Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein

3.0 科研～小助 2025-09-01 4 4 1.36MB 8 页 1知币

侵权投诉

Food Chemistry 401 (2023) 134127

Available online 7 September 2022

Purication identication and function analysis of ACE inhibitory peptide

from Ulva prolifera protein

Zhiyong Li

, Yuan He

, Hongyan He

, Weizhe Zhou

, Mengru Li

, Aiming Lu

, Tuanjie Che

Songdong Shen

School of Biology & Basic Medical Sciences, Soochow University, Suzhou, Jiangsu 215101, China

Key Laboratory of Functional Genomic and Molecular Diagnosis of Gansu Province, Lanzhou 730030, China

ARTICLE INFO

Keywords:

Ulva prolifera protein

ACE-inhibitory peptide

Purication and characterization

Molecular docking

In vitro gastrointestinal digestion

Immunomodulation

ABSTRACT

In the present study, Ulva prolifera, an edible alga, was used to prepare angiotensin-I converting enzyme (ACE)

inhibitory peptide. The algae protein was isolated and later hydrolyzed by ve commercial enzymes (alcalase,

papain, pepsin, trypsin, neutral protease), either individually or in combination. Hydrolysate, with the highest in

vitro ACE inhibitory activity, was processed using the Sephadex-G100, ultraltration, HPLC-Q-TOF-MS, ADMET

screening and molecular docking, respectively. The ACE inhibitory peptide DIGGL with a IC

value of 10.32 ±

0.96

M was then identied. The peptide against ACE by a non-competitive mode and mainly attributable to the

three Conventional Hydrogen Bonds. It could activate Endothelial nitric oxide synthase activity in NO generation

and reduce Endothelin-1 secretion induced by Angiotensin II in Human umbilical vein endothelial cells.

Meanwhile, DIGGL could promote mice splenocytes proliferation, which was also effective when co-incubated

with Con A or LPS, respectively. Besides, the anti-ACE peptide could remain active during the digestion of

gastrointestinal proteases (pepsin-trypsin) in vitro.

1. Introduction

Hypertension, which aficts>20 % of adults worldwide, is the

leading cause of cardiovascular disease and premature death (Mills

et al., 2020). The mechanisms leading to increased blood pressure

mainly comprise the renin angiotensin aldosterone regulatory system

(RAAS) (Azushima et al., 2020), sympathetic activation system (Hart,

2016), endothelial dysfunction (Hall et al., 2019), and inammatory

response (Bartoloni et al., 2018). Angiotensin-I converting enzyme

(ACE), one of the key enzymes in RAAS, catalyzes the conversion of

inactive Angiotensin I (Ang I) into a potent vasoconstrictor Angiotensin

II (Ang II) to inactivate the vasodilator bradykinin (Raghavan & Kris-

tinsson, 2009); therefore, inhibiting ACE activity is considered to be an

effective therapeutic approach for the treatment of hypertension. Until

now, the use of several synthetic ACE inhibitors, such as captopril,

enalapril and lisinopril, in clinical treatments have led to signicant side

effects, such as inammatory response, cough, or renal impairment (Ko

et al., 2012). Therefore, there has been growing interest in the past

decades in extracting ACE peptide inhibitors from natural products. The

proteins of several algae, such as Chlorella vulgaris (Xie et al., 2018) and

Spirulina platensis (Zhang et al., 2022), have been used in isolating ACE

inhibitory peptides.

Ulva prolifera is an edible green algae from the Chlorophyta and Ulva

families. The eutrophication of sea water caused by industrial and do-

mestic sewage has provided favorable living conditions for Ulva prolifera

and has resulted in the excess production of this algae (Ye et al., 2011).

Because of its huge biomass and excellent nutritional composition

(proteins 9–14 %, 2–3.6 % ether extract, 32–36 % ash, and 10.9 g/100 g

of total fatty acids), Ulva prolifera is a promising candidate for com-

mercial applications (Aguilera-Morales et al., 2005). However, it has

shown poor usefulness in human pharmaceutical development. Nearly

all the algae have been used as food, animal feed, and fertilizer or have

even been regarded as industrial waste (Li et al., 2016). Although some

ACE inhibitory peptides from Ulva prolifera protein has been reported

(Pan et al., 2016). However, no peptide inhibitors isolated form this

protein have been synthesized and used in clinical trials.

