Effect of seabuckthorn seed protein and its arginine-enriched peptides on combating memory impairment in mice

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International Journal of Biological Macromolecules 232 (2023) 123409

Available online 24 January 2023

Effect of seabuckthorn seed protein and its arginine-enriched peptides on

combating memory impairment in mice

Xiping Zhu

, Lei Cai

, Jinqi Liu

, Wen Zhu

, Chun Cui

, Daofu Ouyang

, Jianwen Ye

College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu 241000, Anhui, China

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China

Perfect (Guangdong) Daily Necessities Co, Ltd, Zhongshan 528400, Guangdong, China

ARTICLE INFO

Keywords:

Seabuckthorn seed protein

Arginine-rich peptides

Brain aging

Nitric oxide synthase

Anti-inammation

ABSTRACT

The current study characterized the combating memory impairment effect of seabuckthorn seed protein (SSP) and

the arginine (Arg)-enriched peptides (SSPP) on d-galactose-induced brain aging in mice. The Arg content in SSP

and SSPP were 10.11 and 17.82 g/100 g, respectively. Seven Arg peptides (Ile/Leu-Arg, Arg-Glu, Asp-Arg-Pro,

Arg-Try-Ala, Glu-Arg-Ser, Val-Gly-Arg-Pro, and Lys-Thr-Glu-Arg) were identied from SSPP. The animal ex-

periments of the Morris water maze and the step-down test indicated that the oral administration of SSP (0.25,

0.5, 1.0 mg/g⋅d) and SSPP (0.25, 0.5, 1.0 mg/g⋅d) signicantly (p <0.05) reversed the learning and memory

impairment symptoms. The activation of endothelial nitric oxide (NO) synthase and neuronal NO synthase were

increased, and inducible NO synthase decreased after SSP and SSPP in the hippocampus compared to the model

group, with the SSPP being quite effective. Moreover, the treatment signicantly exhibited the ability to

normalize the serum inammatory cytokine levels (NF-ĸB, TNF-

, IL-6) and suppress the Arg-inducible nitric

oxide (Arg-iNO) pathway. Therefore, SSP and SSPP ingestion reversed the behavioral learning and memory

impairment symptoms possibly associated with the anti-inammation and Arg-iNO pathway. Consumption of

SSP and SSPP diets can be benecial to memory impairment.

1. Introduction

Brain aging is accompanied by different stages of memory decline in

the elderly. It is a devastating neurodegenerative ailment characterized

by an irreversible, progressive deterioration in diverse cognitive func-

tions [1,2]. In addition, cognitive dysfunction caused by neurodegen-

erative diseases such as Alzheimer's has become a signicant problem in

older people [1–3]. Currently, acetylcholinesterase (AChE) inhibitors

and N-methyl-D-aspartic acid (NMDA) receptor antagonists are avail-

able for treating amnesia and memory impairment. However, neither

can delay nor block the pathological process of brain aging [3,4].

Therefore, in the absence of an effective treatment module for brain

aging, alternative therapeutic strategies and the biochemical process in

etiopathogenesis are being explored.

Rising evidence depicted that L-arginine (Arg, a semi-essential,

proteinogenic amino acid) and its nitric oxide (NO) production exhibi-

ted multiple functions in human health with a prominent role in age-

related degenerative diseases like Alzheimer's [5,6]. Arg can affect

amnesia and memory impairment pathogenesis since Arg and NO

regulate the physiological functions of the brain and other organs [7,8].

The dysfunction of nitric oxide synthase (NOS) enzyme in the Alz-

heimer's disease brain could be due to Arg depletion caused by Aβ

deposition [9]. However, the relationship of Arg with NOS isoforms is

intricate. There are three closely related NOS isoenzymes (endothelial

NO synthase (eNOS), neuronal NO synthase (nNOS), and inducible NO

synthase (iNOS)) that have been discovered so far [5]. Regarding iNOS,

its expression is induced in the inammatory cell types by cytokine (NF-

ĸB, TNF-

, IL-6) stimulation [10]. iNOS activity is independent of cal-

cium, the inducible NO (iNO) production rate is high, and NO can last

for several hours [11,12]. iNOS, a catalyst of excessive NO and Arg

depletion production, participate in the inammatory process [10,13].

