γ-Cyclodextrin encapsulated thymol for citrus preservation and its possible mechanism against Penicillium digitatum.

The volatility of essential oils greatly limits their industrial applications. Here, we successfully prepared γ-cyclodextrin (γ-CD) inclusion compounds (γ-CDTL) containing thymol (TL) for the control of green mold caused by Penicillium digitatum (P. digitatum) in citrus fruit. In vitro experiment showed that the minimum fungicidal concentration (MFC) of γ-CDTL against the hyphae growth of P. digitatum was 2.0 g/L, and 8 × MFC treatment significantly reduced the occurrence of green mold in citrus fruit and had no adverse effect on fruit quality in vivo test compared to prochloraz. Scanning electron microscopy (SEM), x-ray diffraction (XRD), fourier transform-infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), physical properties and sustained release properties were also performed, results indicated that the hydrogen bonds between TL and γ-CD were the basis for the formation of γ-CDTL. We further investigated the inhibition mechanism of γ-CDTL. SEM and TEM experiments showed that γ-CDTL treatment caused severe damage to the hyphal morphology and cells in 30 min and disrupted the permeability of P. digitatum mycelial cell walls by increasing the chitinase activity, thus accelerating the leakage of intracellular lysates. However, the integrity of the cell membrane was obviously damaged only after 60 min of treatment. In conclusion, we prepared a novel inclusion complex γ-CDTL with obvious antifungal effects and preliminarily elucidated its inclusion mechanism and antifungal mechanism. γ-CDTL might be a potent alternative to chemical fungicides for controlling the postharvest decay of citrus.

Solid γ-Cyclodextrin Inclusion Compound with Gingerols, a Multi-Component Guest: Preparation, Properties and Application in Yogurt.

Gingerols from the rhizome of fresh ginger (Zingiber officinale) were obtained by a simple extraction, followed by purification. The gingerols extract was composed of 6-gingerol (54%), 8-gingerol (20%), and 10-gingerol (26%). It was included into γ-cyclodextrin by classic co-dissolution procedures. Solid-state characterisation of γ-cyclodextrin·gingerols shows that this inclusion compound features 1:1 host-to-guest stoichiometry and that it is a microcrystalline powder with a crystalline cell that belongs to the tetragonal space group 4212, having the host molecules stacked in infinite channels where the gingerols are accommodated. In chimico studies with ABTS•+ scavenging, NO• scavenging, ß-carotene peroxidation, and 5-LOX inhibition show that γ-cyclodextrin is a suitable carrier for gingerols, because it does not alter their reactivity towards these substances. Yogurt was tested as a matrix for the incorporation of gingerols and γ-cyclodextrin·gingerols into foodstuff. The colour of the fortified yogurt suffered little alterations. In the case of yogurt with the inclusion compound, γ-cyclodextrin·gingerols, as fortificant, these alterations were not perceptible to the naked eye. Moreover, yogurt with γ-cyclodextrin·gingerols showed a good antioxidant activity, thus being suitable for use in nutraceutical applications.

Analytical characterization of cyclodextrins: History, official methods and recommended new techniques.

The main goal of this review is to provide a comprehensive overview on the methods used for analysis of cyclodextrins (CDs) and CD-derivatives. The paper intends to act as a guide for the readers in looking around the classical and modern instrumental analytical methods suitable for identification, characterization and determination of CDs themselves, CDs in finished products or even in biological samples. At present, in the European and United States Pharmacopoeias, the three parent CDs and two synthetic derivatives, namely the (2-hydroxypropyl)-beta-CD and sulfobutylether-beta-CD Na salt are official. Besides these modified CDs, two other derivatives are approved as excipients in human pharmaceutical products: the (2-hydroxypropyl)-gamma-CD and the randomly methylated-beta-CD. Although most of the official analysis methods in the pharmacopoeias have been well used for decades, new aspects of the functional excipient CD characterization suggest a need to revisit compendial methods. Comparison of strengths and weaknesses of current official methods with new improved techniques intends to help analysts to decide on changing traditional analytical methods with improved new ones. This review also deals with the analytical aspects of the first single isomer CD derivative approved as a drug active (Sugammadex/Bridion®) as well as analytical considerations of using CDs themselves as active pharmaceutical ingredients. Stability-indicating instrumental methods suitable to adequately follow chemical- and enzymatic degradation of CDs will also be discussed. Challenges in the determination of CDs in different biological matrices will be illustrated on real pharmaco- and toxicokinetic studies of CD-enabled drug formulations.

