Prebiotic Potential of Oligosaccharides Obtained by Acid Hydrolysis of α-(1→3)-Glucan from Laetiporus sulphureus: A Pilot Study

Increasing knowledge of the role of the intestinal microbiome in human health and well-being has resulted in increased interest in prebiotics, mainly oligosaccharides of various origins. To date, there are no reports in the literature on the prebiotic properties of oligosaccharides produced by the hydrolysis of pure fungal α-(1→3)-glucan. The aim of this study was to prepare α-(1→3)-glucooligosaccharides (α-(1→3)-GOS) and to perform initial evaluation of their prebiotic potential. The oligosaccharides were obtained by acid hydrolysis of α-(1→3)-glucan isolated from the fruiting bodies of Laetiporus sulphureus and then, characterized by HPLC. Fermentation of α-(1→3)-GOS and reference prebiotics was compared in in vitro pure cultures of Lactobacillus, Bifidobacterium, and enteric bacterial strains. A mixture of α-(1→3)-GOS, notably with a degree of polymerization of 2 to 9, was obtained. The hydrolysate was utilized for growth by most of the Lactobacillus strains tested and showed a strong bifidogenic effect, but did not promote the growth of Escherichia coli and Enterococcus faecalis. α-(1→3)-GOS proved to be effective in the selective stimulation of beneficial bacteria and can be further tested to determine their prebiotic functionality.     

A Novel Solid Nanocrystals Self-Stabilized Pickering Emulsion Prepared by Spray-Drying with Hydroxypropyl-β-cyclodextrin as Carriers

A drug nanocrystals self-stabilized Pickering emulsion (NSSPE) with a unique composition and microstructure has been proven to significantly increase the bioavailability of poorly soluble drugs. This study aimed to develop a new solid NSSPE of puerarin preserving the original microstructure of NSSPE by spray-drying. A series of water-soluble solid carriers were compared and then Box-Behnken design was used to optimize the parameters of spray-drying. The drug release and stability of the optimized solid NSSPE in vitro were also investigated. The results showed that hydroxypropyl-β-cyclodextrin (HP-β-CD), rather than solid carriers commonly used in solidification of traditional Pickering emulsions, was suitable for the solid NSSPE to retain the original appearance and size of emulsion droplets after reconstitution. The amount of HP-β-CD had more influences on the solid NSSPE than the feed rate and the inlet air temperature. Fluorescence microscopy, confocal laser scanning microscopy and scanning electron microscopy showed that the reconstituted emulsion of the solid NSSPE prepared with HP-β-CD had the same core-shell structure with a core of oil and a shell of puerarin nanocrystals as the liquid NSSPE. The particle size of puerarin nanocrystal sand interfacial adsorption rate also did not change significantly. The cumulative amount of released puerarin from the solid NSSPE had no significant difference compared with the liquid NSSPE, which were both significantly higher than that of puerarin crude material. The solid NSSPE was stable for 3 months under the accelerated condition of 75% relative humidity and 40 °C. Thus, it is possible todevelop the solid NSSPE preserving the unique microstructure and the superior properties in vitro of the liquid NSSPE for poorly soluble drugs.     

Synthesis of Mono-Amino Substituted γ-CD: Host–Guest Complexation and In Vitro Cytotoxicity Investigation

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH₂), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH₂CH₂OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH₂CH₂NH₂). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM⁻¹, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.     

Anti-α-Glucosidase and Antiglycation Activities of α-Mangostin and New Xanthenone Derivatives: Enzymatic Kinetics and Mechanistic Insights through In Vitro Studies

Diabetes mellitus is characterized by chronic hyperglycemia that promotes ROS formation, causing severe oxidative stress. Furthermore, prolonged hyperglycemia leads to glycation reactions with formation of AGEs that contribute to a chronic inflammatory state. This research aims to evaluate the inhibitory activity of α-mangostin and four synthetic xanthenone derivatives against glycation and oxidative processes and on α-glucosidase, an intestinal hydrolase that catalyzes the cleavage of oligosaccharides into glucose molecules, promoting the postprandial glycemic peak. Antiglycation activity was evaluated using the BSA assay, while antioxidant capacity was detected with the ORAC assay. The inhibition of α-glucosidase activity was studied with multispectroscopic methods along with inhibitory kinetic analysis. α-Mangostin and synthetic compounds at 25 µM reduced the production of AGEs, whereas the α-glucosidase activity was inhibited only by the natural compound. α-Mangostin decreased enzymatic activity in a concentration-dependent manner in the micromolar range by a reversible mixed-type antagonism. Circular dichroism revealed a rearrangement of the secondary structure of α-glucosidase with an increase in the contents of α-helix and random coils and a decrease in β-sheet and β-turn components. The data highlighted the anti-α-glucosidase activity of α-mangostin together with its protective effects on protein glycation and oxidation damage.     

