Antioxidant Polyphenols from Lespedeza bicolor Turcz. Honey: Anti-Inflammatory Effects on Lipopolysaccharide-Treated RAW 264.7 Macrophages.

Although the honey produced by Lespedeza bicolor Turcz. is precious because of its medicinal value, its pharmacological mechanism is still unclear. Here, its anti-inflammatory and antioxidant functions on lipopolysaccharide (LPS)-treated murine RAW 264.7 macrophages were analyzed using targeted and non-targeted metabolomics. Results showed that twelve polyphenols were identified in L. bicolor honey using UHPLC-QQQ-MS/MS. L. bicolor honey extract could scavenge the free radicals DPPH• and ABTS+ and reduce Fe3+. Furthermore, pretreatment with L. bicolor honey extract significantly decreased NO production; suppressed the expression of COX-2, IL-10, TNF-α, and iNOS; and upregulated HO-1's expression in the cells with LPS application. UHPLC-Q-TOF-MS/MS-based metabolomics results revealed that L. bicolor honey extract could protect against inflammatory damage caused by LPS through the reduced activation of sphingolipid metabolism and necroptosis pathways. These findings demonstrate that L. bicolor honey possesses excellent antioxidant and anti-inflammatory activities.

Characteristics of gelatin from lizardfish (Saurida micropectoralis) and threadfin bream (Nemipterus hexodon) skins as influenced by extraction conditions.

Gelatins from lizardfish and threadfin bream skins were extracted using distilled water at 45 and 60 °C and 4, 8 and 12 h. Gelatin recovered from both lizardfish and threadfin bream skins was in the range of 63.96-91.46%. As extraction temperature and duration increased, the turbidity of gelatin solution from both species increased. Gelatins isolated from either lizardfish or threadfin bream skins at 45 °C for 4 and 8 h showed the maximum bloom strength (245.03-320.85 g), which were also greater than commercial gelatin from bovine (208.55 g) (P < 0.05). The gelatin gels (6.67%, w/v) could set at 4 °C within 3 min and were able to set at room temperatures within 51.83 min. Gelatins extracted from both fish skins contained α1-, ß- and γ-chains as predominant protein components. The lightness of all gelatin gels faintly declined with an increase in extraction temperature and time. Among the various production conditions explored, lizardfish/threadfin bream skin gelatin developed at 45 °C and 8 h was found to be highly comparable to commercial bovine gelatin. Based on the results obtained, gelatin from both fish species could be used as a replacement for land animal counterparts and can be used in many different food and pharmaceutical products.

Use of batch anion exchange technique for separation of κ-casein glycomacropeptide from bovine whey fraction.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide, which is considered as a health promoting compound found in cheese whey. The study described in this research communication was undertaken to determine whether GMP with undetectable level of contaminating protein or phenylalanine can be isolated from bovine whey fraction using batch anion exchange technique with chitin as an adsorbent. A soluble whey fraction (SWF) prepared from 1 g whey protein isolate (WPI) was mixed with a slurry of 1 g chitin, and the mixture was incubated at pH 3.0. After incubation, the mixture was filtered, and the residue obtained (containing chitin-GMP complex) was washed with water and eluted stepwise with 0.5 M NaCl and 2.0 M NaCl. Most of GMP (corresponding to 75.8% of total sialic acid recovered) was eluted with 0.5 M NaCl. The recovered GMP accounted for 5.4% dry weight of WPI (or 18.9% dry weight of SWF). Amino acid analysis showed that there was no detectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine in the GMP fraction. It was concluded that the batch anion exchange method with chitin developed in this study can be used for the isolation of high purity GMP from bovine SWF.

Extract of Unifloral Camellia sinensis L. Pollen Collected by Apis mellifera L. Honeybees Exerted Inhibitory Effects on Glucose Uptake and Transport by Interacting with Glucose Transporters in Human Intestinal Cells.

