Semi-preparative Reversed-phase High-performance Liquid Chromatography Research Articles

ACE-inhibitory activity of seed storage proteins and hydrolysates from Job’s tears (Coix lacryma-jobi L.)

Though hypertension is a serious health problem, the inhibition of the angiotensin-I converting enzyme (ACE) provides a means to treat and manage it. In the present work work, we investigated the ACE-inhibitory activity of crude proteins and protein hydrolysates from Job’s tears (Coix lacryma-jobi L.). ACE inhibition of 22.15 ± 0.94% was observed with 400 µg of protein from Job’s tears as compared to captopril, a common ACE inhibitor, equalled to 53.7 ± 2.3 nmol captopril equivalent per mg of protein (IC50 of captopril towards ACE was 4.8 nmol). The crude proteins from Job’s tears were enzymatically hydrolysed for 1, 2, and 3 h (E/S of 1:20 by weight) using commercial proteolytic enzymes including Alcalase, Papain, Pronase, and Trypsin. All hydrolysates exhibited increased ACE-inhibitory activity. The protein hydrolysates (400 µg) prepared using Pronase for 2 h (CLPrH-2h) exhibited the highest inhibitory activity (78.38 ± 0.23% or 190.0 ± 0.5 nmol captopril equiv. per mg protein) and were ~3.5 times more active as compared to crude proteins. Fractionation of the peptides was performed using semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC), and all the fractions exhibited ACE-inhibitory activity. The most active fraction was F2 (41.58% inhibition) which was ~7.5 times more active than the crude proteins. These results suggested that seeds from Job’s tears could be an interesting source for developing functional foods with antihypertensive properties.

P076 The promising antimycotic activities of a novel cyclic antifungal lipopeptide against Human Dermatophyte Isolates

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo determine the minimum inhibitory concentrations (MICs) of a novel antifungal lipopeptide against clinical isolates of dermatophytes of human origin.MethodsTo perform antifungal susceptibility testing (AFST) by CLSI microbroth dilution method, a reversed-phase high-performance liquid chromatography (RP-HPLC) purified lipopeptide of 1071.4 Da from a wild-type soil isolate Bacillus subtilis was used and compared with the standard allylamine terbinafine MICs. Briefly, 1200 ml of cell-free culture supernatant was extracted using a solvent mixture and silica gel (230-400 mesh size)-based adsorption chromatography. The semi-preparative RP-HPLC system consisted of an Agilent quaternary pump and a variable wavelength detector equipped with a Phenomenex Luna C18 column (10 mm × 250 mm, 5 μm). The solvent system for RP- HPLC was (A) water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile containing 0.1% TFA. The gradient of solvent B used for purification was as follows: 0%-54% for 0-20 min at the flow rate of 1 ml/min, 54%-60% from 20-48 min at 0.5 ml/min, 60%-100% from 48-58 min at 0.5 ml/min, 100%-0% from 58-65 min at 1 ml/min and monitored at 210 nm.ResultsSuperficial skin infections are caused by dermatophytes including Trichophyton spp. Nowadays, resistance to terbinafine in Trichophyton spp. isolates with higher MICs have been documented in India. We report here the antifungal prowess of a novel small antifungal lipopeptide against 20 clinical isolates of Trichophyton spp. of human origins (from human skin scrapings and nails) with the clinical diagnosis of tinea corporis/cruris and tinea unguium. The representative photographs of T. tonsurans, T. rubrum, and T. mentagrophytes complex are provided below. A total of 6 isolates of T. mentagrophytes and T. rubrum carry point mutations at F397L of squalene epoxidase (SQLE) protein. The in vitro antifungal efficacies determined by AFST revealed that the lipopeptide showed less or equivalent MICs (100% inhibition) in the case of five dermatophytes. The lipopeptide drug has exhibited improved MICs against two T. mentagrophytes complex and two T. rubrum isolates with amino acid substitution F397L in SQLE protein. Trichophyton mentagrophytes complex and T. rubrum with F397L mutations were inhibited at MICs 4-32 μg/mL of terbinafine. In comparison, the lipopeptide showed 4-16 μg/ml (100% inhibition). In the case of all four Trichophyton tonsurans, the MICs ranged between 2-4 μg/ml for the lipopeptide.ConclusionsThe broad-spectrum lipopeptide showed promising antifungal activity against dermatophytes and may be considered for nano-emulsion formulation and tested for topical application in a mice model.

