Valorization of cell wall polysaccharides extracted from Liubao brick tea residues: chemical, structural, and hypoglycemic properties.

BACKGROUND: Tea dregs, typically generated during the production of instant tea or tea beverages, have conventionally been regarded as waste material and routinely discarded. Nevertheless, contemporary research endeavors are concentrating on discovering efficient methods for utilizing the potential of this discarded resource. RESULTS: In this study, we employed a sequential extraction method using chemical chelating agents to extract and isolate four distinct cell wall polysaccharides, designated as CWTPS-1 through CWTPS-4, from the tea dregs of Liubao brick tea. A comprehensive investigation into their physicochemical, structural, and hypoglycemic properties was conducted. The analysis of chemical composition and physicochemical characteristics revealed that all four CWTPSs were characterized as acidic polysaccharides, albeit with varying chemical compositions and physicochemical attributes. Specifically, the xyloglucan fractions, CWTPS-3 and CWTPS-4, were found to be rich in glucose and xylose, displaying a more uniform molecular weight distribution, greater structural stability, and a more irregular surface compared to the others. Moreover, they exhibited a higher diversity of monosaccharide residues. Importantly, our research unveiled that all four CWTPSs exhibited the capacity to modulate key glucose-regulated and antioxidant enzyme activities within HepG2 cells via the IRS-1-PI3K/AKT signaling pathway, thereby ameliorating cellular insulin resistance. Furthermore, our correlation analysis highlighted significant associations between monosaccharide composition and neutral sugar content with the observed hypoglycemic activity of CWTPSs. CONCLUSION: This study highlights the potential of utilizing tea dregs as a valuable resource, making a significant contribution to the advancement of the tea industry. Furthermore, CWTPS-4 exhibits promising prospects for further development as a functional food ingredient or additive. © 2024 Society of Chemical Industry.

Longan Polysaccharides with Covalent Selenylation Combat the Fumonisin B1-Induced Cell Toxicity and Barrier Disruption in Intestinal Epithelial (IEC-6) Cells.

In this study, the soluble, but non-digestible, longan (Dimocarpus longan Lour.) polysaccharides (LP) were extracted from dried longan fruits and then chemically selenylated to produce two selenylated products, namely SeLP1 and SeLP2, with different selenylation extents. The aim was to investigate their protective effects on rat intestinal epithelial (IEC-6) cells exposed to the food toxin fumonisin B1 (FB1). LP only contained total Se content of less than 0.01 g/kg, while SeLP1 and SeLP2 were measured with respective total Se content of up to 1.46 and 4.79 g/kg. The cell viability results showed that these two selenylated products were more efficient than LP in the IEC-6 cells in alleviating FB1-induced cell toxicity, suppressing lactate dehydrogenase (LDH) release, and decreasing the generation of intracellular reactive oxygen species (ROS). These two selenylated products were also more effective than LP in combating FB1-induced barrier disruption via increasing the transepithelial electric resistance (TEER), reducing the paracellular permeability, decreasing the mitochondrial membrane potential (MMP) loss, and maintaining cell barrier integrity by upregulating the tight-junction-related genes and proteins. FB1 caused cell oxidative stress and barrier dysfunction by activating the MAPK and mitochondrial apoptosis signaling pathways, while SeLP1 and SeLP2 could regulate the tMAPK- and apoptosis-related proteins to suppress the FB1-mediated activation of the two pathways. Overall, SeLP2 was observed to be more active than SeLP1 in the IEC-6 cells. In conclusion, the chemical selenylation of LP caused an activity enhancement to ameliorate the FB1-induced cell cytotoxicity and intestinal barrier disruption. Meanwhile, the increased selenylation of LP would endow the selenylated product SeLP2 with more activity.

Activity Changes of the Peptic Lactoferrin Hydrolysate in Human Gastric Cancer AGS Cells in Response to Cu(II) or Mn(II) Addition.