Bee pollen possesses potential hypoglycemic effects but its inhibitory mechanisms on glucose absorption and transportation in intestinal cells still need to be clarified. Here, we determined the inhibitory effects of bee pollen extract originating from Camellia sinensis L. (BP-Cs) as well as its representative phenolic compounds on glucose uptake and transport through a human intestinal Caco-2 cell monolayer model. It showed that three representative phenolic compounds, including gallic acid (GA), 3-O-[6'-O-(trans-p-coumaroyl)-ß-d-glucopyranosyl]kaempferol (K1), and 3-O-[2',6'-di-O-(trans-p-coumaroyl)-ß-d-glucopyranosyl]kaempferol (K2), with contents of 27.7 ± 0.86, 9.88 ± 0.54, and 7.83 ± 0.46 µg/mg in BP-Cs extract, respectively, exerted mutual antagonistic actions interacting with glucose transporters to inhibit glucose uptake and transport based on their combination index (CI) and molecular docking analysis. K1, K2, and GA might compete with d-glucose to form hydrogen bonds with the same active residues including GLU-412, GLY-416, GLN-314, and TRP-420 in GLUT2. These findings provide us a deep understanding of the mechanisms underlying the anti-hyperglycemia by bee pollen, which provide a new sight on dietary intervention strategies against diabetes.

Development of mucoadhesive hydrogels based on polyacrylic acid grafted cellulose nanocrystals for local cisplatin delivery.

To fabricate a mucoadhesive hydrogel with superior properties for local delivery of cisplatin (CDDP) to colorectal cancer, a hardcore bottle-brush polymer (HCBBP) was developed through grafting of poly(acrylic acid) (PAA) on cellulose nanocrystals (CNC) at 6, 9 and 12 CNC:PAA w/w ratios. The developed materials were characterized by acid-base titrations, FT-IR, electron microscopy, muco-rheological behaviour in the presence of mucin, in vitro drug release and anticancer activity against human HCT-116 colorectal cancer cells. The results showed CNC-g-PAA9 to have superior rheological behavior in the presence of mucin compared to CNC and other gels under study indicating beneficial mucoadhesive characteristics. CNC-g-PAA9:CDDP complex showed slow CDDP release causing a significant increase in IC 50 of the drug (> 3-fold) against HCT116 cells. The developed CNC-PAA9 hydrogel showed no intrinsic cytotoxicity on its own. The results point to a great promise for CNC-g-PAA9 as mucoadhesive hydrogels for local platinum delivery in colorectal cancer.

Production of sialic acid rich glycopeptide from bovine κ-casein glycomacropeptide by hydrolyzing with papain.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (<1 and 4.5, respectively), serine (20.3 and 10.3, approximately twice higher in GPF), asparagine/aspartic acid and isoleucine. The contents of the last two amino acids were approximately twice lower in GPF. On gel filtration chromatography with Sephacryl S-100, GMP was eluted as a single peak with elution volume similar to that of dimeric ß-lactoglobulin (36.6 kDa) whereas GPF was eluted in two peaks both with elution volumes greater than that of α-lactalbumin (14.2 kDa). These peak fractions containing high (fraction I) and low (fraction II) molecular size glycopeptides gave different sialic acid to peptide ratio, which was 1.7 times higher in fraction I than in fraction II. Results of size exclusion HPLC on Superdex-75 were consistent with those of gel filtlation chromatography. On cellulose acetate electrophoresis, the mobility of GPF relative to that of GMP as 1.0 was found to average 1.2, suggesting a higher negative charge density in GPF than in GMP. It was concluded that papain digestion of GMP is an efficient method to produce glycopeptide with high sialic acid content.

Isolation of κ-casein glycomacropeptide from bovine whey fraction using food grade anion exchange resin and chitin as an adsorbent.

