RESEARCH ARTICLE

A Novel Peptide from Soybean Protein

Isolate Significantly Enhances Resistance of
the Organism under Oxidative Stress
Heran Ma1, Rui Liu2, Ziyuan Zhao1, Zhixian Zhang1, Yue Cao1, Yudan Ma3, Yi Guo1*,
Li Xu1*
1 Key laboratory for Molecular Enzymology and Engineering, the Ministry of Education, National Engineering
Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, 130012, P.R. China,
2 Department of endocrinology, China-Japan Union Hospital, Jilin University, Changchun, Jilin, 130033, P.R.
China, 3 Sports Science Research Institute of Jilin Province, Changchun, Jilin, 130022, P. R. China
a11111
* guoyi@jlu.edu.cn (YG); xuli@jlu.edu.cn (LX)

Abstract
Recent studies have indicated that protein hydrolysates have broad biological effects. In the
OPEN ACCESS
current study we describe a novel antioxidative peptide, FDPAL, from soybean protein iso-
Citation: Ma H, Liu R, Zhao Z, Zhang Z, Cao Y, Ma late (SPI). The aim of this study was to purify and characterize an antioxidative peptide from
Y, et al. (2016) A Novel Peptide from Soybean
SPI and determine its antioxidative mechanism. LC–MS/MS was used to isolate and identify
Protein Isolate Significantly Enhances Resistance of
the Organism under Oxidative Stress. PLoS ONE 11 the peptide from SPI. The sequence of the peptide was determined to be Phe-Asp-Pro-Ala-
(7): e0159938. doi:10.1371/journal.pone.0159938 Leu (FDPAL, 561 Da). FDPAL can cause significant enhancement of resistance to oxidative
Editor: Ferenc Gallyas, Jr., University of Pecs stress both in cells as well as simple organisms. In Caenorhabditis elegans (C. elegans),
Medical School, HUNGARY FDPAL can up-regulate the expression of certain genes associated with resistance. The
Received: April 10, 2016 antioxidant activity of this peptide can be attributed to the presence of a specific amino acid
sequence. Results from our work suggest that FDPAL can facilitate potential applications of
Accepted: July 11, 2016
proteins carrying this sequence in the nutraceutical, bioactive material and clinical medicine
Published: July 25, 2016
areas, as well as in cosmetics and health care products.
Copyright: © 2016 Ma et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
Introduction
credited. According to the free radical theory proposed by Denham Harman, living organisms can pro-
Data Availability Statement: All relevant data are duce radical species by various metabolic pathways. These include reactive oxygen species
within the paper and its Supporting Information files. (ROS), such as O2-, HO2, H2O2 and OH [1]. Excessive accumulation of free radicals, known as
Funding: This study was supported by grants from
oxidative stress, can be harmful to the cell. Usually, oxidative stress is considered to be a pro-
the National Natural Science Foundation of China moter of chronic diseases, such as cancer, diabetes, Alzheimer’s disease and others, rather than
(no. 81271697, 81571791, and 31571017), the an initiator [2–5]. Extensive investigations have indicated that dietary supplementation of anti-
Specialized Research Fund for the Doctoral Program oxidants can enhance the body’s natural defense mechanism. This appears to be a reasonable
of Higher Education (20100061120077), and the and practical approach to reduce the level of oxidative stress in vivo [6, 7]. Over the past several
Project of Science and Technology Department of
decades, a large number of natural antioxidants have been obtained from animal, plant, and
Jilin Province, China (no. 20130206069GX). The
funders had no role in study design, data collection
even microbial sources [8–12].
and analysis, decision to publish, or preparation of For many years, Soy products have been one of the main sources of dietary protein, and it is
the manuscript. therefore of relevance to investigate the presence of any potential additional bioactivity that

