A novel preparation of methyl-β-cyclodextrin from dimethyl carbonate and β-cyclodextrin

A novel green synthesis process about methyl-β-cyclodextrin has been investigated through the reaction between β-cyclodextrin and dimethyl carbonate by anhydrous potassium carbonate as catalyst in DMF. The influence of experimental factors including the molar ratio of dimethyl carbonate to β-cyclodextrin, reaction temperature, and reaction time on the average degree of substitution of methyl-β-cyclodextrin was studied. The results show that the average degree of substitution of methyl-β-cyclodextrin can be dependent on the reaction temperature and the molar ratio of raw material primarily. The structures of methyl-β-cyclodextrin were characterized by TLC, IR, MS, ¹H NMR, and ¹³C NMR.     

Uptake of a fluorescent methyl-β-cyclodextrin via clathrin-dependent endocytosis

Cyclodextrins (CDs) are widely used both in pharmaceutical applications to improve drug bioavailability and in cell biology as cholesterol-depleting and -delivering agents. Recently, it was shown that β-CD covalently coupled to fluorescent dextran polymers accumulates in cholesterol-enriched lysosomal storage organelles of human fibroblasts (Rosenbaum et al., 2010). By employing a methyl-βCD tagged with fluorescein (FMβCD), we have characterized the cellular trafficking of the CD in mammalian cell lines and its distribution into the endocytic compartments within the first minutes following addition to cells. FMβCD enters mammalian cells via endocytosis. The colocalization of FMβCD with transferrin-containing endosomes and the inhibition of FMβCD internalization by chlorpromazine or by an antisense RNA against clathrin heavy chain indicate that FMβCD is taken up via receptor-mediated, clathrin-dependent endocytosis. These results not only highlight the possibility of using CDs to target drugs intracellularly, but also warn about potential unwanted effects on cell physiology other than cholesterol extraction/loading at high concentrations, high temperatures and prolonged incubation times.     

Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P > 0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P < 0.05) after exposure to 2 mg/mL of MβCD compared to the group exposed to 0 mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P < 0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P < 0.05) by MβCD. The blastocysts rate (P < 0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P > 0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.     

Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P > 0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P < 0.05) after exposure to 2 mg/mL of MβCD compared to the group exposed to 0 mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P < 0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P < 0.05) by MβCD. The blastocysts rate (P < 0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P > 0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.     

Activation of serine protease subtilisin Carlsberg in organic solvents: combined effect of methyl-β-cyclodextrin and water

Methyl -cyclodextrin (MCD) increased the activity and enantioselectivity of lyophilized subtilisin suspended in dry THF and acetonitrile in two transesterification model reactions. These beneficial improvements were diminished by the addition of water, in contrast to the observation that water activates subtilisin lyophilized from buffer alone. For example, the initial rate for the S enantiomer in the transesterification of vinylbutyrate with (±)-1-phenylethanol (sec-phenethylalcohol) decreased ca. 4-fold and the enantioselectivity from 59 to 40 when 0.1% (v/v) of water was added to THF.     

α-Chymotrypsin stabilization by chemical conjugation with O-carboxymethyl-poly-β-cyclodextrin

Bovine pancreatic α-chymotrypsin was covalently modified with the O-carboxymethyl poly-β-cyclodextrin (M=1.3×10⁴, 40% COOH groups) using a water-soluble carbodiimide as a coupling agent. The conjugate prepared by this procedure retained high proteolytic and esterolytic activity and contained about 74% carbohydrate by weight of transformed protein. The optimum temperature for α-chymotrypsin was increased by 5 °C after this transformation. The thermostability of the polymer–enzyme adduct was also increased by 5 °C. The conjugate prepared was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 55 °C. Additionally, the modified enzyme was 11-fold more stable at pH 9.0. The direct influence of supramolecular interactions between the hydrophobic amino acid residues located at the surface of the protease and the attached polycyclodextrin moieties on α-chymotrypsin stabilization was demonstrated.     