Development and validation of a simple high performance liquid chromatography - evaporative light scattering detector method for direct quantification of native cyclodextrins in a cyclization medium.

A simple HPLC-ELSD method was developed for the separation and quantification of native cyclodextrins. The technique was validated in the presence of two interfering matrices composed of byproducts from the cyclization medium. A fast separation of the compounds was achieved (in <20min) using a NUCLEODUR(®) C18 Pyramid column (150mm×4.6mm; particle size 5µm) at 30°C. The analytes were eluted using a linear gradient of acetonitrile and water containing 1% (v/v) of acetic acid at a flow rate of 0.3mL/min. Validation results showed that the method was accurate (93-110%) and selective. The precision was ≤5.7% for a hydrolyzed starch blank matrix spiked with cyclodextrins, and ≤6.2% for a blank matrix composed of a mixture of dextrin and glucose spiked with cyclodextrins. The limit of quantification was 0.05g/L for alpha- and 0.06g/L for beta- and gamma-cyclodextrins. The new HPLC-ELSD method could accurately quantify the three cyclodextrins directly in a cyclization medium, without pretreatment of the samples.

Cyclodextrin-modified micellar electrokinetic chromatography for enantioseparations.

The separation of enantiomers is one of the important fields of modern analytical chemistry, especially for agrochemical and pharmaceutical products because the stereochemistry has a significant influence on the biological activities of compounds. Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) has become an important capillary electrophoresis mode for enantioseparations. Here, we describe an example of a CD-MEKC method using hydroxypropyl-γ-cyclodextrin as chiral selector and sodium dodecyl sulfate as micellar solution for enantioseparation of triazole fungicides and the drug econazole.

Determination of sugammadex in human plasma, urine, and dialysate using a high-performance liquid chromatography/tandem mass spectrometry assay.

Sugammadex (Bridion®, Merck Sharp & Dohme Corp., Oss, The Netherlands) is a modified γ-cyclodextrin which has the ability to reverse the neuromuscular blockade induced by the steroidal neuromuscular blocking agents rocuronium and vecuronium. The objective of the current study is to describe the bioanalytical methods that have been developed and validated according to US Food and Drug Administration guidelines on bioanalytical method validation, and subsequently applied to determine total sugammadex (i.e., free sugammadex plus sugammadex bound to the neuromuscular blocking agent) in human heparinized plasma, urine and dialysate. Sugammadex was extracted from human plasma and urine using solid phase extraction with Isolute HAX 96-well extraction plates; no extraction was performed on dialysate samples. Samples from plasma, urine, and dialysate were analyzed on a Polaris® C18-A PEEK (polyaryletheretherketone) analytical column (50 mm × 4.6 mm internal diameter, 5 µm) with a linear mobile phase gradient of 0.1% (v/v) formic acid in water:methanol from 70:30 to 20:80. The flow rate was 1 mL/min with a total run time for each injection of 6 min. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ion mode with a turbo ion-spray interface to quantify the concentration of sugammadex. Inter- and intra-assay precision and accuracy were within pre-defined acceptance limits. The presence of rocuronium did not interfere with the assay in plasma, urine or dialysate; similarly, vecuronium did not interfere with the plasma assay (not tested for interference in urine or dialysate). Sugammadex was found to be stable in plasma, urine and dialysate in the short-term at room temperature, in the long-term at -20°C, and after several freeze/thaw cycles. The validated bioanalytical methods developed here have been successfully applied in a series of clinical studies for the determination of total sugammadex in plasma, urine and dialysate.

Evaluation of sugammadex self-association.

Sugammadex, a thiolated γCD derivative used as an antagonist of steroidal blockers, was studied with regard to its tendency to self-associate in aqueous solution. Three independent methods - permeation through semi-permeable cellophane membranes, dynamic light scattering, and sedimentation equilibrium analytical ultracentrifugation - were used for this purpose. The results were in agreement with each other and showed no evidence of self-association in a wide sugammadex concentration range from 0.25 to 100mg/ml.

Comparative effect of the addition of α-, ß-, or γ-cyclodextrin on main sensory and physico-chemical parameters.

