ORIGINAL PAPER

Analysis and Enhancement of Nutritional and Antioxidant

Properties of Vigna aconitifolia Sprouts
Rakesh M. Kestwal & Dipali Bagal-Kestwal &
Been-Huang Chiang
#Springer Science+Business Media, Inc. 2012
Abstract Vigna aconitifolia sprouts (Moth bean sprouts,
MBS) were analyzed for their nutritional and antioxi-
dant properties during sprouting. Sprouting for six days
led to a 7.0 fold increase in fresh weight, 2.4 fold
increase in soluble proteins, 3.0 fold increase in carbo-
hydrates, and a 5.5 fold increase in mineral content.
Phenolic content also increased by 28% during germi-
nation. Caffeic acid, ferulic acid, cinnamic acid and
kaempferol were the predominant phenolic compounds
detected in the ethanolic extracts of MBS by HPLC.
Following supplementation with metal ions (200 g ml
1
),
the sprouts demonstrated a considerable increase in metal
ion uptake, with improved phenolic content. MBS etha-
nolic extracts also reduced intracellular oxidative stress in
HepG2 cells.
Keywords Vigna aconitifolia sprouts
.
Metal ions
.
Antioxidant
.
HepG2
Abbreviations
HPLC High performance liquid chromatography
MBS Moth bean sprouts
R
f
Retention factor
SOD Superoxide dismutase
Introduction
Sprouts contain a wide variety of antioxidant compounds,
which in turn provide protection against oxidative damage.
Soybean sprouts contain phenolic compounds, such as caf-
feic acid, ferulic acid, and p-coumaric acid. As a result,
demand for soybean sprouts has been increasing, because
of their potential health promoting function [1]. Broccoli
sprouts, rich in indole compounds and sulforaphane are
efficient inhibitors of rat tumor genesis and UV induced
skin carcinogenesis in mice [2]. Sprouting of seeds can also
lead to a decrease in the concentration of anti-nutrition
factors, such as trypsin inhibitors, phytic acid and saponins,
contributing to their nutritional value [3].
Supplementing the nutrient medium (water/soil) of
sprouting seeds can be of great benefit in improving the
nutritional quality of the sprouts. An increase in total phe-
nolic content and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
based antioxidant activity was observed in pea seedlings
when grown in media supplemented with folic acid and
vitamin-C [4]. Recently, it has been reported that supple-
menting cruciferous sprouts with sulphur salt can increase
glucosinolate content, which in turn enhances anticancer
activity [5].
Vigna aconitifolia (Jacq.) Marchal (Moth bean) is a hot-
weather legume, belonging to the family Fabaceae. The
seeds and sprouts of this plant are rich in protein and other
elements, which make them an excellent supplement to
cereal-based diets [6]. However, changes in biochemical
profiles and antioxidant properties that occur during sprout-
ing have not been fully explored. Therefore, in the present
study, we examined the changes in composition occurring
during sprouting of MBS. Additionally, metal ion uptake by
MBS was investigated to explore their potential as antioxi-
dant and mineral rich functional food.
Electronic supplementary material The online version of this article
(doi:10.1007/s11130-012-0284-2) contains supplementary material,
which is available to authorized users.
R. M. Kestwal
:
D. Bagal-Kestwal
:
B.-H. Chiang (*)
Institute of Food Science and Technology,
National Taiwan University,
No. 1, Roosevelt Road, Sec. 4,
Taipei, Taiwan, Republic of China
e-mail: bhchiang@ntu.edu.tw
Plant Foods Hum Nutr
DOI 10.1007/s11130-012-0284-2
Materials and Methods
Effect of Cultivation Time on Protein, Carbohydrate,
Mineral and Phenolic Content
Vigna aconitifolia (Moth bean) seeds were obtained from
Pune, Maharashtra, India. Seeds sprouting were performed
in a Phytotron facility. Both aqueous and ethanolic extracts
were prepared from moth bean sprouts (MBS) [5]. For
aqueous extract preparation, the harvested MBS were ho-
mogenized with phosphate buffer (pH 7.4) (4 ml g
1
seeds
or sprouts). Later, the homogenate was kept for stirring for
extraction under chilled conditions for 1 h. The homogenate
was then centrifuged at 10,000 rpm for 15 min at 4 C and
the supernatant was preserved at 20 C until further use.
