Exploration of cyclooxygenase-2 inhibitory peptides from walnut dreg proteins based on in silico and in vitro analysis

Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such peptides in WDPHs through a combination of in silico and in vitro analysis.In total,1262 peptide sequences were observed by nano liquid chromatography/tandem mass spectrometry(nano LC-MS/MS)and 4 novel COX-2 inhibitory peptides(AGFP,FPGA,LFPD,and VGFP)were identified.Enzyme kinetic data indicated that AGFP,FPGA,and LFPD displayed mixed-type COX-2 inhibition,whereas VGFP was a non-competitive inhibitor.This is mainly because the peptides form hydrogen bonds and hydrophobic interactions with residues in the COX-2 active site.These results demonstrate that computer analysis combined with in vitro evaluation allows for rapid screening of COX-2 inhibitory peptides in walnut protein dregs.

Introducing New Peptide Extracts from Saccharomyces cerevisiae and Achatina achatina Fluids with Strong Inhibitory Activities on Human α-Amylase

This study aimed at exploring for new natural peptides with strong inhibitory capabilities on α-amylase, the main metabolic enzyme that regulates mellitus diabetes, in order to contribute in controlling this global pandemic. It has consisted in heat shock (to 60°C, 70°C, 80°C, 90°C and 100°C for 10, 20 and 30 minutes) of crude proteins extracted from biomass and extracellular parts of Saccharomyces cerevisiae under cultivation, and from the digestive fluid of the giant snail Achatina achatina, and in-vitro assays of the resulting solutions, as effectors, in human α-amylase catalyzing reactions. The results showed that whatever the temperature and time of treatment, an increase (from 2.65 to 3.98-fold) in proteins concentration was noticed. When blended up to 75 microliters in reaction mixtures, the three peptide extracts showed beyond 11% of inhibition of initial α-amylase activity. By reducing samples volume, only 5 microliters of the studied peptide extracts representing 4.70 μg of S. cerevisiae biomass peptides, 0.55 μg of S. cerevisiae extracellular peptides or 1.05 μg of peptides from the digestive fluid A. achatina were quite sufficient to induce complete (100%) inhibition of the human α-amylase activity. Compared to the inhibitory effect obtained from 2.50 μg of acarbose, a renowned antidiabetic, the studied peptide effectors showed more pronounced inhibitory activities. So, we can positively state that S. cerevisiae as well as A. achatina are both capable of synthesizing proteins made up of small inhibitory peptides which deserve purification and structural analysis for potential exploitation as healthy antidiabetic drugs.

Underlying anti-hypertensive mechanism of the Mizuhopecten yessoensis derived peptide NCW in spontaneously hypertensive rats via widely targeted kidney metabolomics

The angiotensin-converting enzyme(ACE)inhibitory peptide NCW derived from Mizuhopecten yessoensis has been demonstrated to have significant in vivo anti-hypertensive effects,however,its anti-hypertensive mechanism is still not fully clarified.This study established a UPLC-Q-TRAP-MS/MS-based widely targeted kidney metabolomics approach to explore the changes of kidney metabolic profiles and to clarify the antihypertensive mechanism of peptide NCW in spontaneously hypertensive rats(SHRs).Multivariate statistical analysis indicated that the kidney metabolic profiles were clearly separated between the SHR-NCW and SHRUntreated groups.A total of 85 metabolites were differentially regulated,and 16 metabolites were identified as potential kidney biomarkers,e.g.,3-hydroxybutyrate,malonic acid,deoxycytidine,and L-aspartic acid.The peptide NCW might regulate kidney metabolic disorder of SHRs to alleviate hypertension by suppressing inflammation and improving nitric oxide production under the regulation of linoleic acid metabolism,folate related pathways,synthesis and degradation of ketone bodies,pyrimidine metabolism,β-alanine metabolism,and retinal metabolism.

Characterization of SARS-COV-2 main protease inhibitory peptides from Ulva prolifera proteins

