung et
of 5 × 106 CFU/ml (Winn and Koneman, 2006). al. (2006) also reported the strong antioxidant and
antimicrobial activity of Amaranthus cruentus in diabetic
Preparation of resazurin solution rat.

The resazurin solution was prepared by dissolving a 270 mg tablet

in 40 ml of sterile distilled water. A vortex mixer was used to
dissolve tablet and homogenous solution formation. Phytochemical constituents

Preparation of the plates

In plants, the secondary metabolites function to attract
beneficial and repel harmful organisms, serve as
Plates were prepared under aseptic conditions. A sterile 96 well phytoprotectants and respond to environmental changes.
plate was labeled. A volume of 100 µL of test material in 10% (v/v) In humans, however, the compounds have beneficial
dimethylsulphoxide (DMSO)/sterile water (10 mg/ml for crude effects including antioxidant, anti-inflammatory, modu-
extracts) was pipetted into the first row of the plate. To all other
wells, 50 µL of nutrient broth or normal saline was added. Serial lation of detoxification enzymes, stimulation of the
dilutions were performed using a multichannel pipette. Tips were immune system, modulation of steroid metabolism and
discarded after use such that each well had 50 µL of the test antibacterial and antiviral effects (Lipkin et al., 2004).
material in serially descending concentrations. To each well, 10 µL Table 2 shows the qualitative data for the presence of
of resazurin indicator solution was added. Furthermore, using a different phytochemicals in leaves and seeds of A. viridis.
pipette, 30 µL of 3.3 × strength isosensitised broths was added to
The results from the current study indicate that A. viridis
each well to ensure that the final volume was single strength of the
nutrient broth. Finally, 10 µL of bacterial suspension (5 × 106 CFU/ leaves and seed extracts contained varied types of
mL) was added to each well to achieve a concentration of 5 × 105 pharmacologically active compounds with antimicrobial
CFU/ml. Each plate was wrapped loosely with cling film to ensure potential. The commonly identified components in this
that bacteria did not become dehydrated. Each plate had a set of species included flavonoids, cyanogenic glycoside,
controls: a column with a broad-spectrum antibiotic as positive saponins, tannin, and phlobatannins. The results,
control (usually ciprofloxacin in serial dilution), a column with all
solutions with the exception of the test compound, and a column
especially for alkaloids, indicate levels comparatively
with all solutions with the exception of the bacterial solution adding similar to many medicinal plants (Elizbith et al., 1999;
10 µL of nutrient broth instead. The plates were prepared in Okwu and Josiah, 2006), thus supporting the medicinal
triplicate, and placed in an incubator set at 37°C fo r 18 to 24 h. The uses of this species.
color change was then assessed visually. Any color change from
purple to pink or colorless was recorded as positive. The lowest
concentration at which color change occurred was taken as the MIC
value. The average of three values was calculated and that was the Antioxidant activity
MIC for the test material and microbial strain.
DPPH radical scavenging assay
Statistical analysis
We investigated DPPH free radical scavenging activity of
Values are given as means ± standard deviation (SD) of each A. viridis leaves and seeds extracts produced through
 Iqbal et al. 4453

Table 1. Percentage yield extracts from leaves and seeds of Amaranthus viridis.

Plant used Extract Percent yield (g/100 g)

a
100% Methanol 5.4 ± 0.13
Leaves a
80% Methanol 6.0 ± 0.14

ab
100% Methanol 3.7 ± 0.11
Seeds b
80% Methanol 2.4 ± 0.04
Values are mean ± SD of three samples analyzed individually in triplicate. Different letters in superscript indicate significant and
non-significant differences with solvents.

Table 2. Phytochemical constituents of leaf and seed extract of Amaranthus viridis.

Amaranthus viridis
Phytochemical constituent
Leave Seed
Tannins + +
Phlobatannins + +
Saponins - -
Flavonoids + +
Terpenoids - -
Cardiac glycosides + +
+Represents presence of the phytoconstituents; represents absence of the phytoconstituents.

Table 3. Antioxidant activity of Amaranthus viridis leaf and seed methanolic extracts.

Plant used Extracts DPPH, IC50 (µg/ml) TP contents* TF contents**

a a b
100% Methanol 14.25 ± 0.712 1.03 ± 0.5 2.78 ± 0. 20
Leaves a a a
80% Methanol 83.43 ± 3.84 2.89 ± 0.2 18.4 ± 0. 30

a a a
100% Methanol 46.50 ± 2.97 3.64 ± 0.4 5.42 ± 0. 20
Seeds a a b
80% Methanol 75.91 ± 3.03 3.20 ± 0.39 2.51 ± 0.07
Values are mean ± SD of samples analyzed in triplicate. *, Total phenolic contents in gallic acid equivalent; **, Total flavonoid
contents in quercetin equivalent.