For ethanolic extract preparation, fine powder of dehydrated
sprouts was extracted with 95% ethanol (1:30, w/v) at
30 C under constant shaking at 100 rpm overnight. The
supernatant was preserved and the pellet was re-extracted
with ethanol. The two supernatants were combined together,
filtered through Whatman No. 1 filter paper, and concen-
trated in a rotary vacuum evaporator. The residue (etha-
nolic extract) was weighed and stored at 20 C until further
use. The soluble protein and carbohydrate content of the
MBS aqueous extracts were analyzed using the methods
described by Kestwal et al. [7]. The phenol sulphuric acid
method was used to determine the carbohydrate content,
using glucose as standard. Mineral content estimation was
performed using the Association of Official Analytical
Chemists method (AOAC 940.26) [8]. The phenolic content
of ethanolic extract was analyzed using FolinCiocalteus
reagent as described by Burguieres et al. [4] using gallic acid
as standard.
HPLC Analysis of Phenolics
Pure HPLC standard compounds were purchased from
Sigma-Aldrich (St. Louis, MO). Moth bean ethanolic
extracts (50 mg ml
1
) and standards (10 mg ml
1
) were
prepared in methanol and filtered through 0.45 m sy-
ringe filters. Agilent 1100 series HPLC system equipped
with Hypersil Gold C18 column (25005254630: 250
4.6 mm, 5 m) was used for analysis. The mobile phase
consisted of phase A: 2% acetic acid in water and phase
B: 0.5% acetic acid in water and acetonitrile (50:50, v/v).
The gradient program was as follows: 0% B to 75% B
(50 mins), 75% B to 100% B (1 min), and 100% B to
100% B (8 mins) at a flow rate of 1 ml min
1
with 5 L
sample injection volume. MBS extracts and standards
were analyzed at 254 nm using a diode array detector
(DAD) and the retention times were used to calculate R
f
(retention factor) values using pyrogallol as an internal
standard.
Metal Ion Uptake Studies of Sprouts
An effort was made to supplement MBS with different metal
ions (NaCl, KCl, CaCl
2
.2H
2
O, CuCl
2
, MgCl
2
.6H
2
O,
MnCl
2
.4H
2
O, FeCl
2
.4H
2
O and ZnCl
2
) to increase their nu-
tritional value. MBS were grown at 200 and 500 g ml
1
metal ion concentrations and their metal ion uptake was
studied using a Hitachi 18030 atomic absorption spectro-
photometer (AAS). Acid digestion of lyophilized MBS was
performed using Hotplate aqua regia method [9].
Intracellular Superoxide Ion Estimation in HepG2 Cells
Metal ion supplemented MBS were also analyzed for
their antioxidant properties. Intracellular superoxide
anions in HepG2 cells were measured using the fluores-
cent compound dihydroethidium at 605 nm. The cells
were cultured in 60 mm diameter plates at a concentration of
410
6
per plate and incubated overnight at 37 C in 5% CO
2
.
They were treated with ethanolic extract from MBS
(0.2 mg ml
1
) for 6 h, followed by the addition of 2 ml of
10 M dihydroethidium and incubation for 30 min. The cells
were washed and resuspended in 1 ml PBS for analysis.
Superoxide anion measurements were performed using
FACSCalibur flow cytometer; Becton Dickinson, with the
Cell-Quest software.
Intracellular Superoxide Dismutase Activity
HepG2 cells treated with MBS extracts were also analyzed
for intracellular antioxidant superoxide dismutase (SOD)
levels. SOD activity was determined using Cayman chemi-
cal company kit (Ann Arbor, MI) according to the manu-
facturers directions. Values for each treatment group were
expressed as a percentage of the control value.
Statistical Analysis
Statistical analysis was performed using two-way ANOVA
and Tukeys multiple comparison test (SAS Institute Inc.,
Cary, NC) to determine significant differences among means
(P<0.05).
Results and Discussion
Influence of Sprouting on Nutritional Content
The growth profile and antioxidant activities of the MBS
were monitored at regular time intervals for 10 days. Fresh
weight gain was very rapid during the first six days (25 g to
174 g per 25 g of seeds, > 6 fold) (Online Resource 1a).
Initial weight gain can be attributed to water uptake and later
Plant Foods Hum Nutr
on the increase in metabolic activity and synthesis of bio-
mass by the germinating seedlings. A rapid increase in
soluble proteins was observed during the first two days
(1,315 to 2,740 mg per 25 g of seeds, > 2 fold). This is
because during germination the storage proteins found in the
protein bodies of the seeds are broken down, releasing
hydrolytic products including free amino acids, albumin
and globulin fractions. However, after four days, no sig-
nificant increase in protein content was observed (Online
Resource 1a), which is the same as that observed in millet
seeds [10].
