Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment

Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained beta-lactoglobulin and alpha-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that beta-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was beta-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an alpha-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity.     

In Vitro and In Vivo Effects and Safety Assessment of Corn Peptides on Alcohol Dehydrogenase Activities

The in vitro and in vivo effects of corn peptides(CPs) prepared from corn gluten meal by proteolysis with an alkaline protease and fractions of CPs from Sephadex G-15 and G-10 columns on activities of alcohol dehydrogenase(ADH) were studied. The results show that CPs and fraction 3 of CPs from Sephadex G-10 column enhance in vitro ADH activity. Furthermore, the in vitro accelerating effect of the fraction 3 of CPs on ADH activity was superior to that of glutathione, which was also found even in the presence of ADH inhibitor, such as pyrazole. In the in vivo experiments, the animals were fed with different dosages of CPs and with a dose of Chinese distilled spirit orally, and sacrificed for the measurement of ADH activity. In vivo experimental results indicate that CPS enhanced hepatic ADH activities. To test the safety of CPs as health food, 30 d feeding test was performed. No obvious toxic effects were detected in treated Wistar rats.     

Preparation and Anti-oxidative Effects of Corn Peptides

IntroductionBioactive peptides include the natural peptidesfrom organism itself and the active peptide fromhydrolysate of protein. In the pastfew years,someresearchers have found that small peptides derivedfrom the hydrolysates of food proteins play animportant role in regulating autonomic nervoussystem,activating the cellular immunity function,ameliorating the cardiovascular function,antioxidizing and antiaging,etc.[1— 7] . Smallpeptides prepared from food proteins becomenitrogenous source f…     

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC₅₀ of 0.125 μg mL⁻¹). The untargeted characterization revealed that (−)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC₅₀ value equal to 11.62 ± 0.47 μg mL⁻¹) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.     

Isolation of Phlorotannins from Eisenia bicyclis and Their Hepatoprotective Effect against Oxidative Stress Induced by tert-Butyl Hyperoxide

Eisenia bicyclis (Kjellman) Setchell is a common brown alga that inhabits the middle Pacific coast around Korea and Japan. In this study, the ethanol extract and its serial solvent fractions were prepared from fresh E. bicyclis, and their hepatoprotective effects were investigated against hepatotoxicity in tert-butyl hyperoxide(t-BHP)-injured HepG2 cells. When these samples were used at a dose of 10-40 μg/mL?1, they significantly protected the t-BHP-induced cell death in HepG2 cells. Among fractions, ethyl acetate fraction (EF) and n-butanol extract (BF) exhibited potent hepatoprotective activities (62.60% for EF and 64.86% for BF) in t-BHP-injured HepG2 cells at a concentration of 10 μg/mL?1. To find the potential factors for this activity, the samples were characterized on total phenolics, chlorophylls, carotenoids, and radical scavenging activity. Among them, EF showed the highest content of total phenolics and the strongest antioxidant activity both in on- and offline assays. Five phlorotannin compounds, oligomers of phloroglucinol, were isolated chromatographically from this fraction and structurally identified by (1)H-NMR and liquid chromatography-electrospray ionization-mass spectrometry analyses as eckol(1), 6,6'-bieckol(2), 8,8'-bieckol(3), dieckol(4), and phlorofucofuroeckol A(5). Compound 5 among five purified compounds showed the strongest protective activity (45.54%) at a concentration of 10 μM. At the high dose (40 μM), the protective activities of three compounds (compound 2, 4, and 5) were higher than that of quercetin treated with 10 μM concentration. Therefore, we can speculate that they can be developed as potential candidates for natural hepatoprotective agents.     

Effects of eleven flavonoids from the osteoprotective fraction of Drynaria fortunei (KUNZE) J. SM. on osteoblastic proliferation using an osteoblast-like cell line