Octopus-derived antioxidant peptide protects against hydrogen peroxide-induced oxidative stress in IEC-6 cells.

This study aims to find antioxidant peptides from octopus protein hydrolyzates and verify the protective effects against H2O2-induced oxidative stress in IEC-6 cells. After the alcalase hydrolysate was ultrafiltrated, purified by Sephadex G-25 gel fractionation and semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC), 16 peptides were identified, and chemically synthesized. In particular, the peptides AQNY, AMMLAW, FEGAW, GGAW, VDTVVCVW, and VVCLW showed better oxygen radical absorbance capacity (ORAC) and ABTS radical scavenging capacity. Among them, the smallest-molecular-weight peptide GGAW exhibited the best antioxidant activity. Furthermore, GGAW protected IEC-6 cells from H2O2-induced oxidative damage by significantly reducing the generation of reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), thereby improving cell viability. These results indicated that the peptide GGAW possessed the antioxidant capacity to prevent oxidative stress damage.

Isolation and identification of polyphenol monomers from celery leaves and their structure-antioxidant activity relationship

This study aimed to isolate, purify and identify phenols from the celery leaves and further evaluate the antioxidant activities of the monomers in chemical system. Seven polyphenol monomers (a-g) were identified by HPLC-ESI-MS/MS after being separated by D101 macroporous resin, semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and sephadex LH-20. Interestingly, chrysoeriol 7-O-glucoside was identified from celery leaves by UV, IR, one dimensional nuclear magnetic resonance (NMR) and two-dimensional NMR for the first time. In addition, these polyphenol monomers manifested desirable DPPH• scavenging capacity (EC50: 39.55 ± 1.43–612 ± 8.45 μM) and ABTS+• scavenging capacity (EC50: 32.12 ± 0.58–512 ± 25.12 μM), respectively. Among them, the polyphenol monomers (a and b) with two hydroxyl groups (-OH) on the B ring in flavone skeleton had the strongest antioxidant activities. Moreover, the analysis of the structure-activity relationship showed that the position and quantity of -OH on the B-ring and the methylation degree of 3-OH played an important role in regulating antioxidant activities.

Taste-Active Dipeptides from Hydrolyzed Mushroom Protein Enhance Saltiness.

An activity-guided fractionation approach applied to thermally treated, enzymatically hydrolyzed mushroom, Agaricus bisporus L., protein led to the identification of several saltiness- and kokumi-enhancing peptides. The identification was accomplished by employing a combination of solid-phase extraction (SPE), gel-permeation chromatography (GPC), and semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC), coupled with sensory analysis. As a result, this study led to the identification of a collection of common mushroom derived tastants, including 5'-mononucleotides and free amino acids, along with several taste-modulating pyroglutamyl dipeptides, including pyroglutamylcysteine (pGlu-Cys), pyroglutamylvaline (pGlu-Val), pyroglutamylaspartic acid (pGlu-Asp), pyroglutamylglutamic acid (pGlu-Glu), and pyroglutamylproline (pGlu-Pro). The taste-modulating thresholds for the pyroglutamyl dipeptides were calculated in a model mushroom broth containing natural concentrations of guanosine 5'-monophosphate and 14 amino acids, all with dose-over-threshold (DoT) factors ≥1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to quantitate the pyroglutamyl dipeptides, and their concentrations ranged from 2 to 58 μmol/L; however, they were determined to be present in the hydrolysate below their individual taste-modulating thresholds. Despite being present below their individual thresholds, when the dipeptides were collectively added to a model mushroom broth at their natural concentrations (143 μmol/L combined), both salty (p = 0.0061) and kokumi (p = 0.0025) taste attributes were significantly enhanced, demonstrating a synergistic subthreshold taste-modulating effect. This study lays the groundwork for future investigations on the saltiness-enhancing potential of mixtures of subthreshold levels of pyroglutamyl dipeptides found in mushrooms and other sources.

Anti-osteoporosis effect and purification of peptides with high calcium-binding capacity from walnut protein hydrolysates.

