Wheat peptides with different hydrolysis degree have similar relief effect in constipated mice

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Food Bioscience 52 (2023) 102459

Available online 1 February 2023

Wheat peptides with different hydrolysis degree have similar relief effect in

constipated mice

Qianqian Wang

, Fei Shen

, Junhui Zhang

, Jiachen Zhuang

, Fengqin Feng

College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China

National Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China

Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture, Zhejiang University, Hangzhou, 310058, China

ARTICLE INFO

Keywords:

Wheat peptides

Hydrolysis degree

Constipation

Intestinal barrier

Oxidative stress

ABSTRACT

Constipation has become more common in recent decades, affecting people’s quality of life. Natural bioactive

substances could be effective in alleviating constipation. The goal of this study was to assess and compare the

efcacy of low and high hydrolysis degree wheat peptides (WP-L and WP-H) at a dose of 1 mg/g.bw on relieving

loperamide-induced constipation in mice and the underlying mechanisms. Here, we proved that both WP-L and

WP-H treatment showed constipation relieving effect by improving defecation function and small intestinal

propulsion rate while the different hydrolysis degree did not affect the efcacy of WP. Moreover, WP-L and WP-H

could balance the secretion of excitatory and inhibitory factors, and enhance the activity of antioxidant enzyme

(SOD and GSH-Px). Importantly, both of them could accelerate intestinal motility, improve water-salt meta-

bolism and intestinal barrier, and ameliorate oxidative stress. In summary, our ndings indicated that 1 mg/g.bw

WP-L and WP-H effectively relieved loperamide-induced constipation by affecting multiple targets in mice.

1. Introduction

Constipation, a common gastrointestinal disorder, is characterized

by the infrequent passage of stools or difculty with the evacuation of

stools (Longstreth et al., 2006). Despite the signicant global expendi-

ture on constipation treatment research, more than 50% of constipation

patients are still unable to be treated, which signicantly impacting

their quality of life (Nelson et al., 2017). Until now, stool softeners,

osmotic or irritating laxatives, as well as secretagogues and prokinetic

medicines, have been the most common drugs for treating constipation

(Sharma & Rao, 2017; Vazquez Roque & Bouras, 2015). However, these

drugs have signicant drawbacks that limit their usage in the treatment

of constipation, including strong drug dependence, high recurrence rate,

poor long-term effect, and expensive cost (Chang, et al., 2014; Miner

et al., 2014). Hence, developing safe and effective drugs to treat con-

stipation is on the cutting edge. Natural bioactive substances for the

control and prevention of constipation are favored by consumers due to

their low toxicity and less adverse effects (Wang et al., 2022a, 2022b;

Xia et al., 2022). For instance, bioactive peptides generated from food

proteins have been reported to play a prominent role in maintain gut

health and function (Bao & Wu, 2021).

In the previous study, we determined that 2 mg/g wheat peptides

(WP) effectively relieved constipation in loperamide-induced consti-

pated mice (Wang et al., 2022) . However, as patients may respond

differently to WP treatment, an additional dose choice of less than 2 mg

is preferred to guarantee that patients with varying degrees of con-

stipation severity (from mild to severe) have a dosing option with WP.

The degree of hydrolysis (DH) of bioactivity peptides, on the other hand,

may affect their functional characteristics in that peptides with a higher

DH are accompany with a lower molecular weight (MW), which enable

them to pass through the intestinal barrier to exert their functional

properties in vivo (Miao et al., 2018; Sarmadi & Ismail, 2010; You et al.,

2012). For instance, high hydrolysis degree sea cucumber peptides with

a lower molecular weight displayed a better antioxidant effect in

fatigued mice than low hydrolysis degree (Wang et al., 2021). However,

no comparative study of the constipation relief effect of WP with

different DH has been reported. A comprehensive study of WP with

different hydrolysis degree could provide some guidance to the opti-

mization of WP production.

