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Nutritional Quality and Antioxidant Activity of Wheatgrass (Triticum

aestivum) Unwrap by Proteome Profiling and DPPH and FRAP assays

Article  in  Journal of Food Science · July 2018

DOI: 10.1111/1750-3841.14224

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Nutritional Quality and Antioxidant Activity of
Wheatgrass (Triticum aestivum) Unwrap by
Proteome Profiling and DPPH and FRAP assays
Santosh B. Parit, Vishal V. Dawkar , Rahul S. Tanpure, Sandeep R. Pai, and Ashok D. Chougale

Abstract: Precious contribution of plants in the field of medicine is very well known. Wheat (Triticum aestivum) seeds and
seedlings are an important source of food and feed due to the presence of various health-promoting compounds. Proteomic
analysis of three seed developmental stages (0, 8, and 16 days after germination [DAG]) of wheat was carried out using
liquid chromatography-mass spectrometry. A total of 297 proteins were identified and their functional annotation revealed
that a majority of them were involved in preventing many diseases, oxidative stress, primary metabolism, storage, and
energy related mechanisms. Particularly to mention, peroxidases, superoxide dismutases, and cytochromes are abundantly
present in wheatgrass. In the ferric-reducing antioxidant power assay, antioxidant activity was increased by 1.55 times

Food Chemistry
after 16 DAG as compared to 0 DAG, however it was decreased after 8 DAG. The antioxidant activity of the plant extracts
by DPPH had an increasing trend after all the three time points. The percent radical scavenging activity of extract by
DPPH was 15, 22, and 30 after 0, 8, and 16 DAG, respectively. Observations obtained revealed that antioxidant power
of the plants is directly proportional to the age of seedlings. Data attained on wheatgrass showing that it can be a strong
antioxidant agent due to its free radical scavenging activity and could be used in stress and nourishing human health.

Keywords: antioxidant, LC-MSE , proteomics, peroxidase, Triticum aestivum, wheatgrass

Practical Application: Wheatgrass contains minerals, phytochemicals, active enzymes, and vitamins that can be easily
absorbed. The consumption of wheatgrass juice can give better health benefits. Information about beneficial properties of
wheat grass juice is clearly mentioned in this work. Proteins found in wheatgrass are known to be involved in preventing
many diseases, oxidative stress, primary metabolism, storage, and energy-related mechanisms. Results of this work revealed
that Triticum aestivum seedlings can act as an antioxidant agent due to their free radical scavenging activity and can be
constructive to control or treat many health complications. From all these results we believed that wheatgrass can be used
for the nourishment of humans.

Introduction The field of medicine is partial without consideration of plants.

Indians, Greeks, Egyptians, and Chinese have used plants and
their reformulations to treat many diseases and nutrient deficits
from thousands of years back (Boukhris et al., 2012; Kumar et al.,
2012; Shaw, Sicree, & Zimmet, 2010; Wannes & Marzouk, 2016).
Research towards a search for dietary supplements and drugs
derived from plants has increased more recently. In Ayurweda,
medicinal properties in plants have already been highlighted
(Chopra, 2003). From very ancient time, throughout the world,
people have discovered uses of plants to treat many diseases and
deficiencies (Beecher, 1999). Blends of nutrients in the diet
are important for proper growth and maintenance of the body.
Hence, only one diet cannot provide all the required nutrients to
maintain health and to confront the diseases.
JFDS-2017-2067 Submitted 12/21/2017, Accepted 5/25/2018. Authors Parit
and Chougale are with The New College, Shivaji Peth, Kolhapur 416012,
Maharashtra, India. Authors Dawkar and Tanpure are with Plant Molecular Bio-
logy Unit, Div. of Biochemical Sciences, CSIR-Nat. Chemical Laboratory, Dr.
Homi Bhabha Road, Pune 411008, Maharashtra, India. Author Pai is with
Amity Inst. of Biotechnology (AIB), Amity Univ., Mumbai–Pune Expressway,
Bhatan, Post-Somathne, Panvel, Mumbai 410206, Maharashtra, India. Direct in-
quires to authors Chougale and Dawkar (E-mails: ashokdchougale@gmail.com and
vv.dawkar@ncl.res.in).
Their precious contribution in this field is very well-known.
Medicinal effects of the plants especially the antioxidant prop-
erties are very important (Briskin, 2000). Oxidative stress can be
defeated by natural antioxidants present in the plants in reformula-
tions (Zengin, Cakmak, Guler, & Aktumsek, 2011). Plants which
are commonly used in human daily diets are also popular for their
medicinal properties. These plants are used a lot and are gener-
ally accepted for many purposes (Oluyemi, Okwuonu, Baxter,
& Oyesola, 2007; Vongtau et al., 2005). Reactive oxygen species
(ROS) has been known during the last few decades, and researchers
have been trying to find its role in immunity, diseases, and dis-
orders. With this stand point, from last few decades, researchers
are trying to find the role of antioxidants in human health to
control immunity damage (Halliwel & Gutteridge, 1981). Some
research articles proved a great role in health, including antioxi-
dants. Furthermore, antioxidants role in anticancer and anti-aging
mechanisms is proved (Göçer & Gülçin, 2011; Gülçin, 2012). Free
radicals are usually known to attack biological cells but antioxi-
dants can inhibit them. To confront this situation, food containing
antioxidants can be included in the daily diet.
During normal body conditions the scavenging system can
eliminate ROS. Nevertheless, severe oxidative stress conditions can
damage cells and cells containing proteins, lipids, and DNA which
may lead to carcinogenesis (Kasai, Fukada, Yamaizumi, Sugie, &

C 2018 Institute of Food Technologists

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doi: 10.1111/1750-3841.14224 Vol. 0, Iss. 0, 2018 r Journal of Food Science 1

Further reproduction without permission is prohibited
 Antioxidant activity of wheatgrass . . .

Mori, 2000). Body metabolic reactions release free radicals and are
involved in a number of diseases like brain dysfunction, diabetes,
shock, inflammation, infertility, tumors, and cancer. But enzymes
like superoxide dismutase (SOD) and peroxidase are involved in
controlling free radicals and ROS (Ames, 1983; Bandoniene &
Murkovic, 2002; Ben-arye et al., 2002). It is believed that can-
cers, cardiovascular, inflammatory, and neurological diseases can be
treated with polyphenol antioxidants (Bandoniene & Murkovic,
2002). Those plants which contain many antioxidants may play a
central role in confronting many diseases and complications of the
biological systems. Because of their free radical scavenging activ-
ity, antioxidant agents can be constructive to control or treat many
health complications. Plants with high levels of antioxidant con-
stituents, viz. phenolic and polyphenolic compounds have been
proposed as an effective therapeutic approach.
There are very few scientific and clinical studies on the role
Food Chemistry
Sample preparation for proteomic analysis
Proteins were extracted from wheat seedlings according to
Schuster and Davies (1983) with slight modifications. Protein pel-
let was dissolved in 0.1% Rapigest (500 μL) and concentration
was estimated by the Bradford assay (1976). This complex protein
mixture was concentrated to 100 μL using MW 5000 cut off spin
column and reduced with dithiothretol and alkylated with iodoac-
etamide. After overnight digestion with trypsin, digested proteins
were concentrated by using MW 3000 cut off spin column and
used for nano-liquid chromatography-mass spectrometric analysis
(nano-LC-MSE ).
Liquid chromatography-mass spectrometric analysis
Digested protein samples were subjected to a NanoAcquity ultra
performance liquid chromatography (UPLC) coupled to MALDI-