Drynaria fortunei (KUNZE) J. SM. (DFE) is one of the most frequently used traditional Chinese medicines prescribed for the treatment of osteoporosis in China. The present study was designed to investigate the osteoprotective constituents from Drynaria fortunei. The 60% ethanol extract of the rhizome of D. fortunei (DFE) was chromatographed on a D-101 macroporous resin column (psi 25x150 cm); three fractions (DFA eluted with water, DFB eluted with 30% and 50% ethanol, and DFC eluted with 95% ethanol) were obtained and their osteoprotective activity as examined both in vivo and in vitro. DFB showed significant activity on both the proliferation of UMR106 cells and promoting bone mineral density (BMD) in ovariectomized (OVX) mice. A bioactivity-guided method led to the isolation of 11 flavonoids from this fraction (DFB) with antiosteoporotic activity, including two new compounds, kaempferol 3-O-beta-D-glucopyranoside-7-O-alpha-L-arabinofuranoside (1) and (R)-5,7,3',5'-tetrahydroxy-flavanone 7-O-neohesperidoside (2), along with nine known ones: three flavanones (3-5), one flavone (7), one flavonol (6), two chromones (8, 10), maltol glucoside (9), and (-)-epicatechin (11). Compounds 4-11 are reported for the first time from this genus. We investigated the proliferative effects of the 11 compounds in the UMR106 osteoblastic cell line in vitro. All compounds exhibited the proliferative activity in the UMR106 cells at most of the concentrations tested. Most compounds are reported for the first time from the Drynaria genus and this was the first study of their proliferative activity in osteoblast-like cells. The main peaks in the HPLC fingerprint of the active fraction (DFB) were also identified.     

Anti-inflammatory effect of Zingiber cassumunar Roxb. and its active principles.

The present study was carried out to elucidate the anti-inflammatory effect of the methanol extract obtained from the rhizomes of Zingiber cassumunar Roxb. and its active principles. The methanol extract was partitioned between ether and water, and then the ether-soluble fraction was extracted with n-hexane. The n-hexane-soluble fraction was chromatographed and part of the fraction was rechromatographed by silica gel column. Three compounds were isolated from the n-hexane-soluble fraction and the chemical structures of these compounds were identified as (E)-1-(3,4-dimethoxyphenyl)but-1-ene, (E)-1-(3,4-dimethoxyphenyl)butadiene and zerumbone. The anti-inflammatory activity of these fractions was investigated on carrageenin-induced edema in rats, as well as on acetic acid-induced vascular permeability and writhing symptoms in mice. The methanol extract (p.o.) showed both anti-inflammatory activity and analgesic activity. These activities shifted successively to ether-soluble and n-hexane-soluble fractions and to (E)-1-(3,4-dimethoxyphenyl)but-1-ene. These results suggest that the anti-inflammatory action and analgesic action of Zingiber cassumunar is the result of the (E)-1-(3,4-dimethoxyphenyl)but-1-ene that it contains.     

Antimicrobial activity of some new thioureides derived from 2-(4-chlorophenoxymethyl)benzoic acid

We report here the characterisation of eight newly synthesized thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid and the evaluation of the in vitro antimicrobial activity of the new compounds against Gram-positive [Listeria monocytogenes,Staphylococcus aureus, Bacillus subtilis], Gram-negative [Psedomonas aeruginosa,Escherichia coli, Salmonella enteritidis], as well as Candida spp., using both reference and clinical multidrug resistant strains to establish the minimal inhibitory concentration (MIC)values. Our results showed that the tested compounds exhibited specific antimicrobial activities, both concerning the spectrum of antimicrobial activity and the corresponding MIC values, which ranged widely between 1024 and 32 mug/mL, depending on the nature and position of the substituents on the benzene ring. The most active compounds were N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(2,6-dichlorophenyl)-thiourea (5 g) and N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(4-bromophenyl)-thiourea (5h), which showed a broad spectrum of antimicrobial activity against enterobacterial strains (E. coli and S. enteritidis),P. aeruginosa, S. aureus and Candida spp. All the tested compounds except 5f were highly active against S. aureus (MIC=32 mug/mL), suggesting their possible use in the treatment of MRSA infections. Four of compounds also exhibited antifungal activity (MIC =256-32 microg/mL) against C. albicans, but L. monocytogenes as well as B. subtilis were resistant to all tested compounds. Our studies thus demonstrated that among other biological activities,the thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid also exhibit selective and effective antimicrobial properties that could lead to the selection and use of these compounds as efficient antimicrobial agents, especially for the treatment of multidrug resistant infections.     

Bilirubin- and light induced cell death in a murine lymphoma cell line

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.     

Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.     