PLOS ONE | DOI:10.1371/journal.pone.0159938 July 25, 2016 1 / 15

 An Antioxidative Soybean Peptide

Competing Interests: The authors have declared can meet the need to counter the increasingly higher incidence of environmental stress. Nor-
that no competing interests exist. mally, enzymatic hydrolysis or fermentation is used to enhance the functionality of protein
ingredients, which may lead to the production of short peptide sequences with various bioac-
tivities. After enzymatic hydrolysis, the resulting lower molecular mass of peptides will likely
have more potential to exert biological effects in vivo due to their increased permeability
through the intestinal cells [13, 14]. Soybean protein hydrolysates that possess a range of bioac-
tivities including antioxidation, anti-hypertension, anti-hyperlipidemia, cholesterol reduction
and immunity enhancing activities have been extensively reported in literature [15–18].
In this research we propose the hypothesis that peptides from soybean protein possess anti-
oxidant activity and that this activity contributes to its various bioactivities. However, we can-
not investigate the antioxidation mechanism of these peptides unless we determine their
precise amino acid sequences. Liquid chromatography tandem mass spectrometry (LC–MS/
MS) is the preferred method for separation and identification of peptides in complex bioactive
peptide mixtures. In this article, we purified and identified peptide sequences using continuous
chromatography and LC-MS/MS methods. We identified a purified peptide with the sequence
Phe-Asp-Pro-Ala-Leu (FDPAL, 561 Da) from soybean protein isolate (SPI). The antioxidant
activities of FDPAL were subsequently evaluated, and were found to include scavenging free
radicals in vitro, reducing the accumulation of ROS and lipofuscin, up-regulating the expres-
sion of SOD-3 in vivo, improving the survival rate of Hela cell and C. elegans under oxidative
stress. We conclude that the activity of this pentapeptide is related to its amino acid composi-
tion and sequence.
The specific objectives of this study were to: (i) isolate the antioxidant peptide from SPI and
determine its primary structure; (ii) evaluate the antioxidant activities of this peptide both in
vitro and in vivo; (iii) elucidate its antioxidation mechanism at molecular and cellular levels,
and also in multicellular organisms, and subsequently to determine whether these mechanisms
are consistent with each other.

Materials and Methods

Materials
Soybean protein isolate (SPI) was purchased from Daqing Celestial Sun Moon Star Protein
Co., Ltd., China. An enzyme (Alcalase) was purchased from Novo Nordisk, Shenyang Bio-
chemical Processing Co. Ltd., China, which was used in enzymolysis. Sephadex G-10 and
DEAE Sephadex A-25 were purchased from Pharmacia Chemicals Co., Sweden. All other
reagents used were of analytical grade and obtained from Sigma Chemical Co, USA.

Enzyme hydrolysis
10 g SPI was added to 100 mL distilled water in a 250 mL conical flask. The flask was then incu-
bated in a water-bath at a temperature of 50°C, and the pH of the solution was adjusted to 8.0.
After hydrolysis was allowed to occur for the required length of time, the conical flask was
placed in a boiling water bath and incubated for 10 min in order to inactivate the enzyme and
terminate the enzymatic reaction. The aqueous solution was then rapidly cooled to room tem-
perature and centrifuged at 7000 rpm for 15 min. The supernatant was removed till only 30
mL remained. The degree of hydrolysis (DH) was determined using the pH stat method based
on the Eq (1) [19]:
Nb
DH ¼ B   100% ð1Þ
a  Mp  htot

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 An Antioxidative Soybean Peptide

where B is the volume of NaOH in the hydrolysis process (mL), Nb is the normality of NaOH
(mol/L), Mp is the mass of protein used (g), htot is the amount of peptide bonds per gram of
SPI (7.80 mmol/g) [20], α is the degree of dissociation of α-amino and determined by Eq (2):
10pHpKa
a¼ ð2Þ
1 þ 10pHpKa
where pH is the initial pH of enzymatic hydrolysis (8.0) and pKa is a dissociation constant,
which is calculated by Eq (3):
 
298  T
pKa ¼ 7:8 þ  2400 ð3Þ
298  T

where T is the temperature of enzymatic hydrolysis (K).