of wheatgrass (Triticum aestivum) in human health. Wheatgrass is

SYNAPT HDMS (Waters Corp., Milford, MA, U.S.A.). The
used as a source of nutrients (Ben-arye et al., 2002). Furthermore,
nano-LC separation was performed using a bridged-ethyl hybrid
wheatgrass plays a part in increasing amounts of hemoglobin and
(BEH) C18 reversed phase column (1.7 μm particle size) with an
preventing bacterial infections. Vitamin A, C, and E and minerals
internal diameter of 75 μm and length of 150 mm. Each sample of
like Fe, Ca, Mg, and amino acids are present in it. They remove
total digested protein was injected into the trapping column and
toxins from the liver and blood, as well as heavy metals and cancer-
flushed with 0.1% solvent A (0.1% formic acid containing water)
causing agents from the body (Byers et al., 2002; Macintosh, 2008).
for 3 min at a flow rate of 15 μL/min. Upon each injection, pep-
Common cold, fever, bronchitis, cough, cystitis, gout, rheumatic
tides were eluted into the NanoLock-Spray ion source at a flow
pain, chronic skin disorders and even constipation, and inflamma-
rate of 300 μL/min using a gradient of 2% to 40% B (0.1% formic
tion of the mouth and throat can be treated by wheatgrass (Byers
acid containing acetonitrile) for over 110 min. The lockmass cal-
et al., 2002; Gruenwald, Brendler, & Jaenicke, 2004; Macintosh,
ibrant peptide standard, 500 fmol/μL glu-fibrinopeptide B, was
2008). Overall, it is very clear that wheatgrass is a versatile diet and
infused into the nano-lock spray. The mass spectrometer (MS) was
helpful in killing harmful bacteria and strengthening the immune
operated in V-mode at a resolution of at least 9,000 full width
system. To develop an effective natural drug and nutritious diets,
at half height. Full scan (m/z = 50 to 2,000) LC-MSE data were
a complete understanding of the physiological and genetic mech-
acquired using the “expression” mode, which involves alternating
anisms by which wheatgrass become a versatile diet and drug is
1 s scans of normal and elevated collision energy (Silva et al., 2005,
needed. The purpose of this study was to understand the capability
2006). Data were collected at a constant collision energy setting
of wheatgrass in diseases and deficiencies. This is a biochemical
of 4 V during low-energy MS mode scans, whereas a step from
and a proteome-level analysis to investigate the role of wheatgrass
14 to 40 V of collision energy was used during the high-energy
in treating health complications. With this standpoint, we claim
MSE mode scans.
that wheatgrass or wheatgrass diet programs can be helpful for
human beings.

Data processing and database searching

Materials and Methods
Chemicals
Acetonitrile, bovine serum albumin (BSA) and sequencing
grade modified trypsin were procured from Sigma Chemicals,
St Louis, MO, U.S.A. MassPrep predigested standard protein rab-
bit glycogen phosphorylase B (GP), Rapigest and enolase were
purchased from Waters Corp. (www.waters.com). Ascorbic acid,
Trolox, curcuminoids, 2,2-diphenyl-1- picrylhydrazyl (DPPH),
2, 4, 6- tripyridyl-s-triazine (TPTZ), and all other chemicals of
analytical grade were procured locally.
Wheat kernel germination and collection of samples
Wheat kernels (Triticum aestivum) were germinated in the green
house in controlled condition as: approximately 70% relative hu-
midity, the maintained temperature was 24 °C for 0, 8, and 16 days.
Light:dark period was 16:8 hr. The seedlings after 0, 8, and 16 days
were harvested and immediately frozen in liquid nitrogen and
ground to get a fine powder with a mortar and pestle, and the
powder was then kept at −80 °C until used.
The continuum LC-MSE data were processed and analyzed
using Protein Lynx Global Server 2.5.2 software (PLGS; Waters
Corp). Protein identifications were obtained by searching the
UniProt database (www.uniprot.org) for wheat and NCBI
database for flowering plants (Magnoliophyta) which are built sepa-
rately. LC-MSE data were analyzed with a fixed carbamidomethyl
modification for Cys residues, along with a variable modifi-
cation for oxidized Met residues. The ion accounting search
algorithm within PLGS was developed specifically for searching
data-independent MSE data sets, and a detailed description of
the algorithm was recently published by Li et al. (2009). Ion
accounting search parameters used for data independent analysis
(DIA) were precursor and product ion tolerance (automatic set-
ting), minimum number of peptide matches (3), minimum num-
ber of product ion matches per peptide (5), minimum
number of product ion matches per protein (7), and maximum
number of missed tryptic cleavage sites (2). The false-positive rate
was 4%. The results of DIA (proteins and the individual MS/MS
spectra) with confidence level 95% were accepted. Digested
BSA (100 fmol) was spiked in the samples to quantify the digested
protein samples. Replicates were performed for each sample
and analysis of three different wheat samples was done.

2 Journal of Food Science r Vol. 0, Iss. 0, 2018

Antioxidant activity of wheatgrass . . .

Functional classification
Gene Ontology (GO) annotation was performed with
Blast2GO (Conesa et al., 2005) based on sequence similarity. For
annotation, the default configuration settings were used and the
proteins were searched in the NCBI-nr protein database. Proteins
with unknown function or without specific homology or similar-
ity descriptions were BlastP searched in the NCBI-nr database to
confirm their function. The information about subcellular local-
ization was incorporated into protein description.
Ferric reducing antioxidant power activity
Ferric-reducing antioxidant power (FRAP) assay was used to
measure the total antioxidant power of the extract. The FRAP
assay was carried out using the method of Benzie and Strain (1996)
with minor modifications. FRAP reagent formed by assimilation
of the acetate buffer (0.3M, pH 3.6), 10 mM 2,4,6-tripyridyl-s-
triazine (TPTZ) in HCL (40 mM) and FeCl3 6H2 O (20 mM)
as described by Upadhya, Pai, and Hegde (2015). The DPPH
reagent was prepared by dissolving 2.5 mg of DPPH in 100 mL of
methanol. The plant extracts (10 μL) were allowed to react with
290 μL of DPPH reagent. The reaction mixtures were allowed to
interact properly and stand in the dark at room temperature for
30 min. The absorbance at 517 nm was measured using methanol
as blank on a Multiskan GO 1.00.40, UV-Visible spectropho-
tometer (multiskan Go 1510; Thermo Scientific). Ascorbic acid
(10 to 1,000 μM), Trolox (10 to 1,000 μM), and curcuminoids
(10 to 1,000 μM) were used for calibration. Standard curves, y =
0.0005x − 0.0032, R² = 0.997; y = 0.0005x − 0.0007, R² = 0.998
and y = 0.0005x + 0.0091, R² = 0.997, were obtained for the
three standards (ascorbic acid, Trolox, and curcuminoids, respec-
tively). The percent radical scavenged of DPPH was calculated
and the results were expressed as %DPPH quenched. The results
were also represented as μM ascorbic acid/trolox/curcuminoids

