Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

Preparation and characterization of a new open‐tubular capillary column for enantioseparation by capillary electrochromatography

In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open‐tubular (OT) column, in this study, a new OT‐CEC, coated with cationic cyclodextrin (1‐allylimidazolium‐β‐cyclodextrin [AI‐β‐CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI‐β‐CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI‐β‐CD‐coated column were optimized with the orthogonal experiment design L₉(3⁴). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI‐β‐CD‐coated columns was about 0.2 to 0.5 μm. The AI‐β‐CD content in stationary phase based on the EA was approximately 2.77 mmol·m^?2. The AI‐β‐CD‐coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI‐β‐CD, has a great potential for enantioseparation in OT‐CEC.     

Chiral Separation of Uncharged Pomalidomide Enantiomers Using Carboxymethyl‐β‐Cyclodextrin: A Validated Capillary Electrophoretic Method

The racemic mixture of pomalidomide (POM), a second‐generation immunomodulatory uncharged drug, was separated into enantiomers by capillary zone electrophoresis for the first time. Seven different chargeable cyclodextrin (CD) derivatives were screened as complexing agents and chiral selectors, investigating the stability of the POM‐CD inclusion complexes and their enantiodiscriminating capacities. Based on preliminary experiments, carboxymethyl‐β‐CD (CM‐β‐CD) was found to be the most effective chiral selector. Factors influencing enantioseparation were systematically optimized, using an orthogonal experimental design. Optimal parameters (background electrolyte [BGE]: 50 mM Tris‐acetate buffer, pH 6.5, containing 15 mM CM‐β‐CD; capillary temperature: 20°C; voltage applied +15 kV) allowed baseline separation of POM enantiomers with a resolution as high as 4.87. The developed method was validated, in terms of sensitivity (limit of detection and limit of quantification), linearity, accuracy, repeatability, and intermediate precision. Chirality 28:199–203, 2016. © 2015 Wiley Periodicals, Inc.     

Nonaqueous Capillary Electrophoretic Enantioseparation of Water Insoluble Tröger's Base Derivatives Using β‐Cyclodextrin as Chiral Selector

Racemic mixtures of six Tröger's base derivatives were separated by chiral nonaqueous capillary electrophoresis. The separation protocol was optimized first for suitable solvents. Then the applicability of various salts dissolved in organic solvents and their mixtures was evaluated. As chiral selectors β‐cyclodextrin and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin at various concentrations were used. The best enantioselectivity for the studied analytes was obtained utilizing formamide as organic nonaqueous solvent containing a mixture of sodium citrate and tris(hydroxymethyl)aminomethane acetate as electrolytes, and β‐cyclodextrin as chiral additive. The experimental results demonstrated the feasibility of nonaqueous capillary electrophoresis for enantioseparation of Tröger's base derivatives. This technique represents a suitable alternative to more commonly used capillary electrophoresis in aqueous environment. Chirality 25:810–813, 2013. © 2013 Wiley Periodicals, Inc.     

The unexpected increase of clotrimazole apparent solubility using randomly methylated β‐cyclodextrin

Clotrimazole (CTZ) and cyclodextrin (CD) inclusion complexes having improved apparent water solubility were obtained from phase solubility diagrams. β‐CD (1.5% w/w) and hydroxypropyl‐β‐CD (40% w/w) offered poor CTZ solubility enhancements (12 and 384 times, respectively). Unexpectedly, the apparent solubility of CTZ was 9980 times increased from 0.4 µg.mL^?1 (1.42 μM) without CD to 4.89 mg.mL^?1 (14.9 mM) using randomly‐methylated β‐CD (Me‐β‐CD) (40% w/w). This is the highest apparent CTZ solubility improvement ever reported in the literature using conventional CDs. Quantitative nuclear magnetic resonance (¹H‐NMR) coupled with two‐dimensional nuclear Overhauser effect (NOESY) experiments and molecular docking calculations showed that the highest interactions with Me‐β‐CD were reported for CTZ two phenyl groups. A lower interaction was reported for chlorophenyl, while imidazole had the weakest interaction with Me‐β‐CD. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of the interaction of a mono‐6‐thio‐β‐cyclodextrin‐capped CdTe quantum dots–methylene blue/methylene green system with herring sperm DNA using a spectroscopic approach