Lactoferrin is an interesting bioactive protein in milk and can interact with various metal ions of trace elements such as copper, iron, manganese, and others. In this study, a lactoferrin hydrolysate (LFH) was generated from commercial bovine lactoferrin by protease pepsin, fortified with Cu2+ (or Mn2+) at two levels of 0.64 and 1.28 (or 0.28 and 0.56) mg/g protein, respectively, and then measured for the resultant bioactivity changes in the well-differentiated human gastric cancer AGS cells. The assaying results indicated that the LFH and Cu/Mn-fortified products had long-term anti-proliferation on the cells, while the treated cells showed DNA fragmentation and increased apoptotic cell proportions. Regarding the control cells, the cells treated with the LFH and especially Cu/Mn-fortified LFH had remarkably up-regulated mRNA expression of caspase-3 and Bax by respective 1.21-3.23 and 2.23-2.83 folds, together with down-regulated mRNA expression Bcl-2 by 0.88-0.96 folds. Moreover, Western-blot assaying results also indicated that the cells exposed to the LFH and Cu/Mn-fortified LFH (especially Mn at higher level) for 24 h had an enhanced caspase-3 expression and increased ratio of Bax/Bcl-2. It can thus be concluded that the used Cu/Mn-addition to the LFH may lead to increased bioactivity in the AGS cells; to be more specific, the two metal ions at the used addition levels could endow LFH with a higher ability to cause cell apoptosis by activating caspase-3 and increasing the Bax/Bcl-2 ratio.

Amino Acid Composition of a Chum Salmon (Oncorhynchus keta) Skin Gelatin Hydrolysate and Its Antiapoptotic Effects on Etoposide-Induced Osteoblasts.

A gelatin hydrolysate with a hydrolysis degree of 13.7% was generated using the skin gelatin of chum salmon (Oncorhynchus keta) and papain-catalyzed enzymatic hydrolysis. The results of analysis demonstrated that four amino acids, namely Ala, Gly, Pro, and 4-Hyp, were the most abundant in the obtained gelatin hydrolysate with measured molar percentages ranging from 7.2% to 35.4%; more importantly, the four amino acids accounted for 2/3 of the total measured amino acids. However, two amino acids, Cys and Tyr, were not detected in the generated gelatin hydrolysate. The experimental results indicated that the gelatin hydrolysate at a dose of 50 µg/mL could combat etoposide-induced apoptosis in human fetal osteoblasts (hFOB 1.19 cells), causing a decrease in the total apoptotic cells from 31.6% to 13.6% (via apoptotic prevention) or 13.3% to 11.8% (via apoptotic reversal). Meanwhile, the osteoblasts exposed to the gelatin hydrolysate showed expression changes for 157 genes (expression folds > 1.5-fold), among which JNKK, JNK1, and JNK3 were from the JNK family with a 1.5-2.7-fold downregulated expression. Furthermore, the protein expressions of JNKK, JNK1, JNK3, and Bax in the treated osteoblasts showed a 1.25-1.41 fold down-regulation, whereas JNK2 expression was not detected in the osteoblasts. It is thus suggested that gelatin hydrolysate is rich in the four amino acids and has an in vitro antiapoptotic effect on etoposide-stimulated osteoblasts via mitochondrial-mediated JNKK/JNK(1,3)/Bax downregulation.

Low-level Cu-fortification of bovine lactoferrin: Focus on its effect on in vitro anti-inflammatory activity in LPS-stimulated macrophages.