Review: β-glucans as Effective Antibiotic Alternatives in Poultry

The occurrence of microbial challenges in commercial poultry farming causes significant economic losses. Antibiotics have been used to control diseases involving bacterial infection in poultry. As the incidence of antibiotic resistance turns out to be a serious problem, there is increased pressure on producers to reduce antibiotic use. With the reduced availability of antibiotics, poultry producers are looking for feed additives to stimulate the immune system of the chicken to resist microbial infection. Some β-glucans have been shown to improve gut health, to increase the flow of new immunocytes, increase macrophage function, stimulate phagocytosis, affect intestinal morphology, enhance goblet cell number and mucin-2 production, induce the increased expression of intestinal tight-junctions, and function as effective anti-inflammatory immunomodulators in poultry. As a result, β-glucans may provide a new tool for producers trying to reduce or eliminate the use of antibiotics in fowl diets. The specific activity of each β-glucan subtype still needs to be investigated. Upon knowledge, optimal β-glucan mixtures may be implemented in order to obtain optimal growth performance, exert anti-inflammatory and immunomodulatory activity, and optimized intestinal morphology and histology responses in poultry. This review provides an extensive overview of the current use of β glucans as additives and putative use as antibiotic alternative in poultry.     

Sensitization to Drug Treatment in Precursor B-Cell Acute Lymphoblastic Leukemia Is Not Achieved by Stromal NF-κB Inhibition of Cell Adhesion but by Stromal PKC-Dependent Inhibition of ABC Transporters Activity

Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.     

The Effect of α-, β- and γ-Cyclodextrin on Wheat Dough and Bread Properties

Cyclodextrins (CDs) are cyclic oligosaccharides that have found widespread application in numerous fields. CDs have revealed a number of various health benefits, making them potentially useful food supplements and nutraceuticals. In this study, the impact of α-, β-, and γ-CD at different concentrations (up to 8% of the flour weight) on the wheat dough and bread properties were investigated. The impact on dough properties was assessed by alveograph analysis, and it was found that especially β-CD affected the viscoelastic properties. This behavior correlates well with a direct interaction of the CDs with the proteins of the gluten network. The impact on bread volume and bread staling was also assessed. The bread volume was in general not significantly affected by the addition of up to 4% CD, except for 4% α-CD, which slightly increased the bread volume. Larger concentrations of CDs lead to decreasing bread volumes. Bread staling was investigated by texture analysis and low field nuclear magnetic resonance spectroscopy (LF-NMR) measurements, and no effect of the addition of CDs on the staling was observed. Up to 4% CD can, therefore, be added to wheat bread with only minor effects on the dough and bread properties.     

Kinetic Study of 17α-Estradiol Mechanism during Rat Sperm Capacitation

17α-Estradiol (αE2) is a natural diastereoisomer of 17β-estradiol (E2). It is well known that αE2 can bind to estrogen receptors. However, its biological activity is less than that of E2 and is species and tissue specific. The goal of our study was to propose the mechanism of αE2 hormonal response in rat sperm during their capacitation in vitro and compare it with a previously studied mouse model. Concentration changes in externally added αE2 during capacitation of rat sperm were monitored by the high-performance liquid chromatographic method with tandem mass spectrometric detection (HPLC-MS/MS). The calculated values of relative concentrations B_t were subjected to kinetic analysis. The findings indicated that αE2 in rat sperm did not trigger autocatalytic reaction, in contrast to the mouse sperm, and that the initiation of the hormone penetration through the sperm plasma membrane was substantially faster in rats.     

