Preparation Of Peptides Research Articles

Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin 1 Glycosylation in Cell Adhesion.

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.

Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities.

Feather keratin is an underappreciated protein resource of high quality, with limited bioavailability, and it urgently requires eco-friendly methods to enhance its value. Here, we report on the preparation, purification, and identification of novel peptides with antioxidant and xanthine oxidase (XOD) inhibitory activities from fermented feather broth, using Bacillus licheniformis 8-4. Two peptides, namely, DLCRPCGPTPLA (DA-12) and ANSCNEPCVR (AR-10), displayed remarkable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities with half-maximal inhibitory concentrations (IC50) values of 0.048, 0.034, and 0.95, 0.84 mg/mL, respectively. These values exceed those of the previously reported feather keratin-derived antioxidant peptides. Another peptide, GNQQVHLQSQDM (GM-12), demonstrated XOD activity inhibition, with an IC50 value of 12.15 mg/mL, and it quenched the fluorescence of XOD. Furthermore, after simulating gastrointestinal digestion, DA-12, AR-10, and GM-12 retained their biological activities. Meanwhile, DA-12 and GM-12 showed an unexpected synergistic inhibition on XOD activity accompanied by fluorescence quenching. This study provides new insights into the potential applications of feather keratin, including functionalized feed with antioxidative and antigout (anti-hyperuricemia) activities.

Extraction of Novel Bioactive Peptides from Fish Protein Hydrolysates by Enzymatic Reactions

Bioactive peptides derived from fish the byproduct protein hydrolysate have wide potential as functional food ingredients. The preparation of bioactive peptides is commonly achieved via enzymatic hydrolysis; this is the most preferred method because it has high specificity, fewer residual organic solvents in the product, and it is usually carried out in mild conditions. The use of various enzymes such as proteases is widely practiced in the industry, yet there are various limitations as it is of high cost and there is a limited availability of food-grade enzymes in the market. Moreover, high-throughput purification and the identification analysis of these peptides are currently being studied to further understand the functionality and characterization of the bioactive peptides. This review mainly focuses on the novel bioactive peptides derived from fish protein hydrolysates from various fish wastes and byproducts. The hydrolysis conditions, source of hydrolysate, and amino acid sequence of these novel peptides are presented, along with their corresponding methods of analysis in purification and identification. The use of various enzymes yields novel peptides with potent bioactivities, such as antiproliferative, antimicrobial, antihypertensive, antiglycemic, antitumor, and antioxidative biological functions. The increasing interest in proteomics in marine and aquatic waste utilization continues due to these products’ bioactivity and sustainability.

Preparation, purification, and identification of novel antioxidant peptides from red-bellied pacu (Piaractus brachypomus) fish meat protein hydrolysate.

The current study investigates the preparation, purification, and identification of novel antioxidant peptides from Piaractus brachypomus fish (RBPF) meat. Antioxidant peptides from RBPF meat protein hydrolysate (RPMPH) were fractionated by ultrafiltration (3kDa MWCO membrane). RPMPH-IF (MW < 3kDa) fraction displayed significantly higher antioxidant activities (P < 0.05) (DPPH, ABTS, FRAP, and Fe2+chelating activity). RPMPH-IF was purified by Sephadex G-25 gel filtration chromatography, and the RPMPH-1 fraction exhibited significantly higher antioxidant activities (P < 0.05). Subsequently, the RPMPH-1 fraction was purified by reversed-phase high-performance liquid chromatography. RPH-8 showed the highest antioxidant activities. The sequence of peptides of the RPH-8 fraction was later identified by LC-MS/MS and MASCOT software. RPH-8 fraction showed the two peptides with MW of 1105.52 Da and 748.25 Da, and the sequence of peptides was identified as His-Asn-Leu-Gly-Leu-Leu-His-Gly-Asp-Met and Asp-Ala-Pro-Ser-Met-Asn-Asp, respectively. Thus, RPMPH or purified antioxidant peptides produced by probiotic Bacillus strain could be a bio-functional ingredient in food and nutraceutical applications.

Evolution of Peptide Biopharmaceuticals

Peptides are small molecule substances involved in numerous essential physiological functions such as human growth and development, stress, regulation of the emotional state, sexual behavior, and immune responses. Their mechanisms of action are based on receptor-ligand interaction, which leads to highly selective effects. These properties and low toxicity allow them to be considered potent drugs. The production of peptide preparations became possible at the beginning of the 20th century after a method for the selective synthesis of peptides was developed. However, after the successful synthesis of the first peptide drugs, many issues related to increasing stability, bioavailability, half-life, and the ability to move through cell membranes remained unresolved. The review considers the historical path of development of the synthesis and production of peptides, as well as modern approaches to the creation of peptide drugs and their use in biopharmaceutics, including the development of original peptide drugs in Russia.

