Enhancement of selectivity for producing γ-cyclodextrin

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.     

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain No. 37

The enzyme cyclod extringly cosyltransferase (CGTase), EC2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillus firmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl₂, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of β- and γ-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35–60°C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6k Da. Results for CGTase activity as a function of temperature gave maximal activity for the production of β-CD at 65°C, pH 6.0, and 7 1.5 mmol of β-CD/(min·mg of protein), whereas for γ-CD it was 9.1 m mol of γ-CD/(min·mg of protein) at 70°C and pH 8.0. For long contact times, the bestuse of the enzymatic activity occurs at 60°C oratalower temperature, and the reaction pH may be selected to increase the vield of a desired CD.     

Investigation of Drug Nanoparticle Formation by Co-grinding with Cyclodextrins: Studies for Indomethacin, Furosemide and Naproxen

Drugs with poor water solubility were co-ground with cyclodextrins (CDs) to create nanoparticles with improved solubility characteristics. Indomethacin (IDM), furosemide (FRM) and naproxen (NAP) were co-ground with β-CD at the molar ratio of 2:1 (CD:drug). Co-grinding of a drug with CD resulted in not only the formation of drug nanoparticles but also the solubilization of the drug by inclusion complex formation with CD in aqueous media. The nanoparticle fraction of IDM, and FRM from ground mixtures prepared with β-CD was as high as 60–70% while the solubilization fraction was less than 10%. In contrast, β-CD–NAP ground mixture showed a large fraction, 48%, for drug solubilization and only 4% for nanoparticle formation. Furosemide ground mixtures prepared with α-CD, β-CD and γ-CD showed comparatively high nanoparticle fraction while the solubilization fraction was around 10%. Both the nanoparticle fraction and the solubilization fraction were greater in the IDM–β-CD system than those in γ-CD and α-CD systems. The nanoparticle formation of NAP depended on the types of CD used as a co-grinding additive. Naproxen nanoparticles could be prepared by co-grinding NAP and α-CD, while the solubilization of NAP tended to improve when β-CD or γ-CD was used.     

Enhanced conversion of starch to cyclodextrins in ethanolic solutions by bacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase

Improved formation of cyclodextrins (CDs) from starch in ethanolic solutions byBacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase was studied. The β- and γ-CD yields increased and α-CD yield gradually decreased as the ethanol concentration was raised. The ethanol concentration required for maximal CD yield depended essentially on starch concentration. The ethanol's effect was pronounced at high starch concentrations. For example, with 30% (w/v) starch, the CD yield was 2.4-fold (146.5 g/L) in the presence of 15% (v/v) ethanol. The effect of dimethylsulfoxide on the formation of CDs was similar to that of ethanol. The disintegration of β- and γ-CDs were narrowly interdependent on the formation of a α-CD and malto-sugars. The amount of reducing sugars decreased from a dextrose equivalent value of roughly 7.5 to 4.5 in the presence of ethanol at starch concentrations 1-30% (w/v). The effect of ethanol on starchy materials from various sources was similar. It was concluded that ethanol retards the decomposition of β-CD by a general mechanism involving a decreased activity of water.     

A new example of reversal of the order of migration of enantiomers, as a function of cyclodextrin concentration and pH, by cyclodextrin-modified capillary zone electrophoresis: enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol

Enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol (DBBD) by cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was studied using the three native α, β, and γ cyclodextrins, the three hydroxypropylated cyclodextrins (2-hydroxypropyl-α, β, and γ), heptakis-2,6-di-O-methyl-β-CD (DM-β-CD), and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD). First, the acidity constants of DBBD were determined using capillary electrophoresis, before performing enantioseparation. The influence of the concentrations of the studied cyclodextrins on the enantioseparation was explored and the experimental optimal concentrations were determined and compared to the theoretical optimal concentrations. Moreover, the apparent complexation constants between each studied cyclodextrin and the two DBBD enantiomers were evaluated using a non-linear curve fitting method and three linear plotting methods (x-reciprocal, y-reciprocal and double reciprocal). For TM-β-CD, the order of migration of the enantiomers of DBBD reversed as a function of TM-β-CD concentration. The influence of the nature of methylated cyclodextrin derivatives (methyl-β-CD (M-β-CD) and DM-β-CD) was then studied. Inversion of the order of migration of the enantiomers of DBBD was observed for DM-β-CD, whereas the S enantiomer of DBBD always migrated first for M-β-CD.     

Binary Systems of Nifedipine And Various Cyclodextrins in The Solid State. Thermal,FTIR, XRD studies

Nifedipine complexes with β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), 2-hydroxypropyl-β-cyclodextrin (2HP-β-CD), randomly methylated-β-cyclodextrin (RM-β-CD) and heptakis(2,6-O-dimethyl)-β-cyclodextrin (DM-β-CD) have been prepared by both kneading and heating methods and their behaviour studied by differential scanning calorimetry (DSC), diffuse reflectance mid-infrared spectroscopy (FTIR) and X-ray diffractometry (XRD). DSC revealed the nifedipine melting endotherm with onset at approximately 171°C for the kneaded mixtures with β-CD, γ-CD and 2HP-β-CD, thus confirming the presence of nifedipine in the crystalline state, while some decrease in crystallinity was observed in the DM-β-CD kneaded mixture. With RM-β-CD, however, broadening and shifting of the nifedipine endotherm and reduction in its intensity suggested that the kneading could have produced an amorphous inclusion complex. These differing extents of interaction of nifedipine with the cyclodextrins were confirmed by FTIR and XRD studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Study on inclusion complexes of meso-tetrakis(2-thienyl)porphyrin and Cu-meso-tetrakis(2-thienyl)porphyrin with cyclodextrins by spectroscopy method

In phosphate buffer solution of pH5.4, the interaction of meso-tetrakis(2-thienyl)porphyrin(H₂TTP) and Cu-meso-tetrakis(2-thienyl)porphyrin(Cu-TTP) with α-cyclodextrin(α-CD), β-CD, γ-CD, heptakis(2,3,6-tri-O-methyl)-β-CD(TM-β-CD) has been studied by means of UV-vis, fluorescence and ¹HNMR spectroscopy, respectively. The H₂TTP and Cu-TTP can form 1:2 inclusion complexes with TM-β-CD and 1:1 inclusion complexes with the other three cyclodextrins. In this paper, the inclusion constants (K) of H₂TTP and Cu-TTP for the formation of the inclusion complexes have been estimated from the changes of absorbance and fluorescence intensity in phosphate buffer solution. The inclusive capabilities of different kinds of cyclodextrins are compared. The result shows that the inclusion ability of α-CD with H₂TTP and Cu-TTP is the strongest among the three native CDs. The inclusion ability of modified β-CD with H₂TTP and Cu-TTP is stronger, compared to the native β-CD, which indicates that the capacity matching plays a crucial role in the inclusion procedure except for the hydrophobic effect. In addition ¹HNMR spectra supports the inclusion conformation of the TM-β-CD-Cu-TTP inclusion complex, indicating the interaction mechanism of inclusion processes.     

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·g_IF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.     

Influence of substrate and product concentrations on the production of cyclodextrins by CGTase of Bacillus firmus,strain no. 37

The influence of substrate or product level on the initial velocity of cyclodextrin (CD) production by cyclodextringlycosyltransferase from a Brazilian isolate of Bacillus firmus was studied. Our results indicate that the product γ-CD is a stronger inhibitor to the reaction than β-CD. Small saccharides could also inhibit CD production, although to a lesser extent than the products, and maltose was the strongest inhibitor among small saccharides. Increasing substrate concentration resulted in greater reduction on enzyme activity for the formation of β-CD than for γ-CD. We modeled the kinetics of CD production with a set of four reversible reactions including the cyclization/coupling reaction that forms/opens CDs, and three disproportionation reactions. Our model on the initial velocity data explained well the substrate inhibition phenomenon. Kinetic parameters were determined by fitting the initial velocity data into our model.     

