Small Peptides Isolated from Enzymatic Hydrolyzate of Fermented

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Letter

Small peptides isolated from enzymatic hydrolyzate of fermented soybean meal promotes endothelium-independent vasorelaxation and ACE inhibition zhengquan wang, Yunyun Cui, Pengyang Liu, Yong Zhao, Liping Wang, Yuan Liu, and Jing Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05026 • Publication Date (Web): 26 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

PE

ACh Wash PE

PE

10

19 22 25

10

ACh

30 min

(E)

ACh

Wash

PE

Wash 25 27 34 41 47

PE

Wash

PSS buffer (0.1 µM PE)

500-1000 Da F2 (0.1 µM PE)

(F)

ACh

PE

Wash

30 min

Wash

0.22 0.27 0.25 Spike level, 0.10-0.47 g/L PE 0.34 0.410.47 Wash

PE

PE

PE 0.1 0.19

30 min

75%) compared with

210

control (absorbance of peptide), while ACE inhibition ratio was low in GPANV and in

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tetra-peptide PNAV. Nevertheless, no significant corresponding relationship between ACE

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inhibition and vasorelaxation activity was observed.

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DISCUSSION

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The results of this study demonstrated that the filtration fractions obtained from FSM

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hydrolyzate promote vasorelaxation in isolated aorta ring, in a dose-dependent manner.

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High-dose resulted superior to low-dose in reducing the response time of EC50, while the

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two-different dose-to-response curves for contraction to PE provided different EC50 values.

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According to Figure 1, the general similar reduction or increment trends were observed.

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Furthermore, most of the isolated peptides obtained from the smallest molecular weight fraction,

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i.e. F3, showed the potent vasorelaxation activity. A reverse effect of constricted aorta ring was

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observed in one peptide i.e. CQ; this result was similar to F2 (Figure 1B), which may a reason

10 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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for vasoconstriction of F2. The other three isolated peptides, QC, PANV and GPANV, all

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exerted an endothelium-independent vasorelaxation action, which was not produced by the

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constituent amino acids mixture.

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Kamiya et al.

14

have classified the mechanisms of vasorelaxators as endothelium-dependent

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and endothelium-independent. The endothelium-dependent manner involves certain vasoactive

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substances like nitric oxide (NO), prostacyclin and endothelin-1, which are related with

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endothelium synthesizes; while endothelium-independent manner is usually associated with

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vascular smooth muscle cell (VSMC) and it’s related signaling pathways. Most of the published

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vasorelaxing peptides from food source have proven to be endothelium-dependent, like AHIII,2

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RF (EC50 = 0.58 M),14 IVF, RADHPFL, YAEERYPIL,15 RADHPF,1 KTCGY, KRIHF,5

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RGDDNP,16 QK,17 MRW (EC50 = 7.9 M),18 RIY (EC50 = 5.1 M)7 and RPLKPW,8 while their

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underlying mechanisms involve NO,1,2,15 cholecystokinin(CCK),7,14 prostaglandin,16―18 inositol

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polyphosphate (IP) receptor,7,8 bradykinin receptor15 and even [Ca2+]i regulation.5 Some studies

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have revealed that the endothelium-independent vasoactive peptides like HRW,19 AH6 and VY,10

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[Ca2+]i,19 cyclic GMP6 and VSMC10,19,20 are essential in eliciting vasorelaxation effect.

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Importance of endothelium layer for other peptides like VLQRF,3 IHRF4 and WMSLHWSL

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(EC50 = 0.026 M)21 are still unclear, since the vasoactive mechanism investigations have been

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focused on singling pathways, such as the CCK3,4 or [Ca2+]i4. In the present study, QC, PANV

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and GPANV dose-dependently relaxed PE-constricted aorta rings; GPNAV (EC50 = 0.41 M)

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showed to be the most potent vasorelaxation peptide compared with others, even more potent

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than tri-peptide HRW (EC50 = 1.2 mM) in the same experimental system.19 Previous researches

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have reported that the di-peptides, such as VY,10 WH20 and HRW,19 may involve vessel tones

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either through the suppression of extracellular Ca2+ influx into VSMC or by directly binding to

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L-type Ca

2+

channels, which are common signaling pathways in VSMC.10,11,20 Another soy


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peptide may also mediate signaling pathways, like HGK, a tri-peptide isolated from soybean

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11S glycinin hydrolysate, which has exhibited a potent inhibition against [Ca2+]i elevation in

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angiotensin II (Ang II)-stimulated VSMC, and the imino proton of His positioned at C-terminal

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of the peptide has been considered to play a crucial role.11 As shown in Table 2, our vasoactive

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di-peptide QC may have come from glycinin subunit.