Bovine lactoferrin (LF) per 1 g was reacted with 0.16, 0.32, and 0.64 mg CuCl2 to reach 10%, 20%, and 40% copper-saturation, respectively, aiming to assess their anti-inflammatory activities to lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The macrophages treated with CuCl2 at 0.051 µg/mL dose did not have obvious change in cell viability, lactate dehydrogenase (LDH) release, and intracellular reactive oxygen species (ROS) production. However, LF and Cu-fortified LF products (10-80 µg/mL doses) mostly showed inhibitory effects on the stimulated macrophages dose-dependently. Moreover, Cu-fortified LF products of lower Cu-fortifying levels at lower doses exerted weaker inhibition on the stimulated macrophages than LF, leading to higher cell viability but decreased LDH release. Meanwhile, LF and Cu-fortified LF products at 10 and 20 µg/mL doses showed different activities to the stimulated cells, via partly decreasing or increasing the production of inflammatory mediators namely prostaglandin E2 (PGE2), nitric oxide, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, and ROS production, depending on the used Cu-fortifying and dose levels. Compared with LF, Cu-fortified LF product (Cu-fortifying level of 0.16 mg/g LF) at 10 µg/mL dose showed enhanced inhibition on the production of PGE2, ROS, IL-1ß, and TNF-α, evidencing increased anti-inflammatory activity. However, the inhibition of Cu-fortified LF product (Cu-fortifying level of 0.32 mg/g LF) at 20 µg/mL dose on the production of these inflammatory mediators was mostly reduced. It is thus proposed that both Cu-fortifying and dose levels could affect LF's anti-inflammatory activity in LPS-stimulated macrophages, while the Cu-fortifying level of LF could govern activity change.

How does a celiac iceberg really float? The relationship between celiac disease and gluten.

Celiac disease (CD) is an autoimmune intestinal disease caused by intolerance of genetically susceptible individuals after intake of gluten-containing grains (including wheat, barley, etc.) and their products. Currently, CD, with "iceberg" characteristics, affects a large population and is distributed over a wide range of individuals. This present review summarizes the latest research progress on the relationship between CD and gluten. Furthermore, the structure and function of gluten peptides related to CD, gluten detection methods, the effects of processing on gluten and gluten-free diets are emphatically reviewed. In addition, the current limitations in CD research are also discussed. The present work facilitates a comprehensive understanding of CD as well as gluten, which can provide a theoretical reference for future research.

Effect of ferulic acid covalent conjugation on the functional properties and antigenicity of ß-lactoglobulin.

Binding to phenolics can improve the functional properties of proteins. Changes in structure, functional properties, and antigenicity of ß-lactoglobulin (ß-LG) after covalent conjugation with ferulic acid (FA) at different mass ratios were reported here. The results of SDS-PAGE and gel exclusion chromatography confirmed that covalent complexes were formed. When the mass ratio of ß-LG and FA was 10:6, the binding content of FA was the highest. Fluorescence spectroscopy, UV-visible absorption spectrometry, and FTIR analysis showed that the structure of the complexes was more stretched compared to native ß-LG. The addition of FA significantly improved the emulsifying property of ß-LG. When the mass ratio was 10:6, the radical scavenging activities of DPPH and ABTS reached 65.06% and 88.22%, respectively, and the antigenicity of ß-LG reduced by about 35%. This study provides novel ß-LG-FA complexes in food systems to reduce the antigenicity of ß-LG and improve functional properties.

Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation.

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1ß and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-ß (TGF-ß), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity.

Monosaccharide Composition and In Vitro Activity to HCT-116 Cells of Purslane Polysaccharides after a Covalent Chemical Selenylation.

