Absorption of iron from Tegillarca granosa using an in vitro simulated digestion and Caco-2/HepG2 co-culture system.

BACKGROUND: Iron-deficiency anemia is one severe micronutrient malnutrition and has captured worldwide attention. This study evaluated the in vitro iron absorption of two iron-binding proteins (hemoglobin and ferritin) from Tegillarca granosa. In addition, the protein structure-iron absorption relationship and the regulatory effect of hepcidin on cellular iron absorption were explored. RESULTS: Our findings revealed that both hemoglobin and ferritin extracted from T. granosa contained abundant iron-binding sites, as evidenced by stronger peaks in amide I and II regions compared with the two proteins from humans. Less ß-sheet (27.67%) structures were found in hemoglobin compared with ferritin (36.40%), probably contributing to its greater digestibility and more release of available iron. This was confirmed by the results of Caco-2/HepG2 cell culture system that showed iron absorption of hemoglobin was 26.10-39.31% higher than that of ferritin with an iron content of 50-150 µmol L-1 . This high iron absorption of hemoglobin (117.86-174.10 ng mg-1 ) could also be due to more hepcidin produced by HepG2 cells, thereby preventing ferroportin-mediated iron efflux from Caco-2 cells. In addition, the possible risk of oxidative stress was evaluated in cells post-iron exposure. In comparison with ferrous sulfate, a common iron supplement, Caco-2 cells treated with the iron-binding proteins had a 9.50-25.73% lower level of intracellular reactive oxygen species, indicating the safety of hemoglobin and ferritin. CONCLUSION: Collectively, the data of this research would be helpful for understanding the key features and potential of developing hemoglobin and ferritin from T. granosa as novel iron supplements. © 2022 Society of Chemical Industry.

Efficacy of Pneumatophorus japonicus meat as an iron fortificant in whole-wheat flour in preventing iron deficiency.

Iron deficiency anemia (IDA) is a global health concern affecting one-third of the world's population, particularly those dominated by plant-based food. Fortifying staple foods with iron has been an effective strategy for preventing IDA. Pneumatophorus japonicus is an essential economic fish in China. Pneumatophorus japonicus dark meat is usually underutilized as a byproduct, though it contains bounteous nutrients, including heme iron (10.50 mg/100 g). This study aimed to investigate the iron bioavailability of P. japonicus dark meat and to evaluate its potential as an iron fortifier for whole-wheat flour, a typical staple food, using an in vitro digestion/Caco-2 cell culture system. Our results suggested the excellent iron bioavailability of P. japonicus dark meat in comparison with beef (a heme dietary iron reference), whole-wheat flour (a non-heme dietary iron reference), and FeSO4 (a conventional iron supplement). The addition of P. japonicus dark meat notably enhanced iron solubility, bioavailability, and protein digestibility of whole-wheat flour. The flour-dark meat mixture yielded 1.96 times the iron bioavailability compared to beef per gram. The iron bioavailability was further improved by adding vitamin C, a commonly used dietary factor, at the Vc/iron mass ratio of 2:100-5:100. Our findings reveal the promise of P. japonicus dark meat as a significant source of bioavailable iron, providing a basis for developing fish byproducts as alternatives for iron supplementation. PRACTICAL APPLICATION: This study investigated the iron bioavailability of Pneumatophorus japonicus meat using in vitro digestion/Caco-2 cell culture system. These results could be used to improve the utilization of Pneumatophorus japonicus byproduct (dark meat) and develop the potential of the byproduct as an iron fortifier for whole-wheat flour.

High Endogenously Synthesized N-3 Polyunsaturated Fatty Acids in Fat-1 Mice Attenuate High-Fat Diet-Induced Insulin Resistance by Inhibiting NLRP3 Inflammasome Activation via Akt/GSK-3ß/TXNIP Pathway.