UNLABELLED: Although α-, ß-, and γ-cyclodextrins (CDs) have been widely used to improve the color of different fruit juices, a comparative study of the effect of these natural CDs on other properties that also influence pear juice quality, such as odor and aroma, have not been reported yet. In this study, the comparative effect of the addition of α-, ß-, and γ-cyclodextrin, the only CDs authorized to be used in the food industry by U.S. Food and Drug Administration (FDA) and European Union, on the pear juice quality was evaluated for the first time. Several instrumental and sensory properties of this fruit juice, such as color, volatile composition, odor, and aroma have been evaluated in the absence and presence of α-, ß-, and γ-CD. A study of the aroma profile of pear juice showed that esters, aldehydes, alcohols, and terpenes were the most important chemical families. However, the addition of α-, ß-, and γ-CD had different effects on both the concentration of individual volatile compounds and their chemical grouping. Furthermore, a trained sensory panel was used to evaluate color, overall odor, overall aroma, and overall quality of pear juice in the presence or absence of CDs. PRACTICAL APPLICATION: After comparing the effects of the addition of α-, ß-, and γ-CD on pear juice, our final recommendation is to add α-CD (the natural CD formed by 6 units of glucose) to pear juice because it will significantly increase the global quality of the juice by reducing its browning but without producing a significant reduction in the aroma quality.

Chiral separation of econazole using micellar electrokinetic chromatography with hydroxypropyl-gamma-cyclodextrin.

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) method with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selector for the enantiomeric separation of econazole is reported. Enantioseparation of econazole was successfully achieved by the optimized CD-MEKC system containing 40mM HP-gamma-CD, 50mM SDS and 20mM phosphate buffer (pH 8) solution with an analysis time of less than 9min. Calibration curves were linear for the two stereoisomers of econazole (r(2)>0.998). Good repeatabilities in the migration time, peak area and peak height were obtained in terms of RSD% ranging from 0.30 to 7.67%. Combination of solid-phase extraction (SPE) procedure using diol column and the CD-MEKC method was successfully applied to the determination of econazole in a formulated cream sample.

Chiral discrimination between D- and L-tryptophan based on the alteration of the fluorescence lifetimes by the chiral additives.

The chiral microenvironmental properties of bovine serum albumin, cyclodextrins and their mixtures were comparatively investigated based on pyrene as the fluorescence probe. On this basis, a time resolved fluorescence technique to discriminate D- and L-tryptophan (Trp) without separation was developed. This method was based on the quenching rate difference between d- and l-Trp to pyrene in various media including bovine serum albumin, cyclodextrins and their mixtures. Pyrene, tryptophan and bovine serum albumin (or cyclodextrin) could form ternary complexes, which provided a possibility for the discrimination of D- and L-Trp enantiomers. The results indicated that the chiral discrimination of Trp enantiomers could be obtained in the mixture system of gamma-cyclodextrin and BSA system, in which the fluorescence lifetime difference (Deltatau) and the lifetime difference percentage (Deltatau/tau) were 55.8ns and 28.50%, respectively.

Photoinduced DNA cleavage by alpha-, beta-, and gamma-cyclodextrin-bicapped C60 supramolecular complexes.

Water-soluble supramolecular inclusion complexes of alpha-, beta-, and gamma-cyclodextrin-bicapped C60 (CD/C60) have been investigated for their photoinduced DNA cleavage activities, with the aim to assess the potential health risks of this class of compounds and to understand the effect of host cyclodextrins having different cavity dimensions. Factors such as incubation temperature, irradiation time, and concentration of NADH or CDs/ C60 supramolecular inclusion complexes have been examined. The results show that alpha-, beta-, and gamma-CDs/C60 are all able to cleave double-stranded DNA under visible light irradiation in the presence of NADH. However, a difference in the photoinduced DNA cleavage efficiency is observed, where the cleavage efficiency increases in the order of alpha-, beta-, and gamma-CD/C60. The difference is attributed to the different aggregation behavior of the inclusion complexes in aqueous solution, which is correlated to the cavity dimension of the host cyclodextrin molecules.

NMR relaxation interference effects and internal dynamics in gamma-cyclodextrin.