The main protease(M^(pro))is essential for the replication of SARS-COV-2 and therefore represents a promising anti-viral target.In this study,we screened M^(pro)inhibitory peptides from Ulva prolifera protein on in-silico proteolysis.Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all the peptides were non-cytotoxic.The hexapeptide(SSGFID)exhibited high M^(pro)inhibitory activity in molecular docking and its IC_(50)value was 139.40±0.82μmol/L in vitro according to fluorescence resonance energy transfer assay(FRET).Quantitative real-time(qRT-)PCR results show that SSGFID could stimulate the expression of mitosis-related factors,including nuclear factor-κB,cyclin D1,and cyclin-dependent kinase 4,to promote the proliferation of mice splenocytes.Stability study revealed that SSGFID showed resistance against pepsin and trypsin but lost D(Asp)after pretreatment at121℃ for 15 min.Besides,SSGFID was mainly transported through the Caco-2 cell monolayer by the peptide transporter PepT1 and passive-mediated transport during the transport study.Unfortunately,the peptide was also degraded by Caco-2 intracellular enzymes,and the transfer rate of intact peptide was4.2%.Furthermore,Lineweaver–Burk plots demonstrated that SSGFID possessed a mixed inhibitory characteristic with M^(pro).Our study indicated the potential of Ulva prolifera as antiviral and immuneenhancing functional food ingredients and nutraceuticals.

Novel insight into the formation and inhibition mechanism of dipeptidyl peptidase-Ⅳ inhibitory peptides from fermented mandarin fish(Chouguiyu)

Fermented foods are a potential source to produce novel dipeptidyl peptidase-IV inhibitory peptides(D4IPs).In this study,the fermented mandarin fish(Chouguiyu)was used to screen D4IPs and their formation mechanism was studied by metagenomics and peptidomics.A total of 400 D4IPs with DPP-IV inhibition structure and high hydrophobicity were identified.The correlation network map showed that Lactococcus,Bacillus,Lysobacter,Pelagivirga,Kocuria,Escherichia,Streptococcus,and Peptostreptococcus were significantly correlated with the most D4IPs.Four stable D4IPs,including KAGARALTDAETAT,GEKVDFDDIQK,VVDADEMYLKGK,and GQKDSYVGDEAQ were respectively from the precursor proteins parvalbumin,troponin,myosin,and actin,and were mainly formed by the hydrolysis of subtilisin(EC 3.4.21.62),aspartic proteinase(EC 3.4.23.1),thermolysin(EC 3.4.24.27),oligopeptidase B(EC 3.4.21.83),and proteinase P1(EC 3.4.21.96)from Bacillus,Kocuria,Lysobacter,Lactococcus,and Peptostreptococcus.The inhibition mainly resulted from the hydrogen bond and salt bridge between D4IPs and DPP-IV enzyme.This study provides important information on the proteases and related microbial strains to directionally prepare D4IPs in Chouguiyu.

Optimization of Hydrolysis Conditions for the Isolation of Angiotensin-I Converting Enzyme(ACE) Inhibitory Peptides from Rhopilema hispidum

To obtain the maximum angiotensin-I converting enzyme(ACE) inhibitory activity, the protein hydrolysis conditions of the jellyfish Rhopilema hispidum were optimized using response surface methodology(RSM). Trypsin was selected to produce R. hispidum protein hydrolysates(RPH) with ACE inhibitory activity. The optimal parameters for producing protein hydrolysates with the highest ACE inhibitory activity were as follows: hydrolysis time 5 h, hydrolysis temperature 50℃, and the enzyme-to-substrate ratio 6%. Under these conditions, the ACE inhibitory rate of RPH could reach 64.28% ± 5.72%. In addition, RPH contained high levels of Gly, Glu, Pro, Ala, Asp and Arg, with a molecular weight distribution range of 0.32–6.84 kDa. The following three novel ACE inhibitory peptides were isolated and identified: Ile-Gly-Glu-Thr-Gly-Pro, Gly-Ala-Thr-Gly-Pro-Ala-Gly-Tyr-Val and Gly-AlaPhe-Gly-Pro-Gly-Gly-Leu-Val-Gly-Arg-Pro. The IC_(50) values of the ACE inhibitory activity of these three purified peptides were 19.07, 27.42 and 31.26 μmol L^(-1), respectively. These results suggested that proteins and peptides isolated from R. hispidum could be utilized as antihypertensive functional food sources.

A Novel Angiotensin I Converting Enzyme Inhibitory Peptide from the Milk Casein:Virtual Screening and Docking Studies

Angiotensin I converting enzyme （ACE） plays an important physiological role in the regulation of hypertension. In this study, we applied virtual screening to discover a novel angiotensin I converting enzyme inhibitory peptides from milk casein. One potential hit was identified based on docking scores, subsequently confirmed by activity studies in vitro （IC50=20.85 μmol L-1）. The proposed peptide in this study contains a unique sequence, Lys-Val-Leu-Ile-Leu-Ala. Moreover, we performed the docking studies to understand the binding mode between the enzyme and peptide hit.