The walnut protein hydrolysate (WPH) was prepared via simulated gastrointestinal digestion. The degree of hydrolysis (DH), amino acid composition, and relative molecular weight distribution of WPH were analyzed. The results showed that the DH of WPH was 11.6%, WPH was rich in Glu and Pro, and the relative average molecular weight of 572 Da accounted for 59.78%. The effects of WPH on osteoporosis were evaluated using a model of retinoic acid-induced osteoporosis rat. Treatment with WPH effectively increased the serum calcium and phosphorus contents, alleviated calcium loss, and reduced tartrate-resistant acid phosphate and alkaline phosphatase activities and bone gla protein content. WPH treatment significantly improved the biomechanical properties of the bone and increased the value of bone mineral density. In addition, WPH treatment improved the bone microstructure. WPH was isolated and purified by Sephadex G-25 gel filtration chromatography and semi-preparative reversed-phase high-performance liquid chromatography. A fraction with high calcium-binding activity was obtained and 15 peptides were identified.

Two novel potent ACEI peptides isolated from Pinctada fucata meat hydrolysates using in silico analysis: identification, screening and inhibitory mechanisms.

The aim of this study was to discover potent angiotensin-converting enzyme (ACE) inhibitory (ACEI) peptides from Pinctada fucata (P. fucata) for treating hypertension and to characterize them using in silico analysis. The P. fucata proteins were hydrolyzed by Alcalase®, a serine endopeptidase with broad selectivity, at various times (0, 2, 4, 6, 8, 10 h). The degree of hydrolysis (DH) and ACEI activity of the different hydrolysates were measured. Considering the molecular weight and ACEI activity, the 10 h hydrolysate was purified by a series of traditional separation methods, including ultrafiltration, gel G-25 chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC), with ACEI activity as a guide. The results showed two fractions, C17 and C18, eluted by means of semi-preparative RP-HPLC, and showed the highest ACEI activities of 80.33 ± 2.70% and 81.66 ± 0.29%, respectively, at 1 mg mL−1. The two fractions were then identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and their MS/MS spectra data were subjected to de novo sequencing. Subsequently, the potential ACEI peptides were screened by in silico methods, namely, to analyze the average local confidence (ALC) value obtained from the sequencing software and the P-value from the Pepsite 2. In total, 13 potential ACEI peptide sequences were obtained and identified from the two fractions by LC-ESI-MS/MS, and two novel tetrapeptides, FRVW (607.3314 Da) and LPYY (555.2881 Da), were screened for synthesis according to the in silico analysis. The in vitro ACEI tests indicated that FRVW and LPYY had IC50 values of 18.34 and 116.26 μM, respectively. The Lineweaver–Burk plot showed that FRVW was a noncompetitive inhibitor, and LPYY was shown to be a mixed-mode type inhibitor. A stability study against ACE indicated that both peptides were hydrolyzed by ACE to some extent, the higher ACEI activity following incubation with ACE indicating that they should be classified as pro-drug substrates. Molecular docking results showed that hydrophobic amino acids (HAAs) within peptides formed vital interactions including hydrogen bonds, electrostatic forces, van der Waals forces and Pi–Pi interactions with ACE residues, which stabilized the enzyme–peptide complex. Furthermore, the docking results accorded with the inhibition kinetic mode. Our study demonstrated that FRVW and LPYY isolated from P. fucata have potential applications as antihypertensive agents.

New lathyrane diterpenoids with anti-inflammatory activity isolated from the roots of Jatropha curcas L

