The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury

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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,†

Pantian Huang,†

Yingyan Liang,

Qiaoe Wang*

and

Jianyin Miao *

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

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

: 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.

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.

Oxidative stress contributes to many non-communic-

able chronic diseases, including cardiovascular disease, dia-

betes, inflammatory diseases, and aging.

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.

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.

Additionally, food-derived bioactive peptides also possess pre-

ventive and therapeutic functions in a healthy diet.

What

makes bioactive peptides even more appealing is their ability

to exhibit minimal side eﬀects in the human body due to their

natural origin.

Peptide segments with antioxidant properties

can exert their antioxidant eﬀects by scavenging free radicals,

inhibiting peroxides, and chelating metal ions (Fe

/Cu

which endows them with potential characteristics as food pro-

cessing additives. Antioxidant peptides have been reported in

various sources, such as pearl oyster meat,

oysters,

cotton-

seed,

and bitter buckwheat.

Alcoholic liver disease (ALD) is one of the most common

liver diseases worldwide and is mainly caused by chronic

excessive alcohol consumption.

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.

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,

predni-

solone

and pentoxifylline,

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.

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

China Food Flavor and Nutrition Health Innovation Center, Beijing Technology and

Business University, Beijing, 100048, China

This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,5315–5328 | 5315

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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.

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.

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,

anti-inflammatory

and neuro-

protective eﬀects,

and lowering of uric acid.

Feng et al. iso-

lated and identified a tripeptide (FPY) with tyrosinase inhibi-

tory activity from walnut meal.

Wang et al. isolated and

identified an ACE inhibitory peptide (EPNGLLLPQY) from

walnut protein.

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.

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.

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.

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.

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

. 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

, 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

. The DPPH free radical

scavenging rate was computed according to the formula (1).

DPPH free radical scavenging rate ð%Þ

¼½1ðAtArÞ=A0100 ð1Þ

and A

are the absorbance values of the sample, control

and blank groups.

Paper Food & Function

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