Structural requirements and interaction mechanisms of ACE inhibitory peptides:molecular simulation and thermodynamics studies on LAPYK and its modifi ed peptides

The understanding of the structural requirements and the intermolecular-interaction mechanism are important for discovering potent angiotensin-converting enzyme(ACE)inhibitory peptides.In this study,we modifi ed an egg-white derived peptide,LAPYK,using the amino acids with different properties to produce the LAPYK-modified peptides.The ACE inhibitory activities of the modified peptides were determined to explore the structural requirements of ACE inhibitory peptides(ACEIPs).Molecular simulation and isothermal titration calorimetry analysis were used to investigate interactions between the peptides and ACE.We found that hydrophobicity and the amino acids with ring structures were benefi cial for the ACE inhibitory activities of the peptides.The results of the molecular mechanics poisson boltzmann surface area(MMPBSA)binding free energy calculations indicated that the polar solvation free energy(ΔG_(polar))of the charged peptides(LAPYK,LAPYE)were unfavorable for binding to ACE.On the other hand,the results of isothermal titration calorimetry analyses suggested that the enthalpy-driven ACE-peptide interactions were more favorable than the entropy-driven ACE-peptide interaction counterparts.

Identification and dipeptidyl peptidase Ⅳ(DPP-Ⅳ) inhibitory activity verification of peptides from mouse lymphocytes

The objective of this study was to isolate and identify the intracellular bioactive peptides from mouse lymphocytes before and after lipopolysaccharide(LPS)stimulation,to explore novel peptides and to research the bioactive function.Mouse spleen lymphocytes were isolated and cultured with LPS stimulation(experimental group)or not(control group)to collect intracellular peptides.Totally 385 peptides were analyzed by nanoliter liquid phase-Q Exactive quadrupole ultra-high resolution orbitrap mass spectrometer(Nano LC-Q Exactive Plus)and identifi ed by PEAKS X software.After compared with peptides reported,131 novel peptides were discovered,which then were predicted bioactivity by Peptide Ranker and 6 peptides with high bioactivity were predicted function by BIOPEP-UMW database.Prediction data showed that they may have dipeptidyl peptidase IV(DPP-IV)inhibitory activity.Finally,two peptides showed better potent inhibition were verifi ed with competitive and noncompetitive modes.

Interaction mechanism of egg white-derived ACE inhibitory peptide TNGIIR with ACE and its effect on the expression of ACE and AT1 receptor

The egg white-derived hexapeptide TNGIIR inhibits angiotensin-converting enzyme(ACE)activity in vitro.In this work,molecular docking revealed that TNGIIR established hydrogen bonds with the S1(Ala 354),S2(Gln 281,His 513,Tyr 520 and Lys 511)and S1(Glu 162)pockets of ACE.In addition,the potential antihypertensive effect of the oral administration of TNGIIR in spontaneously hypertensive rats(SHR)was investigated,as was the effect of this peptide on the mRNA expression of ACE and angiotensin type 1(AT1)and type 2(AT2)receptors in renal tissue.The oral administration of TNGIIR(2,10 and 50 mg/kg)for up to four weeks did not reduce the blood pressure of SHR,in contrast to captopril(10 mg/kg,orally),but attenuated the mRNA expression of ACE and AT1 receptor(as did captopril).In contrast,both TNGIIR and captopril enhanced the expression of AT2 receptor mRNA.There was no change in the circulating concentration of angiotensin I,but a slight decrease(about 10%)was seen in the concentration of circulating angiotensin II with TNGIIR and captopril.

Drying Technology of Angiotensin Converting Enzyme Inhibitory Peptide Derived from Bovine Casein

Drying technology of angiotensin converting enzyme （ACE） inhibitory peptides derived from bovine casein was investigated. No significance was observed on ACE inhibitory activity of products prepared by spay drying and freeze drying （P〉0.05）. Spay drying was the best drying process for practical industry production. The inlet temperature ranged from 140℃ to 160℃ and the exit temperature ranged from 70 ℃ to 90 ℃ during the spay drying process. Under the optimal conditions, scale-up of angiotensin converted enzyme inhibitory peptide from 1 L to 10 L and the experiment was successively conducted. Peptide yield was 29% and half inhibitory concentration （IC50） was 0.53 g. L^-1.