High-fat (HF) diets and low-grade chronic inflammation contribute to the development of insulin resistance and type 2 diabetes (T2D), whereas n-3 polyunsaturated fatty acids (PUFAs), due to their anti-inflammatory effects, protect against insulin resistance. Interleukin (IL)-1ß is implicated in insulin resistance, yet how n-3 PUFAs modulate IL-1ß secretion and attenuate HF diet-induced insulin resistance remains elusive. In this study, a HF diet activated NLRP3 inflammasome via inducing reactive oxygen species (ROS) generation and promoted IL-1ß production primarily from adipose tissue preadipocytes, but not from adipocytes and induced insulin resistance in wild type (WT) mice. Interestingly, endogenous synthesized n-3 polyunsaturated fatty acids (PUFAs) reversed this process in HF diet-fed fat-1 transgenic mice although the HF diet induced higher weight gain in fat-1 mice, compared with the control diet. Mechanistically, palmitic acid (PA), the main saturated fatty acid in an HF diet inactivated AMPK and led to decreased GSK-3ß phosphorylation, at least partially through reducing Akt activity, which ultimately blocked the Nrf2/Trx1 antioxidant pathway and induced TXNIP cytoplasm translocation and NLRP3 inflammasome activation, whereas docosahexaenoic acid (DHA), the most abundant n-3 PUFA in fat-1 adipose tissue, reversed this process via inducing Akt activation. Our GSK-3ß shRNA knockdown study further revealed that GSK-3ß played a pivot role between the upstream AMPK/Akt pathway and downstream Nrf2/Trx1/TXNIP pathway. Given that NLRP3 inflammasome is implicated in the development of most inflammatory diseases, our results suggest the potential of n-3 PUFAs in the prevention or adjuvant treatment of NLRP3 inflammasome-driven diseases.

Polyphenols extracted from Enteromorpha clathrata alleviates inflammation in lipopolysaccharide-induced RAW 264.7 cells by inhibiting the MAPKs/NF-κB signaling pathways.

ETHNOPHARMACOLOGY RELEVANCE: Enteromorpha has long been recorded in traditional Chinese medicine, with cholesterol-lowering, anti-cancer, anti-inflammatory and antibacterial effects. Recently, we extracted the polyphenol-enriched fraction from Enteromorpha clathrata (E. clathrata) by ethyl acetate (ECPs), and isolated six individual polyphenols from ECPs via high-speed counter-current chromatography (HSCCC) with high-performance liquid chromatography (HPLC). AIM OF THE STUDY: In this study, we explored the anti-inflammatory activity and underlying mechanism of ECPs in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. MATERIALS AND METHODS: ECPs and the six polyphenols were used for nitric oxide (NO) assay to identify the components with potent inflammation inhibitory effect. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), flow cytometry, and Western blot analysis were applied to further investigate their anti-inflammatory effects and underlying mechanism in LPS-stimulated RAW264.7 cells. RESULTS: ECPs and the three individual polyphenols, including (-)-epicatechin, epigallocatechin-3-O-gallate and (-)-epicatechin-3-O-gallate, showed in vitro immunosuppressive activity by altering the cell biology at the gene, protein and functional levels in a dose- and species-dependent manner. Their anti-inflammatory effects were achieved by inhibiting LPS-induced production of nitric oxide and its upstream enzyme inducible nitric oxide synthase (iNOS), the pro-inflammatory cytokines including interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), as well as the phagocytotic capacity, without cytotoxicity. The mechanism study further revealed that these anti-inflammatory properties were, at least partly, attributed to the suppressed activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: These findings indicated for the first time the correlation between the anti-inflammatory activity of ECPs and NF-κB and MAPK signaling pathways, suggesting that polyphenol-enriched organic fraction of E. clathrata could be potential candidate as therapeutic agent for treating inflammatory diseases.

Docosahexaenoic acid monoglyceride induces apoptosis and autophagy in breast cancer cells via lipid peroxidation-mediated endoplasmic reticulum stress.