Ion-exchange chromatography
DEAE Sephadex Fast Flow ion-exchange column (2.0 cm × 20 cm; Pharmacia Fine Chemicals,
Sweden) was used to separate the components of the lyophilized SPI powder with 10 mM phos-
phate buffer (pH 9.0). The column was previously equilibrated with buffer until no absorbance
peaks were observed and then a linear gradient elution of 0–5 M NaCl was performed at a flow
rate of 1 mL/min. The eluate was collected and evaluated at 220 nm. Fractions (fractions 1–3)
with distinct peaks were collected and lyophilized to test for antioxidant activity (S1A and S1B
Fig).

Gel filtration chromatography

As shown in S1A and S1B Fig, fraction 2 was found to have the highest antioxidant activity fol-
lowing ion-exchange chromatography. This fraction was dissolved in distilled water and loaded
onto a Sephadex G-10 gel filtration column (2.0 cm × 100 cm; Pharmacia Fine Chemicals, Swe-
den) and eluted with distilled water at a flow rate of 0.5 mL/min. The eluate was collected and
evaluated at 220 nm. Fractions with distinct peaks were collected and lyophilized to test for
antioxidant activity (S1C and S1D Fig).

Determination of amino acid sequence by LC-MS/MS

The LC-MS/MS system consisted of an Eksigent Ekspert UltraLC100 Reversed Phase High Per-
formance Liquid Chromatography (RP-HPLC) system coupled to an AB SCIEX Triple TOF™
5600 mass spectrometer with Turbo spray. The chromatography column was a reverse phase
C18 column, (serial No. 186003023, 3.5 μm, 2.1 mm × 150 mm Waters XBridge). 10 μL of sam-
ple was injected and subsequently eluted with a gradient elution program, with a linear gradi-
ent of 0.1% methanoic acid in distilled water (A) and 0.1% methanoic acid in acetonitrile (B)
from 10% B to 90% B in 15 min, at a flow rate of 0.25 mL/min. The scan range was set at m/z
100.00–1000.00. The MS/MS spectrum was sequenced by manual calculation and pure peptide
FDPAL was obtained.

Fenton’s reaction
Hydroxyl free radicals generated by Fenton’s reaction were measured by monitoring chemilu-
minescence of the reaction mixture. Reagents were added into a cuvette in the following order:
10 μL 3% H2O2, 10 μL 0.1 mM Fe2+, 10 μL FDPAL, and 970 μL 0.1 mM luminol (in sodium
carbonate buffer, pH 10.2), and were incubated in a water bath at 25°C. A series of reactions

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 An Antioxidative Soybean Peptide

with a final FDPAL concentration 0.05, 0.1, 0.2, 1 and 2 mM were set up and absorbance was
measured at 550 nm. Vitamin C (Vc) group was treated as control.

Pyrogallol self-oxidation assay

The in vitro superoxide anion-scavenging characteristic of FDPAL was measured using a pyro-
gallol autoxidation system with modifications [21]. Reagents were added into a cuvette in the
following order: 10 μL 3 mM pyrogallol, 80 μL 4 mM NaOH, 10 μL FDPAL, and 900 μL 0.1
mM luminol (in sodium carbonate buffer, pH 10.2) and incubated in a water bath at 25°C. A
series of reactions with a final FDPAL concentration of 0.05, 0.1, 0.2, 1 and 2 mM were set up
and absorbance was measured at 325 nm. Vitamin C (Vc) group was treated as control.

MTT assay
HeLa cells were purchased from China General Microbiological Culture Collection Center
(CGMCC, Beijing, CHN), and were grown exponentially for investigation. The cells were
seeded in 96-well plates at a final concentration of 8 × 103 cells per well and incubated with
FDPAL at various concentrations (0, 0.1, 0.2, 1, 2, 10, 20 mM) for 12 h prior to the addition of
500 μM H2O2. The medium was then removed and the cells were washed twice with PBS.
Fresh low serum (5%) medium containing 500 μM H2O2, was then added to the cells and incu-
bated at 37°C, 5% CO2 for 4 h. At the indicated time, MTT assay was used to evaluate the cell
survival rate [22].