Chemoselective, Oxidation-Induced Macrocyclization of Tyrosine-Containing Peptides.

Inspired by nature's wide range of oxidation-induced modifications to install cross-links and cycles at tyrosine (Tyr) and other phenol-containing residue side chains, we report a Tyr-selective strategy for the preparation of Tyr-linked cyclic peptides. This approach leverages N4-substituted 1,2,4-triazoline-3,5-diones (TADs) as azo electrophiles that react chemoselectively with the phenolic side chain of Tyr residues to form stable C-N1-linked cyclic peptides. In the developed method, a precursor 1,2,4-triazolidine-3,5-dione moiety, also known as urazole, is readily constructed at any free amine revealed on a solid-supported peptide. Once prepared, the N4-substituted urazole peptide is selectively oxidized using mild, peptide-compatible conditions to generate an electrophilic N4-substituted TAD peptide intermediate that reacts selectively under aqueous conditions with internal and terminal Tyr residues to furnish Tyr-linked cyclic peptides. The approach demonstrates good tolerance of native residue side chains and enables access to cyclic peptides ranging from 3- to 11-residues in size (16- to 38-atom-containing cycles). The identity of the installed Tyr-linkage, a stable covalent C-N1 bond, was characterized using NMR spectroscopy. Finally, we applied the developed method to prepare biologically active Tyr-linked cyclic peptides bearing the integrin-binding RGDf epitope.

Structural polymorphism and cytotoxicity of brain-derived β-amyloid extracts.

To date, more than 37 amyloidogenic proteins have been found to form toxic aggregates that are implicated in the progression of numerous debilitating protein misfolding diseases including Alzheimer's disease (AD). Extensive literature highlights the role of β-amyloid (Aβ) aggregates in causing excessive neuronal cell loss in the brains of AD patients. In fact, major advances in our understanding of Aβ aggregation process, including kinetics, toxicity, and structures of fibrillar aggregates have been revealed by examining in vitro preparations of synthetic Aβ peptides. However, ongoing research shows that brain-derived Aβ aggregates have specific characteristics that distinguish them from in vitro prepared species. Notably, the molecular structures of amyloid fibrils grown in the human brain were found to be markedly different than synthetic Aβ fibrils. In addition, recent findings report the existence of heterogeneous Aβ proteoforms in AD brain tissue in contrast to synthetically produced full-length aggregates. Despite their high relevance to AD progression, brain-derived Aβ species are less well-characterized compared with synthetic aggregates. The aim of this review is to provide an overview of the literature on brain-derived Aβ aggregates with particular focus on recent studies that report their structures as well as pathological roles in AD progression. The main motivation of this review is to highlight the importance of utilizing brain-derived amyloids for characterizing the structural and toxic effects of amyloid species. With this knowledge, brain-derived aggregates can be adopted to identify more relevant drug targets and validate potent aggregation inhibitors toward designing highly effective therapeutic strategies against AD.

Molecular Docking Revealed the Potential Anti-Oxidative Stress Mechanism of the Walnut Polypeptide on HT22 Cells.

The preparation of novel antioxidant peptides from food raw materials is one of the research focuses, but there are fewer studies on the preparation of antioxidant peptides from walnut meal, a by-product of processing walnuts. This study analyzed the antioxidant properties and protective effects of walnut protein hydrolyzed by alkaline protease and trypsin on the oxidative stress of HT22 cells. The peptides were identified by UPLC-MS/MS, and the anti-oxidative peptides were screened based on virtual computer tools. The potential anti-oxidative stress mechanism of the walnut polypeptide on HT22 cells was explored by molecular docking. The results revealed that walnut protein hydrolysates (WPH) with molecular weights of less than 1 kDa had good antioxidant properties and inhibited oxidative damage of HT22 cells by regulating the levels of reactive oxygen species (ROS) and antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Six of the ninety identified new peptides showed good solubility, non-toxicity, and bioactivity. The molecular docking results showed that the six peptides could dock with Keap1 successfully, and EYWNR and FQLPR (single-letter forms of peptide writing) could interact with the binding site of Nrf2 in the Keap1-Kelch structural domain through hydrogen bonds with strong binding forces. The results of this study provided important information on the antioxidant molecular mechanism of the walnut polypeptide and provided a basis for further development of walnut antioxidant polypeptide products.