Novel, water‐soluble CdTe quantum dots (QDs) capped with β‐cyclodextrin (β‐CD) and ~ 4.0 nm in diameter were synthesized in aqueous solution, and characterized using transmission electron microscopy (TEM). A fluorescence‐sensing system based on the photoinduced electron transfer (PET) of (mono‐6‐thio‐β‐CD)–CdTe QDs was then designed to measure the interaction of phenothiazine dyes [methylene blue (MB) and methylene green (MG)] with herring sperm DNA (hsDNA). This fluorescence‐sensing system was based on a fluorescence “OFF–ON” mode. First, MB/MG adsorbed on the surface of (mono‐6‐thio‐β‐CD)–CdTe QDs effectively quenches the fluorescence of (mono‐6‐thio‐β‐CD)–CdTe QDs through PET. Then, addition of hsDNA restores the fluorescence intensity of (mono‐6‐thio‐β‐CD)–CdTe QDs, because hsDNA can bind with MB/MG and remove it from the as‐prepared (mono‐6‐thio‐β‐CD)–CdTe QDs. In addition, detailed reaction mechanisms of the (mono‐6‐thio‐β‐CD)–CdTe QDs–MB/MG–hsDNA solution system were studied using optical methods, by comparison with the TGA–CdTe QDs–MB/MG–hsDNA solution system. Copyright © 2014 John Wiley & Sons, Ltd.     

Enantioselective separation of (±)‐β‐hydroxy‐1,2,3‐triazoles by supercritical fluid chromatography and high‐performance liquid chromatography

This paper reports the enantioseparation of β‐hydroxy‐1,2,3‐triazole derivatives, which present a broad range of biological properties, by supercritical fluid chromatography (SFC) and high‐performance liquid chromatography techniques (HPLC). Polysaccharide‐based chiral columns (cellulose and amylose) were used to evaluate the separation in SFC and HPLC. Time of analyses, consumption of solvent, and parameter optimization were reduced using SFC technique. The columns based on cellulose chiral stationary phase using 2‐propanol and ethanol as modifiers showed the best results for the enantioresolution of the (±)‐β‐hydroxy‐1,2,3‐triazoles by SFC analyses. These techniques were applied to evaluate the selectivity of biocatalytic reduction of β‐keto‐1,2,3‐triazoles by marine‐derived fungus Penicillium citrinum CBMAI 1186 to obtain the (±)‐β‐hydroxy‐1,2,3‐triazoles.     

Enantioseparation of nine indanone and tetralone derivatives by HPLC using carboxymethyl‐β‐cyclodextrin as the mobile phase additive

High‐performance liquid chromatography (HPLC) is a powerful method in the area of chiral separation. In this study, a method of HPLC using carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) as chiral selector was developed for enantioseparation of nine indanone and tetralone derivatives. The separation was performed on a conventional C₁₈ column. The optimal mobile phase was a mixture of methanol and 0.05 mol/L phosphate buffer at pH 1.8 (55:45, v /v) containing 22.9 mmol/L CM‐β‐CD. Under such conditions, the resolutions of all analytes were over 1.8 except for Compound F. The results of the study indicate the presence of a complex with 1:1 stoichiometry of the inclusion complex. In addition, it can be inferred from thermodynamic analysis that the behavior of formation of the inclusion complex and enantioseparation occurred simultaneously, while they were driven by different forces. The effect of analyte structure is also discussed.     

Separation of Selected Imidazole Enantiomers Using Dual Cyclodextrin System in Micellar Electrokinetic Chromatography

Cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) method was developed for simultaneous enantioseparation of three imidazole drugs namely tioconazole, isoconazole and fenticonazole. Three easily available and inexpensive cyclodextrins namely 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), 2‐hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD) and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DM‐β‐CD) were evaluated to discriminate the six stereoisomers of the drugs. However, none of the three CDs gave a complete enantioseparation of the drugs. Effective enantioseparation of tioconazole, isoconazole and fenticonazole was achieved using a combination of 35 mM HP‐γ‐CD and 10 mM DM‐β‐CD as chiral selectors. The best separation using both HP‐γ‐CD and DM‐β‐CD (35 mM:10 mM) as chiral selectors were accomplished in background electrolyte (BGE) containing 35 mM phosphate buffer (pH 7.0), 50 mM sodium dodecyl sulfate (SDS) and 15% (v/v) acetonitrile at 27 kV and 30 °C with all peaks resolved in less than 15 min with resolutions, R_s 1.90‐27.22 and peak efficiencies, N > 180 000. The developed method was linear over the concentration range of 25–200 mg l^‐1 (r² > 0.998) and the detection limits (S/N = 3) of the three imidazole drugs were found to be 2.7‐7.7 mg l^‐1. The CD‐MEKC method was successfully applied to the determination of the three imidazole drugs in spiked human urine sample and commercial cream formulation of tioconazole and isoconazole with good recovery (93.6‐106.2%) and good RSDs ranging from 2.30‐6.8%. Chirality 25:328–335, 2013. © 2013 Wiley Periodicals, Inc.     