Grafting of cyclodextrins onto filter paper

Grafting of cyclodextrins and cyclodextrins derivatives on cellulosic surface, such as paper or filter paper, provides hosting cavities that can include a large variety of chemicals for specific cellulose finishing. In this study grafting of monocholorotriazinyl-β-cyclodextrin (MCT-β-CD) and β-cyclodextrin (β-CD) to filter paper has been performed. β-cyclodextrin has been bonded to filter paper using 1,4-butanediol diglycidyl ether as the crosslinking agent. The untreated and treated filter papers were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), demonstrating the covalent binding of cyclodextrins to filter paper. The quantification of β-CD and MCT-β-CD grafted to filter paper was determined by the dye extinction method with the inclusion of phenolphthalein. The final β-CD content amounted to 15.9 μmol per gram of support (1.8% by weight), and 72.8 μmol per gram of support (11.3% by weight) for MCT-β-CD.     

Influence of Cyclodextrin Complexation on the in vitro Human Skin Penetration and Retention of the Sunscreen Agent, Oxybenzone

The objective of this study was to investigate the influence of cyclodextrins on the cutaneous availability of the sunscreen oxybenzone. The interaction between oxybenzone and hydrophilic α-, β- and γ-cyclodextrin derivatives was studied in water by phase-solubility analysis. Among the available cyclodextrins, hydroxypropyl-β-cyclodextrin (HP-β-CD) and especially sulfobutylether-β-cyclodextrin (SBE-β-CD) had the greatest solubilizing activity. Ethanol–water solutions containing oxybenzone free or complexed with HP-β-CD or SBE-β-CD were applied to human skin in Franz diffusion cells and the amount of sunscreen permeated into the different cutaneous compartments was determined by HPLC. As much as 20.5% of the oxybenzone applied dose diffused within the skin tissue after 6 h application. Between 39.4% and 54.9% of the penetrated UV filter was localized in the stratum corneum, with no significant difference between uncomplexed oxybenzone or its complex with HP-β-CD. Conversely, the amount retained in the stratum corneum was markedly decreased (ca. 50%) by complexation with SBE-β-CD. Considerable quantities of oxybenzone accumulated into the viable epidermis (5.7% of the applied dose) and dermis (6.2% of the applied dose) from the preparation containing the free UV filter. The sunscreen penetration to the deeper living layers of the skin was remarkably lower (1.0% and 2.0% of applied dose for epidermis and dermis, respectively) upon application of the sunscreen complexed with SBE-β-CD, whereas HP-β-CD had no effect. In addition, photostability experiments demonstrated that SBE-β-CD complexation did not alter the sunscreen photochemical properties.     

Production of Cyclodextrins by CGTase from Bacillus clausii Using Different Starches as Substrates

Cyclodextrins (CDs) are cyclic oligasaccharides composed by d-glucose monomers joined by α-1,4-d glicosidic linkages. The main types of CDs are α-, β- and γ-CDs consisting of cycles of six, seven, and eight glucose monomers, respectively. Their ability to form inclusion complexes is the most important characteristic, allowing their wide industrial application. The physical property of the CD-complexed compound can be altered to improve stability, volatility, solubility, or bio-availability. The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into CD molecules. In this work, the CGTase produced by Bacillus clausii strain E16 was used to produce CD from maltodextrin and different starches (commercial soluble starch, corn, cassava, sweet potato, and waxy corn starches) as substrates. It was observed that the substrate sources influence the kind of CD obtained and that this CGTase displays a β-CGTase action, presenting a better conversion of soluble starch at 1.0%, of which 80% was converted in CDs. The ratio of total CD produced was 0:0.89:0.11 for α/β/γ. It was also observed that root and tuber starches were more accessible to CGTase action than seed starch under the studied conditions.     