Isolation,Structural Elucidation,Antioxidant and Hypoglycemic Activity of Polysaccharides of Brassica rapa L.

The aim of this study was to investigate the effects of microwave ultrasonic-assisted extraction (MUAE) on the content, structure, and biological functions of Brassica rapa L. polysaccharide (BRP). Response surface methodology (RSM) was used to optimize the parameters of MUAE, and it obtained a polysaccharide with yield of 21.802%. Then, a neutral polysaccharide named BRP-1-1 with a molecular weight of 31.378 kDa was isolated and purified from BRP using DEAE-650 M and Sephadex G-100. The structures of the BRP-1-1 were elucidated through a combination of FT-IR, GC-MS, NMR, and methylation analysis. The results showed that BRP-1 consisted of mannose (Man) and glucose (Glu) in a molar ratio of 7.62:1. The backbone of BRP-1-1 mainly consisted of →6)-α-D-Glup-(1→4-β-D-Glup-(1→2)-α-D-Manp-(1→2)-α-D-Glup-(1→, the branch was [T-α-D-Manp-(1]_n→. BRP-1-1 intervention significantly inhibited α-glucosidase activity; an inhibition rate of 44.623% was achieved at a concentration of 0.5 mg/mL. The results of the in vitro biological activity showed that BRP-1-1 has good antioxidant and hypoglycemic activity, suggesting that BRP-1-1 could be developed as a functional medicine.     

The Biological Fate of Pharmaceutical Excipient β-Cyclodextrin: Pharmacokinetics,Tissue Distribution,Excretion, and Metabolism of β-Cyclodextrin in Rats

β-cyclodextrin has a unique annular hollow ultrastructure that allows encapsulation of various poorly water-soluble drugs in the resulting cavity, thereby increasing drug stability. As a bioactive molecule, the metabolism of β-cyclodextrin is mainly completed by the flora in the colon, which can interact with API. In this study, understanding the in vivo fate of β-cyclodextrin, a LC-MS/MS method was developed to facilitate simultaneous quantitative analysis of pharmaceutical excipient β-cyclodextrin and API dextromethorphan hydrobromide. The established method had been effectively used to study the pharmacokinetics, tissue distribution, excretion, and metabolism of β-cyclodextrin after oral administration in rats. Results showed that β-cyclodextrin was almost wholly removed from rat plasma within 36 h, and high concentrations of β-cyclodextrin distributed hastily to organs with increased blood flow velocities such as the spleen, liver, and kidney after administration. The excretion of intact β-cyclodextrin to urine and feces was lower than the administration dose. It can be speculated that β-cyclodextrin metabolized to maltodextrin, which was further metabolized, absorbed, and eventually discharged in the form of CO₂ and H₂O. Results proved that β-cyclodextrin, with relative low accumulation in the body, had good safety. The results will assist further study of the design and safety evaluation of adjuvant β-cyclodextrin and promote its clinical development.     

Euphorbiasteroid Abrogates EGFR and Wnt/β-Catenin Signaling in Non-Small-Cell Lung Cancer Cells to Impart Anticancer Activity

EGFR and Wnt/β-catenin signaling pathways play a prominent role in tumor progression in various human cancers including non-small-cell lung carcinoma (NSCLC). Transactivation and crosstalk between the EGFR and Wnt/β-catenin pathways may contribute to the aggressiveness of cancers. Targeting these oncogenic pathways with small molecules is an attractive approach to counteract various types of cancers. In this study, we demonstrate the effect of euphorbiasteroid (EPBS) on the EGFR and Wnt/β-catenin pathways in NSCLC cells. EPBS induced preferential cytotoxicity toward A549 (wildtype EGFR-expressing) cells over PC-9 (mutant EGFR-expressing) cells. EPBS suppressed the expression of EGFR, Wnt3a, β-catenin, and FZD-1, and the reduction in β-catenin levels was found to be mediated through the activation of GSK-3β. EPBS reduced the phosphorylation of GSK-3β^S9 with a parallel increase in β-TrCP and phosphorylation of GSK-3β^Y216. Lithium chloride treatment increased the phosphorylation of GSK-3β^S9 and nuclear localization of β-catenin, whereas EPBS reverted these effects. Forced expression or depletion of EGFR in NSCLC cells increased or decreased the levels of Wnt3a, β-catenin, and FZD-1, respectively. Overall, EPBS abrogates EGFR and Wnt/β-catenin pathways to impart its anticancer activity in NSCLC cells.     

