Comparison of Alternative Protein Hydrogels for Delivering Myricetin: Interaction Mechanism and Stability Evaluation.

Food protein carriers from different sources might have distinct stabilizing and enhancing effects on the same small molecule. To elucidate the molecular mechanism, five different sourced proteins including soy protein isolates (SPIs), whey protein isolates (WPIs), edible dock protein (EDP), Tenebrio molitor protein (TMP), and yeast protein (YP) were used to prepare protein hydrogels for delivering myricetin (Myr). The results suggested that the loading capacity order of Myr in different protein hydrogels was EDP (11.5%) > WPI (9.3%) > TMP (8.9%) > YP (8.0%) > SPI (7.6%), which was consistent with the sequence of binding affinity between Myr and different proteins. Among five protein hydrogels, EDP had an optimum loading ability since it possessed the highest hydrophobic amino acid content (45.52%) and thus provided a broad hydrophobic cavity for loading Myr. In addition, these protein-Myr composite hydrogels displayed the core-shell structure, wherein hydrogen bonding and hydrophobic interaction were the primary binding forces between proteins and Myr. Moreover, the thermal stability, storage stability, and sustained-release properties of Myr were significantly enhanced via these protein delivery systems. These findings can provide scientific guidance for deeper utilization of food alternative protein sources.

Regulation Mechanism of Phenolic Hydroxyl Number on Self-Assembly and Interaction between Edible Dock Protein and Hydrophobic Flavonoids.

In this study, galangin (Gal), kaempferol (Kae), quercetin (Que), and myricetin (Myr) were chosen as the representative flavonoids with different phenolic hydroxyl numbers in the B-ring. The edible dock protein (EDP) was chosen as the new plant protein. Based on this, the regulation mechanism of the phenolic hydroxyl number on the self-assembly behavior and molecular interaction between EDP and flavonoid components were investigated. Results indicated that the loading capacity order of flavonoids within the EDP nanomicelles was Myr (10.92%) > Que (9.56%) > Kae (6.63%) > Gal (5.55%). Moreover, this order was consistent with the order of the hydroxyl number in the flavonoid's B ring: Myr (3) > Que (2) > Kae (1) > Gal (0). The micro morphology exhibited that four flavonoid-EDP nanomicelles had a core-shell structure. In the meantime, the EDP encapsulation remarkably improved the flavonoids' water solubility, storage stability, and sustained release characteristics. During the interaction of EDP and flavonoids, the noncovalent interactions including van der Waals forces, hydrophobic interaction, and hydrogen bonding were the main binding forces. All of the results demonstrated that the hydroxyl number of bioactive compounds is a critical factor for developing a delivery system with high loading ability and stability.

Self-assembly and interaction mechanisms of edible dock protein and flavonoids regulated by the phenolic hydroxyl position.

In this study, chrysin (Chr), baicalein (Bai), apigenin (Api) and galangin (Gal) were selected as the representative flavonoids with different position of phenolic hydroxyl groups, and edible dock protein (EDP) was used as a material to construct delivery system. Subsequently, the molecular interactions and functional properties of flavonoids-loaded EDP nanomicelles were investigated. Results exhibited that hydrogen bond, hydrophobic interaction and van der Waals force were the main driving forces for self-assembly of flavonoids and EDP molecules. Meanwhile, this self-assembly remarkably enhance the storage and digestion stability of flavonoid compounds. Among four flavonoids, the order of loading ability was: Api > Gal > Bai > Chr. Herein, Api had a largest loading capacity (6.74%) because of its active phenolic hydroxyl group in ring B. These results suggested that the position of phenolic hydroxyl groups in flavonoids is a key factor to regulate its self-assembly with protein molecules.

Quantification of 7-aminoflunitrazepam in human urine by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry.

Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1-50ngmL-1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL-1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL-1 (intra-day/inter-day precision: 0.6-1.8% / 0.1-0.8%; post-spiked/pre-spiked recovery: 95.4-102.9% / 96.3-102.5%). In addition, acceptable relative differences (-24.2 - 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.

Polymer monolith microextraction using poly(butyl methacrylate-co-1,6-hexanediol ethoxylate diacrylate) monolithic sorbent for determination of phenylurea herbicides in water samples.