Ethnopharmacological relevanceJatropha curcas L. (Euphorbiaceae), as a drought resistant shrub mainly cultivated in tropical and subtropical areas worldwide, is widely used as traditional medicine to cure arthritis, dysentery, abscess and pneumonia in Asian, African and South American folklores. The methanolic extracts of the roots have been revealed the anti-inflammatory activity in vivo and vitro. Aim of studyThis research aimed to provide promising anti-inflammatory candidates from the roots of J. curcas. In addition, RNA-Seq was conducted to give targeted genes involved in the anti-inflammatory action. Materials and methodsThe diterpenoids were isolated from the CH2Cl2 fraction of the methanolic extract from the roots of J. curcas by column chromatography (CC): silica gel, Sephadex LH-20, ODS, semi-preparative reversed-phase high-performance liquid chromatography (HPLC). The structures were identified based on HR-ESI-MS and 1D, 2D-NMR spectroscopic analysis. Their anti-inflammatory effects were tested on lipopolysaccharide (LPS, 500 ng/mL)-stimulated murine RAW264.7 macrophages. Furthermore, we conducted transcriptome-wide RNA sequencing to profile gene expression alterations in LPS-induced RAW264.7 cells upon treatment with jatrocurcasenone I (4) and analyzed the underlying genes targeted by this compound. ResultsSix diterpenoids were obtained from J. curcas, and four of them were identified to be new lathyrane diterpenoids: jatrocurcasenones F–I (1–4). Compounds 3 and 4 exhibited potent inhibitory activities against LPS-induced nitric oxide (NO) production in RAW264.7 cells with IC50 values of 11.28 μM and 7.71 μM, respectively. Western blotting analysis showed that the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were suppressed with the supplementation of 3 and 4. The results of RNA-seq showed that 4 (20 μM) exhibited regulation on the 587 differentially expressed genes (DEGs) induced by LPS (500 ng/mL). Transcriptome-wide RNA sequencing indicated that the protective activity of 4 supplementation was most likely driven by modulating expression levels of IL-1α, IL-1β, IL-1f6, IL-6, IL-1rn, IL-27, Ccl2, Ccl5, Ccl7, Ccl9, Ccl22, Cxcl10, Tnfsf12, Tnfsf15, Lta, Trim25, Bcl2a1a, Dusp1, Dusp2, Ptgs2, Edn1 and Nr4a1. ConclusionsThis study offered four new lathyrane diterpenoids, of them, jatrocurcasenone I (4) showed significant anti-inflammatory activity. RNA-Seq suggested that jatrocurcasenone I (4) could be a candidate drug for the prevention inflammation-mediated diseases by modulating 24 candidate DEGs.

Isolation and Characterization of a New Fusaricidin-Type Antibiotic Produced by Paenibacillus bovis sp. nov BD3526.

Paenibacillus bovis sp. nov BD3526, isolated from raw yak (Bos grunniens) milk, was able to produce antibacterial substances against Micrococcus luteus. The antibacterial substances produced by the strain BD3526 in 3% (w/v) wheat bran broth under aerobic conditions were precipitated from the cultivated broth with ammonium sulfate at 60% saturation. Two antibacterial compounds were obtained by Sephadex LH-20 chromatography and semi-preparative reverse-phase high-performance liquid chromatography (Semi-Pre RP-HPLC). The chemical structures of the two antibacterial compounds were further elucidated by means of ultra high-performance liquid chromatography-mass spectrometer/mass spectrometer (UHPLC-MS/MS). One compound, with a molecular mass of 883.56195Da (M + H)+, was determined to be identical in chemical structure with that of the well-known compound fusaricidin A. The other antimicrobial compound with a molecular mass of 911.59393Da (M + H)+ was determined to be a derivative of fusaricidin A by tandem mass spectrometry and amino acid composition analysis and was designed as bovisin. Bovisin possessed the stability against acid/alkali, heat and some proteases treatment, the same with the fusaricidin A. However, the minimal inhibition concentration (MIC) of bovisin on the tested indicator including Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes and Bacillus subtilis were 50, 50, 50, 50μg/mL, respectively, slightly higher than those of fusaricidin A (6.25, 6.25, 6.25, 12.5μg/mL), indicating bovisin with a weaker inhibitory activity.

Cyclic Tripeptide-based Potent and Selective Human SIRT5 Inhibitors.

SIRT5 is one of the seven members (SIRT1-7) of the mammalian sirtuin family of protein acyl-lysine deacylase enzymes. In recent years, important regulatory roles of SIRT5 in (patho)physiological conditions (e.g. metabolism and cancer) have been increasingly demonstrated. For a better biological understanding and therapeutic exploitation of the SIRT5- catalyzed deacylation reaction, more effort on identifying potent and selective SIRT5 inhibitors beyond those currently known would be rewarding. In the current study, we would like to see if it would be possible to develop potent and selective SIRT5 inhibitory lead compounds with a novel structural scaffold than those of the currently known potent and selective SIRT5 inhibitors. In the current study, six N-terminus-to-side chain cyclic tripeptides (i.e. 8-13) each harboring the thiourea-type catalytic mechanism-based SIRT5 inhibitory warhead Nε-carboxyethylthiocarbamoyl- lysine as the central residue were designed, synthesized by the Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis (SPPS) on the Rink amide 4-methylbenzhydrylamine (MBHA) resin, purified by the semi-preparative reversedphase high performance liquid chromatography (RP-HPLC), characterized by the high-resolution mass spectrometry (HRMS); and were evaluated by the in vitro sirtuin inhibition assay and the in vitro proteolysis assay. Among the cyclic tripeptides 8-13, we found that 10 exhibited a potent (IC50 ~2.2 μM) and selective (≥60-fold over the SIRT1/2/3/6-catalyzed deacylation reactions) inhibition against the SIRT5-catalyzed desuccinylation reaction. Moreover, 10 was found to exhibit a ~42.3-fold stronger SIRT5 inhibition and a greater proteolytic stability than its linear counterpart 14. With a novel and modular structural scaffold as compared with those of all the currently reported potent and selective SIRT5 inhibitors, 10 could be also a useful and feasible lead compound for the quest for superior SIRT5 inhibitors as potential chemical/pharmacological probes of SIRT5 and therapeutics for human diseases in which SIRT5 desuccinylase activity is upregulated.