Supramolecular interactions between β‐lapachone with cyclodextrins studied using isothermal titration calorimetry and molecular modeling

Supramolecular interactions between β‐lapachone (β‐lap) and cyclodextrins (CDs) were investigated by isothermal titration calorimetry. The most favorable host: guest interaction was characterized using X‐ray powder diffraction (XRD), differential scanning calorimetry and thermogravimetry (DSC/TG), spectroscopy (FT‐IR), spectroscopy (2D ROESY) nuclear magnetic resonance (NMR), and molecular modeling. Phase solubility diagrams showed β‐, HP‐β‐, SBE‐β‐, γ‐, and HP‐γ‐CDs at 1.5% (w/w) allowed an increase in apparent solubility of β‐lap with enhancement factors of 12.0, 10.1, 11.8, 2.4, and 2.2, respectively. β‐lap has a weak interaction with γ‐ and HP‐γ‐CDs and tends to interact more favorably with β‐CD and its derivatives, especially SBE‐β‐CD (K = 4160 M⁻¹; ΔG = −20.66 kJ·mol⁻¹). Thermodynamic analysis suggests a hydrophobic interaction associated with the displacement of water from the cavity of the CD by the β‐lap. In addition, van der Waals forces and hydrogen bonds were responsible for the formation of complexes. Taken together, the results showed intermolecular interactions between β‐lap and SBE‐β‐CD, thereby confirming the formation of the inclusion complex. Molecular docking results showed 2 main orientations in which the interaction of benzene moiety at the wider rim of the SBE‐β‐CD is the most stable (average docking energy of −7.0 kcal/mol). In conclusion, β‐lap:SBE‐β‐CD is proposed as an approach for use in drug delivery systems in cancer research.     

Combined Use of Ionic Liquid and Hydroxypropyl‐β‐Cyclodextrin for the Enantioseparation of Ten Drugs by Capillary Electrophoresis

In the present study, hydroxypropyl‐β‐cyclodextrin and an ionic liquid (1‐ethyl‐3‐methylimidazolium‐l ‐lactate) were used as additives in capillary electrophoresis for the enantioseparation of 10 analytes, including ofloxacin, propranolol hydrochloride, dioxopromethazine hydrochloride, isoprenaline hydrochloride, chlorpheniramine maleate, liarozole, tropicamide, amlodipine benzenesulfonate, brompheniramine maleate, and homatropine methylbromide. The effects of ionic liquid concentrations, salt effect, cations, and anions of ionic liquids on enantioseparation were investigated and the results proved that there was a synergistic effect between hydroxypropyl‐β‐cyclodextrin and the ionic liquid, and the cationic part of the ionic liquid played an important role in the increased resolution. With the developed dual system, all the enantiomers of 10 analytes were well separated in resolutions of 5.35, 1.76, 1.85, 2.48, 2.88, 1.43, 5.45, 4.35, 2.76, and 2.98, respectively. In addition, the proposed method was applied to the determination of the enantiomeric purity of S‐ofloxacin after validation of the method in terms of selectivity, repeatability, linearity range, accuracy, precision, limit of detection (LOD), and limit of quality (LOQ). Chirality 25:409–414, 2013. © 2013 Wiley Periodicals, Inc.     

Enantiomeric Separation of 13 New Amphetamine‐Like Designer Drugs by Capillary Electrophoresis,Using Modified‐‐Cyclodextrins

An easy‐to‐prepare chiral CE method for the enantiomeric separation of 13 new amphetamine‐like designer drugs, using CDs as chiral selectors, was developed. Sulfated‐β‐CD was found to be the best chiral selector among the three used (sulfated‐β‐CD, caroboxymethyl‐β‐CD, dimethyl‐β‐CD). The separation of the analytes was achieved in a fused‐silica gel capillary at 20 °C using an applied voltage of +25 kV. The optimized background electrolyte consisted of 63.5 mM H₃PO₄ and 46.9 mM NaOH in water. Several electrophoretic parameters such as CD type, CD concentration (1 ? 40 mg/mL), buffer pH (2.6, 3.6, 5.0, 6.0), length of the capillary (70 ? 40 cm total length), amount of the organic solvent (methanol and acetonitrile) were investigated and optimized. Chirality 25:617–621, 2013. © 2013 Wiley Periodicals, Inc.     