Growing evidence suggests that oligopeptides can be directly

absorbed by cells as nutrients to promote cell metabolism (Shen &

Matsui, 2017). A growing number of immune-enhancing peptides have

been isolated from natural products, such as Juglans regia L (Mao

* Corresponding author.

E-mail address: shensongdong@suda.edu.cn (S. Shen).

Contents lists available at ScienceDirect

Food Chemistry

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

https://doi.org/10.1016/j.foodchem.2022.134127

Received 2 June 2022; Received in revised form 1 September 2022; Accepted 2 September 2022

Food Chemistry 401 (2023) 134127

Ruixue et al., 2020), egg (Lozano-Ojalvo et al., 2016), and wheat gluten

(Horiguchi et al., 2005). There is a tremendous amount of Ulva prolifera

available from the ocean; however, very few studies have been con-

ducted on the immunomodulatory peptide from its protein.

The aim of the present study was designed to develop a promising

ACE inhibitory peptide from Ulva prolifera and determine its immuno-

modulatory activity. In addition, its gastrointestinal stability, inhibitory

pattern, and molecular interaction mechanism were explored. Finally,

the cellular mechanism by which the puried peptide regulates blood

pressure was explored in human umbilical vein endothelial cells

(HUVECs).

2. Materials and methods

2.1. Materials and chemicals

Ulva prolifera was obtained from Institute of Oceanography, Chinese

Academy of Sciences (Qingdao, China). Angiotensin I-converting

enzyme (from rabbit lung), N-hippuril-L-histidy-L-leucine (HHL),

Angiotensin II (Ang II), alcalase and papain were purchased from San-

gon Biotech (Shanghai, China). Sephadex G-100 were purchased from

Auyoo Biotechnology Co. (Shanghai, China). Human Endothelin-1 (ET-

1) ELISA Kit was purchased from Sangon Biotech (Shanghai, China).

Nitric Oxide Assay Kit was purchased from Beyotime (shanghai, China).

MTT cell proliferation and cytotoxicity assay kits were purchased from

Sigma-Aldrich (St Louis, MO, USA). HUVECs cell line was purchased

from SSRCC (Shanghai, China). All other reagents were of analytical

reagent grade.

2.2. Preparation of Ulva prolifera protein

The protein was extracted according to the previous report (Li et al.,

2020) with slight modications. Dry powder of Ulva prolifera was soaked

in double-distilled water (1:20, w/v). The pH value of the mixture was

adjusted to 10 (using 1 M NaOH), before stirring at 37 ◦C for 3 h. Then,

the suspension was centrifuged at 1000 r/min for 15 min to obtain the

supernatant. Subsequently, the pH value of above solution was adjusted

to 4.5 (using 1 M HCl) to precipitate the protein. Finally, the puried

protein was freeze-dried and stored at −20 ◦C until further analysis.

2.3. Preparation of hydrolysate of Ulva prolifera protein

Ulva prolifera protein solution (10 %, w/v) was adjusted to the

appropriate pH and temperature, before adding some commercial pro-

teases (1 % enzyme/substrate, E/S, w/w protein). The enzymes and

digestion conditions used in present study were alcalase (pH 9.0,

50.8 ◦C, 3500 U/g), neutral protease (pH 7.4, 47 ◦C, 3500 U/g), papain

(pH 7.2, 45.7 ◦C, 1250 U/g), alcalase-papain (pH 9.0, 50.8 ◦C, 3500 U/g-

pH 7.2, 45.7 ◦C, 1250 U/g), and pepsin-trypsin (Ph 2.0, 37 ◦C, 3500 U/g-

pH 8.1, 37 ◦C, 1250 U/g). The hydrolysis reaction of each enzyme lasted

for 1 h and was then terminated by boiling water bath.

2.4. Degree of hydrolysis and peptide yield

The degree of hydrolysisi (DH) of the Ulva prolifera protein was

measured according to the pH stat method reported by Pan et al (Pan

et al., 2016). Glutathione reduced (GSH) was used to establish the

standard curve for quantication of peptide concentration. A 2.5 mL

solution was mixed with the same volume of trichloroacetic acid (TCA)

(10 %, w/v). Subsequently, the mixture was let stand at room temper-

ature for 10 min and then centrifugated at 4000 r/min, for 15 min. The

supernatant was transferred into a volumetric ask, and diluted with

TCA (5 %, w/v) to 50 mL, Then, 3 mL of the above solution was mixed

with 2 mL biuret reagent and placed as the above steps. Finally, the

mixture was centrifugated (2000r/min, 10 min) and the absorbance of

the supernatant was measured at 540 nm with a UV–visible

spectrophotometer ZG-EU-2600 (ZHUO GUANG Corporation, Shanghai,

China). The peptide yield was determined by the ratio of peptide mass to

protein mass.