Studies have revealed that iNOS expression and activity were signi-

cantly increased in brain aging patients, which produces large amounts

of NO and Arg depletion [5]. However, nNOS and eNOS expression is

constitutive and regulated by calcium or calmodulin. The production

rate of neuronal NO (nNO) and endothelial NO (eNO) is low [14,15].

* Corresponding author at: School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China.

E-mail address: cuichun@scut.edu.cn (C. Cui).

These authors contributed equally.

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

https://doi.org/10.1016/j.ijbiomac.2023.123409

Received 29 November 2022; Received in revised form 19 January 2023; Accepted 20 January 2023

International Journal of Biological Macromolecules 232 (2023) 123409

The reduction of eNOS activity and eNO content may cause the

dysfunction of BBB endothelial cell clearance of Aβ, which could

contribute to brain aging pathogenesis [16–18]. nNO also is an essential

transmitter in various developmental processes in the default neural

network formation and has a signicant role in the learning and memory

process [5]. Studies have reported that nNOS inactivation leads to

working memory decits and cognitive impairments [19]. Therefore,

eNO and nNO, but not iNO, as transmitters, have a neuroprotective ef-

fect and ameliorate cognition in brain-aging patients. Thus, modulating

the relevant NOS isoforms expression and their activity may provide a

new therapeutic to improve brain aging.

Furthermore, Arg is the only nitrogen-supplying precursor within the

highly active molecule NO. Arg and its metabolic fates (metabolic

pathways and metabolites) are critical for monitoring brain aging. It is

involved in various physiological and pathological processes (for

example, cellular redox metabolism [20], redox stress [21], cerebral

blood ow regulation [20,22], neurotransmission, neurogenesis and

neuroplasticity [23], and inammation [24], etc.). Therefore, Arg sup-

plementation and an Arg-enriched diet can provide a new therapeutic

avenue in brain aging. However, very few studies have examined the

effect of Arg-enriched protein and peptides to diminish

neurodegenerative-related states. Seabuckthorn seed protein is an Arg-

enriched protein with signicant anti-inammatory and hypoglycemic

effects [25–27]. In addition, seabuckthorn (Hippophae rhamnoides L.),

known for its nutritional and medicinal properties, is a wild Eurasian

shrub from the Elaeagnaceae family [27,28]. Nonetheless, the anti-

neurodegenerative effect of the seabuckthorn seed protein and its Arg-

enriched peptides has not been properly investigated.

Therefore, this study aimed to obtain Arg-enriched peptides from

seabuckthorn seed protein with controllable enzymolysis technology.

Moreover, Arg-enriched peptides were separated using ion exchange

resin and RP-HPLC. Then, the combating memory impairment effect of

seabuckthorn seed protein and the Arg-enriched peptides on D-galactose-

induced amnesia was investigated in mice. Furthermore, we investi-

gated their correlation with the Arg-iNO pathway closely associated

with the pathogenesis of Arg metabolism and memory impairment.

2. Materials and methods

2.1. Material and chemicals

Seabuckthorn seed meal (crude protein 21.03 %, lipid 0.48 %, car-

bohydrates 11.24 %, on a dry basis) purchased from Qinghai KangPu

Biotechnology Co., Ltd. (Qinghai, China). Flavourzyme from Aspergillus

niger (activity: 48 U/mg) and trypsin from porcine pancreas (activity:

15000 U/mg) were purchased from Guangzhou Qiyun Biotechnology

Co., Ltd. (Guangdong, China). Commercial peptides (as standards for

sensory evaluation) were purchased from Peptide Biological Technology

Co., Ltd. (Nanjing, China). D-galactose was purchased from Chengdu

Nakeli Biological Technology Co., Ltd. (Chengdu, China). iNOS, nNOS

and eNOS assay kits were purchased from Solarbio Science & Technol-

ogy Co., Ltd. (Beijing, China). The remaining reagents and chemicals

above analytical grade used were purchased from Wuhan Seville Bio-

logical Technology Co., Ltd. (Wuhan, China).

2.2. Preparation of seabuckthorn seed protein (SSP) and its Arg-enriched

peptides (SSPP)

2.2.1. Preparation of SSP

Seabuckthorn seed protein (SSP) (protein content was 79.21 ±0.52

%, on a dry basis) was prepared from seabuckthorn seed meal following

the alkali-soluble acid precipitation method [26,28]. Seabuckthorn seed

meals were mashed with a masher system, pushed through 80 mesh

copper sieves. Then, the parameters of alkaline extraction were as fol-

lows: pH 11, a 1:14 ratio of seabuckthorn seed meal powder to water at a

temperature of 60 ◦C for 1 h, then centrifugation and separation to

obtain the supernatant. The parameters for acid precipitation were as

follows: pH 5.0, then centrifuged and separated to obtain the precipitate.