The anti-cancer effects of selenylated plant polysaccharides are a focus of research. As a natural plant with extensive biological effects, there have been few studies related to edible purslane (Portulaca oleracea L.). Thus, in this study, soluble P. oleracea polysaccharides (PPS) were extracted from the dried P. oleracea and then selenylated chemically using the HNO3-Na2SeO3 method to obtain two selenylated products, namely, SePPS1 and SePPS2. Compared with the extracted PPS, SePPS1 and SePPS2 had much higher Se contents (840.3 and 1770.5 versus 66.0 mg/kg) while also showing lower contents in three saccharides-arabinose, fucose, and ribose-and higher contents in seven saccharides including galactose, glucose, fructose, mannose, rhamnose, galacturonic acid, and glucuronic acid, but a stable xylose content demonstrated that the performed chemical selenylation of PPS led to changes in monosaccharide composition. Moreover, SePPS1 and SePPS2 shared similar features with respect to monosaccharide composition and possessed higher bioactivity than PPS in human colon cancer HCT-116 cells. Generally, SePPS1 and SePPS2 were more active than PPS with respect to cell growth inhibition, the alteration of cell morphology, disruption of mitochondrial membrane potential, intracellular reactive oxygen species (ROS) generation, the induction of cell apoptosis, and upregulation or downregulation of five apoptosis-related genes and proteins such as Bax, Bcl-2, caspases-3/-9, and cytochrome C, that cause cell apoptosis and growth suppression via the ROS-mediated mitochondrial pathway. SePPS2 consistently showed the highest capacity to exert these observed effects on the targeted cells, suggesting that the performed chemical selenylation of PPS (in particular when higher degrees of selenylation are reached) resulted in an increase in activity in the cells. It can thus be concluded that the performed selenylation of PPS was able to incorporate inorganic Se into the final PPS products, changing their monosaccharide composition and endowing them with enhanced nutraceutical and anti-cancer effects in the colon.

The Barrier-Enhancing Function of Soluble Yam (Dioscorea opposita Thunb.) Polysaccharides in Rat Intestinal Epithelial Cells as Affected by the Covalent Se Conjugation.

The non-starch yam polysaccharides (YP) are the bioactive substances of edible yam, while Se is an essential nutrient for the human body. Whether a covalent conjugation of Se to YP might cause bioactivity change for the resultant selenylated YP in the intestine is still insufficiently studied, including the critical intestinal barrier function. In this study, two selenylated YP products, namely, YPSe-I and YPSe-II, with corresponding Se contents of 795 and 1480 mg/kg, were obtained by the reaction of YP and Na2SeO3 in the presence of HNO3 and then assessed for their bioactivities to a cell model (i.e., rat intestinal epithelial IEC-6 cells). The results showed that YP, YPSe-I, and YPSe-II at 5-80 µg/mL dosages could promote cell growth with treatment times of 12-24 h. The three samples also could improve barrier integrity via increasing cell monolayer resistance and anti-bacterial activity against E. coli or by reducing paracellular permeability and bacterial translocation. Additionally, the three samples enhanced F-actin distribution and promoted the expression of the three tight junction proteins, namely, zonula occluden-1, occludin, and claudin-1. Meanwhile, the expression levels of ROCK and RhoA, two critical proteins in the ROCK/RhoA singling pathway, were down-regulated by these samples. Collectively, YPSe-I and, especially, YPSe-II were more potent than YP in enhancing the assessed bioactivities. It is thus concluded that this chemical selenylation of YP brought about enhanced activity in the cells to promote barrier integrity, while a higher selenylation extent of the selenylated YP induced much activity enhancement. Collectively, the results highlighted the important role of the non-metal nutrient Se in the modified polysaccharides.

The Impact of Heat Treatment of Quercetin and Myricetin on their Activities to Alleviate the Acrylamide-Induced Cytotoxicity and Barrier Loss in IEC-6 Cells.

Two flavonols quercetin and myricetin were assessed for their in vitro activities to attenuate the acrylamide-induced cytotoxicity and barrier loss in rat intestinal epithelial (IEC-6) cells and to identify whether heat treatment of the flavonols might cause activity changes. The results showed that the flavonols could alleviate the acrylamide-caused cell injury, resulting in higher cell viability, lower lactate dehydrogenase release, and less formation of reactive oxygen species. Meanwhile, the flavonols could antagonize the acrylamide-induced barrier dysfunction via decreasing the paracellular permeability, increasing the transepithelial resistance of cell monolayer, and enhancing the expression of three tight junction proteins namely occludin, claudin-1, and zonula occludens-1. The flavonols also could down-regulate the expression of JNK/Src proteins and thus cause lower relative protein ratios of p-JNK/JNK and p-Src/Src, resulting in a suppressed JNK/Src activation. Totally, quercetin was more potent than myricetin to exert these assessed activities, while the heated flavonols obtained lower activity than the unheated ones. It is thus concluded that the flavonols had beneficial activities towards the intestinal epithelial cells with acrylamide exposure by alleviating the acrylamide-induced cytotoxicity and barrier disruption, while heat treatment of the flavonols was unfavorable because it led to a reduced flavonol activity to the cells.

