Screening for antiradical efficiency of 21 semi-synthetic derivatives of quercetin in a DPPH assay

The group of 21 novel semi-synthetic derivatives of quercetin was screened for the antiradical efficiency in a DPPH assay. The initial fast absorbance decrease of DPPH, corresponding to the transfer of the most labile H atoms, was followed by a much slower absorbance decline representing the residual antiradical activity of the antioxidant degradation products. Initial velocity of DPPH decolorization determined for the first 75-s interval was used as a marker of the antiradical activity. Application of the kinetic parameter allowed good discrimination between the polyphenolic compounds studied. The most efficient chloronaphthoquinone derivative (compound Ia) was characterized by antiradical activity higher than that of quercetin and comparable with that of trolox. Under the experimental conditions used, one molecule of Ia was found to quench 2.6±0.1 DPPH radicals.


Chemicals
Samples of new semi-synthetic derivatives of Qc Ia-Ir ( Figure 1) were synthesized by reaction of appropriate acyl chloride with Qc or the corresponding protected derivative and then purified by repeated column chromatography of the rich reaction mixture. Qc was oxidized to generate heterodimer IIa (Figure 2). Diquercetin was treated with an anhydride to yield corresponding acyl derivatives IIb-IIc (Figure 2; Veverka et al. 2013).
1,1`-Diphenyl-2-picrylhydrazyl (DPPH) radical was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were purchased from local commercial sources and were of analytical grade quality.

DPPH test
To investigate the antiradical activity of the compounds studied, the ethanolic solution of DPPH (50 μM) was incubated in the presence of the given compound tested (50 μM) at laboratory temperature. The absorbance decrease, recorded at λ max = 518 nm, during the first 75-s interval was taken as a marker of the antiradical activity.  interval used, an approximately linear decrease of DPPH absorbance was observed, which was considered as a good assessment of the initial velocity of the radical reaction. The stoichiometry of the radical reaction was determined by spectrophotometric titration of the ethanolic solution of DPPH (50 μM) by increasing concentrations of an antioxidant with the reaction time long enough for completion of the reaction as indicated.
The radical studies were performed at the laboratory temperature.

Results and discussion
As a weak hydrogen atom abstractor, DPPH is considered a good kinetic model for peroxyl ROO· radicals (Blois, 1958;Ratty et al., 1988). DPPH assay is routinely used as a primary screening test of antiradical efficacy. Figure 3 shows UV-VIS spectra of DPPH and compound Ia with characteristic absorbance maxima and their timedependent changes during the first 75 sec after mixing the reactants. The time-dependent decrease of the characteristic absorbance of the ethanolic solution of DPPH at 518 nm in the presence of Qc and one of its derivatives, Ia, is illustrated by Figure 4. As shown in Figure 4, the initial fast absorbance decrease, corresponding to the transfer of the most labile H atoms, is followed by a much slower absorbance decline representing the residual antiradical activity of the antioxidant degradation products. The initial velocity of DPPH decolorization determined for the first 75-s interval was used as a marker of antiradical activity. Based on the kinetic parameter the compounds studied were arranged according to their decreasing activity in comparison with the parent Qc and standard trolox, as shown in Table 1. It is apparent that a group of six new derivatives (Ia-Ie, IIa) exert antioxidant activity comparable with that of Qc and even slightly higher. The antiradical efficacy of the most efficient chloronaphthoquinone derivative Ia was found comparable with that of the standard trolox. The results indicate that application of the initial velocity of DPPH decolorization allows good discrimination between the polyphenolic compounds studied. In addition, the kinetic parameter is considered to be of primary importance in antioxidant evaluation since fast reaction with low concentrations of short-living damaging radicals is of utmost importance for antioxidant protection. Other authors applied the kinetic approach to rank flavonoids according to their antioxidant efficacy (Goupy et al. 2003;Butkovic et al. 2004;Villano et al. 2007).
In general, the antioxidant efficacy is characterized not only by kinetics of free radical quenching but also by stoichiometry of the scavenging reaction. So for the most efficient chloronaphthoquinone derivative Ia, the total stoichiometry of DPPH scavenging was determined in comparison with the parent Qc. The technique of spectrophotometric titration of fixed concentration of DPPH (50 μmol/l) with increasing concentrations of the antioxidant was used to determine the point of equivalence. In this approach the reaction time was set long enough to let the reaction run to completion. Fig. 5 shows the absorbance decrease of the ethanolic solution of DPPH radical in the presence of increasing concentrations of the compounds tested. By analyzing the titration curves, points of equivalence were determined and  corresponding stoichiometric factors were calculated. Under the experimental conditions used, one molecule of Ia was found to quench 2.6±0.1 DPPH radicals, while one molecule of Qc scavenged 5.5±0.2 DPPH radicals. The high stoichiometric ratio found for Qc is in agreement with findings of other authors (Goupy et al. 2003;Villano et al. 2007;Markovic et al. 2012) and indicates high antiradical activity of its decomposition products which is in contrast to compound Ia. To conclude, by using a DPPH assay, 21 novel derivatives of Qc were ranked according to their antiradical efficacy in comparison with the parent Qc and the standard trolox. For the most efficient derivative, stoichiometry of DPPH scavenging was determined.