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According to some studies, the peptide sequence descending order plays a key role in

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vasoactivities, and the retro-sequence peptides and side-chain modified peptides are always

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inactive or have a low effect (RF vs. FR, RA, AF; VY vs. YV; WH vs. HW; HRW vs.

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1-methyl-HRW, 3-methyl-HRW and His-citrulline-Trp).6,9―11,20 Also, the small peptide fragment

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could induce a potent vasorelaxation activity as well as their precursor peptides (RF vs. VLQRF,

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IHRF; AH vs. AHIII; VY vs. angiotensin (1-7); RADHPF vs. RADHPFL),1―4,6,9,15,22 which were

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also observed in this study (QC vs. CQ; PANV vs. GPNAV), and which may be produced by

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thermolysin through cleave at a certain position of peptide chain. The reverse peptide sequence

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induced different vasoactivity behavior, while the precursor peptide was more potent than its’

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fragment, which indicated that along with amino acid compositions, the sequence of small

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peptide managed to enhance vasorelaxation power and showed a structure-function correlations.

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Some vasorelaxing peptides have been initially identified as ACE-inhibitor, like angiotensin

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(3-4) (VY), the shortest Ang II-derived peptide, that was first discovered to induce

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ACE-inhibition, and then was also recognized for potent vasorelaxation activity.9 AHIII induced

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vasoactivity has been proven to be related with the ACE-inhibition and NO-regulation.2 Some

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studies have also reported a contradictory conclusion that the vasorelaxation activity of IHRF

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may be independent of its ACE inhibitory activity (the IC50 for IHRF was more than 100 M

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while EC50 was 0.58 M).4 Moreover, the vasorelaxant dipeptide VY has shown a significant



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anti-proliferative action against Bay K8644-stimulated VSMC, which was independent from

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ACE inhibition, indicating the involvement of VY in VDCC, since Bay K8644 is an agonist of

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VDCC.23 To verify the speculation of these assays, ACE-inhibition activities of filtrate fractions

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obtained from FSM hydrolyzate and their isolated peptides were also performed in this study.

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As shown in Table 2, no direct relationship and involvement of ACE-inhibition to

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vasorelaxation was observed, and the results demonstrated that four isolated peptides

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vasoactivity differences were completely independent. Even though the potent ACE inhibitory

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peptide of QC showed the lowest vasorelaxation among tested four peptides, the potent

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vasorelaxting GPNAV peptide showed the lowest ACE inhibitory activity, indicating that small

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peptides elicit vasoactivities not only through ACE-inhibition but also via other mechanisms,

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such as [Ca2+]i regulation,2,7,8,11 which needs to be further investigated.

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Accordingly,

FSM

isolated

small

peptides

can

induce

endothelium-independent

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vasorelaxation activity, similar to some potent antihypertensive drugs that are associated with

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signaling pathway of VSMC but not with endothelium cell. Additionally, ACE-inhibitory

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activities for three peptides are independent of their vasorelaxation behaviors. Besides, it is

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important to emphasize the advantages of food source bioactive peptides, which do not require

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long time toxicology experiments before being sold as functional food. Moreover, the physical

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processes are very safe, and they may promote food and/or drug development.

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ACKNOWLEDGEMENTS

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This study was financially supported by National Natural Science Foundation of China

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(NSFC) (No. 31401486) and supported by Innovation Program of Shanghai Municipal

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Education Commission (No. ZZHY13018). Support was also provided by Shanghai Ocean 13 ACS Paragon Plus Environment

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University Scientific Research Foundation for Doctor Project (No. A-0209-13-0105345).

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Figure Captions

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Figure 1. Vasorelaxation activities of filtration fractions obtained from FSM hydrolysate. (A) 1000-10000 Da F0; (B) 500-1000 Da F1; (C)

Small Peptides Isolated from Enzymatic Hydrolyzate of Fermented

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