Food Chemistry
equivalents (AEAC, TEAC, and CEAE) for fresh/dry material.
in 10:1:1 ratio. The plant extracts (10 μL) were allowed to react
with 290 μL of the FRAP reagent, the reaction mixture was kept
in room temperature for 15 min and the absorbance was recorded Statistical analysis
at 595 nm using Multiskan GO 1.00.40, UV-Visible spec- Data were analyzed by one-way analysis of variance (ANOVA)
trophotometer (multiskan Go 1510; Thermo Scientific, U.S.A.). with Tukey–Kramer multiple comparisons test. Data points were
Ascorbic acid (10 to 1,000 μM), Trolox (10 to 1,000 μM), and considered significant at p  .05, p  .01, and p  .001.
curcuminoids (10 to 1,000 μM) were used for calibration. Stan-
dard curves, y = 0.001x + 0.026, R² = 0.997; y = 0.001x + 0.007,
R² = 0.997; y = 0.0006x + 0.0357, R² = 0.987, for the three
Results
standards (ascorbic acid, trolox, and curcuminoids, respectively)Diversity of proteins in wheatgrass
were obtained as output. The results of the activity were calcu- In this study, a total of 297 proteins were identified by nano-LC-
lated and represented as μM ascorbic acid/trolox/curcuminoids MSE in all stages of wheatgrass. Among them 91 proteins were
equivalents (AEAC, TEAC, and CEAE) for fresh/dry sample. identified after 0 DAG, 63 proteins after 8 DAG, and 143 pro-
teins after 16 DAG. Representative MS/MS spectrum of glutenin
2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity and α-amylase inhibitor of wheatgrass is as shown in Figure 1.
The antioxidant activities of all the plant extracts were de- It is very clear that fewer proteins were identified after 8 DAG
termined by using 2,2-diphenyl-1- picrylhydrazyl (DPPH) assay (Figure 2) because it was just transition phase between wheat

Figure 1–Representative MS/MS spectrum of wheatgrass (A) glutenin and (B) α-amylase inhibitor. These spectrums observed after MS/MS analysis by
TM TM
ProteinLynx Global SERVER (PLGS) is a fully integrated Mass-Informatics platform for quantitative and qualitative proteomics research.

Vol. 0, Iss. 0, 2018 r Journal of Food Science 3

 Antioxidant activity of wheatgrass . . .
Food Chemistry

Figure 2–GO analysis of proteins identified in wheat seeds and developing wheatgrass: A total of 297 unique proteins were analyzed by ProteinLynx
TM
Global SERVER (PLGS) and annotated by using the Blast2GO program. The annotated proteins are categorized as cellular components (CC), biological
processes (BP), and molecular functions (MF). (A) Proteins identified and annotated in 0 DAG. (B) Proteins identified and annotated in 8 DAG. (C)
Proteins identified and annotated in 16 DAG.

kernel and wheatgrass. In seeds numbers of proteins are present
but after seed germination many of the proteins may be used as
energy source for seed germination. Hence, in newly emerged
seedling, fewer proteins are present as compared to the kernel.
The α-amylase inhibitor was consistently present in all the three
stages of wheatgrass as possibly this is important artillery against
insect pests because majority of the insect pests make their diet di-
gestible by using α-amylase enzyme (Table 1, 2, and 3). The same
pattern was observed for glutenin and globulin; both of these pro-
teins were identified in all the three stages with isoforms (Table
1, 2, and 3). Histone was present in 8 and 16 DAG wheatgrass
whereas glyceraldehyde-3-phosphate dehydrogenase was identi-
fied in 8 DAG wheatgrass (Table 2 and 3).
Serine threonine protein kinase was identified in all stages of
wheatgrass and is known to be involved in the regulation of cell
differentiation, cell proliferation, apoptosis, and embryonic devel-
opment. Even in the regulation of signal transduction pathways,
protein kinase remains present. Oxidoreductase was identified in
all taken experimental time points. Peroxidase was identified in
8 DAG wheatgrass and cytochrome b subunit, cytochrome P450,
and cytochrome b6 were found in 16 DAG seedlings. Gain or loss
of electrons is catalyzed by the action of an oxidoreductase. This
is a very important enzyme for accepting or donating electrons.
However, cofactors such as NADP or NAD+ are required for the
activity of this enzyme. Cytochromes are hem bonded protein and
required for many metabolic pathways.
GO annotations and functional categorization
To understand the biological functions of the expressed
proteins, GO annotation was performed. The identified 297

4 Journal of Food Science r Vol. 0, Iss. 0, 2018

Antioxidant activity of wheatgrass . . .
Figure 2–Continued.
proteins were analyzed using BlastP against the NCBI-nr plant
database, which yielded 1284 GO annotations (Figure 2A, B, and
C). The result shows the distribution of GO annotation in the
three main categories, viz. molecular function, biological process,
and cellular components. According to the GO annotations,
in the first (0 DAG) and second stage (8 DAG), the annotated
proteins in the category of cellular component (CC), molecular
function (MF), and biological processes (BP) assignments were
very low as compared to third stage (16 DAG; Figure 2A, B,
and C). The most important thing which is surprising and can
be highlighted is that, proteins which are involved in immune
system process and locomotion are present only in third stage
(Figure 2C). Cell killing proteins are present only in second and
third stages. In CC, there were eight subcategories seen in all three
stages of proteins. But in MF and BP the scenario was different.
In the first-stage, proteins were annotated in nine subcategories;
however, at the same time, only six and eight subcategories
were in the second and third stage, respectively. In BP, 12, 11,
and 15 subcategories were showed for proteins at 0, 8, and
16 DAG.
Food Chemistry
FRAP values of wheatgrass extract
The FRAP assay was developed to determine the total antiox-
idant power of the sample. In this assay, ability of the sample
(antioxidants) was determined by inhibiting the oxidative effects
of reactive species in the reaction mixture. The FRAP value
of wheatgrass extract after 0, 8, and 16 DAG is as shown in
Figure 3. Among all the wheatgrass extracts tested, 16 DAG had
highest FRAP value, followed by 8 and 0 DAG. The first time
point was 0 DAG, in which seeds directly were used for extraction
to determine the FRAP value. This observation clearly indicates
that germination period was directly proportional to FRAP value.
As germination time of the seeds increases, accordingly antioxidant
power of the extract increases. Upon biochemical analysis five-fold
increased in FRAP value between 0 and 16 DAG were observed.
There was a significant difference (p < .001) in antioxidant power
of the wheatgrass extract between 0, 8, and 16 DAG.
DPPH radical-scavenging activity of wheatgrass
To assess the radical scavenging activity of the samples
numerous methods have been developed, however, DPPH
Vol. 0, Iss. 0, 2018 r Journal of Food Science 5
 Antioxidant activity of wheatgrass . . .
Food Chemistry

Figure 2–Continued.

