Φαινολικά οξέα ως φυσικοί παρεμποδιστές οξείδωσης: αξιολόγηση δραστικότητας, προοπτική αξιοποίησης σε λειτουργικά τρόφιμα

Abstract

Structure-radical scavenging activity relationships (SAR) among a great number of acid phenols and related compounds were studied using the Crocin Bleaching Assay (CBA) and/or other assays (DPPH•, ORAC, lipid oxidation), where necessary. The focus was on the activity of various hydroxybenzoic, hydroxyphenylacetic, hydroxyphenylpropanoic and hydroxycinnamic acids. Prior to estimation of antioxidant activity, the CBA protocol was in-house validated taking into account that reliability of SAR evaluation is often subjected to method limitations (e.g. ease and reproducibility). Consistency of experimental findings with Physical Organic Chemistry principles was also taken into account. A step by step chromatographic (HPLC, GC) and spectrophotometric examination of method performance with regard to probe, azo-initiator and test compound characteristics showed that: a) any authentic commercial saffron (origin, grade) can be used for probe preparation as long as certain precautions are taken (removal of interference, adjustement of crocin concentration, short-time storage of probe solution), b) AAPH-derived radical generation conditions such as preheatment are not critical, c) any phenolic compound (AH) meeting the criterion Log PAH < Log PTrolox may be examined under the current CBA conditions. Reproducibility of CBA measurements throughout a long period of analysis was quite satisfactory. Reaction monitoring through periodic UV-Vis spectra recording was very informative concerning the specificity of the probe solution. Using the ratio [AH]/[Crocin]=1, the “% Inhibition of crocin bleaching value”: % ( ) 100 0 0 ? Δ Δ ? Δ = A Inh A A was calculated instead of the so far used krel values. In this case the analysis time was considerably reduced indicating the suitability of the alternative way of expression for routine analysis. Based on both ways of expression, the activity order between structurally related AHs obeyed classical SAR criteria (e.g. number of phenolic –OHs). A more systematic approach within a series of simple hydroxybenzoic acids differing in the number and position of –OH and/or –OMe substituents indicated a similar trend with that evaluated within a series of phenols. Electronic effects of ring substituents seemed to play a key role to the relative activity of acids belonging to the same group. Thus, gallic acid was the most active one. The position of the –COOH moiety relative to the –OH groups was critical for the activity of isomer acids, as expected based on steric phenomena. Hydroxybenzoic acids were somewhat less active than their parent phenols. The importance of proximity of the –COOH group to the phenyl ring was not clarified. Small differences in the activity of guaiacol acid derivatives indicated a greater potential of phenylpropanoic and phenylacetic acids to that of benzoic acid (dihydroferulic ? homovanillic > vanillic acid). However, the activity order between the respective catechol acid derivatives strongly varied depending on the assay conditions. Homoprotocatechuic acid (-CH2COOH) was most active towards DPPH• in methanol while protocatechuic acid (–COOH) was most active under CBA conditions. CBA findings were meaningful when the acidity of –COOH and 4-OH groups were taken into account. Under all assay conditions, extended conjugation to the side carbon chain of 4- hydroxycinnamic acids was essential for the rapid scavenging of free radicals. Thus, caffeic and ferulic acids were considerably more active than their dihydrocounterparts. Apart from a better stabilization of the phenoxy radicals through resonance structures, the strong acidity of these compounds may contribute to their potency. The results indicate that CBA is suitable for evaluation of SARs among closely related compound bearing small structural differences. The importance of simple plant-derived acid phenols as efficient radical scavengers under conditions that imitate physiological ones is also highlightened. Investigation of the role of the – COOH group showed that reduction to –CHO or esterification with short-chain alcohols may render acid phenols even more potent towards peroxyl radicals. The greater antiradical activity found for some acid derivatives such as alkyl gallates (methyl- and propyl-gallate) and hydroxybenzaldehydes (vanillin, syringic aldehyde), also less hydrophilic than the respective acids, is promising for further studies and exploitation of natural sources.