Bovine κ-casein glycomacropeptide (GMP) found in cheese whey is a sialylated phosphorylated peptide which is thought to be a potential ingredient for functional food as well as dietetic food. This study was undertaken to determine whether high purity GMP can be isolated from soluble whey fraction (SWF) using column chromatography on food grade anion exchange resin and chitin as an adsorbent. Samples of commercially available anion exchange resin (resin A, resin B and resin C) and those of chitin (chitin A, chitin B and chitin C) were examined in this experiment. The GMP fraction obtained from each column was analyzed for amino acid composition which reflects the purity of the peptide. Major findings for commercial anion exchange resin were that: (1) the proportion of GMP monitored as sialic acid in total recovered sialic acid was similar among the three samples of resin accounting for average 78% of recovered sialic acid; (2) the GMP fraction from resin A or resin B contained undetectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine; (3) the GMP fraction from resin C contained small amounts (<1 mol%) of contaminating amino acids, arginine, phenylalanine and tyrosine; and (4) the GMP binding capacity expressed as mg/100 mg dry weight of resin was more than 2.5 times higher in resin C (average 22.9) than in resin A or resin B with no difference between resin A and resin B averaging 8.7. Results obtained for chitin A, chitin B and chitin C were in general similar to those found with resin A and resin B. Since chitin has a remarkable GMP binding capacity averaging 8.6 mg/100 mg dry weight of chitin, it may be a useful adsorbent for whey fractionation. Further research is needed to develop an efficient inexpensive method to purify GMP.

Cold non-enzymatic browning of glucosamine in the presence of metmyoglobin induces glucosone and deoxymyoglobin formation.

Glucosamine (GlcN) and GlcN-myoglobin reaction systems were incubated at 4 °C to verify that GlcN can go through non-enzymatic browning at this low temperature, and to test the hypothesis that certain reductones from GlcN non-enzymatic browning can promote the formation of deoxy- and oxymyoglobin from metmyoglobin reduction. Remarkably, alpha-dicarbonyls and self-condensation products, fructosazine and deoxyfructosazine, were produced at this relatively low temperature. The presence of myoglobin shifted GlcN non-enzymatic browning toward the formation of glucosone and fructosazine. When glucosone (250-2000 mg/L) was incubated with myoglobin it contributed to the formation of deoxymyoglobin, indicating its capacity to reduce metmyoglobin. This study opens the possibility of using GlcN in meat products to increase oxy- and deoxymyoglobin and enhance the color of meat.

The effect of amino acids on non-enzymatic browning of glucosamine: Generation of butterscotch aromatic and bioactive health compounds without detectable levels of neo-formed alkylimidazoles.

A mixture of glucosamine (GlcN, 15% w/v) and different amino acids in 1:1 M ratio was incubated at 70 °C for 12 h. The resulting GlcN-amino acid caramels were analysed for α-dicarbonyl compounds, polyhydroxyalkyl pyrazines, heterocyclic compound and alkylimidazoles. All the analyses were performed by using HPLC-MS/MS followed by pooling the variables with principal component analysis (PCA). GlcN-Gly caramels generated the greatest amount of butterscotch aromatic compound diacetyl and polyhydroxyalkyl pyrazines (fructosazine and deoxyfructosazine). The potentially toxic heterocyclic compound, 5-hydroxymethylfurfural (HMF) was generated in greater amounts with the GlcN-Arg caramels. However, the toxic alkylimidazoles (4-MEI and THI) were not present in any of the GlcN-amino acid caramels. The results suggest that caramel with butterscotch aroma and bioactivity can be produced with GlcN-amino acid at 70 °C. The PCA performed discriminated the majority of the GlcN-amino acid combinations; GlcN-Gly and GlcN-Ser were best discriminated.

Bee Pollen Extracts Modulate Serum Metabolism in Lipopolysaccharide-Induced Acute Lung Injury Mice with Anti-Inflammatory Effects.

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

Improved bactericidal capacity of UV-B radiation against E. coli strains by photosensitizing bacteria with fructosazine - An advanced Maillard reaction product.