Silver Carp (Hypophthalmichthys molitrix) Scales Collagen Peptides (SCPs): Preparation, Whitening Activity Screening and Characterization

This study involves the preparation of scale collagen peptides (SCPs) with whitening activity from silver carp (Hypophthalmichthys molitrix) and their characterization and peptide sequence identification. In this article, scanning electron microscopy (SEM) was used to observe structure changes of sliver carp scales; enzymatic hydrolysis was optimized through protease screening and response surface optimization. The ultrafiltration was used to separate SCPs and the whitening activity was comprehensively evaluated using radical scavenging rate and tyrosinase-inhibiting activity, among others. An optimal component was characterized and identified using various modern spectral analysis techniques. The results showed that the surface of silver carp scales after decalcification was smooth and clear. The pepsin had the highest peptide yield and tyrosinase-inhibiting activity (90.01% and 82.25%, respectively). The optimal enzymatic hydrolysis conditions were an enzyme dosage of 16.1%, a solid-liquid ratio of 1:15.6 and a time of 4.9 h. The proportions of hydrophobic and basic amino acids in the peptide composition were 32.15% and 13.12%, respectively. Compared with SCPs2, SCPs1 (6096.68-9513.70 Da) showed better ·OH scavenging ability, tyrosinase-inhibiting activity and moisture absorption. SCPs1 was a macromolecular fragment of type I collagen with a triple helix structure, containing three peptide sequences with the potential for tyrosinase activity inhibition (AGPPGADGQTGQRGE, SGPAGIAGPAGPRGPAGPNGPPGKD and KRGSTGEQGSTGPLGMRGPRGAA). These results show that SCPs1 is a collagen peptide product with whitening potential.

Preparation of soybean meal peptide for chelation with copper/zinc using Aspergillus oryzae in solid-state fermentation

This study investigated the optimal process of solid-state fermentation (SSF) using soybean meal (SBM) as substrate by Aspergillus oryzae and proteolysis of SBM to prepare SBM-derived peptide (SBMP) for chelating with Cu(Ⅱ)/Zn(Ⅱ). During the SSF of SBM, the alkaline protease activity of A. oryzae was higher than that of two other fungi, reaching maximum activity (574 U/g) at the fermentation time of 86 h, 43% inoculation ratio, and liquid–substrate (I/S) ratio of 0.8. The SBMP yield reached a maximum value at 55 °C for 6 h hydrolysis according to the analysis of the degree of hydrolysis (DH) and total Kjeldahl nitrogen (TKN) content in hydrolysates. After ultrafiltration, the SBMP of 1–5 kDa had the best ability to chelate with Cu(Ⅱ) and Zn(Ⅱ). Moreover, the SBMP displayed a higher capacity to chelate with Zn(Ⅱ) (222.7 mg/g) than Cu(Ⅱ) (68.3 mg/g) under optimal conditions. Aspartic acid, glutamic acid, lysine, arginine and histidine in SBMP played a critical role at the chelating Cu(Ⅱ)/Zn(Ⅱ). UV and FTIR spectrum analysis revealed that the –COOH and –NH2 in SBMP were the main bioactive groups for chelating with Cu(Ⅱ)/Zn(Ⅱ). SEM showed that the surface of SBMP–Cu(Ⅱ)/Zn(Ⅱ) chelate was rough and formed spherical aggregates. These results indicated that combining SSF and proteolysis exhibited great potential for preparing SBMP–metal chelates for developing organometallic supplements.

Preparation of an amphiphilic peptide (P13) with proton sponge effect and analysis of its antitumor activity