Worm strains and their maintenance

C. elegans was grown in a standard nematode growth medium (NGM) in plates maintained at
20°C and fed with live Escherichia coli OP50 bacteria (Brenner 1974). The wild-type strain Bris-
tol N2 and the transgenic strain CF1553 (muIs84) were obtained from Caenorhabditis Genetics
Center; CGC, USA. SOD-3::GFP-linked reporter in CF1553 was used to visualize SOD-3
expression.

Stress resistance assay

Stress resistance assay was performed with two-day-old adult worms. The worms were incu-
bated for two days with FDPAL (10 mM) and were then transferred to plates with 500 μM
juglone. Worm deaths per hour were counted and recorded. Each assay was performed in three
independent parallel experiments in a double-blind manner [23].

Measurement of intracellular ROS in C. elegans

For the ROS test under oxidative stress, two-day-old adult worms were treated with 300 μM
juglone for 1 h and were then transferred to plates with or without FDPAL (10 mM). After two
days of incubation, worms were transferred into a 96-well plate with a transparent bottom and
opaque walls containing M9 buffer in the wells. A fluorescent probe (H2DCF-DA) was
employed to determine the intracellular ROS in worms [24]. Fluorescence intensity correlated
with the ROS level and was measured by a microplate reader at excitation and emission wave-
lengths of 485 and 520 nm respectively.

Fluorescence quantification and visualization

Net fluorescence of worms was assayed using a Thermo Labsystems Fluoroskan Ascent micro-
plate reader (Thermo Fisher, Waltham, MA). Adult worms were treated with or without 10
mM FDPAL for two days. 20 control or treated adult animals of the indicated age were

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 An Antioxidative Soybean Peptide

transferred into each well of a Costar 96-well microtitre plate (black, clear, flat-bottom wells)
containing 100 μL of M9 buffer, after which GFP and ROS fluorescence were measured using
485 nm excitation and 530 nm emission filters. Under ultraviolet light, gut granules emit blue
fluorescence, which has been attributed to lipofuscin, with maximal intensity at λex/λem 340/
430 nm. Four replicates were used for each determination. For fluorescence microscopy, the
worms were suspended in a drop of levamisole (10 mM) and mounted on a cover slip layered
with 3% agarose. The fluorescent images of worms were captured using an AXIO Imager M2
microscope system.

Statistical analysis
Values were expressed as mean ± S.D. The statistical significance between six groups was evalu-
ated by one-way ANOVA followed by Students-'t' test. Results with P-values less than 0.05
were considered to be significant (Graph Pad, San Diego, CA).

Results
Isolation and purification
In our study, the enzymatic reactions were terminated when the DH values reached
24.64 ± 1.77%. DEAE Sephadex A-25 ion-exchange chromatography was then used to separate
the SPI extract into three fractions (fraction 1–3). Fraction 2, which exhibited the strongest
antioxidant activity (Fig 1A and S1A and S1B Fig) was further separated by Sephadex G-10 gel
filtration column into two fractions (fractions I, II). Of these two, fraction I showed a strong
antioxidant activity (Fig 1B and S1C and S1D Fig). LC-MS/MS was used to further purify and
identify the monomer peptide from fraction I. We first eluted fraction I from a low organic
mobile phase (10% acetonitrile) to a high organic mobile phase (90% acetonitrile), following
which the monomer peptide FDPAL was purified from it. Triple TOF-MS was employed to
determine the molecular weight of FDPAL, which revealed the molecular ion mass (M+H+) of
FDPAL to be 562.2860 Da (Fig 1C and 1D).

Characterization of purified peptides

In order to identify the putative active peptide, an MS/MS experiment was performed on an
AB SCIEX Triple TOF™ 5600 mass spectrometer. Fragmentation spectra that matched with the
FDPAL fragment were sequenced manually. As shown in Fig 2, purified peptide FDPAL was
identified to be a pentapeptide with the amino acid composition of Phe-Asp-Pro-Ala-Leu (561
Da).