Monosaccharide composition and in vivo immuno-stimulatory potential of soluble yam (Dioscorea opposita Thunb.) polysaccharides in response to a covalent Se incorporation.

The present study aimed to investigate whether selenylation modification could affect compositional features and in vivo immuno-stimulatory potential of yam polysaccharides. In this study, the soluble yam mucilage polysaccharides (YPS) were prepared and selenylated in the HNO3-Na2SeO3 system, and two selenylated polysaccharide products, namely SeYPS-1 and SeYPS-2 with respective Se contents of 719 and 1585 mg/kg, were thus obtained. GC-MS analysis demonstrated that the compositional features of SeYPS-1 and SeYPS-2 were similar to those of YPS. Meanwhile, the immuno-stimulatory potential of the selenylated products, especially SeYPS-2, in the BALB/c mice model was higher than that of YPS, reflected by the elevated contents of serum immunoglobins and increased percentage of CD4+ splenic lymphocytes. It was thus confirmed that the selenylation did not change the composition of monosaccharides but endowed YPS with greater immuno-stimulation in the mice, while the higher extent of selenylation also caused a much enhanced immuno-stimulatory potential of SeYPS-2.

Activities of the soluble and non-digestible longan (Dimocarpus longan Lour.) polysaccharides against HCT-116 cells as affected by a chemical selenylation.

The soluble and non-digestible longan (Dimocarpus longan Lour.) polysaccharides (LP) with Se content less than 0.01 g/kg were extracted and selenylated chemically with the HNO3-Na2SeO3 system, to prepare two selenylated products namely SeLP1 and SeLP2 with enhanced Se contents of 1.46 and 4.79 g/kg, respectively. LP, SeLP1, and SeLP2 were then measured and compared for their saccharide features and bioactivity in human colon carcinoma HCT-116 cells. Compared with LP, both SeLP1 and SeLP2 contained more neutral saccharides, but showed reduced uronic acid content and undetectable sulfate. Moreover, SeLP1 and especially SeLP2 in the cells showed higher activities than LP, reflected by their enhanced capacity to inhibit cell growth, alter cell morphology, and suppress cell colony formation. Compared with LP, SeLP1 and especially SeLP2 were also more capable of promoting intracellular reactive oxygen species and Ca2+ levels, causing mitochondrial membrane potential loss, or inducing cell apoptosis via up- and down-regulating the eight apoptosis-related genes and proteins. Overall, the performed chemical selenylation of LP resulted in obvious changes in these saccharide features and simultaneously enhanced the anti-cancer activity of the selenylated products against the cells clearly, while a higher selenylation extent of the selenylated products consistently caused higher activity towards the cells. The results of this study thus highlighted that this chemical selenylation is applicable when aiming to enhance the bioactivities of natural polysaccharides.

A Simple and Rapid "Signal On" Fluorescent Sensor for Detecting Mercury (II) Based on the Molecular Beacon Aptamer.