In order to prevent drugs from being captured and degraded by the acidic environment of organelles, such as lysosomes, after entering cells, this study designed and synthesized a novel carrier amphiphilic polypeptide (DGRHHHLLLAAAA), designated P13, for use as a tumor-targeting drug delivery vehicle. The P13 peptide was synthesized by the solid phase synthesis method, and its self-assembly behavior and drug-loading capacity in aqueous solution were studied and characterized in vitro. Doxorubicin (DOX) was loaded by dialysis method, and P13 and DOX were mixed at a mass ratio of 6:1 to form regular rounded globules. The acid–base buffering capacity of P13 was investigated determined by acid–base titration. The results revealed that P13 had excellent acid–base buffering capacity, a critical micelle concentration value of about 0.000 21 g l−1, and the particle size of P13-Dox nanospheres was 167 nm. The drug encapsulation efficiency and drug loading capacity of micelles were 20.40 ± 1.21% and 21.25 ± 2.79%, respectively. At the concentration of 50 μg ml−1 of P13-DOX , the inhibition rate was 73.35%. The results of the in vivo antitumor activity assay in mice showed that P13-DOX also exhibited excellent inhibitory effect on tumor growth, compared with the tumor weight of 1.1 g in the control group, the tumor weight in the P13-DOX-treated group was only 0.26 g. Additionally, the results of hematoxylin and eosin staining of the organs showed that P13-DOX had no damaging effect on normal tissues. The novel amphiphilic peptide P13 with proton sponge effect designed and prepared in this study is expected to be a promising tumor-targeting drug carrier with excellent application potential.

Targeting HER2-receptors with 177Lu-labeled triazole stapled cyclic peptidomimetic

In this study on-resin Cu(I)-catalyzed click reaction was performed to synthesize triazole-stapled cyclic peptidomimetic, DOTA-c[TZ]A9 targeting HER2 receptor expression in breast cancers. Spectroscopic (circular dichroism) and docking analysis provided evidence of enhanced helicity and secondary structure stabilization along with improved HER2 affinity in comparison to the corresponding linear peptide, DOTA-[Pra1, Aza7]A9. 177Lu-labeled cyclic peptide, 177Lu-DOTA-c[TZ]A9 displayed higher in vitro serum stability and in vivo metabolic stability and better HER2 binding affinity {Kd of 16.93 ± 3.02 nM} than the linear counterpart, [177Lu]DOTA-[Pra1, Aza7]A9 {Kd of 26.28 ± 2.87 nM}. Biodistribution profile in SKBR3 tumor bearing SCID mice demonstrated elevated radioactivity levels and prolonged retention of cyclic peptide in the tumor compared to the linear peptide. Thus, solid phase click cyclization technique can be extended towards preparation of triazole-stapled peptides targeting different receptors with improved stability and efficacy.

Advanced Utilization of By-Product from the Sturgeon Aquaculture Industry: Optimum Preparation of Notochord-Derived Multifunctional Peptides by Glycation using the Maillard Reaction

Advanced Utilization of By-Product from the Sturgeon Aquaculture Industry: Optimum Preparation of Notochord-Derived Multifunctional Peptides by Glycation using the Maillard Reaction

Identification and preparation of selenium-containing peptides from selenium-enriched Pleurotus eryngii and their protective effect on lead-induced oxidative damage in NCTC1469 hepatocytes.

Lead (Pb) is a highly toxic and persistent substance that easily accumulates in living organisms, eliciting cellular toxicity and oxidative stress. Some selenium-containing proteins and peptides prepared from plant extracts are beneficial for protecting the body's health and resisting external disturbances. In the present study, selenium-containing peptide species were prepared from selenium-enriched Pleurotus eryngii protein hydrolysates and to evaluate the benefits of selenium-containing peptides on Pb-induced oxidative stress in NCTC1469 hepatocytes. Trypsin was selected as primary enzyme to hydrolyze the selenium-enriched protein (SPH). The optimal hydrolysis conditions were: hydrolysis time, 1.5h; initial pH 8.0. The SPH was digested by trypsin and then purified by ultrafiltration, gel filtration chromatography and reversed-phase HPLC to obtain the selenium-containing peptides SPH-I-2. Furthermore, SPH-I-2 was analyzed and a number of total 12 selenium-containing peptides were identified by liquid chromatography-tandem mass spectroscopy. The NCTC1469 cell culture study showed that selenium-containing peptides were capable of reducing reactive oxygen species levels and regulating the Keap1/Nrf2 pathway by upregulating Nrf2, HO-1, GCLC, GCLM and NQO1 genes and downregulating Keap1 genes. Moreover, selenium-containing peptides were also able to suppress Pb-induced elevated levels of nitric oxide (NO), lactate dehydrogenase (LDH), malondialdehyde (MDA), increase antioxidant enzyme activity and alleviate cell apoptosis. The present study indicated that the selenium-containing peptides could protect cells from Pb2+ -induced oxidative stress. © 2023 Society of Chemical Industry.