Multiple-magnetic field (9.4, 14.1 and 21.1 T) measurements of (13)C spin-lattice and spin-spin relaxation rates, the heteronuclear Overhauser enhancement and cross-correlated relaxation rates (CCRRs) in the methylene groups are reported for gamma-cyclodextrin in water/dimethylsulfoxide solution at 323 and 343K. The CCRRs are obtained from differences in the initial relaxation rates of the components of the CH(2) triplet in the (13)C spectra. The relaxation data are analyzed using the Lipari-Szabo approach and a novel modification of the two-site jump model. According to the latter model, inclusion of the dipolar (CH,CH(')) cross-correlated longitudinal and transverse relaxation is important for estimating the rate of the conformational jumps in the hydroxymethyl group. Using the dynamic information from the jump model, we have also used the differences in the initial relaxation rates for the triplet components to estimate the anisotropy of the chemical shielding tensor.

Enantiomeric discrimination of 1,1'-binaphthol by room temperature phosphorimetry using gamma-cyclodextrin as chiral selector.

In the presence of a small amount of 1,2-dibromopropane (1,2-DBP), 1,1'-binaphthol (BINOL) displays strong room temperature phosphorescence in gamma-cyclodextrin (gamma-CD) solution without deoxygenation. The phosphorescence intensity as well as the phosphorescence lifetime of (S)-BINOL is greater than that of (R)-BINOL, indicating a distinct chiral discrimination of gamma-CD toward this pair of enantiomers. Both (R)-BINOL and (S)-BINOL exhibit a double exponential phosphorescence decay with lifetimes of 5.89 ms and 17.3 ms for (R)-BINOL and 7.58 ms and 23.6 ms for (S)-BINOL, respectively. The association constant obtained for (S)-BINOL/gamma-CD/1,2-DBP ternary complex is larger than that for (R)-BINOL/gamma-CD/1,2-DBP complex. Thus, the observation of RTP lifetime differences between (R)-BINOL and (S)-BINOL can be attributed to their different ability to form complexes with chiral gamma-CD, which is further supported by an analysis of the proton NMR chemical shift differences between (R)-BINOL and (S)-BINOL.

Effects of alcohols on CE enantioseparation of basic drugs with native gamma-CD as chiral selector.

The effects of alcohol on the CE enantioseparation of selected basic drugs with gamma-CD as the chiral selector was investigated. The enantioseparation behavior of the analytes with gamma-CD in the absence and presence of different alcohols specifically methanol, ethanol, 2-propanol (IPA), and 2-methyl-2-propanol (TBA), the relationship of enantiomeric resolution (R(s)) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of these alcohols on interaction of the analytes with gamma-CD were studied. Results showed that hydrophobicity and/or bulkiness of alcohols have an influence on the enantioresolution of most of the analytes based on the relatively high correlation coefficients (R) obtained between R(s) versus log P and between R(s) versus ovality (i.e., parameter to indicate bulkiness of a molecule). Comparison of the values of the average binding constants obtained for each enantiomeric pair in the presence and absence of 5% IPA showed that alcohols can increase, decrease, or give a minimal effect on the analyte-gamma-CD interaction depending on the analyte. Furthermore, the significant enhancement in the enantioresolution of both propranolol and pindolol in the presence of either IPA or TBA led to the baseline enantioresolution of both drugs using 35 mM gamma-CD.

Analysis of the complexation of gemfibrozil with gamma- and hydroxypropyl-gamma-cyclodextrins.

The interactions of gemfibrozil with gamma- and HP-gamma-cyclodextrin (CD) have been studied in aqueous solution by fluorescence and NMR spectroscopy and by solubility measurements and in the solid state by X-ray diffraction, thermal analysis and FTIR spectroscopy. The influence of the technique employed in the analysis of complexation is discussed. The fluorescence of gemfibrozil increased in the presence of gamma- and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD), especially with the later, because the inclusion of the aromatic ring in the cavity, evidenced by 1H NMR, has a protective effect on the excited state of the drug. The fluorescence enhancement allowed the determination of the binding constants at pH 2.8. Complexation was a both entropy and enthalpy driven process. The solubility diagrams obtained with gamma-CD and HP-gamma-CD were B(s) and A(L) type, respectively. The apparent stability constants calculated from the solubility data at 25 degrees C were compared with those obtained from the fluorescence assays. It was found that drug solubilization with gamma-CD involves other contributions together with the inclusion phenomena. Solid complexes of gemfibrozil with gamma-CD (and not with HP-gamma-CD) have been obtained by kneading, coevaporation and coprecipitation methods. The solid complexes crystallised in the channel structure, in a process involving the carboxyl and aryl-ether groups.

