The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury
Food &
Function
PAPER
Cite this: Food Funct., 2024, 15, 5315
Received 6th January 2024,
Accepted 20th March 2024
DOI: 10.1039/d4fo00091a
rsc.li/food-function
The antioxidant peptides from walnut protein
hydrolysates and their protective activity against
alcoholic injury
Peihang Chen,†
a
Pantian Huang,†
a
Yingyan Liang,
a
Qiaoe Wang*
b
and
Jianyin Miao *
a
In this study, walnut protein was hydrolyzed, separated by ultrafiltration, purified by RP-HPLC, identified
by LC-MS/MS, and screened by molecular docking to finally obtain three novel antioxidant peptides
HGEPGQQQR (1189.584 Da), VAPFPEVFGK (1089.586 Da) and HNVADPQR (949.473 Da). These three
peptides exhibited excellent cellular antioxidant activity (CAA) with EC
50
values of 0.0120 mg mL
−1
,
0.0068 mg mL
−1
, and 0.0069 mg mL
−1
, respectively, which were superior to that of the positive control
GSH (EC
50
: 0.0122 mg mL
−1
). In the ethanol injury model, three antioxidant peptides enhanced the survi-
val of cells treated with ethanol from 47.36% to 62.69%, 57.06% and 71.64%, respectively. Molecular
docking results showed that the three antioxidant peptides could effectively bind to Keap1, CYP2E1 and
TLR4 proteins. These results suggested that walnut-derived antioxidant peptides could be potential anti-
oxidants and hepatoprotective agents for application in functional foods.
1. Introduction
In the metabolic processes of living organisms, reactive oxygen
species (ROS) or free radicals are inherently produced through
oxidative reactions involving respiratory mechanisms.
Organisms have evolved their unique antioxidant defense
mechanisms to counterbalance excessive ROS.
1
When the
limited efficiency of the organism does not prevent all oxi-
dative damage related to environmental conditions, the
accumulation of excess free radicals and ROS in the cells
causes oxidative stress, which can lead to organismal
damage.
2
Oxidative stress contributes to many non-communic-
able chronic diseases, including cardiovascular disease, dia-
betes, inflammatory diseases, and aging.
3
Therefore, the devel-
opment of natural antioxidants has great practical significance
in the prevention of diseases.
In recent years, peptides have attracted widespread interest
as one of the most important classes of biologically active
ingredients.
4
Studies have shown that bioactive peptides have
significant physiological benefits, including antioxidant, anti-
hypertensive, anti-thrombotic, antimicrobial, anticancer, anti-
inflammatory, antidiabetic, anti-obesity, cholesterol-lowering,
immunomodulatory, and mineral binding effects.
5
Additionally, food-derived bioactive peptides also possess pre-
ventive and therapeutic functions in a healthy diet.
6
What
makes bioactive peptides even more appealing is their ability
to exhibit minimal side effects in the human body due to their
natural origin.
1
Peptide segments with antioxidant properties
can exert their antioxidant effects by scavenging free radicals,
inhibiting peroxides, and chelating metal ions (Fe
2+
/Cu
2+
),
7
which endows them with potential characteristics as food pro-
cessing additives. Antioxidant peptides have been reported in
various sources, such as pearl oyster meat,
8
oysters,
9
cotton-
seed,
2
and bitter buckwheat.
10
Alcoholic liver disease (ALD) is one of the most common
liver diseases worldwide and is mainly caused by chronic
excessive alcohol consumption.
11
According to the World
Health Organization (WHO), ALD is responsible for hundreds
of thousands of deaths each year, and the progression of ALD
from asymptomatic hepatic steatosis to alcoholic hepatitis,
hepatic fibrosis and cirrhosis is a constant threat to people’s
health and lives.
12
It has been shown that alcohol can cause
the accumulation of intracellular ROS, affect hepatic lipase
activity, and trigger inflammation, leading to cell damage and
death.
13,14
Although there are a number of drugs available that
can effectively treat ALD such as silybum marianum,
15
predni-
solone
16
and pentoxifylline,
17
the side effects associated with
their long-term use are unavoidable. A number of studies have
shown that natural antioxidants can prevent ALD through anti-
†These authors contributed equally to this work and should be considered co-
first authors.
a
Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,
College of Food Science, South China Agricultural University, Guangzhou 510642,
China. E-mail: miaojy8181@scau.edu.cn; Fax: +862085286234;
Tel: +8620 85286234
b
China Food Flavor and Nutrition Health Innovation Center, Beijing Technology and
Business University, Beijing, 100048, China
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oxidant mechanisms such as scavenging of free radicals, res-
toration of antioxidant enzyme activity, and maintenance of
homeostasis of the hepatic antioxidant defense system.
18,19
Antioxidant peptides, as classical natural source actives, have
great research significance and development value in the pre-
vention of alcoholic liver injury.
Walnut is one of the four most consumed dried fruits in
the world, and is considered to be an important oil, economic
and ecological tree species.
20
According to the database of the
Food and Agriculture Organization of the United Nations
(FAO), China’s walnut production in 2022 exceeded 6 million
tons, ranking first in the world. However, currently, most of
the walnut meal is either used as animal feed or discarded
after oil extraction, resulting in a waste of resources.
21
Therefore, efficient utilization of walnut meal protein holds
significant practical importance. Studies have shown that the
peptides isolated from walnut protein have activities such as
reduction of blood pressure,
22
anti-inflammatory
23
and neuro-
protective effects,
24
and lowering of uric acid.