Biosensors for mercury (II) (Hg2+) with high sensitivity are urgently required for food safety, ecosystem protection and disease prevention. In this study, a simple and fast detection method of Hg2+ based on the molecular beacon aptamer was established, according to the principle that Hg2+ could change the structure of the molecular beacon aptamer, resulting in the changed fluorescence intensity. All of the detection conditions were optimized. It was found that an optimal molecular beacon aptamer MB3 showed the optimal response signal in the optimized reaction environment, which was 0.08 µmol/L MB3, 50 mmol/L tris buffer (40 mmol/L NaCl, 10 mmol/L MgCl2, pH 8.1), and a 10 min reaction. Under the optimal detection conditions, the molecular beacon aptamer sensor showed a linear response to Hg2+ concentration within a range from 0.4 to 10 µmol/L and with a detection limit of 0.2254 µmol/L and a precision of 4.9%. The recovery rates of Hg2+ in water samples ranged from 95.00% to 99.25%. The method was convenient and rapid, which could realize the rapid detection of mercury ions in water samples.

Enhanced Growth Inhibition and Apoptosis Induction in Human Colon Carcinoma HT-29 Cells of Soluble Longan Polysaccharides with a Covalent Chemical Selenylation.

The selenylated polysaccharides chemically belong to the organic Se-conjugated macromolecules and have recently been attracting more and more attention due to their potential to promote body health or prevent cancers. Longan (Dimocarpus longan L.), as a subtropical fruit, contains soluble and non-digestible polysaccharides that are regarded with health care functions in the body. In this study, the longan polysaccharides (LP) were obtained via enzyme-assisted water extraction, and then chemically selenylated using a reaction system composed of HNO3-Na2SeO3 to yield two selenylated products, namely, SeLP1 and SeLP2, with Se contents of 1.46 and 4.79 g/kg, respectively. The anti-cancer effects of the three polysaccharide samples (LP, SeLP1, and SeLP2) were thus investigated using the human colon cancer HT-29 cells as the cell model. The results showed that SeLP1 and SeLP2 were more able than LP to inhibit cell growth, alter cell morphology, cause mitochondrial membrane potential loss, increase intracellular reactive oxygen and [Ca2+]i levels, and induce apoptosis via regulating the eight apoptosis-related genes and proteins including Bax, caspases-3/-8/-9, CHOP, cytochrome c, DR5, and Bcl-2. It was thereby proven that the selenylated polysaccharides could induce cell apoptosis via activating the death receptor, mitochondrial-dependent, and ER stress pathways. Collectively, both SeLP1 and SeLP2 showed higher activities than LP in HT-29 cells, while SeLP2 was consistently more active than SeLP1 in exerting these assessed anti-cancer effects on the cells. In conclusion, this chemical selenylation covalently introduced Se into the polysaccharide molecules and caused an enhancement in their anti-cancer functions in the cells, while higher selenylation extent was beneficial to the activity enhancement of the selenylated products.

Bioactivity of Two Polyphenols Quercetin and Fisetin against Human Gastric Adenocarcinoma AGS Cells as Affected by Two Coexisting Proteins.

It is recognized that minor dietary components polyphenols have anticancer effects on digestive tract, lung, leukemia, and other cancers, while polyphenols also can covalently or noncovalently interact with major dietary components proteins such as casein, soybean proteins, whey proteins, and bovine serum albumin. Thus, whether the noncovalent interaction between the molecules of two polyphenols (quercetin and fisetin) and two proteins (bovine serum albumin and casein) has positive or negative impact on anticancer activities of the polyphenols against human gastric adenocarcinoma AGS cells was assessed in this study. The two polyphenols had obvious anticancer activities to the cells, because dose levels as low as 20-160 µmol/L caused reduced cell viability of 30.0-69.4% (quercetin) and 24.6-63.1% (fisetin) (using a cell treatment time of 24 h), or 9.9-48.6% (quercetin) and 6.4-29.9% (fisetin) (using a cell treatment time of 48 h). However, the cell treatments by the polyphenols in the presence of the two proteins mostly caused lower polyphenol activity toward the cells, compared with those treatments by the polyphenols in the absence of the proteins. Specifically, the presence of the proteins led to reduced growth inhibition in the cells, because higher cell viability of 33.2-86.7% (quercetin) and 29.1-77.7% (fisetin) at 24 h, or 14.1-66.8% (quercetin) and 7.9-59.0% (fisetin) at 48 h, were measured in these treated cells. The two coexisting proteins also yielded the polyphenol-treated cells with less mitochondrial membrane potential loss, less formation of reactive oxygen species, and decreased cell apoptosis. Thus, it is highlighted that the noncovalent interaction between dietary polyphenols and proteins resulted in weakened anticancer ability for the polyphenols to the gastric cancer cells.

