3.7. Nitric Oxide Radical Scavenging Assay

The assay is based on the principle that sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent at 540 nm. The NO radical scavenging activity is often expressed as IC₅₀ value (Table 3). p-CA (IC₅₀ = 17 µmol/L) exhibits lower inhibition lipopolysaccharide-stimulated NO production in mouse macrophage like cells (RAW 264.7 cells) compared to CFA (IC₅₀ = 0.5 µmol/L) [118]. They also refer biological activities of p-CA and CFA to the enthalpy of O-H bond dissociation energy (BDA). The IC₅₀ value reported for RA was 43.49 µmol/L. RA showed 3.4× higher inhibition of nitric oxide radical production than Trolox [96].

The review of literature on the antioxidant capacity of NCA showed that information on RA, CFA and p-CA are more available, while on BA, CinA and ChA are very scarce. There are only a few papers in which antioxidant capacity of more than two out of six carboxylic acids described in this work were tested under the same experimental conditions [70,82,85,95,115,116]. Values obtained for four reviewed compounds are reported only in one paper [85]. Comparison of the results obtained using the same experimental protocol could also lead to misleading conclusions, because in the cited papers different concentrations of antioxidants were used. In many papers it was reported that increasing concentration of the tested compounds resulted in their higher reduction activity [84,120,124]. Moreover, the results of analysis could be influenced by the reaction time and the temperature. For instance, ABTS radical scavenging activity of CFA (RSA = 47.98%) obtained by Choi et al. [92] after 2 min of incubation was lower than the RSA value equal 92.9% obtained by Gülcin [76] after 30 min of incubation. The antioxidant activity of p-CA and CFA obtained by CUPRAC assay at room temperature [116] was higher than that achieved at enhanced (50 °C) temperature [66]. Moreover, different values were reported for the antioxidant activity of p-CA and CFA estimated by using the same experimental protocols of ABTS and DPPH [91,99] and by FRAP and CUPRAC methods [84]. It is obvious that the lack of standardized analytical methods for examination of antioxidant capacity and clear and comparable ways of expressing measurement results leads to the inconsistency of the reported data.

However, information gathered in this review allows to describe general trends and categorize the antioxidant activity of the tested compounds. Antioxidant capacity among plant-derived antioxidants decreases in the following order: RA > CFA > p-CA > CinA > BA. The data presented for ChA are very scarce, but it could be placed between p-CA and CFA or CFA and RA (according to the results obtained by DPPH and ABTS methods). It could be concluded that cinnamic acid is more effective antioxidant than its benzoic counterpart. Strong antioxidant properties of CFA and p-CA are due to a phenolic hydroxyl group that reacts with oxidants and free radicals to form the resonance-stabilized phenoxyl radical, and to the presence of a propenoic side chain, whose conjugated double bond could, by resonance, have a stabilizing effect on the phenoxyl radical. However, CFA exhibit stronger antioxidant capacity compared to p-CA, that could be explained by the intramolecular hydrogen bonding in ortho substituted phenols [78]. RA, the dimer of CFA, exhibited the highest antioxidant capacity among hydroxycinnamic acids, because it possesses four phenolic hydroxyl groups whose presence correlate with high antioxidant activity. Moreover, it has a conjugated structure, further stabilizing the aryloxyl radicals produced during the course of RA oxidation [116]. Additionally, ChA possess four phenolic hydroxyl groups and conjugated structure (tartaric acid ester of two caffeic acids) that contribute for the extension of the delocalization of electrons, enhance the stability of the phenoxyl radical, and improve its radical scavenging ability. However, its antioxidant activity is lower comparing to RA.