In-situ and real-time monitoring of two-stage enzymatic preparation of ACE inhibitory peptides from Cordyceps militaris medium residues by ultrasonic-assisted pretreatment

A protocol for the preparation of angiotensin-Ⅰ-converting enzyme (ACE) inhibitory peptides from Cordyceps militaris medium residues (CMMR) was established by two-stage enzymatic hydrolysis (amylase and protease). In a combination (mono-, dual-, and tri-frequency) of five different frequencies (20, 28, 35, 40 and 50 kHz), ultrasound-assisted pretreatment increased ACE inhibition rate in hydrolysate by 63.30 % under the mode of 20/28 kHz. Afterwards, near-infrared (NIR) spectrometer combined with spectral preprocessing methods and multivariate analysis like partial least square (PLS), synergy interval-PLS (Si-PLS), random frog-PLS (RF-PLS) and competitive adaptive reweighted sampling (CARS-PLS) was used to monitor the ACE inhibitory activity. The performance of models was evaluated by the correlation coefficient (Rp) and root mean square error (RMSEP). CARS-PLS models achieved optimal results for both amylase and protease hydrolysis with Rp = 0.9693, RMSEP = 0.01 for the former and Rp = 0.9454, RMSEP = 0.03 for the latter. NIR spectrometer combined with CARS-PLS models may be employed for in-situ and real-time monitoring of the preparation of ACE inhibitory peptides under ultrasonic-assisted pretreatment.

Identification of a Novel Post-digestion Angiotensin I-Converting Enzyme Inhibitory Peptide from Silver Prussian Carp (Carassius auratus gibelio) Roe

Proteins of aquatic products are potential sources of bioactive peptides. This study attempted to screen 29 kinds of aquatic products with the highest angiotensin I-converting enzyme (ACE) inhibitory activity and to identify the active peptide. Results showed that the enzyme hydrolysate of silver Prussian carp roe exhibited the highest ACE inhibitory rate before (64.71%) and after (53.82%) digestion. LAKVAP identified from the enzymatic extract of silver Prussian carp roe was a competitive inhibitor, with an ACE inhibitory rate of 95.33%. The molecular docking study suggested that LAKVAP formed hydrogen bonds with S2 active sites (Gln281, His 353, and His513) and made coordinate bonds with Zn2+ (His383) of ACE, which contributed to the interaction between ACE and peptide. The results suggested that silver Prussian carp roe proteins could be suitable materials for ACE inhibitory peptide preparation, and LP-6 might be potentially beneficial as an antihypertensive agent.

The effect of peptide bioregulators on the resistance of rats to cold-stress factor

BACKGROUND: Biologically active regulatory peptides are the drugs of new type that act selectively on targets, with having no significant side effects.&#x0D; AIM: The aim of the study was to study the potential effect of peptide preparations Cortexin and Semax on the resistance of the rat organism to the cold stress factor in the model of cold stress maladaptation.&#x0D; MATERIALS AND METHODS: White outbred male rats were injected intraperitoneally twice daily with placebo (n = 30), Cortexin 5 mg/kg (n = 30) or Semax 0.3 mg/kg (n = 30). Three days after the start of the injection course, the cold-stress effect was simulated in the climate chamber (Feutron, Germany) by cooling the air to 5C at a relative humidity of 7580%.&#x0D; RESULTS: The use of RP significantly increased the duration of the rectal temperature plateau-period within 35C (moderate hypothermia): in animals of the Cortexin group from 10 days to 12 days, and in animals of the Semax group, from 10 to 14 days. Cortexin demonstrated moderate immunomodulatory properties, and Semax had a more pronounced immunomodulatory effect. Both preparations showed a stress-protective effect. The use of regulatory peptides provided to delay the decrease in the rat general motor activity and exploratory behavior which develops under the influence of a simulated cold-stress factor. A slower rate of decrease in the swimming time of rats under the influence of regulatory peptides was registrated.&#x0D; CONCLUSIONS: The meteoadaptogenic, stress-protective and nootropic effects of the regulatory peptides Cortexin and Semax were confirmed in rats on the model of cold-stress maladaptation.