Antioxidant activity of FDPAL

Free-radical scavenging capacity of FDPAL was evaluated in subsequent experiments with the
Vc group as control. Initially, the ability of FDPAL to remove hydroxyl free radical was investi-
gated in the Fenton’s reaction system. The data revealed that FDPAL had a stronger effect of
scavenging the hydroxyl free radical as compared to Vc (Fig 3A). Subsequently, the ability of
FDPAL to remove superoxide anions was then investigated in a pyrogallol self-oxidation sys-
tem. These data showed that FDPAL could also effectively scavenge the free radicals produced
by pyrogallol self-oxidation (Fig 3B).
The MTT assay was used to assess the protective effect of FDPAL on H2O2-induced injury
in HeLa cells. As shown in Fig 3C, after exposure of HeLa cells to 500 μM H2O2 for 4 h, the cell
survival rate in the FDPAL groups increased significantly when compared with the control
group. Preincubation of the HeLa cell with 0, 0.1, 0.2, 1, 2, 10 or 20 mM of FDPAL inhibited

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 An Antioxidative Soybean Peptide

Fig 1. Purification and identification of FDPAL from Soybean Protein Isolate extraction. (A) Separation of antioxidant peptides from SPI by
using Sephadex A-25. Elutions were performed with 10 mM phosphate buffer (pH 9.0) and a linear gradient of 0–5 M NaCl at a flow rate of 1.0 mL/
min for 8 h. (B) Separation of fraction 2 in (A) by Sephadex G-10. Elutions were performed with distilled water at a flow rate of 0.5 mL/min for 4 h.
(C) TIC spectrum of active fraction I from (B) measured by LC-MS/MS. (D) TOF-MS spectrum of FDPAL.
doi:10.1371/journal.pone.0159938.g001

apoptosis. As shown in the results, FDPAL could significantly increase cell viability up to a
concentration of 10 mM.
Further experiments were performed to evaluate the potential longevity-promoting effect of
FDPAL on wild-type C. elegans N2 under oxidative stress. Worms that had just reached adult-
hood were pretreated with 10mM FDPAL for 48 h and then exposed to juglone (500 μM).
Juglone is a pro-oxidant that can be reduced by diaphorases in the presence of NAD(P)H. It
converts oxygen to superoxide anion and consequently increases intracellular oxidative stress.
Our results showed that pretreatment with 10mM FDPAL had a strong protective effect. As
shown by the statistical analyses, the mean survival rate was significantly increased by 24.2% in
the FDPAL-treated group as compared to that of control (Fig 3D). The results indicated that
FDPAL pretreatment improved resistance towards juglone induced oxidative stress in the
worms.
Additionally, FDPAL was also demonstrated to have ROS-scavenging capability in C. ele-
gans. Our data showed that pretreatment of the worms with 10 mM FDPAL effectively reduced

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 An Antioxidative Soybean Peptide

Fig 2. Identification of molecular mass and amino acid sequence of FDPAL. MS/MS experiments were performed on AB SCIEX
TripleTOF™ 5600 mass spectrometer. The sequence of FDPAL was determined manually.
doi:10.1371/journal.pone.0159938.g002

ROS accumulation in juglone-treated (300 μM) wild-type C. elegans (compared with the con-
trol,  p < 0.0001; Fig 4).
These results suggested that FDPAL was a versatile free radical scavenger both in vitro as
well as in vivo.

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 An Antioxidative Soybean Peptide

Fig 3. Antioxidant effect of FDPAL in vitro and in vivo. Vc group was treated as control. (A) FDPAL
dramatically scavenged the hydroxyl free radical generated by Fenton’s reaction; n = 5 in each group. (B) FDPAL
effectively scavenged the free radicals generated by pyrogallol self-oxidation; n = 5 in each group. (C) HeLa cell
viability under oxidative stress with or without FDPAL. Significance of difference versus control group at
####p < 0.0001. Significance of differences versus 0 mM FDPAL group at *p < 0.05, ***p < 0.001,
****p < 0.0001; n = 5 in each group. (D) FDPAL improves the stress resistance of C. elegans under oxidative
stress. Survival curves are presented based on three individual experiments.
doi:10.1371/journal.pone.0159938.g003

