Four cyclodextrins (CD) including β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), heptakis-O-(2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), and heptakis-O-(2, 6-di-O-methyl)-β-cyclodextrin (DM-β-CD) were used as solubilizer to study the solubility enhancement of myricetin. The results of the phase solubility study showed that the presence of CDs could enhance the solubility of myricetin by forming 1:1 complexes. Among all CDs, HP-β-CD had the highest solubilization effect to myricetin. The concentration of myricetin could be 1.60 × 10⁻⁴ moL/L when the presence of HP-β-CD reached 1.00 × 10⁻² moL/L, which was 31.45 times higher than myricetin’s aqueous solubility. Subsequently, the HP-β-CD:myricetin complex was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). In order to get an insight of the plausible structure of the complex, molecular docking was used to study the complexation process of HP-β-CD and myricetin. In the complex, the A ring and C ring of myricetin were complexed into the hydrophobic cavity of HP-β-CD, while the ring B was located at the wide rim of HP-β-CD. Four hydrogen bonding interactions were found between HP-β-CD and -OH groups of the guest in the HP-β-CD: myricetin complex. The complexation energy (△E) for the host-guest interactions was calculated with a negative sign, indicating the formation of the complex was an exergonic process. A 30-ns molecular dynamics simulation was conducted to the HP-β-CD: myricetin complex. Calculation results showed that no large structural deformation or position change were observed during the whole simulation time span. The average root-mean-square deviation (RMSD) changes of the host and guest were 2.444 and 1.145 Å, respectively, indicating the complex had excellent stability.

Myricetin (MYR) and myricitrin (MYT) are well recognized for their nutraceutical value, such as antioxidant, hypoglycemic, and hypotensive effects. In this work, fluorescence spectroscopy and molecular modeling were adopted to investigate the conformational and stability changes of proteinase K (PK) in the presence of MYR and MYT. The experimental results showed that both MYR and MYT could quench fluorescence emission via a static quenching mechanism. Further investigation demonstrated that both hydrogen bonding and van der Waals forces play significant roles in the binding of complexes, which is consistent with the conclusions of molecular modeling. Synchronous fluorescence spectroscopy, Förster resonance energy transfer, and site-tagged competition experiments were performed to prove that the binding of MYR or MYT to PK could alter its micro-environment and conformation. Molecular docking results revealed that either MYR or MYT spontaneously interacted with PK at a single binding site via hydrogen bonding and hydrophobic interactions, which is consistent with the results of spectroscopic measurements. A 30 ns molecular dynamics simulation was conducted for both PK-MYR and PK-MYT complexes. The calculation results showed that no large structural distortions or interaction changes occurred during the entire simulation time span. The average RMSD changes of PK in PK-MYR and PK-MYT were 2.06 and 2.15 Å, respectively, indicating excellent stability of both complexes. The molecular simulation results suggested that both MYR and MYT could interact with PK spontaneously, which is in agreement with spectroscopic results. This agreement between experimental and theoretical results indicates that the method herein could be feasible and worthwhile for protein–ligand complex studies.

Four cyclodextrins (CDs) including heptakis-O-(2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), heptakis-O-(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD) were evaluated for their ability to enhance the aqueous solubility of kaempferol (Kae). Phase solubility studies indicated that these four CDs can form 1:1 type complexes with Kae and that HP-β-CD demonstrated the most significant solubilizing effect on Kae. Among the CDs tested, HP-β-CD demonstrated the most significant solubilizing effect on Kae. With an HP-β-CD concentration of 5.00 × 10⁻³ mol·L⁻¹, the concentration of Kae reached 4.56 × 10⁻⁵ mol·L⁻¹, which is 12.7 times greater than its solubility in water. Characterization of the HP-β-CD/Kae complex was performed using empirical methods. Molecular docking indicated that the A and C rings of Kae fit into the hydrophobic cavity of HP-β-CD, while the B ring remained at the rim. Six hydrogen bonds were found between HP-β-CD and the -OH groups of Kae. The negative complexation energy (ΔE) suggests the complex formation was exergonic. A 30-ns molecular dynamics simulation revealed no significant structural changes, with average root-mean-square deviation RMSD values of 2.230 Å for HP-β-CD and 0.786 Å for Kae, indicating high stability of the complex.

The effect of anions on the positive electrospray ionization (ESI) of proteins in different strong acids with varying pH values from 3 to 1 is studied using high-pressure ESI. Reducing the pH from ∼2 to 1 caused a drastic shift in charge state from a high-charge-state distribution (HCSD) to a narrow low-charge-state distribution (LCSD). The shift in charge state was consistent with the circular dichroism result that showed a conformational change due to the “acid-induced folding” of proteins from an unfolding state to a compact molten globule state. Acids of different anions produced noticeable differences in the average charge for HCSD and LCSD. For HCSD, the average charge was lower than the value typically observed using formic and acetic acids. As for LCSD, the average charge was lower than the “native” charge. The high abundance of acid anion that induces the protein compaction was believed to play a role in charge reduction. The effectiveness of anions to “refold” a highly unfolded protein to a compact state and the propensity to reduce the charge of HCSD for proteins appeared to follow the selectivity series of anions towards the stationary phase in ion chromatography. However, the propensity of anions to reduce the charge for LCSD follows quite an opposite trend. The presence of ammonium salt in the acidic solution was found to increase the charge of LCSD. The simple mass spectrum with a narrow distribution of charge state obtained with perchloric acid at pH 1 was demonstrated to facilitate the counting of basic sites.