As for the mechanism of wheat peptide in improving constipation,

the aspects of water-salt metabolism, intestinal mobility, and gut

microbiota were analyzed (Wang, Shen, et al., 2022). However, the

etiology of constipation is believed to be multifaceted and has not yet

been elucidated in its entirety (Bharucha & Lacy, 2020). The intestinal

* Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou, 310058, China.

E-mail address: feng_fengqin@hotmail.com (F. Feng).

Contents lists available at ScienceDirect

Food Bioscience

journal homepage: www.elsevier.com/locate/fbio

https://doi.org/10.1016/j.fbio.2023.102459

Received 21 November 2022; Received in revised form 31 January 2023; Accepted 1 February 2023

Food Bioscience 52 (2023) 102459

barrier constituted by intestinal epithelial cells play critical roles on the

electrolyte and nutrient absorption, and inhibiting luminal bacteria and

undigested food particles from entering the body uncontrolledly

(Groschwitz & Hogan, 2009; Turner, 2009). Tight junction proteins are

therefore critical for to the process of preserving the structural integrity

of the intestinal barrier. Additionally, the homeostatic concentrations of

free radicals in intestinal epithelial cells also play essential role in

maintaining the function of intestinal barrier (Chaudhari, et al., 2014).

The two amino acids that were most abundant in WP were glutamic acid

(Glu) and glutamine (Gln) have been closely related to the integrity of

intestinal barrier (Zheng et al., 2017). When Gln was depleted, the

expression of tight junction proteins was drastically downregulated in

Caco-2 cell lines (Li et al., 2004). On the ip side, Glu supplementation

improved intestinal barrier function by upregulating the expression of

tight junction proteins (Jiao et al., 2015). Moreover, WP was able to

exert an effective antioxidant effect and protect against oxidative stress

in ethanol-induced gastric mucosal damage (Yang et al., 2020; Zhang,

Wen, et al., 2019). Hence, we postulated that the relief of constipation

by WP may also be linked to the enhancement of intestinal barrier

function and the suppression of oxidative stress.

This study was aimed at: (i) evaluating the efcacy of 1 mg/g wheat

peptides dose in loperamide-induced mice; (ii) determining whether

there are differences between WP-L and WP-H in alleviating con-

stipation; and (iii) elucidating the possible mechanism of WP-L and WP-

H to relieve constipation from the perspective of water-salt metabolism,

intestinal barrier, oxidative stress, and intestinal motility.

2. Material and methods

2.1. Materials

WP-L (DH =7.31%) was graciously provided by Hangzhou Kan-

gyuan Food Science and Technology Co., Ltd. (Hangzhou, China),

whereas WP-H (DH =25.06%) was obtained locally (Hangzhou, China).

Loperamide was bought from Xi’an Janssen Pharmaceutical Ltd. (Xi’an,

China). The ink preparation operation was executed in accordance with

the previously stated method (Chai et al., 2021).

2.2. Animals and experimental design

The Ethics Committee of Experimental Animals of Zhejiang Chinese

Medical University (20211206-06) authorized the animal experiment

procedure. Forty-eight BALB/c male mice (20 ±2g) were divided into

four groups (n =12 each group) at random: control group (NC), model

group (MC), low-degree hydrolysis wheat peptides treated group (WP-

L), and high-degree hydrolysis wheat peptides treated group (WP–H).

Mice in the MC, WP-L, and WP-H groups were administered with

loperamide (10 mg/kg⋅bw) 0.5 h before daily treatment, while mice in

the NC group were provided with saline. Then, mice in the WP-L and

WP-H groups were gavage administration with the corresponding WP (1

mg/g.bw) per day, while mice in the NC and MC groups were treated

with saline daily.

2.3. Determination of fecal excretion function

2.3.1. Defecation test

On day 15, the mice were fasted for 16 h with water provided. The

next morning, the mice in the MC, WP-L, and WP-H groups were pro-

vided with loperamide (10 mg/kg⋅bw) while the NC group receiving

saline. Thirty minutes later, the ink solution was given to all mice (0.01

mL/g.bw). After that, each mouse was promptly placed in its own in-

dividual container, where it was given unrestricted access to food and

water. The time to the rst black stool defecation after being given ink,

as well as the weight, number, and water content of black feces in 5 h

were meticulously documented (Zhai et al., 2018).

2.3.2. Small intestinal motility test

After the defecation test, the mice were fasted for 16 h with water

provided. The next morning, the mice in the MC, WP-L, and WP-H

groups were provided with loperamide (7 mg/kg⋅bw) while the NC

group receiving saline. Thirty minutes later, the ink solution was given

to all mice (0.01 mL/g.bw). After half an hour, the mice were sacriced

by cervical dislocation. The small intestine propulsion rate was calcu-

lated by recording the total length of the gut and the distance ink

traveled (Eor et al., 2019). The serum and colon tissue were subse-

quently collected and stored at −80 ◦C for subsequent tests.

2.4. Determination of biochemical index

The motilin (MTL), gastrin (GAS), substance P (SP), somatostatin

(SS), vasoactive intestinal peptide (VIP), and endothelin 1 (ET-1) were

measured using commercial ELISA kits (Wuhan ColorfulGene Biological

Technology Co., Ltd., Wuhan, China) following the manufacture’s pro-

tocol. Briey, serum sample (10

L) and sample dilution buffer (40

were mixed in 96-well microelisa stripplate and then the HRP-conjugate

reagent (50

L) was added for incubate 30 min at 37 ◦C. Afterward, the

solution was discarded and washed with wash solution for 5 times. After

washing, the chromogen solution A (50

L) and chromogen solution B

(50

L) were added and incubated for 10 min at 37 ◦C and subsequently

measured at 532 nm.

The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px),

and malondialdehyde (MDA) were measured using commercial assay

kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)

following the manufacture’s protocol. The SOD activity was determined

based on the reaction of xanthine-xanthine oxidase reaction to produce

superoxide radicals and its absorbance was measured at 550 nm. The

GSH-Px was determined by the reaction of GSH and 5,5’-dithionbis (2-

nitrobenzoic acid) to produce a color reaction and its absorbance was

measured at 412 nm. The MDA level was determined by the reaction of

MDA and thiobarbituric acid to produce a color reaction and its absor-

bance was measured at 532 nm.

2.5. Real-time quantitative PCR

The detailed protocol of RT-qPCR was performed as reported pre-

viously in our laboratory (Zhong et al., 2020). The sequences of the

primers were synthesized by Tsingke Biotechnology Co., Ltd. (Hang-

zhou, China) (Table 1). GAPDH was utilized as a reference. The results

were calculated using the method of 2

−ΔΔCt

2.6. Statistical Analysis

Statistical analysis was completed by GraphPad Prism 6.0 with One-

way ANOVA followed by Tukey’s test. The data are presented as means

±SEM, and a P-value <0.05 was considered statistically signicant.

3. Results

3.1. WP-L and WP-H improved defecation function and small intestinal

propulsion rate in constipated mice

After induction of constipation, the food intake and weight gain of

the mice in the MC group were less than those in the NC group, but the

WP-L and WP-H groups reversed the decreasing trends (Fig. 1B and C).

The time to the rst black stool defecation in the WP-L and WP-H groups

were signicantly shortened by 25.42% and 27.64% than that of the MC

group (P <0.01) (Fig. 1D). And the number of fecal pellets, fecal wet

weight, and fecal water content in the WP-L and WP-H groups were

signicantly increased by 76.50% (P <0.01) and 53.30% (P <0.01),

27.20% and 39.84%, 40.70% and 68.45% (P <0.01) (1E-1G).

Furthermore, WP treatment signicantly accelerated the small intestine

propulsion rate in constipated mice by 45.49% and 48.89% (P <0.01,

Q. Wang et al.

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