Isolation, separation and purification of rutin from Banana leaves (Musa balbisiana)

In this study, rutin was detected as a main component after the phytochemical screening of flavonoids from an ethanolic extract of banana leaves (Musa balbisiana) using thin layer chromatography plates (TLC). The rutin from the crude extract was separated using a series of solvent partition separations and was purified by using Sephadex™ column chromatography and semi-preparative reverse phase-high performance liquid chromatography (RP-HPLC). The compounds were characterized and quantified using NMR, Mass Spectroscopy (MS) and HPLC. A crude ethanolic extract of 124 g, containing 5.3 % of rutin, was obtained from 1 kg of dried banana leaf powder. After a series of solvent partition separations, the product from partitions using hexane/dichloromethane and dichloromethane/water in sequence contained a rutin content of 40.6 %. Purification using a Sephadex column yielded fractions with purity of rutin up to 74–84 % and further purification using semi-preparative RP-HPLC produced a final product with a yield of rutin of 32.4 mg/g of the crude extracts and a purity of 98.4 %. The results revealed that banana leaves (M. balbisiana), a food industry by-product and agricultural waste, has the potential for use as an inexpensive and new source of rutin.

Total Synthesis of the Natural Products Ulmoside A and (2R,3R)-Taxifolin-6-C-β-d-glucopyranoside

An efficient first total synthesis of highly polar ulmoside A and (2R,3R)-taxifolin-6-C-β-d-glucopyranoside, useful for the prevention of metabolic disorders, has been described. Key elements of the synthesis include a Sc(OTf)3-catalyzed regio- and stereoselective C-glycosidation on taxifolin in 35% yield with d-glucose and chiral semipreparative reverse-phase high-performance liquid chromatography (HPLC) for the separation of both taxifolins and the diastereomeric mixture of taxifolin-6-C-β-d-glucopyranosides. Correlation of the analytical data of synthetic ulmoside A and its diastereomer with a natural ulmoside A sample confirmed the assigned absolute stereochemistry of the natural products.

Yak (Bos grunniens) bones collagen-derived peptides stimulate osteoblastic proliferation and differentiation via the activation of Wnt/β-catenin signaling pathway.

As the world's population is transitioning gradually to an ageing stage, the incidence of osteoporosis is increasing annually. Yak bone is one of the major components of Tibetan medicine and it has mainly been associated with an improvement in bone health, for example against osteoporosis. However, the functional bioactive ingredients and the underlying mechanisms are still unclear. Sequential purification of yak-bone hydrolysates was achieved by ultrafiltration, size exclusion chromatography, and semi-preparative reverse-phase high-performance liquid chromatography. After this, 35 novel peptides were identified by mass spectrometry analysis, of which peptide GPAGPPGPIGNV (GP-12) displayed the highest osteoblast proliferation-promoting activity, with an increase of 42.7% in cell growth. An in vitro stability study demonstrated that GP-12 was digested into smaller peptides (GP-9, GV-9, AV-10 and GP-11) after simulated gastrointestinal digestion and absorption (Caco-2 cell monolayers) experiments. However, some of them still can be absorbed intact through the (Caco-2 cell monolayers by a paracellular route (Papp: 5.36 ± 0.34 cm s-1 ). Flow cytometry results indicated that GP-12 enhanced osteoblastic proliferation by inducing the alteration of the cell-cycle progression both from the G0/G1 to the S phase and from the S to the G2/M phase. Quantitative real-time polymerase chain reaction (PCR) and western blot results revealed that GP-12 induced osteoblastic proliferation and differentiation in a dose-response manner through the activation of a Wnt/β-catenin signaling pathway. These findings highlighted that such peptides hold the promise of discovering candidates for functional and health-promoting foods, which could be potentially used for the treatment of osteoporosis. © 2020 Society of Chemical Industry.