In this study, recently developed 1,6-hexanediol ethoxylate diacrylate (HEDA)-based polymeric monoliths were utilized as sorbents for efficient extraction of phenylurea herbicides (PUHs) from water samples. The HEDA-based monolithic sorbents were prepared in a fused silica capillary (0.7mm i.d., 4.5-cm long) for polymer monolith microextraction (PMME). The experimental parameters of PMME microextraction including sample loading speed, pH of sample solution, composition of elution solvent, and addition of salt were optimized to efficiently extract PUHs from environmental water samples. The extracted PUHs were determined using ultra-high performance liquid chromatography (UHPLC) with UV-photodiode array detection. The extraction recoveries for PUHs-spiked water samples were 91.1-108.1% with relative standard deviations lower than 5%. The linearity range was 0.025-25ngmL(-1) for each PUH and the detection limits of PUHs were estimated at 0.006-0.019ng mL(-1). In addition, good intra-day/inter-day precision (0.1-8.7%/0.2-8.9%) and accuracy (92.0-108.0%/96.5-105.2%) of the proposed method were obtained. The extraction capacity of the monolith-filled capillary was also determined to be approximately 1µg. Moreover, each monolith-filled capillary could be reused up to 8 times without carry-over. According to the European Union regulations, the allowed permissible limit of any single herbicide in drinking water is 0.1ng mL(-1). This permissible level fell in the linear range examined in this study. In addition, the proposed method provided detection limits lower than the allowed permissible level, which demonstrated the feasibility of utilizing the HEDA-based monolithic sorbent to perform PMME for determining contaminants, such as PUHs, in environmental application.

Preparation and evaluation of poly(alkyl methacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithic columns for separating polar small molecules by capillary liquid chromatography.

In this study, methacrylic acid (MAA) was incorporated with alkyl methacrylates to increase the hydrophilicity of the synthesized ethylene dimethacrylate-based (EDMA-based) monoliths for separating polar small molecules by capillary LC analysis. Different alkyl methacrylate-MAA ratios were investigated to prepare a series of 30% alkyl methacrylate-MAA-EDMA monoliths in fused-silica capillaries (250-µm i.d.). The porosity, permeability, and column efficiency of the synthesized MAA-incorporated monolithic columns were characterized. A mixture of phenol derivatives is employed to evaluate the applicability of using the prepared monolithic columns for separating small molecules. Fast separation of six phenol derivatives was achieved in 5 min with gradient elution using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. In addition, the effect of acetonitrile content in mobile phase on retention factor and plate height as well as the plate height-flow velocity curves were also investigated to further examine the performance of the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. Moreover, the applicability of prepared polymer-based monolithic column for potential food safety applications was also demonstrated by analyzing five aflatoxins and three phenicol antibiotics using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column.

Palmitic acid-induced lipotoxicity and protection by (+)-catechin in rat cortical astrocytes.

BACKGROUND: Astrocytes do not only maintain homeostasis of the extracellular milieu of the neurons, but also play an active role in modulating synaptic transmission. Palmitic acid (PA) is a saturated fatty acid which, when being excessive, is a significant risk factor for lipotoxicity. Activation of astrocytes by PA has been shown to cause neuronal inflammation and demyelination. However, direct damage by PA to astrocytes is relatively unexplored. The aim of this study was to identify the mechanism(s) of PA-induced cytotoxicity in rat cortical astrocytes and possible protection by (+)-catechin. METHODS: Cytotoxicity and endoplasmic reticulum (ER) markers were assessed by MTT assay and Western blotting, respectively. Cytosolic Ca(2+) and mitochondrial membrane potential (MMP) were measured microfluorimetrically using fura-2 and rhodamine 123, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: Exposure of astrocytes to 100µM PA for 24h resulted in apoptotic cell death. Whilst PA-induced cell death appeared to be unrelated to ER stress and perturbation in cytosolic Ca(2+) signaling, it was likely a result of ROS production and subsequent MMP collapse, since ascorbic acid (anti-oxidant, 100µM) prevented PA-induced MMP collapse and cell death. Co-treatment of astrocytes with (+)-catechin (300µM), an anti-oxidant found abundantly in green tea, significantly prevented PA-induced ROS production, MMP collapse and cell death. CONCLUSION: Our results suggest that PA-induced cytotoxicity in astrocytes may involve ROS generation and MMP collapse, which can be prevented by (+)-catechin.

Preparation and evaluation of 1,6-hexanediol ethoxylate diacrylate-based alkyl methacrylate monolithic capillary column for separating small molecules.

Due to the high porosity, good thermal stability, and good physical stability at high pressure, polymer monoliths have been successfully utilized as the stationary phases for capillary liquid chromatography (LC) analysis. In this study, we introduced 1,6-hexanediol ethoxylate diacrylate (HEDA) as a cross-linker to prepare alkyl methacrylate monoliths for efficient separation of polar small molecules. HEDA provided additional dipole-dipole interactions between the monolithic stationary phases and polar analytes. For comparison, ethylene dimethacrylate and 1,6-hexanediol dimethacrylate were also utilized as cross-linkers to prepare alkyl methacrylate monoliths. A series of alkyl methacrylate polymeric monoliths were synthesized in fused-silica capillaries using the three different cross-linkers. The porosity, permeability and column efficiency of the synthesized alkyl methacrylate monoliths were characterized. A mixture of phenol derivatives was employed to evaluate the applicability of the prepared monolithic columns for separating small molecules using capillary LC. The HEDA-based alkyl methacrylate monoliths offered the most efficient chromatographic separation for phenol derivatives. Moreover, the capability of applying the novel HEDA-based alkyl methacrylate monolithic columns for potential environmental analysis was demonstrated by separating eight phenylurea herbicides.