Epidemiologic and preclinical studieshave shown that marine n-3 polyunsaturated fatty acids (n-3 PUFAs) elicit promising chemoprevention against breast cancer. Docosahexaenoic acid monoglyceride (MAG-DHA), a docosahexaenoic acid sn-1-monoacylglycerol does not required pancreatic lipase to be absorbed, eliciting a better bioavailability when compared with other formulations such as DHA-free fatty acid, DHA-triglycerol, or DHA-ethyl ester. However, the anticancer actions and underlying mechanisms of MAG-DHA on breast cancer remain to be assessed. In this study, MAG-DHA induced significant growth inhibition in MCF-7 and MDA-MB-231 breast cancer cells in a dose-dependent manner. MAG-DHA treatment (80 µM) led to 83.8 and 94.3% growth inhibition between MCF-7 and MDA-MB-231 cells, respectively. MAG-DHA-induced growth inhibition was tightly associated with apoptosis, as evidenced by increased active forms of caspase-3, poly (ADP-ribose) polymerase (PARP) and caspase-12. In particular, MAG-DHA-induced apoptosis was triggered by oxidative stress-mediated endoplasmic reticulum (ER) stress, as evidenced by activation of the PERK-eIF2α pathway in ER. MAG-DHA treatment also strongly suppressed the growth of E0771 murine breast cancer xenografts, significant differences of tumor volume were found between MAG-DHA group (0.271 cm3 ) and control group (0.875 cm3 ) after 15 daily MAG-DHA treatments. The in vitro antibreast cancer mechanism of MAG-DHA was supported by the in vivo xenograft model. In addition, MAG-DHA-induced ER stress concomitantly triggered autophagy in these cancer cells, and the induction of autophagy suppressed its ability to induce apoptotic cell death. Our data suggested that MAG-DHA as dietary supplement, in combination with autophagy inhibitors may be a useful therapeutic strategy in treating breast cancer.

Rational formulation engineering of fraxinellone utilizing 6-O-α-D-maltosyl-ß-cyclodextrin for enhanced oral bioavailability and hepatic fibrosis therapy.

Although Fraxinellone (Frax) isolated from Dictamnus albus L. possessed excellent anti-hepatic fibrosis activity, oral administration of Frax suffered from the inefficient therapeutic outcome in vivo due to negligible oral absorption. At present, the oral formulation of Frax is rarely exploited. For rational formulation design, we evaluated preabsorption risks of Frax and found that Frax was rather stable while poorly dissolved in the gastrointestinal tract (78.88 µg/mL), which predominantly limited its oral absorption. Further solubility test revealed the outstanding capacity of cyclodextrin derivatives (CDs) to solubilize Frax (6.8-12.8 mg/mL). This led us to study the inclusion complexes of Frax with a series of CDs and holistically explore their drug delivery performance. Characterization techniques involving 1H-NMR, FT-IR, DSC, PXRD, and molecular docking confirmed the most stable binding interactions when Frax complexed with 6-O-α-D-maltosyl-ß-cyclodextrin (G2-ß-CD-Frax). Notably, G2-ß-CD-Frax exhibited the highest solubilizing capacity, fast dissolution rate, and superior Caco-2 cell internalization with no obvious toxicity. Pharmacokinetic studies demonstrated markedly higher oral bioavailability of G2-ß-CD-Frax (5.8-fold that of free drug) than other Frax-CDs. Further, long-term administration of G2-ß-CD-Frax (5 mg/kg) efficiently inhibited CCl4-induced hepatic fibrosis in the mouse without inducing any toxicity. Our results will inspire the continued advancement of optimal oral Frax formulations for anti-fibrotic therapy.

Structure, functional properties and iron bioavailability of Pneumatophorus japonicus myoglobin and its glycosylation products.

Developing safe and efficient iron supplements is significant for the alleviation of iron-deficient anemia (IDA). Myoglobin (Mb) is a heme-protein rich in bioavailable iron. Pneumatophorus japonicus (P. japonicus), one important economic fish in China, contain a high Mb level in its dark meat normally discarded during processing. The present study aimed to determine the structure, physicochemical properties, and iron bioavailability of Mb extracted from P. japonicus. Meanwhile, the effects of glycosylation, a commonly applied chemical modification of proteins, on these parameters were evaluated. Using Box-Behnken design, the optimal conditions for Mb-chitosan glycosylation were obtained: 45.07 °C, pH 6.10 and Mb/chitosan mass ratio of 6.29. The structure and functional properties of the glycosylated Mb (Mb-gly) were investigated. Compared with the original Mb, Mb-gly obtained a more ordered secondary structure. The surface hydrophobicity of Mb-gly was found to be decreased together with the observations of elevated water solubility. Moreover, glycosylation enhanced the Mb antioxidant capacity, and improved its stability in enzymatic digestion system. Regarding to the iron bioavailability, the cellular uptake of Mb­iron was significantly higher than FeSO4, and further elevated by glycosylation. These results provided a basis for the development of Mb-based iron supplements, promoting the utilization of fish-processing industries wastes.

Determination of the iron bioavailability, conformation, and rheology of iron-binding proteins from Tegillarca granosa.

The increased interest in achieving, solely through diet, the same effect on iron levels with supplementation, leads to numerous studies on iron absorption of iron binding proteins (IBPs). The characteristics of IBPs from Tegillarca granosa (T. granosa) and its iron utilization were determined to analyze their relationship. The results showed in T. granosa, Fe(ӀӀ) was main iron form in hemoglobin (TH) and that Fe(ӀӀ) and Fe(ӀӀӀ) coexisted in ferritin (TF). After in vitro digestion, TH was easier to be digested than TF, bovine hemoglobin, and bovine ferritin. In caco-2 cells model, iron bioavailability of TH also was the best, which related to TH's superior fluid properties, higher ratios of α-helix to ß-sheet and amide I to amide II. These suggest TH could be used as a good source of organic iron and provide references for application of T. granosa in human nutrition. PRACTICAL APPLICATIONS: This research investigated the iron bioavailability and structural properties of iron-binding proteins from Tegillarca granosa (T. granosa). Moreover, the effects of iron absorption in bovine hemoglobin and ferritin were compared with those from T. granosa. The results showed the hemoglobin in T. granosa had better iron bioavailability and it could be a good source of iron. These data could provide a basic instruction of the application of T. granosa in functional food production.

High n-3 fatty acids counteract hyperglycemia-induced insulin resistance in fat-1 mice via pre-adipocyte NLRP3 inflammasome inhibition.

Although n-3 polyunsaturated fatty acids (n-3 PUFAs) have potential anti-insulin resistance activity, the mechanism remains largely unknown. In this study, increased glucose resistance, insulin sensitivity, and lower glycemia were observed upon streptozotocin (STZ) treatment in n-3 PUFA-enriched fat-1 mice compared to wild type (WT) mice. Endogenous n-3 PUFAs in fat-1 mice were found to impair hyperglycemia or high glucose level-induced nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome activation and inhibit IL-1ß secretion in adipose tissues. In addition, endogenous n-3 PUFAs also inhibited high glucose-induced caspase-1 activity and IL-1ß secretion in pre-adipocyte-enriched stromal vascular fractions (SVF) isolated from adipose tissues. Furthermore, in 3T3-L1 pre-adipocytes, high levels of glucose induced thioredoxin interacting protein (TXNIP) expression and activated the NLRP3 inflammasome, which was counteracted by docosahexaenoic acid (DHA), the major n-3 PUFA in fat-1 mice, by downregulating TXNIP via the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Our results suggest that n-3 PUFA-mediated insulin sensitivity is at least partly associated with inflammasome inhibition in pre-adipocytes. Our findings highlight the potential clinical use of dietary n-3 PUFAs in the prevention or intervention of T2D and other NLRP3 inflammasome-driven inflammatory diseases.

Neuroprotective Effects of Lindleyin on Hydrogen Peroxide-Induced Cell Injury and MPTP-Induced Parkinson's Disease in C57BL/6 Mice.

Oxidative stress (OS) is a crucial factor influencing the development of Parkinson's disease (PD). Here we first reported that Lindleyin (Lin), one of the major components of rhubarb, possessed neuroprotective effects against H2O2-induced SH-SY5Y cell injury and MPTP-induced PD of C57BL/6 mice. The results showed that Lin can decrease cell death and apoptotic rate induced by H2O2 through inhibiting mitochondrial apoptotic pathway and increasing the activities of SOD, GSH-Px, and CAT as well as decreasing the level of MDA. In addition, in vivo studies showed that oral administration of Lin (5 or 20 mg/kg) showed significant change in motor function deficits, antioxidant enzyme activities, apoptotic pathway, and tyrosine hydroxylase expression. Our results reveal that Lin might be a promising anti-PD agent by reducing OS and apoptosis.

Comparison of gelling properties and flow behaviors of microbial transglutaminase (MTGase) and pectin modified fish gelatin.

The gelling and structural properties of microbial transglutaminase (MTGase) and pectin modified fish gelatin were compared to investigate their performances on altering fish gelatin properties. Our results showed that within a certain concentration, both MTGase and pectin had positive effects on the gelation point, melting point, gel strength, textural, and swelling properties of fish gelatin. Particularly, low pectin content (0.5%, w/v) could give fish gelatin gels the highest values of gel strength, melting temperature, and hardness. Meantime, flow behavior results showed that both MTGase and pectin could increase fish gelatin viscosity without changing its fluid characteristic, but the latter gave fish gelatin higher viscosity. Both MTGase and pectin could increase the lightness of fish gelatin gels but decreases its transparency. More importantly, fluorescence and UV absorbance spectra, particle size distribution, and confocal microscopy results indicated that MTGase and pectin could change the structure of fish gelatin with the formation of large aggregates. Compared with MTGae modified fish gelatin, pectin could endow fish gelatin had similar gel strength, thermal and textural properties to pig skin gelatin.

Algal oil rich in n-3 polyunsaturated fatty acids suppresses B16F10 melanoma lung metastasis by autophagy induction.

Melanoma is a malignant tumor that arises from epidermal melanocytes with high morbidity and mortality, and currently, there are no effective conventional genotoxic treatments or systematic treatment. Increasing evidence shows that n-3 polyunsaturated fatty acids (PUFAs) exhibit anti-melanoma activity, but their anti-melanoma mechanism remains elusive. Here, C57BL/6 mice were injected with B16F10 melanoma cells via a tail vein to establish a lung metastasis model. n-3 PUFAs were significantly increased in lung metastatic tissues from mice treated with algal oil, especially rich in docosahexaenoic acid (DHA). Algal oil treatment significantly suppressed pulmonary metastases and outgrowth of melanoma cells, which was associated with autophagy induction, as evidenced by an increase in LC3-II levels. In addition, algae oil-triggered autophagy was mediated by inactivation of the mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein (MAP) kinase, and activation of c-Jun N-terminal kinases (JNKs), which led to a decrease in p62 accumulation and decreased secretion of proinflammatory cytokine interleukin-1ß (IL-1ß). These results suggest that algal oil exerts its antitumourigenic activities via autophagy-mediated p62 elimination and anti-inflammatory properties.

Endogenously synthesized n-3 fatty acids in fat-1 transgenic mice prevent melanoma progression by increasing E-cadherin expression and inhibiting ß-catenin signaling.

Malignant melanoma is the most lethal form of skin cancer. Although preclinical studies have shown that n-3 polyunsaturated fatty acids (PUFAs) are beneficial for prevention of melanoma, the molecular mechanisms underlying the protective effects of n­3 PUFAs on melanoma remain largely unknown. In the present study, endogenously increased levels of n-3 PUFAs in the tumor tissues of omega­3 fatty acid desaturase (fat­1) transgenic mice was associated with a reduction in the growth rate of melanoma xenografts. This reduction in tumor growth in fat­1 mice compared with wild­type controls may have been associated, in part, to the: i) Increased expression of E­cadherin and the reduced expression of its transcriptional repressors, the zinc finger E­box binding homeobox 1 and snail family transcriptional repressor 1; ii) significant repression of the epidermal growth factor receptor/Akt/ß­catenin signaling pathway; and iii) formation of significant levels of n­3 PUFA­derived lipid mediators, particularly resolvin D2 and E1, maresin 1 and 15­hydroxyeicosapentaenoic acid. In addition, vitamin E administration counteracted n­3 PUFA­induced lipid peroxidation and enhanced the antitumor effect of n­3 PUFAs, which suggests that the protective role of n­3 PUFAs against melanoma is not mediated by n­3 PUFAs­induced lipid peroxidation. These results highlight a potential role of n­3 PUFAs supplementation for the chemoprevention of melanoma in high­risk individuals, and as a putative adjuvant agent in the treatment of malignant melanoma.

