Extraction optimization and screening of angiotensin-converting enzyme inhibitory peptides from Channa striatus through bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking

Highlights

• An effective approach for screening ACEIPs from hydrolysate was established.

• Seven novel ACEIPs were screened and identified from C. striatus hydrolysates.

• Peptides EYFR and LPGPGP were showed excellent ACE inhibitory activity.

• The interaction between EYFR/LPGPGP and ACE were analyzed.

Abstract

Channa striatus is high-protein food with many health functions. This study aimed to prepare, screen and identify the angiotensin-converting enzyme inhibition peptides (ACEIPs) from C. striatus hydrolysates by response surface methodology and bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking. The optimal conditions for preparing ACEIPs were hydrolysis temperature 55 °C, hydrolysis time 3 h, pH 9, solid–liquid ratio 1:20 g/mL, and enzyme addition 5%, the ACE inhibition and molecular weight distribution of obtained hydrolysate was 54.35% and 8770–160 Da, respectively. Seven novel ACEIPs were screened through the established high-throughput screening approach, among which, EYFR and LPGPGP showed the strongest ACE inhibition with the IC₅₀ value of 179.2 and 186.3 μM, respectively (P > 0.05). Molecular docking revealed that three and ten hydrogen bonds were formed between ACE and LPGPGP and EYFR, respectively, S1 and S2 were the major active pockets, but the major driving forces varied.

Introduction

Hypertension is the major risk factors of cardiovascular and end stage renal diseases (Jimsheena & Gowda, 2011). The World Health Organization (WHO) reported that the prevalence of hypertension in low- and middle-income countries varied from 23% to 52% (Basu & Millett, 2013). Angiotensin-I converting enzyme (ACE) is a key factor in blood pressure regulation, it raises blood pressure by converting the inactive angiotensin-I to the active angiotensin-II in renin-angiotensin system (RAS) or inactivating the bradykinin in kallikrein-kinin system (KKS) (Ryan et al., 2011, Tu et al., 2018). Suppressing the activity of ACE is considered as a promising strategy for blood pressure management. Benazepril, captopril, and enalapril, et al. are the major clinically applied ACEIs. However, these ACE inhibitors (ACEIs) are all chemosynthetic, and have some side effects (Lee & Hur, 2017).

Currently, many peptides with excellent ACE inhibitory activity have been screened from protein hydrolysates, due to its safety and ignorable side effects (Lee & Hur, 2017). But the composition of protein hydrolysates is generally complicated, and the molecular weight distribution is very broad. Conventional procedures for discovering ACE inhibition peptides (ACEIPs) from complicate protein hydrolysates include preparation of hydrolysates, bioassay-guided fractionation, purification, and identification of peptides sequence. Unfortunately, these traditional isolation and purification procedures are time-consuming and labor intensive (Wu et al., 2015, Zhong et al., 2018). Bio-affinity ultrafiltration combined with liquid chromatography-mass spectrometry, basing on the interactions between small molecular ligands and the active sites of enzymes, is a powerful tool for discovering bioactive compounds from complex mixtures (Wang, Liu, Luo, Huang, & Wu, 2018). It has been widely used to screen and identify multiple bioactive compounds from natural extracts and traditional Chinese medicine (Yang et al., 2012, Zhang et al., 2019). But, up to now, no report on the screening of ACEIPs by this method is available.

Molecular docking is a virtual screening method combined by bioinformatics, structural biology, and computational biology techniques. It can screen potential bioactive molecules from large amounts of compounds or complex fractions via simulated calculation and analysis, which can avoid the tedious and time-consuming isolation, purification, and biological evaluation applied in conventional peptides screening approaches. Meanwhile, the possible binding mechanism between ligand and receptor can also be elucidated (Wu et al., 2014, Wu et al., 2019). Wu et al. (2014) established a virtual screening method with Discovery Studio 3.5 software, and found a significant relationship between Libdock score and experimental IC₅₀. Yu et al. (2018) identified three novel ACEIPs EGF, HGR, and VDF by in silico method, the IC₅₀ value was 474.65, 106.21, and 439.27 µM, respectively.

Channa striatus (C. striatus) is both a popular food choice and a natural remedy in traditional medicine, its extracts or hydrolysates were reported to show antioxidant, antibacterial, wound healing, and antinociceptive activities, et al. (Jais, 2007, Galla et al., 2012, Sulaiman et al., 2004, Wei et al., 2010). So far, only Ghassem et al. (2011) identified two ACEIPs (VPAAPPK and NGTWFEPP) from C. striatus myofibrillar protein, no high-throughput approach was applied to screen and identify ACEIPs. Therefore, the aim of this work was to screen potential ACEIPs from C. striatus hydrolysates by bioaffinity ultrafiltration combined with Nano-LC-Q-Orbitrap-MS/MS techniques and molecular docking. The appropriate hydrolysis conditions were optimized by single factor and response surface methodology (RSM) with ACE inhibition as index. The potential ACEIPs in hydrolysate were screened and identified via ultrafiltration and Nano LC Orbitrap-MS/MS. Finally, the binding energy of identified peptides with ACE was measured by molecular docking, which with the lowest binding energy was synthesized and used for bioactivity and mechanism analysis.