2.5. Determination of ACE inhibitory activity.

The ACE inhibitory rate was explored according to previous report

(Pan et al., 2016). Briey, 80

L of 5 mM HHL solution was mixed with

L of peptide solution, followed by incubation for 5 min at 37 ◦C.

Subsequently, 10

L of ACE solution (0.1 U/mL) was added, followed by

incubation at 37 ◦C for 60 min. The reaction was terminated by 200

HCl (1 M). In the blank group, peptide solution was replaced by 0.1 M

sodium borate buffer. The reaction production was extracted with 1500

L of ethyl acetate with slight oscillation for 1 min. Then, the mixture

was centrifuged at 4000 rpm for 15 min, 1 mL of supernatant was

transferred to another test tube, mixed with 1000

L of acetic anhydride

and 2000

L of 0.5% (V/V) p-dimethyl amino benzaldehyde in pyridine,

and then incubated at 40 ◦C for 30 min prior to spectrophotometrical

measurement at 459 nm. The degree of ACE inhibition (in percentage)

was calculated according to Eq. (1):

The inhibition of ACE (%)=(Ab−Aa)/(Ab−Ac) × 100% (1)

where A

represents the mixture of HHL, peptide and ACE; A

represents

the mixture of HHL and ACE; A

represents the mixture of HHL and

inactive ACE. The IC

value was dened as the inhibitor concentration

inhibiting 50 % activity of ACE.

2.6. Purication of ACE-inhibitory peptides from hydrolysates

Hydrolysates were puried using a Sephadex-G100 column (2.5 cm

×70 cm) that was eluted with ultrapure water at a ow rate of 1.0 mL/

min. Fractions were collected at 2 min intervals, and each was explored

for ACE inhibitory activity. The fraction with the highest ACE inhibitory

activity was fractionated using an ultraltration membrane, and yielded

three fractions: <3 kDa, 3–10 kDa, and >10 kDa. Then, the highest

active fraction was frozen at -20 ◦C.

2.7. Determination of peptide sequences

The puried fractions were analyzed by online nano ow liquid

chromatography tandem mass spectrometry performed on an EASY-

nano LC 1200 system (Thermo Fisher Scientic, MA, USA) connected

to a PepMap C18 (75

m ×25 cm) (HPLC-MS/MS) as equilibrated with

solvent A (A: 0.1 % formic acid in water) and solvent B (B: 0.1 % formic

acid in ACN). The amino acid sequences of the peptides were determined

by de novo sequencing using PEAKS Studio version X+(Bioinformatics

Solutions Inc., Waterloo, Canada) and Protein Data Bank (RCSB PDB:

Homepage).

2.8. Screening and synthesis of the potential ACE inhibitory peptides

Using the identied peptides, the reported anti-ACE peptides were

determined by BIOPEP (https://biochemia.uwm.edu.pl/biopep-uwm/)

and eliminated, and the remaining peptides were further evaluated. The

biological activity potential of the peptides was determined at Peptide

Ranker (http://distilldeep.ucd.ie/PeptideRanker/). Solubility was

calculated at (Innovagen AB: Antibodies, proteins, and peptides).

Toxicity was measured at ToxinPred (osdd.net). Human intestinal ab-

sorption (HIA) was evaluated at admetSAR (ecust.edu.cn). Then, the

unreported polypeptides with biological activity potential, good water

solubility, no toxin, high human intestinal absorptivity, high blood–-

brain barrier permeability were obtained. Furthermore, the afnities

between the selected peptides and ACE were evaluated by molecular

docking in Discovery Studio 2020 software (DS 2020, Accelrys, San

Diego, CA, USA). ACE crystal structure (ID: 1O8A) was selected as the

Z. Li et al.

文档加载中……请稍候！
如果长时间未打开，您也可以点击刷新试试。

下载文档到电脑，查找使用更方便

1 知币 4人已下载

立即下载

Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein.pdf

共8页,预览3页

还剩页未读，继续阅读

Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein

相关推荐

开通VIP享超值会员特权

作者详情

相关内容

推荐作者

热门标签

举报选择: