Comparative evaluation of the effect of cyclodextrins and pH on aqueous solubility of apigenin.

The aqueous solubility of a flavonoid, apigenin, was studied in the presence of first generation cyclodextrins (α-CyD, ß-CyD, γ-CyD), ionic and nonionic synthetic derivatives of ß-CyD, namely SBE-ß-CyD, HP-ß-CyD and RM-ß-CyD at various physiological pH. The order of solubility enhancement was as follows: RM-ß-CyD>SBE-ß-CyD>γ-CyD>HP-ß-CyD>ß-CyD>α-CyD. The phase solubility diagrams of HP-ß-CyD and SBE-ß-CyD indicated Higuchi AL subtype behavior, suggesting 1:1 stoichiometry of the complex. In contrast, AP subtype, so higher order complex formation can be assumed in the case of RM-ß-CyD and γ-CyD. The formation of inclusion complexes has been confirmed by absorption and fluorescence spectroscopic measurements. Increased antioxidant activity was observed due to the inclusion complexes. These results prove that synthetic derivatives of ß-CyD will be potentially useful excipients in the development of drug delivery systems for healthcare products containing flavonoids.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin.

Nanoemulsions aimed at dermal drug delivery are usually stabilised by natural lecithins. However, lecithin has a high tendency towards self-aggregation and is prone to chemical degradation. Therefore, the aim of this study was to develop nanoemulsions with improved structure and long-term stability by employing a natural sucrose ester mixture as sole surfactant. A thorough comparison between the novel sucrose stearate-based nanoemulsions and corresponding lecithin-based nanoemulsions revealed that the sucrose ester is superior in terms of emulsifying efficiency, droplet formation as well as physical and chemical stability. The novel formulations exhibited a remarkably homogeneous structure in cryo TEM investigations, as opposed to the variable structure observed for lecithin-based systems. The in vitro skin permeation rates of lipophilic drugs from sucrose stearate nanoemulsions were comparable to those obtained with their lecithin-based counterparts. Furthermore, it was observed that addition of γ-cyclodextrin led to enhanced skin permeation of the steroidal drug fludrocortisone acetate from 9.99±0.46 to 55.10±3.67 µg cm(-2) after 24 h in the case of sucrose stearate-based systems and from 9.98±0.64 to 98.62±24.89 µg cm(-2) after 24 h in the case of lecithin-based systems. This enhancement effect was significantly stronger in formulations based on lecithin (P<0.05), which indicates that synergistic mechanisms between the surfactant and the cyclodextrin are involved. Cryo TEM images suggest that the cyclodextrin is incorporated into the interfacial film, which might alter drug release rates and improve the droplet microstructure.

Cyclodextrins as carriers for kavalactones in aqueous media: spectroscopic characterization of (S)-7,8-dihydrokavain and beta-cyclodextrin inclusion complex.

Kavalactones represent the active constituents of kava-kava (Piper methysticum G. Forster), endowed with sedative and anaesthetic properties. Kavalactones are polar constituents, but poorly soluble in water with a low bioavailability. In this study, the formation of inclusion complexes of one of the most representative kavalactone isolated from kava-kava extract, (S)-7,8-dihydrokavain (DHK), with beta-cyclodextrin (beta-CyD) was investigated mainly by spectroscopic methods. NMR experiments were extensively used for the complete characterization of the complex and included (1)H NMR complexation shifts analysis, (1)H NMR diffusion measurements (DOSY), and ROESY experiments. In particular DOSY experiments demonstrated that in the presence of beta-CyD the translational diffusion of kavalactone is sizably slowed down (2.5x10(-10)m(2)/s) with respect to the free drug (4.4x10(-10)m(2)/s) according to the inclusion of DHK in the cavity of (beta-CyD). ROESY experiments confirmed the inclusion of DHK in the hydrophobic pocket of beta-CyD through the primary hydroxyl rim, being the most relevant interactions between the H3' of beta-CyD and the ortho protons on the phenyl ring of the DHK, and between H5' of beta-CyD and the meta/para protons of DHK phenyl ring. The inclusion of the phenyl ring of DHK, leaving the lactone moiety outside of CyD was also confirmed by the induced CD effects. The binary solution DHK/beta-CyD shows a 50% intensity increase of the negative band of the pi-pi* transitions of the phenyl ring with respect to the absorption observed with DHK alone. Molecular dynamics simulations results corroborated and further clarify observed spectroscopic data. It was found that the phenylethyl substituent at C6 has a preferential equatorial position in the free state, and an axial one in the complex, justifying the large downfield shift experienced by H6 of DHK upon binding. Finally the influence of beta-CyD on water solubility of DHK was investigated by phase-solubility studies. In the range 2-4mM of host, solubility of DHK was increased only two-fold, but being beta-CyD also a penetration enhancer, in vivo studies will be performed to clarify a possible role of the complex on the bioavailability of DHK.

Fast method development and rapid analysis using a screening approach for enantiomeric separations in capillary electrophoresis.

In order to speed up the trial-and-error process during enantioselective capillary electrophoresis methods development, a systemized approach is proposed to develop methods by applying several screening methods in the search for an initial separation. Screening methods combine high selectivity with broad applicability and are applied to find an initial enantiomeric separation during early pharmaceutical development (pre-Phase 1 to Phase 1). The goal is to achieve enantiomeric separation rapidly in order to characterize the chiral purity of pharmaceutical products. Dedicated, highly efficient screening methods are suggested for basic, neutral, and acidic compounds. In these screening methods, multiple chiral selectors are applied in mixtures at different buffer pH values. For the compounds studied, the technique allows fast method development. Furthermore, it is potentially applicable to a wide range of low-molecular-weight compounds and permits rapid analysis at low cost, since runs are performed in inexpensive, bare silica capillaries using ordinary buffer systems with only typical cyclodextrins as the selector. Along with simplicity and robustness, the approach results in sufficient efficacy (i.e., it is easy, straightforward, and reproducible, with a high success rate). Typical pharmaceutical applications are described. The major advantage of the screening approach to methods development is the decrease in development cycle time. The total screening time for one compound was about 5.3 hr on one CE instrument.

[Study on the inclusions of xindonin A-beta-cyclodextrin and xindonin B-beta-cyclodextrin].

The inclusions compounds of Xindonin A-beta-cyclodextrin and Xindonin B-beta-cyclodextrin were prepared by the liquid-phase method in aqueous solution. They were confirmed by thin layer chromatography, differential thermal analysis and specific rotation. Changes of ultraviolet spectrum were determined. The composition ratios of inclusion compounds were obtained by the continuous variation method. Solubility of the inclusions were determined.

Inducing cholesterol precipitation from pig bile with beta-cyclodextrin and cholesterol dietary supplementation.

BACKGROUND/METHODS: In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile. RESULTS: Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0<<10<5% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0<5<10% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet. CONCLUSIONS: We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.