## 4 Discussion
In this work, we took into account previous results which demonstrated that the use of hot water, for the same extraction time, increased the extraction capacity of the solvent [19]. The use of hot water has two important critical issues: first, heating the water has a cost for industries; second, the thermolabile phenolic component is affected [51]. We showed that this is not always true, at least in terms of the yield of total polyphenols, flavonoids and proanthocyanidins. After 4 h of extraction in cold water or one hour of extraction in hot water at 95 \({}^{\circ}\)C, the extracts from the four Sardinian accessions contained statistically non-different quantities of TP and PA. Differently, for the marron, hot water was able to extract a greater quantity of total polyphenols and proanthocyanidins than cold water. In regard to the flavonoid content, exactly the opposite was observed. The DPPH, ABTS and FRAP tests for antioxidant capacity determination showed that the antioxidant activity of the Sardinian accessions was not influenced by the temperature of the aqueous solution. No significant differences were observed by the extraction at different temperatures for MURG, LOCG and ILDP accessions, while a higher antioxidant activity was measured for COEV and MARRV when the extraction was performed at 95 \({}^{\circ}\)C. The extract concentration of MURG, LOCG and ILDP that provide a 50% radical inhibition (EC\({}_{50}\)) were lower than that of COEV and MARV, and both if calculated with DPPH and ABTS. The extraction temperature did not affect the EC\({}_{50}\) of the Sardinian accessions, but affected the marron one, suggesting a higher antioxidant capacity of the hot extract of MARRV. Our results are of the same order of magnitude as those obtained by [PERSON] et al. [52] who carried out the extraction with an ethanol/water mixture at 50 \({}^{\circ}\)C; they are also in line with those of [PERSON] et al. [19] who, with extraction in water at 75 \({}^{\circ}\)C, obtained EC\({}_{50}\) values of 0.031 mg mL\({}^{-1}\) (DPPH method). Two other research groups, following extraction in hot water, obtained EC\({}_{50}\) average values higher than ours: 0.061 mg mL\({}^{-1}\) using water at 90 \({}^{\circ}\)C [23], and 0.027-0.078 mg mL\({}^{-1}\) by extracting with boiling water [22]. The electrochemical tests, however, showed a different trend: the antioxidant capacity of MURG extracts was higher at 20 \({}^{\circ}\)C, the one of LOCG and MARRV was higher at 95 \({}^{\circ}\)C, and ILDP and COEV did not statistically differ. A comparison between DPPH assay and CV cyclic voltammetry can be found in a previous study [24]: what the two works have in common, and what gives us a valid term of comparison, is the threshold of +0.5 V established a priori in order to discriminate the real antioxidant capacity of the extracts. [PERSON]'s group attributes two important limitations to the DPPH assay, long incubation times and difficulties with colored samples: we completely agree and, furthermore, we also believe that the DPPH and ABTS assays tend to overestimate the antioxidant capacity because they take into account species in solution, which oxidize at a potential greater than +0.5 V [33]. [PERSON] and [PERSON] [53], in a dissertation on advantages and pitfalls of methods for antioxidant activity evaluation, affirmed that the capacity of antioxidants to reduce the radical cation ABTS\({}^{\star}\) to ABTS is directly related to the reduction potential of the antioxidants, and only those with \(E^{0}\) values lower than that of the couple ABTS\({}^{\star+}\)/ABTS (\(E^{0}\) = 0.68 V) are able to perform this reduction. Obtaining a good correlation between spectrophotometric and electrochemical methods is possible if similar trends are observed, but this does not mean that comparable antioxidant capacity values are obtained.