Biodegradation of cyclodextrins in soil.

Cyclodextrins, especially random methylated betaCD (RAMEB) and hydroxypropyl betaCD (HPbetaCD), are becoming common enhancing additives in the bioremediation of soils formerly contaminated by hydrocarbons and/or other poorly bioavailable organic pollutants. Therefore, their degradation in the soil, particularly the most persistent RAMEB, has been of great concern. Like oil contaminants, these additives should be biodegradable via an environmentally safe technology. Hence, in this paper, the biodegradability of eight different cyclodextrins (CDs) in four different soils was examined under various treatment conditions in laboratory and pilot scale field experiments. This paper is the first report on the potential biological fate of CDs studied under a large variety of environmental conditions and in different soil ecosystems. Data on the potential relationship between CD biodegradation and the biological removal of hydrocarbons in the CD-amended contaminated soils are also given. All CDs were found to be more or less biodegradable; even the most persistent RAMEB was depleted from soils under favourable conditions. In the field experiments, the depletion of RAMEB to about 40% of its initial level was observed for a period of 2 years in hydrocarbon-contaminated soils of high organic matter and cell concentration.

Inclusion complexes of alpha- and gamma-cyclodextrins and the herbicide norflurazon: I. Preparation and characterisation. II. Enhanced solubilisation and removal from soils.

The interaction of norflurazon with alpha- and gamma-cyclodextrins (CDs) yielded the formation of inclusion complexes at a 1:1 stoichiometric ratio in solution and in the solid state. Apparent stability constants of 50.7+/-1.6 and 37+/-1.7 M(-1) and an increase in herbicide solubility by up to five and fourfold for alpha- and gamma-CD, respectively, were determined from the phase solubility diagrams at 25 degrees C in water. Three processing methods (kneading, spray-drying and vacuum evaporation) were used to prepare norflurazon-CD solid inclusion complexes, which were characterised by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. A high increase in the norflurazon dissolution rate was obtained with all the solid complexes with gamma-CD, but when alpha-CD was used, only the solid system obtained after the vacuum evaporation process showed a higher dissolution rate. This finding is a first step in the development of new, environmentally sound formulations of norflurazon (NFL), due to the capacity for increasing its dissolution rate and hydrosolubility, and thus diminishing the use of organic solvents. On the other hand, the effect of alpha- and gamma-cyclodextrin on the solubility of norflurazon in solution was also considered as a way of modifying its behaviour in the soil environment. Desorption studies of NFL from soils in the presence of alpha- and gamma-cyclodextrin were carried out using a batch equilibration method. The results obtained showed that alpha- and gamma-cyclodextrin greatly increased the removal of norflurazon previously adsorbed, proving the potential use of these CDs for in situ remediation of pesticide-contaminated soils.

Spectrofluorimetric study of eflucimibe-gamma-cyclodextrin inclusion complex.

Eflucimibe, a novel and highly potent acyl-coenzyme A cholesterol O-acyl-transferase (ACAT) inhibitor, is sparingly soluble in aqueous media and exhibits a very weak natural fluorescence. However, when increasing concentrations of gamma-cyclodextrin (gamma-CD) are added, an increase in the fluorescence signal is observed, attesting the formation of a non-covalent inclusion complex between eflucimibe and the gamma-CD. In this work, the stoichiometry of the complex and the corresponding association constant have been determined from fluorescence data by Benesi-Hildebrand's method (double reciprocal plots). As a result, a 1:1 stoichiometric ratio and a 20 M(-1) formation constant were obtained. This apparent formation constant was determined in water containing 10% methanol, which was needed to improve 'aqueous' solubility of the drug in a CD-free medium. Owing to the extreme hydrophobicity of eflucimibe, these results provide valuable information for pharmaceutical formulation studies.

Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii.

The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55 degrees C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS-polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55-70 degrees C and pH 5-10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially alpha-cyclodextrin. With a longer incubation period, the alpha-cyclodextrin to beta-cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5-20.0%) and glucans containing branching points (alpha-1,6 glycosidic linkages) shifted the product pattern to: beta-cyclodextin > alpha-cyclodextrin > gamma-cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl(2) shifted the apparent melting point of the enzyme from 60 degrees C to 69 degrees C.