FDPAL up-regulates SOD-3::GFP expression in transgenic C. elegans

CF1553
To further investigate the mechanisms of the protective effects of FDPAL in C. elegans, SOD-
3::GFP reporter gene expression in transgenic CF1553 was analyzed. The CF1553 worms were
treated with FDPAL or left untreated after stimulation with juglone (300 μM). When compared
with the control group, the FDPAL-treated group demonstrated a higher SOD-3::GFP intensity
as observed with confocal laser scanning microscopy (Fig 5B). The fluorescence intensity was
then quantified by a Thermo Labsystems Fluoroskan Ascent microplate reader. The results
showed that 10mM FDPAL up-regulated SOD-3::GFP expression by 116.06% in transgenic
CF1553 (Fig 5A), indicating that FDPAL could enhance the expression of SOD-3 under oxida-
tive stress in C. elegans.

Discussion
Traditionally, dietary protein is regarded as a potential source of energy and essential amino
acids for humans and animals and has been the subject of numerous investigations. In recent
times there is a growing interest in identifying antioxidative peptides from natural sources,
including plants and animals [11, 25, 26]. These peptides may possess various properties, such

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 An Antioxidative Soybean Peptide

Fig 4. FDPAL at 10 mM reduced ROS accumulation in C. elegans N2 under juglone-generated oxidative

stress. (A) Quantified DCF-DA intensity (±SE) in worms from three individual experiments with 50 worms per
treatment (****p < 0.0001). (B) Image of fluorescence intensity in control worms and 10 mM FDPAL-treated
worms. The ROS level in control worms is higher than that in FDPAL treated worms.
doi:10.1371/journal.pone.0159938.g004

as antioxidative [27, 28], antithrombotic [29], antimicrobial [30], immunomodulatory [31],

opiate-like [32], mineral binding [33], hypocholesterolaemic [34] and antihypertensive [35,
36], owing to their structure, and amino acid composition and sequence. In this study, we puri-
fied a novel active peptide from SPI by chromatographic methods. The amino acid sequence of
this peptide was determined to be Phe-Asp-Pro-Ala-Leu by AB SCIEX Triple TOF™ 5600 mass
spectrometer (Figs 1C, 1D and 2). This novel mass spectrometer provides excellent MS

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 An Antioxidative Soybean Peptide

Fig 5. FDPAL up-regulates SOD-3::GFP expression in transgenic C. elegans CF1553. (A) Quantified
GFP intensity (±SE) in CF1553 from three individual experiments with 50 worms per treatment
(****p < 0.0001). (B) SOD-3::GFP expression in control worms and 10 mM FDPAL-treated worms. The
SOD-3::GFP expression in FDPAL treated worms is higher than that in control worms.
doi:10.1371/journal.pone.0159938.g005

performance combining high resolution, good mass accuracy and high sensitivity in order to
achieve a high rate of success for identification.
We performed in vitro experiments to determine the antioxidant potential of this peptide.
The results reveal that it possesses a high antioxidant efficacy (Fig 3), especially in the hydroxyl
free radical scavenging test, where FDPAL was better in scavenging hydroxyl radicals when

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 An Antioxidative Soybean Peptide

compared to Vc (Fig 3A). To further test whether FDPAL is permeable across the cell membrane
and can exert biological effects, an MTT assay was designed. The H2O2-induced HeLa cells injury
model was chosen for this assay to evaluate the protective action of FDPAL against acute oxida-
tive injury [37, 38]. H2O2 was used as an inducing agent to mimic oxidative stress induced injury.
As expected, exposure of cells to H2O2 resulted in decreased cell viability, an effect that was atten-
uated by FDPAL in a concentration dependent manner. Interestingly, we noticed a distinct drop

Fig 6. FDPAL can significantly reduce lipofuscin content in worms. (A) Lipofuscin fluorescence was
measured by Image J, 30 worms were tested in each group. (B) Lipofuscin fluorescence in worms.
doi:10.1371/journal.pone.0159938.g006