A comprehensive method to explore inhibitory kinetics and mechanisms of an anticoagulant peptide derived from Crassostrea gigas

A comprehensive method was applied to evaluate the anticoagulant activity of a novel anticoagulant peptide(NAESLRK)derived from oyster(Crassostrea gigas).The anticoagulant peptide drastically reduced the extrinsic clotting activity and also impaired the intrinsic clotting activity slightly.Consistent with clotting data,the thrombin peak height was reduced to 84.7 nmol/L from 123.4 nmol/L,and thrombin generation time was delayed to 4.67 min from 4.42 min when the extrinsic trigger was applied.The inhibitory kinetics of FⅪa,FⅨa,FⅩa,FⅡa,and APC in a purified component system rationally explained the reduction of extrinsic clotting activity and impairment of thrombin generation.Besides the inhibition of FⅩa and FⅡa activity,the activation processes of FⅩand FⅡby intrinsic/extrinsic tenase complex and prothrombinase were also damaged.The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors.The research provided a novel method for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Molecular dynamics studies of the inhibitory mechanism of copper(Ⅱ) on aggregation of amyloid β-peptide

The inhibitory mechanism of copper（Ⅱ） on the aggegation of amyloid β-peptide （Aβ） was investigated by molecular dynamics simulations. The binding mode ofcopper（Ⅱ） with Aβ is characterized by the imidazole nitrogen atom, Nπ, of the histidine residue H 13, acting as the anchoring site, and the backbone＇s deprotoned amide nitogen atoms as the main binding sites. Drove by the coordination bonds and their induced hydrogen bond net, the conformations of Aβ converted from β-sheet non-β-sheet conformations, which destabilized the aggregation of Aβ into fibrils.

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.

Suppression of postprandial blood glucose elevation by buckwheat(Fagpopyrum esculentum) albumin hydrolysate and identification of the peptide responsible to the function

The objective of this study is to evaluate the suppressive effect of buckwheat-albumin hydrolysate on postprandial hyperglycemia and identify the peptide responsible to the function. Buckwheat-albumin hydrolysate was prepared by using digestive enzymes and was orally administered to rats together with soluble starch. The blood was taken from the tail vein up to 90 min after oral administration to measure blood-glucose and plasma-insulin levels. The peptide with α-amylase inhibitory activity was purified from the buckwheathydrolysate by gel-filtration chromatography, α-amylase affinity chromatography, and high performance liquid chromatography(HPLC). The amino-acid sequence of the peptide was identified by a protein sequencer and was compared with that in the buckwheat-genome database. Buckwheat-albumin hydrolysate significantly suppressed the elevation of blood glucose level 15 min after starch administration. The amino-acid sequence of the peptide with α-amylase inhibitory activity was YVEPDCGNLGCCYHC in the parental protein of molecular mass 17.8 k Da and theoretical pI 4.77. The amino-acid sequence, molecular weight, and pI of the parental protein in buckwheat albumin were different from those of α-amylase inhibitor in wheat albumin. This study suggests that the novel α-amylase inhibitor identified in buckwheat albumin is a potential candidate for a functional food material to suppress postprandial blood glucose elevation.

Identifi cation and characterization of a novel tetrapeptide from enzymatic hydrolysates of Baijiu byproduct

In order to prepare angiotensin I-converting enzyme(ACE)inhibitory peptides,distilled spent grains of Chinese strong-flavor Baijiu were hydrolyzed by alcalase followed by papain under optimized conditions.A superior ACE inhibitory peptide was separated and purifi ed by ultrafi ltration and high-performance liquid chromatography(HPLC),and its amino acid sequence was further identified as Gln-Gly-Val-Pro(QGVP)by electrospray mass spectrometry(ESI-MS).QGVP formed 6 hydrogen bonds with the active site of ACE,which is responsible for reducingα-helix structure content of ACE causing subsequent inactivation.M oreover,it showed no significant cytotoxicity toward human umbilical vein endothelial cells(HUVECs),a nd signifi cantly i nduced phosphorylation of endothelial nitric oxide synthase(p-e NOS)and decreased endothelin 1(END1)expression in angiotensin I(Ang I)-treated HUVECs,demonstrating the potential antihypertensive effect.The peptide QGVP hydrolyzed from distilled spent grain proteins of Chinese strong-fl avor Baijiu was expected to be used as a food ingredient to prevent or co-treat hypertension with other chemical drugs.