Preparation of mannan modified anionic PCL-PEG-PCL nanoparticles at one-step for bFGF antigen delivery to improve humoral immunity

In this article, blank anionic poly(-caprolactone)-poly(ethylene glycol)-poly(-caprolactone) (PCEC) and anionic mannan modified PCEC (MPCEC) nanoparticles with nearly the same particle size and zeta potential were prepared by emulsion solvent evaporation method. Human basic fibroblast growth factor (bFGF) was absorbed onto anionic nanoparticles surface due to electrostatic interaction. The obtained bFGF-nanoparticles complexes were injected subcutaneously into C57BL/6 mice at 20 μg of bFGF/dose on week 0, 1, 2 and 3. The mice serum was collected on week 4, and bFGF-specific autoantibody total IgG, IgG1 and IgG2a titer in serum was determined by ELISA. The results indicated that the autoantibody IgG, IgG1 and IgG2a titer of the mice immunized by bFGF–MPCEC complexes were higher than that immunized by either bFGF–PCEC or bFGF–Alum. This phenomenon might be due to that mannan functionalized MPCEC nanoparticles could be targeted to dendritic cells (DCs) to improve humoral immunity. The prepared anionic mannan modified PCEC nanoparticles (MPCEC) might have great potential application in vaccine delivery systems.     

Separation of the hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 into mitogenic and antitumor-active subfractions

The hot water extract of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) was divided into representative fractions by ammonium sulfate and ethanol precipitations, and (1----3)-beta-D-glucanase treatment. The ammonium sulfate and ethanol precipitations gave a (1----3)-beta-D-glucan fraction (TSG) and a mannan fraction (TSM). After the degradation of (1----3)-beta-D-glucan in TSHW by (1----3)-beta-D-glucanase treatment, a water-insoluble protein fraction (EDP) and supernatant (EDS) were obtained. Among these fractions, the mitogenic and antitumor activities were mainly observed in EDP and TSG, respectively. On the other hand, the stimulatory effect on the reticuloendothelial system was mainly found in EDP and EDS, and a weak effect was observed in TSG. These findings suggest that the mitogenic and antitumor activities of TSHW were mainly due to the protein and (1----3)-beta-D-glucan, respectively, and that the mitogenic substance (EDP) is tightly bound to (1----3)-beta-D-glucan (TSG) in TSHW, accounting for its solubility in aqueous solution.     

Antiinflammatory effect of Graptophyllum pictum (L.) Griff

The red leaves of Graptophyllum pictum (L.) Griff. (G. pictum) are used in Indonesian folk medicine. The present study was carried out to elucidate the antiinflammatory effect of the ethanol extract obtained from this crude drug. The extract was partitioned between ether and water, and then the water-soluble fraction was extracted with 1-butanol. The 1-butanol-soluble fraction was extracted with chloroform-acetone, hot methanol and water, successively, and the hot methanol-soluble fraction (fr.) was chromatographed (frs. I-III). The antiinflammatory activity of these fractions was investigated on carrageenin-induced edema in rats and acetic acid-induced vascular permeability as well as the writhing symptom in mice. The ethanol extract (p.o.) showed an antiinflammatory activity as well as an analgesic activity and these activities shifted to the water-soluble fraction, 1-butanol-soluble fraction, methanol-soluble fraction and fr. II, successively. It was found that fr. II contained flavonoids. These results suggest that these flavonoids are at least partly responsible for the antiinflammatory effect of the ethanol extract of G. pictum.     

A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins

Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.     

The peptide toxin of the cyanobacterium Microcystis aeruginosa PCC 7941. Isolation and analysis by nuclear magnetic resonance and fast atom bombardment mass spectroscopy

Toxin was obtained from the cyanobacterium Microcystis aeruginosa PCC7941 by extracting freeze-dried cells with water-saturated, acidified n-butanol, diethyl ether-water distribution, reversed-phase thin-layer chromatography and silica high-performance liquid chromatography (HPLC). Two toxic peptide fractions resulted from HPLC. One of these fractions was analyzed by UV and NMR spectroscopy, amino acid analysis and fast atom bombardment mass spectroscopy. The following amino acid analysis and fast atom bombardment mass spectroscopy. The following amino acids were identified: beta-methyl-Asp, Thr, Glu, Ala, Val, Leu, Phe, Arg, N-methyldehydro-Ala and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. Yet the mass spectroscopic data showed that the fraction was still composed of several, most likely cyclic peptides that did not stain with ninhydrin.     