In MBS, an increase in total soluble carbohydrate was
observed during the first two days of sprouting (Online
Resource 1b), which may be due to the hydrolysis of the
stored starch for cellular metabolic activity during germina-
tion. Further increase in soluble carbohydrate content (800
to 1,200 mg per 25 g of seeds) may be due to the photo-
synthetic activity of the sprouts. Similar results have been
reported in germinating amaranth seeds [11]. An increase
in total mineral (ash) content was also observed (0.8 to
6.5 mg per 25 g of seeds) in the MBS during sprouting
(Online Resource 1b). Further, after day 4, due to gen-
eration of the plumule and radicle and uptake of min-
erals from the soil, there was a continuous increase in
mineral content [12].
Fig. 1 Changes in phenolic content during moth bean sprouting. Values
followed by different letters are significantly different (P<0.05) by
Tukeys multiple comparison test (n03)
Fig. 2 HPLC profiles of
standards and moth bean
ethanolic extract at 254 nm: (2a:
Standards and 2b: Moth bean
ethanolic extract). The peak
numbers refer to the following
phenolic compounds detected:
(1) Gallic acid, (2) Pyrogallol
(internal standard), (3) Vanillic
acid, (4) Caffeic acid, (5)
p-Coumaric acid, (6) Ferulic
acid, (7) Rutin, (8) Cinnamic
acid, (9) Quercetin and (10)
Kaempferol
Plant Foods Hum Nutr
Change in Total Phenolic Content during Sprouting
The ethanolic extracts of MBS were analyzed for phenolic
content on a daily basis. During sprouting a slight decrease
in phenolic content was observed during the first two days,
followed by a gradual increase and finally a plateau on day 5
(Fig. 1). The increase may be due to photosynthetic activity
in the sprouts leading to the synthesis of different phyto-
chemicals including polyphenolic compounds. Similar
trends have been observed in Vigna radiate and Glycine
max sprouts [13]. These results indicate that sprouting for
six days can be a very effective means of increasing the
nutritional quality of the sprouts including their protein,
mineral, carbohydrate and phenolic content. Hence, six days
of sprouting was carried out for all further experiments.
HPLC Profile of Phenolic Compounds
HPLC analysis was performed to identify phenolic com-
pounds contributing to the antioxidant properties of MBS.
As shown in Fig. 2, caffeic acid, ferulic acid, cinnamic acid
and kaempferol were identified as key phenolic compounds
in MBS by comparing the retention time and R
f
values with
that of the standards (Online Resource 2). To date, there
have been no published reports on the phenolic compounds
present in moth bean seeds or sprouts. However, it has been
reported that vanillic acid, p-coumaric acid, caffeic acid,
ferulic acid, sinapic acid, kaempferol, quercetin, isorhamnetin,
apigenin and k-3-rutinoside are the predominant phenolic
compounds present in other Vigna species [14].
Metal Ion Uptake by MBS
Following supplementation with 200 g ml
1
of metal
ions, high uptake was observed for Fe
2+
(10.80 fold),
Cu
2+
(8.96 fold), Mn
2+
(6.59 fold) and Zn
2+
(6.10 fold)
(Table 1). However, uptake was relatively low for K
+
(1.94 fold), Na
+
(1.80 fold), Mg
2+
(1.29 fold) and Ca
2+
(1.08 fold). Supplementation of metal ions in the growth
medium at optimum concentration can increase the metal
ion content of MBS, thus enhancing their nutritional value.
Previous attempts have involved the addition of metal ion
supplementation to fertilizers to benefit plant growth and
prevent plant chlorosis [15].
Table 1 Estimation of metal ion
uptake by moth bean sprouts
grown in the media supple-
mented with various types of
metal ions
1
No metal ion supplementation
for the control group
2
A concentration
of 200 g ml
1
of metal ion was
added to the cultivating medium
for the treatment group
Metal ion
supplemented
Type of salt used for
supplementation
Concentration of
metal ion in controls
(g ml
1
)
1
Concentration of metal
ion in treatment
(g ml
1
)
2
Fold increase
in uptake
Sodium NaCl 90454 162933 1.80
Potassium KCl 124837 2418169 1.94
Calcium CaCl
2
.2H
2
O 1938116 2098147 1.08
Copper CuSO
4
.5H
2
O 248 21517 8.96
Magnesium MgCl
2
.6H
2
O 15574623 20058602 1.29
Manganese MnCl
2
.4H
2
O 372 2449 6.59
Iron FeCl
2
.4H
2
O 302 32416 10.80
Zinc ZnCl
2
722 43939 6.10
Table 2 Phenolic content of
moth bean sprouts and their
antioxidant properties at cellular
levels in HepG2 cells
Values followed by different
letters within a column are
significantly different (P<0.05)
by Tukeys multiple comparison
test (n03)
Metal ion supplement Total phenolic content
(gallic acid equivalent
mg g
1
of sprout dry meal)
Relative HepG2 intracellular
superoxide radical
levels (%)
Relative HepG2
intracellular
SOD levels (%)
Untreated Cells 100.002.13
a
100.003.22
a
Seeds 2.810.31
d
92.313.43
ab
102.322.61
a
Control sprout 3.830.23
bcd
69.012.32
c
101.292.56
a
Sodium 4.540.25
a
56.043.15
d
98.321.43
a
Potassium 4.810.11
a
54.725.12
d
104.521.57
a
Calcium 3.040.15
cd
54.135.91
d
99.212.27
a
Copper 3.450.24
bc
86.428.83
b
114.673.55
b
Magnesium 4.830.42
a
53.515.33
d
97.773.21
a
Manganese 2.820.36
d
84.073.48
b
112.813.72
b
Iron 3.810.11
b
75.225.37
bc
102.262.88
a
Zinc 4.840.07
a
52.046.91
d
103.912.92
a
Plant Foods Hum Nutr
Antioxidant Properties of MBS
Ethanolic extracts of moth bean seeds and 6 day-old sprouts
were prepared and checked for phenolic content and anti-
oxidant properties. The phenolic content of the seeds was
2.81 mg g
1
of the dry meal, which is close to that reported
by Siddhuraju et al. [16]. In addition, the phenolic content of
the sprouts was approximately 27% higher (3.83 mg g
1
dry
meal) compared to the seeds (Table 2). The antioxidant
activity at cellular level was checked using HepG2 cells.