绿豆蛋白α-淀粉酶抑制肽的制备与鉴定

采用胃-胰蛋白酶水解绿豆蛋白及其分级蛋白,以水解物α-淀粉酶活性抑制率为主要指标,结合水解度、氨基酸组成及分子质量分析其抑制效果差异及原因。结果表明,绿豆蛋白肽对α-淀粉酶活性抑制率最高,为16.51%;与绿豆分级蛋白相比,绿豆蛋白的疏水氨基酸含量和水解度最高,分别为32.68%和6.28%,水解物的肽分子质量最小,均小于20kDa,因此选用绿豆蛋白制备α-淀粉酶抑制肽。然后分离鉴定绿豆蛋白肽,新发现了17条有潜在α-淀粉酶抑制效果的肽。本研究表明绿豆蛋白较其分级蛋白具有更强的α-淀粉酶抑制效果,能够用于降血糖的功能性食品或药物中。

皖浙花猪干腌火腿源血糖调节肽的分离纯化、鉴定及量子化学表征

为探究小肽抑制糖类消化的机理,制备皖浙花猪干腌火腿肌肉的水提物和胃胰酶消化产物,分离纯化出α-淀粉酶和α-葡萄糖苷酶抑制活性较高的组分,鉴定、筛选其中的肽序列,采用量子化学方法计算分子前线轨道分布和能量、静电荷分布、键长等结构和电荷参数,推测活性位点。结果表明:1)酶解后火腿肌肉的粒径减小、降糖活性提高;2)水提物和胃胰酶消化产物经过葡聚糖凝胶分离后分别获得两个组分(S-Ⅰ、S-Ⅱ)和3个组分(WY-Ⅰ、WY-Ⅱ和WY-Ⅲ);3)利用质谱技术从高活性组分WY-Ⅱ中鉴定出104条长度8~24的肽序列,筛选出Peptide Ranker程序评分大于0.7的5条序列;4)前述序列的最高占据轨道多分布在精氨酸的胍基及氨基端附近基团,而最低未占轨道多分布在羧基端及附近基团;5)能级差ΔEL-H较低的序列GPMGPSGPR、LGFGGPSGPNAGR、APAPAPAPAPAPPK可能具有较高活性。根据库伦定律,其活性位点分别定位于精氨酸的C106H108、亮氨酸的C10H12和赖氨酸的C176H177,都位于C—H键。研究为探究肽的血糖调节机制,证明地方品种猪的营养价值提供了理论支撑。

红毛藻血管紧张素转化酶抑制肽的筛选及其稳定性评价

本实验以红毛藻为原料,利用酶解法及超滤分级制备获得不同分子质量的肽组分,经高效液相色谱法测定体外血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制活性发现F2组分(800~2000 Da)的ACE抑制率最高;采用液相色谱-串联质谱技术和PEAKS Studio软件的de novo从头测序对F2组分进行氨基酸序列鉴定,并结合分子对接筛选出与ACE稳定结合的6个ACE抑制肽。利用固相合成法制备预测肽并经体外ACE抑制活性验证,发现L1(LVLLFLFGE)的ACE抑制活性最高,半抑制浓度(half maximal inhibitory concentration,IC_(50))为14.22μg/mL。分子对接结果表明,L1对ACE的抑制主要归因于其能够与ACE的活性口袋形成氢键相互作用。最后,探讨温度、pH值、金属离子、光照类型和模拟胃肠道酶系消化对L1稳定性的影响,结果表明L1具有较好的热稳定性和离子强度稳定性,pH>2时抑制活性逐步减弱,紫外光处理会影响其抑制活性,体外模拟胃肠液处理后L1的ACE抑制率虽然显著降低,但仍具有较高ACE抑制活性。

人参α-淀粉酶抑制肽的降血糖作用

通过采用高糖高脂饲料联合腹腔注射链脲佐菌素(Streptozotocin,STZ)构建小鼠2型糖尿病(Type 2 Diabetes,T2DM)模型,建模成功后连续4周给药治疗T2DM小鼠,以小鼠体质量、脏器指数、血糖、血脂、肝脏氧化应激反应、肝脏病理学检验结果作为参考指标,探究人参α-淀粉酶抑制肽对T2DM小鼠的降糖作用。结果表明,阿卡波糖阳性组和人参α-淀粉酶抑制肽各剂量组对小鼠的体重下降情况、高血糖情况均有不同程度的改善作用及调控效果,所以人参α-淀粉酶抑制肽各个剂量对T2DM小鼠具有降糖的效果。经过4周的治疗,高剂量组血糖从建模后的15.43 mmol/L下降至12.10 mmol/L与阿卡波糖阳性组血糖从建模后的15.71 mmol/L下降至10.17 mmol/L结果相近,且两组的小鼠肝脏损伤较轻,说明高剂量的人参α-淀粉酶抑制肽对2型糖尿病引发的肝脏损伤可起到明显的防护效果(P<0.01)。因此,人参α-淀粉酶抑制肽具有降血糖的作用和成为辅助治疗2型糖尿病产品或保健品的潜力。