This study investigated the effect of UV-B irradiation and the combinational effect with glucosamine caramel, fructosazine and riboflavin on the antimicrobial activities against Bacillus subtilis (ATCC 6633) and two strains of Escherichia coli (AW 1.7 and ATCC 25922). The quantum yield of fructosazine was two times less than that of tryptophan, indicating its ability to emit fluorescent light but less efficiently than tryptophan. UV-B treatment alone was efficient to achieve a bactericidal effect for both E. coli stains tested, however no effect was found for Bacillus subtilis for up to 80 mJ/cm2 UV-B. The combination of UV-B with photosensitizers fructosazine, glucosamine caramel and riboflavin enhanced the UV-B efficacy against E. coli strains at lower UV-B doses, while Bacillus subtilis ATCC 6633 was more resistant to the treatment combinations. High-performance liquid chromatography showed the production of different fructosazine reaction products occurred during irradiation, including the possible formation of endoperoxides.

Sous-Vide Nonenzymatic Browning of Glucosamine at Different Temperatures.

Sous-vide is an increasingly popular method of cooking under controlled conditions of temperature and time inside vacuumed pouches to preserve the nutritional and sensory qualities of food. Sous-vide nonenzymatic browning of glucosamine (GlcN) was investigated at 50, 60, and 70 °C for 12 h. Changes investigated were pH, color, level of browning, and the concentrations of the key Maillard and caramelization reaction products, including α-dicarbonyls and pyrazines. The concentrations of undesired 4-methylimidazole (4-MEI), 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), and 5-hydroxymethylfurfural (5-HMF) were also determined. Six types of caramels were produced of unique composition with no detectable levels of 4-MEI. GlcN caramels produced under vacuum were more acidic and lighter in color, containing significantly less flavorful diacetyl, but more fructosazine (FR) as compared to nonvacuum caramels. THI concentration was well below the toxicity levels for all studied caramels. Principal component analyses showed that the incubation temperature played a key role in determining the composition of caramels.

Inhibitory activity of a Concanavalin-isolated fraction from a glucosamine-peptides reaction system against heat resistant E. coli.

Alcalase-derived gelatin hydrolysates were glycated with glucosamine in the presence (+) or absence (-) of transglutaminase (TGase), and their antimicrobial activities toward Escherichia coli AW 1.7 were studied. Glycation treatments were subjected to concanavalin A affinity chromatography to selectively collect the glycopeptide-enriched fractions and the changes in antimicrobial activity were determined. The minimum inhibitory concentration of glycated hydrolysates decreased by 1.2 times compared to the native hydrolysate, with no differences between (+) or (-) TGase treatments. No difference was observed in the dicarbonyl compound concentration between the two glycation methods except that 3-deoxyglucosone was greater in the TGase-mediated reaction. Concanavalin A-retentate, but not the flow-through fractions, significantly improved the antimicrobial activity, however there was no difference between +TGase and -TGase glycated treatments. Purification of the retentate fraction from fluorescent compounds did not improve its antimicrobial activity.

Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (Pa,b) of fructosazine was 1.30 ± 0.02 × 10-6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

Both PepT1 and GLUT Intestinal Transporters Are Utilized by a Novel Glycopeptide Pro-Hyp-CONH-GlcN.

Pro-Hyp (PO) accounts for many beneficial biological effects of collagen hydrolysates for skin and bone health. The objective of this study was to conjugate PO with glucosamine (GlcN) to create a novel glycopeptide Pro-Hyp-CONH-GlcN (POGlcN) and then to investigate the potential involvement of multiple transepithelial transport pathways for this glycopeptide. Nuclear magnetic resonance results revealed the amide nature of this glycopeptide with α and ß configurations derived from GlcN. This glycopeptide was very resistant to simulated gastrointestinal digestion. Also, it showed a rate of transepithelial transport [permeability coefficient (Papp) of (2.82 ± 0.15) × 10-6 cm/s] across the Caco-2 cell monolayer superior to those of parental dipeptide PO and GlcN [Papp values of (1.45 ± 0.17) × 10-6 and (1.87 ± 0.15) × 10-6 cm/s, respectively]. A transport mechanism experiment indicated that the improved transport efficiency of POGlcN is attributed to the introduction of glucose transporters.

Transepithelial transport efficiency of bovine collagen hydrolysates in a human Caco-2 cell line model.