Encapsulation of teniposide into albumin nanoparticles with greatly lowered toxicity and enhanced antitumor activity.

Teniposide (VM-26) is a semisynthetic derivative of podophyllotoxin effective for the treatment of many types of tumors. However, the poor water solubility and adverse effects restrict its clinical use. Our study aimed to develop a novel phospholipid complex albumin nanoparticle (VM-E80-AN) to reduce the systemic toxicity and enhance antitumor activity of VM-26. Egg yolk lecithin E80 and human serum albumin (HSA) were used as the main excipients to replace Cremophor EL in the commercial formulation. The physicochemical properties of VM-E80-AN were characterized to optimize the formulation. Cell and animal studies were further carried out to estimate its tumor inhibition efficacy, biodistribution, and toxicity. Comparison between VM-26 solution and VM-E80-AN showed that VM-E80-AN significantly reduced the toxicity of VM-26 and enhanced the anticancer efficacy of the drug. Thus, VM-E80-AN represents a safe and promising formulation of teniposide for clinical application.

Comprehensive determination of polycyclic aromatic hydrocarbons in Chinese herbal medicines by solid phase extraction and gas chromatography coupled to tandem mass spectrometry.

A simple and highly sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method combined with solid-phase extraction cleanup was established for the comprehensive determination of 16 Environmental Protection Agency (EPA) polycyclic aromatic hydrocarbons (PAHs) in various kinds of Chinese herbal medicines (CHMs). A solid-phase extraction (SPE) purification strategy, including three parallel procedures, was developed depending on sample type, and satisfactory purification performances were achieved for all selected CHMs. The limits of detection ranged from 0.12 to 1.08 µg kg(-1) for the analyzed PAHs. The average recoveries were in the range of 65.9 % to 100.8 %, except for naphthalene (43.8 %-75.9 %), and the relative standard deviations were ≤12.8 %. The proposed method was successfully applied to the analysis of PAHs in 24 CHMs including five roots, three stems, four flowers, two fruits, four seeds, three leaves, and three barks. In the samples analyzed, all 16 PAHs are present. Their sum ranges from 21.1 to 2236.3 µg kg(-1). The entire procedure was shown to be effective and conveniently fast, and may serve as an alternative screening protocol for the determination of PAHs in CHMs.

Effects of chitosan, aqueous extract of ginger, onion and garlic on quality and shelf life of stewed-pork during refrigerated storage.

Effects of 1% or 0.5% chitosan (CHI), 10% or 5% aqueous extract of ginger, onion and garlic (GOG) and their composite solutions (1% CHI+10% GOG, 0.5% CHI+5% GOG) on quality and shelf life of stewed-pork were evaluated. Microbiological (total bacterial count), chemical (pH, total volatile basic nitrogen (TVB-N), peroxide value (POV), 2-thiobarbituric acid (TBA)) and sensory characteristics were analysed periodically during refrigerated storage at 4 °C for 12 days. CHI and/or GOG treatments retarded the increases in pH, TVB-N, POV, TBA and total bacterial count. CHI showed better antibacteria but weaker antioxidation than GOG. Composite treatment had possible synergistic effect while the high concentration of composite solution (Mix1) had adverse effect on odour and overall acceptance. Mix2, the diluted solution of Mix1, could be a natural promising preservative for the stewed-pork considering the comprehensive effects of antioxidation, antibacteria and sensory quality, which could extend the shelf life for about 5-6 days.