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.

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.

Evaluation of bioactive flavonoids in Citri Reticulatae Pericarpium from different regions and its association with antioxidant andα-glucosidase inhibitory activities

Background:To explore the differences of 14 batches of Citri Reticulatae Pericarpium from different regions.Methods:The main aim of this study was to develop a high-performance liquid chromatography method-photodiode array detection for determining the contents of five flavonoids and the chromatographic fingerprints of Citri Reticulatae Pericarpium from different regions.Theα-glucosidase inhibitory activities and antioxidant properties of Citri Reticulatae Pericarpium,based on free-radical scavenging assays against 1,1-diphenyl-2-picrylhydrazyl,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),and hydroxyl radicals,were estimated and compared.Results:Among the tested compounds,the content of hesperidin(13.386-68.235 mg/g)was the highest and that of hesperitin(0.045-0.277 mg/g)was the lowest.In comparing different sources of Citri Reticulatae Pericarpium,the contents of narirutin in Citri Reticulatae Pericarpium from Guangdong(0.824-0.851 mg/g)and Sichuan(1.069-1.204 mg/g)provinces of China were lower than in other provinces.In contrast,nobiletin(8.429-12.237 mg/g)and tangeretin(3.947-4.613 mg/g)were most abundant in Guangdong sources of Citri Reticulatae Pericarpium,followed by samples from Sichuan Province(nobiletin:6.761-7.658 mg/g;tangeretin:3.422-3.933 mg/g).Correlation analysis showed that nobiletin and tangeretin were the main contributors to the antioxidant capacity,and narirutin was the main active component inhibiting theα-glucosidase activity of Citri Reticulatae Pericarpium.Conclusion:This work revealed that the intrinsic quality of Citri Reticulatae Pericarpium was affected by different growing regions,which provides a scientific basis for controlling the quality of Citri Reticulatae Pericarpium and rationally developing and utilizing Citri Reticulatae Pericarpium.

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.

Cucurbitane-Type Triterpene Glycosides from Momordica charantia and Their α-Glucosidase Inhibitory Activities

Ten cucurbitane-type triterpene glycosides,including five new compounds named charantosides H(1),J(2),K(3),momor-characoside A(4),goyaglycoside-l(5),and five known compounds(6-10),were isolated from the EtOAc extract of Momor-dica charantia fruits.The chemical structures of these compounds were identified by 1D and 2D NMR and HRESIMS spectroscopic analyses.Configurations of new compounds were determined by ROESY correlations and comparison of their 13C NMR data with literature reported values.All compounds were evaluated for their inhibition againstα-glucosidase,in which compounds 2,5,7,8,9 showed moderate inhibitory activities with IC50 values ranging from 28.40 to 63.26μM comparing with the positive control(acarbose,IC5087.65±6.51μM).

α-Glucosidase inhibitors isolated from medicinal plants

Objective:α-Glucosidase inhibitors can be used as a new class of antidiabetic drug.By competitively inhibiting glycosidase activity,these inhibitors help to prevent the fast breakdown of sugars and thereby control the blood sugar level.This study provides a wealth of information aboutα-glucosidase inhibitors isolated from medicinal plants;this knowledge will be useful in finding more potent antidiabetic candidates from the natural resources for the clinical development of antidiabetic therapeutics.Results:411 compounds exhibitingα-glucosidase inhibitory activity were summarized and isolated them from medicinal plants.The compound classes isolated include:terpenes(61)from 14 genus,alkaloids(37)from 11 genus,quinines(49)from 4 genus,flavonoids(103)from 24 genus,phenols(37)from 9 genus,phenylpropanoids(73)from 20 genus,sterides(8)from 5 genus,and other types of compounds(43).Conclusion:Compounds withα-glucosidase inhibitory activity are abundant in nature and can be obtained from several sources.They have highα-glucosidase inhibitory potential,and can be clinically developed for treating diabetes mellitus.

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.

α-Glucosidase inhibitor produced by an endophytic fungus, Xylariaceae sp. QGS 01 from Quercus gilva Blume

Xylariaceae sp.QGS 01,an endophytic fungus isolated from the stem of Quercus gilva Blume showed high-glucosidase inhibitory activity.α-Glucosidase inhibitor have the role as one of carbohydrate-hydrolyzing enzymes to postpone absorption of glucose in the digestive organs.The α-glucosidase inhibitor constituents were isolated from the ethyl acetate extract of the mycellium of endophytic fungi Xylariaceae sp.QGS 01 using a bioassay-guided fractionation technique.Further separation and purification of the active fraction led to the isolation of constituents with strong inhibitory activities against-glucosidase:8-hydroxy-6,7-dimethoxy-3-methylisocoumarine(1)with inhibitory concentration(IC50)values against-glucosidase from Saccharomyces cerevisiae of 41.75μg/mL,while quercetin as the standard had an IC50 value of 4.80g/mL.The results of the present study showed that the endophytic fungus Xylariaceae sp.QGS 01 is potentially a rich source of antidiabetic medicine.

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.

α-Glucosidase Inhibition by New Schiff Base Complexes of Zn(II)

There are many reports that divalent alkaline earth, first-row transition metals, and Zn(II) ions have α-glucosidase inhibitory effects. Cu(II) and Zn(II) ions, in particular, have strong α-glucosidase inhibitory effects. Several Schiff bases also display α-glucosidase inhibitory effects. In this study, we focused on safe and highly effective complexes including Zn(II) ion. We prepared and characterized the Zn(II) complexes with four different Schiff bases (N-salicylidene-β-alanine (N-sβ), N-N’-bis (salicylidene) ethylenediamine (N-bsE), N, N’-bis (salicylidene)-phenylenediamine (N-bsP), and 1-[(2-dimethylaminoethylimino) methyl]naphtholate (DMN)) and investigated their α-glucosidase inhibitory effects in vitro, using α-glycosidases from Saccharomyces sp. and rat small intestine, and in vivo, using a sucrose tolerance test. The Zn(II) complexes with DMN showed the highest in vitro and in vivo α-glucosidase inhibitory effects in this study.

抗菌肽F1高产菌株筛选鉴定及其对多重耐药菌生物膜形成的抑制作用

该研究以L.paracasei subsp.Tolerans FX-6为出发菌株,经过^(60)Coγ射线辐照诱变,筛选出一株高产抗菌肽F1的突变菌株(菌26)。对菌26进行16S rDNA序列比较分析发现其与FX-6具有高度相似性。进一步对菌26的牛奶发酵粗提物进行分离纯化,成功富集抗菌肽F1。通过抑菌实验发现,菌26的牛奶发酵粗提物对E.coli的最低抑菌质量浓度(Minimum Inhibitory Concentration,MIC)为3.16 mg/mL,较FX-6牛奶发酵粗提物的MIC降低了75%,同时抗菌肽F1的产量也提高了3.03倍,初步推断抗菌肽F1质量浓度与菌株的抑菌活性之间可能存在正相关关系。前期研究已经发现抗菌肽F1对黏菌素耐药大肠杆菌SHP45的MIC为320.0μg/mL,该实验进一步研究表明当抗菌肽F1的质量浓度达到2×MIC时,可抑制50%以上的黏菌素耐药大肠杆菌SHP45生物膜的形成。基于以上研究结果表明,菌26比出发菌株具有更高的产抗菌肽F1能力,且抗菌肽F1对黏菌素耐药大肠杆菌SHP45的生物膜形成具有显著的抑制作用。该研究为高产抗菌肽菌株的获得提供研究思路,并有望为多重耐药细菌感染的防控提供物质基础。