Impact of covalent grafting of two flavonols (kaemperol and quercetin) to caseinate on in vitro digestibility and emulsifying properties of the caseinate-flavonol grafts.

Covalent grafting of one of the two flavonols (kaemperol and quercetin) to caseinate was achieved by a reaction between the heat-oxidized flavonols and caseinate at flavonol-lysine molar ratios of 1:100 and 1:200. Grafted caseinate products (GCPs) showed - NH2 content reduction and respective kaemperol and quercetin contents of 1.08-6.13 and 3.23-6.64 mmol/kg protein. Quercetin was more reactive than kaemperol under the same conditions, while long-time flavonol heat and higher flavonol-lysine molar ratio caused greater flavonol-grafting. GCPs subjected to 180-day storage had further flavonol-grafting, -NH2 content decrease, and weak protein crosslinking. GCPs consistently had higher surface hydrophobicity but lower emulsification and digestibility than caseinate, while greater flavonol-grafting caused a remarkable value change. Meanwhile, the Kjeldahl method was more suitable than the UV-absorption method to evaluate protein digestibility, because the grafted flavonols in this case did not interfere with data results. Collectively, the covalent flavonol-grafting of proteins can impact the assayed protein functionalities.

Casein Oligochitosan-Glycation by Transglutaminase Enhances the Anti-Inflammatory Potential of Casein Hydrolysates to the Lipopolysaccharide-Stimulated IEC-6 Cells.

In this study, milk protein casein was glycated by oligochitosan through the catalysis of transglutaminase (TGase) and then hydrolyzed by trypsin. The obtained glycated casein hydrolysates (GCNH) were assessed for their anti-inflammatory activities, using the lipopolysaccharide (LPS)-stimulated rat intestinal epithelial cells (IEC-6) as cell models and the casein hydrolysates (CNH) without TGase catalysis as controls. The results showed that GCNH had oligochitosan incorporation and thus possessed a glucosamine content of 5.74 g/kg protein. In general, GCNH at dose levels of 25-100 µg/mL could elevate IEC-6 cell growth, and at dose levels of 25-50 µg/mL, they were also able to alleviate the LPS-induced cytotoxicity by increasing cell viability efficiently. Although LPS caused clear inflammation in the LPS-stimulated cells, GCNH were capable of reducing the secretion of three pro-inflammatory mediators including interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α, or promoting the secretion of two anti-inflammatory mediators like IL-10 and transforming growth factor-ß, demonstrating their anti-inflammatory activities to the stimulated cells. Moreover, GCNH also could down-regulate the expression of three inflammation-related proteins including TLR4, p-p38, and p-p65 in the stimulated cells, and thus possessed a capacity to suppress the phosphorylation of p38 and p65 proteins as well as to inactivate the NF-κB and MAPK signaling pathways. Additionally, a higher GCNH dose level consistently led to higher anti-inflammatory effect in the cells, while GCNH were always more potent than CNH at performing anti-inflammatory function targets. It is thus suggested that the TGase-catalyzed casein oligochitosan-glycation could enhance the anti-inflammatory activities of casein hydrolysates efficiently. TGase-catalyzed protein glycation thus might enhance the healthcare function of protein ingredients in the body.

Impact of the Plastein Reaction of Casein Hydrolysates in the Presence of Exogenous Amino Acids on Their Anti-Inflammatory Effect in the Lipopolysaccharide-Stimulated Macrophages.

