Data for antiradical properties of saffron extract and its bioactive constituents (crocins, crocetin) are limited and poorly discussed in comparison with those of extracts containing potent scavengers. Further examination was sought using the Folin-Ciocalteu (F-C) reagent and various free radical species produced in cell-free or cell model systems. Oregano and turmeric methanol extracts, rich in well established scavengers, and also crocetin, rosmarinic acid, and curcumin, representing the major types of constituents in the three studied extracts, were used as "reference". On the same weight basis, saffron extract activity was found to be rather negligible in all cell-free systems with regard to that found for reference ones. On the contrary, in the human monocyte system, saffron extracts or free crocetin were found to reduce ROS production as effectively as the phenolic antioxidants. Our findings point out that saffron extracts exhibit a remarkable intracellular antioxidant activity that cannot be revealed using assays repeatedly applied to the evaluation of phenolic-type antioxidants.

Examination of the crocin bleaching assay performance and in-house validation were focused on probe and test compound characteristics, conditions for peroxyl radical generation, reaction monitoring, and expression of results. HPLC and spectrometric examination showed that any authentic commercial saffron (origin, grade) can be used for probe preparation given that (a) interferences, such as tocopherols, are removed, (b) working solution concentration is adequately adjusted, and (c) stock probe solution changes during storage are not neglected. As suggested by log P values, calculated for a great number of radical scavengers (AHs), any AH more polar than Trolox (common reference compound) can be tested in the aqueous environment of the assay. AH activities order obeyed principles of structure-activity relationships. The assay was robust toward preheating of the azo-initiator (2,2'-azobis(2-aminopropane) dihydrochloride). Reaction monitoring through periodic UV-vis spectra recording was very informative. An alternative expression of results as "percent inhibition of crocin bleaching value", % Inh = [(DeltaA(0) - DeltaA)/DeltaA(0))] x 100, is proposed for [AH]/[crocin] = 1, instead of the so far used k(rel) values. The above findings also lead to analysis cost and time reduction.

Kinetics of individual crocetin ester degradation in aqueous extracts of saffron upon thermal treatment in the dark has been studied. Special attention has been paid to the comparison between saffron extracts and aqueous solutions of a crocetin ester rich fraction, with a lower stability of the latter observed. The degradation reaction was the same for all crocetin esters whether they were in saffron extracts or whether they were purified, although it was affected by external factors that modified their kinetic and thermodynamic parameters, making some of them less stable than others.

The scavenging behavior of a series of catechol and guaiacol acid derivatives toward DPPH(*) was examined having as a starting point the order of activity derived on the basis of theoretically calculated BDE values. The studied compounds were protocatechuic, homoprotocatechuic, dihydrocaffeic, and caffeic acids and also vanillic, homovanillic, dihydroferulic, and ferulic acids. Catechol and guaiacol were used as reference compounds. Observations from the parallel study were made with regard to structural features (number and position of hydroxyl groups and the side-carbon chain characteristics) that regulated the behavior of the compounds experimentally. The exceptional DPPH(*) scavenging behavior observed for homoprotocatechuic acid in ethanol and for caffeic acid in acetonitrile could not be supported by the respective BDE values. Ferulic was the most active among guaiacolic acids, whereas dihydroferulic exhibited the highest stoichiometry. Ionizable carboxylic groups seem to affect considerably the relative order of activity as was also evidenced using the ORAC assay. Questions raised about the validity of widely accepted views on criteria for SARs are discussed with regard to literature findings.

Near-infrared reflectance spectroscopy has been applied for the first time to saffron spice to determine the chemical composition and geographical origin of 111 samples from the there main producers' countries: Iran, Greece, and Spain. The validation procedures with the results obtained by UV-vis and HPLC-DAD measurements demonstrated that this technique is appropriate to determine the following parameters: moisture and volatile content, coloring strength, (250 nm), and (330 nm), established on the ISO 3632 Technical Specification Normative and used to certify saffron quality in the international market. Also, it can be used to estimate the content of the five main crocetin glycosides, the compounds responsible for saffron color, the best correlations being for trans-crocetin di-(beta-D-gentibiosyl) ester (R2= 0.93), trans-crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) (R2= 0.94), and picrocrocin (R2= 0.92), the compound accepted as responsible for saffron bitterness. Finally, a discriminant analysis among the three geographical origins reveals that Iranian samples are the most different, whereas Greek and Spanish samples are more similar. All of these results reveal that NIRS spectroscopy has an enormous potential for its application to saffron quality control as the results are obtained in 2 min and without any sample manipulation.

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The applicability of the crocin bleaching assay (CBA) to structure-activity relationship (SAR) studies of a great number (n = 39) of selected phenolic compounds was thoroughly investigated. The focus was on the activity of hydroxybenzoic, hydroxyphenylacetic, hydroxyphenylpropanoic, and hydroxycinnamic acids. Other assays [oxygen radical absorbance capacity (ORAC), lipid oxidation] were applied when necessary. Hydroxybenzoic acids were less active than the respective simple phenols. The position of the -COOH group relative to hydroxyl substituents was critical. The number and position of the -OH groups governed the order and size of activity within the subgroup of these acids. Gallic acid was the most active, being 1.6- and 3.4-fold superior to protocatechuic and syringic acids, respectively. The effect of proximity of the -COOH group to the phenyl ring was more distinct for 3,4-guaiacol acids (ferulic >> dihydroferulic congruent with homovanillic > vanillic) than for 3,4-catechol ones (caffeic >> protocatechuic > or = dihydrocaffeic congruent with homoprotocatechuic). Compounds such as vanillin, tyrosol, ferulic acid derivatives, rosmarinic acid, and quercetin were examined to reinforce discussion on the basis of physical organic chemistry principles. Taking into account the acidity of most compounds, the CBA-derived order of activity was meaningful.