spectrophotometric method is one of the most widely applied & Kalender, 2010; Borrelli & Ernst, 2008). Because of safe and
due to its consistency. Scavenging power of wheatgrass extract on significant contributions of natural products in drug discovery,
DPPH radical was in the following order: 16 DAG > 8 DAG > 0 there is a need to make herbal drugs. It is also proposed that iso-
DAG (Figure 4). The scavenging DPPH radicals for the TEAC and lated or purified single components from plant crude extracts are
%RSA after 0, 8, and 16 DAG were 222 ± 1, 332.20 ± 1, 445 ± not efficient compared to crude extract to treat diseases or health
2 μM and 30, respectively (Figure 3). The same trend was observed complications. By keeping this point in consideration, in this study,
for AEAC and CEAC. The antioxidant potential of extracts was we have shown that wheatgrass crude extract has good antioxi-
found to be significant at p < .001, for all the values. The study dant power which may not be in purified components (Bar-Sela,
revealed that wheatgrass extract has prominent antioxidant activity Cohen, Ben-Arye, & Epelbaum, 2015). We applied a proteomic
which may be due to the presence of phenolic compounds. approach in wheatgrass to understand the role of proteins as an-
tioxidative agents and many tasks that may be useful for human’s
Discussion healthy life. In the case of wheatgrass, if we incorporate traditional
approaches in new ways as mentioned in this study it will surely
Wheatgrass proteins enriched in variety of critical be beneficial to treat many diseases.
biological functions
Peoples believe that an herbal medicine cure and eradicates dis-
eases. Synergic activity found more in plants crude extracts than Wheatgrass is a rich repository to tackle health
pure compounds (Phytoextrakte-Produkte und Prozesse, 2004). complications
Black cohosh (Cimicifuga racemosa) is effective for menopausal prob- Efficient antioxidant enzyme systems evolved in plants help
lems (Amsterdam et al., 2009; Bebenek, Kemmler, Von Stengel, to scavenge ROS, which develops due to oxidative stress and

6 Journal of Food Science r Vol. 0, Iss. 0, 2018

Antioxidant activity of wheatgrass . . .

Table 1–List of identified proteins by LC-MSE in 0 DAG wheatgrass.

Acc. No. Protein MW (Da) pI PLGS score Peptides Coverage (%)

1 D2TGC2 α-Amylase inhibitor 12,996 5.81 24,205 13 97
2 P16851 α-Amylase trypsin inhibitor 15,449 6.95 17258 12 82
3 Q0Q5D3 HMW glutenin 69,586 7.62 13,280 12 33
4 B5B0D5 Major allergen 15,772 4.66 12,083 13 58
5 M8B9L0 α-Amylase trypsin inhibitor 15,906 4.88 10,429 9 39
6 Q2PCC7 Lipid transfer protein 9,826 8.30 76,186 6 53
7 Q8LKV6 HMW glutenin 89,818 6.09 7,048 5 14
8 C4P5P7 α-Amylase inhibitor 16,591 6.66 6,589 11 60
9 C4P5A1 α-Amylase inhibitor 16,475 6.66 5,022 11 74
10 A4ZIZ0 α-Amylase inhibitor 13,047 5.18 4,964 9 71
11 Q0Q5D9 Globulin 24,534 8.14 4,129 6 32
12 D0PRB4 Peroxiredoxin 23,962 6.33 3,867 6 44
13 A0A0 × 9ARV2 HMW glutenin 89,813 6.13 3,354 7 12
14 A0A059UHD1 HMW glutenin 89,781 5.59 3,283 6 10
15 Q6RS99 Globulin 24,984 7.95 2,358 6 34
16 H9XH65 Globulin 24,984 7.95 2,358 6 34
α-Amylase trypsin inhibitor

Food Chemistry
17 M8BV45 24,232 7.72 2,046 8 52
18 I6QQ39 Globulin 66,284 8.27 1,946 19 43
19 Q53YX8 α-Amylase inhibitor 18,209 7.32 1,767 7 63
20 W5B1E5 Superoxide dismutase 15,111 5.70 1,531 3 29
21 D2KFH2 Gliadin avenin 11,317 7.60 1,479 4 38
22 G8J377 Photosystem II protein 3,505 7.01 994 5 90
23 A0A0M4HM24 LEA3 OS 21,883 9.53 976 4 21
24 B2FH42 Heat shock protein 16,960 5.73 965 4 37
25 A0A0K2BBI4 ABC transporter permease 30,752 10.08 539 10 70
26 M8A8G4 Purothionin 14,635 4.57 528 7 65
27 S4YYX3 NADPH quinone oxidoreductase 40,208 4.61 526 14 63
28 P01543 Purothionin 14,614 4.74 481 7 47
29 A0A0D9AT47 Threonine transporter 22,678 10.75 443 7 65
30 B2VYF7 Isoflavone reductase 34,406 5.09 441 10 60
31 D3UAL7 LMW glutenin 18,579 8.42 431 3 31
32 S4Z8N4 NADPH quinone oxidoreductase 38,342 4.66 389 11 53
33 J3P9H4 Pre mRNA processing protein 1,09,061 6.86 384 34 20
34 L7FQV4 Heme exporter protein 22,227 9.70 365 7 53
35 Q8LPA7 Cold shock protein 21,370 5.67 334 6 53
36 M8B3H7 Glycerol kinase 51,420 5.18 318 3 20
37 N1QVQ6 Peptidyl prolyl cis trans isomerase 1,09,302 4.35 297 8 10
38 A0A0K2BI59 Divalent cation transporter 26,598 6.21 295 7 46
39 L7HE33 Protein export membrane protein 35,248 8.97 288 5 39
40 H1WPG4 Amino acid permease 50,858 10.11 262 7 36
41 A0A0U3IBI7 Chemotaxis protein 56,742 5.44 259 7 21
42 Q0ZH66 HFR 3 20,207 7.16 258 12 89
43 Q08837 Triticin 56,918 9.50 258 8 32
44 M7YIA6 Heat stress transcription factor 19,520 4.80 256 6 43
45 A0A0K2BB49 Sugar ABC transporter permease 33,001 6.15 256 6 55
46 A0A0D9AGA7 ABC transporter permease 33,248 5.82 254 5 34
47 M7Z512 MATE efflux family protein 40,271 10.29 240 12 44
48 W5FG19 Peptidyl prolyl cis trans isomerase 14,446 5.17 239 2 17
49 A0A0K2B909 Methionine transporter permease 22,849 9.25 236 5 52
50 A0A0D9AKJ6 Membrane protein 31,840 10.29 210 8 44
51 A0A0D9AIY0 Murein hydrolase transporter 13,039 11.58 206 3 85
52 M8BPI3 Tumor protein 55,915 5.07 205 9 29
53 A0A089VER5 Transcription factor 31,839 5.53 198 2 36
54 S3MH66 TerB-like domain protein 93,182 5.36 197 9 26
55 A0A0K2BBD5 Glycosyltransferase 63,876 9.69 197 10 43
56 A0A067ZWN0 Boron transporter 73,792 7.39 194 11 43
57 A0A0K2BBB9 C4 dicarboxylate ABC transporter 75,377 7.06 193 9 31
58 W1ID93 NADH ubiquinone oxidoreductase 62,331 8.25 190 14 49
59 H1 × 879 ATP binding cassette transporter permease 66,136 9.79 183 9 30
60 A0A0D9AUI9 Magnesium transporter 53,254 4.45 183 8 44
61 W5H7M6 Serine threonine protein kinase 88,219 7.60 180 16 29
62 B2WHA5 YagE family protein 57,603 5.35 176 6 35
63 M8CDB7 Boron transporter like protein 66,744 6.76 171 11 49
64 A0A0K2BAL6 Cation transporter 1,16,213 8.26 162 23 33
65 M8BN38 Luminal binding protein 66,283 5.07 162 8 26
66 A0A0P9SSE8 Cation efflux family protein 1,13,053 5.79 161 25 41
67 L7H3I7 Methyl accepting chemotaxis protein 83,860 5.34 161 6 18
68 S3NHC1 4 hydroxyphenylacetate permease 47,825 9.65 158 12 42
69 H1 × 8H0 Polyamine organocation transporter 66,997 9.89 154 5 23
70 R7WDS4 B3 domain containing protein 76,219 9.39 150 10 26
(Continued)

Vol. 0, Iss. 0, 2018 r Journal of Food Science 7

 Antioxidant activity of wheatgrass . . .