In this study, papain-generated casein hydrolysates (CH) with a degree of hydrolysis of 13.7% were subjected to a papain-mediated plastein reaction in the absence or presence of one of the exogenous amino acids-Gly, Pro, and Hyp-to prepare four plastein modifiers, or mixed with one of three amino acids to prepare three mixtures. The assay results confirmed that the reaction reduced free NH2 for the modifiers and caused amino acid incorporation and peptide condensation. When RAW264.7 macrophages were exposed to the CH, modifiers, and mixtures, these samples promoted macrophage growth and phagocytosis in a dose-dependent manner. In addition, the CH shared similar activity in the cells as the mixtures, while the modifiers (especially the PCH-Hyp prepared with Hyp addition) exerted higher potential than CH, the mixtures, and PCH (the modifier prepared without amino acid addition). The plastein reaction thus enhanced CH bioactivity in the cells. When RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS), the inflammatory cells produced more lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) formation, and caused more four inflammatory mediators (NO, PGE2, TNF-α, and IL-6) and two anti-inflammatory mediators (TGF-ß1 and IL-10). However, the PCH-Hyp, PCH, and CH at dose levels of 100 µg/mL could combat against the LPS-induced inflammation. Overall, the PCH-Hyp was more active than the CH and PCH in reducing LDH release, ROS formation, and the secretion of these inflammatory mediators, or in increasing the secretion of the anti-inflammatory mediators. The qPCR and Western blot analysis results further confirmed that these samples had anti-inflammatory effects on the stimulated cells by suppressing the LPS-induced activation of the NF-κB signaling pathway, via regulating the mRNA/miRNA expression of iNOS, IL-6, TNF-α, IL-1ß, COX-2, TLR4, IL-10, TGF-ß1, miR-181a, miR-30d, miR-155, and miR-148, as well as the protein expression of MyD88, p-IKKα, p-IκBα, p-NF-κB p65, and iNOS, involved in this signaling pathway. In addition, the immunofluorescence assay results revealed that these samples could block the LPS-mediated nuclear translocation of the p65 protein and displayed the same function as the NF-κB inhibitor BAY 11-7082. It was concluded that CH could be endowed with higher anti-inflammatory activity to the macrophages by performing a plastein reaction, particularly that in the presence of exogenous Hyp.

The anti-inflammatory effects of apigenin and genistein on the rat intestinal epithelial (IEC-6) cells with TNF-α stimulation in response to heat treatment.

The aims of the present study were to investigate the anti-inflammatory function of two flavonoids apigenin and genistein in rat intestinal epithelial (IEC-6) cells stimulated by tumor necrosis factor-alpha (TNF-α) and to clarify whether the heat treatment of the flavonoids might affect flavonoid activity. The flavonoids at lower dosage (e.g. 5 µmol/L) had no toxic effect but growth promotion on the cells. Meanwhile, the flavonoid pretreatment of the cells before TNF-α stimulation could maintain cellular morphology, decrease the production of prostaglandin E2 and two pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-6, but increase the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-ß. Additionally, the flavonoids could block off the nuclear translocation of nuclear factor-kappaB (NF-κB) p65, and suppress the expression of phosphorylated IκBα and p65 induced by TNF-α. Meanwhile, the NF-κB inhibitor BAY 11-7082 shared a similar function with the flavonoids to mediate the production of IL-6/IL-10. Furthermore, in silico analysis also declared that the flavonoids could interact with the IκBα-NF-κB complex at the binding pockets to yield the binding energies ranging from -31.7 to -34.0 kJ/mol. However, the heated flavonoids were consistently less effective than the unheated counterparts to perform these anti-inflammatory effects. It is thus proposed that both apigenin and genistein have anti-inflammatory potential to the TNF-α-stimulated IEC-6 cells by inactivating the NF-κB pathway, while heat treatment of the flavonoids caused a negative impact on these assessed anti-inflammatory effects.