Inclusion complexation of diclofenac with natural and modified cyclodextrins explored through phase solubility, 1H-NMR and molecular modeling studies

Guest–host interactions were examined for neutral diclofenac (Diclo) and Diclofenac sodium (Diclo sodium) with each of the cyclodextrin (CD) derivatives: α-CD, β-CD, γ-CD and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), all in 0.05 M aqueous phosphate buffer solution adjusted to 0.2 M ionic strength with NaCl at 20 °C, and with β-CD at different pHs and temperatures. The pH solubility profiles were measured to obtain the acid–base ionization constants (pK _as) for Diclo in the presence and absence of β-CD. Phase solubility diagrams (PSDs) were also measured and analyzed through rigorous procedures to obtain estimates of the complex formation constants for Diclo/CD and Diclo sodium/CD complexation in aqueous solutions. The results indicate that both Diclo and Diclo sodium form soluble 1:1 complexes with α-, β-, and HP-β-CD. In contrast, Diclo forms soluble 1:1 Diclo/γ-CD complexes, while Diclo sodium forms 1:1 and 2:1 Diclo/γ-CD, but the 1:1 complex saturates at 5.8 mM γ-CD with a solubility product constant (pK _sp = 5.5). Therefore, though overall complex stabilities were found to follow the decreasing order: γ-CD > HP-β-CD > β-CD > α-CD, some complex precipitation problems may be faced with aqueous formulations of Diclo sodium with γ-CD, where the overall concentration of the latter exceeds 5.8 mM γ-CD. Both ¹H-NMR spectroscopic and molecular mechanical modeling (MM⁺) studies of Diclo/β-CD indicate the possible formation of soluble isomeric 1:1 complexes in water.     

β-Cyclodextrin production by simultaneous fermentation and cyclization

Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast,S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE     

Comparative Study of the Inclusion Complexation of Pizotifen and Ketotifen with Native and Modified Cyclodextrins

The interaction of two benzocycloheptanes namely, pizotifen (Pizo) and ketotifen (Keto), with cyclodextrins (CDs: α-, β-, γ-, and HP-β-CDs) has been investigated by several techniques including phase solubility, X-ray powder diffractometry, ¹H-nuclear magnetic resonance and molecular mechanical modeling. The effects of CD type, pH, ionic strength and temperature on complex stability were also explored. The complex formation constant (K ₁₁) values for the Pizo/CD system follows the decreasing order β-CD > γ-CD > HP-β-CD > α-CD. However, for the Keto/CD system it follows the decreasing order γ-CD > β-CD > HP-β-CD > α-CD. The tendency of Pizo and Keto to complex with β-CD is driven to the extent of 70% by the hydrophobic effect. Complex formation of Keto and Pizo was substantially driven by entropy (>100 J⋅mol⁻¹⋅K⁻¹) but slightly retarded by enthalpy (3–8 kJ⋅mol⁻¹). ¹H-NMR and MM⁺ studies indicate multimodal inclusion of the methylpiperadine, thiophene and phenyl moieties into the β-CD cavity.     

Fast purification of cyclodextrin-glucosyltransferase fromBacillus circulans E 192 by affinity chromatography using an epichlorhydrin-reticulated copolymer of beta-cyclodextrin

Summary Bacillus circulans E 192 Cyclodextrin-glucosyltransferase was purified 40 fold to homogeneity on a large scale, by affinity chromatogrpahy on a β-CD copolymer cross-linked with epichlorhydrin. A preliminary step was necessary to remove maltooligosaccharides and cyclomaltooligosaccharides from the crude extract. The simplest method consists in performing the affinity chromatography directly from a redissolved 65% saturation ammonium sulfate precipitate of the crude extract. Five successive affinity runs of Cetavlon — treated concentrated extract gave about one gram of pure CGTase with a 70% yield, using 50 g of a 750 U/g β-CD copolymer. This rapid purification is described and the advantages of the method are discussed.     

Interactions of tetrakis(4-N-methylpyridyl)porphyrin with cyclodextrins and disodium phthalate in aqueous solution

In pH 7.3 buffers, the interactions of a cationic porphyrin, tetrakis(4-N-methylpyridyl)porphyrin (TMPyP), with cyclodextrins (CDs) and disodium phthalate (DSP) have been examined by means of absorption, fluorescence, and induced circular dichroism spectroscopy. α-CD, β-CD, and γ-CD form a 1:1 inclusion complex with a TMPyP monomer, which dimerizes in solution without CD. TMPyP also forms a 1:1 organic cation–organic anion complex with DSP. The 1:1 TMPyP–DSP complex forms a ternary CD–TMPyP–DSP inclusion complex with α-, β-, and γ-CD, in which a DSP molecule is not incorporated into the CD cavity. From the fluorescence intensity change, the␣equilibrium constants have been evaluated for the formation of the inclusion complexes and the organic cation–anion complexes.     