Identification of estrogenic/anti-estrogenic compounds in diesel exhaust particulate extract

Diesel exhaust particulate extract (DEPE) was obtained from diesel exhaust particulates with Soxhlet extraction using dichloromethane. After separating DEPE into 11 fractions by liquid-liquid extraction, the neutral fraction (N) showed anti-estrogenic activity and the weak acid (phenol) fraction (WA(P)) showed estrogenic and anti-estrogenic activities by a yeast two-hybrid assay system expressing human estrogen receptor alpha. Both fractions were thoroughly fractionated by silica gel column chromatography and reversed-phase HPLC. In the WA(P) fraction, 3-methyl-4-nitrophenol and 2,6-dimethyl-4-nitrophenol were identified by LC-MS/MS as estrogenic compounds. This is the first study to identify 2,6-dimethyl-4-nitrophenol in DEPE and the first study to show that it is an estrogenic compound. In the N fraction, 1-hydroxypyrene was also identified by LC-MS/MS as an anti-estrogenic compound.     

Identification of novel angiotensin-converting enzyme-inhibitory peptides from ovine milk proteins by CE-MS and chromatographic techniques

In this report, we present the use of CE-MS as complement to RP separation for the identification of novel angiotensin-converting enzyme-inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to kappa-casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP-HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT-MS (CE-MS). The use of CE-MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP-HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C-terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from kappa-casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual kappa-casein fraction or the total casein fraction from ovine milk.     

Effects on free radicals and inhibition of alpha-amylase of Cardamine battagliae (Cruciferae), an apoendemic Calabrian (southern Italy) plant

The present study showed for the first time the biological properties of different fractions from Cardamine battagliae Cesca & Peruzzi, an apoendemic Calabrian (southern Italy) plant that belongs to the Cruciferae family. The antioxidant activities of the different fractions of C. battagliae were carried out using two different in vitro assays (beta-carotene bleaching test and lipid peroxidation of liposomes assay) while radical scavenging activity was carried out using DPPH test. AcOEt fraction showed the highest activity on DPPH inhibition (IC50 of 0.162 mg mL(-1)) while dichloromethane fraction showed the highest activity on beta-carotene bleaching (IC(50) of 0.004 mg mL(-1)). The assay for alpha-amylase inhibition showed that n-hexane fraction showed the highest activity with an IC50 of 0.055 mg mL(-1).     

Chromatographic characterization of substance P endopeptidase in the rat brain reveals affected enzyme activity following heat stress

This paper describes a study of substance P endopeptidase (SPE)-like activity in various regions of the brain from male rats subjected to heat stress (HS). The enzyme activity was found to be affected in several brain areas including cerebellum, cerebral cortex, hippocampus, hypothalamus[sol ]thalamus and the spinal cord following HS. Significant increases in SPE activity were observed in, for example, hippocampus and the spinal cord. SPE-containing extracts from hippocampus were pooled and subsequently purified by size exclusion chromatography (using a Superdex 75 HR column) and by anion-exchange chromatography (using Resource Q column). The gel permeation chromatography separated the SPE-like activity into two fractions, one of which was suggested to be identical to neutral endopeptidase owing to its molecular size and inhibitory profile. The other active enzyme fraction behaved in conformity with SPE, previously identified in human cerebrospinal fluid. The activity of the purified fraction of these two enzymes was found to be increased (27%) in HS-treated animals.     

Effect of metal ions on the stability of metallothionein in the degradation by cellular fractions in vitro

Metallothioneins (MT), small molecular weight metal binding proteins are known to play an important protective role against heavy metal toxicity, either as antioxidants or pre-oxidants. However, the mode of metabolic fate of MTs in various metal complexes is not clearly understood. This study was carried out to better understand the mode of selective turnover rate of various form of MT in complexes with different metals. The degradation of in vitro translated mouse 35S-cysteine-MT was examined in lysosomal or cytosolic fractions from mouse liver by gel electrophoresis and autoradiography. Overnight incubations of MT showed extensive proteolysis in the lysosomal fraction but not in cytosolic fractions. However, Cu2+-MT was found to be stable under the same experimental condition. In contrast, Zn did not interfere with MT degradation. These results suggest that lysosomes are chiefly responsible for MT removal and appears to be selective on the metals involved in the MT complex. In vitro, translated, radiolabeled MT provides a suitable substrate for investigating the characteristics of MT degradation.