Table 1–Continued.

Acc. No. Protein MW (Da) pI PLGS score Peptides Coverage (%)

71 A0A0L7T8J3 Peptide transporter 37,321 9.61 150 6 30
72 L7GXM7 Alkyl hydroperoxide reductase 14,984 5.16 149 4 31
73 M8BFA5 NADPH dependent diflavin oxidoreductase 59,206 8.06 148 11 30
74 B2VZY4 Mevalonate kinase 54,839 6.61 146 13 49
75 V6J5S4 Citrate transporter family protein 65,723 9.31 146 14 32
76 M7Z2U7 GPI anchored wall transfer protein 18,770 9.74 146 5 44.
77 A0A0P9U2I8 Malate transporter 33,363 9.50 145 12 71
78 A0A0L7SW23 Iron ABC transporter 68,926 11.79 143 21 67
79 J3P9U7 ATP dependent RNA helicase 1,33,639 9.03 142 22 18
80 H1 × 7Q5 Phosphatidylglycerol synthetase 93,869 8.43 141 11 25
81 V6JAN5 Transporter family protein 31,315 10.43 141 11 56
82 R7W8H0 Response regulator 33,167 8.26 140 14 54
83 A0A0F7J4J2 Ca2 permeable channel protein 93,283 9.25 138 19 37
84 W5A3G2 Ca2 permeable channel protein 92,999 9.20 138 18 38
85 A0A0L7T6S7 Iron ABC transporter permease 34,756 11.96 137 7 51
86 A0A0D9BAJ2 Methionyl tRNA formyltransferase 33,515 5.66 137 6 36
Food Chemistry

87 H1WPL9 Ribosome biogenesis GTPase 33,397 8.66 136 8 38

88 A0A0D9B059 4 hydroxybenzoate octaprenyltransferase 33,279 9.41 134 11 41
89 M8A186 phosphoribosylformylglycinamidine synthase 1,47,656 4.81 117 17 24
90 M7Z8F1 RNA binding protein 87,151 6.71 95 14 28
91 W4ZQF9 Diacylglycerol kinase 80,279 6.50 87 10 36

protects themselves from injury (Allen, 1995; Bowler et al., 1991). scavenging power for both the FRAP and DPPH radical test. It
In plants, at specific stages, proteins involved in immune system was found that wheatgrass extract of 16 DAG was abundant in
processes are present. Hence with the present defense system in FRAP and DPPH scavenging activity. From long ago Ayurvedic
humans, there is need to tackle antigens by supporting supple- practitioners were using wheatgrass as medicine to treat diseases
ments. Thus with this scenario, we analyzed the wheatgrass at and health complications (Ben-arye et al., 2002). It is proposed
different time points for proteomic changes. At the third time that in anemia, liver disorders, digestion problems, and cancer-
point (16 DAG) immune system process related proteins were related diseases wheatgrass can be used (Chauhan, 2014; Gore,
found in wheatgrass. According to GO consortium, in BP, im- Palaskar, & Bartake, 2017; Macintosh, 2008). Reports showed
mune system process is divided into nine substeps, among them that wheatgrass was rich in antioxidant properties (Falcioni et. al.
immune response is the important step. Again immune response 2002; Zendehbad, Mehran, & Malla, 2014). This work shows that
can be divided into seven responses. All these steps covered im- wheatgrass is very strong in radical scavenging activity. The pro-
munity mechanisms of the mammalians. Proteins that play the role teomics study showed that peroxidases, SODs, and cytochromes
in these pathways are grouped under immune system process of are abundantly present in wheatgrass. Hence, the plant extracts
the proteins. As many of proteins from wheatgrass are annotated rich in these enzymes can be used in treating health complications
under this category, we hypothesized that wheatgrass can be used such as stomach ailments, cancer, anemia, and other blood-related
as supplementary food components for nourishing human beings. diseases.
In multicellular organisms, controlling the rate of cell division There are multiple roles of peroxidase in human’s healthy life
and cell death is tightly regulated by cell killing proteins. Removal(Ratnasinghe et al., 2000; Valko, Rhodes, & Monco, 2006). For
of infected, mutated, or damaged cells is a “defensive” role in example, good oral health is maintained by salivary peroxidase
organisms (Vaux & Korsmeyer, 1999). Once a single cell is in- system in several ways (Tenovuo & Pruitt, 1984). Furthermore,
duced to die by apoptosis, neighboring, or specialized phagocytic this enzyme is one of the non-immunoglobulin defense factors
cells recognize, internalize, and degrade the cell corpse (Savill which regulate the quantity and species distribution of oral
& Fadok, 2000). These types of instantaneous consent of apop- microorganisms. Moreover, peroxidases inactivate many carcino-
totic cell corpses prevent inflammation and autoimmune disease, genic and mutagenic compounds and prevent toxic accumulations
as dead cells are not able to release harmful intracellular contents of hydrogen peroxide (Mates, Perez-Gomez, & Nunez De Castro,
into the surrounding tissue (Nagata, Hanayama, & Kawane, 2010). 1999; Papp, Lu, Holmgren, & Khanna, 2007; Valko et al., 2006).
Cell killing proteins are present in 8 and 16 DAG wheatgrass only There is a huge interest in peroxidases as therapeutics due to their
meaning after germination of wheat kernel important immunity- use as an antioxidant and antimutagenic role. More attention is
related proteins develop in seedlings. Because of all these aspects, gravitating towards naturally occurring antioxidant compounds.
incorporations of wheatgrass diet is very important. LC-MSE and By using biotechnological approaches overexpression of perox-
Blast2GO analysis by the GO Consortium provided good results idases is possible (Robertson & Harmon, 2007). In wheatgrass
in this study. Further wheatgrass metabolite level analysis and gene abundant quantity of peroxidase is observed, however, there is
expression studies will help to dissect immune system processes need to focus more on specific protein databases because multiple
with the functional output. isoforms of peroxidases as well as other important enzymes may
be there in the extract. SOD have a vital antioxidant role in
Index of antioxidant potential of wheatgrass offers their human health (Johnson & Giulivi, 2005). Different types of
crucial role in oxidative stress SODs are known, and they are classified on the basis of their Cu,
Zn-containing prosthetic group. It is known that SODs played
The wheatgrass extracts were screened for the FRAP and DPPH
an important role in defense and the detoxification of products
radical scavenging activity assay. The extracts showed a significant

8 Journal of Food Science r Vol. 0, Iss. 0, 2018

Antioxidant activity of wheatgrass . . .