The Potential Synergistic Modulation of AMPK by Lippia citriodora Compounds as a Target in Metabolic Disorders.

Lippia citriodora (LC) represents a complex plant-derived source of polyphenols and iridoids that has shown beneficial properties against obesity-related metabolic disorders. The complete extract and its major compound, verbascoside, have shown AMPK-activating capacity in cell and animal models. In this work, we aimed to elucidate the contribution of the different compounds present in the LC extract on the AMPK activation capacity of the whole extract. Semipreparative reversed-phase high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RP-HPLC-ESI-TOF-MS) was used to identify the major compounds with bioassay-guided fractionation in an adipocyte cell model for the measurement of AMPK activity. Twenty-two compounds were identified and purified almost to homogeneity in 16 fractions, and three compounds, namely verbascoside, luteolin-7-diglucuronide and loganic acid, showed the highest AMPK-activating capacity. The synergy study using the checkerboard and fractional inhibitory concentration index (FICI) methods exhibited synergistic behavior between loganic acid and luteolin-7-diglucuronide. Molecular docking experiments revealed that these three compounds might act as direct agonists of AMPK, binding to the AMP binding sites of the gamma subunit and/or the different sites of the interaction zones between the gamma and beta subunits. Although our findings conclude that the bioactivity of the extract is mainly due to verbascoside, the synergy found between loganic acid and luteolin-7-diglucuronide deserves further research aimed to develop optimized combinations of polyphenols as a new nutritional strategy against obesity-related metabolic disorders.

Purification and Identification of Antioxidant Alcalase-Derived Peptides from Sheep Plasma Proteins.

In this study, sheep plasma was submitted to Alcalase-hydrolysis and peptides with better antioxidant properties measured through both the ferric-reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability assays were isolated and identified. After hydrolysate ultrafiltration and semi-preparative reverse-phase high-performance liquid chromatography, nine fractions (F1–F9) were obtained, with the two first (F1 and F2) showing the greatest antioxidant potential. These two fractions were further separated by the AKTA purifier system to generate four (F1-1–F1-4) and five (F2-1–F2-5) fractions, respectively, with two of them (F1-2 and F2-1) exhibiting appreciable FRAP activity and DPPH radical scavenging ability. Using liquid chromatography-tandem mass spectrometry, three antioxidant peptides were identified. From their amino acid sequences (QTALVELLK, SLHTLFGDELCK, and MPCTEDYLSLILNR), which include amino acids that have been previously reported as key contributors to the peptide antioxidant properties, it can be maintained that they come mainly from serum albumin. These results suggested that the sheep plasma protein can be considered as a good source of antioxidant peptides and bring forth new possibilities for the utilization of animal blood by-products.

Purification and Characterization of Peptides Inhibiting MMP-1 Activity with C Terminate of Gly-Leu from Simulated Gastrointestinal Digestion Hydrolysates of Tilapia (Oreochromis niloticus) Skin Gelatin.

Tilapia skin gelatin hydrolysates (TSGHs) were prepared by simulated gastrointestinal digestion and separated by gel filtration and semi-preparative reversed-phase high-performance liquid chromatography. The anti-photoaging effects were evaluated using an ultraviolet radiation B (UVB)-induced mouse embryonic fibroblast (MEF) photoaging model in vitro. Three fractions from TSGHs with high inhibitory intercellular matrix metalloproteinase-1 (MMP-1) activities and reactive oxygen species (ROS) production were obtained. Three key peptides, GYTGL, LGATGL, and VLGL, were identified, and their C terminate was Gly-Leu. Three peptides were synthesized and exhibited a significant inhibition of intercellular MMP-1 activity and ROS production. Furthermore, three peptides inhibiting MMP-1 activities were evaluated through their docking of S1' and S3' active pockets of MMP-1. Hydrogen bonds and C terminate Gly-Leu played important roles. Finally, the protective effects of three peptides on intercellular collagen in UVB-induced MEFs were compared. Our results indicated that tilapia gelatin peptides exhibited potential activities to prevent and regulate photoaging.