When HepG2 cells were incubated with non-supplemented
(control) MBS ethanolic extract at a concentration of
0.2 mg ml
1
for 3 h, the intracellular superoxide was re-
duced by 31% (Table 2). When this process was repeated
with the metal ion supplemented sprouts, we found that the
phenolic content as well as the antioxidant property of
sodium supplemented sprouts was better than that of
the control sprouts. These results are similar to those
of Haidarizadeh et al. [17], who found that sodium supple-
mentation (230 g ml
1
) of wheat seedlings resulted in in-
creased growth including radicle weight, leaf weight and leaf
length. Potassium supplemented MBS also demonstrated im-
proved phenolic content and antioxidant properties than the
control. This may be because potassium is an essential en-
zyme activator in isoflavone synthesis [18]. Calcium supple-
mentation could also result in improved antioxidant activity.
Calcium is a crucial regulator of plant growth and develop-
ment, and the benefits of calcium supplementation have been
observed previously in different leguminous and Chrysanthe-
mum species [19]. Similarly, zinc supplementation resulted in
increased phenolic content and superoxide scavenging prop-
erties in MBS. Zinc plays a fundamental role in protein
metabolism, gene expression, photosynthesis and indole ace-
tic acid metabolism. Zinc protects cells from the damaging
reactions caused by reactive oxygen species as it is directly or
indirectly required for scavenging O
2

-
and H
2
O
2
, and thus
blocking generation of the powerful oxidant OH [20]. Even
magnesium supplementation had some positive effects on the
antioxidant properties of the sprouts.
Experimental studies have shown that iron, copper and
manganese can have slightly inhibitory effects on MBS,
resulting in relatively low phenolic content and reduced
superoxide scavenging properties. It has been reported that
iron and copper can react with intracellular H
2
O
2
to produce
highly toxic hydroxyl radicals, which cause DNA damage
and can partly contribute to H
2
O
2
-mediated cell death [21].
Also, excessive manganese nutrition leads to oxidation of
polyphenols and decreases chlorophyll synthesis, thus
resulting in decreased phenolic content and reduced plant
growth [22]. Our results suggest that the HepG2 cellular
superoxide scavenging activity of MBS extracts could be
linked to their phenolic content. These findings are similar to
studies where sprout extracts have demonstrated decreased
intracellular superoxide levels [23]. From the results of
SOD expression levels (Table 2), it appears that copper
and manganese extract could induce the enzyme to
significant levels in HepG2 cells. These elevated levels
may be contributing, to some extent, for the lower
superoxide levels of these treatment groups as compared
to untreated cells. Also, previous reports have shown
that supplementation with copper and manganese can
induce expression of superoxide dismutase in eukaryotic cell
lines [24, 25].
Conclusions
The results demonstrated that six days of sprouting can
significantly increase the nutrient content and antioxidant
properties of MBS. Caffeic acid, ferulic acid, cinnamic acid
and kaempferol were the major phenolic compounds found
in MBS. An increase in metal ion uptake was observed in
MBS supplemented with different metal chlorides. Further-
more, antioxidant properties improved following supple-
mentation with certain metal chlorides. Mineral fortified
MBS were able to scavenge the intracellular superoxide
radicals in HepG2 cells, demonstrating their potential anti-
oxidant properties at the cellular level. These metal enriched
moth bean sprouts with their antioxidant properties could
effectively serve as potential functional foods with improved
nutritional value.
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