Table 2–List of identified proteins by LC-MSE in 8 DAG wheatgrass.

Acc. No. Protein mW (Da) pI PLGS score Peptides Coverage (%)

1 P01085 α-Amylase inhibitor 13,328 6.80 17,116 12 94
2 M8C3B8 α-Amylase inhibitor 16,570 6.06 13,142 12 75
3 P16159 α-Amylase trypsin inhibitor 15,771 5.13 11,080 12 79
4 M8B5G5 β-Amylase 58,709 5.22 9,374 23 52
5 V9TQA3 HMW glutenin 69,614 7.89 8,899 6 18
6 B5B0D5 Major allergen 15,772 4.66 8,831 9 48
7 C4P5P7 α-Amylase inhibitor 16,591 6.66 8,663 15 65
8 B7U6L4 Globulin 66,309 7.76 7,671 27 33
9 Q6S5B1 α-Amylase inhibitor 18,209 7.32 7,296 9 64
10 A4ZIW6 α-Amylase inhibitor 13,133 5.18 6,406 15 90
11 A4ZIZ0 α-Amylase inhibitor 13,047 5.18 6,384 13 77
12 M8AQX5 β-Amylase 59,918 4.90 6,292 24 49
13 W5EKI0 β-Amylase 61,074 4.82 6,158 24 48
14 I6QQ39 Globulin 66,284 8.27 5,590 26 26
15 M8A380 Globulin 56,880 9.24 5,473 26 41
16 P02861 Glutenin 10,889 8.61 5,203 2 23
α-Amylase trypsin inhibitor

Food Chemistry
17 R7W9W1 15,516 7.41 5,137 4 49
18 D2TGC2 α-Amylase inhibitor 12,996 5.81 4,593 7 93
19 P08489 Glutenin 89,119 5.77 4,543 5 8
20 Q0PW13 HMW glutenin 89,778 6.49 4,529 5 8
21 P16851 α-Amylase trypsin inhibitor 15,449 6.95 4,074 7 79
22 M8C629 Serpin 43,143 5.31 3,541 12 51
23 Q0Q5D9 Globulin 24,534 8.14 3,123 9 24
24 M8BGV8 Globulin 62,630 9.74 3,070 26 33
25 B7U6L3 Globulin 38,405 9.23 2,755 13 27
26 C0LF32 Serpin 43,113 5.46 2,734 12 51
27 Q0Q5E3 Globulin 24,983 8.29 2,663 8 19
28 M8C8G6 Glyceraldehyde 3 phosphate dehydrogenase 36,504 6.78 2,544 6 29
29 C7C4 × 1 Glyceraldehyde 3 phosphate dehydrogenase 36,504 6.78 2,304 6 29
30 Q8LK23 Peroxidase 38,798 7.85 2,214 16 46
31 Q41518 Nucleic acid binding protein 16,245 4.97 1,953 5 64
32 M7Z0R3 Histone 15,523 11.94 1,926 6 33
33 M8AVT1 Heat shock cognate 76,854 5.17 1,428 20 40
34 M8CK39 Aspartic proteinase oryzasin 57,293 7.96 1,381 6 21
35 M8BBI3 Heat shock cognate 70,986 4.94 1,227 16 37
36 N1QVN1 Histone 16,830 10.60 885 3 40
37 M8B8C6 Globulin 51,555 6.86 800 8 25
38 Q07810 rRNA N glycosidase 29,594 10.12 788 7 38
39 W5FYY0 rRNA N glycosidase 23,331 9.97 662 6 53
40 P12783 Phosphoglycerate kinase 42,095 5.51 634 5 23
41 W5H9U3 Histone 17,904 10.43 580 3 38
42 M8B0N7 Enolase 38,775 4.85 329 9 55
43 M4Q9V0 Enolase 48,060 5.34 315 6 29
44 S4YYX3 NADPH quinone oxidoreductase 40,208 4.61 304 8 40
45 J3P6E3 mRNA cleavage and polyadenylation factor 51,398 5.68 242 6 28
46 M7Z1R4 Auxin induced protein 62,268 9.58 204 7 22
47 W5G9F9 Alpha amylase 47,348 5.80 169 6 25
48 N1R5M9 Disease resistance protein 82,150 9.17 157 9 18
49 J3NLH9 MFS transporter 57,576 8.36 127 11 42
50 M7YQ54 Serine threonine protein kinase 93,091 6.15 109 12 28
51 J3P2K7 COP9 signalosome complex subunit 30,788 8.80 107 2 15
52 W5DL54 Endoglucanase 69,098 9.13 106 9 18
53 V6J3B9 Transporter gluconate H symporter family protein 47,529 9.03 105 6 42
54 S3MIH1 NADH quinone oxidoreductase 51,851 8.97 104 7 35
55 M7Z0K7 Pectinesterase inhibitor 1,14,264 5.89 104 15 18
56 N1R1I9 Pectinesterase inhibitor 1,18,722 5.41 102 15 14
57 M8CRZ1 Cytokinin O glucosyltransferase 35,870 4.85 96 5 28
58 A0A090MB51 WGS 1,08,885 8.80 91 12 11
59 A0A0D9AM29 Polyamine ABC transporter substrate binding protein 45,765 9.40 90 6 32
60 F9XPS1 FACT complex protein 1,15,841 5.25 83 18 26
61 N1QPD5 Subtilisin like protease 79,004 7.71 79 10 31
62 N1R2U9 Protein FAR1 62,873 9.40 78 7 10
63 M8APF2 Furry homolog like protein 2,40,845 6.05 53 25 25

resulting from oxidative stress (Vouldoukis, Conti, & Krauss, Conclusion

2004). Wheatgrass proteomics study showed the presence of SOD For years, scientists tried to find a way to boost the body’s
and hence will be beneficial for human health and we need to primary internal antioxidant defenses to reduce oxidative stress
incorporate wheatgrass extract in daily diet. and risk of other life-threatening diseases. Studies have shown

Vol. 0, Iss. 0, 2018 r Journal of Food Science 9

 Antioxidant activity of wheatgrass . . .

Table 3–List of identified proteins by LC-MSE in 16 DAG wheatgrass.