The precipitate was washed with deionized water at pH 7, then vacuum

freeze-dried as seabuckthorn seed protein (SSP).

2.2.2. Preparation of SSPP

Then, Arg-enriched peptides were prepared using the above sea-

buckthorn seed protein were performed under conditions as follows: pH

7.0 for trypsin and avourzyme complex (trypsin:avourzyme =1:3 (w/

w)), seabuckthorn seed protein to water ratio 1:9, at temperature 55 ◦C,

incubation time 12 h. The supernatant was obtained by centrifuging the

reaction mixture in a centrifuge (GL-21 M, Changsha Xiangzhi Centri-

fuge Instrument Co., Ltd., Changsha China) at 8000 rpm and 4 ◦C for 20

min. Arginine-rich peptides were separated and puried according to

Zhu et al. [29], an aliquot (200 mL) of the supernatant was carefully

loaded onto the glass laminated column (2.6 cm ×60 cm, Shanghai

Jiapeng Technology CO., Ltd., Shanghai, China), which was packed with

strongly acidic ion exchange resins (001 ×7(732), Sanat chemical CO.,

Ltd., Hebei, China); pre-equilibrated with HCl (4 %, pH 6.1); and kept at

4 ◦C overnight. Then the column was eluted with 500 mL of deionized

water at 25 ◦C and 2 mL/min to obtain the arginine-rich fraction. Then,

The lyophilized arginine-rich fraction was redissolved by using Milli-Q

water (10 mg/mL) [30] and the solution was loaded onto an

XBridge™ BEH130 semipreparative C

RP-HPLC column (10 mm ×

150 mm, 10

m; Waters, Milford, MA, USA) according to previous

studies [31–33]. The elution was programmed at a ow rate of 3 mL/

min, at 25 ◦C for 25 min, with Milli-Q water (95–50 %) and a linear

gradient of acetonitrile (5–50 %), which was monitored at the wave-

length of 220 nm with a UV detector (UV201, USA). The corresponding

peak fractions of arginine-rich peptides were collected and the charac-

terization was conducted using an ultraperformance liquid chromatog-

raphy coupled with mass spectrometry (UPLC-Q-TOF MS/MS) (Bruker

Daltonics, Beijing, China) as described by Yang et al. [34]. The collected

fractions of arginine-rich peptides were freeze-dried for further studies.

2.3. Proximate analysis of SSP and SSPP

The total nitrogen content was obtained by using a nitrogen deter-

mination apparatus (conversion factor is 6.25) (SKD-1000, Shanghai

Peiou Analytical Instrument Co., Ltd., Shanghai, China) according to

China National Standard (GB 18186-2000). Amino acid proles of SSP

and SSPP were analyzed using an A300 auto amino acid analyzer

(membraPure, Bodenheim, Germany) according to the method of Zhu

et al. [32]. After the samples had been hydrolyzed at 110 ◦C for 24 h

with 6 mol/L hydrochloric acid (HCl), the total amino acids (reported as

g/100 g) of the samples were measured. The dissociative amino acids

(expressed as g/100 g) of the samples were determined by directly

injected into the analyzer after ltrating through a 0.22

m membrane

lter.

2.4. Animal trials

2.4.1. Animals

A total of one hundred 4-week-old, male SPF KM mice (weight: 18 ±

2 g) were provided by Changzhou Cavens Laboratory Animal Co. Ltd.

(Changzhou, China). Mice were maintained in cages (6 animals per cage:

320 ×215 ×170 mm) under a 12 h light-dark cycle at 24 ±2 ◦C with

free access to the normal chow diet and water in the laboratory animal

center of Anhui Polytechnic University. The experimental protocols

were approved by the Animal Experiment Committee of the laboratory

animal center of Anhui Polytechnic University and followed the guide-

lines of the National Institutes of Health for the Care and Use of Labo-

ratory Animals.

2.4.2. D-galactose-induced amnesia mice and different treatments

Mice were randomly assigned to 8 experimental groups with a total

X. Zhu et al.

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