α-Amyrin and β-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant,Anti-Xanthine Oxidase,and Anti-Tyrosinase Potentials

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and β-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and β-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and β-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC₅₀ of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC₅₀ = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and β-amyrin mixture (IC₅₀ = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and β-amyrin. Furthermore, this was the first study indicating that α-amyrin and β-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.     

Galangin/β-Cyclodextrin Inclusion Complex as a Drug-Delivery System for Improved Solubility and Biocompatibility in Breast Cancer Treatment

The purpose of this study was to evaluate the potential of a newly modified cyclodextrin derivative, water-soluble β-cyclodextrin–epichlorohydrin (β-CD), as an effective drug carrier to enhance the poor solubility and bioavailability of galangin (GAL), a poorly water-soluble model drug. In this regard, inclusion complexes of GAL/β-CDP were prepared. UV-VIS spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), X-ray crystallography (XRD), zeta potential analysis, particle size analysis, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) were applied to characterize the synthesized GAL/β-CD. Michigan Cancer Foundation-7 (MCF-7; human breast cancer cells) and rat embryo fibroblast (REF; normal cells) were employed to examine the in vitro cytotoxic effects of GAL/β-CD using various parameters. The dye-based tests of MTT and crystal violet clearly exhibited that GAL/β-CD-treated cells had a reduced proliferation rate, an influence that was not found in the normal cell line. The cells’ death was found to follow apoptotic mechanisms, as revealed by the dye-based test of acridine orange/ethidium bromide (AO/EtBr), with the involvement of the mitochondria via caspase-3-mediated events, as manifested by the Rh 123 test. We also included a mouse model to examine possible in vivo toxic effects of GAL/β-CD. It appears that the inclusion complex does not have a significant influence on normal cells, as indicated by serum levels of kidney and liver enzymatic markers, as well as thymic and splenic mass indices. A similar conclusion was reached on the histological level, as manifested by the absence of pathological alterations in the liver, kidney, thymus, spleen, heart, and lung.     

Metagenomics Approach to the Intestinal Microbiome Structure and Abundance in High-Fat-Diet-Induced Hyperlipidemic Rat Fed with (−)-Epigallocatechin-3-Gallate Nanoparticles

The effects of nanoparticles (NPs) on microbiota homeostasis and their physiological relevance are still unclear. Herein, we compared the modulation and consequent pharmacological effects of oral administration of (−)-epigallocatechin-3-gallate (EGCG)-loaded β-cyclodextrin (β-CD) NPs (EGCG@β-CD NPs) and EGCG on gut microbiota. EGCG@β-CD NPs were prepared using self-assembly and their influence on the intestinal microbiome structure was analyzed using a metagenomics approach. The “Encapsulation efficiency (EE), particle size, polydispersity index (PDI), zeta potential” of EGCG@β-CD NPs were recorded as 98.27 ± 0.36%, 124.6 nm, 0.313 and –24.3 mV, respectively. Surface morphology of EGCG@β-CD NPs was observed as spherical. Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and molecular docking studies confirmed that EGCG could be well encapsulated in β-CD and formed as EGCG@β-CD NPs. After being continuously administered EGCG@β-CD NPs for 8 weeks, the serum cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and liver malondialdehyde (MDA) levels in the rats were significantly decreased, while the levels of catalase (CAT) and apolipoprotein-A1 (apo-A1) in the liver increased significantly in the hyperlipidemia model of rats, when compared to the high-fat-diet group. Furthermore, metagenomic analysis revealed that the ratio of Verrucomicrobia/Bacteroidetes was altered and Bacteroidetes decreased in the high-fat diet +200 mg/kg·bw EGCG@β-CD NPs group, while the abundance of Verrucomicrobia was significantly increased, especially Akkermansia muciniphila in rat feces. EGCG@β-CD NPs could be a promising EGCG delivery strategy to modulate the gut microbiota, enhancing its employment in the prevention of hyperlipidemia.     