Acc. No. Protein mW (Da) pI PLGS score Peptides Coverage (%)

1 Q4U1A4 α-Amylase inhibitor 15,036 4.79 26,339 7 44
2 Q7M219 α-Amylase inhibitor 2,760 8.28 21,261 1 44
3 M8BV45 α-Amylase/trypsin inhibitor. 24,232 7.72 16,568 7 45
4 Q53YX8 α-Amylase inhibitor protein. 18,209 7.32 11,098 6 50
5 D7REK2 HMW glutenin 88,829 6.49 8,558 7 7
6 Q41540 CM 17 protein 15,978 4.88 6,150 5 35
7 M7Z0R3 Histone 15,523 11.95 5,515 3 19
8 D1MJA1 Glutenin 91,419 5.87 5,327 7 9
9 M8AQX5 Beta amylase 59,918 4.90 4,874 6 15
10 M8B9L0 α-amylase trypsin inhibitor 15,906 4.88 3,729 4 35
11 W5EKI0 β-Amylase 61,074 4.82 3,633 6 14
12 R7W4S0 α-Amylase subtilisin inhibitor 11,933 7.31 3,367 3 22
13 N1R1Z3 Heat shock cognate 71,778 4.97 3,350 7 20
14 C4P5P7 α-Amylase inhibitor 16,591 6.66 3,329 6 34
15 P16347 α-Amylase subtilisin inhibitor 19,620 6.80 3,243 3 13
16 P16851 α-Amylase trypsin inhibitor 15,449 6.95 2,731 2 31
Food Chemistry

17 M7ZPC7 Heat shock cognate 71,788 4.97 2,722 6 18

18 M8BBI3 Heat shock cognate 70,986 4.94 2,616 6 16
19 W5A8H1 Histone 16,493 11.03 2,275 6 23
20 Q41593 Serpin 43,091 5.50 2,152 2 6
21 M8BJK1 Lipid transfer protein 9,826 8.30 1,794 2 22
22 W5FIQ1 rRNA N glycosidase 16,686 10.02 1,649 2 16
23 Q9SQG4 PR 4 Fragment 13,088 6.28 1,601 1 13
24 Q8S4P7 Thaumatin like protein 23,582 7.49 1,477 7 47
25 A0A0D9B2T1 Potassium ABC transporter ATPase 3,100 6.84 1,241 1 82
26 O64392 Wheatwin. 15,624 7.57 1,190 1 10
27 Q07810 rRNA N glycosidase 29,594 10.13 1,153 2 9
28 B7U6L5 Globulin 56,896 7.57 1,114 7 8
29 J7K291 α-Purothionin fragment 11,926 4.83 1,100 4 49
30 A0A075TNA6 Cytochrome b6 17,505 7.15 998 3 36
31 M8B5L7 Histone 14,065 10.47 968 6 37
32 N1QPV1 α-2 purothionin 18,552 6.07 953 3 16
33 Q6UJY8 Globulin 24,486 8.14 930 5 29
34 M7ZQM3 Globulin 55,300 7.80 819 5 10
35 Q2QL53 α-Gliadin storage protein 31,070 7.10 814 1 4
36 W5D5L4 Fructose bisphosphate aldolase 38,786 6.99 754 4 18
37 Q08837 Triticin fragment 56,918 9.51 649 4 8
38 I6QQ39 Globulin 66,284 8.27 630 9 13
39 M8C2Y1 Serpin 42,969 5.01 612 1 3
40 A0A0U3HXU4 CDP diacylglycerol glycerol 3 20,556 9.84 561 7 67
phosphate 3 phosphatidyltransferase
41 M8AL81 Serine threonine protein kinase 61,994 9.67 499 9 16
42 N1QSF9 Calcium binding protein 20,503 4.25 496 7 18
43 N1R1P9 Histone 16,482 10.63 487 6 24
44 A5PI89 NADH ubiquinone oxidoreductase 13,032 3.91 481 2 38
45 P12783 Phosphoglycerate kinase 42,095 5.51 424 7 16
46 R7W5A9 Low temperature induced protein 5,833 4.26 422 2 40
47 A7UME2 Xylanase inhibitor 41,218 7.35 387 2 8
48 A0A0K2B909 DL methionine transporter permease 22,849 9.25 380 4 43
49 E7CWA8 Cytochrome P450 15,479 11.07 329 4 24
50 H1WRY8 YihY family protein 32,701 9.65 329 4 46
51 A0A0L7SZZ8 Sec independent protein translocase 9,105 5.65 321 3 34
52 M8A1S2 Trypsin α-amylase inhibitor 16,460 8.20 307 2 15
53 K4Q5U9 Protein disulphide isomerase 4,180 11.93 305 2 64
54 D3JUT3 Xylanase inhibitor 33,287 8.56 293 4 16
55 M8CTA1 Lectin domain containing receptor 37,868 6.18 282 6 26
kinase
56 K9N9F0 NADH ubiquinone oxidoreductase 13,033 3.82 281 4 38
57 A0A0U2YLX0 ABC transporter permease 34,950 11.38 266 8 56
58 A0A0L7TF43 Iron ABC transporter 15,573 8.30 261 6 24
59 L7GG80 Phosphatidylglycerophosphate 21,384 10.18 260 2 37
synthase
60 A0A0L7T0P7 ABC transporter permease 23,047 9.50 260 4 29
61 M7ZZS0 Pre mRNA splicing factor 63,613 4.84 254 11 21
62 H1 × 6V3 Putative fluoride ion transporter 13,264 9.81 252 1 29
63 W5AQD7 HVA22 like protein 25,431 8.68 248 3 18
64 A0A0D9AUN9 Pilus assembly protein 25,619 10.13 248 5 14
65 A0A0D9AYJ7 Exclusion suppressor 17,384 10.68 244 6 62
66 M8C6M9 Sec1 family domain containing 1E+05 4.87 240 7 9
protein
(Continued)

10 Journal of Food Science r Vol. 0, Iss. 0, 2018

Antioxidant activity of wheatgrass . . .

Table 3–Continued.

Acc. No. Protein mW (Da) pI PLGS score Peptides Coverage (%)

67 S3MWH3 Cytoplasmic membrane protein 28,913 9.08 230 10 53
68 A0A0D9AQA2 ABC transporter permease 35,405 9.89 221 4 28
69 M8A5L4 4 hydroxyphenylacetaldehyde oxime 52,691 6.34 218 5 13
monooxygenase
70 L7HF78 NaH antiporter 59,366 9.04 214 7 28
71 M8B9V0 Auxin responsive protein 16,462 5.62 208 4 53
72 A0A0L7TJ41 Osmoprotectant uptake system 41,361 10.63 205 9 29
permease
73 A0A0L7T5E6 Carbonate dehydratase. 50,544 7.98 204 3 12
74 A0A0P9SQJ9 Gluconate transporter family protein 50,410 9.15 201 10 51
75 H9BUT2 Trehalose 6 phosphate synthase 21,443 6.57 201 5 17
76 M8ACW5 Homeobox leucine zipper protein 11,682 5.73 198 2 46
77 A0A0L7T5U6 ABC transporter permease 36,683 8.50 198 3 9
78 H1WP29 TMnm-related protein 14,136 10.41 191 8 39
79 M7Z226 Protein disulfide isomerase 56,253 5.88 184 5 12
80 H1WPL9 Ribosome biogenesis GTPase. 33,397 8.66 183 2 10

