engleski

Lipophilicity characterization of flavonoids by reversed-phase high performance liquid chromatograhy

Flavonoids are a broad class of low molecular weight secondary plant phenolics, almost ubiquitous in plant kingdom. Those polyphenolic compounds have the diphenylpropane (C3C6C3) skeleton. Different positions of hydroxylation of main (flavan) nucleus result in variety of flavonoid structures. Over 4000 structurally unique flavonoids have been identified until now. Numerous positive health effects of those compounds have been described, such as antioxidant, antiviral, antimicrobic, antiimflamatory and immunomodulatory. They have been proposed to exert beneficial effects in a multitude disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. Many conducted studies, in which listed pharmacological activities have been investigated, have ignored the questions of flavonoids' achievable concentrations in the circulation after ingestion, together with their distribution, excretion, and metabolism in human organism. Recent papers demonstrate that their bioavailability is much greater than previously believed, but existing data are still unconclusive. Molecular lipophilicity is physico-chemical property, which describes oral absorption, cell uptake, protein binding, blood-brain penetration, metabolism and toxicity (ADME/Tox processes) of the bioactive substances. The reversed-phase HPLC method is a promising alternative to other experimental methods, having such advantages as a higher throughput, an insensitivity to impurities or degradation products and a broader lipophilicity range. In our work we have investigated the lipophilicity of fourteen different flavonoids using RP-HPLC method: quercetin, hesperetin, galangin, pinocembrine (purchased from Sigma-Aldrich), flavone, morin, myricetin, chrysin, kaempferol, apigenin, acacetin, luteolin, naringenin (purchased from Fluka) and flavanone (purchased from ChromaDex). All compounds were HPLC grade. The HPLC measurements were performed using Agilent 1100 LC System. The column used was ZORBAX SB-C18, 4.6 mm x 150 mm, particle size 3.5 μ m (Agilent Technologies). Analytical Guard Column SB-C18, 4.6 mm x 12.5 mm, particle size 5 μ m was purchased from Agilent Technologies. The binary solvent system, methanol (gradient grade for liquid chromatography, Merck, Germany) – 50 mM phosphate buffer pH = 2.5 (gradient grade for liquid chromatography, Merck, Germany), was used as a mobile phase with a varying content of organic modifier (80 - 25%, increment 5%). The solvents were filtered with sterile membrane filter (0.45 μ m, Whatman, England). The flow-rate was 1 mL/min at constant temperature (25º ; C). The samples were prepared as solutions of 0.1 mg/mL in methanol and the injection volume was 5 μ L. The void volume was measured using acetone (Kemika, Croatia). The optimum wavelength was set for each flavonoid, according to maximum absorption in the UV spectra in the range 200 - 450 nm. The linear regression analysis was carried out by STATISTICA v6.0 software package (StatSoft). The capacity factors were calculated from experimentally determined retention data: k = (tr – t0)/t0, where tr and t0 are the retention times of the solute and of an unretained compound, respectively. The logarithm of capacity factors ranged from – 1.088 to 1.768. The linear relationship between the retention parameter (log k) and the organic modifier concentration (φ o.m.) in the mobile phase was extrapolated to pure water as mobile phase (log kw). To test the linearity of regression curves following statistical parameters were used: number of data points (n), correlation coefficient (r), Means Squares Residual (MS) and F-ratio between the variances of observed and calculated values (F). All experiments were performed in triplicate and the mean log kw values were used in further calculations (log k qurecetin = 3.2499, log k morin = 3.0792, log k myricetin = 2.9644, log k kaempferol = 3.3741, log k apigenin = 3.4086, log k acacetin = 3.7861, log k chrysin = 3.7326, log k flavone = 3.6695, log k flavanone = 3.7342, log k galangin = 3.9095, log k pinocembrine = 3.6711, log k naringenin = 3.1530, log k hesperetin = 3.3437 and log k luteolin = 3.2499). Standard deviations for all extrapolated values were below 0.03. The extrapolated values characterize the partition of the compound between the non-polar stationary phase and water. Log P values were calculated using different computer programs based on data for either molecular fragments or atomic contributions, or on molecular properties (ALOGPs v2.1, IAlogP, CLOGP, miLogP, KowWin, XLOGP v2.0, ChemSilico, MLOGP, HyperChem v7.0, Osiris clogP and SPARC). Calculated log P values in most cases were lower than the extrapolated capacity factors. The best correlation with extrapolated capacity factors was found for CLOGP program (n = 14, r = 0.86, MS = 0.0244 and F = 34.8785).

lipophilicity; RP-HPLC; flavonoids

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