Complex effects of 17β-estradiol on mitochondrial function

Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17β-estradiol (βE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (≥30μM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that βE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that βE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

High-performance liquid chromatographic assay for methyl-β-cyclodextrin in plasma and cell lysate

This paper describes a high-performance liquid chromatographic method with fluorescence detection for the analysis of methyl-β-cyclodextrin (MEBCD) in plasma and cell lysate, after in situ complexation with 1-naphthol. The size-exclusion HPLC column packed with TSK 3000 SW gel, was equilibrated with an eluent mixture composed of methanol and purified water (2:98, v/v) containing 10⁻⁴ M 1-naphthol as a fluorophore. The detection is based on fluorescence enhancement caused by the formation of inclusion complexes and was performed at 290 and 360 nm for excitation and emission, respectively. The method involved a simple treatment of the samples with chloroform. Daunorubicin was used as internal standard. Limits of quantitation were 0.8 μM in plasma and 0.5 μM in cell lysate. Detection limits of 0.5 μM (50 pmol) and 0.3 μM (30 pmol) were obtained for MEBCD in the two media, respectively. Linear detection response was obtained for concentrations ranging from 1 to 100 μM in plasma and cell lysate. Recovery from plasma proved to be more than 40%. Precision, expressed as C.V. was in the range of 4 to 11%. Accuracy ranged from 89 to 105%.     

Influence of nonionic surfactants and hydroxypropyl-β-cyclodextrin on the biodegradation of nitrobenzene

This paper investigates the effect of two nonionic surfactants (Tween 80 and Triton X-100) and hydroxypropyl--cyclodextrin (HP--CD) on the biodegradation of nitrobenzene (NB) by Acinetobacter sp. in liquid cultures at different dosages as well as the fate of both surfactants. When the initial concentration of NB was about 400 mg/l, neither Tween 80 nor HP--CD had any effect on the degradation of NB. However, Triton X-100 retarded the full removal of NB and the bacterial growth entering the stationary phase. While the initial concentration of NB was increased to about 850 mg/l, they all significantly enhanced the extent and rate of biodegradation if they were added at concentrations above 2000 mg/l. HP--CD could not be utilized by Acinetobacter sp. as the sole carbon source whereas both surfactants could, but no surfactant depletion was observed during the biodegradation of NB. So the rapid bacterial growth observed in the presence of each additive should be attributed to the rapid metabolism of NB. Both surfactants would promote the degradation of NB more than HP--CD would do if their dosages were increased properly.     

Effects of hydroxypropyl-β-cyclodextrin on the growth and morphology of Absidia coerulea

Cyclodextrin has been found to be an attractive novel solubilizer due to its unique material properties. Absidia coerulea is widely used in steroid bioconversion. The effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, morphology, and steroid-converting activity of A. coerulea CICC 40302 were systematically studied. HP-β-CD affected A. coerulea growth, resulting in changes in its spore morphology and mycelial morphology. It induced an increase in the spore germination rate and a decrease in cell biomass at the stationary phase. Optical microscopy revealed that HP-β-CD altered the mycelial morphology and reduced the pellet compactness of A. coerulea. A convenient and feasible computing method was used to measure pellet compactness, and it demonstrated that the compactness degree of the pellet decreased as HP-β-CD increased, which could be attributed to the modification of the physical properties of the fermentation medium. Moreover, the changing of mycelial morphology influenced steroid-converting activity. The results showed that HP-β-CD had multiple concentration-dependent effects on A. coerulea cells. HP-β-CD in the proper concentration range holds great potential as a biocompatible solubilizer.     

Cholesterol depletion by methyl-β-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl--cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric -actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and -catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.This work was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Programas de Apoio aos Núcleos de Excelência (Pronex)     

Binding of Sulfamethazine to β-cyclodextrin and Methyl-β-cyclodextrin

β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) complexes with sulfamethazine (SMT) were prepared and characterized by different experimental techniques, and the effects of βCD and MβCD on drug solubility were assessed via phase-solubility analysis. The phase-solubility diagram for the drug showed an increase in water solubility, with the following affinity constants calculated: 40.4 ± 0.4 (pH 2.0) and 29.4 ± 0.4 (pH 8.0) M⁻¹ with βCD and 56 ± 1 (water), 39 ± 3 (pH 2.0) and 39 ± 5 (pH 8.0) M⁻¹ with MβCD. According to ¹H NMR and 2D NMR spectroscopy, the complexation mode involved the aromatic ring of SMT included in the MβCD cavity. The complexes obtained in solid state by freeze drying were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and thermal analysis. The amorphous complexes obtained in this study may be useful in the preparation of pharmaceutical dosage forms of SMT.     

Influence of hydroxypropyl-β-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-β-cyclodextrin (HP-β-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-β-CD, conversion of 0.5?g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9?% compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-β-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ(1)-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-β-CD.     