Food Chemistry
81 S4Z027 NADPH quinone oxidoreductase 40,196 4.61 181 3 12
82 H1WNG4 UPF0118 membrane protein 40,846 10.06 178 4 26
83 H1 × 7D2 Di- and tricarboxylate transporter 40,571 10.11 176 3 28
84 A0A0L7TC61 Osmoprotectant uptake system 28,682 10.1 176 5 24
permease
85 A0A0P9SR10 Nickel oligopeptide ABC type 38,724 9.48 174 5 25
transport system permease
86 A0A0D9AQ43 Sodium proton antiporter 45,322 5.02 174 7 26
87 M8B1 × 0 Serine threonine protein kinase 68,858 5.47 172 8 12
88 V6JHC6 EamA like transporter family protein. 34,507 9.83 167 4 23
89 A0A0D9AVH3 Metal ABC transporter permease 23,580 5.67 166 2 30
90 A0A0D9AKI5 L dehydroascorbate transporter large 44,030 9.51 164 2 22
permease
91 V6JEZ7 Binding dependent transport system 23,950 7.17 163 3 49
inner membrane component family
protein
92 A0A0K2BE94 Chemotaxis 26,996 9.72 163 4 16
93 H1 × 8R8 Multidrug resistance protein 48,462 10.51 161 6 29
94 S3MG07 Achromobactin ABC type transport 35,934 11.18 161 6 32
system permease
95 A0A0P9T059 MFS transporter 44,125 9.36 160 2 17
96 N1R3U5 Heparan α-glucosaminide N 87,502 8.83 160 8 15
acetyltransferase
97 R7WDX6 Protein gar2 72,025 4.57 159 22 26
98 A0A0D9AZ10 Formate dehydrogenase 54,890 4.86 159 5 28
99 A0A0L7TIL6 Membrane protein 35,235 10.43 157 4 25
100 M7YAE7 Cinnamyl alcohol dehydrogenase 40,992 5.63 156 3 18
101 V6J738 β-Carotene 15 15 monooxygenase. 29,524 9.35 155 4 31
102 V6J4T0 Binding dependent transport system 30,227 10.36 154 2 12
protein
103 A0A0D9ARC4 Paraquat inducible protein 22,535 6.57 154 2 28
104 A0A0L7SYV1 Multidrug resistance protein 1E+05 5.26 153 7 19
105 A7UG09 NADH ubiquinone oxidoreductase 41,654 4.45 153 5 12
106 A0A0D9AKJ6 Membrane protein 31,840 10.30 152 6 39
107 Q9SNM0 Os06g0132100 protein 50,088 5.27 151 2 9
108 H1ADY3 RNA polymerase I 45,276 4.78 150 3 11
109 A0A0L7T3C0 Permease 48,443 9.32 150 4 28
110 M7ZZM7 Transporter protein 26,533 6.84 149 6 51
111 M8A0C1 Equilibrative nucleoside transporter 40,412 8.86 149 4 15
112 A0A0D9B467 Spermidine putrescine ABC 29,195 9.64 149 2 14
transporter permease
113 L7HD95 Permease OS Xanthomonas 40,058 9.96 146 7 32
translucens
114 M8AS63 1 acyl sn glycerol 3 phosphate 39,224 10.12 145 6 21
acyltransferase
115 A0A0D9AQU5 Lipoprotein signal peptidase 19,084 7.73 143 2 11
116 A0A0D9AUK3 Type II secretion system protein 34,582 9.41 142 8 28
117 B2WDA2 1 3 4 β-Glucanase 53,133 7.84 141 1 10
118 L7GZW8 UPF0056 membrane protein 21,370 9.63 141 4 27
119 A0A0U2YPR9 RNA binding protein 17,224 11.40 140 3 14
120 T1MD83 Formin like protein 51,993 6.81 139 2 9
121 L7HAD8 General secretion pathway protein 43,880 9.25 137 3 13
122 W5I0R4 Serine threonine protein kinase 93,276 4.75 137 9 7
123 A0A0D9AXN7 Membrane protein 8,609 11.83 137 1 48
(Continued)

Vol. 0, Iss. 0, 2018 r Journal of Food Science 11

 Antioxidant activity of wheatgrass . . .

Table 3–Continued.

Acc. No. Protein mW (Da) pI PLGS score Peptides Coverage (%)

124 H1WNG1 Integral membrane protein 32,369 9.83 136 10 45
125 J3NRM0 ATP dependent RNA helicase 62,375 8.26 136 8 28
126 V6J5 × 6 Glycosyl transferase 2 family protein 42,791 9.25 136 4 29
127 A0A0P9U364 Iron ABC transporter permease 35,945 11.41 134 6 33
128 A0A0D9AIW0 Leucine isoleucine valine transporter 45,583 9.66 134 4 14
permease
129 H1WTC7 Regulatory protein 36,143 11.01 134 6 38
130 B2VY37 1 3 β-Glucanosyltransferase 47,375 4.39 133 2 5
131 H1WNE9 Membrane associated phospholipid 25,273 10.54 133 4 21
phosphatase
132 W5ELM3 DNA topoisomerase 50,217 9.03 132 2 10
133 H1WRU2 Cytochrome b subunit 65,126 10.09 132 7 29
134 N1QY32 Serine threonine protein kinase 99,941 6.61 131 4 12
135 H1 × 890 High affinity gluconate transporter 46,437 9.89 131 4 35
136 M8BA43 FACT complex subunit 70,034 5.44 121 4 18
137 R7WDY1 Histone. 20,431 11.25 109 2 20
Food Chemistry

138 M8ADD0 Serine threonine protein kinase 88,548 6.22 105 9 11

139 V6J701 Methyl accepting chemotaxis MCP 68,539 4.89 89 5 14
signaling domain protein
140 W5A8N1 Potassium transporter 87,765 7.81 85 4 17
141 Q6RUK8 Synaptobrevin 24,657 8.27 73 2 9
142 M8BBS2 CHD3 type chromatin remodeling 2E+05 5.58 60 9 6
factor
143 N1R4A8 Cytochrome P450 52,277 6.39 41 2 8

Figure 4–The antioxidant activities of the wheatgrass extract were deter-

mined by using 2,2-diphenyl-1- picrylhydrazyl (DPPH) after 0, 8, and 16
DAG. Standard mean errors are indicated. ∗ indicate that DPPH values
are significantly different from each other at p < .001 for all time points
Figure 3–Ferric reducing antioxidant power (FRAP) of the wheatgrass ex- selected.
tract after 0, 8, and 16 DAG. Standard mean errors are indicated. ∗ indicate
that FRAP values are significantly different from each other at p < .001 nourishment of humans. Furthermore upon case to case studies
for all time points selected. wheatgrass may be used for prevention of diseases.

that antioxidant radicals and some enzymes such as peroxi-
dases, cytochrome P450, and SODs can play a critical role in
reducing oxidative stress. This study employed a nontargeted
proteomic approach and investigated the proteome of wheat-
grass. Upon detailed investigation by using LC-MSE and GO
analysis, cytochrome, peroxidase, heme exporter protein, and
glyceraldehydes-3-phosphate dehydrogenase were identified in
wheatgrass extract. FRAP and DPPH scavenging activity assay
showed powerful activity in wheatgrass. Data from present results
revealed that Triticum aestivum seedlings can act as an antioxidant
agent due to their free radical scavenging activity and can be
constructive to control or treat many health complications. From
all these results we believed that wheatgrass can be used for the
Acknowledgments
VVD is thankful to the Science and Engineering Research
Board, Dept. of Science and Technology, Government of India,
New Delhi, India, for funding under Young Scientist Scheme
(SB/YS/LS-260/2013). Technical assistance of Miss Shraddha La-
hoti is highly acknowledged.
Conflicts of Interest
The authors declare that they have no conflict of interest.
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Supporting Information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
Figure S1. GO analysis of proteins identified in developing
wheatgrass and wheat seeds: A total of 297 unique proteins were
analyzed by ProteinLynx Global SERVERTM (PLGS) and anno-
tated by using the Blast2GO program.
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