Collagen was extracted from raw bovine hide and hydrolyzed by one of three enzymes - Alcalase, Flavourzyme, or trypsin - or by using a combination of two or three of these enzymes. The Alcalase-containing enzymatic hydrolysis treatments generated a greater proportion of hydrolysates with molecular weight (MW) <2kDa (79.8-82.7%). Flavourzyme-containing hydrolysis treatments exhibited the greatest proportion of free amino acids (686-740nmol/mg). The hydrolysates were then subjected to a simulated gastrointestinal (GI) digestion, and transport studies were conducted using a Caco-2 cell model. Due to the lower MW profile, the hydrolyzed collagen showed greater resistance to GI digestion and greater transport efficiency than the unhydrolyzed collagen control. Hydrolysates from a dual enzyme mixture - the Alcalase/Flavourzyme combination - generated the greatest transport efficiency across Caco-2 cell monolayers (21.4%), two-fold more than that of the collagen control.

Sulfated glycosaminoglycan-derived oligosaccharides produced from chicken connective tissue promote iron uptake in a human intestinal Caco-2 cell line.

Food grade sulfated glycosaminoglycan (GAG) polysaccharides were successfully extracted from chicken cartilage and skin. Their respective oligosaccharides were obtained by bovine testicular hyaluronidase digestion. The antioxidant capacity was analyzed to determine the possible mechanism explaining how GAGs promote iron uptake by the Caco-2 cells. The sulfated GAG oligosaccharides derived from cartilage possessed the greatest DPPH scavenging and ferric reducing activities (p<0.05), but limited ferrous chelating activities. Both the cartilage and skin sulfated GAG polysaccharides showed greater ferritin formation compared to the negative control (p<0.05). Depolymerisation of GAG polysaccharides to oligosaccharides further improved ferritin formation by twofold. This research establishes that chicken sulfated GAG polysaccharides can enhance iron uptake by Caco-2 cells. The enhanced iron uptake through enzymatic GAG depolymerisation could be due to the combined effects of reduced molecular weight, increased amount of hydroxyl terminal groups and ferric reducing activities.

Transglutaminase-catalyzed amination of pea protein peptides using the biogenic amines histamine and tyramine.

BACKGROUND: Biogenic amines (BAs) are produced by the enzymatic decarboxylation of amino acids, and are well-known for their toxicity to humans. This study describes a new method using microbial transglutaminase (MTGase) to covalently link BAs such as histamine (HIS) and tyramine (TYR) to the glutamine residues of alcalase-hydrolyzed pea protein (PPH). RESULTS: The incubation of PPH and HIS and TYR in the presence of MTGase at 37 °C led to the formation of conjugates, as determined by liquid chromatography, after derivatization with dansyl chloride. Seventy-six % of HIS and 65% of TYR were covalently incorporated to PPH by MTGase. The incubation of PPH and TYR in the presence of MTGase exhibited a 52% DPPH radical scavenging activity at 10 mg mL-1 . Conjugation via MTGase improved the antioxidant status by reducing lipid peroxidation. CONCLUSION: This study emphasizes that the application of MTGase can effectively reduce histamine and tyramine content while simultaneously enhancing antioxidative capacity of PPH. © 2016 Society of Chemical Industry.

Fructosazine, a Polyhydroxyalkylpyrazine with Antimicrobial Activity: Mechanism of Inhibition against Extremely Heat Resistant Escherichia coli.

Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity against heat-resistant Escherichia coli AW 1.7. This study investigated fructosazine's antimicrobial mechanism of action and compared it to that of riboflavin. Fructosazine-acetic acid was effective in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity using 1-N-phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by E. coli was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h, except 2, 3, and 10 h. Fructosazine generates 1O2, which is partially why it damages E. coli. DNA fragmentation was confirmed by fluorescence microscopy, and the fructosazine-acetic acid was the second most intense treatment after riboflavin-acetic acid. Electron microscopy revealed membrane structural damage by fructosazine at pH 5 and 7. This study provides evidence that fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging membrane integrity, and fragmenting DNA.