Cyclodextrins Binding to Paeonol and Two of Its Isomers in Aqueous Solution. Isothermal Titration Calorimetry and 1H NMR Investigations of Molecular Recognition

Interactions between CDs with three substituted phenols, paeonol (Pae), acetovanillone (Ace) and 2-hydroxyl-5-methoxy-acetophone (Hma), which are isomers, have been determined by isothermal titration calorimetry (ITC) and ¹H NMR in aqueous solution at 298.2 K. Both the binding thermodynamics and ¹H NMR spectra show that the interaction between α-cyclodextrin (α-CD) molecule and each guest molecule is extremely weak. The thermodynamic parameters indicate that the binding processes of β-cyclodextrin (β-CD) with the isomers are mainly entropy driven and that β-CD binds with Pae or Ace in 1:1 stoichiometry, whereas with Hma binds in 1:1 and 2:1 stoichiometries. The thermodynamic parameters also suggest that γ-cyclodextrin (γ-CD) binds each isomer in the same 1:1 stoichiometry. The binding processes of Pae and Hma with γ-CD are enthalpy driven whereas Ace with γ-CD is predominantly driven by entropy. The ¹H NMR spectra reveal that the three isomers were trapped into the torus cavity of the β-CD molecule from the narrow side during the binding process. Pae penetrates into the γ-CD cavity from the primary rim of the macrocycle whereas Ace does so from the secondary rim, but Hma appears not interact with the internal cavity of γ-CD at all.     

Elucidation of the Role of the Complex in Hydride Transfer Reaction between Methylene Blue and 1-Benzyl-1,4-dihydronictinamide by Effect of γ-Cyclodextrin

The kinetics of the hydride transfer reaction between Methylene Blue (MB⁺) and 1-benzyl-1,4-dihydronicotinamide (BNAH) were studied in 10% ethanol-90% water mixed solvents containing β- and γ-cyclodextrins (β-CD and γ-CD). The pseudo-first order rate constant shows kinetic saturation at high initial concentration of BNAH. This indicates the formation of a complex between MB⁺ and BNAH. The reaction was suppressed by addition of β-CD, but enhanced by addition of γ-CD. MB⁺ and BNAH were separately accommodated within the β-CD cavity and the cavity walls may protect the activity site of the reactants. On the other hand, in the MB⁺-BNAH-γ-CD system, the inclusion of the complex between MB⁺ and BNAH with γ-CD occurred. This effect of γ-CD can distinguish between the productive and non-productive nature of the complex.     

Study on the Inclusion Compounds of Eugenol with α-, β-, γ- and Heptakis (2,6-di-O-methyl)-β-cyclodextrins

Several host–guest inclusion compounds of eugenol as a guest molecule and cyclodextrins (α-,β-,γ-CD) and heptakis (2,6-di-O-methyl)-β-cyclodextrin (DMβ-CD) as hosts were investigated in the solid state and in aqueous solution. The one-to-one solid inclusion compounds of eugenol and β-CD or γ-CD were prepared, but those of eugenol with α- or DMβ-CD were not obtained under the same condition. However, the UV-visible absorption spectroscopy data indicated that the liquid guest could form a 1:1 inclusion compound with all four hosts respectively in aqueous solution. The two solid inclusion compounds were characterized by powder X-ray diffraction (XRD), infrared spectroscopy (IR), thermogravimetric analysis (TG), differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The association constants (K), calculated from the modified Benesi–Hidebrand equation, of eugenol with α-, β-, γ- and DMβ-CD is 4.95 × 10⁴, 3.96 × 10⁵, 1.47 × 10⁵ and 9.33 × 10⁴ mol⁻¹ dm³, respectively.