Stabilization of trypsin by chemical modification with β-cyclodextrin monoaldehyde

The monoaldehyde derivative of -cyclodextrin was attached to trypsin via reductive alkylation with NaBH₄. The thermostability was enhanced from 49.5 °C to 60 °C for modified trypsin. The activation free energy of thermal inactivation at 50 °C was increased by 3.2 kJ mol^–1. The conjugated enzyme retained 100% of its initial activity after 3 h incubation at pH 9.     

DFT study on the effects of β-cyclodextrin in synthesis of 2-phenylbenzimidazole via benzaldehyde and o-phenylenediamine

The conversion of 2-phenylbenzimidazole using o-phenylenediamine and benzaldehyde can be improved significantly under β-cyclodextrin (β-CD). The density functional theory (DFT) method was applied to study the whole process. According to energy parameters (binding energy, deformation energy) and structural deformation, entry models and the reaction process can be pinpointed, with o-phenylenediamine embedding β-CD from a wide rim, and then benzaldehyde passing into the inclusion from the narrow rim. Subsequently, natural bonding orbital (NBO), Mulliken charge, frontier orbital, FuKui function and nuclear magnetic resonance (NMR) methods were employed to reveal the mechanism of electron transfer. The results illustrate that β-CD plays a catalytic role in synthesis reaction mechanism on the secondary side, improving the reactivity and selectivity of the process.

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Graphical Abstract Density functional theory study of the effects of β-cyclodextrin in synthesis of 2-phenylbenzimidazole via benzaldehyde and o-phenylenediamine

     

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-β-cyclodextrin. Role of low-density caveolae-like membranes

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5′nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-β-cyclodextrin (MβCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.     

The beneficial effects of α-cyclodextrin on blood lipids and weight loss in healthy humans

α‐Cyclodextrin (α‐CD) is a soluble fiber derived from corn. It has previously been reported that early intervention with Mirafit fbcx, a trademarked name for α‐CD, has beneficial effects on weight management in obese individuals with type 2 diabetes, and that it preferentially reduces blood levels of saturated and trans fats in the LDL receptor knockout mice. The current investigation involves overweight but not obese nondiabetic individuals and was intended to confirm the effects of α‐CD on both weight management and improving blood lipid levels. Forty‐one healthy adults (age: 41.4 ± 13.6 years) participated in this 2‐month, double‐blinded, crossover study. In 28 compliant participants (8 males and 20 females), when the active phase was compared to the control phase, there were significant decreases in body weight (?0.4 ± 0.2 kg, P < 0.05), serum total cholesterol (mean ± s.e.m., ?0.295 ± 0.10 mmol/l, 5.3%, P < 0.02) and low‐density lipoprotein (LDL) cholesterol (?0.23 ± 0.11 mmol/l, ?6.7%, P < 0.05). Apolipoprotein B (Apo B) (?0.0404 ± 0.02 g/l, ?5.6%, P = 0.06) and insulin levels also decreased by 9.5% (?0.16 ± 0.08 pmol/l, P = 0.06) while blood glucose and leptin levels did not change. These results suggest that α‐CD exerts its beneficial health effects on body weight and blood lipid profile in healthy nonobese individuals, as previously reported in obese individuals with type 2 diabetes.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.     

Apolipoprotein E isoform-dependent effects on the processing of Alzheimer's amyloid-β

Since the identification of the apolipoprotein E (apoE) *ε4 allele as a major genetic risk factor for late-onset Alzheimer's disease, significant efforts have been aimed at elucidating how apoE4 expression confers greater brain amyloid-β (Aβ) burden, earlier disease onset and worse clinical outcomes compared to apoE2 and apoE3. ApoE primarily functions as a lipid carrier to regulate cholesterol metabolism in circulation as well as in the brain. However, it has also been suggested to interact with hydrophobic Aβ peptides to influence their processing in an isoform-dependent manner. Here, we review evidence from in vitro and in vivo studies extricating the effects of the three apoE isoforms, on different stages of the Aβ processing pathway including synthesis, aggregation, deposition, clearance and degradation. ApoE4 consistently correlates with impaired Aβ clearance, however data regarding Aβ synthesis and aggregation are conflicting and likely reflect inconsistencies in experimental approaches across studies. We further discuss the physical and chemical properties of apoE that may explain the inherent differences in activity between the isoforms. The lipidation status and lipid transport function of apoE are intrinsically linked with its ability to interact with Aβ. Traditionally, apoE-oriented therapeutic strategies for Alzheimer's disease have been proposed to non-specifically enhance or inhibit apoE activity. However, given the wide-ranging physiological functions of apoE in the brain and periphery, a more viable approach may be to specifically target and neutralise the pathological apoE4 isoform.