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 An Antioxidative Soybean Peptide

in the surviving fraction when the concentration reached 20 mM (Fig 3C). This could be more
acidic in 20 mM solution will injure cell, due to the presence of carboxyl group in FDPAL.
Thus we showed that FDPAL possesses a strong antioxidant activity in vitro. In order to test
whether it has similar activity in vivo, we used Caenorhabditis elegans to investigate antioxidant
activity in vivo under both normal and stress conditions. We found that FDPAL could significantly
extend the life expectancy of C. elegans under oxidative stress conditions (Fig 3D), and reduce
ROS and lipofuscin accumulation under normal conditions (Figs 4 and 6). We believe that this
activity of FDPAL in vivo is related to its molecular weight, amino acid composition and sequence.
An obvious drawback of protein and polypeptide drugs is their lower oral bioavailability due to
enzymatic degradation in the gastrointenstinal tract and the impenetrable barrier of the intestinal
epithelium. Generally in the gastrointestinal tract, most of the protein and polypeptide drugs are
degraded to oligopeptides (2–6 amino acid residues) rather than free amino acids [39]. Thus
FDPAL, due to its small size and low molecular weight (562 Da), can easily cross the intestinal bar-
rier, thereby exerting its biological effects [40, 41]. In addition, the presence of hydrophobic groups
at both the C-terminus (Leu) and N-terminus (Phe) renders FDPAL more soluble in lipids. This
special feature of hydrophobic regions on both sides can also increase its affinity and reactivity to
the intestinal cell membrane [6, 42]. In FDPAL, Phe-Asp are present as direct proton-donators,
which, owing to their ability to quench unpaired electrons or radicals by supporting protons, are
very important for the radical-scavenging activity of this peptide [43, 44]. Nonetheless, we believe
that the activity of FDPAL does not completely depend on its own direct antioxidant capacity or
its indirect oxidation resistance. We speculate that FDPAL influences the expression of genes asso-
ciated with resistance in C. elegans and thereby enhances oxidative stress resistance. Our work
indicates that FDPAL increases the expression of SOD-3::GFP under oxidative stress conditions in
C. elegans (Fig 5). We speculate that FDPAL may activate certain regulatory pathways conferring
resistance (such as the well-known insulin signaling pathway) in C. elegans, which then causes
increased expression of SOD-3 [45, 46]. However, the exact mechanism by which FDPAL up-reg-
ulates SOD-3 expression is unclear, and we do not know yet whether FDPAL is released by enzy-
matic hydrolysis in vivo or not. These issues need further investigation to be resolved.
In summary, we present a novel antioxidant peptide, FDPAL, from SPI. The presence of
specific amino acids and their sequence is responsible for the antioxidant activity of this pep-
tide. Moreover, we found a longevity-promoting effect of FDPAL in C. elegans under oxidative
stress conditions, which might be attributed to its direct ROS-scavenging activity and indirect
free radical-scavenging activity, via up-regulation of expression of resistance associated genes
such as SOD-3. In this study we describe, from the perspective of antioxidant activity, a series
of influences that FDPAL has on organisms. Above all, these interesting findings suggest that
FDPAL is an important peptide having potential applications in the nutraceutical, bioactive
material and Clinical Medicine areas, as well as in cosmetics and health care products.

Supporting Information
S1 Fig. Antioxidant activities of the fractions in vitro. (A) Scavenging activities of fractions
1, 2 and 3 on superoxide anion. (B) Scavenging activities of fractions 1, 2 and 3 on hydroxyl
radical. (C) Scavenging activities of fractions I and II on superoxide anion. (D) Scavenging
activities of fractions I and II on hydroxyl radical.
(TIF)

Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China
(No. 81271697, 81571791, and 31571017), the Specialized Research Fund for the Doctoral

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 An Antioxidative Soybean Peptide

Program of Higher Education (20100061120077), and the Project of Science and Technology
Department of Jilin Province, China (No. 20130206069GX). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions
Conceived and designed the experiments: YG LX. Performed the experiments: HRM RL. Ana-
lyzed the data: HRM RL ZYZ ZXZ YC YDM. Contributed reagents/materials/analysis tools:
YDM YG LX. Wrote the paper: HRM RL.

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