Preformulation and mechanistic studies on inclusion complexes of selected flavonoids with beta-cyclodextrin and its water-soluble derivatives

The present project aimed to investigate the feasibility of utilizing unsubstituted beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) to enhance the stability and aqueous solubilities of quercetin and other selected bioactive flavonoids (as a possible means of improving their bioavailability) and to elucidate the modes of complexation between the beta-CDs and flavonoids. Being important determinants in subsequent formulation development work, the antioxidant activity of the flavonoids and the impact of the beta-CDs on intestinal membrane permeability were also studied. Antioxidant activity was determined using an in vitro Cu2+-mediated low-density lipoprotein (LDL) oxidation assay. Phase solubility analysis, stability assessment and solution nuclear magnetic resonance (NMR) spectroscopy were employed in conjunction with molecular modeling to establish a flavonoid/beta-CD inclusion complex model. Spray-dried complexes of quercetin with HP-beta-CD/SBE-beta-CD were prepared and characterized by laser-diffraction particle sizing, scanning electron microscopy, BET nitrogen adsorption, thermal analysis, powder X-ray diffractometry, and dissolution testing. Effects of beta-CDs on transport of selected markers through rat intestinal epithelium were determined using an Ussing Chamber. Antioxidative activity determination demonstrated that the presence of two adjacent hydroxyl groups in the B-ring of the flavonoid structure is essential for antioxidative activity while other potential reactive sites e.g. the keto group at C4, and the double bond between C2 and C3, are least important. Phase solubility and stability studies revealed that the solubility- and stability-enhancing effects of beta-CDs on quercetin follow the rank order: SBE-beta-CD > HP-beta-CD > beta-CD. Replacement of the proton in the OH group by a sugar moiety and loss of the planar structure in flavonoids tends to reduce their binding to beta-CDs. An inclusion complex model deduced from stability, NMR and molecular modeling data suggested that the B-ring, C-ring and at least part of the A-ring of the flavonoids (except C6) are enclosed within the beta-CD hydrophobic cavity. Physical characterization studies indicated that the spray-dried complexes are amorphous and hygroscopic, consistent in particulate properties between batches and highly water-soluble. Intestinal permeability measurements showed no significant impact on both transcellular and paracellular absorption routes with all three beta-CDs, attesting to their role as carriers rather than penetration enhancers for the substrates.