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Physico-Chemical Properties of Polyphenols Characterized by Reversed-phase Thin Layer Chromatography

The number of research papers, reviews and commercially available software packages relating to the prediction of absorption, distribution, metabolism and elimination (ADME) of pharmaceutical compounds has increased dramatically in recent years. Molecular lipophilicity is a major physico-chemical property, which has impact on oral absorption, as well as permeability, cell uptake, protein binding, blood-brain penetration and metabolism of bioactive substances. The ability to predict drug absorption through the gastro-intestinal barrier is a key issue in the selection of new drug candidates for oral delivery. Passive diffusion is the major mechanism for drug uptake through the intestinal epithelium, driven by a concentration gradient. Lipophilic compounds cross the plasma membrane easily and therefore are mainly transported transcellularly. Cell membrane is relatively impermeable for hydrophilic compounds, and they are transported predominantly via the paracellular route. Besides, excessive lipophilicity is common cause of poor solubility of substances and can lead to incomplete absorption after oral administration. Futhermore, it is generally belived that very lipophilic compounds have greater affinity for plasma-protein binding and are easily transported across blood-brain barrier. Widespread applications of lipophilicity to model biological processes explains the need for rapid and valid procedures to quantify that physico-chemical property. The aim of this work was to investigate the chromatographic behavior of polyphenols (flavonoids and phenolic acids) by reversed-phase thin-layer chromatography (RP-TLC), in order to establish the relationship between chromatographic indices and selected ADME parameters. RP-TLC is rapid, easy to perform technique, which requires small quantities of the sample and allows simultaneous analysis of several compounds. Moreover, RP-TLC allows an easy measurement of a variety of indices (RMw and φ 0), which provide information on the lipophilic behavior of compounds. Chromatography was performed on 10 x 20 cm glass TLC plates precoated with RP-18F254s stationary phase (Merck, Germany). Binary solvent system, methanol – phosphate buffer, was used as a mobile phase. pH of the mobile phase was at least 2 log units lower than pKa values of all investigated compounds (in order to avoid pKa correction, phosphate buffer pH = 2.5 with ionic strength 36 mM was used). Content of organic modifier was varied from 30 – 80% (v/v) in 5% increment. 5 μ L of standard solutions were applied as 8 mm bands by means of 100 μ L microsyringe (Hamilton, USA), mounted on a Linomat 5 applicator (Camag, Switzerland). Ascending development was performed at the room temperature in the saturated, glass chamber (Desaga, Germany). Plates were developed to the distance of 80 mm and after development were dried at the room temperature. Detection was performed at λ = 254 nm using TLC Scanner 3 (Camag, Switzerland). Data acquisition and processing were performed with Wincats software v1.4 (Camag, Switzerland). ADME parameters of selected polyphenols were calculated using several computer programs that cover the main currently used theoretical approaches: CLOGP, KowWin, molinspiration, MolSoft, XLOGP, OSIRIS, DRAGON, ALOGPS v2.1, ChemSilico, Interactive Analysis, VEGA ZZ and SPARC v3. Correlations between RMw and φ 0 values, obtained by RP-TLC, and calculated ADME parameters were analyzed. The solubility parameters calculated with the ALOGPS (r = 0.934), ChemSilico (r = 0.936), Osiris (r = 0.964) and MolSoft (r = 0.965) programs correlate very well with RMw values of the investigated polyphenols. In general, aqueous solubility decreases with increasing RMw values. According to correlation between RMw values and plasma-protein binding data predicted by ChemSilico program, RMw is a useful parameter for evaluation of polyphenols´ plasma-protein binding properties (r = 0.937). Correlations between RMw and calculated log P values are surprisingly poor. The best correlation is found for OSIRIS program (r = 0.890). Comparing the calculated log P values with φ 0 values, obtained by chromatographic analysis, better correlations are found for molinspiration (r = 0.925) and ChemSilico (r = 0.907). Apparently, the φ 0 parameter is more suitable for determination of the polyphenols´ lipophilicity than RMw. Moderate correlations are found between chromatographic indices (RMw and φ 0) and other predicted ADME parameters (polar surface area, blood-brain barrier penetration and human intestinal absorption).

ADME; RP-TLC; flavonoids

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