Production of Ethanol and Feed by High Dry Matter Hydrolysis and Fermentation of Palm Kernel Press Cake

Palm kernel press cake (PKC) is a residue from palm oil extraction presently only used as a low protein feed supplement. PKC contains 50% fermentable hexose sugars present in the form of glucan and mainly galactomannan. This makes PKC an interesting feedstock for processing into bioethanol or in other biorefinery processes. Using a combination of mannanase, β-mannosidase, and cellulases, it was possible without any pretreatment to hydrolyze PKC at solid concentrations of 35% dry matter with mannose yields up to 88% of theoretical. Fermentation was tested using Saccharomyces cerevisiae in both a separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) setup. The hydrolysates could readily be fermented without addition of nutrients and with average fermentation yields of 0.43?±?0.02 g/g based on consumed mannose and glucose. Employing SSF, final ethanol concentrations of 70 g/kg was achieved in 216 h, corresponding to an ethanol yield of 70% of theoretical or 200 g ethanol/kg PKC. Testing various enzyme mixtures revealed that including cellulases in combination with mannanases significantly improved ethanol yields. Processing PKC to ethanol resulted in a solid residue enriched in protein from 17% to 28%, a 70% increase, thereby potentially making a high-protein containing feed supplement.     

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1–4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2, reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2–4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1–4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1–4 expression by TDEE is related to the inhibition of PKCβ activation.     

Purification,Structural Characterization,and Anti-Inflammatory Effects of a Novel Polysaccharide Isolated from Orostachys fimbriata

The present study elucidated the structural characteristics and anti-inflammatory activity of a novel polysaccharide isolated from Orostachys fimbriata, which is a traditional Chinese medicinal plant. O. fimbriata polysaccharide (OFP) was extracted and subsequently purified by chromatography using a DEAE cellulose-52 and Sephadex G-75 column. The molecular weight was determined as 6.2 kDa. HPGPC and monosaccharide composition analysis revealed a homogeneous polysaccharide containing only Glc. Chromatography and spectral analysis showed that the possible chemical structure consisted of →4)-α-Glcp-(1→ and a small quantity of →4,6)-β-Glcp-(1→ in the main chain and →6)-β-Glcp-(1→, α-Glcp-(1→, and β-Glcp-(1→ in the side chain. Morphological analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM) indicated that OFP had a multi-branched structure, and the sugar chain molecules of polysaccharide appeared aggregated. OFP was found to exhibit anti-inflammatory activity by reducing the secretion of inflammatory factors in RAW264.7 cells and by decreasing the extent of xylene-induced ear swelling in mice.     

Structural Changes of β-Casein Induced by Temperature and pH Analysed by Nuclear Magnetic Resonance,Fourier-Transform Infrared Spectroscopy,and Chemometrics

This study investigated structural changes in β-casein as a function of temperature (4 and 20 °C) and pH (5.9 and 7.0). For this purpose, nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy were used, in conjunction with chemometric analysis. Both temperature and pH had strongly affected the secondary structure of β-casein, with most affected regions involving random coils and α-helical structures. The α-helical structures showed great pH sensitivity by decreasing at 20 °C and diminishing completely at 4 °C when pH was increased from 5.9 to 7.0. The decrease in α-helix was likely related to the greater presence of random coils at pH 7.0, which was not observed at pH 5.9 at either temperature. The changes in secondary structure components were linked to decreased hydrophobic interactions at lower temperature and increasing pH. The most prominent change of the α-helix took place when the pH was adjusted to 7.0 and the temperature set at 4 °C, which confirms the disruption of the hydrogen bonds and weakening of hydrophobic interactions in the system. The findings can assist in establishing the structural behaviour of the β-casein under conditions that apply as important for solubility and production of β-casein.     

Exploring In Vitro Biological Cellular Responses of Pegylated β-Cyclodextrins

βCDPEG5 and βCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to βCD. As βCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of βCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO₂⁻ overproduction in macrophages, we found that βCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to βCDPEGs and the parent compounds, a similar pattern was observed: the effect of βCDPEG5 on cell viability and cell cycle progression was larger than that of βCDPEG2; PEG2 affected cell viability and cell cycle more than βCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of βCDPEG5 with cell migration was smaller than that of βCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of βCD and PEGs, particularly to yield βCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of βCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from βCD and PEGs.