extraction by pure and aqueous methanol. The free reported previously (Amin et al., 2006). The betalians
radical scavenging capacity increased with increasing from plants in the family Amaranthaceae exhibit strong
extracts concentrations. The leaves and seed extracts antiradical activity, with IC50 values ranging from 3.4 to
showed good hydrogen-donating ability in the presence 8.4 µM, and representing a new class of dietary
of DPPH stable radicals (Table 3), with IC50 (the extract antioxidants (Cai et al., 2005). These results showed that
concentration providing 50% inhibition) values ranging the methanol leave extract contained strongest DPPH
from 14.25 – 83.43 and 46.50 – 75.91 µg/ml, free radical scavenging compounds; the efficacy of those
respectively. When compared with the synthetic anti- was quite comparable with the positive control BHT (15.7
oxidant BHT (15.7 µg/mL), the tested extracts offered µg/ml).
slightly lower activity except 100% methanol leaf extract
(14.25 µg/ml).
These results were consistent with previous Total phenolic and total flavonoid contents
observation that Amaranthus varieties contained radical
scavenging agents that could directly react with and The total phenolic content (TPC) and total flavonoid
quench stable DPPH radicals (Oboh, 2005). The ability of content (TFC) of A. viridis leaf and seed extracts are
an Amaranthus paniculatus extracts to act as a free presented in Table 3. The differences in the amount of
radical scavenger or hydrogen donor has also been TP and TF may be due to varied efficacy of the extracting
4454 J. Med. Plants Res.

Table 4. Antimicrobial activity and minimum inhibitory concentration of Amaranthus viridis leaf and seed methanolic extracts against the selected strains of bacterial and fungal
strain.

S. aureus E. coli F. solani R. oligosporus

Part of plant used Extracts
Zone size MIC Zone size MIC Zone size MIC Zone size MIC
ab
100% Methanol 24 ± 1.9 179 ± 1.28ab 16 ± 1.4a
398 ± 1.26a 17 ± 1.8 ab
436 ± 1.46 c
19.0 ± 1.4 302 ± 1.36c
Leaves
80% Methanol 23 ± 2.4ab 182 ± 1.68ab 12 ± 0.3b 603 ± 2.36b 15 ± 2.2b 491±1.76b 18.0b ± 0.9 352 ± 2.42b

100% Methanol 16 ± 2.1c 428 ± 1.36c 11 ± 1.5b 639 ±1.26b 13 ± 1.7c 602 ± 1.89c 15.0bc ± 1.0 482 ±1.27bc
Seeds
80% Methanol 18 ± 1.5c 403 ± 2.36c 10 ± 1.2b 645 ±1.48b 10 ± 1.5c 641 ± 2.38c 13.0c ± 1.6 547 ± 2.38c

Control Methanol 26 ± 3.1a 141 ± 1.31a 17 ± 1.7a 381 ± 2.39a 18 ± 1.9a 391 ± 2.48a 16a ± 2.1 436 ± 2.17a
Values are mean ± SD of three samples analyzed individually in triplicate. Diameter of inhibition zone (mm) including disc diameter of 6 mm and MIC in µg/ml. Controls used are
rifampicin and fulconazole for bacterial and fungal strains, respectively. Different letters in superscript indicate significant differences within solvents.

solvents to dissolve endogenous compounds. E. coli was most sensitive microbe tested, A. viridis leaves and seeds extracts contained
The ability of different solvents to extract TP showing the largest inhibition zones (24 mm) for varied types of pharmacologically active
and TF contents was of the order: for leaf 80%> leaves and minimum (18 mm) for seeds compounds with antioxidant and antimicrobial
100% methanol and for seed 100% > 80% extracts. The least activity is inhibited by 80% activities which differed between the two parts
methanol. These values were higher than the methanol seeds extract against R. oligosporus, and extraction solvents used. Further research
reported values of A. cruentus (0.3 g/100 g) with the smallest zone (13 mm). work involving more detailed in vitro and in vivo
(Nisimba et al., 2007). Gorinstien et al. (2007) In general, the antimicrobial activity of the investigations to establish which component of
showed from the values obtained in their work, tested A. viridis leaves and seeds extracts was the extracts offer best antioxidant and
less phenolic content compared to the four comparable with the standard drugs, antimicrobial activity is needed. Detail toxi-
reported Amaranthus verities (107 g/kg). streptomycin and mecanozol. In support to our cological studies are also recommended to
Amaranth plants have been reported as one of present data, in a previous study, isolation of explore the uses this plant extracts as natural
many vegetables rich in antioxidant compounds the antifungal peptide from the A. viridis seed food preservative. The production of bioactive
(Obadoni and Ochuko, 2001). The other extracts has been done (Lipkin et al., 2004). components from such indigenous resources
reported total phenolic contents (TPC) for Resazurin is an oxidation-reduction indicator and their utilization as potential natural food
Amaranthus species ranged from 2.95 - 3.75 used for the evaluation of cell growth. The preservatives could be of high economic value.
GAE, mg/100 g (Pasko et al., 2009). effectiveness of resazurin oxidation compound
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