Functional mechanism of tracheal relaxation, antiasthmatic, and toxicological studies of 6-hydroxyflavone.

Previously, we described tracheal rat rings relaxation by several flavonoids, being 6-hydroxyflavone (6-HOF) the most active derivative of the series. Thus, its mechanism of action was determined in an ex vivo tracheal rat ring bioassay. The anti-asthmatic effect was assayed in in vivo OVAlbumin (OVA)-sensitized guinea pigs. Finally, the toxicological profile of 6-HOF was studied based on Organization of Economic Cooperation and Development guidelines with modifications. 6-HOF-induced relaxation appears to be related with receptor-operated calcium channel and voltage-operated calcium channel blockade as the main mechanism of action, and also through the production of relaxant second messengers NO and cGMP. Molecular docking supports that 6-HOF acts as calcium channel blocker and by activation of nitric oxide synthase. In addition, the in vivo anti-asthmatic experiments demonstrate the dose-dependent significant anti-allergic effect of 6-HOF induced by OVA, with best activity at 50 /kg. Finally, toxicological studies determined a LD50 > 2,000 mg/kg and, after 28 day of treatment with 6-HOF (50 mg/kg) by intragastric route, mice did not exhibit evidence of any significant toxicity. In conclusion, experiments showed that 6-HOF exerts significant relaxant activity through calcium channel blockade, and possibly, by NO/cGMP-system stimulation on rat trachea, which interferes with the contraction mechanism of smooth muscle cells in the airways. In addition, the flavonoid shows potential anti-asthmatic properties in an anti-allergic pathway. Furthermore, because the pharmacological and safety evidence, we propose this flavonoid as lead for the development of a novel therapeutic agent for the treatment of asthma and related respiratory diseases.

Biotransformations of Flavones and an Isoflavone (Daidzein) in Cultures of Entomopathogenic Filamentous Fungi.

Entomopathogenic filamentous fungi of the genus Isaria are effective biocatalysts in the biotransformation of flavonoids as well as steroids. In the present study, the species Isariafumosorosea and Isariafarinosa isolated from the environment were used. Their catalytic capacity to carry out biotransformations of flavones-unsubstituted, with hydroxy- and amino-substituents as well as a hydroxylated isoflavone-was investigated. Biotransformations of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and daidzein resulted in the formation of O-methylglucosides, in the case of flavone and 5-hydroxyflavone with additional hydroxylations. 7-Aminoflavone was transformed into two acetamido derivatives. The following products were obtained: From flavone⁻flavone 2'-O-ß-d-(4''-O-methyl)-glucopyranoside, flavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside and 3'-hydroxyflavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside; from 5-hydroxyflavone⁻5-hydroxyflavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside; from 6-hydroxyflavone⁻flavone 6-O-ß-d-(4''-O-methyl)-glucopyranoside; from 7-hydroxyflavone⁻flavone 7-O-ß-d-(4''-O-methyl)-glucopyranoside; from daidzein⁻daidzein 7-O-ß-d-(4''-O-methyl)-glucopyranoside; and from 7-aminoflavone⁻7-acetamidoflavone and 7-acetamido-4'-hydroxyflavone. Seven of the products obtained by us have not been previously reported in the literature.

Influence of amide versus ester linkages on the anticancer properties of the new flavone-biotin conjugates.

Novel biotinylated C-6 substituted flavones were synthesised by a one-step method that connects biotin to 6-hydroxyflavone and 6-aminoflavone by esterification and amidation of hydroxyl and amino groups, respectively. The obtained compounds, 6-O-biotinylflavone and 6-biotinylamidoflavone, are the bifunctional molecules composed of a flavone moiety as a fluorescent reporter and biotin as a cancer-targeting unit. Antiproliferative activity was evaluated using SRB assays in MCF-7, MCF-10A, HepG2, MDA-MB-231, 4T1, and Balb/3T3 cell lines. In vitro evaluation revealed that compounds with biotin moiety displayed better cell selectivity between the cancer and normal cells than the parental substrates. These results indicate that anticancer effect is not related to the position of biotin moiety, but it is related to the presence of ester or amide bond. 6-O-Biotinylflavone was more active than 6-hydroxyflavone against human breast (MDA-MB-231) and liver (HepG2) cancer cells with IC50 (concentration of tested agent that inhibits proliferation of the cell population by 50%) values equal to 78.5 ± 18.8 µM and 133.2 ± 14.2 µM, respectively. Non biotinylated 6-aminoflavone was more active than 6-biotinylamidoflavone against all tested cell lines, with IC50 values between 34.3 ± 9.1 µM (4T1) and 173.86 ± 24.3 µM (MCF-7).

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies.

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV-vis absorption studies. The binding constant (Kb) of the HEWL-6HF complex was observed to be 6.44 ± 0.09 × 104 M-1 at 293 K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = -11.91 ± 1.02 kJ mol-1) along with a positive entropy change (ΔS = +51.36 ± 2.43 J K-1 mol-1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster's theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL. Communicated by Ramaswamy H. Sarma.

Characterization of non-covalent binding of 6-hydroxyflavone and 5,7-dihydroxyflavone with bovine hemoglobin: Multi-spectroscopic and molecular docking analyses.

Flavonoids are biologically imperative compounds used as anti-oxidants, anti-cancer, anti-bacterial agents etc. The current work reports comprehensive binding studies of two important flavonoids, 6-hydroxyflavone and 5,7-dihydroxyflavone (chrysin) with bovine hemoglobin (BHb) at 298K and 308K, in aqueous medium using UV-vis spectroscopy, steady state fluorescence, circular dichroism (CD) measurements, Fourier Transform infrared spectroscopy (FT-IR) and molecular docking studies. Both 6-hydroxyflavone and chrysin can quench the intrinsic fluorescence intensity of BHb via static quenching mechanism. The values of binding constant (Kb) for BHb-chrysin complex (3.177±0.992×104M-1, at 298K) was found to be greater than that of BHb-6-hydroxyflavone complex (2.874±0.863×104M-1, at 298K) and the Kb values decreased with the rise in temperature. The thermodynamic parameters indicated that hydrophobic forces and H-bonding play crucial role in BHb-6-hydroxyflavone complexation whereas electrostatic interaction plays the major role in the binding of BHb and chrysin. The binding distances from donor BHb to the acceptor ligands (6-hydroxyflavone and chrysin) were estimated using the Föster's theory and the possibility of non-radiative energy transfer from BHb to 6-hydroxyflavone/chrysin was observed. The ligands, 6-hydroxyflavone and chrysin induced conformational change around Trp residues in BHb as confirmed by synchronous and 3D fluorescence results. CD and FT-IR studies indicated that the % α-helicity of BHb was enhanced due to 6-hydroxyflavone/chrysin binding. Both the flavonoids showed remarkable inhibitory effect towards BHb glycation. Hydrophobic probe (8-anilino-1-naphthalenesulfonic acid, ANS) displacement and molecular docking studies revealed that the ligands bind within the hydrophobic pocket of BHb.