25
Feng et al. iso-
lated and identified a tripeptide (FPY) with tyrosinase inhibi-
tory activity from walnut meal.
26
Wang et al. isolated and
identified an ACE inhibitory peptide (EPNGLLLPQY) from
walnut protein.
22
Although walnut hydrolysates/peptides have
been shown to have a variety of biological activities and appli-
cations, there have been fewer studies on the structural charac-
terization (especially peptide sequences with antioxidant
effects) and conformational relationships of walnut peptides,
as well as a lack of studies on the potential for applications in
oxidative stress-related diseases such as alcoholic liver injury.
In our previous study, we extracted proteins from walnut
meal and investigated the process of hydrolysis for the prepa-
ration of antioxidant peptides.
27
In this study, based on the
previous foundation, the walnut antioxidant hydrolysate was
isolated and purified by ultrafiltration and reversed-phase high
performance liquid chromatography (RP-HPLC), the peptide
sequences were identified, and the potential antioxidant pep-
tides were screened by molecular docking. Finally, the anti-
oxidant peptides were evaluated for their cellular antioxidant
activity and protection against ethanol damage, with a view to
providing new ideas for the high-value utilization of walnut
meal resources and potential natural alternatives for the treat-
ment of alcoholic liver injury.
2. Materials and methods
2.1. Materials
Trypsin (2500 USP mg
−1
) was purchased from Nanning Pangbo
Biological Engineering Co., Ltd (Nanning, China). Glutathione
(GSH) and metadoxine were purchased from Shanghai Yuanye
Biotechnology Co., Ltd (Shanghai, China). Dulbecco’s modi-
fied Eagle’s medium (DMEM), fetal bovine serum (FBS), phos-
phate buffer solution (PBS), and penicillin–streptomycin anti-
biotic mixture were purchased from Thermo Fisher Scientific
Co., Ltd (Shanghai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-
diphenyl tetrazolium bromide (MTT) was purchased from
Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). All
other reagents were analytically pure.
2.2. Preparation of walnut antioxidant peptide hydrolysates
Antioxidant peptide hydrolysates of walnut protein were pre-
pared using a pre-determined hydrolysis process.
27
The walnut
protein was hydrolyzed with trypsin, and the hydrolysis con-
ditions were set at a substrate concentration of 5.8%, pH 8.0,
trypsin 0.1%, 39.4 °C, and 3.5 h. The hydrolysate was heated at
90 °C for 15 min to terminate the protease activity. After the
hydrolysate was cooled to room temperature, it was centrifuged
at 2852gfor 10 min, and the supernatant was collected and
stored lyophilized at −20 °C.
2.3. Purification of walnut antioxidant peptides
2.3.1. Ultrafiltration. The hydrolysate was separated using
an ultrafiltration membrane (3 kDa). Two fractions (>3 kDa
and <3 kDa) with molecular weights above and below 3 kDa
were obtained and freeze-dried to measure their in vitro anti-
oxidant activity.
2.3.2. Purification by RP-HPLC. A preparative RP-HPLC
system (LC-8, Shimadzu, Kyoto, Japan) was used to purify the
high antioxidant activity part of the ultrafiltration results by refer-
ring to the method of Huang et al.
8
The high-activity fraction was
loaded onto a well-equilibrated reverse C18 column (20 mm ×
450 mm, 10 µm, Shimadzu, Japan) at a volume of 3 mL. The
mobile phases were eluent A (ultrapure water + 0.1% trifluoroace-
tic acid (TFA)) and eluent B (methanol + 0.1% TFA). The flow rate
was 10 mL min
−1
and the detection wavelength was 214 nm.
Gradient elution conditions were as follows: 1–65 min, 5–25% B;
65–75 min, 25–50% B; 75–85 min, 50–95%B.Eachpeakwascol-
lected and freeze-dried as a separate fraction, and the antioxidant
activity of each component was determined. Finally, the fractions
with the highest antioxidant activity were screened for LC-MS/MS
to identify their peptide sequences.
2.3.3. Antioxidant activity assay. Antioxidant activity was
determined according to the method of Miao et al.
28
Different
concentrations of sample solution (100 μL) and 1,1-diphenyl-2-
picrylhydrazyl (DPPH) (100 μL of 0.2 mmol L
−1
) solution were
mixed (DPPH dissolved in 95% ethanol) and reacted for
30 min at room temperature against light. Then, they were
placed in a microplate reader (Enspire2300, PerkinElmer, MA,
USA) at 517 nm and recorded as A
t
. At the same time, the
absorbance value of a mixture of 100 μL of sample solution
and 100 μL of 95% ethanol at 517 nm was measured and
recorded as A
r
, and then the absorbance value of a mixture of
100 μL of DPPH solution and 100 μL of 95% ethanol at 517 nm
was measured and recorded as A
0
. The DPPH free radical
scavenging rate was computed according to the formula (1).
DPPH free radical scavenging rate ð%Þ
¼½1ðAtArÞ=A0100 ð1Þ
A
t
,A
r
and A
0
are the absorbance values of the sample, control
and blank groups.
Paper Food & Function
5316 |Food Funct.,2024,15,5315–5328 This journal is © The Royal Society of Chemistry 2024
Published on 27 March 2024. Downloaded by ZHENG ZHOU UNIVERSITY on 7/8/2024 9:34:45 AM.
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