Chiral recognition of verapamil by cyclodextrins studied with capillary electrophoresis,NMR spectroscopy,and electrospray ionization mass spectrometry

Capillary electrophoresis (CE) allows the observation of the opposite affinities of the enantiomers of (±)‐verapamil [2‐isopropyl‐2,8‐bis(3,4‐dimethoxyphenyl)‐6‐methyl‐6‐azaoctannitrile, VP] toward β‐cyclodextrin (β‐CD) and heptakis(2,3,6‐tri‐O‐methyl)‐β‐CD (TM‐β‐CD). In addition, in the presence of β‐CD in the background electrolyte, longer migration times and lower separation factors were observed compared to TM‐β‐CD. The binding constants of (+)‐ and (−)‐VP with β‐CD and TM‐β‐CD determined using ¹³C NMR spectroscopy explain the results observed in CE. Electrospray ionization mass spectrometry (ESI‐MS) was used as an alternative technique for the characterization of VP‐CD complexes. Chirality 11:635–644, 1999. © 1999 Wiley‐Liss, Inc.     

A chiral enantioseparation generic strategy for anti‐Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach

The present study describes a generic strategy using capillary electrophoretic (CE) method for chiral enantioseparation of anti‐Alzheimer drugs, namely, donepezil (DON), rivastigmine (RIV), and antifungal drugs, namely, ketoconazole (KET), Itraconazole (ITR), fluconazole (FLU), and sertaconazole (SRT) in which these drugs have different basic and acidic properties. Several modified cyclodextrins (CDs) were applied for enantioseparation of racemates such as highly sulfated α, γ CDs, hydroxyl propyl‐β‐CD, and Sulfobutyl ether‐β‐CD. The starting screening conditions consist of 50‐mM phosphate‐triethanolamine buffer at pH 2.5, an applied voltage of 15 kV, and a temperature of 25°C. The CE strategy implemented in the separation starts by screening prior to the optimization stage in which an experimental design is applied. The design of experiment (DOE) was based on a full factorial design of the crucial two factors (pH and %CD) at three levels, to make a total of nine (3²) experiments with high, intermediate, and low values for both factors. Evaluation of the proposed strategy pointed out that best resolution was obtained at pH 2.5 for five racemates using low percentages of HS‐γ‐CD, while SBE‐β‐CD was the most successful chiral selector offering acceptable resolution for all the six racemates, with the best separation at low pH values and at higher %CD within 10‐min runtime. Regression study showed that the linear model shows a significant lack of fit for all chiral selectors, anticipating that higher orders of the factors are most likely to be present in the equation with possible interactions.     

Effect of 2‐hydroxypropyl‐β‐cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP‐β‐CD is destabilization of the protein molecule under action of HP‐β‐CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP‐β‐CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP‐β‐CD was substantiated by the data on the enzyme inhibition by HP‐β‐CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010.     

Enantioseparation of ketoconazole and miconazole by capillary electrophoresis and a study on their inclusion interactions with β‐cyclodextrin and derivatives

A chiral separation method coupled with capillary electrophoresis (CE) analysis for ketoconazole and miconazole enantiomers using chiral selectors such as β‐cyclodextrin (β‐CD) and hydroxypropyl‐β‐CD (HP‐β‐CD) was developed in this study, which included the optimisation, validation and application of the method on the antifungal cream samples. The formation of inclusion complex between the hosts (β‐CD and HP‐β‐CD) and guests (ketoconazole and miconazole) were compared and analysed using ultraviolet–visible spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy and molecular docking methods. Results from the study showed that in a concentration that ranged between 0.25 and 50 mg L^?1, the linear calibration curves of each enantiomer had a high coefficient of regression (R² > 0.999), low limit of detection (0.075 mg L^?1) and low limit of quantification (0.25 mg L^?1). The relative standard deviation (RSD) of the intraday and interday analyses ranged from 0.79% to 8.01% and 3.30% to 11.43%, respectively, while the recoveries ranged from 82.0% to 105.7% (RSD < 7%, n = 3). The most probable structure of the inclusion complexes was proposed based on the findings from the molecular docking studies conducted using the PatchDock server.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

Formation of dityrosine (DT) cross‐linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein‐polyphenol cross‐linkages. To prevent the formation of such cross‐linkages we have used β‐cyclodextrin (β‐CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β‐cyclodextrin (β‐CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β‐CD and are no longer available for protein cross‐linking.