Characterization and Immunomodulatory Activity of a Novel Peptide, ECFSTA, from Wheat Germ Globulin.

A novel peptide was extracted from wheat germ globulin and purified using ion-exchange chromatography, gel filtration chromatography, and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The sequence of the peptide was found to be Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA). Its immunomodulatory effects were evaluated, and the results showed that ECFSTA could enhance phagocytosis of RAW 264.7 cells and significantly increase their secretion of nitric oxide (NO), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and reactive oxygen species (ROS). ECFSTA activated macrophages mainly through the pattern recognition receptors (PRRs) of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4), and the production of ROS simultaneously stimulated macrophages to produce TNF-α. Thus, ECFSTA could be used as an immunomodulator and might be a promising component of functional foods.

ACE- inhibitory and radical scavenging activities of bioactive peptides obtained from camel milk casein hydrolysis with proteinase K

The aim of this study was to evaluate the effects of enzymatic hydrolysis of camel whole casein on their antioxidant and angiotensin-converting enzyme (ACE)-inhibitory properties. Whole camel casein was hydrolyzed by proteinase K (PK), and the hydrolysates were fractionized by ultrafiltration membranes into three fractions. Semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used to differentiate the mixture of peptides in the 3 kDa permeate fractions. A fraction (F4) with potentials of ACE-inhibitory activity (IC50 = 73 μg.mL−1) and radical scavenging activity (IC50 = 6.8 μg.mL−1) was selected for further purification and fractionation. The fraction F4C obtained from a second step purification of F4 showed strong ACE-inhibitory activity (IC50 = 36 μg.mL−1) as well as radical scavenging activity (IC50 = 3.3 μg.mL−1). The results of this study suggest that whole camel casein can be considered as a promising source for the production of peptides with potential of ACE-inhibitory and antioxidant activities.

In vitro antithrombotic activities of peanut protein hydrolysates

The antithrombotic activities of peanut protein hydrolysates were investigated using a microplates assay. When peanut proteins were hydrolyzed to a limited extent by various enzymes, their thrombin inhibitory abilities were significantly enhanced. However, the resultant hydrolysates showed significantly different activities even at the same degrees of hydrolysis. The hydrolysates generated by Alcalase 2.4L displayed the best antithrombotic activities and the hydrolysis process was further optimized by response surface methodology. The antithrombotic activities were increased to 86% based on a protein concentration of 50mg/ml under the optimal conditions: pH 8.5, enzyme concentration of 5000IU/g of peanut proteins, and 2h hydrolysis time at 50°C. The Alcalase 2.4L crude hydrolysates were then fractionated successively by preparative and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide fraction collected inhibited thrombin-catalyzed coagulation of fibrinogen completely at a concentration of 0.4mg/ml, with an antithrombotic activity close to that of heparin at quite a low concentration (0.2mg/ml). This peptide fraction was further analyzed by online reverse-phase ultra-performance liquid chromatography (RP-UPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and three new peptides were identified as Ser-Trp-Ala-Gln-Leu, Gly-Asn-His-Glu-Ala-Gly-Glu and Cys-Phe-Asn-Glu-Tyr-Glu, respectively. This research provided an effective way to produce antithrombotic peptides from peanut proteins, and also helped to elucidate the structure–function relationships of peanut peptides.

Preparation of morusin from Ramulus mori and its effects on mice with transplanted H22 hepatocarcinoma.

In this study, we used branches Ramulus mori from cultivated mulberry Husang-32 (Morus multicaulis Perry) as the experimental material and anhydrous ethanol as the extraction solution to obtain the crude extract from the branch bark. The ethanolic extract was successively purified through a macroporous resin, Sephadex LH-20, and semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC). The high-purity monomer was identified as morusin by HPLC with diode array detection (HPLC-DAD) and its UV spectrum. The contents of morusin exhibited almost no difference between the root and branch bark in Husang-32, and morusin was not detected in the leaves. Morusin is able to inhibit the tumor growth of transplanted H22 hepatocarcinoma in mice and has no side effects. The fluorescence quantitative real-time PCR (qRT-PCR) results indicate that morusin has a marked inhibitory effect on liver cancer cells through a mechanism that may be related increases in the expression of p53, Survivin, CyclinB1, and Caspase-3 and a decrease in NF-κ B gene expression. The influence of this compound is more apparent in the Caspase-3 and the NF-κ B genes.