Protective Effect of Peptides from Pinctada Martensii Meat on the H2O2-Induced Oxidative Injured HepG2 Cells

Pinctada martensii is a major marine pearl cultured species in southern China, and its meat is rich in protein, which is an excellent material for the preparation of bioactive peptides. In this study, the peptides from Pinctada martensii meat were prepared by simulated gastrointestinal hydrolysis, and after multistep purification, the structures of the peptides were identified, followed by the solid-phase synthesis of the potential antioxidant peptides. Finally, the antioxidant activities of the peptides were verified using HepG2 cells, whose oxidative stress was induced by hydrogen peroxide (H2O2). It was shown that the antioxidant peptide (S4) obtained from Pinctada martensii meat could significantly increase the cell viability of HepG2 cells. S4 could also scavenge reactive oxygen species (ROS) and reduce the lactate dehydrogenase (LDH) level. In addition, it could enhance the production of glutathione (GSH) and catalase (CAT) in HepG2 cells, as well as the expression of key genes in the Nrf2 signaling pathway. Three novel antioxidant peptides, arginine-leucine (RL), arginine-glycine-leucine (RGL), and proline-arginine (PR), were also identified. In conclusion, peptides from Pinctada martensii meat and three synthetic peptides (RGL, RL, PR) showed antioxidant activity and could have the potential to be used as antioxidant candidates in functional foods.

Solid-Phase Synthesis of the Bicyclic Peptide OL-CTOP Containing Two Disulfide Bridges, and an Assessment of Its In Vivo μ-Opioid Receptor Antagonism after Nasal Administration.

New strategies facilitate the design of cyclic peptides which can penetrate the brain. We have designed a bicyclic peptide, OL-CTOP, composed of the sequences of a selective μ-opioid receptor antagonist, CTOP (f-cyclo(CYwOTX)T) (X = penicillamine, Pen; O = ornithine) and odorranalectin, OL (YASPK-cyclo(CFRYPNGVLAC)T), optimized its solid-phase synthesis and demonstrated its ability for nose-to-brain delivery and in vivo activity. The differences in reactivity of Cys and Pen thiol groups protected with trityl and/or acetamidomethyl protecting groups toward I2 in different solvents were exploited for selective disulfide bond formation on the solid phase. Both the single step and the sequential strategy applied to macrocyclization reactions generated the desired OL-CTOP, with the sequential strategy yielding a large quantity and better purity of crude OL-CTOP. Importantly, intranasally (i.n.s.) administered OL-CTOP dose-dependently antagonized the analgesic effect of morphine administered to mice through the intracerebroventricular route and prevented morphine-induced respiratory depression. In summary, the results demonstrate the feasibility of our solid-phase synthetic strategy for the preparation of the OL-CTOP bicyclic peptide containing two disulfide bonds and reveal the potential of odorranalectin for further modifications and the targeted delivery to the brain.

Preparation and activity evaluation of angiotensin-I converting enzyme inhibitory peptides from protein hydrolysate of mulberry leaf.

Angiotensin-I converting enzyme (ACE) inhibitory peptides drew wide attention in the food industry because of their natural reliability, non-toxicity, and safety. However, the characteristics of ACE inhibitory peptides obtained from protein hydrolysate of mulberry leaf prepared by Flavourzyme were still unclear. Based on the single-factor test, the Plackett-Burman test and response surface test were used to determine the key factors affecting the ACE inhibition rate in mulberry leaf protein hydrolysate and the optimum conditions of enzymatic hydrolysis. The results showed that the optimum technical parameters were as follows: the ratio of material to liquid is 1: 25 (w / v, g/mL), the Flavourzyme to substrate ratio was 3,000 U/g, the temperature of enzymatic hydrolysis was 50°C, pH was 6.3, and the time of enzymatic hydrolysis was 2.9 h. The ACE inhibitory peptides in the mulberry leaf protein hydrolysates were purified by ultrafiltration and gel filtration, aiming to obtain the highest active component. The 12 peptide sequences were identified by reverse liquid chromatography-mass spectrometry, and then, they were docked to the crystal structure of human angiotensin-I converting enzyme (1O8A), and the interaction mechanisms of 12 peptide sequences and 1O8A were analyzed. The docking results showed that among the 12 peptide sequences, ERFNVE (792.37 Da), TELVLK (351.72 Da), MELVLK (366.72 Da), and FDDKLD (376.67 Da), all had the lowest docking energy, and inhibition constant. The chemosynthetic ERFNVE (IC50: 2.65 mg/mL), TELVLK (IC50: 0.98 mg/mL), MELVLK (IC50:1.90 mg/mL) and FDDKLD (IC50:0.70 mg/mL) demonstrated high ACE-inhibitory activity with competitive inhibition mode. These results indicated that the ACE-inhibiting peptides from mulberry leaf protein hydrolyzed (FHMP) had the potential activities to inhibit ACE and could be used as functional food or drugs to inhibit ACE. This work provides positive support for mining the biological activity of mulberry leaves in the treatment of hypertension.